WO2017026718A1 - Novel 3-(isoxazol-3-yl)-pyrazolo[3,4-d]pyrimidin-4-amine compound, which is ret kinase inhibitor - Google Patents

Abstract

The present invention relates to a novel 3-(isoxazol-3-yl)-pyrazolo[3,4-d]pyrimidin-4-amine compound, a preparation method for the compound, and a pharmaceutical use of the compound to be used in the prevention and treatment of various cancer diseases caused by the activity of RET kinases.

Description

Novel 3- (isoxazol-3-yl) -pyrazolo [3,4-di] pyrimidin-4-amine compounds as RET kinase inhibitors

The present invention relates to novel 3- (isoxazol-3-yl) -pyrazolo [3,4-d] pyrimidin-4-amine compounds, methods for preparing the compounds, and various cancer diseases caused by RET kinase activity of the compounds. It relates to a medicinal use for the prevention and treatment of medicament.

RET (Rearranged during transfaction) kinase is one of the tyrosine kinase receptors (RTK; Receptor tyrosine kinase) required for the development of sympathetic and parasympathetic nerves. It has been reported that the RET gene causes various types of thyroid cancers (MEN2A, MEN2B, FMTC, PTC, etc.) when they have abnormal functions such as point mutations, chromosomal inversions and gene amplification. Among the thyroid cancers, MEN2A, MEN2B, and FMTC are caused by point mutations of the RET gene, and PTC has been reported that the RET gene is caused by internal chromosomal translocation. [Non-Patent Document 1. J. Med . Chem . 2012, 55, 4872-4876]

Recently, RET kinase has attracted attention as a lung cancer causing factor. [Non-Patent Document 2. Genome Res 2012, 22, 436-445] Non-small cell lung cancer is induced by the expression of the fusion type tyrosine kinase KIF5B-RET or CCDC6-RET with cancerous ability by fusion of the RET gene with the KIF5B gene or the CCDC6 gene. It has been reported. In addition, in non-small cell lung cancer patients, fusion tyrosine kinases NCOA4-RET and TRIM33-RET, in which the RET gene is fused with NCOA4 or TRIM33 (tripartite motif-containing 33) gene, have been reported. [Non-Patent Document 3. Cancer Discov 2013 3 (6), 630-635]

Therefore, compounds having an inhibitory activity of RET kinase are very useful for the prevention and treatment of various cancers.

Examples of RET tyrosine kinase inhibitors include Sorafenib, Sunitinib, XL184, Vandetanib, and Ponatinib, which are cell proliferations for cell lines expressing KIF5B-RET. Inhibitory activity has been reported. In addition, cabozantinib as a RET tyrosine kinase inhibitor has been reported to have a partial effect on non-small cell lung cancer due to RET fusion gene expression.

On the other hand, compounds having pyrazolo [3,4-d] pyrimidin-4-amine nuclei have been reported to exhibit excellent inhibitory effects on various protein kinases. As an example, International Patent Publication No. WO 2013/010136 (Patent Document 1) discloses a compound represented by the following formula (A).

[Formula A]

Patent document 1 relates to a pyrazolo [3,4-d] pyrimidin-4-amine-based compound having an inhibitory effect on Btk (Bruton's Tyrosine Kinase) protein, and a pyrazolo [3,4-d] pyridine. Compounds in which various aryl or heteroaryl groups are substituted at the C3 position of the midin-4-amine nucleus are disclosed, and as the heteroaryl group, an isoxazol-5-yl group is substituted at the C3 position of the mother nucleus as represented by Formula A above. The chemical structure of substituted compounds may also be described. However, Patent Document 1 is effective for the treatment of lymphoma tumors by confirming the synthesis of the compound substituted with the phenoxyphenyl group at the C3 position of the pyrazolo [3,4-d] pyrimidin-4-amine parent nucleus and Btk inhibitory activity. To reveal That is, Patent Document 1 discloses only the chemical structure of the compound in which the isoxazol-5-yl group is substituted at the C3 position of the pyrazolo [3,4-d] pyrimidin-4-amine parent nucleus represented by the formula (A). Only a specific synthesis example of the compound represented by the formula (A) and Btk inhibitory activity are not disclosed at all.

In contrast, the 3- (isoxazol-3-yl) -pyrazolo [3,4-d] pyrimidin-4-amine compound characterized by the present invention has an isoxazol-3-yl ring in the C3 position of the parent nucleus. It is substituted, and the structure is substituted with-(CH ₂ ) _n -A ( _wherein A is an alkyl, aryl or heteroaryl group, n is an integer of 0 to 4) at the C5 position of the isoxazol-3-yl ring It is a novel compound whose chemical structure has not been revealed to date. In addition, on the basis of experimentally confirming that the novel compound characterized by the present invention has an effect of inhibiting the activity of the RET gene among protein kinases, it is useful for treating thyroid cancer, non-small cell lung cancer, and breast cancer caused by RET gene expression. Has excellent medicinal uses

(Prior art document)

(Patent literature)

(Patent Document 1) WO 2013/010136 "Inhibitor of Bruton's Tyrosine Kinase (Btk)"

(Non-patent literature)

(Non-Patent Document 1) “Preparation of 3-substituted-1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amines as RET Kinase Inhibitors”, J. Med. Chem. 2012, 55, 4872-4876

(Non-Patent Document 2) “A transforming KIF5B and RET gene fusion in lung adenocarcinoma revealed from whole-genome and transcriptome sequencing”, Genome Res 2012, 22, 436-445

(Non-Patent Document 3) “Response to Cabozantinib in Patients with RET Fusion-Positive Lung Adenocarcinomas”, Cancer Discov 2013, 3 (6), 630-635

Accordingly, an object of the present invention is to provide a novel compound having excellent inhibitory activity and selectivity against RET kinase among protein kinases.

Another object of the present invention is to provide a pharmaceutical composition for preventing and treating cancer diseases caused by abnormal expression of RET kinase containing the novel compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a method for preparing the novel compound.

It is another object of the present invention to provide intermediate compounds useful for the synthesis of the novel compounds described above.

In order to solve the above problems, the present invention is a 3- (isoxazol-3-yl) -pyrazolo [3,4-d] pyrimidin-4-amine compound represented by the following general formula (1), pharmaceutically acceptable And a compound selected from the group consisting of salts thereof, hydrates thereof, solvates thereof and isomers thereof.

[Formula 1]

In Chemical Formula 1,

Represents a single bond or a double bond;

A is a hydrogen atom; C ₁ -C ₁₀ alkyl group; C ₆ -C ₁₅ aryl group; Or a 5 to 6 heteroaryl group containing 1 to 2 heteroatoms selected from S and N;

R is a hydrogen atom; C ₁ -C ₁₀ alkyl group; C ₁ -C ₁₀ hydroxyalkyl group; Benzyl groups; Or a 5- to 6-membered saturated or partially saturated heterocycloalkyl group containing 1 to 2 heteroatoms selected from O and N;

n represents an integer of 0 to 4;

The aryl group or heterocycloalkyl group is halo, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ alkoxy, R ¹ -C (O)-(wherein R ¹ is C ₁ -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl), or R ² -S (O) _2- , wherein R ² is C ₁ -C ₁₀ alkyl.

3- (isoxazol-3-yl) -pyrazolo [3,4-d] pyrimidin-4-amine compounds according to the present invention exhibit excellent inhibitory activity against RET protein kinases and are therefore induced by abnormal cell growth. It can be used for the purpose of preventing or treating a disease.

Diseases caused by abnormal cell growth in the present invention may include cancer diseases such as thyroid cancer, lung cancer, breast cancer and the like.

Pharmaceutically acceptable salts of the compounds represented by Formula 1 according to the present invention may be prepared by conventional methods in the art. Pharmaceutically acceptable salts are of low toxicity to humans and should not adversely affect the biological activity and physicochemical properties of the parent compound. Pharmaceutically acceptable salts include pharmaceutically usable free acids and acid addition salts of base compounds of formula (1), alkali metal salts (such as sodium salts) and alkaline earth metal salts (such as calcium salts), and organic salts and carboxyl compounds of formula (1). Organic base addition salts of acids and amino acid addition salts. Free acids that can be used for the preparation of pharmaceutically acceptable salts can be divided into inorganic acids and organic acids. The inorganic acid may be hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, bromic acid, or the like. Organic acids include acetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, fumaric acid, maleic acid, malonic acid, phthalic acid, succinic acid, lactic acid, citric acid, citric acid, gluconic acid, tartaric acid, salicylic acid, malic acid, oxalic acid, Benzoic acid, embonic acid, aspartic acid, glutamic acid and the like can be used. Organic bases that can be used for the preparation of organic base addition salts are tris (hydroxymethyl) methylamine, dicyclohexylamine and the like. Amino acids that can be used to prepare amino acid addition bases are natural amino acids such as alanine, glycine and the like.

The compound represented by Chemical Formula 1 according to the present invention includes all hydrates and solvates in addition to the pharmaceutically acceptable salts described above. The pharmaceutically acceptable salts described above may be prepared by conventional methods, for example, in a solvent that may be mixed with water such as methanol, ethanol, acetone, 1,4-dioxane, and the like. It may be prepared by melting and then crystallizing or recrystallizing after adding a free acid or free base.

In addition, the compound represented by Formula 1 according to the present invention may have one or more asymmetric centers, and in the case of such compounds, enantiomers or diastereomers may be present. Accordingly, the present invention includes each isomer or a mixture of these isomers. Different isomers can be separated or resolved by conventional methods, or any given isomer can be obtained by conventional synthesis or by stereospecific or asymmetric synthesis.

In addition, the present invention includes a radioactive derivative of the compound represented by Formula 1 according to the present invention, these radioactive compounds are useful in the field of biological research.

The substituents used to define the compounds according to the invention are explained in more detail as follows.

The term "halo" or "halogen atom" in the present invention is interchangeable with each other and means chloro, fluoro, bromo, and iodo.

'Alkyl' in the present invention has 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, and refers to a linear, pulverized or cyclic aliphatic saturated hydrocarbon group. . Specific examples of such alkyl groups include methyl group, ethyl group, normal propyl group, isopropyl group, cyclopropyl group, cyclopropylmethyl group, normal butyl group, isobutyl group, tert-butyl group, cyclobutyl group, normal pentyl group and iso Pentyl group, neopentyl group, tert-pentyl group, cyclopentyl group, normal hexyl group, isohexyl group, cyclohexyl group, normal heptyl group, normal octyl group and the like can be included.

In the present invention, 'hydroxyalkyl' refers to an aliphatic saturated hydrocarbon group in which at least one hydroxy group (—OH) is bonded to a linear or pulverized carbon chain as defined above. Specific examples of such hydroxyalkyl groups include hydroxymethyl group, 2-hydroxyethyl group, 3-hydroxypropyl group, 1-hydroxy-isopropyl group, 2-hydroxybutyl group, 4-hydroxybutyl group, 2-methyl-2-hydroxypropyl group and the like can be included.

'Heterocycloalkyl' in the present invention refers to an aliphatic ring group containing 5 to 6, saturated or partially saturated hetero atoms selected from O and N. Specific examples of such heterocycloalkyl groups include tetrahydrofuranyl group, 2,3-dihydrofuranyl group, 2,5-dihydrofuranyl group, pyrrolidinyl group, 2,3-dihydropyrrolidinyl group, 2 , 5-dihydropyrrolidinyl group, tetrahydro-2H-pyranyl group, 3,4-dihydro-2H-pyranyl group, 4H-pyranyl group, piperidinyl group, 1,2,3,4-tetrahydropyripy Denyl groups, 1,4-dihydropyridinyl groups, piperazinyl, N-protected piperazinyl, morpholino groups, and the like. The N-protecting group of piperazinyl may typically contain an alkyl group.

"Aryl" in the present invention means a monocyclic, bicyclic, or tricyclic aromatic hydrocarbon group having 6 to 15 carbon atoms. Specific examples of such an aryl group may include a phenyl group, naphthyl group, anthracenyl group, phenanthrenyl group, and the like.

“Heteroaryl” in the present invention means an aromatic ring group containing 1 to 2 heteroatoms selected from S and N, and having 4 to 13 carbon atoms. Such heteroaryl includes pyrrolyl group, pyrazolyl group, imidazolyl group, thiazolyl group, isothiazolyl group, pyridinyl group, pyrazinyl group, pyridazinyl group, pyrimidinyl group, indolyl group, isoindoleyl group, Indazolyl group, benzimidazolyl group, benzothiazolyl group, benzisothiazolyl group, quinolinyl group, isoquinolinyl group, phthalazinyl group, quinazolinyl group and the like can be included.

Compounds according to the invention can be represented by the following formula (1).

[Formula 1]

In Chemical Formula 1,

Represents a single bond or a double bond;

A is a hydrogen atom; C ₁ -C ₁₀ alkyl group; C ₆ -C ₁₅ aryl group; Or a 5 to 6 heteroaryl group containing 1 to 2 heteroatoms selected from S and N;

R is a hydrogen atom; C ₁ -C ₁₀ alkyl group; C ₁ -C ₁₀ hydroxyalkyl group; Benzyl groups; Or a 5- to 6-membered saturated or partially saturated heterocycloalkyl group containing 1 to 2 heteroatoms selected from O and N;

n represents an integer of 0 to 4;

The aryl group or heterocycloalkyl group is halo, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ alkoxy, R ¹ -C (O)-(wherein R ¹ is C ₁ -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl), or R ² -S (O) _2- , wherein R ² is C ₁ -C ₁₀ alkyl.

In the compound represented by Formula 1, Preferably, A is a hydrogen atom; Methyl group, ethyl group, normal propyl group, isopropyl group, cyclopropyl group, cyclopropyl methyl group, normal butyl group, isobutyl group, tert-butyl group, cyclobutyl group, normal pentyl group, isopentyl group, neopentyl group, tert An alkyl group selected from a pentyl group, a cyclopentyl group, a normal hexyl group, an isohexyl group, a cyclohexyl group and a normal heptyl group; Phenyl group unsubstituted or substituted with a substituent selected from halo, methyl, ethyl, normal propyl, isopropyl, cyclopropyl, methoxy and ethoxy; Thiopennyl group; Or a compound representing a pyridinyl group.

In the compound represented by Formula 1, preferably R is a hydrogen atom; Methyl group, ethyl group, normal propyl group, isopropyl group, cyclopropyl group, cyclopropyl methyl group, normal butyl group, isobutyl group, tert-butyl group, cyclobutyl group, normal pentyl group, isopentyl group, neopentyl group, tert An alkyl group selected from a pentyl group, a cyclopentyl group, a normal hexyl group, an isohexyl group, a cyclohexyl group and a normal heptyl group; Hydroxyalkyl groups selected from 2-methyl-2-hydroxypropyl group, 2-hydroxybutyl group and 4-hydroxybutyl group; Benzyl groups; Tetrahydro-2H-pyranyl group; Or a piperidinyl group unsubstituted or substituted with R ¹ -C (O)-or R ² -S (O) _2- , wherein R ¹ is a C ₁ -C ₆ alkyl group or a C ₂ -C ₆ alkenyl group , R ² is a compound representing a C ₁ to C ₆ alkyl group.

In the compound represented by the formula (1), particularly preferably

Represents a double bond; A represents a methyl group, an ethyl group, a normal propyl group, an isopropyl group, a cyclopropyl group, a thiophen-2-yl group or a thiophen-2-yl group; R is methyl group, ethyl group, normal propyl group, isopropyl group, cyclopropyl group, cyclopropyl methyl group, normal butyl group, isobutyl group, tert- butyl group, cyclobutyl group, normal pentyl group, isopentyl group, neophene An alkyl group selected from a methyl group, tert-pentyl group, cyclopentyl group, normal hexyl group, isohexyl group, cyclohexyl group and normal heptyl group; Hydroxyalkyl groups selected from 2-methyl-2-hydroxypropyl group, 2-hydroxybutyl group and 4-hydroxybutyl group; Benzyl groups; Or a tetrahydro-2H-pyranyl group; N is a compound representing 0.

More specifically exemplified by the compound represented by Formula 1 is as follows:

1-isopropyl-3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 1);

1-isopropyl-3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 2);

1-isopropyl-3- (5-isopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 3);

3- (5- (tert-butyl) isoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 4);

3- (5-cyclopentylisoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 5);

3- (5-cyclohexylisoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 6);

1-isopropyl-3- (5- (thiophen-3-yl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 7);

1-isopropyl-3- (5- (thiophen-2-yl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 8);

3- (5- (4-fluorophenyl) isoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 9);

1-isopropyl-3- (5- (4-methoxyphenyl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 10);

3- (5- (3-chlorophenyl) isoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 11);

3- (5- (2-chlorophenyl) isoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 12);

1-isopropyl-3- (5- (pyridin-2-yl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 13);

1-isopropyl-3- (5- (pyridin-3-yl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 14);

3- (5-benzylisoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 15);

1-isopropyl-3- (5- (phenyl-4,5-dihydroxyisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 16) ;

3- (5-phenylisoxazol-3-yl) -1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 17);

1- (1- (methylsulfonyl) piperidin-4-yl) -3- (5- (phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidine-4 Amines (Compound No. 18);

1- (4- (4-amino-3- (5- (phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl) piperidin-yl) Ethanone (Compound No. 19);

(±) -1- (3- (4-amino-3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl) piperidine -1-yl) prop-2-en-1-one (Compound No. 20);

(R) -1- (3- (4-amino-3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl) piperidine -1-yl) prop-2-en-1-one (Compound No. 21);

3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 22);

1-cyclohexyl-3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 23);

3- (5-cyclopropylisoxazol-3-yl) -1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (compound Number 24);

3- (5-cyclopropylisoxazol-3-yl) -1-heptyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 25);

3- (5-cyclopropylisoxazol-3-yl) -1-methyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 26);

1-benzyl-3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 27);

1- (4-amino-3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl) -2-methylpropan-2-ol (Compound number 28);

3- (5-ethylisoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 29);

1-isopropyl-3- (isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 30).

 On the other hand, the present invention is characterized by a method for producing a compound represented by the formula (1). Hereinafter, the manufacturing method according to the present invention will be described in detail.

Manufacturing Method 1

According to the preparation method according to Scheme 1 below, using the compound represented by the following formula (2) as a starting material to perform a four-step manufacturing process can be prepared a compound represented by the formula (1).

 Scheme 1

(In Scheme 1,

, A, R and n are as defined in Formula 1, respectively)

Hereinafter, the preparation method according to Scheme 1 will be described in more detail for each reaction.

i) The reaction is a reaction of 3-bromo-1H-pyrazolo [3,4-d] pyrimidin-4-amine represented by Chemical Formula 2 with a compound represented by RX (wherein X represents a halogen atom). By doing so, it is a process for preparing a compound represented by the formula (3) wherein various R is introduced. The i) reaction may proceed under conditions of heating to 70 ℃ to 80 ℃ in the presence of an inorganic base. In this case, the inorganic base may be selected from hydroxides, oxides, carbonates, sulfates, and the like of alkali metals or earth metals, and preferably carbonates of alkali metals such as potassium carbonate. As the reaction solvent, a conventional organic solvent may be used, and the examples of the present invention mainly dimethylformamide (DMF) are specifically illustrated, but the solvent of the present invention is not limited thereto.

ii) The reaction is a process of preparing the compound represented by Chemical Formula 4, wherein a vinyl group is introduced into the compound represented by Chemical Formula 3 by a Stille reaction. The stille reaction is carried out under reflux conditions using a metal catalyst such as palladium (Pd) or nickel (Ni) and an organic tin compound as a coupling reagent. As the metal catalyst, tetrakis (triphenylphosphine) palladium [Pd (PPh ₃ ) ₄ ] may be typically used, and tributylvinyl tin may be typically used as the organic tin compound. As the reaction solvent, aromatic hydrocarbons such as toluene may be used, and the reaction solvent may be in the range of about 120 ° C. to 150 ° C. as a reflux temperature of the solvent used.

iii) The reaction is a process of preparing the compound represented by Chemical Formula 5 by converting the vinyl group of the compound represented by Chemical Formula 4 to an aldehyde group by oxidative cleavage and then to an oxime group. Oxidative cleavage for converting vinyl groups to aldehyde groups can be carried out using ozone (O ₃ ). In addition, the oxidative cleavage reaction was carried out after a hydroxide reaction using osmium tetroxide (OsO ₄ ) as a catalyst and N-methylmorpholine-N-oxide (NMO) as an oxidant, followed by sodium periodate (NaIO). ₄ ) can be carried out under the conditions of the reaction with. The aldehyde compound is then reacted with hydroxylamine (NH ₂ OH) under an ethanol solvent to introduce an oxime group.

iv) The reaction is a process for preparing the compound represented by Chemical Formula 1 by nitrile oxide cycloaddition of nitrile oxide. The cycloaddition reaction is carried out using a hypervalent iodine compound such as (diacetoxyiodo) benzene or phenyliodine bis (trifluoroacetate) as a reaction reagent, under a mixed solvent of alcohol and water. It is carried out at room temperature (20 ℃ to 30 ℃) conditions.

Compounds in which the vinyl functional group represented by Chemical Formula 4 is introduced and the oxime functional group represented by Chemical Formula 5 are introduced as core intermediate compounds synthesized during the preparation method according to Scheme 1 are novel compounds. Therefore, the present invention is also characterized by an intermediate compound represented by the following formula (4) or (5).

[Formula 4]

(In Formula 4, R is a hydrogen atom; C ₁ -C ₁₀ alkyl group; C ₁ -C ₁₀ hydroxyalkyl group; benzyl group; or 5 to 6 each of 1 to 2 hetero atoms selected from O and N containing Saturated or partially saturated heterocycloalkyl groups.)

[Formula 5]

(In Formula 5, R is a hydrogen atom; C ₁ -C ₁₀ alkyl group; C ₁ -C ₁₀ hydroxyalkyl group; benzyl group; or 5 to 6 each of 1 to 2 hetero atoms containing 1 to 2 hetero atoms selected from N and Saturated or partially saturated heterocycloalkyl groups.)

The intermediate compound represented by Formula 4 may typically include 1-isopropyl-3-vinyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine.

The intermediate compound represented by Chemical Formula 5 may typically include (E) -4-amino-1-isopropyl-1H-pyrazolo [3,4-d] pyrimidine-3-carbaaldehyde oxime.

Manufacturing Method 2

According to the preparation method according to Scheme 2, a compound represented by the following Chemical Formula 1 may be prepared by performing a three-step preparation process using the malononitrile compound represented by the following Chemical Formula 6 as a starting material.

Scheme 2

(In Scheme 2,

, A, R and n are as defined in Formula 1, respectively)

Hereinafter, the preparation method according to Scheme 2 will be described in more detail for each reaction.

a) Reaction is a process for producing a 1H-pyrazole-4-carbonitrile compound represented by the formula (7) by cyclization reaction with the malononitrile compound represented by the formula (6) and hydrazine (NH ₂ NH ₂ ) hydrate . The a) reaction may be carried out under conditions of room temperature (20 ° C. to 30 ° C.) in alcohol solvents such as methanol and ethanol.

b) The reaction is carried out by the cyclization reaction of the 1H-pyrazole-4-carbonitrile compound represented by the formula (7) and the formamide compound (HC (O) NH ₂ ), and the 1H-pyrazolo [3] represented by the formula (8). , 4-d] pyrimidin-4-amine compound. The b) reaction may be carried out under the conditions of heating to a temperature of 130 ℃ to 180 ℃.

c) The reaction is a reaction of the 1H-pyrazolo [3,4-d] pyrimidin-4-amine compound represented by Formula 8 with the alcohol compound represented by ROH, where various R are introduced The process of preparing the compound. The c) reaction proceeds through a Mitsunobu reaction using triphenylphosphine (PPh ₃ ) and an azodicarboxylate compound. The azodicarboxylate compound may include, for example, diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD). The Mitsunobu reaction may be performed under conditions of room temperature (20 ° C. to 30 ° C.) using a polar solvent such as tetrahydrofuran.

On the other hand, the present invention includes a compound represented by the formula (1), a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof is a pharmaceutical composition containing the active ingredient.

Since the compound represented by Formula 1 exhibits excellent inhibitory activity against RET kinase, it may be used as a prophylactic or therapeutic agent for diseases caused by abnormal cell growth.

Cancer diseases caused by abnormal cell growth in the present invention may specifically include thyroid cancer, lung cancer, breast cancer. In particular, since the effect of inhibiting the activity of RET kinase is excellent, it is useful as a prophylactic and therapeutic agent for medullary thyroid cancer, non-small cell lung cancer, breast cancer.

The pharmaceutical composition of the present invention contains a compound represented by the formula (1), a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof as an active ingredient, and a conventional non-toxic pharmaceutically acceptable carrier, adjuvant and excipient And the like can be formulated into conventional formulations in the pharmaceutical field, for example, oral or parenteral formulations such as tablets, capsules, troches, solutions, suspensions and the like.

Excipients that may be used in the pharmaceutical compositions of the present invention may include sweeteners, binders, solubilizers, solubilizers, wetting agents, emulsifiers, isotonic agents, adsorbents, disintegrants, antioxidants, preservatives, lubricants, fillers, fragrances and the like. For example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, talc, stearic acid, sterin, magnesium stearate, magnesium aluminum silicate, starch, gelatin, tragacanth rubber, arginine acid, sodium Alginate, methyl cellulose, sodium carboxymethyl cellulose, agar, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, vanilla flavor and the like.

In addition, the dosage of the compound according to the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and is generally 0.01 based on an adult patient having a weight of 70 kg. It is-1,000 mg / day, and may be dividedly administered once to several times a day at regular time intervals depending on the judgment of the doctor or pharmacist.

The present invention as described above will be described in more detail based on the following Examples, Preparation Examples, and Experimental Examples. The following Examples, Preparation Examples, and Experimental Examples are merely illustrative of the present invention and the scope of the present invention. Is not limited by these.

EXAMPLES Synthesis of Compound of Formula 1

Example 1 Synthesis of 1-isopropyl-3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 1)

Step 1: 3-Bromo-1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine

3-bromo-1-H-pyrazolo [3,4-d] pyrimidin-4-amine (3 g, 14.02 mmol) and potassium carbonate (7.75 g, 56.1 mmol) were added to a round bottom flask, Filled with nitrogen. Dimethylformamide (93 mL) was added to the filled flask followed by 2-bromopropane (1.45 mL, 15.4 mmol). After stirring at 80 ° C. for 12 hours, the reaction mixture was diluted with ethyl acetate, terminated with water, and extracted with ethyl acetate and water. The combined organic layers were washed several times with water, dried over anhydrous magnesium sulfate and filtered, and the obtained residue was subjected to column chromatography (EtOAc: n-Hexane = 1: 2) to obtain the title compound (2.4 g, 67% yield). Got it.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.32 (s, 1H), 5.84 (bs, 2H), 5.09 (septet, J = 6.8 Hz, 1H), 1.54 (d, J = 6.8 Hz, 6H)

Step 2: 1-isopropyl-3-vinyl-1 H-pyrazolo [3,4-d] pyrimidin-4-amine

In a round bottom flask, 3-bromo-1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (3.99 g, 15.6 mmol) and Pd (PPh ₃ ) ₄ (3.6 g, 3.12 mmol) was added and the interior was filled with nitrogen. Toluene (156 mL) and tributylvinyltin (5.57 mL, 18.7 mmol) were added and then refluxed at 150 ° C. for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, the solvent was distilled off under reduced pressure, and tetrahydrofuran / 1 N hydrochloric acid (3/1, 160 mL) was added thereto, followed by stirring at room temperature for 12 hours. After diluting with ethyl acetate, the organic layer was extracted several times with 1 N HCl, and the combined aqueous layers were placed in a round bottom flask and neutralized with sodium hydrogen carbonate. The resulting aqueous layer was then extracted with methylene chloride solvent, dried over anhydrous magnesium sulfate and filtered, and the residue was subjected to column chromatography (EtOAc: n-Hexane = 1: 1) to give the title compound (2.59 g, 82% yield). Got.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.33 (s, 1H), 7.25 (s, 1H), 6.97 (dd, J = 11.2 Hz, 18.0 Hz, 1H), 5.93 (d, J = 18.0 Hz, 1H ), 5.65-5.62 (m, 3H), 5.10 (septet, J = 6.8 Hz, 1H), 1.54 (d, J = 6.8 Hz, 6H)

Step 3: (E) -4-amino-1-isopropyl-1H-pyrazolo [3,4-d] pyrimidine-3-carbaaldehyde oxime

To a round bottom flask was placed 1-isopropyl-3-vinyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (2.07 g, 10.19 mmol) and tetrahydrofuran / water (3/1, 140 mL) was added to dissolve. Then at 0 ° C. N-methylmorpholine-N-oxide (4.77 mL, 20.38 mmol, 50 w / w% in water) and osmium tetroxide (OsO ₄ ; 6.5 mL, 1.02 mmol, 4 w / w% in H ₂ O) was added. After stirring at room temperature for 3 hours, the reaction was terminated with sodium sulfite solution, and then the aqueous layer was extracted several times using chloroform / isopropyl alcohol (4/1) solvent. The collected organic solvent was distilled off under reduced pressure and dried, and the obtained residue was dissolved by adding tetrahydrofuran / water (3/1, 140 mL). Thereafter, sodium periodate (NaIO ₄ ; 4.36 g, 20.38 mmol) was added thereto, followed by stirring at room temperature for 2 hours. After the reaction was completed, the suspension was filtered using Celite, and then ethanol (16 mL) was added to the obtained residue. After adding sodium acetate (2 g, 24.46 mmol) and hydroxylamine hydrochloride (841 mg, 25.48 mmol), the mixture was stirred at room temperature for 12 hours. The solvent was distilled off under reduced pressure, and then extracted with ethyl acetate and water. The combined organic layers were washed several times with water, dried over anhydrous magnesium sulfate and filtered, and the obtained residue was subjected to column chromatography (EtOAc: n-Hexane = 2: 1) to obtain the title compound (2 g, 89% yield). Got it.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.39 (s, 1H), 8.31 (s, 1H), 8.17 (bs, 1H), 5.81 (bs, 1H), 5.12 (septet, J = 6.8 Hz, 1H) , 1.54 (d, J = 6.8 Hz, 6H)

Step 4: 1-isopropyl-3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine

(E) -4-amino-1-isopropyl-1H-pyrazolo [3,4-d] pyrimidine-3-carbaaldehyde oxime (220 mg, 1 mmol) was added to a round bottom flask, followed by methanol / water. (8.0 mL / 1.6 mL) was added to dissolve the mixed solvent. Then ethynylbenzene (0.15 mL, 1.1 mmol) was added, followed by phenyliodine bis (trifluoroacetate) (PIFA) three times over two hours (215 mg, 0.5 mmol each). It was then stirred for 4 hours and extracted with ethyl acetate. The combined organic layers were washed several times with water, dried over anhydrous magnesium sulfate and filtered, and the obtained residue was subjected to column chromatography (EtOAc: n-Hexane = 1: 2) to obtain the title compound (134 mg, 42% yield). .

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.91 (bs, 1H), 8.32 (s, 1H), 7.88-7.86 (m, 2H), 7.54-7.26 (m, 3H), 7.19 (s, 1H), 6.91 (bs, 1H), 5.19 (septet, J = 6.8 Hz, 1H), 1.61 (d, J = 6.8 Hz, 6H)

Examples 2-16.

Based on the method illustrated in step 4 of Example 1 above, (E) -4-amino-1-isopropyl-1H-pyrazolo [3,4-d] pyrimidine-3-carbaaldehyde oxime and various acetylenes Nitrile oxide cycloaddition was carried out using the compounds to synthesize each title compound.

Example 2. Synthesis of 1-isopropyl-3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 2)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.72 (bs, 1H), 8.27 (s, 1H), 6.86 (bs, 1H), 6.52 (s, 1H), 5.11 (sep, J = 6.8 Hz, 1H) , 2.11-2.04 (m, 1H), 1.53 (d, J = 6.8 Hz, 6H), 1.13-0.99 (m, 4H)

Example 3. Synthesis of 1-isopropyl-3- (5-isopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 3)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.66 (bs, 1H), 8.33 (s, 1H), 6.64 (d, J = 0.8 Hz, 1H), 5.88 (bs, 1H), 5.15 (sep, J = 6.8 Hz, 1H), 3.16 (sep, J = 7.2 Hz, 1H), 1.58 (d, J = 6.8 Hz, 6H), 1.39 (d, J = 6.8 Hz, 6H)

Example 4 of 3- (5- (tert-butyl) isoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 4) synthesis

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.67 (bs, 1H), 8.33 (s, 1H), 6.63 (s, 1H), 5.85 (bs, 1H), 5.17 (sep, J = 6.8 Hz, 1H) , 3.16 (sep, J = 7.2 Hz, 1H), 1.58 (d, J = 6.8 Hz, 6H), 1.43 (s, 9H)

Example 5. Synthesis of 3- (5-cyclopentylisoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 5)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.67 (bs, 1H), 8.33 (s, 1H), 6.64 (s, 1H), 5.93 (bs, 1H), 5.15 (sep, J = 6.4 Hz, 1H) , 3.27 (quin, J = 7.2 Hz, 1H), 2.18-2.12 (m, 2H), 1.82-1.72 (m, 6H), 1.57 (d, J = 6.4 Hz, 6H)

Example 6 Synthesis of 3- (5-cyclohexylisoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 6)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.67 (bs, 1H), 8.33 (s, 1H), 6.62 (s, 1H), 5.94 (bs, 1H), 5.15 (sep, J = 6.8 Hz, 1H) , 2.85 (tt, J = 3.6 Hz, 10.8 Hz, 1H), 2.14-2.11 (m, 2H), 1.85 (dt, J = 3.6 Hz, 9.6 Hz, 2H), 1.77-1.73 (m, 1H), 1.58 (d, J = 6.8 Hz, 6H), 1.56-1.25 (m, 5H)

Example 7. 1-isopropyl-3- (5- (thiophen-3-yl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 7) Synthesis

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.61 (bs, 1H), 8.35 (s, 1H), 7.87 (dd, J = 1.2 Hz, 2.8 Hz, 1H), 7.49 (dd, J = 1.2 Hz, 4.8 Hz, 1H), 7.46 (dd, J = 2.8 Hz, 4.8 Hz, 1H), 7.04 (s, 1H), 5.18 (sep, J = 6.4 Hz, 1H), 1.59 (d, J = 6.4 Hz, 6H)

Example 8. 1-isopropyl-3- (5- (thiophen-2-yl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 8) Synthesis

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.57 (bs, 1H), 8.35 (s, 1H), 7.61 (dd, J = 0.8 Hz, 4.0 Hz, 1H), 7.52 (dd, J = 1.2 Hz, 5.2 Hz, 1H), 7.18 (dd, J = 4.0 Hz, 5.2 Hz, 1H), 5.87 (bs, 1H), 5.18 (sep, J = 6.8 Hz, 1H), 1.60 (d, J = 6.8 Hz, 6H)

Example 9. 3- (5- (4-fluorophenyl) isoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 9 ) Synthesis

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.59 (bs, 1H), 8.35 (s, 1H), 7.88-7.84 (m. 2H), 7.21 (t, J = 8.8 Hz, 2H), 7.14 (s, 1H), 7.02 (d, J = 8.8 Hz, 2H), 5.84 (bs, 1H), 5.18 (sep, J = 6.8 Hz, 1H), 1.59 (d, J = 6.8 Hz, 6H)

Example 10. 1-isopropyl-3- (5- (4-methoxyphenyl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 10 ) Synthesis

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.68 (bs, 1H), 8.35 (s, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.08 (s, 1H), 7.02 (d, J = 8.8 Hz, 2H), 5.83 (bs, 1H), 5.18 (sep, J = 6.8 Hz, 1H), 3.89 (s, 3H), 1.60 (d, J = 6.4 Hz, 6H)

Example 11. 3- (5- (3-chlorophenyl) isoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 11) Synthesis of

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.55 (bs, 1H), 8.35 (s, 1H), 7.86 (s, 1H), 7.77-7.74 (m, 1H), 7.48-7.45 (m, 2H), 7.23 (s, 1H), 5.90 (bs, 1H), 5.18 (sep, J = 6.8 Hz, 1H), 1.59 (d, J = 6.8 Hz, 6H)

Example 12. 3- (5- (2-chlorophenyl) isoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 12) Synthesis of

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.59 (bs, 1H), 8.36 (s, 1H), 7.99-7.98 (m, 1H), 7.58-7.56 (m, 2H) 7.48-7.42 (m, 2H) , 5.82 (bs, 1H), 5.18 (sep, J = 6.8 Hz, 1H), 1.61 (d, J = 6.8 Hz, 6H)

Example 13. 1-isopropyl-3- (5- (pyridin-2-yl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine trifluoroacetic acid Synthesis of Salts (Compound No. 13)

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.67 (bs, 1H), 8.75-8.74 (m, 1H), 8.30 (s, 1H), 7.99-7.97 (m, 1H), 7.89 (td, J = 1.6 Hz, 8.0 Hz, 1H), 7.57 (s, 1H), 7.43-7.39 (m, 1H), 5.18 (sep, J = 6.8 Hz, 1H), 1.61 (d, J = 6.8 Hz, 6H)

Example 14. 1-isopropyl-3- (5- (pyridin-3-yl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 14 ) Synthesis

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.13 (dd, J = 0.8 Hz, 2.0 Hz, 1H), 8.73 (dd, J = 1.6 Hz, 4.8 Hz, 1H), 8.52 (bs, 1H), 8.36 ( s, 1H), 8.16 (dt, J = 2.0 Hz, 8.0 Hz, 1H), 7.47 (ddd, J = 0.4 Hz, 4.8 Hz, 7.6 Hz, 1H), 7.30 (s, 1H), 5.83 (bs, 1H ), 5.19 (sep, J = 6.8 Hz, 1H), 1.60 (d, J = 6.8 Hz, 6H)

Example 15 Synthesis of 3- (5-benzylisoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 15)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.60 (bs, 1H), 8.31 (s, 1H), 7.39-7.26 (m, 5H), 6.61 (s, 1H), 5.81 (bs, 1H), 5.14 ( sep, J = 6.8 Hz, 1H), 4.15 (s, 2H), 1.54 (d, J = 6.4 Hz, 6H)

Example 16. 1-Isopropyl-3- (5- (phenyl-4,5-dihydroxyisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine ( Synthesis of Compound No. 16)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.42 (bs, 1H), 8.32 (s, 1H), 7.41-7.34 (m, 5H), 5.94 (bs, 1H), 5.78 (dd, J = 8.8 Hz, 11.2 Hz, 1H), 5.11 (sep, J = 6.8 Hz, 1H), 3.98 (dd, J = 11.2 Hz, 17.6 Hz, 1H), 3.57 (dd, J = 8.4 Hz, 17.6 Hz, 1H), 1.52 ( t, J = 7.2 Hz, 6H)

Example 17. 3- (5-phenylisoxazol-3-yl) -1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-d] pyrimidine-4- Synthesis of Amine (Compound No. 17)

Step 1: 2- (methoxy (5-phenylisoxazol-3-yl) methylene) malononitrile

5-phenylisoxazole-3-carboxylic acid (2 g, 10.57 mmol) was added to a round bottom flask, and the inside was filled with nitrogen. To the packed flask was added methylene chloride (35 mL) and catalytic amount of dimethylformamide (5 drops), followed by the slow addition of oxalyl chloride (4.53 mL, 52.85 mmol). After stirring for 2 hours at room temperature, the residue obtained by drying under reduced pressure was used directly in the next reaction without purification.

To the round bottom flask was added sodium hydride (845 mg, 21.14 mmol) and malononitrile (768 mg, 11.63 mmol), filled with nitrogen and then tetrahydrofuran (35 mL). After cooling to 0 ° C., the previously obtained residue was dissolved in tetrahydrofuran solution (20 mL) and slowly added dropwise to the reaction solution. After stirring for 22 hours, diisopropylethylamine (7.3 mL, 42.28 mmol) and dimethylsulfate (8 mL, 84.56 mmol) were added, followed by warming and stirring to 90 ° C. After stirring for 12 hours, the reaction was terminated with water and extracted with ethyl acetate and water. The combined organic layers were washed several times with water, dried over anhydrous magnesium sulfate and filtered to obtain the title compound.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.81-7.79 (m, 2H), 7.51-7.26 (m, 3H), 6.88 (s, 1H), 4.20 (s, 3H)

Step 2: 5-Amino-3- (5-phenylisoxazol-3-yl) -1 H-pyrazole-4-carbonitrile

2- (methoxy (5-phenylisoxazol-3-yl) methylene) malononitrile (2.66 g, 10.57 mmol) was added to a round bottom flask, followed by ethanol (30 mL) at room temperature. Hydrazine hydrate (0.4 mL, 11.1 mmol) was added to the reaction solution, followed by stirring for 12 hours. Thereafter, water was added to terminate the reaction, followed by extraction using an isopropyl alcohol / chloroform solution, and dried over anhydrous magnesium sulfate. The obtained residue was washed several times with ethyl acetate and methylene chloride to obtain the title compound.

¹ H NMR (400 MHz, DMSO) δ 12.61 (bs, 2H), 7.94 (dd, J = 2.0 Hz, 8.0 Hz, 2H), 7.57-7.52 (m, 3H), 7.29 (s, 1H), 6.60 ( bs, 1 H)

Step 3: 3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine

To a round bottom flask was added 5-amino-3- (5-phenylisoxazol-3-yl) -1H-pyrazole-4-carbonitrile (220 mg, 0.88 mmol) and formamide (4 mL), Stir at 160 ° C. for 6 hours. When the solid gradually formed, the temperature was lowered to room temperature and water was added to terminate the reaction. The obtained solid was washed several times with water, chloroform and ethanol to obtain the title compound.

¹ H NMR (400 MHz, DMSO) δ 14.04 (bs, 1H), 8.22 (s, 1H), 8.05 (bs, 2H), 8.00 (dd, J = 2.0 Hz, 8.0 Hz, 2H), 7.63 (s, 1H), 7.60-7.53 (m, 3H)

Step 4: 3- (5-phenylisoxazol-3-yl) -1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine

In a round bottom flask, 4-hydroxypyrane (17 mg, 0.108 mmol) and 3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine ( 15 mg, 0.054 mmol) and triphenylphosphine (21 mg, 0.081 mmol) were added followed by addition of tetrahydrofuran (2 mL) to dissolve. Diisopropyl azodicarboxylate (DIAD; 0.002 mL, 0.081 mmol) was then added slowly. After stirring for 2 hours, water was added to terminate the reaction, and then extracted with ethyl acetate. The combined organic layers were washed several times with water, dried over anhydrous magnesium sulfate and filtered, and the obtained residue was subjected to column chromatography (EtOAc: n-Hexane = 2: 1) to obtain the title compound.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.46 (bs, 1H), 8.34 (s, 1H), 7.88-7.84 (m, 2H), 7.53-7.47 (m, 3H), 7.16 (s, 1H), 5.81 (bs, 1H), 5.01 (tt, J = 4.0 Hz, 12.0 Hz, 1H), 4.15 (dd, J = 4.0 Hz, 11.2 Hz, 2H), 3.63 (td, J = 2.0 Hz, 12.0 Hz, 2H ), 2.42 (qd, J = 4.4 Hz, 12.4 Hz, 2H), 2.03-1.95 (m, 2H)

Examples 18-20.

According to the method illustrated in step 4 of Example 17 above, 3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine and various alcohols Each title compound was synthesized by performing a Mitsunobu reaction using the compound.

Example 18. 1- (1- (methylsulfonyl) piperidin-4-yl) -3- (5- (phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] Synthesis of Pyrimidin-4-amine (Compound No. 18)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.68 (bs, 1H), 8.34 (s, 1H), 7.87-7.85 (m, 2H), 7.54-7.49 (m, 3H), 7.16 (s, 1H), 5.88 (bs, 1H), 4.92 (tt, J = 4.4 Hz, 11.6 Hz, 1H), 4.01 (d, J = 12.0 Hz, 2H), 2.99 (td, J = 2.4 Hz, 12.4 Hz, 2H), 2.87 (s, 3H), 2.47 (qd, J = 4.4 Hz, 12.0 Hz, 2H), 2.16-2.14 (m, 2H)

Example 19. 1- (4- (4-amino-3- (5- (phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl) piperi Synthesis of din-yl) ethanone (Compound No. 19)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.66 (bs, 1H), 8.35 (s, 1H), 7.87-7.84 (m, 2H), 7.53-7.48 (m, 3H), 7.15 (s, 1H), 5.87 (bs, 1H), 5.02 (tt, J = 4.4 Hz, 7.2 Hz, 1H), 4.82-4.79 (m, 2H), 4.03-3.99 (m, 2H), 3.37-3.29 (m, 2H), 2.85 (td, J = 2.8 Hz, 13.2 Hz, 2H), 2.26 (qd, J = 4.8 Hz, 11.6 Hz, 2H), 2.17 (s, 3H), 2.16-1.99 (m, 3H)

Example 20. (±) -1- (3- (4-amino-3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl Synthesis of Piperidin-1-yl) prop-2-en-1-one (Compound No. 20)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.67 (bs, 1H), 8.36 (s, 1H), 7.88-7.86 (m, 2H), 7.54-7.50 (m, 3H), 7.16 (s, 1H), 6.63-6.53 (m, 1H), 6.30-6.27 (m, 1H), 5.90 (bs, 1H), 5.74-5.64 (m, 1H), 4.93-4.88 (m, 1H), 4.87-4.45 (m, 1H ), 4.19-4.02 (m, 1H), 3.84-3.45 (m, 1H), 3.32-3.11 (m, 1H), 2.36-2.25 (m, 2H), 1.81-1.72 (m, 2H)

Example 21. (R) -1- (3- (4-amino-3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl Synthesis of Piperidin-1-yl) prop-2-en-1-one (Compound No. 21)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.67 (bs, 1H), 8.36 (s, 1H), 7.88-7.86 (m, 2H), 7.54-7.50 (m, 3H), 7.16 (s, 1H), 6.63-6.53 (m, 1H), 6.30-6.27 (m, 1H), 5.90 (bs, 1H), 5.74-5.64 (m, 1H), 4.93-4.88 (m, 1H), 4.87-4.45 (m, 1H ), 4.19-4.02 (m, 1H), 3.84-3.45 (m, 1H), 3.32-3.11 (m, 1H), 2.36-2.25 (m, 2H), 1.81-1.72 (m, 2H)

Example 22. Synthesis of 3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 22)

¹ H NMR (400 MHz, DMSO) δ 13.94 (bs, 1H), 8.20 (s, 1H), 8.06 (bs, 1H), 7.99 (bs, 1H), 6.77 (s, 1H), 2.27-2.21 (m , 1H), 1.15-1.10 (m, 2H), 1.03-0.99 (m, 2H)

Example 23. Synthesis of 1-cyclohexyl-3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 23)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.62 (bs, 1H), 8.32 (s, 1H), 6.54 (s, 1H), 5.89 (bs, 1H), 4.78-4.70 (m, 1H), 2.14- 2.07 (m, 1H), 2.05-1.99 (m, 3H), 1.94-1.91 (m, 1H), 1.76 (dt, J = 3.2 Hz, 12.8 Hz, 1H), 1.56-1.45 (m, 2H), 1.34 (tt, J = 3.2 Hz, 12.8 Hz, 1H), 1.28-1.25 (m, 1H), 1.16-1.11 (m, 2H), 1.08-1.02 (m, 2H)

Example 24. 3- (5-cyclopropylisoxazol-3-yl) -1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-d] pyrimidine-4 Of amines (Compound No. 24)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.65 (bs, 1H), 8.33 (s, 1H), 6.54 (s, 1H), 5.91 (bs, 1H), 4.99 (tt, J = 4.4 Hz, 11.6 Hz , 1H), 4.15 (dd, J = 4.4 Hz, 11.6 Hz, 2H), 3.63 (td, J = 1.6 Hz, 12.0 Hz, 2H), 2.39 (qd, J = 4.4 Hz, 12.0 Hz, 2H), 2.16 -2.09 (m, 1H), 1.96 (dd, J = 2.4 Hz, 12.8 Hz, 2H), 1.17-1.11 (m, 2H), 1.09-1.04 (m, 2H)

Example 25. Synthesis of 3- (5-cyclopropylisoxazol-3-yl) -1-heptyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 25)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.63 (bs, 1H), 8.33 (s, 1H), 6.52 (s, 1H), 6.10 (bs, 1H), 4.39 (t, J = 7.2 Hz, 2H) , 2.16-2.08 (m, 1H), 1.92 (quin, J = 7.2 Hz, 2H), 1.33-1.24 (m, 8H), 1.15-1.12 (m, 2H), 1.04-1.02 (m, 2H), 0.85 (t, J = 6.4 Hz, 3H)

Example 26. Synthesis of 3- (5-cyclopropylisoxazol-3-yl) -1-methyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 26)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.60 (bs, 1H), 8.32 (s, 1H), 6.48 (s, 1H), 6.31 (bs, 1H), 4.02 (s, 3H), 2.14-2.05 ( m, 1H), 1.14-1.03 (m, 2H), 1.02-1.01 (m, 2H)

Example 27. Synthesis of 1-benzyl-3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 27)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.64 (bs, 1H), 8.37 (s, 1H), 7.34-7.26 (m, 5H), 6.52 (s, 1H), 5.87 (bs, 1H), 5.60 ( s, 2H), 2.13-2.07 (m, 1H), 1.16-1.05 (m, 2H), 1.05-1.00 (m, 2H)

Example 28. 1- (4-amino-3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl) -2-methylpropane Synthesis of 2-ol (Compound No. 28)

¹ H NMR (400 MHz, MeOD) δ 8.33 (s, 1H), 6.73 (s, 1H), 4.45 (s, 2H), 2.29-2.22 (m, 1H), 1.29 (s, 6H), 1.24-1.18 (m, 2H), 1.08-1.05 (m, 2H)

Example 29. Synthesis of 3- (5-ethylisoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 29)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.64 (bs, 1H), 8.33 (s, 1H), 6.66 (s, 1H), 5.90 (bs, 1H), 5.15 (sep, J = 6.8 Hz, 1H) , 2.85 (q, J = 7.6 Hz, 2H), 1.57 (d, J = 6.8 Hz, 6H), 1.38 (t, J = 7.6 Hz, 3H)

Example 30. Synthesis of 1-isopropyl-3- (isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine (Compound No. 30)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.55 (bs, 1H), 8.47 (d, J = 1.6 Hz, 1H), 8.35 (s, 1H), 7.03 (d, J = 1.6 Hz, 1H), 6.01 (bs, 1H), 5.17 (sep, J = 6.8 Hz, 1H), 1.58 (d, J = 6.8 Hz, 6H)

On the other hand, the novel compound represented by Formula 1 according to the present invention can be formulated in various forms according to the purpose. The following illustrates some formulation methods containing the compound represented by Formula 1 according to the present invention as an active ingredient, but the present invention is not limited thereto.

Preparation Example

Formulation Example 1 Tablet (Direct Pressing)

After sifting 5.0 mg of active ingredient, 14.1 mg of lactose, 0.8 mg of crospovidone USNF, and 0.1 mg of magnesium stearate were mixed and pressed to form a tablet.

Formulation Example 2 Tablet (Wet Granulation)

After sifting 5.0 mg of the active ingredient, 16.0 mg of lactose and 4.0 mg of starch were mixed. 0.3 mg of polysorbate 80 was dissolved in pure water and then an appropriate amount of this solution was added and then atomized. After drying, the fine particles were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The granules were pressed into tablets.

Formulation Example 3 Powders and Capsules

5.0 mg of the active ingredient was sieved, followed by mixing with 14.8 mg of lactose, 10.0 mg of polyvinyl pyrrolidone, and 0.2 mg of magnesium stearate. No. solid the mixture using a suitable device. Filled in 5 gelatin capsules.

Formulation Example 4 Injection

Injectables were prepared by containing 100 mg as active ingredient, as well as 180 mg of mannitol, 26 mg of Na ₂ HPO ₄ 12H ₂ O and 2974 mg of distilled water.

Experimental Example Biological Activity Test of Compound

Experimental Example 1. Measurement of RET kinase inhibitory activity

The inhibitory activity (% inhibition) of RET (Rearranged during transfaction) kinase of the compound represented by Chemical Formula 1 according to the present invention was shown in Table 1 below.

division	RET (uM)	division	RET (uM)
Compound number 1	<1	Compound number 16	<1
Compound number 2	<0.01	Compound number 17	<1
Compound number 3	<0.1	Compound number 18	<1
Compound number 4	<1	Compound number 19	<1
Compound number 5	<1	Compound no.20	<1
Compound number 6	<1	Compound number 21	<1
Compound number 7	<0.1	Compound number 22	<0.1
Compound number 8	<0.1	Compound number 23	<0.1
Compound number 9	<1	Compound number 24	<0.1
Compound number 10	<1	Compound number 25	<0.1
Compound number 11	<1	Compound number 26	<0.1
Compound number 12	<5	Compound number 27	<0.1
Compound number 13	<1	Compound number 28	<0.1
Compound number 14	<5	Compound number 29	<0.1
Compound number 15	<1	Compound number 30	<1

According to the above Table 1, the compound of the present invention was generally excellent in RET kinase inhibitory activity, and the compound of the compounds Nos. 2, 3, 7, 8, 22 to 29 can be confirmed that the inhibitory effect is excellent.

Experimental Example 2. Measurement of inhibitory activity against RET mutant species

As a representative compound according to the present invention, the inhibitory activity of RET variant kinase was evaluated for the compound of Compound No. 2. In order to evaluate the inhibitory activity of RET variant kinase, the inhibitory activity of the phosphorylation reaction of biotinylated peptide (EGPWLEEEEEAYGWMDF) using RET variant kinase with GST tag at the N-terminus of RET kinase domain (Carna Biosciences, Millipore) was used as an index. It was. Phosphorylated biotinylated peptide was detected by TR-FRET method using antiphosphorylated antibody bound with Europium label. The inhibitory activity for each variant of RET kinase was calculated as an IC ₅₀ value and is shown in Table 2 below.

RET	IC 50 (nM)
	Cabozantinib (control)	Compound number 2
RET (wt)	9	<10
RET (G691S)	14	<10
RET (Y791F)	7	<10
RET (V804L)	230	<30
RET (V804M)	358	<10
RET (S891A)	5	<10
RET (M918T)	18	<10

Experimental Example 3. Comparative measurement of inhibitory activity against thyroid cancer cell line and normal cell line

In the present experiment, the thyroid medulla cancer cell line TT (American Type Culture Collection) having a normal thyroid epithelial cell line (Nthy-ori 3-1) and a RET kinase activation mutation (C634W) for the compound represented by Formula 1 according to the present invention. The cell proliferation inhibitory activity was compared, and cell proliferation inhibitory activity of parental Ba / F3 cell line without modification and RET-Ba / F3 cell line with RET was measured.

F-12K Nutrient Mixture containing 10% FBS in a 96 well plate containing 5,000 cells per well of Nthy-ori 3-1 cells, TT cells, Parental Ba / F3 cells, and RET-Ba / F3 cells (Life Technologies Corporation) ) Was incubated overnight at 37 ° C., 5% CO ₂ . Then, the test compound was added to dimethyl sulfoxide, diluted to a final concentration of 1 nM to 10 μM, and added to a 96 well plate. After culturing for 5 days at 37 ° C. in the presence of 5% CO ₂ , the cell number measuring reagent (CellTiter-Glo (R) Luminescent Cell Viability Assay) was added thereto, followed by stirring, and the luminescence intensity was measured using an luminescence measuring device (Envision). . Table 3 shows the results of measuring the GI ₅₀ value of each test compound.

division	GI 50 (uM)
	Nthy ori-3-1	TT	Parental Ba / F3	RET-Ba / F3
Cabozantinib	4.9	0.115	> 10	0.05
Compound number 2	> 10	<0.3	> 10	<0.1
Compound number 8	> 10	<5	N.A.	N.A.
Compound number 13	> 10	<10	<10	<3
Compound number 14	> 10	<10	> 10	<10
Compound number 16	> 10	<5	> 10	<10
Compound number 19	> 10	<5	N.A.	N.A.
Compound no.20	> 10	<10	N.A.	N.A.
Compound number 23	> 10	<1	> 10	<1
Compound number 24	> 2	<1	> 10	<1
Compound number 26	> 10	<5	> 10	<3
Compound number 28	<10	<5	> 10	<3
Compound number 29	> 10	<5	> 10	<3
Compound number 30	> 10	<10	> 10	<5

According to the results of Table 3, the compound of the present invention does not show inhibitory activity against normal cell lines (Nthy-ori 3-1 cells, Parental Ba / F3 cells), abnormally expressed cell line (TT cells or RET- Ba / F3 cells) can be confirmed that the selective inhibitory activity was excellent.

Claims (16)

1. 3- (isoxazol-3-yl) -pyrazolo [3,4-d] pyrimidin-4-amine compound represented by the following Chemical Formula 1, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof and its Compounds selected from the group consisting of isomers:

   [Formula 1]

   

   In Chemical Formula 1,

   

   Represents a single bond or a double bond;

   A is a hydrogen atom; C ₁ -C ₁₀ alkyl group; C ₆ -C ₁₅ aryl group; Or a 5 to 6 heteroaryl group containing 1 to 2 heteroatoms selected from S and N;

   R is a hydrogen atom; C ₁ -C ₁₀ alkyl group; C ₁ -C ₁₀ hydroxyalkyl group; Benzyl groups; Or a 5- to 6-membered saturated or partially saturated heterocycloalkyl group containing 1 to 2 heteroatoms selected from O and N;

   n represents an integer of 0 to 4;

   The aryl group or a heteroaryl cycloalkyl groups are each halo, C ₁ ~ C ₁₀ alkyl, C ₁ ~ C ₁₀ ^{alkoxy, R 1 -C (O) -} ( wherein, R ¹ is C ₁ ~ C ₁₀ alkyl or C ₂ ~ C ₁₀ alkenyl), or R ² -S (O) _2- , wherein R ² is C ₁ -C ₁₀ alkyl.
2. The method of claim 1,

   A is a hydrogen atom; Methyl group, ethyl group, normal propyl group, isopropyl group, cyclopropyl group, cyclopropyl methyl group, normal butyl group, isobutyl group, tert-butyl group, cyclobutyl group, normal pentyl group, isopentyl group, neopentyl group, tert An alkyl group selected from a pentyl group, a cyclopentyl group, a normal hexyl group, an isohexyl group, a cyclohexyl group and a normal heptyl group; Phenyl group unsubstituted or substituted with a substituent selected from halo, methyl, ethyl, normal propyl, isopropyl, cyclopropyl, methoxy and ethoxy; Thiopennyl group; Or a pyridinyl group.
3. The method of claim 1,

   R is a hydrogen atom; Methyl group, ethyl group, normal propyl group, isopropyl group, cyclopropyl group, cyclopropyl methyl group, normal butyl group, isobutyl group, tert-butyl group, cyclobutyl group, normal pentyl group, isopentyl group, neopentyl group, tert An alkyl group selected from a pentyl group, a cyclopentyl group, a normal hexyl group, an isohexyl group, a cyclohexyl group and a normal heptyl group; Hydroxyalkyl groups selected from 2-methyl-2-hydroxypropyl group, 2-hydroxybutyl group and 4-hydroxybutyl group; Benzyl groups; Tetrahydro-2H-pyranyl group; Or a piperidinyl group unsubstituted or substituted with R ¹ -C (O)-or R ² -S (O) _2- , wherein R ¹ is a C ₁ -C ₆ alkyl group or a C ₂ -C ₆ alkenyl group , R ² represents a C ₁ to C ₆ alkyl group.
4. The method of claim 1,

   remind

   

   Represents a double bond,

   A represents a methyl group, an ethyl group, a normal propyl group, an isopropyl group, a cyclopropyl group, a thiophen-2-yl group or a thiophen-2-yl group,

   R is methyl group, ethyl group, normal propyl group, isopropyl group, cyclopropyl group, cyclopropyl methyl group, normal butyl group, isobutyl group, tert- butyl group, cyclobutyl group, normal pentyl group, isopentyl group, neophene An alkyl group selected from a methyl group, tert-pentyl group, cyclopentyl group, normal hexyl group, isohexyl group, cyclohexyl group and normal heptyl group; Hydroxyalkyl groups selected from 2-methyl-2-hydroxypropyl group, 2-hydroxybutyl group and 4-hydroxybutyl group; Benzyl groups; Or a tetrahydro-2H-pyranyl group,

   N represents 0.
5. The method of claim 1,

   1-isopropyl-3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   1-isopropyl-3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   1-isopropyl-3- (5-isopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   3- (5- (tert-butyl) isoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   3- (5-cyclopentylisoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   3- (5-cyclohexylisoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   1-isopropyl-3- (5- (thiophen-3-yl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   1-isopropyl-3- (5- (thiophen-2-yl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   3- (5- (4-fluorophenyl) isoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   1-isopropyl-3- (5- (4-methoxyphenyl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   3- (5- (3-chlorophenyl) isoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   3- (5- (2-chlorophenyl) isoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   1-isopropyl-3- (5- (pyridin-2-yl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   1-isopropyl-3- (5- (pyridin-3-yl) isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   3- (5-benzylisoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   1-isopropyl-3- (5- (phenyl-4,5-dihydroxyisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   3- (5-phenylisoxazol-3-yl) -1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   1- (1- (methylsulfonyl) piperidin-4-yl) -3- (5- (phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidine-4 Amines;

   1- (4- (4-amino-3- (5- (phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl) piperidin-yl) Ethanone;

   1- (3- (4-amino-3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl) piperidin-1-yl ) Prop-2-en-1-one;

   (±) -1- (3- (4-amino-3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl) piperidine -1-yl) prop-2-en-1-one;

   (R) -1- (3- (4-amino-3- (5-phenylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl) piperidine -1-yl) prop-2-en-1-one;

   3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   1-cyclohexyl-3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   3- (5-cyclopropylisoxazol-3-yl) -1- (tetrahydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   3- (5-cyclopropylisoxazol-3-yl) -1-heptyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   3- (5-cyclopropylisoxazol-3-yl) -1-methyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   1-benzyl-3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine;

    1- (4-amino-3- (5-cyclopropylisoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-1-yl) -2-methylpropan-2-ol ;

   3- (5-ethylisoxazol-3-yl) -1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-4-amine;

   1-isopropyl-3- (isoxazol-3-yl) -1H-pyrazolo [3,4-d] pyrimidin-4-amine.
6. 3- (isoxazol-3-yl) -pyrazolo [3,4-d] pyrimidin-4-amine compound represented by the following Chemical Formula 1, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof and its A pharmaceutical composition containing a compound selected from the group consisting of isomers as an active ingredient.

   [Formula 1]

   

   In Chemical Formula 1,

   

   , A, R and n are as defined in claim 1, respectively.
7. 3- (isoxazol-3-yl) -pyrazolo [3,4-d] pyrimidin-4-amine compound represented by the following Chemical Formula 1, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof and its A thyroid cancer therapeutic agent containing a compound selected from the group consisting of isomers as an active ingredient.

   [Formula 1]

   

   In Chemical Formula 1,

   

   , A, R and n are as defined in claim 1, respectively.
8. 3- (isoxazol-3-yl) -pyrazolo [3,4-d] pyrimidin-4-amine compound represented by the following Chemical Formula 1, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof and its A lung cancer therapeutic agent containing a compound selected from the group consisting of isomers as an active ingredient.

   [Formula 1]

   

   In Chemical Formula 1,

   

   , A, R and n are as defined in claim 1, respectively.
9. 3- (isoxazol-3-yl) -pyrazolo [3,4-d] pyrimidin-4-amine compound represented by the following Chemical Formula 1, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof and its A breast cancer therapeutic agent containing a compound selected from the group consisting of isomers as an active ingredient.

   [Formula 1]

   

   In Chemical Formula 1,

   

   , A, R and n are as defined in claim 1, respectively.
10. i) reacting the compound represented by Formula 2 with the compound represented by R-X to prepare a compound represented by Formula 3 in which various Rs are introduced;

    

    (Wherein R is as defined in claim 1; X represents a halogen atom)

    ii) preparing a compound represented by the following Chemical Formula 4 by introducing a vinyl group by a Stille reaction of the compound represented by the following Chemical Formula 3;

    

    (Wherein R is as defined in claim 1)

    iii) After converting the vinyl group of the compound represented by the following formula (4) into an aldehyde group by oxidative cleavage, it is converted into an oxime group by reaction with hydroxylamine (NH ₂ OH), Preparing a compound represented by Formula 5;

    

    (Wherein R is as defined in claim 1)

    iv) preparing a compound represented by the following Chemical Formula 1 by nitrile oxide cycloaddition of nitrile oxide to the compound represented by the following Chemical Formula 5;

    

    (In the above scheme,

    

    , A, R and n are as defined in claim 1, respectively)

    Method for preparing 3- (isoxazol-3-yl) -pyrazolo [3,4-d] pyrimidin-4-amine compound comprising a.
11. The method of claim 10,

    The i) reaction is carried out under the conditions of heating to 70 ℃ to 80 ℃ in the presence of a base of alkaline earth metal carbonate.
12. The method of claim 10,

    The ii) steel reaction is carried out under the conditions of refluxing at a temperature of 120 ℃ to 150 ℃ using a metal catalyst of palladium (Pd) or nickel (Ni), and the organic tin compound coupling reagent Manufacturing method.
13. The method of claim 10,

    Wherein iii) the oxidative cleavage reaction is reacted in the presence of an osmium tetroxide (OsO ₄ ) catalyst and an N-methylmorpholine-N-oxide oxidant, followed by addition of sodium periodate (NaIO ₄ ). Method for producing a compound, characterized in that the.
14. The method of claim 10,

    Said iv) ring addition reaction is carried out under the conditions using a hypervalent iodine compound selected from (diacetoxyiodo) benzene or phenyliodine bis (trifluoroacetate).
15. Intermediate compound represented by the following formula (4):

    [Formula 4]

    

    In Chemical Formula 4,

    R is a hydrogen atom; C ₁ -C ₁₀ alkyl group; C ₁ -C ₁₀ hydroxyalkyl group; Benzyl groups; Or a five to six-membered saturated or partially saturated heterocycloalkyl group containing 1 to 2 heteroatoms selected from O and N.
16. Intermediate compound represented by the following formula (5):

    [Formula 5]

    

    In Chemical Formula 5,

    R is a hydrogen atom; C ₁ -C ₁₀ alkyl group; C ₁ -C ₁₀ hydroxyalkyl group; Benzyl groups; Or a five to six-membered saturated or partially saturated heterocycloalkyl group containing 1 to 2 heteroatoms selected from O and N.