HK1243067B - Crystals of azabicyclic compound - Google Patents

Description

Crystallization of azabicyclic compounds

Technical Field

The present invention relates to a novel crystal of an azabicyclic compound which is stable and has excellent oral absorbability and is useful as an antitumor agent.

Background Art

Generally, when compounds are used as active ingredients in pharmaceuticals, chemical and physical stability is required to maintain consistent quality and/or facilitate storage and management. Therefore, it is preferred that the compound be obtained in a stable crystalline form, and there are many precedents for selecting the most stable crystalline form as the API for pharmaceuticals.

Currently, many HSP90 inhibitors have been reported as antitumor agents. Patent Documents 1 and 2 describe 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide (hereinafter also referred to as "Compound 1") as a compound having excellent HSP90 inhibitory action and exhibiting antitumor activity.

On the other hand, pharmaceutical compositions for oral administration generally require not only the stability of the active ingredient but also excellent absorbability upon oral administration. Patent Documents 1 and 2 contain no description of the crystals of Compound 1, the stability of the crystals, or the oral absorbability.

Prior art literature

Patent Literature

Patent Document 1: International Publication No. 2012/093708

Patent Document 2: International Publication No. 2011/004610

Summary of the Invention

Technical problem to be solved by the invention

An object of the present invention is to provide a stable crystal of Compound 1 which is useful as an antitumor agent and has excellent oral absorbability.

Means for solving technical problems

To address the above-mentioned technical problems, the inventors of the present invention synthesized Compound 1 according to the production method described in Patent Document 1, resulting in Type I crystals of Compound 1. However, as described in the Examples below, oral absorption of Type I crystals was problematic, leading to further investigations into crystallization conditions. As a result, they discovered that Type II crystals could be obtained by suspending Compound 1 in a specific organic solvent. Furthermore, they discovered that Type II crystals exhibited superior stability and oral absorption compared to Type I crystals, leading to the completion of the present invention.

That is, the present invention provides the following [1] to [15].

[1] A type II crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide, characterized in that it has at least three characteristic peaks selected from the group consisting of diffraction angles (2θ±0.2°) of 7.7°, 8.0°, 11.1°, 12.5°, 12.9°, 15.2°, 15.8°, 17.2°, 19.0°, 22.5°, 26.1°, and 27.4° in its powder X-ray diffraction pattern.

[2] The type II crystal as described in [1], which has at least five characteristic peaks selected from the group consisting of diffraction angles (2θ ± 0.2°) of 7.7°, 8.0°, 11.1°, 12.5°, 12.9°, 15.2°, 15.8°, 17.2°, 19.0°, 22.5°, 26.1°, and 27.4° in its powder X-ray diffraction pattern.

[3] The type II crystal as described in [1] or [2], wherein the endothermic peak determined by differential scanning calorimetry is near 270°C.

[4] A pharmaceutical composition comprising the type II crystal according to any one of [1] to [3].

[5] A pharmaceutical composition for oral administration containing the type II crystal according to any one of [1] to [3].

[6] An antitumor agent comprising the type II crystal according to any one of [1] to [3].

[7] Use of the type II crystal according to any one of [1] to [3] in the manufacture of a pharmaceutical composition.

[8] The use according to [7], wherein the pharmaceutical composition is a pharmaceutical composition for oral administration.

[9] Use of the type II crystal according to any one of [1] to [3] in the production of an antitumor agent.

[10] The type II crystal according to any one of [1] to [3], which is used as a medicine.

[11] The type II crystal according to any one of [1] to [3], which is used for treating tumors.

[12] A method for treating tumors, comprising administering an effective amount of the type II crystal according to any one of [1] to [3] to a subject in need of tumor treatment.

[13] A method for producing the type II crystal according to any one of [1] to [3], comprising:

(1) a step of suspending 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide in an organic solvent by heating to obtain a suspension; and

(2) A step of obtaining solid 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide from the suspension obtained in (1).

[14] The method for producing type II crystals as described in [13], wherein the organic solvent is 2-propanol, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, cyclopentyl methyl ether, methyl ethyl ketone, methyl isobutyl ketone, acetone, acetonitrile or a mixed solvent thereof.

[15] A method for producing the type II crystal according to any one of [1] to [3], comprising:

(1) a step of suspending 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide in an organic solvent selected from 2-propanol, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, cyclopentyl methyl ether, methyl ethyl ketone, methyl isobutyl ketone, acetone, acetonitrile, and a mixed solvent thereof to obtain a suspension; and

(2) A step of obtaining solid 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide from the suspension obtained in (1).

Effects of the Invention

According to the present invention, the Type II crystal of Compound 1 has high stability and excellent oral absorbability and is useful as an oral pharmaceutical.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows a powder X-ray diffraction pattern of type I crystal of Compound 1 (the vertical axis represents intensity (cps), and the horizontal axis represents diffraction angle (2θ±0.2°)).

FIG2 shows a powder X-ray diffraction pattern of type II crystal of Compound 1 (the vertical axis represents intensity (cps), and the horizontal axis represents diffraction angle (2θ±0.2°)).

FIG3 shows the differential scanning calorimetry (DSC) curve of Type II crystal of Compound 1.

FIG4 shows the results of a test for measuring the blood concentration of Form II crystals of Compound 1.

DETAILED DESCRIPTION

Compound 1 of the present invention is 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide (formula shown below). Compound 1 is known to have HSP90 inhibitory activity and exhibit excellent antitumor activity. Compound 1 can be synthesized according to the production methods described in Patent Documents 1 and 2.

The crystals of the present invention may contain Form II crystals of Compound 1 and may be single Form II crystals or a polymorphic mixture containing crystals other than Form II crystals. Among the crystals of the present invention, high-purity Form II crystals are preferred. Specifically, Form II crystals having a chemical purity of 90% or greater are preferred, more preferably 95% or greater, and particularly preferably 98% or greater.

The Type II crystal of the present invention can be obtained by adding Compound 1 to a specific organic solvent to suspend the compound. Specifically, the Type II crystal can be obtained by a production method comprising the following steps (1) and (2):

(1) a step of suspending compound 1 in an organic solvent to obtain a suspension,

(2) A step of obtaining a solid form of Compound 1 from the suspension obtained in (1).

Here, the compound 1 added to the organic solvent may be crystalline or not, but from the perspective of obtaining high-purity Type II crystals, it is preferred to use crystalline compound 1, and particularly preferred to use Type II crystals of compound 1. Furthermore, seed crystals may be used during the crystallization of the present invention. From the perspective of obtaining high-purity Type II crystals, Type II crystals are preferably used as seed crystals.

Examples of the organic solvent used in the crystallization of the present invention include alcohols such as methanol, n-propanol, 2-propanol, and ethylene glycol; aliphatic carboxylic acid esters such as methyl acetate, ethyl acetate, propyl acetate, and butyl acetate; ethers such as diethyl ether, methyl tert-butyl ether, cyclopentyl methyl ether, 1,4-dioxane, and tetrahydrofuran; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, and cyclohexanone; aromatic solvents such as toluene, xylene, and chlorobenzene; and aprotic polar organic solvents such as acetonitrile, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, 1,3-dimethyl-2-imidazolidinone, and dimethyl sulfoxide, or mixed solvents thereof. Preferred solvents include ketones, monohydric alcohols having 3 or more carbon atoms, dihydric alcohols, aliphatic carboxylic acid esters, ethers, aprotic polar organic solvents, or mixed solvents thereof. More preferred solvents include 2-propanol, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, cyclopentyl methyl ether, methyl ethyl ketone, methyl isobutyl ketone, acetone, acetonitrile, or mixed solvents thereof. From the perspective of purity and yield of Type II crystals, methyl acetate, methyl ethyl ketone, acetone, or mixed solvents thereof are particularly preferred.

The amount (v/w) of the organic solvent used in the crystallization of the present invention is preferably 2 to 30 times, more preferably 3 to 20 times, and particularly preferably 4 to 15 times the amount of Compound 1 from the viewpoint of purity and yield of Form II crystals.

During crystallization in the present invention, Compound 1 is preferably added to an organic solvent, heated and suspended, and then refluxed for a long period of time. The heating temperature during crystallization in the present invention is not particularly limited as long as it allows reflux and can be appropriately set depending on the organic solvent used. It is preferably 52°C to 126°C.

Regarding the reflux time during crystallization of the present invention, if it is too short, crystallization may not proceed sufficiently and high-purity crystals cannot be obtained; whereas if it is too long, decomposition of the crystals may occur and the yield may decrease. Therefore, it is preferably 12 to 60 hours, more preferably 16 to 48 hours.

During the crystallization of the present invention, precipitated type II crystals can be obtained by cooling after reflux. The cooling temperature can be set as appropriate, but is preferably room temperature.

The precipitated crystals can be separated and purified from the above-mentioned dissolving solution or mixed solution by known separation and purification means such as filtration, washing with an organic solvent, and drying under reduced pressure.

Examples of the organic solvent used for washing include lower alcohols, acetone, acetonitrile, etc. The organic solvent used for type II crystallization can also be used for washing.

The Type II crystal of the present invention obtained as described above has three or more, preferably five or more, more preferably eight or more, and even more preferably twelve characteristic peaks selected from the group consisting of diffraction angles (2θ ± 0.2°) of 7.7°, 8.0°, 11.1°, 12.5°, 12.9°, 15.2°, 15.8°, 17.2°, 19.0°, 22.5°, 26.1°, and 27.4° in its powder X-ray diffraction pattern, as shown in Figure 2. Furthermore, as shown in the results of differential scanning calorimetry (DSC measurement) shown in Figure 3, the Type II crystal has an endothermic peak near 270°C.

In contrast, type I crystals have characteristic peaks at diffraction angles (2θ±0.2°) of 8.1°, 12.1°, 14.0°, 16.2°, 21.5°, 25.4°, and 28.3° in the powder X-ray diffraction pattern as shown in FIG1 .

Peak values in a powder X-ray diffraction pattern may have slight errors due to measurement conditions such as the measuring instrument and peak reading conditions. Peak values in this specification may have a measurement error within the range of approximately ±0.2°.

In DSC measurement, the measurement temperature of the endothermic peak (peak top value) may vary depending on the temperature increase per minute, sample amount, purity, etc. The term "near" in this specification means ±5.0°C.

As shown in the examples below, there is a significant difference in the oral absorption of Form I and Form II crystals of Compound 1. Generally, the solubility of metastable forms is higher than that of stable forms (Akira Tsuji, New Pharmacy, Minamikodo). Therefore, the higher oral absorption of the stable Form II crystals compared to the metastable Form I crystals is unexpected.

Furthermore, Type I crystals of Compound 1 transform into Type II crystals upon refluxing. Type II crystals are therefore stable under high temperature and high humidity conditions. Therefore, Type II crystals are more stable than Type I crystals and are useful as pharmaceutical raw materials.

Therefore, the Type II crystals of the present invention are useful as active ingredients of pharmaceutical compositions, particularly as active ingredients of pharmaceutical compositions for oral administration. Since Compound 1 has excellent HSP90 inhibitory activity, the Type II crystals of the present invention are useful as antitumor agents. The target cancer is not particularly limited, and examples include head and neck cancer, digestive system cancer (esophageal cancer, gastric cancer, gastrointestinal stromal tumors, duodenal cancer, liver cancer, biliary tract cancer (gallbladder and bile duct cancer, etc.), pancreatic cancer, small intestine cancer, large intestine cancer (colorectal cancer, colon cancer, rectal cancer, etc.), lung cancer, breast cancer, ovarian cancer, uterine cancer (cervical cancer, uterine body cancer, etc.), kidney cancer, bladder cancer, prostate cancer, urothelial cancer, bone and soft tissue sarcoma, blood cancer (B-cell lymphoma, chronic lymphocytic leukemia, peripheral T-cell lymphoma, myelodysplastic syndrome, acute myeloid leukemia, acute lymphocytic leukemia, etc.), multiple myeloma, skin cancer, mesothelioma, etc.

When the Form II crystals of the present invention are used as the active ingredient of a pharmaceutical composition, they may be combined with a pharmaceutically acceptable carrier as needed, and various dosage forms may be adopted depending on the purpose of prevention or treatment. Preferred dosage forms include oral preparations such as tablets, capsules, granules, fine granules, and powders. These dosage forms can be prepared using conventional formulation methods known to those skilled in the art.

Example

The present invention is further described below with reference to the following examples, but the present invention is not limited to these examples. While the present invention has been described in detail with reference to the examples, those skilled in the art will appreciate that various changes and modifications may be made. Therefore, as long as such changes and modifications do not depart from the scope of the present invention, they are intended to be included within the scope of the present invention.

All reagents used in the Examples were commercially available unless otherwise specified. NMR spectra were measured using an AL400 (400 MHz, JEOL Ltd.), Mercury 400 (400 MHz, Agilent Technologies) spectrometer, or an Inova 400 (400 MHz, Agilent Technologies) spectrometer equipped with a 400 M NMR probe (Protasis). Tetramethylsilane was used as the internal standard when the deuterated solvent contained tetramethylsilane; otherwise, the NMR solvent was used as the internal standard. All δ values are expressed in ppm.

The meanings of the abbreviations are as follows.

s: single peak

d: Twin Peaks

t: triplet

q: quartet

dd: doublet

dt: double triplet

td: triple double peaks

tt: triple triplet

ddd: double double peak

DDT: Double Double Triple

DTD: Double Triple Doublet

tdd: triplet doublet

m: multiple peaks

br: broad peak

brs: broad single peak

Powder X-ray diffraction measurement

For powder X-ray diffraction, an appropriate amount of a test substance was lightly pulverized in an agate mortar as needed, and then measured under the following test conditions.

Device: PANalytical EMPYREAN

Target: Cu

X-ray output setting: 40mA, 45kV

Scanning range: 2.0-40.0°

Step size: 0.026°

Divergence slit: Automatic

Irradiation width: 10.00mm

Sample width: 10.00mm

The operations of the devices including data processing are performed according to the methods and steps indicated in each device.

Thermal analysis measurement (differential scanning calorimetry (DSC measurement))

The DSC measurement was performed under the following test conditions.

Device: TA Instruments Q1000

Sample: about 1 mg

Sample container: Aluminum

Heating rate: 10°C/min to 300°C

Atmosphere gas: Nitrogen

Nitrogen flow rate: 50mL/min.

The operations of the devices including data processing are performed according to the methods and steps indicated in each device.

Comparative Example 1 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)- Synthesis of Type I Crystals of 1H-Imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (3.58 g) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide, obtained according to the production methods described in International Publication No. 2012/093708 and International Publication No. 2011/004610, was added to ethanol (7.84 mL) and stirred at room temperature for 2 hours. The solid was collected by filtration, washed with ethanol (7.84 mL), and then dried under reduced pressure at 70-80°C for 20 hours to obtain Type I crystals (yield: 2.40 g, yield: 61.2%, purity: 98.21%).

In addition, as shown in Figure 1, the type I crystal shows characteristic peaks at diffraction angles (2θ) of 8.1°, 10.9°, 12.1°, 14.0°, 14.9°, 16.2°, 17.7°, 20.2°, 21.0°, 21.5°, 22.6°, 24.3°, 25.4°, 26.4°, 27.0°, 28.3°, 30.2°, 30.9°, 31.5°, 32.7°, 34.7°, 35.4°, and 36.6° in the powder X-ray diffraction pattern.

¹ H-NMR (DMSO-d ₆ ): δppm 9.35 (1H, d, J = 4.88Hz), 8.93 (1H, d, J = 1.22Hz), 8.84 (1H, brs), 8.72 (1H, d, J = 1.95Hz), 8.70 (1H, s), 8.63 (1H, d, J = 1.22Hz), 8.60 (1H, dd, J = 8.29, 1.95Hz), 8.46 (1H, s), 8.25 (1H, d, J = 8.29Hz), 8.22 (1H, brs), 8.12 (1H, d, J = 4.88Hz), 4.59 (3H, s), 3.95 (1H, tt, J = 6.83, 6.83Hz), 3.21 (2H, q, J=7.56Hz), 1.83 (6H, d, J=6.83Hz), 1.75 (3H, t, J=7.56Hz): LRMS (ESI) m/z 455[M+H]

Example 1 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)- Synthesis of Type II Crystals of 1H-Imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (4.0 g) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide, obtained according to the production methods described in International Publication No. 2012/093708 and International Publication No. 2011/004610, was added to acetone (19.54 mL) and stirred under reflux for 16 hours. After cooling to room temperature, the solid was collected by filtration, washed with acetone (8.4 mL), and dried under reduced pressure at 70-80°C for 16-24 hours to obtain Type II crystals (yield: 1.59 g, yield: 57.0%, purity: 98.37%).

In addition, the Type II crystal exhibits characteristic peaks at diffraction angles (2θ) of 7.7°, 8.0°, 11.1°, 12.5°, 12.9°, 14.2°, 15.2°, 15.8°, 17.2°, 17.7°, 19.0°, 20.2°, 21.1°, 22.5°, 22.8°, 23.5°, 24.5°, 26.1°, 26.7°, 27.4°, 28.0°, 28.7°, 29.4°, 30.0°, 31.7°, 35.1°, 36.2°, 36.9°, and 37.6° in its powder X-ray diffraction pattern as shown in FIG2. Furthermore, as shown in FIG3, differential scanning calorimetry (DSC) analysis reveals that the Type II crystal exhibits an endothermic peak at approximately 270°C.

¹ H-NMR (DMSO-d ₆ ): δppm 9.35 (1H, d, J = 4.88Hz), 8.93 (1H, d, J = 1.22Hz), 8.84 (1H, brs), 8.72 (1H, d, J = 1.95Hz), 8.70 (1H, s), 8.63 (1H, d, J = 1.22Hz), 8.60 (1H, dd, J = 8.29, 1.95Hz), 8.46 (1H, s), 8.25 (1H, d, J = 8.29Hz), 8.22 (1H, brs), 8.12 (1H, d, J = 4.88Hz), 4.59 (3H, s), 3.95 (1H, tt, J = 6.83, 6.83Hz), 3.21 (2H, q, J=7.56Hz), 1.83 (6H, d, J=6.83Hz), 1.75 (3H, t, J=7.56Hz): LRMS (ESI) m/z 455[M+H]

Example 2 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)- Synthesis of Type II Crystals of 1H-Imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide, obtained according to the production methods described in International Publication No. 2012/093708 and International Publication No. 2011/004610, was added to methyl ethyl ketone (2.8 mL) and stirred under reflux for 16 hours. After cooling to room temperature, the solid was collected by filtration, washed with methyl ethyl ketone (1.2 mL), and dried under reduced pressure at 70-80°C for 16-24 hours to obtain Type II crystals (yield: 197 mg, yield: 60.9%, purity: 98.83%).

Example 3 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)- Synthesis of Type II Crystals of 1H-Imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide, obtained according to the production methods described in International Publication No. 2012/093708 and International Publication No. 2011/004610, was added to acetonitrile (4.0 mL) and stirred under reflux for 3 hours. After cooling to room temperature, the solid was collected by filtration, washed with acetonitrile (1.2 mL), and dried under reduced pressure at 70-80°C for 3 hours to obtain Type II crystals (yield: 120 mg, yield: 43.0%, purity: 98.25%).

Example 4 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)- Synthesis of Type II Crystals of 1H-Imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide, obtained according to the production methods described in International Publication No. 2012/093708 and International Publication No. 2011/004610, was added to methyl isobutyl ketone (4.0 mL) and stirred under reflux for 3 hours. After cooling to room temperature, the solid was collected by filtration, washed with methyl isobutyl ketone (1.2 mL), and dried under reduced pressure at 70-80°C for 3 hours to obtain Type II crystals (yield: 154 mg, yield: 55.3%, purity: 96.89%).

Example 5 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)- Synthesis of Type II Crystals of 1H-Imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide, obtained according to the production methods described in International Publication No. 2012/093708 and International Publication No. 2011/004610, was added to 2-propanol (4.0 mL) and stirred under reflux for 3 hours. After cooling to room temperature, the solid was collected by filtration, washed with 2-propanol (1.2 mL), and dried under reduced pressure at 70-80°C for 3 hours to obtain Type II crystals (yield: 108 mg, yield: 38.8%, purity: 96.83%).

Example 6 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)- Synthesis of Type II Crystals of 1H-Imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide, obtained according to the production methods described in International Publication No. 2012/093708 and International Publication No. 2011/004610, was added to ethyl acetate (4.0 mL) and stirred under reflux for 3 hours. After cooling to room temperature, the solid was collected by filtration, washed with ethyl acetate (1.2 mL), and dried under reduced pressure at 70-80°C for 3 hours to obtain Type II crystals (amount: 156 mg, yield: 56.0%, purity: 96.45%).

Example 7 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)- Synthesis of Type II Crystals of 1H-Imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide, obtained according to the production methods described in International Publication No. 2012/093708 and International Publication No. 2011/004610, was added to butyl acetate (4.0 mL) and stirred under reflux for 3 hours. After cooling to room temperature, the solid was collected by filtration, washed with butyl acetate (1.2 mL), and dried under reduced pressure at 70-80°C for 3 hours to obtain Type II crystals (amount: 164 mg, yield: 58.8%, purity: 96.04%).

Example 8 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)- Synthesis of Type II Crystals of 1H-Imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide, obtained according to the production methods described in International Publication No. 2012/093708 and International Publication No. 2011/004610, was added to cyclopentyl methyl ether (4.0 mL) and stirred under reflux for 3 hours. After cooling to room temperature, the solid was collected by filtration, washed with cyclopentyl methyl ether (1.2 mL), and dried under reduced pressure at 70-80°C for 3 hours to obtain Type II crystals (yield: 192 mg, yield: 68.8%, purity: 95.68%).

Example 9 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)- Synthesis of Type II Crystals of 1H-Imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

A white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide, obtained according to the production methods described in International Publication No. 2012/093708 and International Publication No. 2011/004610, was added to propyl acetate (4.0 mL) and stirred under reflux for 3 hours. After cooling to room temperature, the solid was collected by filtration, washed with propyl acetate (1.2 mL), and dried under reduced pressure at 70-80°C for 3 hours to obtain Type II crystals (amount: 172 mg, yield: 61.5%, purity: 96.77%).

Example 10 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)- Synthesis of Type II Crystals of 1H-Imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

Methyl acetate (2.2 mL) was added to a white solid (400 mg) of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide obtained according to the production methods described in International Publication No. 2012/093708 and International Publication No. 2011/004610, and the mixture was stirred under reflux for 16 hours. After cooling to room temperature, the solid was collected by filtration, washed with methyl acetate (0.94 mL), and dried under reduced pressure at 80°C to obtain Type II crystals (yield: 215.5 mg, yield: 68.6%, purity: 98.06%).

Test Example 1 Blood Concentration Measurement Test

Prepare a dosing solution (50 mg/10 mL/kg) for each of Type I and Type II crystals. Using an oral dosing probe, administer these solutions orally to mice (Balb/cA) maintained under feeding conditions at a volume of 10 mL per kg of body weight. After dosing, return the mice to their cages and confirm their condition. Water and feed are freely available within the cages. One, two, three, four, eight, and 24 hours after dosing, anesthetize the mice with isoflurane, and collect 60 μL of blood from the orbital venous plexus using a capillary blood collection tube. The collected blood is ice-cooled, and the plasma is separated by centrifugation.

AUC 0-24hr was calculated from the concentration of Compound 1 in each plasma measured by the Multiple Reaction Monitoring method using LC-MS/MS, and using the software Phoenix WinNonlin (v6.3.0) manufactured by Pharsight, using the log-linear trapezoidal _method .

The results are shown in Figure 4 and Table 1. This test revealed that Type II crystals exhibited approximately three times the value of Type I crystals in AUC _0-24hr (area under the blood concentration-time curve from 0 to 24 hours after administration). Therefore, the Type II crystals of the present invention exhibit significantly superior oral absorbability and are useful as oral pharmaceutical compositions.

[Table 1]

Test Example 2 Solid Stability Test (Accelerated Test)

The solid stability of the type II crystal obtained in Example 1 was measured under the following conditions when stored at 40°C ± 2°C/75% RH ± 5% RH for 1 month, 3 months, and 6 months.

Storage conditions: 40°C ± 2°C/75% RH ± 5% RH

Measurement time points: 1 month, 3 months and 6 months

Storage capacity: 6g

Storage container: Double polyethylene bag, strapping fastener + plastic bucket

Preparation of the sample solution: Accurately weigh 100 mg of Form II crystals and dissolve them in a 4:1 mixture of acetonitrile and water (if the solution is difficult to dissolve, dissolve it by ultrasonication). Accurately weigh 10 mL of this solution and add a 1:1 mixture of water and acetonitrile to a volume of 20 mL to prepare the sample solution.

HPLC measurement was carried out under the following conditions.

Column: National Institute for the Evaluation of Chemical Substances L-column2 ODS

Particle size: 3μm, inner diameter: 4.6mm, length: 15cm

Measurement wavelength: 220nm

Mobile phase: Mobile phase A: 10 mmol/L phosphate buffer (pH 6.9), Mobile phase B: acetonitrile

Flow rate: about 1.0mL/min

During the delivery of the mobile phase, the mixing ratio of mobile phases A and B was changed as follows to control the concentration gradient.

[Table 2]

Time from injection (minutes) Mobile phase A (%) Mobile phase B (%) 0～25 80 20 25～45 80→65 20→35 45～55 65 35 55～70 65→10 35→90 70～80 10 90 80～80.1 10→80 90→20 80.1～95 80 20

The results of the measurement and evaluation of the mass of the analogues in the sample solution by HPLC analysis are shown in Table 3.

Among them, similar substances are substances detected other than 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide.

[Table 3]

From these results, it can be seen that the formation of similar substances to the type II crystal of compound 1 is small, and it shows excellent solid stability.

Claims (9)

1. A type II crystal of 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide, characterized in that: The powder X-ray diffraction pattern shows 12 characteristic peaks selected from diffraction angles (2θ±0.2°) of 7.7°, 8.0°, 11.1°, 12.5°, 12.9°, 15.2°, 15.8°, 17.2°, 19.0°, 22.5°, 26.1°, and 27.4°. 2. The type II crystallization as described in claim 1, characterized in that: The endothermic peak, determined by differential scanning calorimetry, is around 270℃. 3. A pharmaceutical composition comprising type II crystals as described in claim 1 or 2. 4. An oral pharmaceutical composition comprising the type II crystals as described in claim 1 or 2. 5. An antitumor agent comprising type II crystals as described in claim 1 or 2. 6. The use of the type II crystal as described in claim 1 or 2 in the manufacture of pharmaceutical compositions. 7. The use as described in claim 6, characterized in that: The pharmaceutical composition is a pharmaceutical composition for oral administration. 8. The use of the type II crystal as described in claim 1 or 2 in the manufacture of antitumor agents. 9. A method for manufacturing type II crystals as described in claim 1 or 2, characterized in that it comprises: (1) The step of suspending 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide in an organic solvent selected from 2-propanol, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, cyclopentyl methyl ether, methyl ethyl ketone, methyl isobutyl ketone, acetone, acetonitrile, and mixtures thereof to obtain a suspension; and (2) The process of obtaining solid 3-ethyl-4-{3-isopropyl-4-(4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide from the suspension obtained in (1) above.