RU2828232C1 - Pyrimidine compound crystal - Google Patents

Abstract

FIELD: chemistry; pharmaceutics.

SUBSTANCE: invention relates to a crystal having peaks at diffraction angles (2θ±0.2°) selected from 5.5°, 6.8°, 9.3°, 13.4°, 15.3°, 16.3°, 18.5°, 19.8°, 22.0° and 24.5° on powder X-ray diffraction spectrum, which is a crystal of type II 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide with fumaric acid, where the molar ratio is 1:1. Invention relates to a pharmaceutical composition having HER2 inhibitory activity, containing an effective amount of a crystal according to the invention. Invention also relates to an anticancer agent for treating a tumour associated with HER2, containing a crystal according to the invention.

EFFECT: crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide with fumaric acid as a HER2 inhibitor, which has stability, good oral absorption.

6 cl, 32 dwg, 19 tbl, 27 ex

Description

Field of technology

[0001]

The present invention relates to a crystal of a pyrimidine compound useful as an antitumor agent and to a pharmaceutical composition containing the crystal.

State of the art

[0002]

HER2 (also referred to as "ErbB2") is a receptor tyrosine kinase that belongs to the ErbB family.

HER2 is considered to be a proto-oncogene. It has been reported that HER2 gene amplification, overexpression, mutation, etc. occur in various types of malignant tumors. Based on non-clinical and clinical studies, it has been found that HER2 activation and the downstream signals play an important role in the survival and/or proliferation, etc. of malignant cells associated with HER2 genetic abnormality, overexpression, etc. (Non-patent Document 1).

Therefore, it is expected that an inhibitor capable of regulating HER2 kinase activity inhibits HER2 and downstream signals in malignant cells having HER2 gene amplification, overexpression or mutation, showing antitumor effects on malignant cells. Accordingly, it is considered that such an inhibitor will be useful for treating, prolonging life or improving QOL of patients with malignant tumor.

[0003]

It has been reported that brain metastasis occurs in approximately 25%-40% of lung cancer cases, approximately 15%-30% of breast cancer cases, and certain percentages of other multiple malignancies (Non-Patent Documents 2 and 3). In fact, it has been reported that brain metastasis occurs in approximately 20%-30% of HER2-positive breast cancer cases (Non-Patent Document 4).

[0004]

Compounds with HER2 inhibitory activity, such as lapatinib and neratinib, have been approved as therapeutic agents for HER2-positive breast cancer. However, it has been reported that since all of these therapeutic agents are p-gp or Bcrp substrates, the brain penetration properties of these agents in preclinical tests were limited (Non-Patent Document 5). In fact, in clinical tests using lapatinib or neratinib, sufficient effects of these agents on malignant tumor metastasis to the brain could not be achieved (Non-Patent Documents 6, 7, 8, and 9).

From the point of view of controlling pathological conditions involving metastatic foci in the brain, it is desirable to develop a HER2 inhibitor that has HER2 inhibitory activity and also has brain penetrating properties.

[0005]

Generally, when a compound is used as an active ingredient in a pharmaceutical product, chemical and physical stability of the compound is required to maintain stable quality and/or facilitate storage control. It is desirable to develop a HER2 inhibitor that has chemical and physical stability and inhibitory activity against HER2, and also has brain penetration properties.

References

Non-patent documents

[0006]

Non-Patent Document 1: Cancer Treatment Reviews, 40, pp. 770-780 (2014)

Non-Patent Document 2: Current Oncology, 25, pp. S103-S114 (2018)

Non-Patent Document 3: Breast Cancer Research, 18(1), 8, pp. 1-9 (2016)

Non-Patent Document 4: Journal of Clinical Oncology, 28, pp. 3271-3277 (2010)

Non-Patent Document 5: Journal of Medicinal Chemistry, 59, pp. 10030-10066 (2016)

Non-Patent Document 6: Journal of Medicinal Chemistry, 26, pp. 2999-3005 (2008)

Non-Patent Document 7: Journal of Clinical Oncology, 26, pp. 1993-1999 (2008)

Non-Patent Document 8: Journal of Clinical Oncology, 28, pp. 1301-1307 (2010)

Non-Patent Document 9: Journal of Clinical Oncology, 34, pp. 945–952 (2016).

The essence of the invention

Technical problem

[0007]

The object of the present invention is to provide a HER2 inhibitor having chemical and physical stability and HER2 inhibition activity, and also having brain penetration properties. In particular, the object of the present invention is to provide a HER2 inhibitor crystal that has excellent stability, has good oral absorption, and can be produced with good reproducibility.

Solution to the problem

[0008]

As a result of detailed studies to achieve the above objects, the present inventors found that 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide represented by the following formula (I) (hereinafter also referred to as "compound (I)") has HER2 inhibiting activity and brain penetration properties, and is useful as a therapeutic agent for diseases involving HER2 (particularly malignant tumor) by inhibiting HER2.

[0009]

(I)

[0010]

In addition, the present inventors have found that there are two types of free-form crystals (type I crystal and type II crystal) and crystals with acids (salt crystals or co-crystals) of compound (I), including crystals with hydrochloric acid (type I crystal with hydrochloric acid, type II crystal with hydrochloric acid, type III crystal with hydrochloric acid), crystals with hydrobromic acid, crystals with 1 equivalent of tartaric acid, crystals with fumaric acid (type I crystal with 0.5 equivalent of fumaric acid, type II crystal with 0.5 equivalent of fumaric acid, type I crystal with 1 equivalent of fumaric acid, type II crystal with 1 equivalent of fumaric acid, type III crystal with 1 equivalent of fumaric acid, type IV crystal with 1 equivalent of fumaric acid, type V crystal with 1 equivalent of fumaric acid) and crystals with succinic acid.

The present inventors have found that among these crystals, the type I free-form crystal, the type II free-form crystal, the type II crystal with 1 equivalent of fumaric acid, the type V crystal with 1 equivalent of fumaric acid have advantageous properties for pharmaceutical production such as the property of non-hygroscopicity and excellent solid stability, and also have excellent oral absorption. This has led to the completion of the present invention.

[0011]

In particular, the present invention includes the following embodiments.

[1] A crystal having peaks at three or more diffraction angles (2Ɵ ± 0.2°) selected from 5.5°, 6.8°, 9.3°, 13.4°, 15.3°, 16.3°, 18.5°, 19.8°, 22.0° and 24.5° in an X-ray powder diffraction spectrum, which is a type II crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide with fumaric acid, wherein the molar ratio 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide and fumaric acid is 1:1.

[2] Crystal according to [1], which has peaks at diffraction angles (2Ɵ±0.2°) of 5.5°, 6.8°, 9.3°, 13.4°, 15.3°, 16.3°, 18.5°, 19.8°, 22.0° and 24.5° in the powder X-ray diffraction spectrum.

[3] The crystal according to [1] or [2], which is a crystal having the powder X-ray diffraction spectrum shown in Fig. 1.

[4] A crystal according to any one of [1]-[3], which has an endothermic peak, as determined by simultaneous thermogravimetric-differential thermal analysis, at approximately 178°C.

[5] The crystal according to any one of [1] to [4], wherein the purity of the crystal is 90% by weight or more.

[6] A crystal having peaks at three or more diffraction angles (2Ɵ±0.2°) selected from 8.3°, 14.8°, 17.3°, 18.0°, 19.1°, 20.3°, 21.0°, 22.5°, 23.0° and 26.2° in an X-ray powder diffraction spectrum, which is a type II crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide.

[7] Crystal according to [6], which has peaks at diffraction angles (2Ɵ±0.2°) 8.3°, 14.8°, 17.3°, 18.0°, 19.1°, 20.3°, 21.0°, 22.5°, 23.0° and 26.2° in the powder X-ray diffraction spectrum.

[8] The crystal according to [6] or [7], which is a crystal having the powder X-ray diffraction spectrum shown in Fig. 3.

[9] A crystal according to any one of [6]-[8], which has an endothermic peak, determined by simultaneous thermogravimetric-differential thermal analysis, at approximately 182°C.

[10] The crystal according to any one of [6] to [9], wherein the purity of the crystal is 90% by weight or more.

[11] A crystal having peaks at four or more diffraction angles (2Ɵ±0.2°) selected from 9.9°, 11.7°, 13.2°, 17.7°, 18.1°, 18.8° and 20.8° in an X-ray powder diffraction spectrum, which is a type I crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide.

[12] Crystal according to [11], which has peaks at diffraction angles (2Ɵ±0.2°) 9.9°, 11.7°, 13.2°, 17.7°, 18.1°, 18.8° and 20.8° in the powder X-ray diffraction spectrum.

[13] The crystal according to [11] or [12], which is a crystal having the powder X-ray diffraction spectrum shown in Fig. 5.

[14] A crystal according to any one of [11]-[13], which has an endothermic peak, as determined by simultaneous thermogravimetric-differential thermal analysis, at approximately 182°C.

[15] The crystal according to any one of [11] to [14], wherein the purity of the crystal is 90% by weight or more.

[16] A crystal having peaks at four or more diffraction angles (2Ɵ±0.2°) selected from 6.9°, 9.4°, 10.2°, 13.7°, 21.1°, 23.6° and 26.5° in an X-ray powder diffraction spectrum, which is a type V crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide with fumaric acid, wherein the molar ratio 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide and fumaric acid is 1:1.

[17] Crystal according to [16], which has peaks at diffraction angles (2Ɵ±0.2°) 6.9°, 9.4°, 10.2°, 13.7°, 21.1°, 23.6° and 26.5° in the powder X-ray diffraction spectrum.

[18] The crystal according to [16] or [17], which is a crystal having the powder X-ray diffraction spectrum shown in Fig. 7.

[19] A crystal according to any one of [16]-[18], which has an endothermic peak, as determined by simultaneous thermogravimetric-differential thermal analysis, at approximately 151°C.

[20] The crystal according to any one of [16] to [19], wherein the purity of the crystal is 90% by weight or more.

[21] A crystal having peaks at four or more diffraction angles (2Ɵ±0.2°) selected from 6.4°, 10.3°, 12.8°, 15.0°, 20.7°, 23.4° and 26.6° in an X-ray powder diffraction spectrum, which is a type I crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide with fumaric acid, wherein the molar ratio 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide and fumaric acid is 1:1.

[22] The crystal according to [21] has peaks at diffraction angles (2Ɵ±0.2°) 6.4°, 10.3°, 12.8°, 15.0°, 20.7°, 23.4° and 26.6° in the powder X-ray diffraction spectrum.

[23] The crystal according to [21] or [22], which is a crystal having the powder X-ray diffraction spectrum shown in Fig.9.

[24] A crystal according to any one of [21]-[23], which has an endothermic peak, determined by simultaneous thermogravimetric-differential thermal analysis, at approximately 153°C.

[25] The crystal according to any one of [21] to [24], wherein the purity of the crystal is 90% by weight or more.

[26] A crystal having peaks at four or more diffraction angles (2Ɵ±0.2°) selected from 6.4°, 7.5°, 9.7°, 11.7°, 15.1°, 19.6° and 23.9° in an X-ray powder diffraction spectrum, which is a type I crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide with fumaric acid, wherein the molar ratio 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide and fumaric acid is 1:0.5.

[27] Crystal according to [26], which has peaks at diffraction angles (2Ɵ±0.2°) 6.4°, 7.5°, 9.7°, 11.7°, 15.1°, 19.6° and 23.9° in the powder X-ray diffraction spectrum.

[28] The crystal according to [26] or [27], which is a crystal having the powder X-ray diffraction spectrum shown in Fig. 11.

[29] A crystal according to any one of [26]-[28], which has an endothermic peak, determined by simultaneous thermogravimetric-differential thermal analysis, at approximately 161°C.

[30] The crystal according to any one of [26] to [29], wherein the purity of the crystal is 90% by weight or more.

[31] A crystal having peaks at four or more diffraction angles (2Ɵ±0.2°) selected from 5.4°, 6.4°, 7.3°, 12.8°, 13.4°, 14.7° and 15.4° in an X-ray powder diffraction spectrum, which is a type I crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide with fumaric acid, wherein the molar ratio 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide and fumaric acid is 1:0.5.

[32] Crystal according to [31], which has peaks at diffraction angles (2Ɵ±0.2°) 5.4°, 6.4°, 7.3°, 12.8°, 13.4°, 14.7° and 15.4° in the powder X-ray diffraction spectrum.

[33] The crystal according to [31] or [32], which is a crystal having the powder X-ray diffraction spectrum shown in Fig. 13.

[34] A crystal according to any one of [31]-[33], which has an endothermic peak, as determined by simultaneous thermogravimetric-differential thermal analysis, at approximately 162°C.

[35] The crystal according to any one of [31] to [34], wherein the purity of the crystal is 90% by weight or more.

[36] A pharmaceutical composition comprising a crystal according to any one of [1] to [35].

[37] A pharmaceutical composition for oral administration comprising a crystal according to any one of [1] to [35].

[38] An antitumor agent comprising a crystal according to any one of [1] to [35].

[39] A crystal according to any one of [1] to [35] for use as a medicine.

[40] Use of a crystal according to any one of [1] to [35] for the production of an antitumor agent for oral administration.

[41] A crystal according to any one of [1] to [35] for use in treating a tumor.

[42] The crystal according to any one of [1] to [35] for use in treating a tumor by oral administration thereof.

[43] A method of treating a tumor comprising administering an effective amount of a crystal according to any one of [1]-[12] to an individual in need thereof.

[44] Use of a crystal according to any of [1] to [35] for the production of a pharmaceutical composition.

[45] Use of a crystal according to any of [1] to [35] for the production of an antitumor agent.

Effects of invention

[0012]

The type I free crystal, the type II free crystal, the type II crystal with 1 equivalent of fumaric acid, the type V crystal with 1 equivalent of fumaric acid of compound (I) (7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide) of the present invention are superior to other crystal forms in terms of high stability, convenience of processing (lower hygroscopicity), quality control capability, etc. Further, the type II free crystal and the type II crystal with 1 equivalent of fumaric acid have excellent absorbability in the oral cavity, and thus they are useful forms when using the compound as an active pharmaceutical ingredient. Type I crystal with 1 equivalent of fumaric acid has a form which can be isolated as a solid from acetone (solvent) (after isolation, it is dried to obtain type V crystal), it is also suitable as an intermediate material for obtaining type V crystal.

According to the present invention, there is provided a novel crystal of the compound (I) or a novel crystal of the compound (I) with an acid and a pharmaceutical composition, an antitumor agent or an antitumor agent for oral administration containing the novel crystal.

In preferred aspects, the type II crystal with 1 equivalent of fumaric acid, the free-form crystal of type II, the free-form crystal of type I, the type V crystal with 1 equivalent of fumaric acid, the type I crystal with 0.5 equivalent of fumaric acid, and the type II crystal with 0.5 equivalent of fumaric acid of compound (I) have at least one of the advantageous properties in pharmaceutical production such as non-hygroscopic property, reproducible production capability, solid stability, and oral absorbability.

Brief description of the drawings

[0013]

[Figure 1] Figure 1 shows the powder X-ray diffraction spectrum of the type II crystal of compound (I) with 1 equivalent of fumaric acid obtained according to Example 1 (the vertical axis shows the intensity (count units), and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 2] Figure 2 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the type II crystal of compound (I) with 1 equivalent of fumaric acid obtained according to Example 1 (the left vertical axis represents the mass (mg) in the TG curve, the right vertical axis represents the heat flux (μV) in the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 3] Figure 3 shows the powder X-ray diffraction spectrum of the type II crystal of compound (I) obtained according to Example 2 (the vertical axis shows the intensity (count units) and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 4] Figure 4 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the type II crystal of compound (I) obtained according to Example 2 (the left vertical axis represents the mass (mg) in the TG curve, the right vertical axis represents the heat flux (μV) in the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 5] Figure 5 shows the powder X-ray diffraction spectrum of the type I crystal of compound (I) obtained according to Example 3 (the vertical axis shows the intensity (count units) and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 6] Figure 6 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the type I crystal of compound (I) obtained according to Example 3 (the left vertical axis represents the mass (mg) of the TG curve, the right vertical axis represents the heat flux (μV) of the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 7] Figure 7 shows the powder X-ray diffraction spectrum of the type V crystal of compound (I) with 1 equivalent of fumaric acid obtained according to Example 4 (the vertical axis shows the intensity (count units), and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 8] Figure 8 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the type V crystal of compound (I) with 1 equivalent of fumaric acid obtained according to Example 4 (the left vertical axis represents the mass (mg) in the TG curve, the right vertical axis represents the heat flux (μV) in the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 9] Figure 9 shows the powder X-ray diffraction spectrum of the type I crystal of compound (I) with 1 equivalent of fumaric acid obtained according to Example 5 (the vertical axis shows the intensity (count units) and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 10] Figure 10 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the type I crystal of compound (I) with 1 equivalent of fumaric acid obtained according to Example 5 (the left vertical axis represents the mass (mg) in the TG curve, the right vertical axis represents the heat flux (μV) in the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 11] Figure 11 shows the powder X-ray diffraction spectrum of the type I crystal of compound (I) with 0.5 equivalent of fumaric acid obtained according to Example 6 (the vertical axis shows the intensity (count units), and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 12] Figure 12 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the type I crystal of compound (I) with 0.5 equivalent of fumaric acid obtained according to Example 6 (the left vertical axis represents the mass (mg) of the TG curve, the right vertical axis represents the heat flux (μV) of the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 13] Figure 13 shows the powder X-ray diffraction spectrum of the type II crystal of compound (I) with 0.5 equivalent of fumaric acid obtained according to Example 7 (the vertical axis shows the intensity (count units), and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 14] Figure 14 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the type II crystal of compound (I) with 0.5 equivalent of fumaric acid obtained according to Example 7 (the left vertical axis represents the mass (mg) of the TG curve, the right vertical axis represents the heat flux (μV) of the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 15] Figure 15 shows the powder X-ray diffraction spectrum of the type III crystal of compound (I) with 1 equivalent of fumaric acid obtained according to Reference Example 1 (the vertical axis shows the intensity (count units), and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 16] Figure 16 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the type III crystal of compound (I) with 1 equivalent of fumaric acid obtained according to Reference Example 1 (the left vertical axis represents the mass (mg) of the TG curve, the right vertical axis represents the heat flux (μV) of the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 17] Figure 17 shows the powder X-ray diffraction spectrum of the type IV crystal of compound (I) with 1 equivalent of fumaric acid obtained according to Reference Example 2 (the vertical axis shows the intensity (count units), and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 18] Figure 18 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the type IV crystal of compound (I) with 1 equivalent of fumaric acid obtained according to Reference Example 2 (the left vertical axis represents the mass (mg) of the TG curve, the right vertical axis represents the heat flux (μV) of the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 19] Figure 19 shows a powder X-ray diffraction spectrum of a type I crystal of compound (I) with hydrochloric acid obtained according to Reference Example 3 (the vertical axis shows the intensity (count units), and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 20] Figure 20 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the type I crystal of compound (I) with hydrochloric acid obtained according to Reference Example 3 (the left vertical axis represents the mass (mg) of the TG curve, the right vertical axis represents the heat flux (μV) of the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 21] Figure 21 shows a powder X-ray diffraction spectrum of a type II crystal of compound (I) with hydrochloric acid obtained according to Reference Example 4 (the vertical axis shows the intensity (count units), and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 22] Figure 22 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the type II crystal of compound (I) with hydrochloric acid obtained according to Reference Example 4 (the left vertical axis represents the mass (mg) of the TG curve, the right vertical axis represents the heat flux (μV) of the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 23] Figure 23 shows a powder X-ray diffraction spectrum of a type III crystal of compound (I) with hydrochloric acid obtained according to Reference Example 5 (the vertical axis shows the intensity (count units), and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 24] Figure 24 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the type III crystal of compound (I) with hydrochloric acid obtained according to Reference Example 5 (the left vertical axis represents the mass (mg) of the TG curve, the right vertical axis represents the heat flux (μV) of the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 25] Figure 25 shows the powder X-ray diffraction spectrum of the crystal of compound (I) with hydrobromic acid obtained according to Reference Example 6 (the vertical axis shows the intensity (count units), and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 26] Figure 26 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the crystal of compound (I) with hydrobromic acid obtained according to Reference Example 6 (the left vertical axis represents the mass (mg) of the TG curve, the right vertical axis represents the heat flux (μV) of the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 27] Figure 27 shows a powder X-ray diffraction spectrum of a crystal of compound (I) with L-tartaric acid obtained according to Reference Example 7 (the vertical axis shows the intensity (count units), and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 28] Figure 28 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the crystal of compound (I) with L-tartaric acid obtained according to Reference Example 7 (the left vertical axis represents the mass (mg) of the TG curve, the right vertical axis represents the heat flux (μV) of the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 29] Figure 29 shows the powder X-ray diffraction spectrum of the crystal of compound (I) with succinic acid obtained according to Reference Example 8 (the vertical axis shows the intensity (count units), and the horizontal axis shows the diffraction angle (2Ɵ)).

[Figure 30] Figure 30 shows the results of simultaneous thermogravimetric-differential thermal analysis (TG-DTA) for the crystal of compound (I) with succinic acid obtained according to Reference Example 8 (the left vertical axis represents the mass (mg) of the TG curve, the right vertical axis represents the heat flux (μV) of the DTA curve, and the horizontal axis represents the temperature (°C)).

[Figure 31] Figure 31 shows the antitumor effects of compounds (I) in models involving direct transplantation into the brain of a HER2-expressing cell line with an integrated luciferase gene (NCI-N87-luc).

[Figure 32] Figure 32 shows the percentage of compound (I)-induced body weight reduction in models involving direct transplantation of a HER2-expressing cell line with an integrated luciferase gene (NCI-N87-luc) into the brain.

Description of embodiments

[0014]

The present invention relates to a crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide and a crystal with an acid thereof (a crystal of its salt or co-crystal).

(I)

In particular, the present invention relates to a type II crystal with 1 equivalent of fumaric acid, a free-form crystal of type II, a free-form crystal of type I, a type V crystal with 1 equivalent of fumaric acid, a type I crystal with 1 equivalent of fumaric acid, a type I crystal with 0.5 equivalent of fumaric acid, a type II crystal with 0.5 equivalent of fumaric acid of compound (I).

In the present description, type I, type II, type III, type IV and type V are convenient names for distinguishing the crystal forms, and the crystals according to the present invention are not limited to these names.

[0015]

In the present description, a crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound (I)) with an acid means a salt crystal or a cocrystal with an acid. A salt crystal is a crystal in which the compound (I) and an acid molecule are bonded by an ionic bond, and a cocrystal is a crystal in which the compound (I) and an acid molecule are bonded by a non-ionic interaction. In the context of the present invention, a crystal of the compound (I) with an acid may be a salt crystal or a cocrystal, and it includes both meanings. For example, a type II crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide with fumaric acid means a crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide fumarate or a cocrystal 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide with fumaric acid.

[0016]

A crystal is a solid in which the atoms and molecules have regular repeating structures, and is different from an amorphous solid that has no repeating structures. Crystalline or amorphous solids can be examined by methods such as powder X-ray diffraction analysis (XRD analysis), differential scanning calorimetry (DSC analysis), simultaneous thermogravimetric-differential thermal analysis (TG-DTA analysis), and single crystal analysis. It is known that the polymorph of a crystal means that the molecules are the same, but the arrangement of atoms and molecules is different in the crystal, and the peaks obtained by XRD analysis are different among the crystal polymorphs. It is also known that each polymorph has different solubility, oral absorption, stability, etc.

[0017]

In this description, the terms "crystal" and "amorphous" are used in their ordinary meaning.

[0018]

In the present specification, the description "compound (I)" simply means 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide, which is used in a meaning that includes both "amorphous" and "crystal".

In the present description, the description "crystal of compound (I)" is used in the meaning including any of the free form crystals of compound (I) and crystals of compound (I) with an acid (a salt crystal of compound (I) and a co-crystal of compound (I)).

In the present description, a crystal for which molecules other than the compound (I) constituting the crystal (other molecules constituting the salt crystal or co-crystal) are not indicated means a free-form crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound (I)).

In this description, the term "equivalent" means "molar equivalent".

In a crystal of compound (I) with an acid (salt form crystal or co-crystal), the equivalent of the acid to compound (I) can be analyzed, for example, by NMR or ion chromatography.

In the present description, crystals may be hydrates.

[0019]

In addition, the present invention also encompasses a labeled compound (I) or a salt thereof, i.e. a compound obtained by replacing one or more atoms of the compound (I) or its salt with radioactive or non-radioactive isotopes.

[0020]

If the crystal includes a crystal of the compound (I), it may be only one type of crystal of the compound (I), or it may be a polymorphic mixture containing another type of crystal of the compound (I) in addition to one type of crystal of the compound (I). In particular, it is preferable that the purity of the crystal is 50% by mass or more, in other words, 50% by mass or more is a single crystal. It is more preferable that the purity of the crystal is 75% by mass or more, in other words, 75% by mass or more is a single crystal. It is still more preferable that the purity of the crystal is 90% by mass or more, in other words, 90% by mass or more is a single crystal. It is still more preferable that the purity of the crystal is 95% by mass or more, in other words, 95% by mass or more is a single crystal. It is particularly preferable that the purity of the crystal is 99% by mass or more, in other words, 99% by mass or more is a single crystal.

[0021]

In the present specification, the chemical purity is the purity measured by high performance liquid chromatography (HPLC), and when it is described as the chemical purity of the compound (I), it means the purity of the compound (I) measured by high performance liquid chromatography. At the same time, the wavelength of the detector used for measuring the purity can be set appropriately. In particular, the chemical purity of the compound (I) is preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more.

[0022]

For X-ray powder diffraction patterns, the diffraction angle and overall pattern are important in establishing the identity of crystals due to the nature of the data. The relative intensity of the X-ray powder diffraction pattern may vary slightly depending on the crystal growth direction, particle size, and measurement conditions, and thus should not be interpreted strictly.

[0023]

The numerical values obtained from different patterns may have some errors depending on the crystal growth direction, particle size, measurement conditions, etc. Therefore, in the present description, the numerical value of the diffraction angle (2Ɵ) in the powder X-ray diffraction pattern may have a measurement error in the range of approximately ±0.2°.

[0024]

In the present description, "room temperature" generally means from about 10°C to about 35°C.

[0025]

In addition, the endothermic peak in the simultaneous thermogravimetric-differential thermal analysis (TG-DTA) curve mainly means a value with a measurement error in the range of approximately ±5.0°C, since the measurement temperature may vary depending on the range of the temperature increase per minute, the chemical purity of the sample, etc. Therefore, when the measurement is carried out for the crystal according to the present invention (TG-DTA), an error of the peak (peak value) of ±5.0°C is taken into account. The term "approximately" used in such a case means ±5.0°C.

[0026]

One embodiment of the present invention relates to a free-form crystal (type I crystal or type II crystal) of compound (I), a crystal (type I crystal, type II crystal, type III crystal, type IV crystal or type V crystal) of compound (I) with 1 equivalent of fumaric acid, and a crystal (type I crystal or type II crystal) of compound (I) with 0.5 equivalent of fumaric acid.

[0027]

(Type II crystal of compound (I) with 1 equivalent of fumaric acid)

The type II crystal of compound (I) with 1 equivalent of fumaric acid has the powder X-ray diffraction spectrum shown in Fig. 1 and also has the simultaneous thermogravimetric-differential thermal analysis (TG-DTA) curve shown in Fig. 2.

Furthermore, in one embodiment of the present invention, the type II crystal of compound (I) with 1 equivalent of fumaric acid has the diffraction angle (2Ɵ) and intensity (count units) shown in Table 2 in powder X-ray diffraction.

[0028]

Here, the characteristic peaks in the powder X-ray diffraction spectrum of the type II crystal of compound (I) with 1 equivalent of fumaric acid can have diffraction angles (2Ɵ±0.2°) of 5.5°, 6.8°, 9.3°, 13.4°, 15.3°, 16.3°, 18.5°, 19.8°, 22.0° and 24.5°.

The type II crystal of compound (I) with 1 equivalent of fumaric acid according to the present invention is a crystal having three or more peaks selected from the above-mentioned characteristic peaks. Preferably, it is a crystal having four or more peaks selected from said characteristic peaks, more preferably a crystal having five or more peaks selected from said characteristic peaks, even more preferably a crystal having six or more peaks selected from said characteristic peaks, even more preferably a crystal having seven or more peaks selected from said characteristic peaks, even more preferably a crystal having eight or more peaks selected from said characteristic peaks, even more preferably a crystal having nine or more peaks selected from said characteristic peaks, and particularly preferably a crystal having all of the peaks.

[0029]

The endothermic peak (peak apex value) of the type II crystal of compound (I) with 1 equivalent of fumaric acid, determined by simultaneous thermogravimetric-differential thermal analysis, can be from 173°C to 183°C, in other words, approximately 178°C.

[0030]

(Free form crystal of type II compound (I))

The type II crystal of compound (I) has a powder X-ray diffraction spectrum shown in Fig. 3 and also has a simultaneous thermogravimetric-differential thermal analysis (TG-DTA) curve shown in Fig. 4.

Furthermore, in one embodiment of the present invention, the free form crystal of type II of compound (I) has the diffraction angle (2Ɵ) and intensity (count units) shown in Table 3 in powder X-ray diffraction.

[0031]

Here, the characteristic peaks in the powder X-ray diffraction spectrum of the type II crystal of compound (I) can have diffraction angles (2Ɵ±0.2°) of 8.3°, 14.8°, 17.3°, 18.0°, 19.1°, 20.3°, 21.0°, 22.5°, 23.0° and 26.2°.

The type II crystal of the compound (I) according to the present invention is a crystal having three or more peaks selected from the above characteristic peaks. Preferably, it is a crystal having four or more peaks selected from said characteristic peaks, more preferably a crystal having five or more peaks selected from said characteristic peaks, even more preferably a crystal having six or more peaks selected from said characteristic peaks, even more preferably a crystal having seven or more peaks selected from said characteristic peaks, even more preferably a crystal having eight or more peaks selected from said characteristic peaks, even more preferably a crystal having nine or more peaks selected from said characteristic peaks, and particularly preferably a crystal having all of the characteristic peaks.

[0032]

The endothermic peak (peak apex value) of the type II crystal of compound (I), determined by simultaneous thermogravimetric-differential thermal analysis, can be from 177°C to 187°C, in other words, approximately 182°C.

[0033]

(Free-form crystal of type I compound (I))

The type I crystal of compound (I) has a powder X-ray diffraction spectrum shown in Fig. 5 and also has a simultaneous thermogravimetric-differential thermal analysis (TG-DTA) curve shown in Fig. 6.

Furthermore, in one embodiment of the present invention, the free-form crystal of type I of compound (I) has the diffraction angle (2Ɵ) and intensity (count units) shown in Table 4 in powder X-ray diffraction.

[0034]

Here, the characteristic peaks in the powder X-ray diffraction spectrum of the type I crystal of compound (I) can have diffraction angles (2Ɵ±0.2°) of 9.9°, 11.7°, 13.2°, 17.7°, 18.1°, 18.8° and 20.8°.

The type I crystal of the compound (I) according to the present invention is a crystal having three or more peaks selected from the above characteristic peaks. Preferably, it is a crystal having four or more peaks selected from said characteristic peaks, more preferably a crystal having five or more peaks selected from said characteristic peaks, even more preferably a crystal having six or more peaks selected from said characteristic peaks, and particularly preferably a crystal having all of the characteristic peaks.

Here, the characteristic peaks in the powder X-ray diffraction spectrum of the type I crystal of compound (I) can have diffraction angles (2Ɵ±0.2°) of 9.9°, 11.7°, 13.2°, 18.8° and 20.8°.

The type I crystal of the compound (I) according to the present invention is a crystal having three or more peaks selected from the above characteristic peaks. Preferably, it is a crystal having four or more peaks selected from the said characteristic peaks, and particularly preferably a crystal having all of the characteristic peaks.

[0035]

The endothermic peak (peak apex value) of the type I crystal of compound (I), determined by simultaneous thermogravimetric-differential thermal analysis, can be from 178°C to 188°C, in other words, approximately 183°C.

[0036]

(Type V crystal of compound (I) with 1 equivalent of fumaric acid)

The type V crystal of compound (I) with 1 equivalent of fumaric acid has the powder X-ray diffraction spectrum shown in Fig. 7 and also has the simultaneous thermogravimetric-differential thermal analysis (TG-DTA) curve shown in Fig. 8.

Furthermore, in one embodiment of the present invention, the type V crystal of compound (I) with 1 equivalent of fumaric acid has the diffraction angle (2Ɵ) and intensity (count units) shown in Table 5 in powder X-ray diffraction.

[0037]

Here, the characteristic peaks in the powder X-ray diffraction spectrum of the type V crystal of compound (I) with 1 equivalent of fumaric acid can have diffraction angles (2Ɵ±0.2°) of 6.9°, 9.4°, 10.2°, 13.7°, 21.1°, 23.6° and 26.5°.

The type V crystal of the compound (I) with 1 equivalent of fumaric acid according to the present invention is a crystal having three or more peaks selected from the above characteristic peaks. Preferably, it is a crystal having four or more peaks selected from said characteristic peaks, more preferably a crystal having five or more peaks selected from said characteristic peaks, even more preferably a crystal having six or more peaks selected from said characteristic peaks, and particularly preferably a crystal having all of the characteristic peaks.

Here, the characteristic peaks in the powder X-ray diffraction spectrum of the type V crystal of compound (I) with 1 equivalent of fumaric acid can have diffraction angles (2Ɵ±0.2°) of 6.9°, 13.7°, 21.1°, 23.6° and 26.5°.

The type V crystal of the compound (I) with 1 equivalent of fumaric acid according to the present invention is a crystal having three or more peaks selected from the above characteristic peaks. Preferably, it is a crystal having four or more peaks selected from the said characteristic peaks, and particularly preferably a crystal having all of the characteristic peaks.

[0038]

The endothermic peak (peak apex value) of the type V crystal of compound (I) with 1 equivalent of fumaric acid, determined by simultaneous thermogravimetric-differential thermal analysis, can be between 146°C and 156°C, in other words, approximately 151°C.

[0039]

(Type I crystal of compound (I) with 1 equivalent of fumaric acid)

The type I crystal of compound (I) with 1 equivalent of fumaric acid has the powder X-ray diffraction spectrum shown in Fig. 9 and also has the simultaneous thermogravimetric-differential thermal analysis (TG-DTA) curve shown in Fig. 10.

Furthermore, in one embodiment of the present invention, the type I crystal of compound (I) with 1 equivalent of fumaric acid has the diffraction angle (2Ɵ) and intensity (count units) shown in Table 6 in powder X-ray diffraction.

[0040]

Here, the characteristic peaks in the powder X-ray diffraction spectrum of the type V crystal of compound (I) with 1 equivalent of fumaric acid can have diffraction angles (2Ɵ±0.2°) of 6.4°, 10.3°, 12.8°, 15.0°, 20.7°, 23.4° and 26.6°.

The type I crystal of the compound (I) with 1 equivalent of fumaric acid according to the present invention is a crystal having three or more peaks selected from the above characteristic peaks. Preferably, it is a crystal having four or more peaks selected from said characteristic peaks, more preferably a crystal having five or more peaks selected from said characteristic peaks, even more preferably a crystal having five or more peaks selected from said characteristic peaks, and particularly preferably a crystal having all of the characteristic peaks.

The endothermic peak (peak apex value) of type I crystal of compound (I) with 1 equivalent of fumaric acid, determined by simultaneous thermogravimetric-differential thermal analysis, can be between 148°C and 158°C, in other words, approximately 153°C.

Here, the characteristic peaks in the powder X-ray diffraction spectrum of the type V crystal of compound (I) with 1 equivalent of fumaric acid can have diffraction angles (2Ɵ±0.2°) of 6.4°, 10.3°, 12.8°, 20.7°, 23.4° and 26.6°.

The type I crystal of the compound (I) with 1 equivalent of fumaric acid according to the present invention is a crystal having three or more peaks selected from the above characteristic peaks. Preferably, it is a crystal having four or more peaks selected from said characteristic peaks, more preferably a crystal having five or more peaks selected from said characteristic peaks, and particularly preferably a crystal having all of the characteristic peaks.

The endothermic peak (peak apex value) of type I crystal of compound (I) with 1 equivalent of fumaric acid, determined by simultaneous thermogravimetric-differential thermal analysis, can be between 148°C and 158°C, in other words, approximately 153°C.

[0041]

(Type I crystal of compound (I) with 0.5 equivalent of fumaric acid)

The type I crystal of compound (I) with 0.5 equivalent of fumaric acid has the powder X-ray diffraction spectrum shown in Fig. 11 and also has the simultaneous thermogravimetric-differential thermal analysis (TG-DTA) curve shown in Fig. 12.

[0042]

Here, the characteristic peaks in the powder X-ray diffraction spectrum of the type I crystal of compound (I) with 0.5 equivalent of fumaric acid can have diffraction angles (2Ɵ±0.2°) of 6.4°, 7.5°, 9.7°, 11.7°, 15.1°, 19.6° and 23.9°.

The type I crystal of the compound (I) with 0.5 equivalent of fumaric acid according to the present invention is a crystal having four or more peaks selected from the above characteristic peaks. Preferably, it is a crystal having five or more peaks selected from said characteristic peaks, more preferably a crystal having six or more peaks selected from said characteristic peaks, and particularly preferably a crystal having all of the characteristic peaks.

[0043]

The endothermic peak (peak apex value) of type I crystal of compound (I) with 0.5 equivalent of fumaric acid, determined by simultaneous thermogravimetric-differential thermal analysis, can be between 156°C and 166°C, in other words, approximately 161°C.

[0044]

(Type II crystal of compound (I) with 0.5 equivalent of fumaric acid)

The type II crystal of compound (I) with 0.5 equivalent of fumaric acid has the powder X-ray diffraction spectrum shown in Fig. 13 and also has the simultaneous thermogravimetric-differential thermal analysis (TG-DTA) curve shown in Fig. 14.

[0045]

Here, the characteristic peaks in the powder X-ray diffraction spectrum of the type II crystal of compound (I) with 0.5 equivalent of fumaric acid can have diffraction angles (2Ɵ±0.2°) of 5.4°, 6.4°, 7.3°, 12.8°, 13.4°, 14.7° and 15.4°.

The type II crystal of the compound (I) with 0.5 equivalent of fumaric acid according to the present invention is a crystal having four or more peaks selected from the above characteristic peaks. Preferably, it is a crystal having five or more peaks selected from said characteristic peaks, more preferably a crystal having six or more peaks selected from said characteristic peaks, and particularly preferably a crystal having all of the characteristic peaks.

[0046]

The endothermic peak (peak apex value) of the type II crystal of compound (I) with 0.5 equivalent of fumaric acid, determined by simultaneous thermogravimetric-differential thermal analysis, can be between 157°C and 167°C, in other words, approximately 162°C.

[0047]

(Type III crystal of compound (I) with 1 equivalent of fumaric acid)

The type III crystal of compound (I) with 1 equivalent of fumaric acid has the powder X-ray diffraction spectrum shown in Fig. 15 and also has the simultaneous thermogravimetric-differential thermal analysis (TG-DTA) curve shown in Fig. 16.

[0048]

Here, the characteristic peaks in the powder X-ray diffraction spectrum of the type III crystal of compound (I) with 1 equivalent of fumaric acid can have diffraction angles (2Ɵ±0.2°) of 8.1°, 13.2°, 20.4°, 23.1°, 24.7° and 26.1°.

The type III crystal of the compound (I) with 1 equivalent of fumaric acid according to the present invention is a crystal having three or more peaks selected from the above characteristic peaks. Preferably, it is a crystal having four or more peaks selected from said characteristic peaks, more preferably a crystal having five or more peaks selected from said characteristic peaks, and particularly preferably a crystal having all of the characteristic peaks.

The exothermic peak (peak apex value) of the type III crystal of compound (I) with 1 equivalent of fumaric acid, determined by simultaneous thermogravimetric-differential thermal analysis, can be between 148°C and 158°C, in other words, approximately 153°C.

[0049]

(Type IV crystal of compound (I) with 1 equivalent of fumaric acid)

The type IV crystal of compound (I) with 1 equivalent of fumaric acid has the powder X-ray diffraction spectrum shown in Fig. 17 and also has the simultaneous thermogravimetric-differential thermal analysis (TG-DTA) curve shown in Fig. 18.

[0050]

Here, the characteristic peaks in the powder X-ray diffraction spectrum of the type IV crystal of compound (I) with 1 equivalent of fumaric acid can have diffraction angles of (2Ɵ±0.2°) 6.0°, 15.7° and 18.8°.

The type IV crystal of compound (I) with 1 equivalent of fumaric acid according to the present invention is a crystal having all of the above characteristic peaks.

The exothermic peak (peak apex value) of the type IV crystal of compound (I) with 1 equivalent of fumaric acid, determined by simultaneous thermogravimetric-differential thermal analysis, can be between 125°C and 135°C, in other words, approximately 130°C.

[0051]

(Method for obtaining type II crystal with 1 equivalent of fumaric acid)

In one embodiment of the present disclosure, a type II crystal of compound (I) with 1 equivalent of fumaric acid can be obtained by a method comprising, for example, the following two steps.

Step 1: A step of adding fumaric acid and then a solvent to the type I crystal of compound (I) and optionally adding a seed crystal.

Step 2: Step of stirring the suspension obtained in the above step to obtain a crystal of compound (I) with 1 equivalent of fumaric acid.

The resulting solid is then isolated by filtration. Preferably, the isolated solid is washed with water and dried.

In step 1, 1.5 to 10 equivalents (more preferably 2 to 5 equivalents and most preferably 3 equivalents) of fumaric acid are preferably added relative to 1 equivalent of compound (I) in free form.

The solvent in step 1 is added in an amount preferably greater than 5 to 35 times (v/w) (more preferably 5 to 30 times (v/w) and most preferably 10 times (v/w)).

Examples of the solvent used in step 1 may include simple solvents or mixed solvents of alcohols such as methanol, ethanol, isopropanol and tert-butanol, ethers such as 1,4-dioxane and tetrahydrofuran, ketones such as acetone and methyl ethyl ketone, and aprotic polar solvents such as acetonitrile.

A type I crystal of the compound (I) can be obtained by the method of the section "(Method for Obtaining a Free-Form Crystal of Type I)" or by the method described below in Example 3.

The temperature during the reaction in step 2 is preferably room temperature or higher and lower than the boiling point of each solvent as described above, but it is not particularly limited and can be appropriately set to preferably 25°C-70°C (more preferably 40°C-60°C and most preferably 50°C).

The stirring time during the reaction in step 2 is preferably 24 to 124 hours (more preferably 48 to 72 hours and most preferably 60 to 65 hours) to stir the suspension.

[0052]

(Method for producing type II free-form crystal)

(Method A-1)

In one embodiment of the present disclosure, a free-form crystal of type II of compound (I) can be obtained by a method comprising, for example, the following two steps (method A-1).

Step 1: A step of adding one of succinic acid, phosphoric acid and adipic acid and a mixed solvent of good solvent/poor solvent to the compound (I) and optionally adding a seed crystal.

Step 2: A step of stirring the suspension obtained in the above step to obtain a crystal of compound (I) in solid form.

During the suspending and mixing process described above, additional solvent (e.g. 1-propanol as a good solvent) may be added repeatedly if necessary, depending on the degree of evaporation of the solvent.

After this, the resulting solid substance is isolated by filtration and dried.

Preferably, in step 1, 0.5-2 equivalents (more preferably 0.75-1.5 equivalents and particularly preferably 1 equivalent) of acid is added relative to 1 equivalent of compound (I).

The mixed solvent of the good solvent/poor solvent in step 1 (e.g. 1-propanol/water) is added in an amount of preferably 5 to 50 times (v/w) (more preferably 10 to 30 times (v/w) and particularly preferably 20 times (v/w)) the amount of compound (I) in free form.

The temperature during the reaction in step 2 is preferably 25°C-70°C (more preferably 40°C-60°C and particularly preferably 50°C).

The stirring time during the reaction in step 2 is preferably 2 to 7 days (more preferably 3 to 5 days and particularly preferably 4 days) for stirring the suspension.

[0053]

(Method A-2)

In another embodiment of the present disclosure, a free-form crystal of type II of compound (I) can be obtained by a method comprising, for example, the following two steps (method A-2).

Step 1: A step of adding a mixed solvent of a good solvent/poor solvent and a seed crystal to the compound (I) and optionally adding any one of succinic acid, phosphoric acid and adipic acid.

Step 2: A step of stirring the suspension obtained in the above step to obtain a crystal of compound (I) in solid form.

During the suspension and mixing process described above, additional solvent (e.g. 1-propanol as a good solvent) may be added repeatedly if necessary, depending on the degree of evaporation of the solvent.

After this, the resulting solid substance is isolated by filtration and dried.

Preferably, in step 1, 0.5-2 equivalents (more preferably 0.75-1.5 equivalents and particularly preferably 1 equivalent) of succinic acid is added relative to 1 equivalent of compound (I).

The mixed solvent of the good solvent/poor solvent in step 1 (for example, a mixed solvent of 1-propanol/water) is added in an amount of preferably 1 to 10 times (v/w) (more preferably 2 to 5 times (v/w) and particularly preferably 3 times (v/w)) the amount of the compound (I) in free form, and a seed crystal (for example, a seed crystal obtained in either (i) or (ii)) is added thereto.

The reaction temperature in step 2 is preferably from 25°C to 70°C (more preferably from 40°C to 60°C and particularly preferably 50°C).

The reaction time in step 2 is preferably 6 to 48 hours (more preferably 12 to 24 hours, and most preferably 17.5 to 24 hours).

[0054]

(Method B)

In another embodiment of the present disclosure, a free form crystal of type II of compound (I) can be obtained by a method comprising, for example, the following two steps (method B).

Step 1: The step of adding a poor solvent to compound (I) and optionally adding a seed crystal.

Step 2: The step of adding a good solvent to the solution obtained in the above step and stirring the resulting suspension to obtain a crystal of compound (I) in solid form.

A poor solvent (e.g. diisopropyl ether) is added to step 1 in an amount preferably 2-10 times (v/w) (more preferably 3-7 times (v/w) and particularly preferably 5 times (v/w)) greater than the amount of compound (I) in free form.

The reaction time in step 1 is preferably 24 to 96 hours (more preferably 36 to 72 hours, and particularly preferably 44.5 hours) for suspending and stirring.

The reaction temperature in step 2 is preferably from 25°C to 70°C (more preferably from 40°C to 60°C and particularly preferably 50°C).

A good solvent in step 2 (e.g. ethanol) is added in an amount preferably 0.5-3 times (v/w) (more preferably 0.8-2 times (v/w) and particularly preferably 1 time (v/w)) greater than the amount of the free form of compound (I.

The stirring time in step 2 is preferably 6 to 72 hours (more preferably 12 to 48 hours and particularly preferably 21 hours), after which the solid is isolated by filtration and dried.

During the suspending and stirring process described above, additional solvent (e.g. diisopropyl ether as a poor solvent and/or ethanol as a good solvent) may be added repeatedly if necessary, depending on the degree of evaporation of the solvent.

[0055]

Examples of the combination of a good solvent and a poor solvent used in preparing the type II free form crystal of compound (I) include methanol (good solvent) and water (poor solvent), methanol (good solvent) and diisopropyl ether (IPE) (poor solvent), methanol (good solvent) and heptane (poor solvent), ethanol (good solvent) and IPE (poor solvent), ethanol (good solvent) and heptane (poor solvent), 1-propanol (good solvent) and water (poor solvent), 1-propanol (good solvent) and IPE (poor solvent), 1-propanol (good solvent) and heptane (poor solvent), 2-propanol (good solvent) and heptane (poor solvent), acetone (good solvent) and IPE (poor solvent), acetone (good solvent) and heptane (poor solvent), dimethyl sulfoxide (DMSO) (good solvent) and water (poor solvent), dimethylacetamide (DMA) (good solvent) and water (poor solvent), tetrahydrofuran (THF) (good solvent) and IPE (poor solvent), and THF (good solvent) and heptane (poor solvent).

Among them, the preferred combination of a good solvent and a poor solvent is ethyl acetate (a good solvent) and n-heptane (a poor solvent), ethanol (a good solvent) and water (a poor solvent), 1-propanol (a good solvent) and water (a poor solvent), or ethanol (a good solvent) and diisopropyl ether (a poor solvent). The amount of the poor solvent is preferably 1 to 20 times (v/v) and more preferably 1 to 10 times (v/v) greater than the amount of the good solvent.

[0056]

In the above-described method, the added amount of the seed crystal may preferably be 0.5% to 30% (w/w), more preferably 1% to 5% (w/w) of the added amount of the compound (I).

[0057]

(Method for producing type I free-form crystal)

In one embodiment of the present disclosure, a free form crystal of type I of compound (I) can be obtained by a method comprising, for example, a step of suspending a crude product of compound (I) in a solvent and optionally adding a seed crystal to obtain a crystal of compound (I) in a solid form.

A seed crystal is optionally added to promote the crystallization of the free-form crystal of type I, and an appropriate amount of the crystal of type I of the compound (I) or a mixed crystal including the crystal of type I may be added as the seed crystal. The crystal of type I of the compound (I) can be obtained by the above-described method without adding the seed crystal. However, by adding the seed crystal, the time for obtaining the compound can be reduced. In addition, the crystallization can be carried out under stirring to reduce the crystallization time and control the particle size.

The seed crystal is added in an amount of 0.5%-30% (w/w) and preferably 1%-10% (w/w) relative to the amount of the compound (I) added.

The temperature can be set accordingly, but it is preferably between 50°C and 65°C.

Compound (I) may precipitate at the above dissolution temperature. However, if it does not precipitate at the dissolution temperature, a free-form crystal of type I can be obtained by cooling to 25°C.

[0058]

Ethyl acetate, ethanol, acetonitrile, acetone, tert-butyl methyl ether, etc. can be used as a solvent.

[0059]

(Method for obtaining type V crystal with 1 equivalent of fumaric acid)

In one embodiment of the present disclosure, a type V crystal of compound (I) with 1 equivalent of fumaric acid can be obtained by a method comprising, for example, the following two steps.

Step 1: a step of adding to the compound (I) 1-2 equivalents of fumaric acid and acetone as a solvent in an amount greater than 40-60 times (v/w) relative to 1 equivalent of the compound (I) in free form, and optionally adding a seed crystal.

Stage 2: stage of stirring the suspension obtained in the above stage, filtering the suspension and isolating the solid.

Step 3: a step of drying the solid obtained in the above step under reduced pressure (preferably at a pressure of 2 kPa or less) at preferably 20°C to 60°C to obtain a crystal of compound (I) with 1 equivalent of fumaric acid.

Fumaric acid in step 1 is more preferably added in an amount of 2 equivalents to 1 equivalent of compound (I) in free form.

In step 1, acetone is preferably added in an amount 40 to 60 times (v/w) (more preferably 50 times (v/w)) greater than the amount of compound (I) in free form.

The reaction temperature in step 2 is preferably from 10°C to 30°C (preferably 25°C).

The reaction time in step 2 is preferably 12 to 96 hours (more preferably 71 hours).

The reaction temperature in step 3 is from 20°C to 60°C (e.g. room temperature) for the obtained solid.

The time of reduced pressure in stage 3 (for example, a pressure of 2 kPa or less) comprises drying for preferably 6-24 hours (more preferably 7.5 hours).

[0060]

In one embodiment of the present disclosure, a type V crystal of compound (I) with 1 equivalent of fumaric acid can be obtained by a method comprising, for example, the following step.

Step 1: Step of drying the Type I crystal with 1 equivalent of fumaric acid under reduced pressure (preferably at a pressure of 2 kPa or less).

The reaction temperature in step 1 is from 20°C to 60°C.

[0061]

(Method for obtaining type I crystal with 1 equivalent of fumaric acid)

In one embodiment of the present disclosure, a type I crystal of compound (I) with 1 equivalent of fumaric acid can be obtained by a method comprising, for example, the following two steps.

Step 1: a step of adding to the compound (I) 1-2 equivalents of fumaric acid and acetone as a solvent in an amount greater than 40-60 times (v/w) relative to 1 equivalent of the compound (I) in free form, and optionally adding a seed crystal.

Step 2: a step of stirring the suspension obtained in the above step at 30°C or less to obtain a crystal of compound (I) with fumaric acid.

After this, the resulting solid substance is isolated by filtration.

Fumaric acid in step 1 is more preferably added in an amount of 2 equivalents to 1 equivalent of compound (I) in free form.

Acetone in step 1 is preferably added in an amount 40 to 60 times (more preferably 50 times) greater than the amount of compound (I) in free form.

The reaction temperature in step 2 is preferably from 10°C to 30°C (preferably 25°C).

The reaction time in step 2 is preferably 12 to 24 hours (more preferably 19.5 hours).

[0062]

(Method for obtaining type III crystal with 1 equivalent of fumaric acid)

In one embodiment of the present disclosure, a type III crystal of compound (I) with 1 equivalent of fumaric acid can be obtained by a method comprising, for example, the following two steps.

Step 1: A step of adding fumaric acid and acetonitrile as a solvent to the compound (I) and optionally adding a seed crystal.

Step 2: A step of stirring the suspension obtained in the above step for a short period of time of 1.5 hours or less to obtain a crystal of compound (I) with 1 equivalent of fumaric acid.

After this, the resulting solid substance is isolated by filtration.

To 1 equivalent of compound (I) in free form in step 1, 1 to 5 equivalents (more preferably 3 equivalents) of fumaric acid are preferably added.

The solvent (e.g. acetonitrile) in step 1 is preferably added in an amount 25 to 35 times (v/w) (more preferably 30 times (v/w)) greater than the amount of compound (I) in free form.

The reaction temperature in step 2 is preferably from 45°C to 55°C (particularly preferably 50°C).

The reaction time in step 2 is preferably 0.5 to 1.5 hours (more preferably 1 hour).

[0063]

(Method for obtaining type IV crystal with 1 equivalent of fumaric acid)

In one embodiment of the present disclosure, a type IV crystal of compound (I) with 1 equivalent of fumaric acid can be obtained by a process comprising, for example, the following two steps.

Step 1: The step of adding fumaric acid and water as a solvent to the compound (I) and optionally adding a seed crystal.

Step 2: A step of stirring the suspension obtained in the above step for a short period of time of 2 hours or less to obtain a crystal of compound (I) with 1 equivalent of fumaric acid.

After this, the resulting solid substance is isolated by filtration.

In step 1, preferably 1-5 equivalents (more preferably 3 equivalents) of fumaric acid are added to 1 equivalent of compound (I) in free form.

Water as a solvent in step 1 is added in an amount preferably 15-25 times (more preferably 20 times) greater than the amount of compound (I) in free form.

The reaction temperature in step 2 is preferably from 45°C to 55°C (particularly preferably 50°C).

The reaction time in step 2 is preferably 1 to 2 hours (more preferably 1.5 hours).

[0064]

(Method for obtaining type I crystal with 0.5 equivalent of fumaric acid)

In one embodiment of the present disclosure, a type I crystal of compound (I) with 0.5 equivalent of fumaric acid can be obtained by a method comprising, for example, the following two steps.

Step 1: The step of adding fumaric acid and water as a solvent to the compound (I) and optionally adding a seed crystal.

Step 2: a step of stirring the suspension obtained in the above step at a temperature preferably of room temperature or higher and lower than the boiling point of the solvent to obtain a crystal.

After this, the resulting solid substance is isolated by filtration and dried.

In step 1, preferably 0.5-1 equivalent (more preferably 0.5-0.75 equivalent and most preferably 0.5 equivalent) of fumaric acid is added relative to 1 equivalent of compound (I) in free form.

Water as a solvent in step 1 is added in an amount preferably 10 to 40 times (v/w) (more preferably 15 to 25 times (v/w) and most preferably 20 times (v/w)) greater than the amount of compound (I) in free form.

The reaction temperature in step 2 is preferably 40°C to 60°C (more preferably 45°C to 55°C and particularly preferably 50°C).

The reaction time in step 2 is preferably 48 to 120 hours (more preferably 72 to 96 hours, and most preferably 93.5 hours).

[0065]

(Method for obtaining type II crystal with 0.5 equivalent of fumaric acid)

In one embodiment of the present disclosure, a type II crystal of compound (I) with 0.5 equivalent of fumaric acid can be obtained by a method comprising, for example, the following two steps.

Step 1: The step of adding fumaric acid and ethanol to the compound (I) and optionally adding a seed crystal.

Step 2: a step of stirring the suspension obtained in the above step at a temperature preferably of room temperature or higher and lower than the boiling point of the solvent to obtain a crystal.

After this, the resulting solid substance is isolated by filtration and dried.

In step 1, 0.5-1 equivalent (more preferably 1 equivalent) of fumaric acid is preferably added to 1 equivalent of compound (I) in free form.

Ethanol as a solvent in step 1 is added in an amount preferably 10 to 40 times (v/w) (more preferably 15 to 25 times (v/w) and most preferably 20 times (v/w)) greater than the amount of compound (I) in free form.

The reaction temperature in step 2 is preferably 40°C to 60°C (more preferably 45°C to 55°C and particularly preferably 50°C).

The reaction time in step 2 is preferably 6 to 48 hours (more preferably 12 to 24 hours, and most preferably 19.5 hours).

[0066]

The type II crystal with 1 equivalent of fumaric acid, the type II free-form crystal, the type I free-form crystal, the type V crystal with 1 equivalent of fumaric acid, the type I crystal with 0.5 equivalent of fumaric acid and the type II crystal with 0.5 equivalent of fumaric acid of the compound (I) have at least one of the advantageous properties in pharmaceutical production such as non-hygroscopic property, reproducible production capability, solid stability and oral absorbability.

Among them, the type II crystal with 1 equivalent of fumaric acid, the type II free-form crystal, the type I free-form crystal and the type V crystal with 1 equivalent of fumaric acid of compound (I) have advantageous properties in pharmaceutical production such as non-hygroscopic property, reproducible production capability, solid stability and oral absorbability, compared with other crystal forms of compound (I).

[0067]

The type II crystal of compound (I) with 1 equivalent of fumaric acid is less hygroscopic and the possibility of obtaining it so that it is stable is lower. It is important for the industrial production of pharmaceutical products that candidate compounds for drug development that are less hygroscopic can be stably obtained. In addition, it has properties that facilitate processing as pharmaceutical products, such as solid stability and solubility, and excellent oral absorption. Therefore, the type II crystal of compound (I) with 1 equivalent of fumaric acid has excellent properties required for a pharmaceutical product or an active pharmaceutical ingredient.

[0068]

The free-form crystal of type II of compound (I) is less hygroscopic. For industrial production of pharmaceutical products, it is important to have stable quality of compounds that are candidates for drug development. In addition, it has properties that facilitate processing as pharmaceutical products, such as solid stability and solubility, and excellent oral absorption. Therefore, the free-form crystal of compound (I) has excellent properties required for a pharmaceutical product or active pharmaceutical ingredient.

[0069]

The free-form crystal of type I of compound (I) is less hygroscopic and has excellent ability to be stably obtained. For industrial production of pharmaceutical products with stable quality, it is also important that the compounds that are candidates for drug development are less hygroscopic and can be stably obtained. Therefore, the crystal of type I of compound (I) has excellent properties required for a pharmaceutical product or an active pharmaceutical ingredient.

[0070]

The type V crystal of compound (I) with 1 equivalent of fumaric acid is less hygroscopic and can be stably obtained. For industrial production of pharmaceutical products with stable quality, it is also important that the compounds that are candidates for drug development are less hygroscopic and can be stably obtained. Therefore, the type V crystal of compound (I) has excellent properties required for a pharmaceutical product or an active pharmaceutical ingredient.

[0071]

The type I crystal of compound (I) with 1 equivalent of fumaric acid has excellent ability to be stably obtained. It is also important for industrial production of pharmaceutical products with stable quality that the compounds that are candidates for drug development can be stably obtained. Therefore, the type I crystal of compound (I) with 1 equivalent of fumaric acid has excellent properties required for a pharmaceutical product or active pharmaceutical ingredient.

Since the type I crystal of compound (I) with 1 equivalent of fumaric acid can be isolated in solid form, it is also useful as an intermediate for obtaining the type V crystal with 1 equivalent of fumaric acid.

[0072]

The type III crystal of compound (I) with 1 equivalent of fumaric acid has excellent ability to be stably obtained. It is also important for industrial production of pharmaceutical products with stable quality that the compounds that are candidates for drug development can be stably obtained. Therefore, the type III crystal of compound (I) with 1 equivalent of fumaric acid has excellent properties required for a pharmaceutical product or active pharmaceutical ingredient.

[0073]

The type IV crystal of compound (I) with 1 equivalent of fumaric acid has excellent ability to be stably obtained. It is also important for industrial production of pharmaceutical products with stable quality that the compounds that are candidates for drug development can be stably obtained. Therefore, the type IV crystal of compound (I) with 1 equivalent of fumaric acid has excellent properties required for a pharmaceutical product or active pharmaceutical ingredient.

[0074]

The type I crystal of compound (I) with 0.5 equivalent of fumaric acid has excellent ability to be stably obtained. It is also important for industrial production of pharmaceutical products with stable quality that the compounds that are candidates for drug development can be stably obtained. Therefore, the type I crystal of compound (I) with 0.5 equivalent of fumaric acid has excellent properties required for a pharmaceutical product or active pharmaceutical ingredient.

[0075]

The type II crystal of compound (I) with 0.5 equivalent of fumaric acid has an excellent ability to be stably obtained. It is also important for industrial production of pharmaceutical products with stable quality that compounds that are candidates for drug development can be stably obtained. Therefore, the type II crystal of compound (I) with 0.5 equivalent of fumaric acid according to the present invention has excellent properties required for a pharmaceutical product or an active pharmaceutical ingredient.

[0076]

(Crystals with other acids)

Another embodiment of the present invention relates to a crystal of compound (I) with hydrochloric acid (type I crystal, type II crystal or type III crystal), a crystal of compound (I) with hydrobromic acid, a crystal of compound (I) with 1 equivalent of L-tartaric acid or a crystal of compound (I) with succinic acid.

The X-ray powder diffraction spectra and simultaneous thermogravimetric-differential thermal analysis (TG-DTA) curves for these crystals are shown in Figs. 19–30.

The characteristic peaks in the powder X-ray diffraction spectra of these crystals are described in Reference Examples 3 to 7. These crystals are crystals having three or more (preferably four or more, and, if present, more preferably 5 or more) peaks selected from said characteristic peaks described in the Reference Examples, and, in particular, they are crystals having all of the characteristic peaks.

In addition, the endothermic peak temperatures determined by simultaneous thermogravimetric-differential thermal analysis for these crystals are also described in the Reference Examples.

[0077]

Regarding the free-form crystals of the compound (I) and the crystals of the compound (I) with an acid (salt crystal or co-crystal), as described above, the precipitated crystal can be separated and purified from a solution in which the crystal is dissolved, mixed with, etc., by known separation and purification methods such as filtration, washing with water, and drying under reduced pressure.

[0078]

The free-form crystals or the acid crystals (salt crystals or co-crystals) of the compound (I) of the present invention have excellent HER2 inhibition activity. Moreover, the free-form crystals or the acid crystals (salt crystals or co-crystals) of the compound (I) of the present invention have excellent HER2 selectivity. Therefore, the free-form crystals or the acid crystals (salt crystals or co-crystals) of the compound (I) of the present invention are useful as an antitumor agent for diseases or malignancies having HER2 overexpression, HER2 gene amplification, HER2 mutation, etc. In addition, the free-form crystals or the acid crystals (salt crystals or co-crystals) of the compound (I) of the present invention are advantageous in that they have few side effects.

As used herein, the term "HER2" includes HER2 from a human or non-human mammal, and is preferably HER2 from a human. Furthermore, the term "HER2" includes isoforms.

Since the free crystals or crystals with acids (salt crystals or co-crystals) of the compound (I) of the present invention have excellent HER2 inhibiting activity, they are useful as a drug for the treatment of HER2-associated disease.

"HER2-associated disease" means a disease in which a reduction in the incidence, or remission, alleviation and/or complete elimination of its symptoms is achieved by eliminating, suppressing and/or inhibiting the function of HER2. Examples of such a disease may include, but are not limited to, malignant tumors. Preferred examples of a disease may include malignant tumors having HER2 overexpression, HER2 gene amplification or HER2 mutation.

[0079]

Free-form crystals or crystals with acids (salt crystals or co-crystals) of the compound (I) of the present invention selectively inhibit wild-type HER2 and mutant HER2 having one or more insertion mutations, point mutations, deletion mutations, etc. in its HER2 domain, such as an insertion mutation in exon 20.

One embodiment of the present invention relates to: free-form crystals or crystals with acids (salt crystals or co-crystals) of compound (I) having an activity of inhibiting wild-type HER2 and mutant HER2 having an insertional mutation YVMA, which is one of the insertional mutations of exon 20, or a salt thereof; or a medicinal product or pharmaceutical composition containing them.

One embodiment of the present invention relates to an inhibitor of wild-type and mutant HER2, including HER2 having a YVMA insertional mutation, etc., wherein the inhibitor comprises free-form crystals or crystals with acids (salt crystals or co-crystals) of compound (I) of the present invention.

[0080]

The human HER2 gene is represented, for example, by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. The wild-type HER2 protein consists of the amino acid sequence indicated, for example, by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. Information on the nucleotide sequence of the human HER2 gene and information on the amino acid sequence of the wild-type HER2 protein can be obtained, for example, from the sequences under accession number NM_004448, NM_001289936, NM_001005862, etc.

[0081]

In some embodiments, free form crystals or acid crystals (salt crystals or co-crystals) of compound (I) of the present invention exhibit inhibitory activity against mutant HER2 comprising one or more of G309A, S310F, R678Q, L755S, L755_T759del, D769H, A775_G776insYVMA, V777L, V842I and R896C, using the amino acid sequence set forth in SEQ ID NO: 2 as a reference. In another embodiment, free form crystals or acid crystals (salt crystals or co-crystals) of compound (I) of the present invention exhibit inhibitory activity against mutant HER2 comprising A775_G776insYVMA, using the amino acid sequence set forth in SEQ ID NO: 2 as a reference.

In some embodiments, free form crystals or acid crystals (salt crystals or co-crystals) of compound (I) of the present invention exhibit inhibitory activity against mutant HER2 comprising one or more of G294A, S295F, R663Q, L740S, L740_T744del, D754H, A760_G761insYVMA, V762L, V827I and R881C, using the amino acid sequence set forth in SEQ ID NO: 4 as a reference. In another embodiment, free form crystals or acid crystals (salt crystals or co-crystals) of compound (I) of the present invention exhibit inhibitory activity against mutant HER2 comprising A760_G761insYVMA, using the amino acid sequence set forth in SEQ ID NO: 4 as a reference.

In some embodiments, free form crystals or acid crystals (salt crystals or co-crystals) of compound (I) of the present invention exhibit inhibitory activity against mutant HER2 comprising one or more of G279A, S280F, R648Q, L725S, L725_T729del, D739H, A745_G746insYVMA, V747L, V812I and R866C, using the amino acid sequence set forth in SEQ ID NO: 6 as a reference. In another embodiment, free form crystals or acid crystals (salt crystals or co-crystals) of compound (I) of the present invention exhibit inhibitory activity against mutant HER2 comprising A745_G746insYVMA, using the amino acid sequence set forth in SEQ ID NO: 6 as a reference.

[0082]

In addition, in several embodiments, with respect to a mutation in a particular HER2 isoform, even when the position of the mutation differs from the amino acid position shown by SEQ ID NO: 2 due to a deletion or insertion of an amino acid(s), it is understood that the mutation is the same as a mutation at a position corresponding to the amino acid position shown by SEQ ID NO: 2. Thus, for example, glycine at position 309 in HER2 shown by SEQ ID NO: 2 corresponds to glycine at position 294 in HER2 consisting of the amino acid sequence set forth by SEQ ID NO: 4. For example, the term "G309A" means that glycine at position 309 in HER2 shown by SEQ ID NO: 2 is replaced with alanine. Since such "G309" is at a position corresponding to the amino acid at position 294 in HER2 consisting of the amino acid sequence set forth in SEQ ID NO: 4, "G294A" in HER2 consisting of the amino acid sequence set forth in SEQ ID NO: 4 corresponds to "G309A" in HER2 shown in SEQ ID NO: 2. In addition, the amino acid position in SEQ ID NO: 2 that corresponds to a specific amino acid in a specific HER2 isoform can be confirmed by multiple alignment using BLAST.

[0083]

List of sequences

SEQ ID NO:1

Accession No: NM_004448

CDS: 262..4029

1 gcttgctccc aatcacagga gaaggaggag gtggaggagg agggctgctt gaggaagtat

61 aagaatgaag ttgtgaagct gagattcccc tccattggga ccggagaaac caggggagcc

121 ccccgggcag ccgcgcgccc cttcccacgg ggccctttac tgcgccgcgc gcccggcccc

181 cacccctcgc agcaccccgc gccccgcgcc ctcccagccg ggtccagccg gagccatggg

241 gccggagccg cagtgagcac catggagctg gcggccttgt gccgctgggg gctcctcctc

301 gccctcttgc cccccggagc cgcgagcacc caagtgtgca ccggcacaga catgaagctg

361 cggctccctg ccagtcccga gacccacctg gacatgctcc gccacctcta ccagggctgc

421 caggtggtgc agggaaacct ggaactcacc tacctgccca ccaatgccag cctgtccttc

481 ctgcaggata tccaggaggt gcagggctac gtgctcatcg ctcacaacca agtgaggcag

541 gtcccactgc agaggctgcg gattgtgcga ggcacccagc tctttgagga caactatgcc

601 ctggccgtgc tagacaatgg agacccgctg aacaatacca cccctgtcac aggggcctcc

661 ccaggaggcc tgcgggagct gcagcttcga agcctcacag agatcttgaa aggaggggtc

721 ttgatccagc ggaaccccca gctctgctac caggacacga ttttgtggaa ggacatcttc

781 cacaagaaca accagctggc tctcacactg atagacacca accgctctcg ggcctgccac

841 ccctgttctc cgatgtgtaa gggctcccgc tgctggggag agagttctga ggattgtcag

901 agcctgacgc gcactgtctg tgccggtggc tgtgcccgct gcaaggggcc actgcccact

961 gactgctgcc atgagcagtg tgctgccggc tgcacggggcc ccaagcactc tgactgcctg

1021 gcctgcctcc acttcaacca cagtggcatc tgtgagctgc actgcccagc cctggtcacc

1081 tacaacacag acacgtttga gtccatgccc aatcccgagg gccggtatac attcggcgcc

1141 agctgtgtga ctgcctgtcc ctacaactac ctttctacgg acgtgggatc ctgcaccctc

1201 gtctgccccc tgcacaacca agaggtgaca gcagaggatg gaacacagcg gtgtgagaag

1261 tgcagcaagc cctgtgcccg agtgtgctat ggtctgggca tggagcactt gcgagaggtg

1321 agggcagtta ccagtgccaa tatccaggag tttgctggct gcaagaagat ctttgggagc

1381 ctggcatttc tgccggagag ctttgatggg gacccagcct ccaacactgc cccgctccag

1441 ccagagcagc tccaagtgtt tgagactctg gaagagatca caggttacct atacatctca

1501 gcatggccgg acagcctgcc tgacctcagc gtcttccaga acctgcaagt aatccgggga

1561 cgaattctgc acaatggcgc ctactcgctg accctgcaag ggctgggcat cagctggctg

1621 gggctgcgct cactgaggga actgggcagt ggactggccc tcatccacca taacacccac

1681 ctctgcttcg tgcacacggt gccctgggac cagctctttc ggaacccgca ccaagctctg

1741 ctccacactg ccaaccggcc agaggacgag tgtgtgggcg agggcctggc ctgccaccag

1801 ctgtgcgccc gagggcactg ctggggtcca gggcccaccc agtgtgtcaa ctgcagccag

1861 ttccttcggg gccaggagtg cgtggaggaa tgccgagtac tgcaggggct ccccagggag

1921 tatgtgaatg ccaggcactg tttgccgtgc caccctgagt gtcagcccca gaatggctca

1981 gtgacctgtt ttggaccgga ggctgaccag tgtgtggcct gtgcccacta taaggaccct

2041 cccttctgcg tggcccgctg ccccagcggt gtgaaacctg acctctccta catgcccatc

2101 tggaagtttc cagatgagga gggcgcatgc cagccttgcc ccatcaactg cacccactcc

2161 tgtgtggacc tggatgacaa gggctgcccc gccgagcaga gagccagccc tctgacgtcc

2221 atcatctctg cggtggttgg cattctgctg gtcgtggtct tgggggtggt ctttgggatc

2281 ctcatcaagc gacggcagca gaagatccgg aagtacacga tgcggagact gctgcaggaa

2341 acggagctgg tggagccgct gacacctagc ggagcgatgc ccaaccaggc gcagatgcgg

2401 atcctgaaag agacggagct gaggaaggtg aaggtgcttg gatctggcgc ttttggcaca

2461 gtctacaagg gcatctggat ccctgatggg gagaatgtga aaattccagt ggccatcaaa

2521 gtgttgaggg aaaacacatc ccccaaagcc aacaaagaaa tcttagacga agcatacgtg

2581 atggctggtg tgggctcccc atatgtctcc cgccttctgg gcatctgcct gacatccacg

2641 gtgcagctgg tgacacagct tatgccctat ggctgcctct tagaccatgt ccgggaaaac

2701 cgcggacgcc tgggctccca ggacctgctg aactggtgta tgcagattgc caaggggatg

2761 agctacctgg aggatgtgcg gctcgtacac agggacttgg ccgctcggaa cgtgctggtc

2821 aagagtccca accatgtcaa aattacagac ttcgggctgg ctcggctgct ggacattgac

2881 gagacagagt accatgcaga tgggggcaag gtgcccatca agtggatggc gctggagtcc

2941 attctccgcc ggcggttcac ccaccagagt gatgtgtgga gttatggtgt gactgtgtgg

3001 gagctgatga cttttggggc caaaccttac gatgggatcc cagcccggga gatccctgac

3061 ctgctggaaa agggggagcg gctgccccag ccccccatct gcaccattga tgtctacatg

3121 atcatggtca aatgttggat gattgactct gaatgtcggc caagattccg ggagttggtg

3181 tctgaattct cccgcatggc cagggacccc cagcgctttg tggtcatcca gaatgaggac

3241 ttgggcccag ccagtccctt ggacagcacc ttctaccgct cactgctgga ggacgatgac

3301 atgggggacc tggtggatgc tgaggagtat ctggtacccc agcagggctt cttctgtcca

3361 gaccctgccc cgggcgctgg gggcatggtc caccacaggc accgcagctc atctaccagg

3421 agtggcggtg gggacctgac actagggctg gagccctctg aagaggaggc ccccaggtct

3481 ccactggcac cctccgaagg ggctggctcc gatgtatttg atggtgacct gggaatgggg

3541 gcagccaagg ggctgcaaag cctccccaca catgacccca gccctctaca gcggtacagt

3601 gaggacccca cagtacccct gccctctgag actgatggct acgttgcccc cctgacctgc

3661 agcccccagc ctgaatatgt gaaccagcca gatgttcggc cccagccccc ttcgccccga

3721 gagggccctc tgcctgctgc ccgacctgct ggtgccactc tggaaaggcc caagactctc

3781 tccccaggga agaatggggt cgtcaaagac gtttttgcct ttgggggtgc cgtggagaac

3841 cccgagtact tgacacccca gggaggagct gcccctcagc cccaccctcc tcctgccttc

3901 agcccagcct tcgacaacct ctattactgg gaccaggacc caccagagcg gggggctcca

3961 cccagcacct tcaaagggac acctacggca gagaacccag agtacctggg tctggacgtg

4021 ccagtgtgaa ccagaaggcc aagtccgcag aagccctgat gtgtcctcag ggagcaggga

4081 aggcctgact tctgctggca tcaagaggtg ggagggccct ccgaccactt ccaggggaac

4141 ctgccatgcc aggaacctgt cctaaggaac cttccttcct gcttgagttc ccagatggct

4201 ggaaggggtc cagcctcgtt ggaagaggaa cagcactggg gagtctttgt ggattctgag

4261 gccctgccca atgagactct agggtccagt ggatgccaca gcccagcttg gccctttcct

4321 tccagatcct gggtactgaa agccttaggg aagctggcct gagaggggaa gcggccctaa

4381 gggagtgtct aagaacaaaa gcgacccatt cagagactgt ccctgaaacc tagtactgcc

4441 ccccatgagg aaggaacagc aatggtgtca gtatccaggc tttgtacaga gtgcttttct

4501 gtttagtttt tacttttttt gttttgtttt tttaaagatg aaataaagac ccagggggag

4561 aatgggtgtt gtatggggag gcaagtgtgg ggggtccttc tccacaccca ctttgtccat

4621 ttgcaaatat attttggaaa acagctaaaa aaaaaaaaaa aaaa

SEQ ID NO:2

Accession No: NM_004448

MELAALCRWG LLLALLPPGA ASTQVCTGTD MKLRLPASPE THLDMLRHLY QGCQVVQGNL 60

ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNG 120

DPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA 180

LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC 240

AAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYT FGASCVTACP 300

YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL REVRAVTSAN 360

IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF ETLEEITGYL YISAWPDSLP 420

DLSVFQNLQV IRGRILHNGA YSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV 480

PWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQEC 540

VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC 600

PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVG 660

ILLVVVLGVV FGILIKRRQQ KIRKYTMRRL LQETELVEPL TPSGAMPNQA QMRILKETEL 720

RKVKVLGSGA FGTVYKGIWI PDGENVKIPV AIKVLRENTS PKANKEILDE AYVMAGVGSP 780

YVSRLLGICL TSTVQLVTQL MPYGCLLDHV RENRGRLGSQ DLLNWCMQIA KGMSYLEDVR 840

LVHRDLAARN VLVKSPNHVK ITDFGLARLL DIDETEYHAD GGKVPIKWMA LESILRRRFT 900

HQSDVWSYGV TVWELMTFGA KPYDGIPARE IPDLLEKGER LPQPPICTID VYMIMVKCWM 960

IDSECRPRFR ELVSEFSRMA RDPQRFVVIQ NEDLGPASPL DSTFYRSLLE DDDMGDLVDA 1020

EEYLVPQQGF FCPDPAPGAG GMVHHRHRSS STRSGGGDLT LGLEPSEEEA PRSPLAPSEG 1080

AGSDVFDGDL GMGAAKGLQS LPTHDPSPLQ RYSEDPTVPL PSETDGYVAP LTCSPQPEYV 1140

NQPDVRPQPP SPREGPLPAA RPAGATLERP KTLSPGKNGV VKDVFAFGGA VENPEYLTPQ 1200GGAAPQPHPP PAFSPAFDNL YYWDQDPPER GAPPSTFKGT PTAENPEYLG LDVPV 1255

SEQ ID NO:3

Accession No: NM_001289936

CDS: 583..4305

1 aagttcctgt gttctttatt ctactctccg ctgaagtcca cacagtttaa attaaagttc

61 ccggattttt gtgggcgcct gccccgcccc tcgtccccct gctgtgtcca tatatcgagg

121 cgatagggtt aagggaaggc ggacgcctga tgggttaatg agcaaactga agtgttttcc

181 atgatctttt ttgagtcgca attgaagtac cacctcccga gggtgattgc ttccccatgc

241 ggggtagaac ctttgctgtc ctgttcacca ctctacctcc agcacagaat ttggcttatg

301 cctactcaat gtgaagatga tgaggatgaa aacctttgtg atgatccact tccacttaat

361 gaatggtggc aaagcaaagc tatattcaag accacatgca aagctactcc ctgagcaaag

421 agtcacagat aaaacggggg caccagtaga atggccagga caaacgcagt gcagcacaga

481 gactcagacc ctggcagcca tgcctgcgca ggcagtgatg agagtgacat gtactgttgt

541 ggacatgcac aaaagtgaga tacttcaaag attccagaag atatgccccg ggggtcctgg

601 aagccacaag tgtgcaccgg cacagacatg aagctgcggc tccctgccag tcccgagacc

661 cacctggaca tgctccgcca cctctaccag ggctgccagg tggtgcaggg aaacctggaa

721 ctcacctacc tgcccaccaa tgccagcctg tccttcctgc aggatatcca ggaggtgcag

781 ggctacgtgc tcatcgctca caaccaagtg aggcaggtcc cactgcagag gctgcggatt

841 gtgcgaggca cccagctctt tgaggacaac tatgccctgg ccgtgctaga caatggagac

901 ccgctgaaca ataccacccc tgtcacaggg gcctccccag gaggcctgcg ggagctgcag

961 cttcgaagcc tcacagagat cttgaaagga ggggtcttga tccagcggaa cccccagctc

1021 tgctaccagg acacgatttt gtggaaggac atcttccaca agaacaacca gctggctctc

1081 acactgatag acaccaaccg ctctcggggcc tgccacccct gttctccgat gtgtaagggc

1141 tcccgctgct ggggagagag ttctgaggat tgtcagagcc tgacgcgcac tgtctgtgcc

1201 ggtggctgtg cccgctgcaa ggggccactg cccactgact gctgccatga gcagtgtgct

1261 gccggctgca cggggccccaa gcactctgac tgcctggcct gcctccactt caaccacagt

1321 ggcatctgtg agctgcactg cccagccctg gtcacctaca acacagacac gtttgagtcc

1381 atgcccaatc ccgagggccg gtatacattc ggcgccagct gtgtgactgc ctgtccctac

1441 aactaccttt ctacggacgt gggatcctgc accctcgtct gccccctgca caaccaagag

1501 gtgacagcag aggatggaac acagcggtgt gagaagtgca gcaagccctg tgcccgagtg

1561 tgctatggtc tgggcatgga gcacttgcga gaggtgaggg cagttaccag tgccaatatc1621 caggagtttg ctggctgcaa gaagatcttt gggagcctgg catttctgcc ggagagcttt

1681 gatggggacc cagcctccaa cactgccccg ctccagccag agcagctcca agtgtttgag

1741 actctggaag agatcacagg ttacctatac atctcagcat ggccggacag cctgcctgac

1801 ctcagcgtct tccagaacct gcaagtaatc cggggacgaa ttctgcacaa tggcgcctac

1861 tcgctgaccc tgcaagggct gggcatcagc tggctggggc tgcgctcact gagggaactg

1921 ggcagtggac tggccctcat ccaccataac acccacctct gcttcgtgca cacggtgccc

1981 tgggaccagc tctttcggaa cccgcaccaa gctctgctcc acactgccaa ccggccagag

2041 gacgagtgtg tgggcgaggg cctggcctgc caccagctgt gcgcccgagg gcactgctgg

2101 ggtccagggc ccacccagtg tgtcaactgc agccagttcc ttcggggcca ggagtgcgtg

2161 gaggaatgcc gagtactgca ggggctcccc agggagtatg tgaatgccag gcactgtttg

2221 ccgtgccacc ctgagtgtca gccccagaat ggctcagtga cctgttttgg accggaggct

2281 gaccagtgtg tggcctgtgc ccactataag gaccctccct tctgcgtggc ccgctgcccc

2341 agcggtgtga aacctgacct ctcctacatg cccatctgga agtttccaga tgaggagggc

2401 gcatgccagc cttgccccat caactgcacc cactcctgtg tggacctgga tgacaagggc

2461 tgccccgccg agcagagagc cagccctctg acgtccatca tctctgcggt ggttggcatt

2521 ctgctggtcg tggtcttggg ggtggtcttt gggatcctca tcaagcgacg gcagcagaag

2581 atccggaagt acacgatgcg gagactgctg caggaaacgg agctggtgga gccgctgaca

2641 cctagcggag cgatgcccaa ccaggcgcag atgcggatcc tgaaagagac ggagctgagg

2701 aaggtgaagg tgcttggatc tggcgctttt ggcacagtct acaagggcat ctggatccct

2761 gatggggaga atgtgaaaat tccagtggcc atcaaagtgt tgagggaaaa cacatccccc

2821 aaagccaaca aagaaatctt agacgaagca tacgtgatgg ctggtgtggg ctccccatat

2881 gtctcccgcc ttctgggcat ctgcctgaca tccacggtgc agctggtgac acagcttatg

2941 ccctatggct gcctcttaga ccatgtccgg gaaaaccgcg gacgcctggg ctcccaggac

3001 ctgctgaact ggtgtatgca gattgccaag gggatgagct acctggagga tgtgcggctc

3061 gtacacaggg acttggccgc tcggaacgtg ctggtcaaga gtcccaacca tgtcaaaatt

3121 acagacttcg ggctggctcg gctgctggac attgacgaga cagagtacca tgcagatggg

3181 ggcaaggtgc ccatcaagtg gatggcgctg gagtccattc tccgccggcg gttcacccac

3241 cagagtgatg tgtggagtta tggtgtgact gtgtgggagc tgatgacttt tggggccaaa

3301 ccttacgatg ggatcccagc ccgggagatc cctgacctgc tggaaaaggg ggagcggctg

3361 ccccagcccc ccatctgcac cattgatgtc tacatgatca tggtcaaatg ttggatgatt

3421 gactctgaat gtcggccaag attccgggag ttggtgtctg aattctcccg catggccagg

3481 gacccccagc gctttgtggt catccagaat gaggacttgg gcccagccag tcccttggac

3541 agcaccttct accgctcact gctggaggac gatgacatgg gggacctggt ggatgctgag

3601 gagtatctgg taccccagca gggcttcttc tgtccagacc ctgccccggg cgctgggggc

3661 atggtccacc acaggcaccg cagctcatct accaggagtg gcggtgggga cctgacacta

3721 gggctggagc cctctgaaga ggaggccccc aggtctccac tggcaccctc cgaaggggct

3781 ggctccgatg tatttgatgg tgacctggga atggggggcag ccaaggggct gcaaagcctc

3841 cccacacatg accccagccc tctacagcgg tacagtgagg accccacagt acccctgccc

3901 tctgagactg atggctacgt tgcccccctg acctgcagcc cccagcctga atatgtgaac

3961 cagccagatg ttcggcccca gcccccttcg ccccgagagg gccctctgcc tgctgcccga

4021 cctgctggtg ccactctgga aaggcccaag actctctccc cagggaagaa tggggtcgtc

4081 aaagacgttt ttgcctttgg gggtgccgtg gagaaccccg agtacttgac accccaggga

4141 ggagctgccc ctcagcccca ccctcctcct gccttcagcc cagccttcga caacctctat

4201 tactgggacc aggacccacc agagcggggg gctccaccca gcaccttcaa agggacacct

4261 acggcagaga acccagagta cctgggtctg gacgtgccag tgtgaaccag aaggccaagt

4321 ccgcagaagc cctgatgtgt cctcagggag cagggaaggc ctgacttctg ctggcatcaa

4381 gaggtgggag ggccctccga ccacttccag gggaacctgc catgccagga acctgtccta

4441 aggaaccttc cttcctgctt gagttcccag atggctggaa ggggtccagc ctcgttggaa

4501 gaggaacagc actggggagt ctttgtggat tctgaggccc tgcccaatga gactctaggg

4561 tccagtggat gccacagccc agcttggccc tttccttcca gatcctgggt actgaaagcc

4621 ttagggaagc tggcctgaga ggggaagcgg ccctaaggga gtgtctaaga acaaaagcga

4681 cccattcaga gactgtccct gaaacctagt actgcccccc atgaggaagg aacagcaatg

4741 gtgtcagtat ccaggctttg tacagagtgc ttttctgttt agtttttact ttttttgttt

4801 tgttttttta aagatgaaat aaagacccag ggggagaatg ggtgttgtat ggggaggcaa

4861 gtgtgggggg tccttctcca cacccacttt gtccatttgc aaatatattt tggaaaacag

4921 ctaaaaaaaa aaaaaaaaaa

SEQ ID NO:4

Accession No: NM_001289936

MPRGSWKPQV CTGTDMKLRL PASPETHLDM LRHLYQGCQV VQGNLELTYL PTNASLSFLQ 60

DIQEVQGYVL IAHNQVRQVP LQRLRIVRGT QLFEDNYALA VLDNGDPLNN TTPVTGASPG 120

GLRELQLRSL TEILKGGVLI QRNPQLCYQD TILWKDIFHK NNQLALTLID TNRSRACHPC 180

SPMCKGSRCW GESSEDCQSL TRTVCAGGCA RCKGPLPTDC CHEQCAAGCT GPKHSDCLAC 240

LHFNHSGICE LHCPALVTYN TDTFESMPNP EGRYTFGASC VTACPYNYLS TDVGSCTLVC 300

PLHNQEVTAE DGTQRCEKCS KPCARVCYGL GMEHLREVRA VTSANIQEFA GCKKIFGSLA 360

FLPESFDGDP ASNTAPLQPE QLQVFETLEE ITGYLYISAW PDSLPDLSVF QNLQVIRGRI 420

LHNGAYSLTL QGLGISWLGL RSLRELGSGL ALIHHNTHLC FVHTVPWDQL FRNPHQALLH 480

TANRPEDECV GEGLACHQLC ARGHCWGPGP TQCVNCSQFL RGQECVEECR VLQGLPREYV 540

NARHCLPCHP ECQPQNGSVT CFGPEADQCV ACAHYKDPPF CVARCPSGVK PDLSYMPIWK 600

FPDEEGACQP CPINCTHSCV DLDDKGCPAE QRASPLTSII SAVVGILLVV VLGVVFGILI 660

KRRQQKIRKY TMRRLLQETE LVEPLTPSGA MPNQAQMRIL KETELRKVKV LGSGAFGTVY 720

KGIWIPDGEN VKIPVAIKVL RENTSPKANK EILDEAYVMA GVGSPYVSRL LGICLTSTVQ 780

LVTQLMPYGC LLDHVRENRG RLGSQDLLNW CMQIAKGMSY LEDVRLVHRD LAARNVLVKS 840

PNHVKITDFG LARLLDIDET EYHADGGKVP IKWMALESIL RRRFTHQSDV WSYGVTVWEL 900

MTFGAKPYDG IPAREIPDLL EKGERLPQPP ICTIDVYMIM VKCWMIDSEC RPRFRELVSE 960

FSRMARDPQR FVVIQNEDLG PASPLDSTFY RSLLEDDDMG DLVDAEEYLV PQQGFFCPDP 1020

APGAGGMVHH RHRSSSTRSG GGDLTLGLEP SEEEAPRSPL APSEGAGSDV FDGDLGMGAA 1080

KGLQSLPTHD PSPLQRYSED PTVPLPSETD GYVAPLTCSP QPEYVNQPDV RPQPPSPREG 1140

PLPAARPAGA TLERPKTLSP GKNGVVKDVF AFGGAVENPE YLTPQGGAAP QPHPPPAFSP 1200

AFDNLYYWDQ DPPERGAPPS TFKGTPTAEN PEYLGLDVPV 1240

SEQ ID NO:5

Accession No: NM_001005862

CDS: 577..4254

1 aagttcctgt gttctttatt ctactctccg ctgaagtcca cacagtttaa attaaagttc

61 ccggattttt gtgggcgcct gccccgcccc tcgtccccct gctgtgtcca tatatcgagg

121 cgatagggtt aagggaaggc ggacgcctga tgggttaatg agcaaactga agtgttttcc

181 atgatctttt ttgagtcgca attgaagtac cacctcccga gggtgattgc ttccccatgc

241 ggggtagaac ctttgctgtc ctgttcacca ctctacctcc agcacagaat ttggcttatg

301 cctactcaat gtgaagatga tgaggatgaa aacctttgtg atgatccact tccacttaat

361 gaatggtggc aaagcaaagc tatattcaag accacatgca aagctactcc ctgagcaaag

421 agtcacagat aaaacggggg caccagtaga atggccagga caaacgcagt gcagcacaga

481 gactcagacc ctggcagcca tgcctgcgca ggcagtgatg agagtgacat gtactgttgt

541 ggacatgcac aaaagtgagt gtgcaccggc acagacatga agctgcggct ccctgccagt

601 cccgagaccc acctggacat gctccgccac ctctaccagg gctgccaggt ggtgcaggga

661 aacctggaac tcacctacct gcccaccaat gccagcctgt ccttcctgca ggatatccag

721 gaggtgcagg gctacgtgct catcgctcac aaccaagtga ggcaggtccc actgcagagg

781 ctgcggattg tgcgaggcac ccagctcttt gaggacaact atgccctggc cgtgctagac

841 aatggagacc cgctgaacaa taccacccct gtcacagggg cctccccagg aggcctgcgg

901 gagctgcagc ttcgaagcct cacagagatc ttgaaaggag gggtcttgat ccagcggaac

961 ccccagctct gctaccagga cacgattttg tggaaggaca tcttccacaa gaacaaccag

1021 ctggctctca cactgataga caccaaccgc tctcgggcct gccacccctg ttctccgatg

1081 tgtaagggct cccgctgctg gggagagagt tctgaggatt gtcagagcct gacgcgcact

1141 gtctgtgccg gtggctgtgc ccgctgcaag gggccactgc ccactgactg ctgccatgag

1201 cagtgtgctg ccggctgcac gggccccaag cactctgact gcctggcctg cctccacttc

1261 aaccacagtg gcatctgtga gctgcactgc ccagccctgg tcacctacaa cacagacacg

1321 tttgagtcca tgcccaatcc cgagggccgg tatacattcg gcgccagctg tgtgactgcc

1381 tgtccctaca actacctttc tacggacgtg ggatcctgca ccctcgtctg ccccctgcac

1441 aaccaagagg tgacagcaga ggatggaaca cagcggtgtg agaagtgcag caagccctgt

1501 gcccgagtgt gctatggtct gggcatggag cacttgcgag aggtgagggc agttaccagt

1561 gccaatatcc aggagtttgc tggctgcaag aagatctttg ggagcctggc atttctgccg

1621 gagagctttg atggggaccc agcctccaac actgccccgc tccagccaga gcagctccaa

1681 gtgtttgaga ctctggaaga gatcacaggt tacctataca tctcagcatg gccggacagc

1741 ctgcctgacc tcagcgtctt ccagaacctg caagtaatcc ggggacgaat tctgcacaat

1801 ggcgcctact cgctgaccct gcaagggctg ggcatcagct ggctggggct gcgctcactg

1861 agggaactgg gcagtggact ggccctcatc caccataaca cccacctctg cttcgtgcac

1921 acggtgccct gggaccagct ctttcggaac ccgcaccaag ctctgctcca cactgccaac

1981 cggccagagg acgagtgtgt gggcgagggc ctggcctgcc accagctgtg cgcccgaggg

2041 cactgctggg gtccaggggcc cacccagtgt gtcaactgca gccagttcct tcggggccag

2101 gagtgcgtgg aggaatgccg agtactgcag gggctcccca gggagtatgt gaatgccagg

2161 cactgtttgc cgtgccaccc tgagtgtcag ccccagaatg gctcagtgac ctgttttgga

2221 ccggaggctg accagtgtgt ggcctgtgcc cactataagg accctccctt ctgcgtggcc

2281 cgctgcccca gcggtgtgaa acctgacctc tcctacatgc ccatctggaa gtttccagat

2341 gaggagggcg catgccagcc ttgccccatc aactgcaccc actcctgtgt ggacctggat

2401 gacaagggct gccccgccga gcagagagcc agccctctga cgtccatcat ctctgcggtg

2461 gttggcattc tgctggtcgt ggtcttgggg gtggtctttg ggatcctcat caagcgacgg

2521 cagcagaaga tccggaagta cacgatgcgg agactgctgc aggaaacgga gctggtggag

2581 ccgctgacac ctagcggagc gatgcccaac caggcgcaga tgcggatcct gaaagagacg

2641 gagctgagga aggtgaaggt gcttggatct ggcgcttttg gcacagtcta caagggcatc

2701 tggatccctg atggggagaa tgtgaaaatt ccagtggcca tcaaagtgtt gagggaaaac

2761 acatccccca aagccaacaa agaaatctta gacgaagcat acgtgatggc tggtgtgggc

2821 tccccatatg tctcccgcct tctgggcatc tgcctgacat ccacggtgca gctggtgaca

2881 cagcttatgc cctatggctg cctcttagac catgtccggg aaaaccgcgg acgcctgggc

2941 tcccaggacc tgctgaactg gtgtatgcag attgccaagg ggatgagcta cctggaggat

3001 gtgcggctcg tacacaggga cttggccgct cggaacgtgc tggtcaagag tcccaaccat

3061 gtcaaaatta cagacttcgg gctggctcgg ctgctggaca ttgacgagac agagtaccat

3121 gcagatgggg gcaaggtgcc catcaagtgg atggcgctgg agtccattct ccgccggcgg

3181 ttcacccacc agagtgatgt gtggagttat ggtgtgactg tgtgggagct gatgactttt

3241 ggggccaaac cttacgatgg gatcccagcc cgggagatcc ctgacctgct ggaaaagggg

3301 gagcggctgc cccagccccc catctgcacc attgatgtct acatgatcat ggtcaaatgt

3361 tggatgattg actctgaatg tcggccaaga ttccgggagt tggtgtctga attctcccgc

3421 atggccaggg acccccagcg ctttgtggtc atccagaatg aggacttggg cccagccagt

3481 cccttggaca gcaccttcta ccgctcactg ctggaggacg atgacatggg ggacctggtg

3541 gatgctgagg agtatctggt accccagcag ggcttcttct gtccagaccc tgccccgggc

3601 gctggggggca tggtccacca caggcaccgc agctcatcta ccaggagtgg cggtggggac

3661 ctgacactag ggctggagcc ctctgaagag gaggccccca ggtctccact ggcaccctcc

3721 gaaggggctg gctccgatgt atttgatggt gacctgggaa tggggggcagc caaggggctg

3781 caaagcctcc ccacacatga ccccagccct ctacagcggt acagtgagga ccccacagta

3841 cccctgccct ctgagactga tggctacgtt gcccccctga cctgcagccc ccagcctgaa

3901 tatgtgaacc agccagatgt tcggccccag cccccttcgc cccgagaggg ccctctgcct

3961 gctgcccgac ctgctggtgc cactctggaa aggcccaaga ctctctcccc agggaagaat

4021 ggggtcgtca aagacgtttt tgcctttggg ggtgccgtgg agaaccccga gtacttgaca

4081 ccccagggag gagctgcccc tcagccccac cctcctcctg ccttcagccc agccttcgac

4141 aacctctatt actgggacca ggacccacca gagcgggggg ctccacccag caccttcaaa

4201 gggacaccta cggcagagaa cccagagtac ctgggtctgg acgtgccagt gtgaaccaga

4261 aggccaagtc cgcagaagcc ctgatgtgtc ctcagggagc agggaaggcc tgacttctgc

4321 tggcatcaag aggtgggagg gccctccgac cacttccagg ggaacctgcc atgccaggaa

4381 cctgtcctaa ggaaccttcc ttcctgcttg agttcccaga tggctggaag gggtccagcc

4441 tcgttggaag aggaacagca ctggggagtc tttgtggatt ctgaggccct gcccaatgag

4501 actctagggt ccagtggatg ccacagccca gcttggccct ttccttccag atcctgggta

4561 ctgaaagcct tagggaagct ggcctgagag gggaagcggc cctaagggag tgtctaagaa

4621 caaaagcgac ccattcagag actgtccctg aaacctagta ctgcccccca tgaggaagga

4681 acagcaatgg tgtcagtatc caggctttgt acagagtgct tttctgttta gtttttactt

4741 tttttgtttt gtttttttaa agatgaaata aagacccagg gggagaatgg gtgttgtatg

4801 gggaggcaag tgtggggggt ccttctccac acccactttg tccatttgca aatatatttt

4861 ggaaaacagc taaaaaaaaa aaaaaaaaa

SEQ ID NO:6

Accession No: NM_001005862

MKLRLPASPE THLDMLRHLY QGCQVVQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ 60

VRQVPLQRLR IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLREL QLRSLTEILK 120

GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSR ACHPCSPMCK GSRCWGESSE 180

DCQSLTRTVC AGGCARCKGP LPTDCCHEQC AAGCTGPKHS DCLACLHFNH SGICELHCPA 240

LVTYNTDTFE SMPNPEGRYT FGASCVTACP YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR 300

CEKCSKPCAR VCYGLGMEHL REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTA 360

PLQPEQLQVF ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGA YSLTLQGLGI 420

SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPH QALLHTANRP EDECVGEGLA 480

CHQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ 540

NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC 600

THSCVDLDDK GCPAEQRASP LTSIISAVVG ILLVVVLGVV FGILIKRRQQ KIRKYTMRRL 660

LQETELVEPL TPSGAMPNQA QMRILKETEL RKVKVLGSGA FGTVYKGIWI PDGENVKIPV 720

AIKVLRENTS PKANKEILDE AYVMAGVGSP YVSRLLGICL TSTVQLVTQL MPYGCLLDHV 780

RENRGRLGSQ DLLNWCMQIA KGMSYLEDVR LVHRDLAARN VLVKSPNHVK ITDFGLARLL 840

DIDETEYHAD GGKVPIKWMA LESILLRRRFT HQSDVWSYGV TVWELMTFGA KPYDGIPARE 900

IPDLLEKGER LPQPPICTID VYMIMVKCWM IDSECRPRFR ELVSEFSRMA RDPQRFVVIQ 960

NEDLGPASPL DSTFYRSLLE DDDMGDLVDA EEYLVPQQGF FCPDPAPGAG GMVHHRHRSS 1020

STRSGGGDLT LGLEPSEEEA PRSPLAPSEG AGSDVFDGDL GMGAAKGLQS LPTHDPSPLQ 1080

RYSEDPTVPL PSETDGYVAP LTCSPQPEYV NQPDVRPQPP SPREGPLPAA RPAGATLERP 1140

KTLSPGKNGV VKDVFAFGGA VENPEYLTPQ GGAAPQPHPP PAFSPAFDNL YYWDQDPPER 1200

GAPPSTFKGT PTAENPEYLG LDVPV 1225

[0084]

One embodiment of the present invention relates to an antitumor agent comprising the above-described crystal of compound (I) (i.e., a free crystal of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)). Furthermore, one embodiment of the present invention relates to a method for treating a tumor, comprising administering an effective amount of the above-described crystal of compound (I) (i.e., a free crystal of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)) to an individual in need thereof. Moreover, one embodiment of the present invention relates to the use of the above-described crystal of compound (I) (i.e., a free crystal of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)) for the manufacture of an antitumor agent. Moreover, one embodiment of the present invention relates to the described crystal of compound (I) (i.e., a free crystal of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)) for use in treating a tumor.

[0085]

The crystals of the present invention can be used in postoperative adjuvant chemotherapy performed to prevent recurrence after surgical removal of a tumor, or can be used in neoadjuvant chemotherapy performed in advance of surgical removal of a tumor.

[0086]

The tumor which is the target of the present invention is not particularly limited. Examples of tumors may include brain tumor, head and neck cancer, gastrointestinal cancer (esophageal cancer, stomach cancer, duodenal cancer, liver cancer, biliary tract cancer (gallbladder cancer and/or bile duct cancer, etc.), pancreatic cancer, colorectal cancer (colon cancer, rectal cancer, etc.), etc.), lung cancer (non-small cell lung cancer, small cell lung cancer, mesothelioma, etc.), breast cancer, genital cancer (ovarian cancer, endometrial cancer (cervical cancer, endometrial cancer, etc.), etc.), genitourinary cancer (kidney cancer, bladder cancer, prostate cancer, testicular cancer, etc.), hematopoietic tumor (leukemia, malignant lymphoma, multiple myeloma, etc.), bone tumor and/or soft tissue, and skin cancer. Among them, lung cancer, breast cancer, stomach cancer, colorectal cancer, bladder cancer, biliary tract cancer or uterine cancer are preferred, and lung cancer, breast cancer, stomach cancer, bladder cancer or biliary tract cancer are more preferred.

[0087]

In one embodiment, the tumor is a brain tumor. The compound of the present invention may be useful for treating brain symptoms that require crossing the blood-brain barrier. The compound of one embodiment has good ability to penetrate the blood-brain barrier for delivery to the brain, namely, excellent brain penetration properties. The concentration of the compound in the brain or the Kp value (the ratio of the concentration of the drug in the brain and plasma) is used as an indicator of the brain penetration properties of the compound.

The brain tumor treated with the compound of the present invention includes a metastatic brain tumor and a primary brain tumor.

Examples of brain tumor may include, but are not limited to, metastatic brain tumor (e.g., brain metastasis from lung cancer, breast cancer, gastric cancer, colorectal cancer, bladder cancer, biliary tract cancer, endometrial cancer, etc. (preferably, lung cancer, breast cancer, or gastric cancer)), pilocytic astrocytoma, diffuse astrocytoma, oligodendroma and/or oligodendroastrocytoma, anaplastic astrocytoma and/or anaplastic oligodendroglioma, anaplastic oligodendroastrocytoma, glioblastoma, ependymoma, anaplastic ependymoma, ganglioglioma, central neurocytoma, medulloblastoma, germinoma, malignant lymphoma of the central nervous system, meningioma, neurilemoma, GH-secreting adenoma pituitary, PRL-secreting pituitary adenoma, ACTH-secreting pituitary adenoma, nonfunctional pituitary adenoma, craniopharyngioma, chordoma, hemanioblastoma, and epidermoid tumor.

[0088]

As used herein, the term "effective amount" of a compound means an amount of a compound of the present invention that induces a biological or medical response in an individual, such as, for example, reducing or inhibiting the activity of an enzyme or protein; or alleviating symptoms, alleviating a condition, and slowing or delaying the progression of a disease, etc. (therapeutically effective amount).

As used herein, the term "individual" includes mammals and non-mammals. Examples of a mammal may include, but are not limited to, a human, a chimpanzee, an ape, a macaque, a bovine, a horse, a sheep, a goat, a pig, a rabbit, a dog, a cat, a rat, a mouse, a guinea pig, a hedgehog, a kangaroo, a mole, a boar, a bear, a tiger, and a lion. Examples of a non-mammal may include, but are not limited to, birds, fish, and reptiles. In one embodiment, the individual is a human, and may be a human diagnosed as being in need of treatment for the symptoms, conditions, or disease described herein.

[0089]

When using the compound (I) or its salt crystal or its co-crystal as a medicine, various types of dosage forms can be used depending on the therapeutic purpose, with or without breaking the crystal. Examples of the dosage form can include all of oral agents such as tablets, capsules, granules, fine granules, powders and dry syrups, suppositories, inhalers, nasal drops, ointments, patches and injections. Pharmaceutical compositions suitable for these dosage forms can be prepared by widely used preparation methods known to those skilled in the art, using pharmaceutically acceptable carriers.

[0090]

One embodiment of the present invention relates to an antitumor agent comprising the above-described crystal of compound (I) (i.e., a free crystal of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)). One embodiment of the present invention also relates to a method for treating a tumor, comprising administering an effective amount of a crystal of compound (I) (i.e., a free crystal of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)) to an individual in need thereof. One embodiment of the present invention also relates to the use of a crystal of compound (I) (i.e., a free crystal of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)) for producing an antitumor agent. One embodiment of the present invention also relates to a crystal of compound (I) (i.e., a free crystal of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)) for use in treating a tumor by administering it.

One embodiment of the present invention relates to an antitumor agent for oral administration comprising the above-described crystal of compound (I) (i.e., a free crystal of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)). One embodiment of the present invention also relates to a method for treating a tumor, comprising orally administering an effective amount of a crystal of compound (I) (i.e., a free crystal of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)) to an individual in need thereof. One embodiment of the present invention also relates to the use of a crystal of compound (I) (i.e., a free crystal of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)) for producing an antitumor agent for oral administration. One embodiment of the present invention also relates to a crystal of compound (I) (i.e. a crystal of the free form of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)) for use in treating a tumor by orally administering it.

[0091]

One embodiment of the present invention relates to a pharmaceutical composition comprising a crystal of compound (I) (i.e. a crystal of the free form of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)). The pharmaceutical composition in one embodiment of the present invention comprises a crystal of compound (I) (i.e. a crystal of the free form of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)) and a pharmaceutically acceptable carrier. One embodiment of the present invention also relates to the use of a crystal of compound (I) (i.e. a crystal of the free form of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)) for the preparation of a pharmaceutical composition. One embodiment of the present invention relates to a crystal of compound (I) (i.e. a crystal of the free form of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)) for use as a medicine. One embodiment of the present invention relates to a kit comprising (i) a pharmaceutical composition comprising a crystal of compound (I) (i.e. a crystal of the free form of compound (I) or a crystal of compound (I) with an acid (a salt crystal or co-crystal)) and (ii) instructions for use of the pharmaceutical composition.

[0092]

As pharmaceutically acceptable carriers, various types of organic or inorganic carriers are used, which are widely used as production materials. When the compound of the present invention is formed into a solid preparation, examples of the pharmaceutically acceptable carrier mixed with the compound of the present invention may include an excipient, a binder, a disintegrant, a lubricant, and a coating agent. When the compound of the present invention is formed into a liquid preparation, examples of the pharmaceutically acceptable carrier mixed with the compound of the present invention may include a solvent, a solubilizer, a suspending agent, an isotonicity agent, a buffer, and an emollient. In addition, additives for the preparation such as an antiseptic, an antioxidant, a colorant, a sweetener, and a stabilizer may also be used as necessary.

[0093]

Examples of the excipient may include lactose, sucrose, D-mannitol, starch, crystalline cellulose, and calcium silicate.

Examples of a binder may include hydroxypropyl cellulose, methylcellulose, polyvinylpyrrolidone, powdered sugar, and hypromellose.

Examples of disintegrating agents may include sodium starch glycolate, calcium carmellose, sodium croscarmellose, crospovidone, low-substituted hydroxypropyl cellulose, and partially pregelatinized starch.

Examples of a lubricant may include talc, magnesium stearate, sucrose fatty acid ester, stearic acid, and sodium stearyl fumarate.

Examples of the coating substance may include ethylcellulose, aminoalkyl methacrylate copolymer RS, hypromellose and sucrose.

Examples of solvent may include water, propylene glycol, and saline.

Examples of the solubilizer may include polyethylene glycol, ethanol, α-cyclodextrin, macrogol 400, and polysorbate 80.

Examples of suspending agents may include carrageenan, crystalline cellulose/carmellose sodium, and polyoxyethylene hydrogenated castor oil.

Examples of isotonicity agents may include sodium chloride, glycerin, and potassium chloride.

Examples of pH adjusting/buffering agent may include sodium citrate, hydrochloric acid, lactic acid, phosphoric acid, and sodium dihydrogen phosphate.

Examples of emollients may include procaine hydrochloride and lidocaine.

Examples of antiseptic may include ethyl paraoxybenzoate, cresol, and benzalkonium chloride.

Examples of antioxidants may include sodium sulfite, ascorbic acid, and natural vitamin E.

Examples of colorant may include titanium oxide, iron sesquioxide, food blue #1, and copper chlorophyll.

Examples of flavor enhancers may include aspartame, saccharin, sucralose, l-menthol, and mint flavor.

Examples of stabilizer may include sodium pyrosulfite, sodium edetate, erythorbic acid, magnesium oxide, and dibutylhydroxytoluene.

[0094]

In the case of producing a solid preparation for oral administration, an excipient and, if necessary, a binder, a disintegrant, a lubricant, a colorant, a corrective agent, etc. are added to a crystal of compound (I) (i.e., a free-form crystal of compound (I) or a crystal of compound (I) with an acid (a salt crystal or a co-crystal)), and then a tablet, a coated tablet, a granule, a powder, a capsule, etc. can be obtained by conventional methods.

In the case of preparing an injection form, a pH adjusting agent, a buffer, a stabilizer, an isotonicity agent, a local anesthetic, etc. are added to a crystal of compound (I) (i.e., a crystal of a free form of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)), and then subcutaneous, intramuscular and intravenous injection forms can be prepared by conventional methods.

[0095]

The amount of the crystal of compound (I) (i.e., a free-form crystal of compound (I) or a crystal of compound (I) with an acid (salt crystal or co-crystal)) to be mixed into each unit dosage form described above depends on the symptoms of a patient to whom the crystal is to be used, the dosage form, etc., and thus the amount of the compound of the present invention is not constant. In general, it is preferable that the dose to be used be selected so as to be about 0.05 to 1,000 mg per unit dosage form in the case of an oral agent, about 0.1 to 500 mg per unit dosage form in the case of an injection form, and about 1 to 1,000 mg per unit dosage form in the case of a suppository or a topical agent, based on the free-form compound (I).

[0096]

The daily dose of the crystal of compound (I) (i.e., the free-form crystal of compound (I) or the crystal of compound (I) with an acid (salt crystal or co-crystal)) in the drug having the above-described dosage form differs depending on the symptoms, body weight, age, sex, etc. of the patient, and thus cannot be determined. However, the crystal can be administered to an adult (body weight: 50 kg) in a daily dose of usually about 0.05 to 5,000 mg, and preferably 0.1 to 1,000 mg, based on the free-form compound (I), and it is preferably administered once a day or in 2 to 3 divided doses.

EXAMPLES

[0097]

The present invention will now be described specifically by way of examples given below. However, these examples are not intended to limit the scope of the present invention. Although the present invention has been fully described by way of examples, it will be understood that various changes and/or modifications can be made by those skilled in the art. Accordingly, such changes and/or modifications are included in the invention unless they depart from the scope of the present invention.

[0098]

In the examples below concerning compounds, "%" indicates percentage by mass unless otherwise stated.

[0099]

The various types of reagents used in the Examples were commercially available products unless otherwise indicated. Silica gel chromatography was performed using Purif-Pack (registered trademark) SI manufactured by MORITEX Corporation, KP-Sil (registered trademark) silica-packed column manufactured by Biotage Japan Ltd., or HP-Sil (registered trademark) silica-packed column manufactured by Biotage Japan Ltd. Silica gel base column chromatography was performed using Purif-Pack (registered trademark) NH manufactured by MORITEX Corporation or KP-NH (registered trademark) packed column manufactured by Biotage Japan Ltd. Preparative thin layer chromatography was performed using Kieselgel TM60F254, art. 5744 manufactured by Merck or NH2 Silicagel 60F254 Plate manufactured by Wako Pure Chemical Industries, Ltd. For spectral analysis using NMR, an AL400 spectrometer (400 MHz; JEOL Ltd. (JEOL)), a Mercury400 type spectrometer (400 MHz; Agilent Technologies) or an Inova400 type spectrometer (400 MHz; Agilent Technologies) equipped with a 400 MNMR probe (Protasis) were used. When tetramethylsilane was present in a heavy solvent, tetramethylsilane was used as an internal standard, and in other cases, the NMR solvent was used as an internal standard for the measurement and the total δ value was reported in ppm.

[0100]

In addition, LCMS spectral measurement was performed using ACQUITY SQD (quadrupole type) manufactured by Waters under the following conditions.

Column: XSelect CSH C18 (4.6 x 150 mm, 3.5 µm) by Waters

MS detection: ESI positive

UV detection: 246 nm

Column temperature: 40°C

Column flow rate: 1.0 ml/min

Mobile phase: water/acetonitrile (0.1% formic acid)

Injection amount: 5 µl

Sample cooling device: 5°C

Sample concentration: 1 mg/ml

Gradient (table 1)

[0101]

[Table 1]

Time (min) Water Acetonitrile 0-0.1 95% 5% 0.1-2.1 95%→5% 5%→95% 2.1-3.1 5% 95%

[0102]

Purification by reversed-phase preparative HPLC was carried out under the following conditions using a preparative system manufactured by WATERS.

Column: A column obtained by combining YMC-Actus Triart C18 (20×50 mm, 5 μm) manufactured by YMC and YMC-Actus Triart C18 (20×10 mm, 5 μm) manufactured by YMC was used.

UV detection: 254 nm

MS detection: ESI positive

Column flow rate: 25 ml/min

Mobile phase: water/acetonitrile (0.1% formic acid)

Injected amount: 0.1-0.5 ml

[0103]

Abbreviations have the following meanings.

c: singlet

d: doublet

t: triplet

dt: double triplet

m: multiplet

DMSO-d6: deuterated dimethyl sulfoxide

CDCl ₃ : deuterated chloroform

THF: tetrahydrofuran

DMA: N,N-dimethylacetamide

NMP: 1-methyl-2-pyrrolidinone

DMSO: dimethyl sulfoxide

[0104]

Analysis using powder X-ray diffraction

Powder X-ray diffraction analysis was carried out under any of the following test conditions after lightly pulverizing an appropriate amount of the test substance in an agate mortar if necessary.

[0105]

Device: EMPYREAN (Method A) manufactured by PANalytical

Reflection method (concentration method)

Target: Cu

X-ray tube current: 40 mA

Voltage in X-ray tube: 45 kV

Scanning range: 2Ɵ=5.0° to 40.0°

Stage: 2Ɵ=0.0131°

Average time/stage: 8.670 sec

Scanning speed: 0.0015°/s

Divergence slit: 1°

Scattering aperture: 2.0 mm

Light receiving slit: 8.0 mm

Device: EMPYREAN (Method B) manufactured by PANalytical

Method of penetration

Target: Cu

X-ray tube current: 40 mA

Voltage in X-ray tube: 45 kV

Scanning range: 2Ɵ=2.0° to 40.0°

Stage: 2Ɵ=0.0066°

Average time/stage: 8.670 sec

Scanning speed: 0.0008°/s

Divergence slit: 1/2°

Scattering aperture: 2.0 mm

Light receiving slot: no

[0106]

The use of the devices, including data processing, was carried out in accordance with the methods and techniques specified in the instructions for each device. The numerical values obtained for different spectra may vary slightly depending on the direction of crystal growth, particle size, analytical conditions, etc. Therefore, these numerical values should not be understood as strictly specific.

[0107]

For simultaneous thermogravimetric-differential thermal analysis (TG-DTA), the analysis was carried out using 2-3 mg of the test substance according to the following test conditions.

Device: TG/DTA7200, manufactured by Hitachi High-Tech Science Corporation

Sample container: made of aluminum

Rate of temperature increase: The temperature was increased from 25°C to 290°C at 10°C/min.

Atmospheric gas: air (200 ml/min)

Control substance: empty container

The use of the devices, including data processing, was carried out in accordance with the methods and techniques specified in the instructions for each device.

[0108]

Synthesis example 1

Synthesis of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound (I))

Synthesis example 1 (1)

Synthesis of tert-butyl (2S,4R)-4-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-methylpyrrolidine-1-carboxylate

Tert-butyl (2S,4S)-4-hydroxy-2-methylpyrrolidine-1-carboxylate (19.0 g) and 4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (13.1 g) were dissolved in THF (190 mL), and then the resulting solution was cooled to 0°C. Thereafter, triphenylphosphine (37.2 g) and diisopropyl azodicarboxylate (28.1 mL) were added to the reaction solution, and then the temperature of the mixture was raised to room temperature and then stirred for 1 hour. Thereafter, the reaction mixture was concentrated under reduced pressure, and then the resulting residue was purified by silica gel chromatography (hexane:ethyl acetate) to obtain the corresponding coupling base. The resulting compound was used in the next reaction without further purification.

The obtained coupling base, THF (114 ml) and ammonia water (114 ml) were added to a pressure-resistant test tube, and then the resulting mixture was stirred at 100°C for 14 hours. After that, the reaction mixture was cooled to room temperature and then poured into water (285 ml). The mixture thus obtained was stirred at room temperature for 5 hours. After that, the precipitated solid was collected by filtration, then washed with water, and then dried to obtain the product of interest (34.5 g).

¹ H-NMR (CDCl ₃ ) δ: 8.27 (s, 1H) 7.15 (s, 1H) 5.55-5.73 (m, 2H) 5.12-5.25 (m, 1H) 3.86-4.18 (m, 2H) 3.43-3.57 (m, 1H) 2.59-2.69 (m, 1H) 1.92-2.03 (m, 1H) 1, 48 (s, 9H) 1.30-1.40 (m, 3H)

ESI-MS m/z 444 (MH ⁺ )

[0109]

Example of synthesis 1(2)

Synthesis of 4-amino-7-((3R,5S)-1-(tert-butoxycarbonyl)-5-methylpyrrolidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxylic acid

The compound of Synthesis Example 1(1) (28.0 g), 10% palladium carbon catalyst (720 mg), NMP (84 mL), methanol (26 mL), and triethylamine (17.6 mL) were added to a pressure-resistant tube, followed by displacement with carbon monoxide, and the resulting mixture was stirred at 100°C for 2 hours. Thereafter, the reaction mixture was cooled to room temperature, then 2 M aqueous sodium hydroxide solution (79 mL) was added thereto, and then the resulting mixture was stirred at 80°C for 2 hours. Thereafter, the reaction mixture was cooled to room temperature, then filtered, and then washed with methanol. Then, methanol in the filtrate was concentrated under reduced pressure. Then, water was added, and the aqueous layer was washed with tert-butyl methyl ether. To the aqueous layer was added 1 M aqueous potassium hydrogen sulfate solution to adjust the pH to approximately 3. The precipitated solid was collected by filtration, then washed with water, and then dried to obtain the product of interest (23.4 g).

^1H -NMR (400 MHz, DMSO-d6) δ: 8.14 (s, 1H) 8.08 (s, 1H) 5.16-4.93 (m, 1H) 4.07-3.79 ( m, 2H) 3.61-3.45 (m, 1H) 2.53 (m, 1H) 2.33-2.02 (m, 1H) 1.42 (s, 9H) 1.29 (d, J=6.1 Hz, 3H) ESI-MS m/z 362 (MH ⁺ )

[0110]

Example of synthesis 1(3)

Synthesis of tert-butyl (2S,4R)-4-(4-amino-6-bromo-5-(((R)-1-phenylethyl)carbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-methylpyrrolidine-1-carboxylate

To the compound of Synthesis Example 1(2) (1.00 g) were added (R)-(+)-1-phenylethylamine (0.503 g), diisopropylethylamine (1.79 g) and N,N-dimethylformamide (10 ml), followed by the addition of HATU (1.58 g). The resulting mixture was stirred at room temperature overnight. Thereafter, ethyl acetate and a saturated aqueous solution of sodium hydrogen carbonate were added to the reaction mixture, and then the resulting mixture was extracted with ethyl acetate. The collected organic layer was washed with water and then with saturated brine. The resulting material was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (hexane:acetone) to give the amide form (1.53 g). The resulting compound was used in the next reaction without further purification.

Chloroform (15 ml) was added to the amide form (1.53 g), and then the resulting mixture was cooled to 0°C. Thereafter, N-bromosuccinimide (0.88 g) was added to the reaction mixture, and then the resulting mixture was stirred at 0°C for 1 hour. Thereafter, the reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel chromatography (hexane:ethyl acetate) to obtain the product of interest (1.39 g).

¹ H-NMR (CDCl ₃ ) δ: 8.21 (s, 1H) 7.42-7.28 (m, 5H) 6.97 (d, J =7.3 Hz, 1H) 5.36-5 .29 (m, 1H) 5.20-5.07 (m, 1H) 4.30 (t, J =10.3 Hz, 1H) 4.04-3.72 (m, 2H) 3.00- 2.86 (m, 1H) 2.38 (dt, J =14.3.6.0 Hz, 1H) 1.63 (d, J =7.0 Hz, 3H) 1.53-1.43 ( m, 12H)

ESI-MS m/z 543.545 (MH ⁺ )

[0111]

Example of synthesis 1(4)

7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-bromo-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide

Chloroform (3 ml) was added to the compound of Synthesis Example 1(3) (600 mg), and then the resulting mixture was cooled to 0°C. Thereafter, trifluoroacetic acid (4.44 g) was added to the reaction mixture, and then the thus obtained mixture was stirred at room temperature for 1 hour. Thereafter, the reaction mixture was concentrated under reduced pressure, and then acetonitrile (5 ml) was added to the residue. The resulting mixture was concentrated again under reduced pressure to obtain an amine form. The resulting compound was used in the next reaction without further purification.

Acetonitrile (3 ml) was added to the obtained amine form, and then the resulting mixture was cooled to 0°C. Thereafter, acryloyl chloride (99.9 mg) and diisopropylethylamine (713 mg) were added, and then the resulting mixture was stirred at 0°C for 1 hour. Thereafter, the reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel chromatography (ethyl acetate:methanol) to obtain the product of interest (281 mg).

¹ H-NMR (CDCl ₃ ) δ: 8.20 (d, J = 7.3 Hz, 1H) 7.42-7.36 (m, 4H) 7.32-7.28 (m, 1H) 7 .00-6.94 (m, 1H) 6.57-6.33 (m, 2H) 5.76-5.66 (m, 1H) 5.36-5.29 (m, 1H) 5.14 -5.08 (m, 1H) 4.71 (t, J =9.9 Hz, 0.7H) 4.42-4.23 (m, 1.6H) 3.83 (t, J =8, 6 Hz, 0.7H) 3.03-2.92 (m, 1H) 2.60-2.57 (m, 0.3H) 2.44-2.40 (m, 0.7H) 1.64 (d, J = 6.6 Hz, 3H) 1.56 (dd, J = 11.7.6, 2Hz, 3H)

ESI-MS m/z 497.499 (MH ⁺ )

[0112]

Example of synthesis 1(5)

Synthesis of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound (I))

To the compound of Synthesis Example 1(4) (65 mg) were added dichlorobis(triphenylphosphine)dipalladium (9.2 mg), copper(I) iodide (5.0 mg), cyclopropylacetylene (13.0 mg), triethylamine (39.7 mg) and N,N-dimethylformamide (1.3 ml), and then the inside of the reaction system was subjected to nitrogen displacement. Thereafter, the mixture was stirred at 70°C for 2.5 hours. Thereafter, ethyl acetate and a saturated aqueous ammonium chloride solution were added to the reaction mixture, and then the resulting mixture was extracted with ethyl acetate. The collected organic layer was washed with water and then with saturated brine. The resulting material was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (chloroform:methanol) to obtain the product of interest (50 mg).

¹ H-NMR (CDCl ₃ ) δ: 8.22 (d, J =5.1 Hz, 1H) 7.82 (d, J =7.3 Hz, 1H) 7.43-7.35 (m, 4H) 7.30 (t, J =6.8 Hz, 1H) 6.58-6.34 (m, 2H) 5.77-5.66 (m, 1H) 5.35-5.20 (m , 2H) 4.54 (t, J =10.1 Hz, 0.7H) 4.35-4.25 (m, 1.6H) 3.88 (t, J =8.8 Hz, 0.7H ) 2.90-2.78 (m, 1H) 2.65-2.56 (m, 0.3H) 2.49-2.40 (m, 0.7H) 1.63 (d, J =7.0 Hz, 3H) 1.56-1.45 (m, 4H) 1.03-0.91 (m , 2H) 0.84-0.69 (m, 2H)

ESI-MS m/z 483 (MH ⁺ )

[0113]

Example 1

Preparation of Type II Crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Compound (I)) with 1 Equivalent of Fumaric Acid

Type II crystals of compound (I) with fumaric acid were obtained by two methods described below in Production Method 1 and Production Method 2. A free-form crystal of type I of compound (I) was synthesized by the same method as in Example 3 described below.

(i) Method of obtaining 1

Fumaric acid (3 equivalents) was added to 30 mg of the free form type I crystal of compound (I), 0.3 ml of tert-butanol was added, the suspension was stirred at 25°C for about 124 hours, and then the solid was collected by filtration, isolated and dried to obtain the crystal of interest.

(ii) Method of obtaining 2

Fumaric acid (1.06 g) and 22 ml of acetone were added to 2.20 g of the free form crystal of type I compound (I), the suspension was stirred at 50°C for about 62 hours, cooled naturally for 30 minutes, and then the solid was collected by filtration and recovered. It was washed with 2-propanol, and then the solid was collected by filtration, recovered and dried to obtain 2.27 g of the crystal of interest.

[0114]

Powder X-ray diffraction spectrum (method A): See Fig. 1. The powder X-ray diffraction spectral data are presented in Table 2.

[Table 2]

Table 2: Powder X-ray diffraction spectral data of type II crystal of compound (I) with 1 equivalent of fumaric acid

Peak position [°2Ɵ] Total intensity (counting units) Peak position [°2Ɵ] Total intensity (counting units) Peak position [°2Ɵ] Total intensity (counting units) 5.5 1065 18.5 1706 27.3 358 6.8 489 19.8 516 27.9 144 8.1 187 20.5 236 28.1 137 9.3 370 22.0 1123 29.0 154 10.8 54 22.6 101 29.9 183 12.1 107 23.3 129 30.9 106 13.4 564 23.8 151 32.0 73 14.0 319 24.5 929 32.9 104 14.6 171 25.1 676 34.3 174 15.3 771 25.5 774 35.1 125 16.3 602 26.2 432 38.2 105 17.1 101 27.2 455 39.1 114

In addition, the characteristic diffraction angles are as follows.

Characteristic diffraction angle (2Ɵ±0.2°):

5.5°, 6.8°, 9.3°, 13.4°, 15.3°, 16.3°, 18.5°, 19.8°, 22.0° and 24.5°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.2.

Endothermic peak (peak value) in the simultaneous thermogravimetric-differential thermal analysis curve: approximately 182°C

[0115]

Example 2

Preparation of a Type II free form crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound (I))

Free-form crystals of type II were prepared by four methods, described below in Preparation Methods 1–4.

(i) Method of obtaining 1

Succinic acid (1 equivalent) and 0.3 ml of 1-propanol/water (1:3 v/v) were added to 15 mg of compound (I) obtained in the same manner as Synthesis Example 1, the mixture was suspended and stirred at 50°C for 4 days, and then the solid was collected by filtration, isolated and dried to obtain a crystal of interest.

(ii) Method of obtaining 2

Succinic acid (1 equivalent) and 1.5 ml of 1-propanol/water (1:3 v/v) were added to 300 mg of the compound (I) obtained in the same manner as Synthesis Example 1, a small amount of the crystals obtained in (i) was added as a seed crystal, the mixture was suspended and stirred at 50°C for 5 hours, then 0.3 ml of 1-propanol was added, the mixture was further suspended and stirred at 50°C for 2 hours, and then 0.3 ml of 1-propanol was added, the mixture was suspended and stirred at 50°C for 17 hours, and then the solid was collected by filtration, recovered and dried to obtain 84.5 mg of a crystal of interest.

(iii) Method of obtaining 3

Succinic acid (1 equivalent) and 3 ml of 1-propanol/water (1:1 v/v) were added to 1000 mg of compound (I) obtained in the same manner as Synthesis Example 1, a small amount of the crystal obtained in (ii) was added as a seed crystal, the mixture was suspended and stirred at 50°C for 17.5 hours, and then the solid was collected by filtration (washing with water during filtration), recovered and dried to obtain 487.6 mg of the crystal of interest.

(iv) Method of obtaining 4

Diisopropyl ether (5 ml) was added to 1000 mg of the compound (I) obtained in the same manner as Synthesis Example 1, a small amount of the crystal obtained in (iii) was added as a seed crystal, and the mixture was suspended and stirred at 50°C. After 42.5 hours, since the solvent had evaporated, 5 ml of diisopropyl ether was added. The mixture was suspended and stirred at 50°C for 2 hours, and then 0.5 ml of ethanol was added. The mixture was further suspended and stirred at 50°C for 2 hours, and then 0.5 ml of ethanol was added. The mixture was further suspended and stirred at 50°C for 19 hours and naturally cooled for 30 minutes, and then the solid was collected by filtration, recovered and dried to obtain 798.8 mg of a crystal of interest.

[0116]

Powder X-ray diffraction spectrum (method A): see Fig. 3. Powder X-ray diffraction spectral data are presented in Table 3.

[Table 3]

Table 3: X-ray powder diffraction spectral data of the free form crystal of type II compound (I)

Peak position [°2Ɵ] Total intensity (counting units) Peak position [°2Ɵ] Total intensity (counting units) Peak position [°2Ɵ] Total intensity (counting units) 8.3 1844 19.4 154 27.6 218 9.7 179 20.3 952 28.3 100 10.2 531 21.0 963 28.6 101 11.5 248 21.6 271 29.7 374 12.5 660 21.9 520 30.9 113 13.6 440 22.5 957 31.7 261 14.8 2512 23.0 1442 32.3 148 15.8 530 23.6 559 34.0 229 16.0 105 23.9 980 34.6 159 16.5 641 24.7 418 35.3 67 16.9 117 25.0 367 36.4 59 17.3 2130 25.5 208 37.0 123 18.0 945 26.2 1551 37.7 60 18.3 269 26.8 323 38.6 53 19.1 844 27.2 771 39.0 55

In addition, the characteristic diffraction angles are as follows.

[0117]

Characteristic diffraction angle (2Ɵ±0.2°):

8.3°, 14.8°, 17.3°, 18.0°, 19.1°, 20.3°, 21.0°, 22.5°, 23.0° and 26.2°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.4.

Endometric peak (peak apex value) on the simultaneous thermogravimetric-differential thermal analysis curve: approximately 182°C.

[0118]

Example 3

Preparation of the free form crystal of type I 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound (I))

The crude product (100 mg) of compound (I) was suspended in ethanol (500 μl) and stirred at 50°C overnight. The solution was cooled to 25°C and the resulting suspension was filtered to obtain a crystal of type I free form of compound (I) (31 mg).

[0119]

Powder X-ray diffraction spectrum (method A): See Fig. 5. The powder X-ray diffraction spectral data are presented in Table 4.

[Table 4]

Table 4: X-ray powder diffraction spectral data of the free form crystal of type I compound (I)

Peak position [°2Ɵ] Total intensity (counting units) Peak position [°2Ɵ] Total intensity (counting units) Peak position [°2Ɵ] Total intensity (counting units) 9.9 623 21.5 481 30.3 208 10.4 278 22.4 437 31.0 72 11.7 1459 23.3 198 31.8 113 12.0 457 23.9 297 32.2 114 13.2 618 24.3 230 32.5 108 14.6 74 24.9 293 33.0 126 15.7 588 25.5 379 33.6 171 16.0 555 26.1 321 34.7 68 17.7 730 27.1 99 35.5 82 18.1 1070 28.0 183 36.9 80 18.8 1539 28.3 545 37.2 55 19.7 120 28.9 391 38.0 50 20.1 382 29.5 168 20.8 2893 29.8 105

In addition, the characteristic diffraction angles are as follows.

Characteristic diffraction angle (2Ɵ±0.2°): 9.9°, 11.7°, 13.2°, 17.7°, 18.1°, 18.8° and 20.8°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.6.

Endothermic peak (peak value) in the simultaneous thermogravimetric-differential thermal analysis curve: approximately 183°C.

[0120]

Example 4

Preparation of Type V Crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Compound (I)) with 1 Equivalent of Fumaric Acid

Fumaric acid (481 mg) and 50 ml of acetone were added to 1000 mg of compound (I) obtained in the same manner as Synthesis Example 1, the mixture was suspended and stirred at 25°C for 71 hours, and then the solid was collected by filtration and recovered. It was dried under room temperature and reduced pressure for 7.5 hours to obtain 695.0 mg of an interesting crystal.

[0121]

Powder X-ray diffraction spectrum (method A): see Fig. 7. The powder X-ray diffraction spectral data are presented in Table 5.

[Table 5]

Table 5: Powder X-ray diffraction spectral data of type V crystal of compound (I) with 1 equivalent of fumaric acid

Peak position [°2Ɵ] Total intensity (counting units) Peak position [°2Ɵ] Total intensity (counting units) Peak position [°2Ɵ] Total intensity (counting units) 6.9 180 18.1 201 27.5 235 8.7 184 18.7 278 28.2 140 9.4 470 20.1 319 29.0 103 10.2 450 21.1 481 31.0 92 11.9 197 21.7 215 32.4 65 13.7 219 23.1 343 33.0 54 14.5 199 23.6 573 34.4 74 15.9 350 24.6 206 17.5 356 26.5 519

In addition, the characteristic diffraction angles are as follows.

Characteristic diffraction angle (2Ɵ±0.2°): 6.9°, 9.4°, 10.2°, 13.7°, 21.1°, 23.6° and 26.5°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.8.

Endothermic peak (peak value) in the simultaneous thermogravimetric-differential thermal analysis curve: approximately 151°C

[0122]

Example 5

Preparation of Type I Crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Compound (I)) with 1 Equivalent of Fumaric Acid

Fumaric acid (481 mg) and 50 ml of acetone were added to 1000 mg of compound (I) obtained in the same manner as Synthesis Example 1, the mixture was suspended and stirred at 25°C for 19.5 hours, and then the solid was collected by filtration and recovered to obtain 868.8 mg of the crystal of interest.

[0123]

Powder X-ray diffraction spectrum (method A): see Fig.9. The powder X-ray diffraction spectral data are presented in Table 6.

[Table 6]

Table 6: Powder X-ray diffraction spectral data of type I crystal of compound (I) with 1 equivalent of fumaric acid

Peak position [°2Ɵ] Total intensity (counting units) Peak position [°2Ɵ] Total intensity (counting units) Peak position [°2Ɵ] Total intensity (counting units) 6.4 637 17.8 129 26.6 931 8.5 145 18.4 290 28.1 112 8.9 115 19.2 145 28.9 164 10.3 461 19.4 151 30.3 70 11.4 321 20.7 938 30.8 75 12.8 279 21.4 195 31.6 77 14.4 172 22.1 146 32.9 138 15.0 393 22.9 451 33.4 61 15.7 326 23.4 690 34.6 57 16.0 422 24.2 157 16.9 206 24.8 146

In addition, the characteristic diffraction angles are as follows.

Characteristic diffraction angle (2Ɵ±0.2°): 6.4°, 10.3°, 12.8°, 15.0°, 20.7°, 23.4° and 26.6°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.10.

Endothermic peak (peak value) on the simultaneous thermogravimetric-differential thermal analysis curve: approximately 153°C

[0124]

Example 6

Preparation of Type I crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound (I)) with 0.5 equivalent of fumaric acid

Fumaric acid (0.5 equivalent) and 0.3 ml of water were added to 15 mg of compound (I) obtained in the same manner as Synthesis Example 1, the mixture was suspended and stirred at 50°C for 93.5 hours, and then the solid was collected by filtration, recovered and dried to obtain an interesting crystal.

[0125]

Powder X-ray diffraction spectrum (method B): see Fig.11.

Characteristic diffraction angle (2Ɵ±0.2°): 6.4°, 7.5°, 9.7°, 11.7°, 15.1°, 19.6° and 23.9°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.12.

Endothermic peak (peak value) in the simultaneous thermogravimetric-differential thermal analysis curve: approximately 161°C

[0126]

Example 7

Preparation of Type II Crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Compound (I)) with 0.5 Equivalent of Fumaric Acid

Fumaric acid (1 equivalent) and 6 ml of ethanol were added to 300 mg of the compound (I) obtained in the same manner as Synthesis Example 1, the mixture was suspended and stirred at 50°C for 19.5 hours and naturally cooled for 3 hours, and then the solid was collected by filtration and recovered. It was washed with water, and then the solid was collected by filtration, recovered and dried to obtain 224.6 mg of the crystal of interest.

[0127]

Powder X-ray diffraction spectrum: see Fig. 13.

Characteristic diffraction angle (2Ɵ±0.2°): 5.4°, 6.4°, 7.3°, 12.8°, 13.4°, 14.7° and 15.4°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.14.

Endothermic peak (peak value) on the simultaneous thermogravimetric-differential thermal analysis curve: approximately 162°C.

[0128]

Example 8

Analysis of the molar ratio of compound (I) and fumaric acid

The molar ratio of compound (I) and fumaric acid and the molar ratio of compound (I) and L-tartaric acid were confirmed by 1H-NMR. The results of measuring the NMR spectrum of the type I crystal of compound (I) with 1 equivalent of fumaric acid, the type II crystal of compound (I) with 1 equivalent of fumaric acid, and the type V crystal of compound (I) with 1 equivalent of fumaric acid are shown below. The results of measuring the NMR spectrum of the free form crystal of compound (I) and the free form II crystal of compound (I) are also shown below.

(Free form crystal of compound (I))

¹ H-NMR(400 MHz, DMSO-d6) δ: 8.32 (bd, J=7.38 Hz, 1H) 8.14 (s, 1H) 7.25-7.50 (m, 5H ) 6.54-6.72 (m, 1H) 6.13-6.23 (m, 1H) 5.65-5.76 (m, 1H) 5.12-5.39 (m, 2H) 4 .33-4.40 (m, 1H) 4.00-4.22 (m, 2H) 2.44-2.70 (m, 2H) 1.73 (tt, J=4.99.8.27 Hz, 1H) 1.51 (d, J=6.88 Hz, 3H) 1.36-1.43 (m, 3H) 0.78-1.03 (m, 4H)

(Free form crystal of compound (I))

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 8.32 (b, J = 7.38 Hz, 1H) 8.14 (s, 1H) 7.27-7.46 (m, 5H) 6.54-6.72 (m, 1H) 6.13-6.23 (m, 1H) 5.65-5.76 (m, 1H) 5.12-5.39 (m, 2H) 4.32-4.40 (m, 1H) 4.00-4.22 (m, 2H) 2.44-2.69 (m, 2H) 1.73 (tt, J = 4.99.8, 27 Hz, 1H) 1.51 (d, J =6.88 Hz, 3H) 1.40-1.43 (m, 3H) 0.78-1.03 (m, 4H)

(Type I crystal of compound (I) with 1 equivalent of fumaric acid)

¹ H-NMR (400 MHz, DMSO-d6) δ: 13.13 (br s, 2H) 8.32 (br d, J=7.38 Hz, 1H) 8.14 (s, 1H) 7 .24-7.48 (m, 5H) 6.46-6.78 (m, 3H) 6.06-6.30 (m, 1H) 5.63-5.77 (m, 1H) 5.11 -5.38 (m, 2H) 4.27-4.44 (m, 1H) 3.98-4.23 (m, 2H) 2.42-2.73 (m, 2H) 1.73 (tt , J=5.02.8.30 Hz, 1H) 1.51 (d, J=6.88 Hz, 3H) 1.32-1.45 (m, 3H) 0.77-1.02 (m, 4H).

(Type II crystal of compound (I) with 1 equivalent of fumaric acid)

¹ H-NMR (400 MHz, DMSO-d6) δ: 13.14 (br s, 2H) 8.32 (br d, J=7.38 Hz, 1H) 8.14 (s, 1H) 7 .25-7.49 (m, 5H) 6.39-6.89 (m, 3H) 6.07-6.28 (m, 1H) 5.62-5.77 (m, 1H) 5.12 -5.39 (m, 2H) 4.27-4.45 (m, 1H) 3.99-4.24 (m, 2H) 2.42-2.75 (m, 2H) 1.74 (tt , J=5.02.8.30 Hz, 1H) 1.51 (d, J=6.88 Hz, 3H) 1.37-1.46 (m, 3H) 0.78-1.03 (m, 4H)

(Type V crystal of compound (I) with 1 equivalent of fumaric acid)

¹ H-NMR (400 MHz, DMSO-d6) δ: 13.13 (br s, 2H) 8.32 (br d, J=7.38 Hz, 1H) 8.14 (s, 1H) 7 .21-7.49 (m, 5H) 6.47-6.80 (m, 3H) 6.09-6.26 (m, 1H) 5.63-5.77 (m, 1H) 5.11 -5.38 (m, 2H) 4.27-4.44 (m, 1H) 3.98-4.23 (m, 2H) 2.42-2.72 (m, 2H) 1.73 (tt , J=4.99,8.27 Hz, 1H) 1.51 (d, J=6.88 Hz, 3H) 1.34-1.46 (m, 3H) 0.77-1.02 (m, 4H)

[0129]

Reference example 1

Preparation of Type III Crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Compound (I)) with 1 Equivalent of Fumaric Acid

Fumaric acid (3 equivalents) and 9 ml of acetonitrile were added to 300 mg of compound (I) obtained in the same manner as Synthesis Example 1, the mixture was suspended and stirred at 50°C for 1 hour and naturally cooled, and then the solid was collected by filtration and recovered. It was dried to obtain 303.7 mg of an interesting crystal.

[0130]

Powder X-ray diffraction spectrum (method B): see Fig.15.

Characteristic diffraction angle (2Ɵ±0.2°): 8.1°, 13.2°, 20.4°, 23.1°, 24.7° and 26.1°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.16.

Exothermic peak (peak apex value) on the simultaneous thermogravimetric-differential thermal analysis curve: approximately 153°C.

[0131]

Reference example 2

Preparation of Type IV Crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Compound (I)) with 1 Equivalent of Fumaric Acid

Fumaric acid (3 equivalents) and 6 ml of water were added to 300 mg of the compound (I) obtained in the same manner as Synthesis Example 1, the mixture was suspended and stirred at 50°C for 1.5 hours and naturally cooled for 3 hours, and then the solid was collected by filtration and recovered. It was washed with water, and then the solid was collected by filtration, recovered and dried to obtain 311.2 mg of the crystal of interest.

[0132]

Powder X-ray diffraction spectrum (method B): see Fig. 17.

Characteristic diffraction angle (2Ɵ±0.2°): 6.0°, 15.7° and 18.8°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.18.

Exothermic peak (peak apex value) on the simultaneous thermogravimetric-differential thermal analysis curve: approximately 130°C.

[0133]

Reference example 3

Preparation of Type I Crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Compound (I)) with Hydrochloric Acid

2 M hydrochloric acid/ethanol solution (15 μL) and 0.3 mL of 2-propanol/heptane (1:3 v/v) mixed liquid were added to 15 mg of compound (I) obtained in the manner of Synthesis Example 1, the mixture was suspended and stirred for 4 days and naturally cooled, and then the solid was collected by filtration and recovered. It was dried to obtain an interesting crystal.

[0134]

Powder X-ray diffraction spectrum (method B): see Fig.19.

Characteristic diffraction angle (2Ɵ±0.2°): 7.0°, 7.4°, 12.6°, 19.0° and 25.4°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.20.

Endothermic peak (peak apex value) on the simultaneous thermogravimetric-differential thermal analysis curve: approximately 51°C and 179°C.

[0135]

Reference example 4

Preparation of Type II Crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Compound (I)) with Hydrochloric Acid

2 M hydrochloric acid/ethanol solution (15 μL) and 0.3 mL of acetone/heptane (1:3 v/v) mixed liquid were added to 15 mg of compound (I) obtained in the same manner as Synthesis Example 1, the mixture was suspended and stirred for 4 days and naturally cooled, and then the solid was collected by filtration and recovered. It was dried to obtain an interesting crystal.

[0136]

Powder X-ray diffraction spectrum (method B): see Fig.21.

Characteristic diffraction angle (2Ɵ±0.2°): 6.3°, 7.4°, 8.0°, 17.6°, 22.0° and 23.6°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.22.

Endothermic peak (peak value) on the simultaneous thermogravimetric-differential thermal analysis curve: approximately 147°C.

[0137]

Reference example 5

Preparation of Type III Crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Compound (I)) with Hydrochloric Acid

2 M hydrochloric acid/ethanol solution (15 μl) and 0.3 ml ethyl acetate were added to 15 mg of compound (I) obtained in the same manner as Synthesis Example 1, the mixture was suspended and stirred for 4 days and naturally cooled, and then the solid was collected by filtration and recovered. It was dried to obtain an interesting crystal

[0138]

Powder X-ray diffraction spectrum (method B): see Fig.23.

Characteristic diffraction angle (2Ɵ±0.2°): 5.3°, 6.5°, 8.1°, 15.3° and 24.2°

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.24.

Endothermic peak (peak value) on the simultaneous thermogravimetric-differential thermal analysis curve: approximately 156°C.

[0139]

Reference example 6

Preparation of a crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound (I)) with hydrobromic acid

Hydrobromic acid (1 equivalent) and 0.3 ml of 2-propanol/heptane (1:3 v/v) mixed liquid were added to 15 mg of compound (I) obtained in the same manner as Synthesis Example 1, the mixture was suspended and stirred for 5 days and naturally cooled, and then the solid was collected by filtration and recovered. It was dried to obtain an interesting crystal.

[0140]

Powder X-ray diffraction spectrum (method B): see Fig.25.

Characteristic diffraction angle (2Ɵ±0.2°): 7.0°, 7.4°, 13.4°, 15.6°, 19.2° and 25.6°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.26.

Endothermic peak (peak value) on the simultaneous thermogravimetric-differential thermal analysis curve: approximately 204°C.

[0141]

Reference example 7

Preparation of Type IV Crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (Compound (I)) with 1 Equivalent of L-tartaric Acid

L-tartaric acid (466 mg) and 10 ml of acetone were added to 500 mg of compound (I) obtained in the manner of Synthesis Example 1, and the mixture was stirred at 50°C for 40 minutes to dissolve. After that, the dissolved mixture was stirred at room temperature for 17 hours, the suspension was filtered, and the resulting solid was recovered. It was dried to obtain 506.3 mg of an interesting crystal.

[0142]

Powder X-ray diffraction spectrum (method A): see Fig.27.

Characteristic diffraction angle (2Ɵ±0.2°): 7.2°, 12.6°, 17.3° and 23.4°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.28.

Endothermic peak (peak value) on the simultaneous thermogravimetric-differential thermal analysis curve: approximately 121°C.

[0143]

Reference example 8

Preparation of a crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (compound (I)) with succinic acid

Succinic acid (3 equivalents) and 0.3 ml of acetonitrile were added to 15 mg of compound (I) obtained in the same manner as Synthesis Example 1, the mixture was suspended and stirred at 50°C for 3 days and naturally cooled, and then the solid was collected by filtration and recovered. It was dried to obtain an interesting crystal.

[0144]

Powder X-ray diffraction spectrum (method B): see Fig.29.

Characteristic diffraction angle (2Ɵ±0.2°): 6.5°, 10.7°, 13.2°, 14.6°, 18.7° and 23.9°.

Simultaneous thermogravimetric-differential thermal analysis curve: see Fig.30.

Endothermic peak (peak value) on the simultaneous thermogravimetric-differential thermal analysis curve: approximately 148°C and 159°C.

[0145]

Test example 1

Measuring the inhibitory effect ( in vitro ) on HER2 phosphorylation activity

To determine the conditions of the method for measuring the activity of a compoundin vitroo in relation to inhibition of HER2 phosphorylation activity, based on a report concerning the HER2 kinase reaction, using as a peptide substrate a peptide having the same sequence (5-FAM-EEPLYWSFPAKKK-CONH₂), as well as ProfilerPro Peptide 22 from PerkinElmer (Xie H et al., PLoS One.2011; 6(7): e21487), ProfilerPro Peptide 22 was used as a substrate. The purified recombinant human HER2 protein in the present test was purchased from Carna Biosciences, Inc. In determining the inhibitory activity of the compound, first, the compound (I) obtained by the method of Synthesis Example 1 was diluted gradually with dimethyl sulfoxide (DMSO). Then, HER2 protein, peptide substrate (final concentration: 1 μM), manganese chloride (final concentration: 10 mM), ATP (final concentration: 5 μM), and compound (I) in DMSO solution (DMSO final concentration: 5%) were added to the kinase reaction buffer (13.5 mM Tris (pH 7.5), 2 mM dithiothreitol, and 0.009% Tween 20), and then the resulting mixture was incubated at 25°C for 30 minutes to allow the kinase reaction to proceed. EDTA was added to the reaction solution to a final concentration of 30 mM to terminate the reaction. Finally, using LabChip (registered trademark) EZ Reader II (PerkinElmer), the non-phosphorylated peptide substrate (S) and phosphorylated peptide (P) were separated and detected by microchannel capillary electrophoresis. From the peak heights of S and P, the phosphorylation amount was determined, and the concentration of the compound capable of inhibiting the phosphorylation reaction by 50% was determined as the IC50 value (nM). The results are shown in Table 1.

[0146]

Test example 2

Determination of inhibitory action ( in vitro ) against the phosphorylation activity of the insertional mutant of exon 20 HER2 (HER2ex20insYVMA)

To determine the conditions for the method for determining the in vitro activity of a compound in relation to the inhibition of phosphorylation activity of HER2 with an insertion mutation in exon 20, as in the case of HER2, ProfilerPro Peptide 22 was used as a substrate. Purified recombinant human HER2 protein with an insertion mutation in exon 20 (A775_G776insYVMA) was purchased from SignalChem. In determining the inhibition activity of the compound, first, the compound (I) obtained by the method of Synthesis Example 1 was diluted gradually with dimethyl sulfoxide (DMSO). Then, the HER2 protein with an insertion mutation in exon 20 and compound (I) in DMSO solution (final concentration of DMSO: 5%) were added to the kinase reaction buffer (13.5 mM Tris (pH 7.5), 2 mM dithiothreitol, and 0.009% Tween 20), and then the resulting mixture was preincubated at 25°C for 30 minutes. Thereafter, the peptide substrate (final concentration: 1 μM), manganese chloride (final concentration: 25 mM), magnesium chloride (final concentration: 20 mM), and ATP (final concentration: 200 μM) were added to the reaction mixture, and then the thus obtained mixture was incubated at 25°C for 220 minutes so that the kinase reaction occurred. To this reaction solution, EDTA was added to a final concentration of 30 mM to terminate the reaction. Finally, using a LabChip (registered trademark) EZ Reader II (PerkinElmer), the non-phosphorylated peptide substrate (S) and phosphorylated peptide (P) were separated and detected by microchannel capillary electrophoresis. From the peak heights of S and P, the phosphorylation amount was determined, and the concentration of the compound capable of inhibiting the phosphorylation reaction by 50% was determined as the IC50 value (nM). The results are shown in Table 7.

[0147]

[Table 7]

HER2 inhibitory activity,
IC50 value (nM) HER2ex20insYVMA inhibitory activity,
IC50 value (nM) 3.2 < 0.30

[0148]

From the above results, it was found that compound (I) has excellent activity in inhibiting HER2 phosphorylation and HER2 phosphorylation with insertion mutation in exon 20.

[0149]

Test example 3

Determination of growth inhibitory activity of HER2-expressing cell line

SK-BR-3 cells as a HER2-overexpressing human breast cancer cell line were suspended in McCoy's 5a medium (manufactured by Life Technologies) supplemented with 10% fetal bovine serum. The cell suspension was seeded into each well of a 384-well flat-bottomed microplate and then cultured in a 5% carbon dioxide gas-containing container at 37°C for 1 day. Thereafter, the compound (I) obtained by the method of Synthesis Example 1 was dissolved in DMSO, and the compound was diluted to a 500-fold final concentration in DMSO. The compound in the DMSO solution was diluted with the DMSO solution or the medium used in the cell suspension, and then the resulting solution was added to each well of the culture plate so that the final concentration of DMSO became 0.2%. The resulting mixture was then cultured in a culture vessel containing 5% carbon dioxide gas at 37°C for 3 days. After completion of the 3-day culture in the presence of the compound, the cells were counted using CellTiter-Glo 2.0 (manufactured by Promega), and then the growth inhibition percentage was calculated using the following equation. The concentration of the compound that inhibited cell growth by 50% was defined as _IC50 (nM).

Percent growth inhibition (%) = (C-T)/(C)×100

T: Emission intensity in the well to which the test compound was added

C: Emission intensity in the well to which the test compound was not added

The results are shown in Table 8 below.

[0150]

Test example 4

Determination of growth inhibitory activity of a cell line expressing HER2 with an insertional mutation in exon 20

The growth inhibitory activity against HER2 with an insertion mutation in exon 20 was determined using Ba/F3 cells, which are a murine B lymphocyte progenitor cell line that contains human HER2 with an insertion mutation in exon 20. Ba/F3 cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin (Thermo Fisher Scientific), and 1 ng/ml mouse interleukin-3 (mIL-3) (CST). Subsequently, the pCDNA3.1-hyg(+) vector, which incorporated the human HER2 gene with an insertion mutation in exon 20 (A775_G776insYVMA (HER2ex20insYVMA)), internal ribosome entry sequence (IRES) and Kusabira orange gene, was introduced into Ba/F3 cells by electroporation using the Cell Line Nucleofector (registered trademark) Kit V from Amaxa (registered trademark). Ba/F3 cells expressing HER2 with an insertion mutation in exon 20 (Ba/F3-HER2insYVMA), which were selected with hygromycin B (Nacalai Tesque), demonstrated mIL-3-independent growth.

To evaluate the cell growth inhibitory activity, Ba/F3-HER2insYVMA cells were suspended in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cell suspension was seeded into each well of a 96-well flat-bottomed microplate and then cultured in a 5% carbon dioxide gas-containing container at 37°C for 1 day. Compound (I) obtained in Synthesis Example 1 was dissolved in DMSO and then diluted with DMSO or the medium used to suspend the cells. Then, the resulting solution was added to each well of the culture plate so that the final concentration of DMSO became 0.2%. The resulting mixture was then cultured in a culture vessel containing 5% carbon dioxide gas at 37°C for 3 days. After completion of the 3-day culture in the presence of the compound, the cells were counted using CellTiter-Glo 2.0 (manufactured by Promega), and then the growth inhibition percentage was calculated using the following equation. The concentration of the compound that inhibited cell growth by 50% was defined as IC50 (nM).

Percent growth inhibition (%) = (C-T)/(C)×100

T: Emission intensity in the well to which the test compound was added

C: Emission intensity in the well to which the test compound was not added

The results are shown in Table 8 below.

[0151]

[Table 8]

Cell growth inhibitory activity of SK-BR-3
IC50 value (nM) HER2ex20insYVMA cell growth inhibitory activity
IC50 value (nM) 6.6 29

[0152]

From the above results, it was found that compound (I) has excellent cell growth inhibitory activity equally against the HER2-expressing cell line (SK-BR-3) and the HER2-exon 20 insertion mutation-expressing cell line (Ba/F3-HER2insYVMA).

[0153]

Test example 5

Determination of growth inhibitory activity of HER2 expressing cell line (NCI-N87)

NCI-N87 cells as a HER2-overexpressing human gastric cancer cell line (American Type Culture Collection, catalog number ATCC (registered trademark) CRL-5822) were suspended in RPMI1640 medium (Wako Pure Chemical Industries, Ltd.) supplemented with 10% fetal bovine serum. Then, the cell suspension was seeded in each well of a 96-well flat-bottomed microplate and then cultured in a 5% carbon dioxide gas-containing container at 37°C for 1 day. Thereafter, the compound (I) obtained in the manner of Synthesis Example 1 was dissolved in DMSO, and the compound was diluted to 1,000-fold final concentration in DMSO. Compound (I) in DMSO solution was diluted with the medium used for cell suspension, and then the resulting solution was added to each well of the culture plate so that the final concentration of DMSO became 0.1%. As for the control well, DMSO was diluted with the medium used for cell suspension, and then the resulting solution was added to each well of the culture plate so that the final concentration of DMSO became 0.1%. After adding the drug solution, the resulting mixture was cultured in a container containing 5% carbon dioxide gas at 37°C for 3 days. After completion of the culture for 3 days in the presence of the compound, the cells were counted using CellTiter-Glo 2.0 (manufactured by Promega), and then the growth inhibition percentage was calculated according to the following equation. The concentration of the compound at which cell growth was inhibited by 50% was defined as _IC50 (nM).

Percent growth inhibition (%) = (C-T)/(C)×100

T: Emission intensity in the well to which the test compound was added

C: Emission intensity in the well to which the test compound was not added

The results are shown in Table 9 below.

[0154]

[Table 9]

NCI-N87 cell growth inhibitory activity
IC50 value (nM) 9.9

[0155]

From the above results, it was found that compound (I) has excellent cell growth inhibitory activity against HER2-overexpressing cell line (NCI-N87).

[0156]

Test example 6

Evaluation of oral absorption

The compound (I) obtained by the method of Synthesis Example 1 was suspended or dissolved in 0.5% HPMC aqueous solution and 0.1 N hydrochloric acid, and the resulting suspension or solution was orally administered to BALB/cA mice (CLEA Japan, Inc.) at a dose of 50 mg/kg/day. At 0.5, 1, 2, 4 and 6 hours after completion of the oral administration, blood was collected from the facial vein gradually to obtain plasma. The concentration of the compound in the obtained plasma was determined by LC-MS/MS, and the absorbability of the compound of the present invention in the oral cavity was evaluated.

The results are shown in Table 10 below.

[0157]

[Table 10]

AUC 0-6 h (μM ∙ h) 31

[0158]

From the above results, it was found that the compound of the present invention was contained in sufficient concentration in plasma, so that the compound (I) showed good absorbability in the oral cavity.

[0159]

Test example 7

Evaluation of brain penetration properties

The compound (I) obtained by the method of Synthesis Example 1 was suspended or dissolved in 0.5% HPMC aqueous solution and 0.1 N hydrochloric acid, and the resulting suspension or solution was orally administered to BALB/cA mice (CLEA Japan, Inc.) at a dose of 50 mg/kg/day. After 0.5 hour from the completion of the oral administration, blood was collected from the facial vein, and then the whole brain was removed to obtain plasma and brain samples. Water was added to the obtained brain sample in an amount of 3 times the volume of the brain sample, and then the resulting material was homogenized using an ultrasonic homogenizer to obtain a brain homogenate. The concentration of the compound in the obtained plasma and brain homogenate was determined by LC-MS/MS, and the brain penetration property of the compound of the present invention was estimated from the concentration of the compound in the brain/plasma.

The results are shown in Table 11 below.

[0160]

[Table 11]

Plasma concentration of the compound
(µM) Concentration of the compound in the brain
(µM) The value of Kp
(Compound concentration in brain/plasma) 12 2.7 0.23

[0161]

From the above results, it was found that compound (I) showed favorable brain penetration properties.

[0162]

Test example 8

Test to confirm antitumor effect ( in vivo ) in direct brain transplantation models in which a HER2-expressing cell line with an introduced luciferase gene (NCI-N87-luc) was directly transplanted

To confirm the antitumor effects of the test compound in the direct brain transplantation model, NCI-N87-Luc, which was obtained by introducing the luciferase gene into NCI-N87, which is a human gastric cancer tumor cell line purchased from the American Type Culture Collection, was used. NCI-N87-Luc was added to 10% fetal bovine serum (FBS)-containing RPMI-1640 medium (supplemented with 4.5 g/L glucose, 10 mM HEPES, and 1 mM sodium pyruvate) (Wako Pure Chemical Industries, Ltd.), and then the cell line was cultured in a 5% CO2 incubator at 37°C.

NCI-N87-Luc cells were resuspended in PBS at a concentration of 6.25× ¹⁰⁷ cells/ml.

Using a mouse ear fixator, 6-7-week-old nude mice (BALB/cAJcl-nu/nu, CLEA Japan, Inc.) were fixed in a brain stereotaxic device, and the skin over the upper part of the brain was disinfected with alcohol-soaked cotton wool and then cut with a surgical knife.

A microdrill was used to drill a hole into the skull, and then using a needle, manipulator and syringe pump, 4 μl of the cell suspension was transplanted into the brain at a rate of 0.8 μl/min.

As a reference for brain tumor size, the total flux (photon/s) was determined in all surviving animals approximately 3 weeks after transplantation using IVIS (PerkinElmer, Inc., Model: Lumina II). Based on the results obtained, 6 animals were assigned to each group using the MiSTAT group assignment program (Ver. 2.00).

The test compound was administered orally to mice once daily for 21 days, starting on the day following group assignment (days 1–21).

To determine the presence or absence of effects, the value (Log10) obtained by logarithmic transformation of the total flux on the determination date was used. Compound (I) obtained by the method of Synthesis Example 1 was administered to mice at a dose of 25 mg/kg/day as a test compound.

A graph was plotted with the log10 transformed mean total flux on the vertical axis and the number of days (Days) after transplantation on the horizontal axis. The change in total flux over time during drug administration was observed.

0.1 N HCl and 0.5% aqueous HPMC solution were used as controls.

[0163]

The results are shown in Fig. 31 and Fig. 32 below. The value obtained by logarithmic transformation (Log10) of the total flux on day 22 in each group was analyzed by significance test. As a result, it was demonstrated that the above value for the compound was statistically significantly lower than that of the control group (significance level (both sides): 5%). An electronic animal scale was used to measure the body weight. The percentage change in body weight (body weight change; BWCn) relative to the body weight on day n (BWn) was calculated by the following equation:

BWCn (%) = [(body weight on day n) - (body weight on day of distribution into groups)] / [(body weight on day of distribution into groups)] × 100.

From the results of this test, it was found that compound (I) had excellent antitumor effects against the HER2 overexpressing cell line (NCI-N87-luc) transplanted into nude mice. Moreover, none of the mice administered showed a decrease in body weight of -20% or more. Thus, it was found that there were no serious side effects.

[0164]

Test example 9

Solid Stability Test

The free-form type II crystals of compound (I) and its type II crystals with 1 equivalent of fumaric acid obtained according to Examples and Comparative Examples were evaluated for solid state stability after storage for 4 weeks.

Storage conditions: 60°C (closed system)

Shelf life: 4 weeks

Stored quantity: approximately 30 mg

Storage container: glass bottle

The results are shown in Table 12 below.

[Table 12]

Type II free form crystal Type II crystal with 1 equivalent of fumaric acid Chemical purity before storage (%) 99.87 99.85 Chemical purity after storage (%) 99.88 99.85 Amount of related substances before storage (%) 0.13 0.15 Amount of related substances after storage (%) 0.12 0.15

Approximately 1 mg of the sample was weighed, dissolved in approximately 1 ml of a liquid mixture of water/acetonitrile (1:1), and 5 μl of this solution was accurately measured. The changes in the amount of related substances (the amount of detected substances other than compound (I)) were analyzed using HPLC by the following method.

HPLC Analysis Method (Stability Test)

The amount of related substances in the sample solution was measured by HPLC analysis. The use of the devices, including data processing, followed the methods and techniques specified in the instructions for each device.

Column: XSelect CSH C18 (4.6 x 150 mm, 3.5 µm) by Waters

UV detection: 246 nm

Column temperature: 40°C

Flow rate: 1.0 ml/min

Sample cooling device: 5°C

Sample concentration: 1 mg/ml

Mobile phase A: liquid mixture of 5 mmol/L ammonium formate buffer (pH 6.5)/acetonitrile (9:1)

Mobile phase B: acetonitrile

The gradients are presented in Table 13.

[Table 13]

Time (min) Mobile phase A (vol.%) Mobile phase B (vol.%) 0-31 90→30 10→70 31-36 30 70 36-37 30→90 70→10

As a result, no change in the powder X-ray diffraction pattern was observed in either the free-form type II crystal of compound (I) or the type II crystal of the said compound with 1 equivalent of fumaric acid, and virtually no increase in the level of related substances was observed. They were found to be extremely stable crystals.

[0165]

Test Example 10

Dynamic Vapor Sorption (DVS) Test

Dynamic vapor sorption tests were performed using a type II crystal of compound (I) with 1 equivalent of fumaric acid, a free-form type II crystal of said compound, a free-form type I crystal of said compound, a type V crystal of said compound with 1 equivalent of fumaric acid, and a crystal of said compound with 1 equivalent of L-tartaric acid, prepared according to the examples and reference examples.

The dynamic vapor sorption test was carried out to measure under the following conditions.

Approximately 10 mg of each sample was placed in a dedicated quartz cassette and the sample mass at each humidity was continuously measured and recorded under the following conditions. The use of the devices, including data processing, followed the methods and techniques specified in the instructions for each device.

Device: VTI SA+ (manufactured by TA Instruments)

Drying temperature: 60°C

Temperature increase rate: 1°C/min

Drying equilibrium: It was confirmed that a decrease of 0.01 wt% in 5 minutes did not occur within a range not exceeding 300 minutes.

Temperature measurement: 25°C

Hydration equilibrium: It was confirmed that an increase of 0.01 wt% in 5 minutes did not occur within a range not exceeding 120 minutes.

Relative Humidity Program: Increase by 5% RH from 5% RH to 95% RH and decrease by 5% RH from 95% RH to 5% RH

The mass changes over the range of measurement conditions obtained in these tests are presented in Tables 14–18.

[Table 14]

Table 14: Dynamic Vapour Sorption Test Results for Type II Crystal of Compound (I) with 1 Equivalent of Fumaric Acid

Rate of mass change (%) Relative humidity (%) Adsorption Desorption 5 0.01 0.04 20 0.11 0.13 40 0.18 0.20 60 0.27 0.30 80 0.34 0.34 95 0.38 0.38

[Table 15]

Table 15: Dynamic Vapour Sorption Test Results for Type II Free Form Crystal of Compound (I)

Rate of mass change (%) Relative humidity (%) Adsorption Desorption 5 0.01 0.01 20 0.02 0.03 40 0.05 0.06 60 0.08 0.08 80 0.10 0.11 95 0.12 0.12

[Table 16]

Table 16: Dynamic vapor sorption test results for the free form crystal of type I compound (I)

Rate of mass change (%) Relative humidity (%) Adsorption Desorption 5 0.01 0.01 20 0.04 0.04 40 0.10 0.13 60 0.26 0.60 80 0.50 0.69 95 0.72 0.72

[Table 17]

Table 17: Results of the dynamic vapor sorption test for type V crystal of compound (I) with 1 equivalent of fumaric acid

Rate of mass change (%) Relative humidity (%) Adsorption Desorption 5 0.01 0.06 20 0.11 0.16 40 0.21 0.27 60 0.33 0.39 80 0.47 0.51 95 0.59 0.59

[Table 18]

Table 18: Results of the dynamic vapor sorption test for a crystal of compound (I) with 1 equivalent of L-tartaric acid

Rate of mass change (%) Relative humidity (%) Adsorption Desorption 5 0.50 0.54 20 1.05 1.16 40 1.60 1.67 60 2.16 2.38 80 3.26 3.64 95 6.41 6.41

[0166]

As shown in Table 14, the type II crystal of compound (I) with 1 equivalent of fumaric acid had a mass increase of 0.38% in the dynamic vapor sorption test at 95% relative humidity, which was less than 1%. It was determined that the crystal was substantially non-hygroscopic. Similarly, the type II free-form crystal of compound (I), type I free-form crystal of the compound and type V crystal of the compound with 1 equivalent of fumaric acid also had a mass increase of less than 1% in the dynamic vapor sorption test at 95% relative humidity, which was less than 1%. It was determined that the crystals were substantially non-hygroscopic (Tables 15-17). Meanwhile, as shown in Table 18, the crystal of compound (I) with 1 equivalent of L-tartaric acid had a mass increase of as much as 6.41% in the dynamic vapor sorption test at 95% relative humidity, which was less than 1%. The crystal was found to be highly hygroscopic.

Therefore, since the type II crystal of compound (I) with 1 equivalent of fumaric acid, the type II free crystal of said compound, the type I free crystal of said compound and the type V crystal with 1 equivalent of fumaric acid are less hygroscopic than the crystal of compound (I) with 1 equivalent of L-tartaric acid, they can be regarded as excellent candidate compounds for drug development from the viewpoint of industrial production of pharmaceutical products with stable quality. It was confirmed that the type II crystal of compound (I) with 1 equivalent of fumaric acid, the type II free crystal of said compound, the type I free crystal of said compound and the type V crystal of said compound with 1 equivalent of fumaric acid have excellent properties as pharmaceutical products or active pharmaceutical ingredients.

[0167]

Test Example 11

Blood concentration test

Blood concentration tests were performed for the type I crystal of compound (I), the type II crystal of compound (I), and the type II crystal of compound (I) with 1 equivalent of fumarate obtained according to the examples. Specifically, 0.5% HPMC containing 0.1 N hydrochloric acid was used for the type I crystal of compound (I), and 0.5% HPMC was used for the type II crystal of compound (I) and the type II crystal of compound (I) with 1 equivalent of fumaric acid to convert each crystal into compound (I) (free form) in molecular weight units, to obtain suspensions of 100 mg/10 ml and 300 mg/10 ml. These solutions for administration were orally administered to female rats (Crl:CD(SD), Charles River Laboratories Japan, Inc.) which were kept under conditions with access to food in a volume of 10 ml/kg body weight using an oral gavage. After administration, the rats were returned to the rat cage and their condition was checked. Water and food were freely available in the cage. At 0.5, 1, 2, 4, and 6 hours after administration, the rats were anesthetized with isoflurane and approximately 80 μl of blood was collected from the jugular vein using a heparin-coated syringe and injection needle (25 G).

The collected blood was cooled on ice and the plasma was separated by centrifugation. After blood collection was complete, the rats were returned to the rat cage and checked for recovery from anesthesia. After the last blood collection, the depth of anesthesia was checked with isoflurane. The limbs were then restrained in a supine position using a small experimental restraint device and the abdomen was opened to sacrifice the rat by exsanguination through an incision in the abdominal aorta and vena cava.

AUC0-6h (area under the blood concentration-time curve during 0-6 hours after administration, calculated by the trapezoidal method), Cmax (maximum blood concentration) and Tmax (time to reach maximum blood concentration) were calculated from the concentration of compound (I) in each plasma sample measured by the MRM method using LC/MS/MS and quantified from the calibration curve using Phoenix WinNonlin (v6.4.0. Certara USA, Inc.).

[0168]

The results are shown in Table 19. When the dose increased from 100 mg/kg to 300 mg/kg, for the type I crystal of compound (I) with hydrochloric acid and the type II crystal of compound (I), Cmax increased by 0.8 times and 1.2 times, respectively, and AUC0-6h increased by 1.0 times and 1.5 times, respectively. Meanwhile, for the type II crystal of compound (I) with 1 equivalent of fumaric acid, _Cmax and AUC0-6h each increased by 1.9 times. In addition, at a dose of 300 mg/kg, _Cmax of the type II crystal of compound (I) with 1 equivalent of fumaric acid was 1.7 times higher than the value of type I crystal of compound (I) with acid and 1.4 times higher than the value of type II crystal of compound (I), and AUC _0-6h of type II crystal of compound (I) with 1 equivalent of fumaric acid was 2.4 times higher than the values of type I crystal of compound (I) with acid and type II crystal of compound (I). Thus, it was confirmed that the type II crystal of compound (I) with 1 equivalent of fumaric acid according to the present invention exhibits more favorable absorption in the oral cavity.

[Table 19]

Dose Parameter Oral administration Crystal of type I compound (I)
(with hydrochloric acid) Type II crystal of compound (I) Type II crystal of compound (I)
with 1 equivalent of fumaric acid 100
mg/kg AUC _0-6h (μm⋅h) 7.42 5.79 6.14 C _max (µm) 2.67 1.88 2.74 T _max (h) 0.75 1.50 1.25 300
mg/kg AUC _0-6hr (μm⋅h) 7.11 8.53 11.9 C _max (µm) 2.25 2.25 5.34 T _max (h) 3.25 3.33 0.5

[0169]

It should be noted that all documents and publications cited in this specification are incorporated herein by reference in their entirety, regardless of their purpose. Furthermore, this specification includes the content disclosed in the claims, description and drawings of Japanese Patent Application No. 2020-121520 (filed on July 15, 2020), from which this application claims priority.

Some embodiments of the present invention have been described above. However, these embodiments are provided for illustrative purposes only and are thus not intended to limit the scope of the present invention. These new embodiments can be implemented in various forms, and various reductions, substitutions and changes can be made as long as they do not deviate from the spirit of the invention. These embodiments and their modifications are included in the scope or spirit of the invention, and they are also included in the invention in accordance with the claims and in an equivalent scope.

Claims (6)

1. A crystal having peaks at diffraction angles (2θ±0.2°) selected from 5.5°, 6.8°, 9.3°, 13.4°, 15.3°, 16.3°, 18.5°, 19.8°, 22.0° and 24.5° in an X-ray powder diffraction spectrum, which is a type II crystal of 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide with fumaric acid, wherein the molar ratio 7-((3R,5S)-1-acryloyl-5-methylpyrrolidin-3-yl)-4-amino-6-(cyclopropylethynyl)-N-((R)-1-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide and fumaric acid is 1:1. 2. The crystal according to claim 1, which is a crystal having a powder X-ray diffraction spectrum shown in Fig. 1. 3. A crystal according to claim 1 or 2, which has an endothermic peak, determined by simultaneous thermogravimetric-differential thermal analysis, at a level of approximately 178°C±5.0°C. 4. A pharmaceutical composition having HER2 inhibitory activity, containing an effective amount of a crystal according to any one of claims 1-3. 5. A pharmaceutical composition for oral administration, having HER2 inhibitory activity, containing an effective amount of a crystal according to any one of claims 1-3. 6. An antitumor agent for treating a tumor associated with HER2, comprising a crystal according to any one of claims 1 to 3.