KR20190043842A - Pyrimidine-2-amine derivative, a method for producing the same, and an anticancer drug containing the same - Google Patents

Abstract

The present invention relates to a pyrimidine-2-amine derivative, a method for manufacturing the same, and an anticancer drug containing the same. The pyrimidine-2-amine derivative represented by chemical formula 1 according to the present invention has an excellent effect of inhibiting aurora A kinase activity, thereby being useful as the anticancer drug.

Description

Pyrimidine-2-amine derivative, a method for producing the same, and an anticancer agent containing the same,

The present invention relates to a pyrimidin-2-amine derivative, a process for producing the same, and an anticancer agent containing the same.

Colorectal cancer is one of the diseases of developed countries. As the country enters the advanced countries, the eating habits are becoming westernized and the incidence is rising. More than one million new cases are reported annually, and more than 600,000 of these deaths are attributed to the disease. In Korea, the mortality rate for women is the first and the third for men. As with other cancers, the use of surgery, radiation therapy, chemotherapy, etc. is often used to cure cancer patients after surgery, and a new cancer drug has yet to be developed to cure the cancer.

In addition, since colorectal cancer has no early symptoms, it is necessary to develop a preventive agent because it is often found after progressing considerably.

Aurora kinase is a serine / threonine protein kinase that is involved in cell mitosis. It is known to be over-expressed in cancer cells such as breast, colon, stomach, pancreas, ovary, etc. and is suspected as an oncoprotein. There are three types of Aurora kinases: A, B, and C, and Aurora A and B are associated with the proliferation of many cell types. Aurora C, on the other hand, is not known for its normal function, but its expression is known to be restricted to testicular tissue. Aurora-A is activated only when it phosphorylates threonine-288, aurora-B is threonine-232, and aurora-C is threonine-198 residue.

It is known that suppression of aurora kinase activity in normal cancer cells suppresses cell cycle progression and induces apoptosis (programmed cell death), leading to anticancer effects (Bioorg Med Chem. 2013; 21 (22): 7018- 24). Therefore, when the activity of aurora kinase is decreased, abnormal growth of cancer cells can be prevented and thus, cancer treatment can be applied.

Thus, the present inventors have found that the pyrimidine-2-amine derivatives represented by the formula (I) inhibit the activity against Aurora A kinase only, while the Aurora B kinase inhibits the activity.

Korean Patent Publication No. 10-2006-0011891

An object of the present invention is to provide a pyrimidin-2-amine derivative represented by the formula (1), or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a process for preparing the compound represented by the above formula (1).

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises the pyrimidin-2-amine derivative represented by the above formula (1), or a pharmaceutically acceptable salt thereof.

In order to achieve the above object,

The present invention provides a pyrimidin-2-amine derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

(In the formula 1,

R ¹ is C _6-10 aryl, wherein said aryl may be substituted with one or more substituents selected from the group consisting of hydroxy, C _1-6 straight or branched alkoxy and C _6-10 aryl;

R ² is C _6-12 aryl, wherein said aryl may be substituted with one or more C _1-6 straight chain or branched alkoxy.

Also, as shown in the following Reaction Scheme 1,

Dissolving the compound represented by the general formula (2) and the compound represented by the general formula (3) in an organic solvent and adding and stirring an aqueous alkali solution to obtain a compound represented by the general formula (4) (step 1); And

Dissolving the compound of Formula 4 obtained in Step 1 in an organic solvent, adding guanidine hydrochloride represented by Formula 5 and refluxing to obtain a compound represented by Formula 1 (Step 2);

(1), wherein R < 1 >

[Reaction Scheme 1]

(In the above Reaction Scheme 1,

Wherein R < ¹ > and R < ² > are the same as defined in formula (1).

Further, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises the pyrimidin-2-amine derivative represented by the above formula (1) or a pharmaceutically acceptable salt thereof.

The pyrimidin-2-amine derivative represented by the formula (1) according to the present invention has an excellent effect of inhibiting the Aurora A kinase activity, and thus may be useful as an anticancer agent.

FIG. 1 shows the effect of the pyrimidin-2-amine derivatives of the present invention on cancer cell growth inhibition using a clonogenic long-term survival assay.
FIG. 2 shows the inhibitory concentration (GI ₅₀ ) of the pyrimidin-2-amine derivative of the present invention at 50% of the colon cancer cell line.
FIG. 3 shows the results of inhibiting Aurora A kinase phosphorylation of the pyrimidin-2-amine derivatives of the present invention.
4 shows the results of inhibiting the cell cycle G2 / M phase progression of the pyrimidin-2-amine derivatives of the present invention.

Hereinafter, the present invention will be described in detail.

Compound or a pharmaceutically acceptable salt thereof.

The present invention provides a pyrimidin-2-amine derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

(In the formula 1,

R ¹ is C _6-10 aryl, wherein said aryl may be substituted with one or more substituents selected from the group consisting of hydroxy, C _1-6 straight or branched alkoxy and C _6-10 aryl;

R ² is C _6-12 aryl, wherein said aryl may be substituted with one or more C _1-6 straight chain or branched alkoxy.

Preferably,

Wherein R ¹ is C _6-8 aryl, wherein said aryl may be substituted with one or more substituents selected from the group consisting of hydroxy, C _1-3 linear or branched alkoxy and C _6-8 aryl, ;

R ² is C _6-10 aryl, wherein said aryl may be substituted with one or more C _1-3 straight-chain or branched alkoxy.

More preferably,

Wherein R < ¹ > is phenyl, wherein said phenyl may be substituted with hydroxy, methoxy, dimethoxy or phenyl;

R ² is phenyl or naphthalenyl wherein said phenyl may be substituted with a 2 to 3-methoxy.

Preferred examples of the compound represented by the formula (1) according to the present invention include the following compounds, and their chemical structural formulas are shown in Table 1 below.

1) 4 - ([1,1'-biphenyl] -4-yl) -6- (2,4,6-trimethoxyphenyl) pyrimidin-2-amine;

10) 4 - ([1,1'-biphenyl] -4-yl) -6- (3,5-dimethoxyphenyl) pyrimidin-2-amine;

11) 4 - ([1,1'-biphenyl] -4-yl) -6- (2,4,5-trimethoxyphenyl) pyrimidin-2-amine;

12) 2- (2-Amino-6- (2,4-dimethoxyphenyl) pyrimidin-4-yl) phenol;

18) 4- (3,5-dimethoxyphenyl) -6- (2-methoxyphenyl) pyrimidin-2-amine;

21) 4- (3,5-dimethoxyphenyl) -6- (4-methoxyphenyl) pyrimidin-2-amine; And

24) 4- (3,4-Dimethoxyphenyl) -6- (naphthalen-1-yl) pyrimidin-2-amine.

The compound represented by the formula (1) of the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. The term pharmaceutically acceptable salt means a concentration that is relatively non-toxic to a patient and has a beneficial effect that is harmless to the patient, such that the side effects caused by the salt are any organic or inorganic salt of the base compound of Formula 1 that does not impair the beneficial effects of the base compound of Formula An inorganic addition salt. Examples of the inorganic acid include hydrochloric acid, bromic acid, nitric acid, sulfuric acid, perchloric acid and phosphoric acid. Examples of the organic acid include citric acid, acetic acid, lactic acid, maleic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, maleic acid, tartaric acid, succinic acid, malonic acid, succinic acid, malonic acid, glutamic acid, aspartic acid, oxalic acid, P-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid or malonic acid. These salts also include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.). For example, the acid addition salt may be selected from the group consisting of acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camylate, citrate, eddylate, Hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, malate, glucoside, gluconate, gluconate, glucuronate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / Hydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate, dihydroxyacetate, Lactate, stearate, succinate, tartrate, tosylate, trifluoroacetate Diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, zinc salts, and the like. Preferred is hydrochloride or trifluoroacetate.

The acid addition salt according to the present invention can be obtained by a conventional method, for example, by dissolving the compound represented by the formula (1) in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile and the like, Followed by filtration and drying. Alternatively, the solvent and excess acid may be distilled off under reduced pressure, followed by drying or crystallization in an organic solvent.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Furthermore, the present invention encompasses the compounds of formula (I) and pharmaceutically acceptable salts thereof as well as possible solvates, hydrates, isomers, optical isomers and the like which can be prepared therefrom.

Manufacturing method

As shown in the following Reaction Scheme 1,

Dissolving the compound represented by the general formula (2) and the compound represented by the general formula (3) in an organic solvent and adding and stirring an aqueous alkali solution to obtain a compound represented by the general formula (4) (step 1); And

Dissolving the compound of Formula 4 obtained in Step 1 in an organic solvent, adding guanidine hydrochloride represented by Formula 5 and refluxing to obtain a compound represented by Formula 1 (Step 2);

(1), wherein R < 1 >

[Reaction Scheme 1]

(In the above Reaction Scheme 1,

Wherein R < ¹ > and R < ² > are the same as defined in formula (1).

In one embodiment of the present invention, the organic solvents of step 1 and step 2 are independently selected from the group consisting of ethanol, anhydrous tetrahydrofuran (THF), benzene, KOH / MeOH, MeOH, toluene, CH ₂ Cl ₂ , hexane, (DMF), diisopropyl ether, diethyl ether, dioxane, dimethylacetamide (DMA), dimethylsulfoxide (DMSO), acetone and chlorobenzene, and preferably The organic solvent in step 1 may be ethanol, and the organic solvent in step 2 may be anhydrous dimethylformamide, but is not limited thereto.

Pharmaceutical composition for preventing or treating cancer

The present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a pyrimidin-2-amine derivative represented by the following general formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

(In the formula 1,

R ¹ is C _6-10 aryl, wherein said aryl may be substituted with one or more substituents selected from the group consisting of hydroxy, C _1-6 straight or branched alkoxy and C _6-10 aryl;

R ² is a C _6-12 aryl, wherein said aryl substituent is a straight or branched chain alkyl, at least one member selected from straight-chain or branched alkoxy, nitro and the group consisting of halogen, C _1-6 of C _1-6 substituted .

Preferably,

Wherein R ¹ is C _6-8 aryl, wherein said aryl may be substituted with one or more substituents selected from the group consisting of hydroxy, C _1-3 linear or branched alkoxy and C _6-8 aryl, ;

R ² is a C _6-10 aryl, wherein said aryl substituent is a straight or branched chain alkyl, at least one member selected from straight-chain or branched alkoxy, nitro and the group consisting of halogen, C _1-3 of C _1-3 substituted .

More preferably,

Wherein R < ¹ > is phenyl, wherein said phenyl may be substituted with hydroxy, methoxy, dimethoxy or phenyl;

R ² is phenyl or naphthalenyl, wherein said phenyl may be substituted with methyl, methoxy, dimethoxy, trimethoxy, nitro, chloro, fluoro or bromo.

Preferable examples of the compound represented by the above formula (1) include the following compounds, and their chemical structures are shown in Table 1 below.

1) 4 - ([1,1'-biphenyl] -4-yl) -6- (2,4,6-trimethoxyphenyl) pyrimidin-2-amine;

2) 4 - ([1,1'-biphenyl] -4-yl) -6- (4-chlorophenyl) pyrimidin-2-amine;

3) 4 - ([1,1'-biphenyl] -4-yl) -6- (4-nitrophenyl) pyrimidin-2-amine;

4) 4 - ([1,1'-biphenyl] -4-yl) -6- (4-fluorophenyl) pyrimidin-2-amine;

5) 4 - ([1,1'-biphenyl] -4-yl) -6- ( p -tolyl) pyrimidin-2-amine;

6) 4 - ([1,1'-biphenyl] -4-yl) -6- (3-methoxyphenyl) pyrimidin-2-amine;

7) 4 - ([1,1'-biphenyl] -4-yl) -6- (4-methoxyphenyl) pyrimidin-2-amine;

8) 4 - ([1,1'-biphenyl] -4-yl) -6- (2,3-dimethoxyphenyl) pyrimidin-2-amine;

9) 4 - ([1,1'-biphenyl] -4-yl) -6- (3,4-dimethoxyphenyl) pyrimidin-2-amine;

10) 4 - ([1,1'-biphenyl] -4-yl) -6- (3,5-dimethoxyphenyl) pyrimidin-2-amine;

11) 4 - ([1,1'-biphenyl] -4-yl) -6- (2,4,5-trimethoxyphenyl) pyrimidin-2-amine;

12) 2- (2-Amino-6- (2,4-dimethoxyphenyl) pyrimidin-4-yl) phenol;

13) 4- (4-Chlorophenyl) -6- (4-methoxyphenyl) pyrimidin-2-amine;

14) 4- (4-Propylphenyl) -6- (3,4-dimethoxyphenyl) pyrimidin-2-amine;

15) 4- (4-bromophenyl) -6- (4-methoxyphenyl) pyrimidin-2-amine;

16) 4- (4-Chlorophenyl) -6- (3,4-dimethoxyphenyl) pyrimidin-2-amine;

17) 4- (4-methoxyphenyl) -6- ( p -tolyl) pyrimidin-2-amine;

18) 4- (3,5-dimethoxyphenyl) -6- (2-methoxyphenyl) pyrimidin-2-amine;

19) 4- (3,4-dimethoxyphenyl) -6- (4-fluorophenyl) pyrimidin-2-amine;

20) 4- (3,4-dimethoxyphenyl) -6- ( p -tolyl) pyrimidin-2-amine;

21) 4- (3,5-dimethoxyphenyl) -6- (4-methoxyphenyl) pyrimidin-2-amine;

22) 4,6-bis (3,4-dimethoxyphenyl) pyrimidin-2-amine;

23) 4- (3,4-dimethoxyphenyl) -6- (4-nitrophenyl) pyrimidin-2-amine;

24) 4- (3,4-dimethoxyphenyl) -6- (naphthalen-1-yl) pyrimidin-2-amine; And

25) 4- (3,4-Dimethoxyphenyl) -6- (naphthalen-2-yl) pyrimidin-2-amine.

The cancer may be a colorectal cancer, a breast cancer, a pancreatic cancer, a bone marrow cancer, and the like, preferably a colon cancer.

The compound of the present invention may be administered orally or parenterally in various dosage forms during clinical administration. In the case of formulation, the diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, .

Solid form preparations for oral administration include tablets, patients, powders, granules, capsules, troches and the like, which may contain one or more excipients such as starch, calcium carbonate, It is prepared by mixing sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like are included in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

The effective dose of the compound of the present invention on the human body may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity, and is generally about 0.001 to 100 mg / kg / 0.0 > mg / kg / day. ≪ / RTI > It is generally 0.07 to 7000 mg / day, preferably 0.7 to 2500 mg / day, based on an adult patient weighing 70 kg, and may be administered once a day, It may be divided into several doses.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1 Preparation of Pyrimidin-2-amine Derivative

Example 1-1 Preparation of 4 - ([1,1'-biphenyl] -4-yl) -6- p -Tolyl) pyrimidin-2-amine (4 - ([1,1'-biphenyl] -4-yl) -6- p -tolyl) pyrimidin-2-amine)

The process for the preparation of 4 - ([1,1'-biphenyl] -4-yl) -6- ( p -tolyl) pyrimidin-2-amine as a fifth compound in the pyrimidin- .

2 mmol, 250 쨉 l) of 4-acetylbiphenyl (I; 2 mmol, 392 mg) and p -methylaldehyde (II (2 mmol, 250 쨉 L) were dissolved in 20 ml of ethanol. To the mixture was added 2 ml of 50% aqueous solution of potassium hydroxide The mixture was stirred at room temperature for 20 hours. After the reaction was completed, 30 mL of ice water was poured into the reaction mixture, and the mixture was acidified with 3N hydrochloric acid solution to obtain a precipitate.

The precipitate was purified by ethanol to give pure chalcone compound (III). The chalcone (0.5 mmol, 150 mg) was dissolved in 5 mL anhydrous N, N-dimethylformamide, and guanidine hydrochloride (1.5 mmol, 143 mg) and sodium ethoxide (3 mmol, 150 mg) were added. The mixture was refluxed for 3 hours. After completion of the reaction, 20 mL of ice water was poured into the reaction mixture, and the mixture was acidified with 1N hydrochloric acid solution to obtain a precipitate.

The crude solid was purified by recrystallization from ethanol to give 4 - ([1,1'-biphenyl] -4-yl) -6- ( p- tolyl) pyrimidin- .

[Reaction Scheme 2]

Example 1-2 Synthesis of 4- (3,5-dimethoxyphenyl) -6- (2-methoxyphenyl) pyrimidin- -methoxyphenyl) pyrimidin-2-amine)

Aminopyrimidine-2-amine derivative, which is the 18th compound, is shown in the following Reaction Scheme 3.

2.4 mmol, 400 mg) of 2-methoxyacetophenone (V; 2 mmol, 286)) and 1.2 equivalent of 3,5-dimethoxybenzaldehyde (VI) were dissolved in 20 ml of ethanol. The mixture was stirred at 0-4 < 0 > C under ice-cooling. To the reaction mixture was added 2 ml of a 50% aqueous solution of potassium hydroxide and the mixture was stirred at a low temperature (10 ° C or less) for 3 hours. After completion of the reaction, 30 ml of ice water was poured into the reaction mixture, and the mixture was acidified with 3N hydrochloric acid solution to obtain a precipitate.

The precipitate was purified by ethanol to give pure chalcone compound (VII). The chalcone compound (1 mmol, 298 mg) was dissolved in 5 ml of anhydrous dimethylformamide, and guanidine hydrochloride (3 mmol, 285 mg) and sodium ethoxide (6 mmol, 318 mg)

The mixture was refluxed for 6 hours. After completion of the reaction, 30 ml of ice water was poured into the reaction mixture, and the mixture was acidified with 2N hydrochloric acid solution to obtain a precipitate.

The crude solid was purified by recrystallization from methanol to give 4- (3,5-dimethoxyphenyl) -6- (2-methoxyphenyl) pyrimidin-2-amine (VIII).

[Reaction Scheme 3]

The remaining derivatives were also synthesized by methods similar to those of Examples 1 and 2 in the same manner as in Reaction Scheme 1 below.

[Reaction Scheme 1]

The structure and names of the pyrimidin-2-amine derivatives obtained by the synthesis are shown in the table below.

Pyrimidine-2-amine derivative derivative R ₁ R ₂ Mass
(calcd./
found) Name One

414.1812 / 414.1824 4 - ([1,1'-biphenyl] -4-yl) -6- (2,4,6-trimethoxyphenyl) pyrimidin-2-amine 2

358.1106 // 358.1125 4 - ([1,1'-biphenyl] -4-yl) -6- (4-chlorophenyl) pyrimidin-2-amine 3

369.1346 // 369.1377 4 - ([1,1'-biphenyl] -4-yl) -6- (4-nitrophenyl) pyrimidin-2-amine 4

342.1401 / 342.1433 4 - ([1,1'-biphenyl] -4-yl) -6- (4-fluorophenyl) pyrimidin-2-amine 5

338.1652 / 338.1671 4 - ([1,1'-biphenyl] -4-yl) -6- ( p -tolyl) pyrimidin-2-amine 6

354.1601 / 354.1623 4 - ([1,1'-biphenyl] -4-yl) -6- (3-methoxyphenyl) pyrimidin-2-amine 7

354.1601 / 354.1621 4 - ([1,1'-biphenyl] -4-yl) -6- (4-methoxyphenyl) pyrimidin-2-amine 8

384.1707 / 384.1731 4 - ([1,1'-biphenyl] -4-yl) -6- (2,3-dimethoxyphenyl) pyrimidin-2-amine 9

384.1707 / 384.1732 4 - ([1,1'-biphenyl] -4-yl) -6- (3,4-dimethoxyphenyl) pyrimidin-2-amine 10

384.1707 / 384.1724 4 - ([1,1'-biphenyl] -4-yl) -6- (3,5-dimethoxyphenyl) pyrimidin-2-amine 11

414.1812 / 414.1826 4 - ([1,1'-biphenyl] -4-yl) -6- (2,4,5-trimethoxyphenyl) pyrimidin-2-amine 12

324.1343 / 324.1357 2- (2-amino-6- (2,4-dimethoxyphenyl) pyrimidin-4-yl) phenol 13

312.0898 / 312.0911 4- (4-chlorophenyl) -6- (4-methoxyphenyl) pyrimidin-2-amine 14

386.0499 / 386.0515 4- (4-bromophenyl) -6- (3,4-dimethoxyphenyl) pyrimidin-2-amine 15

356.0393 / 356.0431 4- (4-bromophenyl) -6- (4-methoxyphenyl) pyrimidin-2-amine 16

342.1004 / 342.1026 4- (4-chlorophenyl) -6- (3,4-dimethoxyphenyl) pyrimidin-2-amine 17

292.1444 / 292.1440 4- (4-methoxyphenyl) -6- ( p -tolyl) pyrimidin-2-amine 18

338.1499 / 338.1519 4- (3,5-dimethoxyphenyl) -6- (2-methoxyphenyl) pyrimidin-2-amine 19

326.1299 / 326.1320 4- (3,4-dimethoxyphenyl) -6- (4-fluorophenyl) pyrimidin-2-amine 20

322.1550 / 322.1562 4- (3,4-dimethoxyphenyl) -6- ( p -tolyl) pyrimidin-2-amine 21

338.1499 / 338.1517 4- (3,5-dimethoxyphenyl) -6- (4-methoxyphenyl) pyrimidin-2-amine 22

368.1605 / 368.1622 4,6-bis (3,4-dimethoxyphenyl) pyrimidin-2-amine 23

353.1244 / 353.1272 4- (3,4-dimethoxyphenyl) -6- (4-nitrophenyl) pyrimidin-2-amine 24

358.1550 / 358.1567 4- (3,4-dimethoxyphenyl) -6- (naphthalen-1-yl) pyrimidin-2-amine 25

358.1550 / 358.1577 4- (3,4-dimethoxyphenyl) -6- (naphthalen-2-yl) pyrimidin-2-amine

Experimental Example 1 Spectrophotometric Experiment of Pyrimidin-2-amine Derivative

In order to confirm the structure of the pyrimidin-2-amine derivative of the present invention, nuclear magnetic resonance (NMR) spectroscopy experiments were performed. The concentration of the derivative was adjusted to about 50 mM by dissolving in a deuterated dimethyl sulfoxide-d6 and transferred to a 2.5-mm NMR tube. However, the derivatives 9 and 25 were dissolved in pyridine-d5. In the present invention, an Avance 400 spectrometer system (9.4 T; Bruker, Karlsruhe, Germany) was used. In addition, high resolution mass spectrometry (HRMS) was performed to confirm the structure of the derivatives identified by nuclear magnetic resonance spectroscopy. The instrument used was ultraperformance liquid chromatography-hybrid quadrupole-time-of-flight mass spectrometry system, Waters Corp., Milford, Mass.). The results of the high resolution mass spectrometry are summarized by comparing the experimental values and the theoretical values in Table 1 above. The results of the hydrogen nuclear magnetic resonance spectroscopy are shown in Tables 2 to 6 below, and the results of the carbon nuclear magnetic resonance spectroscopy are summarized in Tables 7 to 9 below.

Results of hydrogen nuclear magnetic resonance spectroscopy of pyrimidin-2-amine derivatives 1 to 5 ^{δ of 1 H (J, Hz} ) Position One 2 3 4 5 H-2 7.78 (ddd, 8.4, 2.1, 1.7) 7.82 (ddd, 8.4, 1.9, 1.7) 7.83 (ddd, 8.4, 2.0, 1.9) 7.82 (ddd, 8.5, 2.1, 1.7) 7.82 (ddd, 8.4, 1.7, 1.3) H-3 8.17 (ddd, 8.4, 2.1, 1.7) 8.33 (ddd, 8.4, 1.9, 1.7) 8.34 (ddd, 8.4, 2.0, 1.9) 8.33 (ddd, 8.5, 2.1, 1.7) 8.32 (ddd, 8.4, 1.7, 1.3) H-5 7.78 (ddd, 8.4, 2.1, 1.7) 7.82 (ddd, 8.4, 1.9, 1.7) 7.83 (ddd, 8.4, 2.0, 1.9) 7.82 (ddd, 8.5, 2.1, 1.7) 7.82 (ddd, 8.4, 1.7, 1.3) H-6 8.17 (ddd, 8.4, 2.1, 1.7) 8.33 (ddd, 8.4, 1.9, 1.7) 8.34 (ddd, 8.4, 2.0, 1.9) 8.33 (ddd, 8.5, 2.1, 1.7) 8.32 (ddd, 8.4, 1.7, 1.3) H-2 ' 7.74 (ddd, 7.8, 1.9, 1.4) 7.76 (ddd, 7.7, 1.9, 1.5) 7.76 (ddd, 7.9, 1.8, 1.3) 7.76 (ddd, 7.8, 1.6, 1.2) 7.76 (ddd, 7.2, 2.1, 1.2) H-3 ' 7.49 (ddd, 7.8, 7.4, 1.5) 7.50 (ddd, 7.7, 7.4, 1.5) 7.50 (ddd, 7.9, 7.3, 1.4) 7.50 (ddd, 7.8, 7.4, 1.4) 7.50 (ddd, 7.6, 7.2, 1.2) H-4 ' 7.39 (dddd, 7.4, 7.4, 1.4, 1.4) 7.40 (dddd, 7.4, 7.4, 1.5, 1.5) 7.40 (dddd, 7.3, 7.3, 1.3, 1.3) 7.40 (dddd, 7.4, 7.4, 1.2, 1.2) 7.40 (dddd, 7.6, 7.6, 1.2, 1.2) H-5 ' 7.49 (ddd, 7.8, 7.4, 1.5) 7.50 (ddd, 7.7, 7.4, 1.5) 7.50 (ddd, 7.9, 7.3, 1.4) 7.50 (ddd, 7.8, 7.4, 1.4) 7.50 (ddd, 7.6, 7.2, 1.2) H-6 ' 7.74 (ddd, 7.8, 1.9, 1.4) 7.76 (ddd, 7.7, 1.9, 1.5) 7.76 (ddd, 7.9, 1.8, 1.3) 7.76 (ddd, 7.8, 1.6, 1.2) 7.76 (ddd, 7.2, 2.1, 1.2) NH ₂ 6.60 (s) 6.80 (s) 6.93 (s) 6.77 (s) 6.71 (s) H-py-5 7.02 (s) 7.79 (s) 7.90 (s) 7.77 (s) 7.73 (s) H-2 " - 8.28 (d, 8.6) 8.50 (d, 8.7) 8.31 (dd, 8.9, 5.7) 8.15 (d, 8.2) H-3 " 6.32 (s) 7.59 (d, 8.6) 8.36 (d, 8.7) 7.35 (dd, 8.9, 8.9) 7.33 (d, 8.2) H-5 " 6.32 (s) 7.59 (d, 8.6) 8.36 (d, 8.7) 7.35 (dd, 8.9, 8.9) 7.33 (d, 8.2) H-6 '' - 8.28 (d, 8.6) 8.50 (d, 8.7) 8.31 (dd, 8.9, 5.7) 8.15 (d, 8.2) 2 '' - OCH ₃ 3.69 (s) - - - - 4 '' - OCH ₃ 3.83 (s) - - - - 6 '' - OCH ₃ 3.69 (s) - - - - 4 '' - CH ₃ - - - - 2.38 (s)

Results of hydrogen nuclear magnetic resonance spectroscopy of pyrimidin-2-amine derivatives 6 to 10 ^{δ of 1 H (J, Hz} ) Position 6 7 8 9 10 H-2 7.81 (ddd, 8.4, 2.0, 1.8) 7.82 (ddd, 8.4, 2.1, 1.8) 7.82 (ddd, 8.5, 2.1, 1.8) 7.86 (ddd, 8.4, 2.1, 1.7) 7.82 (ddd, 8.4, 2.1, 1.8) H-3 8.31 (ddd, 8.4, 2.0, 1.8) 8.32 (ddd, 8.4, 2.1, 1.8) 8.18 (ddd, 8.5, 2.1, 1.8) 8.53 (ddd, 8.4, 2.1, 1.7) 8.35 (ddd, 8.4, 2.1, 1.8) H-5 7.81 (ddd, 8.4, 2.0, 1.8) 7.82 (ddd, 8.4, 2.1, 1.8) 7.82 (ddd, 8.5, 2.1, 1.8) 7.86 (ddd, 8.4, 2.1, 1.7) 7.82 (ddd, 8.4, 2.1, 1.8) H-6 8.31 (ddd, 8.4, 2.0, 1.8) 8.32 (ddd, 8.4, 2.1, 1.8) 8.18 (ddd, 8.5, 2.1, 1.8) 8.53 (ddd, 8.4, 2.1, 1.7) 8.35 (ddd, 8.4, 2.1, 1.8) H-2 ' 7.74 (ddd, 7.9, 2.1, 1.6) 7.76 (ddd, 7.8, 1.7, 1.2) 7.74 (ddd, 7.9, 1.8, 1.5) 7.77 (ddd, 7.1, 2.1, 1.4) 7.76 (ddd, 7.8, 2.0, 1.3) H-3 ' 7.49 (ddd, 7.9, 7.4, 1.3) 7.50 (ddd, 7.8, 7.4, 1.6) 7.49 (ddd, 7.9, 7.4, 1.5) 7.51 (ddd, 7.5, 7.1, 1.4) 7.50 (ddd, 7.8, 7.4, 1.5) H-4 ' 7.39 (dddd, 7.4, 7.4, 1.6, 1.6) 7.40 (dddd, 7.4, 7.4, 1.2, 1.2) 7.40 (dddd, 7.4, 7.4, 1.5, 1.5) 7.41 (dddd, 7.5, 7.5, 1.4, 1.4) 7.40 (dddd, 7.4, 7.4, 1.3, 1.3) H-5 ' 7.49 (ddd, 7.9, 7.4, 1.3) 7.50 (ddd, 7.8, 7.4, 1.6) 7.49 (ddd, 7.9, 7.4, 1.5) 7.51 (ddd, 7.5, 7.1, 1.4) 7.50 (ddd, 7.8, 7.4, 1.5) H-6 ' 7.74 (ddd, 7.9, 2.1, 1.6) 7.76 (ddd, 7.8, 1.7, 1.2) 7.74 (ddd, 7.9, 1.8, 1.5) 7.77 (ddd, 7.1, 2.1, 1.4) 7.76 (ddd, 7.8, 2.0, 1.3) NH ₂ 6.71 (s) 6.67 (s) 6.72 (s) 6.06 (s) 6.76 (s) H-py-5 7.729 (s) 7.71 (s) 7.51 (s) 7.97 (s) 7.75 (s) H-2 " 7.728 (dd, 1.0, 1.0) 8.23 (d, 8.8) - 8.23 (d, 2.0) 7.41 (d, 2.3) H-3 " - 7.07 (d, 8.8) - - H-4 '' 7.09 (ddd, 8.2, 2.6, 1.0) - 7.179 (dd, 8.1, 2.9) - 6.67 (dd, 2.3, 2.3) H-5 " 7.44 (dd, 8.2, 8.2) 7.07 (d, 8.8) 7.177 (dd, 8.1, 6.2) 7.15 (d, 8.4) - H-6 '' 7.81 (ddd, 8.4, 2.0, 1.8) 8.23 (d, 8.8) 7.32 (dd, 6.2, 2.9) 8.10 (dd, 8.4, 2.0) 7.41 (d, 2.3) 2 '' - OCH ₃ - - 3.76 (s) - - ₃ '' - OCH 3 3.85 (s) - 3.86 (s) 3.87 (s) 3.85 (s) 4 '' - OCH ₃ - 3.84 (s) - 3.83 (s) - 5 " -OCH ₃ - - - - 3.85 (s)

Results of hydrogen nuclear magnetic resonance spectroscopy of pyrimidin-2-amine derivatives 11 to 15 ^{δ of 1 H (J, Hz} ) Position 11 12 13 14 15 H-2 7.82 (ddd, 8.4, 1.9, 1.8) - 8.20 (d, 8.9) 7.80 (d, 2.0) 8.20 (d, 8.9) H-3 8.15 (ddd, 8.4, 1.9, 1.8) 6.91 (d, 8.0) 7.06 (d, 8.9) - 7.06 (d, 8.9) H-4 - 7.34 (ddd, 8.0, 7.1, 1.6) - - - H-5 7.82 (ddd, 8.4, 1.9, 1.8) 6.92 (dd, 8.7, 7.1) 7.06 (d, 8.9) 7.08 (d, 8.6) 7.06 (d, 8.9) H-6 8.15 (ddd, 8.4, 1.9, 1.8) 7.88 (dd, 8.7, 1.6) 8.20 (d, 8.9) 7.87 (dd, 8.6, 2.0) 8.20 (d, 8.9) H-2 ' 7.74 (ddd, 7.8, 2.0, 1.3) - - - - H-3 ' 7.50 (ddd, 7.8, 7.3, 1.6) - - - - H-4 ' 7.40 (dddd, 7.3, 7.3, 1.3, 1.3) - - - - H-5 ' 7.50 (ddd, 7.8, 7.3, 1.6) - - - - H-6 ' 7.74 (ddd, 7.8, 2.0, 1.3) - - - - NH ₂ 6.61 (s) 7.04 (s) 6.69 (s) 7.04 (br s) 6.70 (s) H-py-5 7.71 (s) 7.71 (s) 7.67 (s) 7.72 (s) 7.67 (s) H-1 " - - - - - H-2 " - - 8.24 (d, 8.6) 8.18 (d, 8.6) 8.17 (d, 8.6) H-3 " 6.82 (s) 6.70 (d, 2.3) 7.57 (d, 8.6) 7.72 (d, 8.6) 7.71 (d, 8.6) H-4 '' - - - - - H-5 " - 6.67 (dd, 8.6, 2.3) 7.57 (d, 8.6) 7.72 (d, 8.6) 7.71 (d, 8.6) H-6 '' 7.61 (s) 7.90 (d, 8.6) 8.24 (d, 8.6) 8.18 (d, 8.6) 8.17 (d, 8.6) _3-OCH 3 - - - 3.88 (s) - 4-OCH ₃ - - 3.83 (s) 3.84 (s) 3.83 (s) 2 '' - OCH ₃ 3.92 (s) 3.84 (s) - - - 4 '' - OCH ₃ 3.88 (s) 3.91 (s) - - - 5 " -OCH ₃ 3.77 (s) - - - -

Results of hydrogen nuclear magnetic resonance spectroscopy of pyrimidin-2-amine derivatives 16 to 20 ^{δ of 1 H (J, Hz} ) Position 16 17 18 19 20 H-2 7.78 (d, 1.9) 8.19 (d, 8.9) - 7.87 (d, 2.2) 7.78 (d, 2.0) H-3 - 7.05 (d, 8.9) 7.16 (d, 8.4) - - H-4 - - 7.45 (ddd, 8.4, 7.5, 1.7) - - H-5 7.07 (d, 8.4) 7.05 (d, 8.9) 7.06 (dd, 7.5, 7.5) 7.16 (d, 8.6) 7.07 (d, 8.3) H-6 7.84 (dd, 8.4, 1.9) 8.19 (d, 8.9) 7.76 (dd, 7.5, 1.7) 7.98 (dd, 8.6, 2.2) 7.83 (dd, 8.3, 2.0) NH ₂ 6.67 (s) 6.59 (s) 6.65 (s) - 6.60 (s) H-py-5 7.67 (s) 7.61 (s) 7.49 (s) 7.90 (s) 7.63 (s) H-2 " 8.24 (d, 8.7) 8.11 (d, 8.2) 7.18 (d, 2.2) 8.38 (dd, 8.9, 5.4) 8.12 (d, 8.2) H-3 " 7.57 (d, 8.7) 7.31 (d, 8.2) - 7.45 (dd, 8.9, 8.9) 7.32 (d, 8.2) H-4 '' - - 6.64 (dd, 2.2, 2.2) - - H-5 " 7.57 (d, 8.7) 7.31 (d, 8.2) - 7.45 (dd, 8.9, 8.9) 7.32 (d, 8.2) H-6 '' 8.24 (d, 8.7) 8.11 (d, 8.2) 7.18 (d, 2.2) 8.38 (dd, 8.9, 5.4) 8.12 (d, 8.2) 2-OCH ₃ - - 3.85 (s) - - _3-OCH 3 3.87 (s) - - 3.91 (s) 3.88 (s) 4-OCH ₃ 3.83 (s) 3.83 (s) - 3.87 (s) 3.83 (s) ₃ '' - OCH 3 - - 3.82 (s) - - 5 " -OCH ₃ - - 3.82 (s) - - 4 '' - CH ₃ - 2.37 (s) - - 2.37 (s)

Results of hydrogen nuclear magnetic resonance spectroscopy of pyrimidin-2-amine derivatives 21 to 25 ^{δ of 1 H (J, Hz} ) Position 21 22 23 24 25 H-2 8.22 (d, 8.9) 7.74 (d, 2.0) 7.85 (d, 2.0) 7.76 (d, 1.9) 8.25 (d, 2.0) H-3 7.06 (d, 8.9) - - - - H-4 - - - - - H-5 7.06 (d, 8.9) 7.07 (d, 8.6) 7.13 (d, 8.5) 7.06 (d, 8.4) 7.15 (d, 8.4) H-6 8.22 (d, 8.9) 7.81 (dd, 8.6, 2.0) 7.94 (dd, 8.5, 2.0) 7.78 (dd, 8.4, 1.9) 8.13 (dd, 8.4, 2.0) NH ₂ 6.64 (s) 6.99 (br s) - 6.75 (s) 6.56 (s) H-py-5 7.63 (s) 7.61 (s) 7.91 (s) 7.37 (s) 8.10 (s) H-1 " - - - - 9.06 (d, 1.4) H-2 " 7.36 (d, 2.3) 7.74 (d, 2.0) 8.49 (d, 9.0) 7.70 (dd, 7.2, 1.5) - H-3 " - - 8.37 (d, 9.0) 7.62 (dd, 7.4, 7.2) 8.57 (dd, 8.6, 1.4) H-4 '' 6.64 (dd, 2.3, 2.3) - - 8.02 (dd, 7.4, 1.5) 8.05 (d, 8.6) H-5 " - 7.07 (d, 8.6) 8.37 (d, 9.0) 8.01 (dd, 7.2, 2.2) 7.96 (dd, 6.3, 1.0) H-6 '' 7.36 (d, 2.3) 7.81 (dd, 8.6, 2.0) 8.49 (d, 9.0) 7.54 (ddd, 7.2, 7.2, 2.3) 7.57 (ddd, 6.3, 6.3, 1.0) H-7 '' - - - 7.55 (ddd, 7.2, 7.2, 2.2) 7.54 (ddd, 6.3, 6.3, 1.0) H-8 '' - - - 8.21 (dd, 7.2, 2.3) 8.06 (dd, 6.3, 1.0) _3-OCH 3 - 3.85 (s) 3.90 (s) 3.85 (s) 3.88 (s) 4-OCH ₃ 3.835 (s) 3.82 (s) 3.86 (s) 3.83 (s) 3.83 (s) ₃ '' - OCH 3 3.841 (s) 3.85 (s) - - - 4 '' - OCH ₃ - 3.82 (s) - - - 5 " -OCH ₃ 3.841 (s) - - - -

Results of Carbon Nuclear Magnetic Resonance Spectroscopy of Pyrimidin-2-amine Derivatives 1 to 8 [delta] of ¹³ C ( J , Hz) Position One 2 3 4 5 6 7 8 C-1 141.7 142.1 142.3 142.0 141.9 142.0 141.9 142.0 C-2 126.8 126.80 126.84 126.79 126.8 126.80 126.8 127.0 C-3 127.3 127.6 127.7 127.6 127.6 127.6 127.5 127.4 C-4 136.3 136.2 135.9 136.2 136.4 136.3 136.5 136.4 C-5 127.3 127.6 127.7 127.6 127.6 127.6 127.5 127.4 C-6 126.8 126.80 126.84 126.79 126.8 126.80 126.8 127.0 C-1 ' 139.4 139.3 139.3 139.4 139.4 139.4 139.4 139.4 C-2 ' 126.7 126.75 126.76 126.75 126.7 126.75 126.7 126.8 C-3 ' 129.0 129.0 129.0 129.0 129.0 129.0 129.0 129.1 C-4 ' 127.8 127.9 127.9 127.9 127.8 127.9 127.9 127.9 C-5 ' 129.0 129.0 129.0 129.0 129.0 129.0 129.0 129.1 C-6 ' 126.7 126.75 126.76 126.75 126.7 126.75 126.7 126.8 C-py-2 163.9 164.0 164.1 163.8 164.0 164.0 163.9 163.9 C-py-4 162.5 164.6 165.1 164.5 164.2 164.4 164.1 163.5 C-py-5 108.3 101.7 102.7 101.6 101.4 102.0 101.0 106.0 C-py-6 164.4 163.6 162.6 164.0 164.8 164.7 164.5 164.8 C-1 " 110.7 136.2 143.5 133.8
(d, 2.7) 134.6 138.9 129.6 132.7 C-2 " 158.0 128.8 128.2 129.3
(d, 8.5) 126.9 119.4 128.6 147.2 C-3 '' 90.8 128.6 123.7 115.5
(d, 21.4) 129.2 159.6 113.9 152.9 C-4 '' 161.1 135.2 148.5 163.6
(d, 247.8) 140.2 116.2 161.3 114.1 C-5 '' 90.8 128.6 123.7 115.5
(d, 21.4) 129.2 129.7 113.9 124.0 C-6 '' 158.0 128.8 128.2 129.3
(d, 8.5) 126.9 112.2 128.6 121.6 2 '' - OCH ₃ 55.6 - - - - - - 60.8 ₃ '' - OCH 3 - - - - - 55.3 - 55.9 4 '' - OCH ₃ 55.4 - - - - - 55.3 - 6 '' - OCH ₃ 55.6 - - - - - - - 4 '' - CH ₃ - - - - 20.9 - - -

The results of carbon nuclear magnetic resonance spectroscopy of pyrimidin-2-amine derivatives 9 to 16 δ of ¹³ C Position 9 10 11 12 13 14 15 16 C-1 143.7 142.0 141.7 117.7 129.5 129.2 129.5 129.7 C-2 128.04 126.8 126.9 160.3 128.6 110.4 128.6 110.3 C-3 128.8 127.7 127.3 118.1 113.9 148.9 114.0 148.8 C-4 138.0 136.3 136.7 132.4 161.3 151.5 161.3 151.1 C-5 128.8 127.7 127.3 118.9 113.9 111.6 114.0 111.5 C-6 128.04 126.8 126.9 127.2 128.6 120.7 128.6 120.4 C-1 ' 141.1 139.4 139.4 - - - - - C-2 ' 127.97 126.8 126.7 - - - - - C-3 ' 129.9 129.0 129.0 - - - - - C-4 ' 128.7 127.9 127.8 - - - - - C-5 ' 129.9 129.0 129.0 - - - - - C-6 ' 127.97 126.8 126.7 - - - - - C-py-2 165.6 163.9 163.8 161.1 163.9 163.2 163.9 163.9 C-py-4 165.9 164.4 163.1 164.3 164.7 164.7 164.7 164.9 C-py-5 102.9 102.1 106.1 103.9 101.0 101.5 101.0 101.3 C-py-6 166.2 164.7 163.5 163.7 163.2 163.6 163.3 163.3 C-1 " 131.5 139.6 117.7 118.8 136.3 136.3 136.7 136.3 C-2 " 111.9 105.0 153.2 162.3 128.7 129.3 129.0 128.8 C-3 '' 150.6 160.7 98.5 98.0 128.6 131.8 131.6 128.7 C-4 '' 152.8 102.4 151.4 159.3 135.1 124.5 124.0 135.2 C-5 '' 112.6 160.7 142.7 105.6 128.6 131.8 131.6 128.7 C-6 '' 121.5 105.0 113.7 131.5 128.7 129.3 129.0 128.8 _3-OCH 3 - - - - - 55.9 - 55.7 4-OCH ₃ - - - - 55.3 55.8 55.3 55.6 2 '' - OCH ₃ - - 56.6 55.5 - - - - ₃ '' - OCH 3 56.5 55.4 - - - - - - 4 '' - OCH ₃ 56.4 - 55.8 55.9 - - - - 5 " -OCH ₃ - 55.4 56.2 - - - - -

Pyrimidin-2-amine derivatives 17 to 25 were subjected to carbon nuclear magnetic resonance spectroscopy [delta] of ¹³ C ( J , Hz) Position 17 18 19 20 21 22 23 24 25 C-1 129.7 127.0 126.3 129.9 129.6 128.8 128.2 129.7 131.4 C-2 128.5 157.4 110.8 110.2 128.6 110.4 110.6 110.1 112.0 C-3 114.0 112.0 149.5 148.8 113.9 148.8 149.2 148.8 150.6 C-4 161.2 131.0 153.0 150.9 161.2 151.4 152.3 151.0 152.9 C-5 114.0 120.4 111.7 111.4 113.9 111.5 112.0 111.5 112.6 C-6 128.5 130.2 122.4 120.2 128.6 120.8 121.6 120.1 121.7 C-py-2 163.9 163.8 163.8 163.8 163.8 162.2 163.0 163.6 165.5 C-py-4 164.3 164.5 159.4 164.4 164.4 163.8 164.8 164.1 166.1 C-py-5 100.8 106.9 102.1 101.0 101.4 101.0 103.3 106.1 103.4 C-py-6 164.5 163.4 163.5 164.5 164.3 163.8 162.4 167.5 166.2 C-1 " 134.7 139.8 131.3
(d, 2.9) 134.7 139.7 128.8 142.7 137.2 128.4 C-2 " 126.9 104.7 131.0
(d, 9.1) 126.9 104.9 110.4 129.1 126.9 129.6 C-3 '' 129.2 160.7 116.6
(d, 21.7) 129.2 160.7 148.8 124.3 125.7 125.7 C-4 '' 140.1 102.1 165.1
(d, 250.7) 140.1 102.3 151.4 149.3 129.1 129.2 C-5 '' 129.2 160.7 116.6
(d, 21.7) 129.2 160.7 111.5 124.3 128.3 128.7 C-6 '' 126.9 104.7 131.0
(d, 9.1) 126.9 104.9 120.8 129.1 126.0 128.1 C-7 '' - - - - - - - 126.5 127.5 C-8 '' - - - - - - - 125.6 129.9 C-9 '' - - - - - - - 130.2 134.6 C-10 '' - - - - - - - 133.3 135.5 2-OCH ₃ - 55.7 - - - - - - - _3-OCH 3 - - 56.4 55.7 - 55.8 56.24 55.57 56.6 4-OCH ₃ 55.3 - 56.3 55.6 55.3 55.7 56.18 55.61 56.5 ₃ '' - OCH 3 - 55.3 - - 55.4 55.8 - - - 4 '' - OCH ₃ - - - - - 55.7 - - - 5 " -OCH ₃ - 55.3 - - 55.4 - - - - 4 '' - CH ₃ 21.0 - - 21.0 - - - - -

<Experimental Example 2> Clonogenic long-term survival assay

There are various methods for measuring the anticancer effect of the compound, which is a method of measuring the effect of inhibiting the growth of cancer cells by a universal method. This method is also used in various methods including MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay, which inhibits the growth of cancer cells Clonogenic long-term survival assay (CSA) is used to measure the effect. This method has the disadvantage that the experiment period is 7 days, whereas CSA is used in the present invention because it has the advantage of distinguishing the cancer cell growth inhibiting effect of the derivatives having the similar structure

HCT116 colon cancer cells were purchased from the American Type Culture Collection (ATTC) and cultured in DMEM (Invitrogen Life Technologies) containing 10% fetal bovine serum (Invitrogen Life Technologies) and Antibiotic-Antimycotic solution (Invitrogen Life Technologies) The cells were cultured in a 5% CO ₂ incubator at 37 ° C in a 100-mm cell culture dish at 2 × 10 ⁶ seed density.

HCT116 colon cancer cells were divided into 3,000 cells per well in a 24-well culture dish and treated with pyrimidin-2-amine derivatives at concentrations of 0, 1, 5, 10 and 20 μM, After adding 6% glutaraldehyde and 0.5% crystal violet solution to the cells, the mixture was allowed to react for 15 minutes and stained with the remaining cells. As a result of measuring the colony forming ability of cancer cells showing 25 kinds of pyrimidin-2-amine derivatives, it can be seen that cell growth is decreased depending on treatment concentration as shown in FIG. These results suggest that the proliferation of HCT116 colon cancer cells was inhibited by the compounds according to the present invention.

<Experimental Example 3> Inhibitory effect of cancer cell colonization

Inhibition of cancer cell colony formation was measured using densitometry and half maximal cell growth inhibitory concetrations (GI ₅₀ ) values were obtained using the SigmaPlot program (SYSTAT, Chicago, IL). Standard deviation error bars were together with GI ₅₀ values are listed in the Figure 2, GI ₅₀ values from 1.45 showed a value of between 40.82uM (see Table 10).

The half-maximal cell growth inhibitory concentration (GI50) derivative GI50 / uM STD One 18.55 0.278 2 40.82 0.537 3 21.60 0.100 4 15.55 0.086 5 33.82 0.165 6 16.50 0.046 7 17.08 0.066 8 18.17 0.075 9 6.75 0.253 10 15.25 0.142 11 11.19 0.056 12 1.45 0.010 13 34.54 0.933 14 29.21 0.244 15 15.63 0.063 16 15.93 0.086 17 22.70 0.077 18 4.21 0.027 19 15.88 0.086 20 12.00 0.056 21 5.32 0.021 22 19.52 0.165 23 9.89 0.118 24 1.92 0.004 25 10.51 0.114

Experimental Example 4 Evaluation of inhibition of Aurora A kinase

As in Experimental Examples 2 to 3, kinase assays were performed on Compound No. 12, which exhibited the best cancer cell growth inhibitory effect among 25 kinds of pyrimidin-2-amine derivatives.

Kinase assays were performed on more than 10 kinases associated with inhibition of cancer cell growth. The inhibitory effect of Aurora A kinase at a concentration of 10 μM was 81%, respectively.

Human Aurora A kinase is incubated in a buffer solution containing 8 mM 3- (N-morpholino) propanesulfonic acid (MOPS) pH 7.0 in a solution containing 0.2 mM EDTA, 10 mM MgAcetate and γ-33 P-ATP . The reaction starts with MgATP, incubates at room temperature for 40 minutes, and terminates the reaction with 3% phosphoric acid solution. 200 μM LRRASLG (Kemptide) is used as a substrate for Aurora A kinase. Detailed experimental methods are based on the Millipore Kinase Assay Protocol. The pyrimidin-2-amine derivative 12 was then treated to measure Aurora A kinase inhibition.

<Experimental Example 5> Western blot analysis

As shown in Experimental Example 4, the pyrimidine-2-amine derivative 12 showed excellent inhibitory effect against Aurora A kinase, so that it was confirmed by Western blot analysis.

HCT116 cells were cultured in a 60-mm cell culture dish at 37 ° C in a 5% CO ₂ incubator at 1 × 10 ⁶ seed density. The cultured cells were treated with pyrimidin-2-amine derivative 12, and the cells were harvested after various treatments. Cell lysis buffer containing 20 mM HEPES (pH 7.2), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 μg / ml leupeptin and 1 mM PMSF was added to the harvested cells for 30 minutes The cells were lysed by high-speed centrifugation, and the cell lysate was harvested. SDS-polyacrylamide gel electrophoresis was performed on the protein lysate prepared to contain the same amount of protein, Were isolated. After transferring the proteins separated by electrophoresis to a polystyrene membrane, a primary antibody that binds to phospho-Aurora A (Thr288) / Aurora B (Thr232) / Aurora C (Thr198) ) And a primary antibody (purchased from Santa Cruz technology) that recognizes GAPDH that does not change protein expression as a control were reacted for 5 hours each, and a secondary antibody (purchased from Cell Signaling Technology) Lt; / RTI &gt; The expression of each protein was analyzed on an X-ray film using a chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ).

As a result, treatment of HCT116 colon cancer cells with 50 μM pyrimidin-2-amine derivative 12 as shown in FIG. 3 reduced the degree of phosphorylation of Aurora A kinase (Thr 288) within 15 minutes. However, the degree of phosphorylation of Aurora B kinase (Thr232) and Aurora C kinase (Thr198) did not change. These results indicate that the derivative 12 of the present invention specifically inhibits Aurora kinase A enzyme activity.

<Experimental Example 6> Inhibitory effect on cell cycle progression in colon cancer cells

The Aurora kinase A enzyme is located in the centrosome and spindle poles of the dividing cells, regulates the maturity of the centrosome, and regulates chromosome alignment, separation, and spindle assembly [ Cell Motil Cytoskeleton; 55: 134; Nat Rev Mol Cell Biol; 4: 842]. Therefore, when the Aurora kinase A enzyme activity is inhibited, the spindle formation is not performed properly in the mitosis process, and the M phase of the cell cycle is not properly progressed. Amine derivative 12 of the present invention inhibited the activity of Aurora kinase A enzyme to affect cell cycle progression.

Cell cycle progression assays are typically analyzed by measuring intracellular DNA content. The HCT116 colon cancer cells were treated with the derivative 12 of the present invention at a concentration of 50 [mu] M. After 24 hours, the cells were harvested and fixed with 70% ethanol. After DNA was stained with PI (Propidium Iodine) for 30 minutes, the DNA content was measured with a NucleoCounter NC-300 image cytometer (ChemoMetec, Allerød, Denmark).

As a result, as shown in Fig. 4, the cells having G1, S and G2 / M cell cycles were 54.4%, 21.1% and 22.7% in the normally growing HCT116 colon cancer cells, respectively, but the pyrimidine- 12, the number of G1 and G2 cells decreased to 28.3% and 16.1%, respectively, after 24 hours, and to 50.1%, respectively. Subcellular cells with progression of apoptosis were increased from 1.3% of control cells to 5.4% after 24 hours of treatment with derivative 12.

Thus, the pyrimidin-2-amine derivative 12 of the present invention inhibits the Aurora kinase A enzyme activity and inhibits the progression of mitosis of cancer cells, thereby inhibiting the M-phase progression of the cell cycle and consequently inducing apoptosis I could.

Meanwhile, the compound represented by Formula 1 according to the present invention can be formulated into various forms according to the purpose. Hereinafter, some formulation methods in which the compound represented by Formula 1 according to the present invention is contained as an active ingredient are exemplified, and the present invention is not limited thereto.

&Lt; Formulation Example 1 > Preparation of powders

The compound of formula (1) 2 g

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

&Lt; Formulation Example 2 > Preparation of tablet

The compound of formula (1) 100 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; Formulation Example 3 > Preparation of capsules

The compound of formula (1) 100 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; Formulation Example 4 > Preparation of injection

The compound of formula (1) 100 mg

Mannitol 180 mg

Na ₂ HPO ₄ .2H ₂ O 26 mg

Distilled water 2974 mg

According to the conventional method for preparing an injectable preparation, an injectable preparation was prepared by incorporating the aforementioned components in the amounts indicated.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (12)

Claims 1. A pyrimidin-2-amine derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

(In the formula 1,
R ¹ is C _6-10 aryl, wherein said aryl may be substituted with one or more substituents selected from the group consisting of hydroxy, C _1-6 straight or branched alkoxy and C _6-10 aryl;
R ² is C _6-12 aryl, wherein said aryl may be substituted with one or more C _1-6 straight chain or branched alkoxy. The method according to claim 1,
Wherein R ¹ is C _6-8 aryl, wherein said aryl may be substituted with one or more substituents selected from the group consisting of hydroxy, C _1-3 linear or branched alkoxy and C _6-8 aryl, ;
R ² is C _6-10 aryl, wherein said aryl can be substituted with one or more C _1-3 linear or branched alkoxy, or a pharmaceutically acceptable salt thereof, salt. The method according to claim 1,
Wherein R &lt; ¹ &gt; is phenyl, wherein said phenyl may be substituted with hydroxy, methoxy, dimethoxy or phenyl;
R ² is phenyl or naphthalenyl, wherein said phenyl can be substituted with 2 to 3 methoxy. The compound represented by the formula (1) is any one selected from the group consisting of the following compounds: or a pharmacologically acceptable salt thereof:
1) 4 - ([1,1'-biphenyl] -4-yl) -6- (2,4,6-trimethoxyphenyl) pyrimidin-2-amine;
10) 4 - ([1,1'-biphenyl] -4-yl) -6- (3,5-dimethoxyphenyl) pyrimidin-2-amine;
11) 4 - ([1,1'-biphenyl] -4-yl) -6- (2,4,5-trimethoxyphenyl) pyrimidin-2-amine;
12) 2- (2-Amino-6- (2,4-dimethoxyphenyl) pyrimidin-4-yl) phenol;
18) 4- (3,5-dimethoxyphenyl) -6- (2-methoxyphenyl) pyrimidin-2-amine;
21) 4- (3,5-dimethoxyphenyl) -6- (4-methoxyphenyl) pyrimidin-2-amine; And
24) 4- (3,4-Dimethoxyphenyl) -6- (naphthalen-1-yl) pyrimidin-2-amine. As shown in Scheme 1 below,
Dissolving the compound represented by the general formula (2) and the compound represented by the general formula (3) in an organic solvent and adding and stirring an aqueous alkali solution to obtain a compound represented by the general formula (4) (step 1); And
Dissolving the compound of Formula 4 obtained in Step 1 in an organic solvent, adding guanidine hydrochloride represented by Formula 5 and refluxing to obtain a compound represented by Formula 1 (Step 2);
(1), wherein R &lt; 1 &gt; and R &lt; 2 &gt;
[Reaction Scheme 1]

(In the above Reaction Scheme 1,
Wherein R &lt; ¹ &gt; and R &lt; ² &gt; are the same as defined in formula (1). 6. The method of claim 5,
The organic solvents of step 1 and step 2 are independently selected from the group consisting of ethanol, anhydrous tetrahydrofuran (THF), benzene, KOH / MeOH, MeOH, toluene, CH ₂ Cl ₂ , hexane, dimethylformamide (DMF) , At least one member selected from the group consisting of diethyl ether, dioxane, dimethylacetamide (DMA), dimethylsulfoxide (DMSO), acetone and chlorobenzene.
Claims 1. A pharmaceutical composition for preventing or treating cancer comprising a pyrimidin-2-amine derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

(In the formula 1,
R ¹ is C _6-10 aryl, wherein said aryl may be substituted with one or more substituents selected from the group consisting of hydroxy, C _1-6 straight or branched alkoxy and C _6-10 aryl;
R ² is a C _6-12 aryl, wherein said aryl substituent is a straight or branched chain alkyl, at least one member selected from straight-chain or branched alkoxy, nitro and the group consisting of halogen, C _1-6 of C _1-6 substituted . 8. The method of claim 7,
Wherein R ¹ is C _6-8 aryl, wherein said aryl may be substituted with one or more substituents selected from the group consisting of hydroxy, C _1-3 linear or branched alkoxy and C _6-8 aryl, ;
R ² is a C _6-10 aryl, wherein said aryl substituent is a straight or branched chain alkyl, at least one member selected from straight-chain or branched alkoxy, nitro and the group consisting of halogen, C _1-3 of C _1-3 substituted &Lt; / RTI &gt;
8. The method of claim 7,
Wherein R &lt; ¹ &gt; is phenyl, wherein said phenyl may be substituted with hydroxy, methoxy, dimethoxy or phenyl;
R ² is phenyl or naphthalenyl, wherein said phenyl may be substituted with methyl, methoxy, dimethoxy, trimethoxy, nitro, chloro, fluoro or bromo.
8. The method of claim 7,
Wherein the compound represented by the formula (1) is any one selected from the group consisting of the following compounds:
1) 4 - ([1,1'-biphenyl] -4-yl) -6- (2,4,6-trimethoxyphenyl) pyrimidin-2-amine;
2) 4 - ([1,1'-biphenyl] -4-yl) -6- (4-chlorophenyl) pyrimidin-2-amine;
3) 4 - ([1,1'-biphenyl] -4-yl) -6- (4-nitrophenyl) pyrimidin-2-amine;
4) 4 - ([1,1'-biphenyl] -4-yl) -6- (4-fluorophenyl) pyrimidin-2-amine;
5) 4 - ([1,1'-biphenyl] -4-yl) -6- ( p -tolyl) pyrimidin-2-amine;
6) 4 - ([1,1'-biphenyl] -4-yl) -6- (3-methoxyphenyl) pyrimidin-2-amine;
7) 4 - ([1,1'-biphenyl] -4-yl) -6- (4-methoxyphenyl) pyrimidin-2-amine;
8) 4 - ([1,1'-biphenyl] -4-yl) -6- (2,3-dimethoxyphenyl) pyrimidin-2-amine;
9) 4 - ([1,1'-biphenyl] -4-yl) -6- (3,4-dimethoxyphenyl) pyrimidin-2-amine;
10) 4 - ([1,1'-biphenyl] -4-yl) -6- (3,5-dimethoxyphenyl) pyrimidin-2-amine;
11) 4 - ([1,1'-biphenyl] -4-yl) -6- (2,4,5-trimethoxyphenyl) pyrimidin-2-amine;
12) 2- (2-Amino-6- (2,4-dimethoxyphenyl) pyrimidin-4-yl) phenol;
13) 4- (4-Chlorophenyl) -6- (4-methoxyphenyl) pyrimidin-2-amine;
14) 4- (4-Propylphenyl) -6- (3,4-dimethoxyphenyl) pyrimidin-2-amine;
15) 4- (4-bromophenyl) -6- (4-methoxyphenyl) pyrimidin-2-amine;
16) 4- (4-Chlorophenyl) -6- (3,4-dimethoxyphenyl) pyrimidin-2-amine;
17) 4- (4-methoxyphenyl) -6- ( p -tolyl) pyrimidin-2-amine;
18) 4- (3,5-dimethoxyphenyl) -6- (2-methoxyphenyl) pyrimidin-2-amine;
19) 4- (3,4-dimethoxyphenyl) -6- (4-fluorophenyl) pyrimidin-2-amine;
20) 4- (3,4-dimethoxyphenyl) -6- ( p -tolyl) pyrimidin-2-amine;
21) 4- (3,5-dimethoxyphenyl) -6- (4-methoxyphenyl) pyrimidin-2-amine;
22) 4,6-bis (3,4-dimethoxyphenyl) pyrimidin-2-amine;
23) 4- (3,4-dimethoxyphenyl) -6- (4-nitrophenyl) pyrimidin-2-amine;
24) 4- (3,4-dimethoxyphenyl) -6- (naphthalen-1-yl) pyrimidin-2-amine; And
25) 4- (3,4-Dimethoxyphenyl) -6- (naphthalen-2-yl) pyrimidin-2-amine.
8. The method of claim 7,
Wherein said cancer is at least one selected from the group consisting of colon cancer, breast cancer, pancreatic cancer and bone marrow cancer.
12. The method of claim 11,
Wherein the cancer is a colon cancer.

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