KR20220147511A - Composition for inhibiting cyclin-dependent kinase and medical uses thereof - Google Patents

Abstract

The present invention relates to a novel compound exhibiting an inhibitory effect on cyclin-dependent kinase activity and a medical use thereof, wherein the compound inhibits cyclin-dependent kinase 9 activity and regulates cell division of cancer, resulting in cell death. Therefore, the present invention is to provide a composition containing the compound as an active component as a cancer treatment agent and a cyclin-dependent kinase 9 inhibitor.

Description

Composition for inhibiting cyclin-dependent kinase and medical uses thereof

The present invention relates to a novel compound exhibiting an effect of inhibiting cyclin-dependent kinase activity, and to a medical use thereof.

Cancer exhibits uncontrolled cell division, and cell cycle control to regulate cell division in cancer can be a strategy for effective cancer treatment. In particular, CDK (cyclin-dependent kinase), which promotes cell cycle progression, is an important regulatory enzyme that induces cell cycle progression, and is considered an important therapeutic target in cancer treatment.

Over the past 20 years, many CDK inhibitors have been developed as potential cancer therapeutics and have been used as therapeutics for multiple tumors. The first-generation CDK inhibitors developed are called pan-CDK inhibitors because they are relatively non-specific. Among the first-generation inhibitors, flavopyridol was the most extensively studied CDK inhibitor so far and showed significant activity in vitro, but did not meet the initial expectations for CDK inhibitors in vivo. , showed low-level results in phase 2 clinical trials for several solid tumors.

CDK9 forms a complex with cyclin T or K to phosphorylate the C terminus of RNA polymerase II, thereby controlling the process of mRNA transcription. Since inhibition of CDK9 lowers the overall rate of protein synthesis, proteins with short half-lives tend to disappear rapidly upon inhibition of CDK9. Examples of such proteins include Myc, Cyclin D1, and Mcl-1. Since proteins necessary for the growth and survival of many cancer cells are composed of proteins with such short half-lives, if CDK9 inhibitors are discovered, there is a sufficient possibility of using them as anticancer agents.

Republic of Korea Patent Publication No. 10-2013-0098151 (published on 04.09.2013)

The present invention provides a novel compound that inhibits cyclin-dependent kinase 9 activity, and an object of the present invention is to provide a composition containing the compound as an active ingredient as a preventive or therapeutic agent for cancer diseases.

The present invention provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1,

A, X and Y may each be the same or different, and are selected from CH or N,

R ¹ and R ² may each be the same or different, and may be any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen.

The present invention provides a pharmaceutical composition for preventing or treating cancer diseases, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a composition for inhibiting cyclin-dependent kinase 9 containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention confirmed a novel compound exhibiting an effect of inhibiting cyclin-dependent kinase 9 (cyclin-dependent kinase 9) activity, and as it was confirmed that the compound induces apoptosis by regulating cancer cell division, the compound An object of the present invention is to provide a composition containing as an active ingredient as a cancer disease treatment agent and a cyclin-dependent kinase 9 inhibitor.

1 shows the compound N-(3-(1,4-diazepan-1-yl)phenyl)-4-(2-methoxyphenyl)pyridin-2-amine (hereinafter referred to as A09-003), N-( 3-(1,4-diazepan-1-yl)phenyl)-4-(pyrazin-2-yl)pyrimidin-2-amine (hereinafter referred to as A09-004) and N-(3-(1,4) -diazepan-1-yl) phenyl) -4- (4-fluoro-2-methoxyphenyl) pyridin-2-amine (hereinafter referred to as A09-005 (AE09-005)) CDK9 activity inhibitory effect was confirmed This is the result of the kinase assay.
2 is a kinase analysis result confirming the inhibitory effect on FLT3 activity of compounds A09-003, A09-004 and A09-005 (AE09-005).
3 shows the results of confirming the cell viability and IC ₅₀ values by treatment with the compounds A09-003 and A09-004 in the leukemia cell lines BDCM, Molm14, MV4-11, THP-1 and U937.
Figure 4 is the result of confirming the cell viability and IC ₅₀ values by treatment with the A09-005 (AE09-005) compound in the leukemia cell lines BDCM, Molm14, MV4-11, THP-1 and U937.
5 is a flow cytometric analysis result confirming the apoptosis effect of compound A09-003 treatment in leukemia cell lines Molm14 and MV4-11 cells.
6 is a fluorescence image showing the apoptosis effect of compound A09-003 treatment in leukemia cell lines Molm14 and MV4-11 cells.
7 is a result of confirming the effect of compound A09-003 treatment on the cell cycle in leukemia cell lines Molm14 and MV4-11 cells using a FACS device.
8 is a Western blot analysis result confirming the protein expression change by treatment with the compounds A09-003 and A09-005 (AE09-005) in the leukemia cell line MV4-11.

Hereinafter, the present invention will be described in more detail.

The present invention identifies a novel compound exhibiting an effect of inhibiting cyclin-dependent kinase 9 activity, and provides a composition containing the compound as an active ingredient as a therapeutic agent for cancer diseases and a cyclin-dependent kinase 9 inhibitor do.

The present invention may provide a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1,

A, X and Y may each be the same or different, and are selected from CH or N,

Each of R ¹ and R ² may be the same or different, and may be any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen.

The compound or a pharmaceutically acceptable salt thereof may be a compound represented by Formula 1-1.

[Formula 1-1]

In Formula 1,

A, X and Y may each be the same or different, and are selected from CH or N,

Each of R ¹ and R ² may be the same or different, and may be any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen.

Wherein R ¹ may be hydrogen or halogen, and R ² may be any one of hydrogen or C1 to C4 alkoxy.

The compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is N-(3-(1,4-diazepan-1-yl)phenyl)-4-(2-methoxyphenyl)pyridin-2-amine [N-(3-(1,4-diazepan-1-yl)phenyl)-4-(2-methoxyphenyl)pyridin-2-amine], N-(3-(1,4-diazepan-1-yl) )phenyl)-4-(pyrazin-2-yl)pyrimidin-2-amine [N-(3-(1,4-diazepan-1-yl)phenyl)-4-(pyrazin-2-yl)pyrimidin- 2-amine] or N-(3-(1,4-diazepan-1-yl)phenyl)-4-(4-fluoro-2-methoxyphenyl)pyridin-2-amine [N-(3- (1,4-diazepan-1-yl)phenyl)-4-(4-fluoro-2-methoxyphenyl)pyridin-2-amine].

The compound represented by Formula 1 or a pharmaceutically acceptable salt thereof may inhibit cyclin-dependent kinase 9 (CDK9).

The present invention can provide a pharmaceutical composition for the prevention or treatment of cancer diseases containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In Formula 1,

A, X and Y may each be the same or different, and are selected from CH or N,

Each of R ¹ and R ² may be the same or different, and may be any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen.

The compound or a pharmaceutically acceptable salt thereof may be a compound represented by Formula 1-1.

[Formula 1-1]

In Formula 1,

A, X and Y may each be the same or different, and are selected from CH or N,

Each of R ¹ and R ² may be the same or different, and may be any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen.

Wherein R ¹ may be hydrogen or halogen, and R ² may be any one of hydrogen or C1 to C4 alkoxy.

The compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is N-(3-(1,4-diazepan-1-yl)phenyl)-4-(2-methoxyphenyl)pyridin-2-amine [N-(3-(1,4-diazepan-1-yl)phenyl)-4-(2-methoxyphenyl)pyridin-2-amine], N-(3-(1,4-diazepan-1-yl) )phenyl)-4-(pyrazin-2-yl)pyrimidin-2-amine [N-(3-(1,4-diazepan-1-yl)phenyl)-4-(pyrazin-2-yl)pyrimidin- 2-amine] or N-(3-(1,4-diazepan-1-yl)phenyl)-4-(4-fluoro-2-methoxyphenyl)pyridin-2-amine [N-(3- (1,4-diazepan-1-yl)phenyl)-4-(4-fluoro-2-methoxyphenyl)pyridin-2-amine].

The compound represented by Formula 1 or a pharmaceutically acceptable salt thereof may inhibit cyclin-dependent kinase 9.

The cancer disease is acute myeloid leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, breast cancer, ovarian cancer, uterine cancer, pancreatic cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, skin cancer, bladder cancer, thyroid cancer, brain cancer and prostate cancer It may be selected from the group consisting of.

In one embodiment of the present invention, the pharmaceutical composition for the prevention or treatment of cancer containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient is prepared by injection, granule, powder, or tablet according to a conventional method. , pills, capsules, suppositories, gels, suspensions, emulsions, drops or liquids may be used in any one formulation selected from the group consisting of.

In another embodiment of the present invention, the pharmaceutical composition comprises a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, flavoring agent, antioxidant, buffer, bacteriostatic agent, diluent, It may further include one or more additives selected from the group consisting of dispersants, surfactants, binders and lubricants.

Specifically, carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agent and the like, and these solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, and the like, and various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

According to an embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject.

The preferred dosage of the compound of Formula 1 may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to an embodiment of the present invention, although not limited thereto, the daily dose may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be administered in several divided doses, thereby not limiting the scope of the present invention.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

In addition, the present invention may provide a composition for inhibiting cyclin-dependent kinase 9 containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In Formula 1,

A, X and Y may each be the same or different, and are selected from CH or N,

Each of R ¹ and R ² may be the same or different, and may be any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Synthesis example> compound synthesis

[Scheme 1]

1-1. Synthesis of 2-chloro-4-(2-methoxyphenyl)pyridine [2-chloro-4-(2-methoxyphenyl)pyridine (3)]

4-bromo-2-chloropyridine (2.00 g, 10.40 mmol), (2-methoxyphenyl)broonic acid [(2-methoxyphenyl)boronic acid; 1.58 g, 10.40 mmol), tetrakis (triphenylphosphine) palladium [Tetrakis (triphenylphosphine) palladium (0); 0.60 g, 0.52 mmol], sodium carbonate (2.20 g, 20.80 mmol) was added to a solution of 1,4-dioxane (1,4-dioxane, 30 ml) and water (10 mL) and stirred under reflux for 12 hours. . The reaction mixture was layer-separated with ethyl acetate (Ethyl acetate, 20 mL), and the organic layer was washed with distilled water, dried over anhydrous Na ₂ SO ₄ , and filtered. After the filtrate was concentrated under reduced pressure, the concentrate was purified by column chromatography to obtain compound (3) (1.56 g, 68%).

1-2. tert-butyl 4-(3-((4-(2-methoxyphenyl)pyridin-2yl)amino)phenyl-1,4-diazepane-1-carboxylate [tert-butyl 4-(3-(( 4-(2-methoxyphenyl)pyridin-2-yl)amino)phenyl)-1,4-diazepane-1-carboxylate (5)] synthesis

2-chloro-4-(2-methoxyphenyl)pyridine [2-chloro-4-(2-methoxyphenyl)pyridine; 100 mg, 0.46 mmol], tert-butyl 4-(3-aminophenyl)-1,4-diazepine-1-carboxylate [tert-butyl 4-(3-aminophenyl)-1,4-diazepane-1- carboxylate; 132 mg, 0.46 mmol], tri(dibenzylideneacetone)dipalladium [Tris(dibenzylideneacetone)dipalladium(0); 21 mg, 0.03 mmol], X-phos (22 mg, 0.05 mmol), potassium carbonate [potassium carbonate; 125 mg, 0.91 mmol] was put into a toluene (toluene, 2 ml) solution and a microwave reactor was used at 150° C. for 3 hours. The reaction mixture was layered with water and ethyl acetate (Ethyl acetate, 5 mL), the organic layer was washed with distilled water, dried over anhydrous Na ₂ SO ₄ , and filtered. After the filtrate was concentrated under reduced pressure, the concentrate was purified by column chromatography to obtain compound (5) (60 mg, 27%).

1-3. N-(3-(1,4-diazepan-1-yl)phenyl)-4-(2-methoxyphenyl)pyridin-2-amine [N-(3-(1,4-diazepan-1-yl) )phenyl)-4-(2-methoxyphenyl)pyridin-2-amine (A9-003)]

tert-butyl 4-(3-((4-(2-methoxyphenyl)pyridin-2-yl)amino)phenyl)-1,4-diazepane-1-carboxylate [tert-butyl 4-( 3-((4-(2-methoxyphenyl)pyridin-2-yl)amino)phenyl)-1,4-diazepane-1-carboxylate; 60 mg, 0.13 mmol] was dissolved in dichloromethane (2 ml), and trifluoroacetic acid (trifluoroacetic acid; 0.03 ml, 0.39 mmol) was added thereto, followed by stirring for 12 hours. The reaction mixture was layer-separated with water and dichloromethane, and the organic layer was washed with sat. After washing with NaHCO ₃ , dried over anhydrous Na ₂ SO ₄ and filtered. The filtrate was concentrated under reduced pressure and recrystallized from ethyl ether to obtain compound (A9-003) (27 mg, 57%).

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.19 (d, 1H, J=4.0 Hz), 7.35 (m, s), 7.30 (dd, 1H, J=2.0 and 8.0 Hz), 7.13 (m, 2H) ), 7.02 (dd, 1H, J=2.0 and 8.0 Hz), 6.98 (d, 1H, J=8.0 Hz), 6.90 (dd, 1H, J=2.0 and 8.0 Hz), 6.66 (m, 2H), 6.53 (bs, 1H), 6.42(dd, 1H, J=2.0 and 8.0Hz), 3.82(s, 3H), 3.53(m, 4H), 3.03(m, 2H), 2.83(m, 2H) and 1.89( m, 2H).

[Scheme 2]

2-1. tert-butyl 4-(3-((4-(pyrizin-2-yl)pyrimidin-2-yl)amino)phenyl)-1,4-diazepane-1-carboxylate [tert-butyl 4-( 3-((4-(pyrazin-2-yl)pyrimidin-2-yl)amino)phenyl)-1,4-diazepane-1-carboxylate (8)] synthesis

2-chloro-4-(pyrazin-2-yl)pyrimidine [2-chloro-4-(pyrazin-2-yl)pyrimidine; 100 mg, 0.52 mmol], tert-butyl 4-(3-aminophenyl)-1,4-diazepane-1-carboxylate [tert-butyl 4-(3-aminophenyl)-1,4-diazepane-1- carboxylate; 151 mg, 0.52 mmol], tris(dibenzylideneacetone)dipalladium [Tris(dibenzylideneacetone)dipalladium(0); 24 mg, 0.03 mmol], X-phos (25 mg, 0.05 mmol), and potassium carbonate (143 mg, 1.04 mmol) were added to tert-butanol (2 ml) and stirred under reflux for 12 hours. . The reaction mixture was layered with water and ethyl acetate (Ethyl acetate, 5 mL), the organic layer was washed with distilled water, dried over anhydrous Na ₂ SO ₄ , and filtered. After the filtrate was concentrated under reduced pressure, the concentrate was purified by column chromatography to obtain compound (8) (60 mg, 25%).

2-2. N-(3-(1,4-diazepan-1-yl)phenyl)-4-(pyrazin-2-yl)pyrimidin-2-amine [N-(3-(1,4-diazepan-1-yl) yl)phenyl)-4-(pyrazin-2-yl)pyrimidin-2-amine (A9-004)] synthesis

tert-butyl 4-(3-((4-(pyrazin-2-yl)pyrimidin-2-yl)amino)phenyl)-1,4-diazepane-1-carboxylate [tert-butyl 4- (3-((4-(pyrazin-2-yl)pyrimidin-2-yl)amino)phenyl)-1,4-diazepane-1-carboxylate; 60 mg, 0.13 mmol] was dissolved in dichloromethane (dichloromethane, 2 ml) and trifluoroacetic acid (trifluoroacetic acid, 0.03 ml, 0.40 mmol) was added thereto, followed by stirring for 12 hours. The reaction mixture was layer-separated with water and dichloromethane, and the organic layer was washed with sat. Washed with NaHCO ₃ , dried over anhydrous Na ₂ SO ₄ , and filtered. The filtrate was concentrated under reduced pressure and recrystallized from diethyl ether to obtain compound A9-004 (30 mg, 65%).

¹ H NMR (CDCl ₃ , 400 MHz): δ 9.65 (d, 1H, J=2.0 Hz), 8.67 (m, 2H, J=4.0 Hz), 8.58 (d, 1H, J=4.0 Hz), 7.70 ( d, 1H, J=8.0 Hz), 7.22 (m, 4H), 6.93 (dd, 1H, J=2.0 and 8.0 Hz), 6.46 (dd, 1H, J=2.0 and 8.0 Hz), 3.62 (m, 4H) ), 3.08(m, 2H), 2.87(m, 2H) and 1.95(m, 2H).

[Scheme 3]

3-1. Synthesis of 2-chloro-4- (4-fluoro-2-methoxyphenyl) pyridine [2-chloro-4- (4-fluoro-2-methoxyphenyl) pyridine (9)]

4-bromo-2-chloropyridine (5.00 g, 25.98 mmol), (4-fluoro-2-methoxyphenyl)bronic acid [(4-fluoro-2-methoxyphenyl) at room temperature ) boronic acid; 4.90 g, 28.58 mmol], tetrakis(triphenylphosphine)palladium [Tetrakis(triphenylphosphine)palladium(0); 1.50 g, 1.30 mmol], sodium carbonate; 5.60 g, 51.96 mmol) was added to a solution of 1,4-dioxane (1,4-dioxane, 60 ml) and water (20 mL) and stirred under reflux for 12 hours. . The reaction mixture was layered with ethyl acetate (Ethyl acetate, 60 mL), the organic layer was washed with distilled water, dried over anhydrous Na ₂ SO ₄ and filtered. After the filtrate was concentrated under reduced pressure, the concentrate was purified by column chromatography to obtain compound (9) (5.00 g, 66%).

3-2. Tert-Butic 4-(3-((4-(4-fluoro-2-methoxybenzyl)pyridin-2-yl)amino)phenyl)-1,4-diazepane-1-carboxylate [tert-butyl 4-(3-((4-(4-fluoro-2-methoxyphenyl)pyridin-2-yl)amino)phenyl)-1,4-diazepane-1-carboxylate (10)] synthesis

2-chloro-4-(4-fluoro-2-methoxyphenyl)pyridine [2-chloro-4-(4-fluoro-2-methoxyphenyl)pyridine at room temperature; 1.80 g, 7.57 mmol], tert-butyl 4-(3-aminophenyl)-1,4-diazepane-1-carboxylate [tert-butyl 4-(3-aminophenyl)-1,4-diazepane-1- carboxylate; 2.70 g, 9.09 mmol], tri(dibenzylideneacetone)dipalladium Tris(dibenzylideneacetone)dipalladium(0); 0.35 g, 0.38 mmol), X-phos (0.37 g, 0.76 mmol) and potassium carbonate (2.10 g, 15.15 mmol) were added to tert-butanol (tert-butanol, 20 ml) and stirred under reflux for 12 hours. . The reaction mixture was layered with water and ethyl acetate (Ethyl acetate, 20 mL), the organic layer was washed with distilled water, dried over anhydrous Na ₂ SO ₄ , and filtered. After the filtrate was concentrated under reduced pressure, the concentrate was purified by column chromatography to obtain compound (10) (2.5 g, 67%).

3-3. N-(3-(1,4-diazepan-1-yl)phenyl)-4-(4-fluoro-2-methoxyphenyl)pyridin-2-amine [N-(3-(1,4- diazepan-1-yl)phenyl)-4-(4-fluoro-2-methoxyphenyl)pyridin-2-amine (AE09-005)] synthesis

tert-Butyl 4-(3-((4-(4-fluoro-2-methoxyphenyl)pyridin-2-yl)amino)phenyl)-1,4-diazepane-1-carboxylate [tert at room temperature -butyl 4-(3-((4-(4-fluoro-2-methoxyphenyl)pyridin-2-yl)amino)phenyl)-1,4-diazepane-1-carboxylate; 2.5 g, 5.08 mmol] was dissolved in dichloromethane (25 ml) and trifluoroacetic acid (1.10 ml, 15.23 mmol) was added thereto, followed by stirring for 12 hours.

The reaction mixture was layer-separated with water and dichloromethane, and the organic layer was washed with sat. Washed with NaHCO ₃ , dried over anhydrous Na ₂ SO ₄ , and filtered. The filtrate was concentrated under reduced pressure and recrystallized from diethyl ether to obtain compound AE09-005 (1.90 g, 95%).

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.83 (dd, 1H, J=2.0 and 8.0 Hz), 7.53 (dd, 1H, J=4.0 and 8.0 Hz), 7.40 (m, 1H), 7.36 (d , 1H, J=2.0Hz), 7.21(dd, 1H, J=2.0 and 12.0Hz), 6.88(m, 2H), 6.81(m, 1H), 6.76(m, 1H), 3.90(s, 3H) , 3.86(m, 2H), 3.64(m, 2H), 3.41(m, 2H), 3.31(m, 2H), and 2.22(m, 2H).

<Example 1> Kinase inhibitory activity and IC of the compound ₅₀ Confirm

The CDK2, CDK4, CDK5, CDK6, CDK7, CDK9 and FLT3 activity inhibitory ability of compounds A09-003, A09-004 and A09-005 (AE09-005) was confirmed.

In order to determine the inhibitory effect on kinase activity and IC ₅₀ of compounds A09-003, A09-004 and A09-005 (AE09-005), each compound was treated with 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and CDK2/cyclinE (h), CDK4/cyclinD3 (h), CDK5/p25 (h), CDK6/cyclinD3 (h), CDK7/cyclinH/MAT1 (h), CDK9 at concentrations of 0 μM (10 total). /cyclinT1 (h) and FLT3 (h) kinase activity assays were performed.

As a result, as shown in Figure 1, all of the compounds A09-003, A09-004 and A09-005 (AE09-005) showed a concentration gradient-dependent decrease in CDK9 activity, in particular, A09-003 > A09-005 > A09-004. CDK9 activity reduction effect was confirmed. In addition, as shown in FIG. 2 , it was confirmed that the inhibitory effect of FLT3 activity decreased in the order of A09-003 > A09-004 > A09-005.

On the other hand, the kinase activity IC ₅₀ of each compound was confirmed as shown in Table 1.

IC ₅₀ (nM) Compound A09-003 Compound A09-004 Compound A09-005 (AE09-005) CDK2 1205 2895 5286 CDK4 1662 9107 4209 CDK5 574 4386 1287 CDK6 9483 > 100,000 20380 CDK7 N/A N/A 19986 CDK9 16 345 43 FLT3 25 81

<Example 2> Apoptosis effect and IC on leukemia cell lines ₅₀ Confirm

1. Check cell viability

CellTiter-Glo Luminescent Cell Viability Assay The apoptotic effect of compounds A09-003, A09-004 and A09-005 (AE09-005) on 5 leukemia cell lines (BDCM, Molm14, MV4-11, THP-1 and U937) (Promega, Cat.# G7572).

Each compound was diluted by 1/2 from 50 μM to check the remaining survival of the cells compared to a total of 9 concentrations (no control material treatment).

First, 80 μL of a cell culture solution containing three thousand cells each was dispensed into a 96-well plate and cultured in an incubator at 37° C. for one day.

On the second day of culture, 20 μL of cell culture solution was prepared to contain the compound 5 times (5X) diluted under the above 9 concentration conditions, and then the cells were treated (final volume 100 μL). Compound-treated cells were cultured in an incubator at 37° C. for 72 hours. The final concentration of DMSO to be treated to the cells was not to exceed 0.2%.

After 72 hours of incubation, 20 mL of CellTiter-Glo solution was treated in each well, and the amount of ATP in living cells was checked after 20 minutes of reaction (Perkin Elmer Victor 3 plate reader). The ATP values derived from the untreated group were calculated as 100% survival rate, and the ATP values of the 9 treatment groups were calculated as relative viability and derived as a graph (GraphPad Prism 5).

As a result, as shown in FIGS. 3 and 4, cell proliferation inhibition and apoptosis effects were confirmed in a concentration gradient in all leukemia cell lines. In particular, compound A09-003 exhibited the lowest IC ₅₀ value, and it was confirmed that an effective apoptosis effect was shown in all five leukemia cell lines. In addition, the apoptosis IC ₅₀ of each compound was confirmed as shown in Table 2.

IC ₅₀ (μM) BDCM Molm14 MV4-11 THP-1 U937 Compound A09-003 1.90 0.86 0.48 2.49 1.84 Compound A09-004 7.26 3.62 1.40 10.19 8.89 Compound A09-005 (AE09-005) 6.71 1.49 0.71 6.47 5.75

2. Confirmation of apoptosis effect through flow cytometry

The apoptotic effect of compound A09-003, which previously exhibited an excellent apoptotic effect on leukemia cell lines, was confirmed by flow cytometry.

A cell culture solution containing 500,000 each of the leukemia cell lines Molm14 and MV4-11 cells was dispensed into a culture dish and cultured in an incubator at 37° C. for 3 hours.

Thereafter, Compound A09-003 was diluted to 0 μM, 1 μM, 2 μM and 3 μM concentration conditions and treated with cells. Compound-treated cells were cultured in an incubator at 37° C. for 72 hours.

After incubation, the cells were harvested, washed with cold phosphate buffered saline, centrifuged to discard the supernatant, and the cells were prepared to 100 µL per assay tube after diluting the cells with 1X Annexin binding buffer.

5 μL Alexa FluorAdd 488 Annexin V and 5 μL 100 μg/mL PI mixture to each 100 μL of cell suspension, incubate at room temperature for 15 minutes, add 300 μL 1X Annexin binding buffer, and perform flow cytometry to analyze the stained cells and measure the fluorescence. measured.

As a result of analyzing cells stained with Annexin V-positive/PI-positive in two types of leukemia cell lines (Molm14, MV4-11), as shown in FIG. 5, compound A09-003 caused apoptosis in both cell lines in a concentration gradient. It could be seen that this increased.

3. Confirmation of apoptosis image through staining

The apoptotic effect of compound A09-003 on leukemia cell lines Molm-14 and MV4-11 was confirmed by fluorescence images.

300,000 Molm-14 and MV4-11 cell culture solutions were dispensed into 60 mm culture dishes, respectively, and cultured in an incubator at 37° C. for 3 hours.

Thereafter, compound A09-003 was diluted to 0 μM, 1 μM, 2 μM and 3 μM concentration conditions and treated with cells. Compound-treated cells were cultured in an incubator at 37° C. for 72 hours.

After incubation, the cells are harvested, washed with cold phosphate buffered saline, centrifuged to discard the supernatant, and 1 μl of calcein AM to check live cells, edium homodimer to check dead cells- 1 (ethidium homodimer-1) 4μl and 2ml PBS mixture 100μl with 1μl of Hoechst 33342 solution capable of staining cell nuclei was added and reacted at room temperature for 30 minutes. was measured.

As a result of analyzing the number of green stained with calcein AM and red stained with edium homodimer-1 in two types of leukemia cell lines (Molm-14, MV4-11), as shown in FIG. 6, compound A09-003 was It could be seen that the number of greens decreased and the number of reds increased according to the concentration, which confirmed that apoptosis was increased in a concentration gradient.

4. Cell cycle analysis using FACS (Fluorescence activated cell sorter) device

In order to see the effect of the compound A09-003 on the cell cycle, DNA was stained with propidium iodide (hereinafter referred to as PI), and the cell cycle was confirmed using a FACS device.

500,000 each of Molm-14 and MV4-11 cell culture solutions were dispensed in a 100 mm culture dish and cultured for 15 hours in an incubator at 37° C. without nutrients.

A09-003 was diluted to a concentration of 0 μM, 1 μM, 2 μM and 3 μM and cultured in an incubator at 37° C. for 30 hours while treating the cells with 2% nutrients.

After culturing, the cells were harvested, washed with cold phosphate buffered saline, centrifuged, and the supernatant was discarded and left as a cell pellet. To fix the cells, 70% ethanol was added to loosen the cells, and the cells were fixed by shaking at a cold temperature for 24 hours.

After 24 hours, centrifuge cleanly remove ethanol, and dissolve 100 μl of 100 μg/ml of ribonuclease A capable of cleaving single-stranded RNA in the cell pellet. Then, 500 μl of a mixture of 300 μl of PI and 5.7 ml of cell staining buffer was added, reacted at room temperature for 30 minutes, and the cell cycle was analyzed using a FACS instrument.

As a result, as shown in FIG. 7 , it was confirmed that apoptosis was induced by confirming that the subG1 value was increased by up to 60% by treatment with A09-003 by concentration, and at the same time, it was confirmed that subG1 arrest occurred by A09-003.

<Example 3> Confirmation of protein expression in leukemia cells

A cell culture solution containing one million leukemia cell line MV4-11 was dispensed into a culture dish and cultured in an incubator at 37° C. for 3 hours.

Compounds A09-003 and A09-005 were diluted to concentrations of 0 μM, 1 μM, 2 μM and 4 μM, respectively, and treated with the cells, and the compound-treated cells were cultured in an incubator at 37° C. for 24 hours.

After culturing for 24 hours, the MV4-11 cells were harvested, the protein was extracted, and the protein concentration was measured using the BCA protein quantification method, and the protein was separated by molecular weight using SDS-PAGE (Sodium Dodecyl sulfate-polyacylamide gel elecrophoresis). .

The separated protein inside the gel was transferred to the membrane by the transfer process, and blocked with 5% Skim milk/T-TBS. After that, the primary antibody (antibody to protein (primary; p-RNA polymerase II ser2, RNA polymerase II, Mcl-1, XIAP, Cyclin D1, Cleaved caspase-3, β-Actin) is bound to the protein and HRP (horseradish) peroxidase) attached secondary was reacted.

Secondary HRP oxidized Luminol in ECL (Enhanced chemiluminescence) solution, and the amount of protein expression was confirmed by sensitizing the emitted light to the X-ray film.

As a result, as shown in FIG. 8 , it was confirmed that Caspase-3 activity was increased in a concentration gradient in both compounds A09-003 and A09-005-treated leukemia cell line MV4-11, thereby promoting apoptosis.

In addition, in the case of compound A09-003, the dephosphorylation of serine 2 residue of RNA polymerase II, and reduction of Mcl-1, XIAP, and Cyclin D1 proteins were significantly compared to compound A09-005.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

A compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
[Formula 1]

In Formula 1,
A, X and Y may each be the same or different, and are selected from CH or N,
R ¹ and R ² may each be the same or different, and may be any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound or a pharmaceutically acceptable salt thereof is a compound represented by Formula 1-1.
[Formula 1-1]

In Formula 1,
A, X and Y may each be the same or different, and are selected from CH or N,
R ¹ and R ² may each be the same or different, and may be any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ¹ is hydrogen or halogen, and R ² is hydrogen or C1 to C4 alkoxy. The method according to claim 1, wherein the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is N-(3-(1,4-diazepan-1-yl)phenyl)-4-(2-methoxyphenyl) Pyridin-2-amine [N-(3-(1,4-diazepan-1-yl)phenyl)-4-(2-methoxyphenyl)pyridin-2-amine], N-(3-(1,4-dia Zepan-1-yl)phenyl)-4-(pyrazin-2-yl)pyrimidin-2-amine [N-(3-(1,4-diazepan-1-yl)phenyl)-4-(pyrazin-2 -yl)pyrimidin-2-amine] or N-(3-(1,4-diazepan-1-yl)phenyl)-4-(4-fluoro-2-methoxyphenyl)pyridin-2-amine [ N-(3-(1,4-diazepan-1-yl)phenyl)-4-(4-fluoro-2-methoxyphenyl)pyridin-2-amine], or a pharmaceutically acceptable salt thereof . The method according to claim 1, wherein the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is a compound or a pharmaceutically acceptable salt thereof, characterized in that it inhibits cyclin-dependent kinase 9 (cyclin-dependent kinase 9). A pharmaceutical composition for preventing or treating cancer, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

In Formula 1,
A, X and Y may each be the same or different, and are selected from CH or N,
R ¹ and R ² may each be the same or different, and may be any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen. The pharmaceutical composition for preventing or treating cancer diseases according to claim 6, wherein the compound or a pharmaceutically acceptable salt thereof is a compound represented by Formula 1-1.
[Formula 1-1]

In Formula 1,
A, X and Y may each be the same or different, and are selected from CH or N,
R ¹ and R ² may each be the same or different, and may be any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen. The pharmaceutical composition for preventing or treating cancer diseases according to claim 6, wherein R ¹ is hydrogen or halogen, and R ² is hydrogen or C1 to C4 alkoxy. The method according to claim 6, wherein the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is N-(3-(1,4-diazepan-1-yl)phenyl)-4-(2-methoxyphenyl) Pyridin-2-amine [N-(3-(1,4-diazepan-1-yl)phenyl)-4-(2-methoxyphenyl)pyridin-2-amine], N-(3-(1,4-dia Zepan-1-yl)phenyl)-4-(pyrazin-2-yl)pyrimidin-2-amine [N-(3-(1,4-diazepan-1-yl)phenyl)-4-(pyrazin-2 -yl)pyrimidin-2-amine] or N-(3-(1,4-diazepan-1-yl)phenyl)-4-(4-fluoro-2-methoxyphenyl)pyridin-2-amine [ N-(3-(1,4-diazepan-1-yl)phenyl)-4-(4-fluoro-2-methoxyphenyl)pyridin-2-amine], a pharmaceutical composition for preventing or treating cancer diseases. The pharmaceutical composition for preventing or treating cancer diseases according to claim 6, wherein the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof inhibits cyclin-dependent kinase 9. The method according to claim 6, wherein the cancer disease is acute myeloid leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, breast cancer, ovarian cancer, uterine cancer, pancreatic cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, skin cancer, bladder cancer, thyroid cancer , A pharmaceutical composition for preventing or treating cancer, characterized in that it is selected from the group consisting of brain cancer and prostate cancer. A composition for inhibiting cyclin-dependent kinase 9 comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

In Formula 1,
A, X and Y may each be the same or different, and are selected from CH or N,
R ¹ and R ² may each be the same or different, and may be any one of hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen.

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