WO2022257965A1 - Cyclin-dependent kinase 9 inhibitor in solid form and use thereof - Google Patents

Abstract

Provided are a compound of formula (A) in a solid form, in particular in a crystalline form, a specific crystal form, and the use thereof. The compound represented by formula (A) has excellent cyclin-dependent kinase 9 in vitro inhibitory activity, excellent tumor cell in vitro inhibitory activity, excellent in vivo antitumor activity, and better safety, and the obtained crystal form of the compound represented by formula (A) has the characteristics of good crystallinity, low hygroscopicity, and good stability, and has good druggable potential.

Description

Cyclin-dependent kinase 9 inhibitors in solid form and uses thereof

related application

This application claims the priority of Chinese Patent Application No. 202110644313.0 filed on June 9, 2021, which is hereby incorporated by reference in its entirety for all purposes.

technical field

The application relates to a compound in solid form as a cyclin-dependent kinase 9 (CDK9) inhibitor, and its application in the preparation of drugs for treating CDK9-related diseases.

Background technique

Cyclin-dependent kinases (CDKs) are a class of serine/threonine protein kinases that play key roles in regulating the cell cycle and transcription. Up to now, there are more than 20 human CDK subtypes and about 30 cell cycle chaperones known. These CDKs can be activated by cell cycle proteins and play different biological functions. CDKs can be divided into two types according to their functions. One controls the cell cycle and the other regulates cell transcription. For example, CDK1, 2, 3, 4, and 6 directly intervene in the cell cycle; CDK5 does not regulate the cell cycle, but plays a key role in the complex migration of neurons after mitosis; CDK7 acts indirectly as an activator of these CDKs; CDK9 acts only in the cell transcription play a role in the regulation of the cell cycle.

CDK9 is an important member of the transcriptional CDKs subfamily, a group of kinases whose function is to control the main steps in the synthesis and processing of mRNA by eukaryotic RNA polymerase II (Pol II). CDK9 is present in all mammalian cells. The activation of CDK9 in vivo depends on its binding to the corresponding cyclin (Cyclin T/K) to form a heterodimer, that is, positive transcription elongation factor b (P-TEFb). When negative transcriptional elongation factors (NELFs) are involved in the negative regulation of cellular transcription, transcription is repressed and P-TEFb is recruited into the system in which negative transcriptional elongation factors inhibit transcriptional elongation, catalyzing the carbon-terminal domain of RNA polymerase II (CTD) phosphorylation, and simultaneously catalyze the phosphorylation of the SPT5 subunit of NELFs and the RD subunit of NELF, resulting in the detachment of the negative transcription elongation factor from the transcription complex, so that the transcription can continue.

Tumors are usually caused by the loss of expression of cyclin-dependent kinase inhibitors (CDKI) or the overexpression of cyclins, which leads to unregulated excessive proliferation of cells. In view of the above regulatory mechanism, the use of CDK9 inhibitors will be able to prevent P-TEFb from phosphorylation of the carbon-terminal domain of RNA polymerase II, further hinder the departure of NEFL, strengthen the negative inhibitory effect, cause transcription to stop, and make intracellular mRNA and The level of proteins with short half-lives decreases rapidly, which can induce apoptosis of tumor cells. CDK9 has become a potential protein target for the development of effective cancer therapy. Recently, pharmaceutical companies have conducted research on CDK9 inhibitors for cancer treatment, for example, AZD4573 of AstraZeneca and BAY-1251152 of Bayer, both of which are in clinical I phase test phase.

Although some small molecules of CDK9 inhibitors have been disclosed (for example, WO2009047359, WO2014076091, etc.), no drugs have been approved for marketing, and it is still necessary to develop new compounds with good efficacy, good safety, and good drug-making potential. Benefit more patients clinically.

Contents of the invention

On the one hand, the application provides the compound represented by formula (A) in solid form,

In another aspect, the present application provides a compound represented by formula (A) in crystalline form.

In some solutions of the present application, the above-mentioned crystalline form is characterized in that the crystalline form is a solvent-free and anhydrous crystalline form or a hydrate crystalline form, preferably a solvent-free and anhydrous crystalline form.

On the other hand, the application provides the crystal form I of the compound represented by formula (A), using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.3±0.2°, 19.4± 0.2°, 20.1±0.2°.

In some schemes of the present application, the above crystal form I uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.3±0.2°, 19.4±0.2°, 20.1±0.2°, 23.0±0.2°.

In some schemes of the present application, the above crystal form I uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.3±0.2°, 19.4±0.2°, 20.1±0.2°, 21.5±0.2°, 23.0±0.2°.

In some schemes of the present application, the above crystal form I uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.3±0.2°, 19.4±0.2°, 20.1±0.2°, 23.0±0.2°, 26.1±0.2°.

In some schemes of the present application, the above crystal form I uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.3±0.2°, 19.4±0.2°, 20.1±0.2°, 20.7±0.2°, 21.5±0.2°, 23.0±0.2°, 26.1±0.2°.

In some schemes of the present application, the above crystal form I uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.3±0.2°, 19.4±0.2°, 20.1±0.2°, 20.7±0.2°, 21.5±0.2°, 23.0±0.2°, 26.1±0.2°, 26.6±0.2°.

In some schemes of the present application, the above crystal form I uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.3±0.2°, 13.3±0.2°, 19.4±0.2°, 20.1±0.2°, 20.7±0.2°, 21.5±0.2°, 23.0±0.2°, 26.1±0.2°, 26.6±0.2°.

In some schemes of the present application, the above crystal form I uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.3±0.2°, 12.4±0.2°, 13.3±0.2°, 18.6±0.2°, 19.4±0.2°, 20.1±0.2°, 20.7±0.2°, 21.5±0.2°, 23.0±0.2°, 26.1±0.2°, 26.6±0.2°.

In some schemes of the present application, the above crystal form I uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.3±0.2°, 12.4±0.2°, 13.3±0.2°, 18.6±0.2°, 19.4±0.2°, 20.1±0.2°, 20.4±0.2°, 20.7±0.2°, 21.5±0.2°, 22.0±0.2°, 23.0±0.2°, 26.1±0.2°, 26.6±0.2°.

In some schemes of the present application, the above crystal form I uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.3±0.2°, 12.4±0.2°, 13.1±0.2°, 13.3±0.2°, 15.4±0.2°, 18.6±0.2°, 19.4±0.2°, 19.5±0.2°, 20.1±0.2°, 20.4±0.2°, 20.7±0.2°, 21.5±0.2°, 22.0±0.2°, 23.0±0.2°, 26.1±0.2°, 26.6±0.2°.

In some schemes of the present application, the above crystal form I uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks (±0.2°) at the following 2θ angles:

Peak position (2θ)°	Relative Strength%	Peak position (2θ)°	Relative Strength%	Peak position (2θ)°	Relative Strength%
9.3	16.2	19.4	100	22.0	27.6
12.4	14.5	19.5	69.7	23.0	61.0
13.1	39.7	20.1	71.3	26.1	61.2
13.3	67.3	20.4	48.1	26.6	39.7
15.4	16.8	20.7	46.2	the	the
18.6	25.6	21.5	43.2	the	the

In some solutions of the present application, the above-mentioned crystal form I, using Cu-Kα radiation, has an X-ray powder diffraction pattern substantially as shown in FIG. 1 .

In some aspects of the present application, the differential scanning calorimetry curve of the above crystal form I has an endothermic peak at 204.33±5°C.

In some solutions of the present application, the above-mentioned crystal form I has a DSC spectrum substantially as shown in FIG. 2 .

In some aspects of the present application, the thermogravimetric analysis curve of the above crystal form I has a weight loss of 0.565%±0.2% between room temperature and 180°C±5°C.

In some solutions of the present application, the above-mentioned crystal form I has a TGA spectrum substantially as shown in FIG. 2 .

On the other hand, the application provides the crystal form V of the compound represented by formula (A), using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 11.3±0.2°, 18.3± 0.2°, 22.8±0.2°.

In some schemes of the present application, the above crystal form V uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 11.3±0.2°, 16.2±0.2°, 18.3±0.2°, 21.0±0.2°, 22.8±0.2°.

In some schemes of the present application, the above crystal form V uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 11.3±0.2°, 16.2±0.2°, 18.3±0.2°, 21.0±0.2°, 22.8±0.2°, 23.5±0.2°.

In some schemes of the present application, the above crystal form V uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.4±0.2°, 11.3±0.2°, 16.2±0.2°, 18.3±0.2°, 19.7±0.2°, 21.0±0.2°, 22.8±0.2°, 23.5±0.2°.

In some schemes of the present application, the above crystal form V uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 6.5±0.2°, 9.4±0.2°, 11.3±0.2°, 16.2±0.2°, 16.7±0.2°, 18.3±0.2°, 19.7±0.2°, 21.0±0.2°, 22.8±0.2°, 23.5±0.2°.

In some schemes of the present application, the above crystal form V uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 6.5±0.2°, 9.4±0.2°, 11.3±0.2°, 16.2±0.2°, 16.7±0.2°, 17.0±0.2°, 18.3±0.2°, 19.7±0.2°, 20.7±0.2°, 21.0±0.2°, 22.8±0.2°, 23.5±0.2°.

In some schemes of the present application, the above crystal form V uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 6.5±0.2°, 9.4±0.2°, 11.3±0.2°, 13.1±0.2°, 16.2±0.2°, 16.7±0.2°, 17.0±0.2°, 18.3±0.2°, 19.7±0.2°, 20.7±0.2°, 21.0±0.2°, 22.8±0.2°, 23.5±0.2°, 24.4±0.2°.

In some schemes of the present application, the above crystal form V uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks (±0.2°) at the following 2θ angles:

Peak position (2θ)°	Relative Strength%	Peak position (2θ)°	Relative Strength%	Peak position (2θ)°	Relative Strength%
6.5	7.9	16.7	17.8	21.0	34.3
9.4	14.6	17.0	11.3	22.8	100.0
11.3	83.5	18.3	30.3	23.5	44.1
13.1	5.2	19.7	9.4	24.4	5.6
16.2	32.2	20.7	8.0	the	the

In some solutions of the present application, the above-mentioned crystal form V, using Cu-Kα radiation, has an X-ray powder diffraction pattern substantially as shown in FIG. 3 .

In some aspects of the present application, the differential scanning calorimetry curve of the above crystal form V has an endothermic peak at 185.87±5°C.

In some solutions of the present application, the above-mentioned crystal form V has a DSC spectrum substantially as shown in FIG. 4 .

In some aspects of the present application, the thermogravimetric analysis curve of the above crystal form V has a weight loss of 0.926%±0.2% between room temperature and 170±5°C.

In some solutions of the present application, the above-mentioned crystal form V has a TGA spectrum substantially as shown in FIG. 4 .

On the other hand, the application provides the crystal form XIII of the compound represented by formula (A), using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 15.8±0.2°, 19.1± 0.2°, 20.8±0.2°.

In some schemes of the present application, the above crystal form XIII uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 14.3±0.2°, 15.8±0.2°, 17.8±0.2°, 19.1±0.2°, 20.8±0.2°.

In some schemes of the present application, the above crystal form XIII uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.5±0.2°, 14.3±0.2°, 15.8±0.2°, 17.8±0.2°, 19.1±0.2°, 20.8±0.2°, 24.4±0.2°.

In some schemes of the present application, the above crystal form XIII uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.5±0.2°, 14.3±0.2°, 15.8±0.2°, 17.8±0.2°, 18.2±0.2°, 19.1±0.2°, 20.8±0.2°, 24.4±0.2°, 26.3±0.2°.

In some schemes of the present application, the above crystal form XIII uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.5±0.2°, 14.3±0.2°, 15.8±0.2°, 16.6±0.2°, 17.8±0.2°, 18.2±0.2°, 19.1±0.2°, 20.8±0.2°, 24.4±0.2°, 26.3±0.2°, 26.9±0.2°.

In some schemes of the present application, the above crystal form XIII uses Cu-Kα radiation, and its X-ray powder diffraction pattern has characteristic diffraction peaks (±0.2°) at the following 2θ angles:

Peak position (2θ)°	Relative Strength%	Peak position (2θ)°	Relative Strength%
9.5	8.2	19.1	33.4
14.3	20.7	20.8	100.0
15.8	92.8	24.4	19.2
16.6	9.2	26.3	12.1
17.8	35.6	26.9	11.9
18.2	21.5	the	the

In some aspects of the present application, the above crystal form XIII, using Cu-Kα radiation, has an X-ray powder diffraction pattern substantially as shown in FIG. 5 .

In some aspects of the present application, the differential scanning calorimetry curve of the above crystal form XIII has two endothermic peaks at 194.07±5°C and 200.51±5°C respectively.

In some solutions of the present application, the above crystal form XIII has a DSC spectrum substantially as shown in FIG. 6 .

In some aspects of the present application, the thermogravimetric analysis curve of the above crystal form XIII has a weight loss of 0.216%±0.2% between room temperature and 180±5°C.

In some solutions of the present application, the above crystal form XIII has a TGA spectrum substantially as shown in FIG. 6 .

Another object of the present application is to provide a crystalline composition comprising Form I, Form V, Form XIII or two or more of the compound of formula (A).

In some embodiments of the present application, the crystal form I accounts for more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or more than 95% of the weight of the crystalline composition.

In some solutions of the present application, the crystal form V accounts for more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or more than 95% of the weight of the crystalline composition.

In some solutions of the present application, the crystal form XIII accounts for more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or more than 95% of the weight of the crystalline composition.

Another object of the present application is to provide a pharmaceutical composition, which comprises the compound represented by formula (A) in solid form, the compound represented by formula (A) in crystal form or its crystal composition.

In some aspects of the present application, the above-mentioned pharmaceutical composition comprises the crystal form I, crystal form V, or crystal form XIII of the compound represented by formula (A).

Another object of the present application is to provide a pharmaceutical composition, which comprises a compound represented by formula (A) in solid form, a compound represented by formula (A) in crystal form or a crystal composition thereof, and a pharmaceutically acceptable carrier .

In some aspects of the present application, the above-mentioned pharmaceutical composition comprises crystalline form I, crystalline form V, or crystalline form XIII of the compound of formula (A), and a pharmaceutically acceptable carrier.

On the other hand, the present application also provides the above-mentioned compound represented by formula (A) in solid form, the compound represented by formula (A) in crystalline form, crystal form I, crystal form V, and crystal form of the compound represented by formula (A) XIII. Use of the above-mentioned crystalline composition or the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing and/or treating a disease mediated by CDK9.

In some aspects of the present application, the above-mentioned use, wherein the CDK9-mediated disease includes a cell proliferative disease or an inflammatory disease.

On the other hand, the present application also provides the above-mentioned compound represented by formula (A) in solid form, the compound represented by formula (A) in crystalline form, crystal form I, crystal form V, and crystal form of the compound represented by formula (A) XIII. Use of the above crystalline composition or the above pharmaceutical composition in the preparation of a medicament as a CDK9 inhibitor.

In some aspects of the present application, the drug is used to treat diseases mediated by CDK9.

In some aspects of the present application, the CDK9-mediated diseases include cell proliferative diseases or inflammatory diseases.

In some aspects of the present application, the aforementioned CDK9-mediated disease refers to a disease involving changes in CDK9 gene, protein, activity or expression thereof.

In some aspects of the present application, the aforementioned cell proliferative disease or inflammatory disease involves changes in CDK9 gene, protein, activity or expression thereof.

In some schemes of the present application, the aforementioned cell proliferative disease is a tumor; preferably, the tumor is a hematological tumor or a solid tumor; preferably a relapsed or refractory hematological tumor or a solid tumor; preferably a malignant hematological tumor or an advanced solid tumor Tumor; more preferably advanced hematological malignancy or advanced malignant solid tumor; more preferably relapsed or refractory advanced hematological malignancy or advanced malignant solid tumor.

In some schemes of the present application, the blood tumor is leukemia, lymphoma or myeloma; preferably, the leukemia is acute myeloid leukemia; preferably, the lymphoma is non-Hodgkin's lymphoma; cell lymphoma or diffuse large B-cell lymphoma; preferably, the myeloma is multiple myeloma.

In some aspects of the present application, the solid tumor is selected from liver cancer, breast cancer and prostate cancer; preferably, the breast cancer is triple-negative breast cancer.

On the other hand, the present application also provides the use of the compound represented by the formula (A) in solid form or the compound represented by formula (A) in crystalline form in the preparation of crystal form I, crystal form V, or crystal form XIII.

On the other hand, the present application also provides the above-mentioned compound represented by formula (A) in solid form, the compound represented by formula (A) in crystalline form, crystal form I, crystal form V, and crystal form of the compound represented by formula (A) XIII. The above crystalline composition or the above pharmaceutical composition, which is used for preventing and/or treating CDK9-mediated diseases.

On the other hand, the present application also provides the above-mentioned compound represented by formula (A) in solid form, the compound represented by formula (A) in crystalline form, crystal form I, crystal form V, and crystal form of the compound represented by formula (A) XIII. The above crystalline composition or the above pharmaceutical composition for use as a CDK9 inhibitor.

In some aspects of the present application, the CDK9 inhibitor is used to treat CDK9-mediated diseases.

In some aspects of the present application, the aforementioned CDK9-mediated diseases include cell proliferative diseases or inflammatory diseases.

In some aspects of the present application, the aforementioned CDK9-mediated disease refers to a disease involving changes in CDK9 gene, protein, activity or expression thereof.

In some aspects of the present application, the aforementioned cell proliferative disease or inflammatory disease involves changes in CDK9 gene, protein, activity or expression thereof.

In some schemes of the present application, the aforementioned cell proliferative disease is a tumor; preferably, the tumor is a hematological tumor or a solid tumor; preferably a relapsed or refractory hematological tumor or a solid tumor; preferably a malignant hematological tumor or an advanced solid tumor Tumor; more preferably advanced hematological malignancy or advanced malignant solid tumor; more preferably relapsed or refractory advanced hematological malignancy or advanced malignant solid tumor.

In some schemes of the present application, the blood tumor is leukemia, lymphoma or myeloma; preferably, the leukemia is acute myeloid leukemia; preferably, the lymphoma is non-Hodgkin's lymphoma; cell lymphoma or diffuse large B-cell lymphoma; preferably, the myeloma is multiple myeloma.

In some aspects of the present application, the solid tumor is selected from liver cancer, breast cancer and prostate cancer; preferably, the breast cancer is triple-negative breast cancer.

On the other hand, the present application also provides a method for treating a disease mediated by CDK9, the method comprising administering a therapeutically effective amount of the compound represented by the above formula (A) in solid form, the formula (A) in crystal form to an individual in need The compound shown in A), the crystal form I, the crystal form V, the crystal form XIII of the compound shown in formula (A), the above crystal composition or the above pharmaceutical composition.

In some aspects of the present application, the CDK9-mediated diseases include cell proliferative diseases or inflammatory diseases.

In some aspects of the present application, the CDK9-mediated disease refers to a disease involving changes in CDK9 gene, protein, activity or expression thereof.

In some aspects of the present application, the cell proliferative disease or inflammatory disease involves changes in CDK9 gene, protein, activity or expression thereof.

In some schemes of the present application, the cell proliferative disease is a tumor; preferably, the tumor is a hematological tumor or a solid tumor; preferably a relapsed or refractory hematological tumor or a solid tumor; preferably a malignant hematological tumor or an advanced Solid tumor; more preferably advanced hematological malignancy or advanced malignant solid tumor; more preferably relapsed or refractory advanced hematological malignancy or advanced malignant solid tumor.

In some schemes of the present application, the blood tumor is leukemia, lymphoma or myeloma; preferably, the leukemia is acute myeloid leukemia; preferably, the lymphoma is non-Hodgkin's lymphoma; cell lymphoma or diffuse large B-cell lymphoma; preferably, the myeloma is multiple myeloma.

In some aspects of the present application, the solid tumor is selected from liver cancer, breast cancer and prostate cancer; preferably, the breast cancer is triple-negative breast cancer.

Definition and Description

Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A specific phrase or term should not be considered indeterminate or unclear if it is not specifically defined, but should be understood according to its ordinary meaning.

The "compound represented by formula (A) in solid form" mentioned in the present application refers to the compound represented by formula (A) in solid state, including its crystalline form and amorphous form.

The "compound represented by formula (A) in crystalline form" mentioned in this application refers to the compound represented by formula (A) in crystalline form, including the anhydrous and solvent-free form and hydrate form of the compound represented by formula (A) , solvate form and co-crystal form; preferably include anhydrous and solvent-free form, hydrate form and solvate form of the compound shown in formula (A); preferably include anhydrous and anhydrous of the compound shown in formula (A) Solvent form and hydrate form.

The term "solvate" or "solvate" refers to a complex with variable stoichiometry formed by a solute (in this application, a compound represented by formula (A)) and a solvent. The solvents used for the purposes of this application must not interfere with the biological activity of the solute. Suitable solvates include pharmaceutically acceptable solvates, and also include both stoichiometric solvates and non-stoichiometric solvates. However, solvates with non-pharmaceutically acceptable solvents are within the scope of the present application, for example, they can be used as intermediates for the preparation of compounds represented by formula (A) or other crystal forms. Preferably, the solvent used is water, and the solvate obtained at this time may also be called a hydrate. As used herein and unless otherwise indicated, the term "hydrate" refers to a compound provided herein that further includes stoichiometric or non-stoichiometric amounts of water bound by non-covalent intermolecular forces.

The "solvent-free and anhydrous crystalline form" means that the sample does not contain solvent molecules or water molecules that are combined with the compound represented by formula (A) by intermolecular forces. For example, a sample contains not more than 3.0%, such as not more than 1.5%, such as not more than 1%, by weight of water or solvent when measured by thermogravimetric analysis (TGA).

The term "crystalline composition" refers to a solid form comprising the crystalline form I, crystalline form V, crystalline form XIII or two or more thereof mentioned in this application. Moreover, in addition to the crystal forms mentioned in this application, the crystalline composition may optionally contain other crystal forms or other amorphous forms of the compound represented by formula (A) or its salt, or impurities other than these substances. Those skilled in the art should understand that the sum of the contents of each component in the crystalline composition should be 100%.

The term "optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that description includes that said event or circumstance occurs and that it does not. The "room temperature" is the room temperature in the conventional sense in the field, generally 10-30°C, preferably 25°C±5°C.

In the X-ray powder diffraction pattern, the terms "substantially" or "substantially as shown" refer to a substantially pure crystalline form having at least 50% of the powder X-ray diffraction pattern, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% of the peaks present in the given spectrum . Furthermore, when the content of a certain crystalline form in the product gradually decreases, some diffraction peaks in the X-ray powder diffraction pattern attributed to the crystalline form may decrease due to the detection sensitivity of the instrument. Furthermore, for any given crystalline form, there may be slight errors in the position of the peaks, as is well known in the art of crystallography. For example, due to temperature changes, sample movement, or instrument calibration when analyzing samples, the position of the peak can move, and the measurement error of the 2θ value is sometimes about ±0.2°. Therefore, when determining the structure of each crystal form, this error should be taken into consideration, and the term "substantially" or "substantially as shown" is also intended to cover such differences in the positions of diffraction peaks.

In the DSC spectrum or TGA spectrum, the term "substantially" or "substantially as shown in the figure" means that for the same crystal form of the same compound, in continuous analysis, the thermal transition onset temperature, endothermic The error of peak-to-peak temperature, exothermic peak-to-peak temperature, melting point, weight loss onset temperature or weight loss end temperature etc. is typically within about 5°C, usually within about 3°C. When describing that a compound has a given thermal transition onset temperature, endothermic peak peak temperature, exothermic peak peak temperature, melting point, weight loss onset temperature or weight loss end point temperature, etc., it refers to the temperature ± 5°C.

As used herein, the term "cell proliferative disorder" refers to a disorder in which a population of cells grows at a rate that is either slower or higher than expected for a given physiological state and conditions.

The term "tumor" includes benign tumors, malignant tumors and borderline tumors, where malignant tumors are collectively referred to as cancer.

As used herein, the term "prevention" means, when used for a disease or disorder (eg, cancer), that the A compound or drug is capable of reducing the frequency or delaying the onset of symptoms of a medical condition in an individual or subject.

As used herein, the term "treating" means alleviating, alleviating or ameliorating the symptoms of a disease or disorder, ameliorating an underlying metabolically caused symptom, inhibiting a disease or symptom, e.g., arresting the development of a disease or disorder, alleviating a disease or disorder, causing a disease Regression of a disease or disorder, alleviation of a condition caused by a disease or disorder, or arrest of symptoms of a disease or disorder.

As used herein, the term "individual" or "subject" refers to a cell or a mammal, such as a human, but can also be other mammals, such as livestock or experimental animals (for example, including but not limited to mice, rats, rabbits, dogs, monkeys, sheep, etc.) etc.

The pharmaceutical composition of the present application can be prepared by conventional methods in the art.

In the context of this application, the term "pharmaceutically acceptable carrier" or "excipient" or "pharmaceutically acceptable adjuvant" or "pharmaceutically acceptable adjuvant" means no significant irritation to the organism, and Those excipients that do not impair the biological activity and performance of the active compound. The term "pharmaceutically acceptable excipients" includes: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, Stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers Agents, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc. Those skilled in the art can select specific pharmaceutically acceptable excipients according to actual needs. Knowledge about excipients is well known to those skilled in the art, for example, reference can be made to "Pharmacy" (Edited by Cui Fude, 5th edition, People's Medical Publishing House, 2003).

The word "comprise" or "comprise" and its English variants such as comprises or comprising should be interpreted in an open and non-exclusive sense, ie "including but not limited to".

Within the scope of the application, various options of any one feature can be combined with various options of other features to form many different embodiments. The present application intends to include all possible implementations consisting of various options of all technical features, as long as such a new technical solution is technically feasible.

technical effect

The solid form of the compound represented by the formula (A), the crystalline form and the specific crystal form of the compound represented by the formula (A) provided by the application have one or more of the following beneficial effects:

The crystal form and specific crystal form of the compound represented by formula (A) mentioned in this application have good crystallinity and are easy to purify, filter and separate;

It is easy to be drugged and has good stability (for example, solid stability, solution stability), especially crystal form I, crystal form V and crystal form XIII;

It has no obvious hygroscopicity and has a good medicinal prospect;

The results of in vitro kinase activity test and cell test show that the compound (A) of the present application has good in vitro kinase inhibitory activity against CDK9, good selectivity for other CDK subtypes, and strong inhibitory effect on various tumor cells;

The hERG inhibitory activity test in vitro shows that the compound represented by formula (A) has a lower risk of cardiotoxicity;

The results of in vivo anti-tumor test and safety test show that, compared with the control compound, the compound represented by formula (A) has better in vivo anti-tumor effect, less toxicity, and higher possibility of becoming a drug. a better choice.

Description of drawings

Figure 1: X-ray powder diffraction pattern of Form I of Example 1.

Figure 2: DSC-TGA spectrum of Form I of Example 1.

Figure 3: X-ray powder diffraction pattern of Form V of Example 2.

Figure 4: DSC-TGA spectrum of Form V of Example 2.

Figure 5: X-ray powder diffraction pattern of Form XIII of Example 3.

Figure 6: DSC-TGA spectrum of Form XIII of Example 3.

Detailed ways

1. X-ray powder diffraction (X-ray powder diffractometer, XRPD)

Instrument model: Bruker D8 Advance X-ray powder diffractometer (Bruker, GER)

Test method: About 2~10mg sample is used for XRPD detection

The detailed XRPD parameters are as follows:

X-ray generator: Cu-Kα

Phototube voltage: 40kV, phototube current: 40mA

Scanning range: 3°-45°(2θ)

Scan step: 0.02°

Exposure time: 0.12 seconds

Sample Tray: Zero Background Sample Tray

Acquisition software: DIFFRAC.Measurement Center (Bruker, GER)

Analysis software: DIFFRAC.EVA (Bruker, GER).

2. Differential Scanning Calorimeter (DSC)

Instrument model: TA Discovery 2500 Differential Scanning Calorimeter (TA, US)

Test method: Take the sample and place it in the DSC sample tray, cover the sample tray and punch holes, equilibrate the sample at 25°C and heat it to the final temperature at a heating rate of 10°C/min.

Sample size: 1~2mg

Gas flow type: nitrogen

Flow rate: 50mL/min

Heating start temperature: 25°C

Termination temperature: 250°C.

3. Thermal Gravimetric Analyzer (TGA)

Instrument model: TA Discovery 55 thermogravimetric analyzer (TA, US)

Test method: Place the sample in a balanced open aluminum sample pan. After the sample quality is automatically weighed in the TGA heating furnace, the sample is heated to the final temperature at a rate of 10°C/min.

Sample size: 2~5mg

Gas flow type: nitrogen

Flow rate: 60mL/min

Heating start temperature: room temperature

Termination temperature: 300°C.

4. Dynamic moisture adsorption and desorption analysis (DVS)

Instrument model: DVS Intrinsic dynamic water vapor adsorption instrument (SMS, UK)

Test method: add enough sample (15mg) into the instrument to simulate dynamic water vapor adsorption, and record the different humidity in the range of 0%-90% at 25°C (the humidity change of each gradient is 10%, the humidity change : 50%-95%-0%-50%) weight change during balance. The end point of the gradient is judged by the dm/dt method, and the end point of the gradient is defined as the dm/dt is less than 0.002% and maintained for 10 minutes.

Classify the hygroscopicity of the sample:

(1) Deliquescence: Absorb enough water to form liquid

(2) Extremely hygroscopic: moisture absorption weight gain is not less than 15%

(3) Hygroscopicity: moisture absorption weight gain is less than 15% but not less than 2%

(4) Slight hygroscopicity: moisture absorption weight gain is less than 2% but not less than 0.2%

(5) No hygroscopicity: moisture absorption weight gain is less than 0.2%.

5. High performance liquid chromatography (HPLC)

Instrument model: Waters Acquity Arc high performance liquid chromatography (Waters, US)

Chromatographic column: Waters CORTECS C18, 4.6mm×150mm, 2.7μm

Test conditions: wavelength 250nm; column temperature 30°C

Mobile phase: A: 0.1% acetic acid in water; B: 0.05% TFA in acetonitrile

Injection volume: 5 μL.

6. Nuclear Magnetic Resonance Spectroscopy (NMRS)

Instrument model: Bruker AVANCE III 400 (Bruker, GER)

Contents and test solvent: ¹ H-NMR, the test solvent is DMSO-d6.

7. Crystal stability test in different pH solutions

The preparation process of the pH buffer is shown in the table below. Add the sample of the crystal form to be tested to 2.0mL of different pH 2-7 buffer media to prepare a suspension, shake at a constant temperature of 25°C for 24 hours, and centrifuge the suspension; take the remaining supernatant to test its pH value , carry out XRPD test on remaining solid.

Instrument: TG16.5 desktop high-speed centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.)

Conditions: 10000rpm, centrifuge for 4 minutes.

8. Crystal stability test in water and biological media

The preparation process of the biological medium is shown in the table below. Samples of different crystal forms were added to 4.0mL of water and biological medium (FaSSIF, FeSSIF, FaSSGF, FaSSIF/FeSSIF/FaSSGF powder manufacturer Biorelevant, powder product number FFF01, batch number FFF-1219-A) to prepare a suspension, in Shake at a constant temperature of 37°C for 24 hours, test the pH value of the supernatant at the time point of 24 hours, and carry out XRPD test on the remaining solid.

Remarks: FaSSIF: Simulates the intestinal juice in the small intestine under the state of hunger before a meal; FeSSIF: simulates the intestinal juice in the small intestine under the state of satiety after a meal; FaSSGF: simulates the gastric juice in the empty stomach under the state of human starvation.

The intermediate compounds and/or compounds of the present application can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the methods in the art The equivalent replacements known to the skilled person, and preferred implementations include but not limited to the examples of the present application. When a trade name appears herein, it is intended to refer to its corresponding trade name or its active ingredient.

The chemical reactions in the specific embodiments of the present application are completed in a suitable solvent, and the solvent must be suitable for the chemical changes of the present application and the reagents and materials required therefor. In order to obtain the compounds of the present application, it is sometimes necessary for those skilled in the art to modify or select synthetic steps or reaction schemes on the basis of existing embodiments.

All solvents used in this application were commercially available and used without further purification.

In order to better understand the content of the present application, further description will be made below in conjunction with specific examples, but the specific implementation manners are not intended to limit the content of the present application. For the test methods that do not indicate specific conditions in the following preparation examples, examples, comparative examples, test examples or test examples, select according to conventional methods and conditions, or according to the product description.

Preparation Example 1: Preparation of Formula (A) Compound

Synthesis of Intermediate 1a:

5-Fluoro-4-iodopyridin-2-amine (1.00 g, 4.20 mmol) was dissolved in ethylene glycol dimethyl ether (20 mL) and water (4 mL), followed by the addition of 4-fluoro-2-methoxybenzene Boronic acid (0.71g, 4.20mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (0.31g, 0.42mmol) and potassium carbonate (1.70g, 12.60mmol), with Nitrogen was replaced three times to make the whole system under nitrogen atmosphere. The system was refluxed and stirred at 100° C., and reacted for 4 hours, and there was no remaining raw material as monitored by TLC. Concentrate under reduced pressure, separate and purify by column chromatography (dichloromethane:methanol=50:1-10:1, v/v) to obtain 1a (0.80g, yield 81%).

Synthesis of intermediate 1b:

1a (0.80 g, 3.40 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of (1S,3R)-3-[(tert-butoxycarbonyl)amino]cyclohexanecarboxylic acid (0.83 g, 3.40mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.56g, 4.10mmol) and N,N- Diisopropylethylamine (0.88 g, 6.80 mmol). The whole system was stirred overnight at room temperature, and there was no remaining starting material as monitored by TLC. Add water (100mL) to the reaction solution, extract with ethyl acetate (50mL×3), combine the organic phases, wash with saturated sodium chloride (50mL×2), dry over anhydrous sodium sulfate, remove the solvent under reduced pressure, and perform column chromatography Separation and purification (dichloromethane:methanol=50:1-20:1, v/v) gave 1b (0.90 g, yield 58%).

Synthesis of intermediate 1c:

1b (0.90 g, 1.95 mmol) was dissolved in dichloromethane (30 mL), followed by addition of trifluoroacetic acid (2 mL) under an ice-water bath. The whole system was stirred overnight at room temperature, and detected by TLC until no raw material remained. Add water (100 mL) to the reaction solution, then adjust the pH=9-10 with saturated aqueous sodium bicarbonate solution, extract with dichloromethane (50 mL×3), combine the organic phases, wash with saturated sodium chloride (50 mL×2), Dry over anhydrous sodium sulfate, remove the solvent under reduced pressure, and separate and purify by column chromatography (dichloromethane:methanol=50:1-8:1, v/v) to obtain 1c (0.61g, yield 87%).

Synthesis of final product A:

1c (50 mg, 0.138 mmol) was dissolved in N,N-dimethylformamide (5 mL), followed by the addition of glycolic acid (16 mg, 0.21 mmol), 2-(7-azobenzotriazole)-N , N,N',N'-tetramethyluronium hexafluorophosphate (80 mg, 0.21 mmol) and N,N-diisopropylethylamine (69 μL, 0.42 mmol). The whole system was stirred at room temperature and reacted for 10 hours, monitored by TLC until no raw material remained. Water (50 mL) was added to the reaction solution, and extracted with ethyl acetate (50 mL×3). Combine the organic phases, wash with saturated sodium chloride (50mL×2), dry over anhydrous sodium sulfate, remove the solvent under reduced pressure, separate and purify by column chromatography (dichloromethane:methanol=40:1-20:1, v/v) , to obtain the final product A (30 mg, yield 52%). MS m/z (ESI): 420.2 [M+H] ⁺ .

¹ H NMR (600MHz,DMSO-d ₆ )δ10.56(s,1H),8.31(s,1H),8.06(d,J=5.4Hz,1H),7.56(d,J=9.0Hz,1H) ,7.34-7.31(m,1H),7.09-7.07(m,1H),6.91-6.88(m,1H),5.38-5.36(m,1H),3.77-3.75(m,5H),3.66-3.63( m,1H),2.61-2.57(m,1H),1.83-1.68(m,4H),1.34-1.26(m,4H).

Embodiment 1: Preparation of formula (A) compound crystal form I

At room temperature, weigh about 200 mg of the sample of Preparation Example 1 and add it to a glass bottle of appropriate volume, add ethyl acetate (5 mL), stir at room temperature for 3 days, centrifuge to obtain a solid, and dry the obtained solid under vacuum at 50°C. The sample was taken for X-ray powder diffraction, and it was shown as a crystalline solid (crystalline form I, anhydrous crystal) with good crystallinity. The spectrum is shown in FIG. 1 , and the XRPD diffraction peak data is shown in Table 1. The sample was taken for DSC-TGA test. The DSC graph showed an endothermic peak at 204.33°C, and the TGA graph showed that the sample had a weight loss of 0.565% between room temperature and 180°C, as shown in Figure 2.

Table 1: XRPD diffraction peak data table of the crystal form I of Example 1

Peak position (2θ)°	Relative Strength%	Peak position (2θ)°	Relative Strength%	Peak position (2θ)°	Relative Strength%
3.813	8.5	19.374	100.0	23.532	26.1
6.404	13.9	19.530	69.7	24.119	21.0
9.254	16.2	19.787	22.2	24.728	12.3
11.605	8.8	20.099	71.3	25.298	10.9
12.395	14.5	20.351	48.1	25.648	17.1
13.062	39.7	20.695	46.2	26.083	61.2
13.301	67.3	21.228	11.4	26.556	39.7
14.709	14.6	21.500	43.2	27.297	14.8
15.377	16.8	21.977	27.6	28.201	13.8
15.754	17.3	22.476	15.2	28.985	8.3
17.923	11.1	22.982	61.0	the	the
18.613	25.6	23.267	23.8	the	the

Note: Select peaks with relative peak intensity >8.0% to be listed in the table.

Embodiment 2: Preparation of formula (A) compound crystal form V

In a suitable container at room temperature, weigh about 200 mg of the sample of Preparation Example 1 and dissolve it in dioxane (4 mL), stir until it dissolves, add the dissolved solution into water (40 mL), continue stirring for 1 h, and centrifuge A solid was isolated and dried under vacuum at 50°C. The sample was taken for X-ray powder diffraction, and it was shown as a crystalline solid (form V, anhydrous crystal) with good crystallinity. The spectrum is shown in FIG. 3 , and the XRPD diffraction peak data is shown in Table 2. The sample was taken for DSC-TGA test. The DSC graph showed an endothermic peak at 185.87°C, and the TGA graph showed that the sample had a weight loss of 0.926% between room temperature and 170°C, as shown in Figure 4.

Table 2: XRPD diffraction peak data table of Form V in Example 2

Peak position (2θ)°	Relative Strength%	Peak position (2θ)°	Relative Strength%	Peak position (2θ)°	Relative Strength%
6.512	7.9	17.015	11.3	21.024	34.3
9.371	14.6	18.270	30.3	22.823	100.0
11.337	83.5	18.615	7.2	23.541	44.1
13.115	5.2	18.920	5.1	24.436	5.6
16.235	32.2	19.740	9.4	29.455	7.4
16.711	17.8	20.657	8.0	the	the

Note: Select peaks with relative peak intensity >5.0% to list in the table.

Embodiment 3: Preparation of formula (A) compound crystal form XIII

In a suitable container at room temperature, weigh about 80 mg of the sample of Preparation Example 1 and dissolve it in dimethylformamide (0.2 mL), stir until it dissolves, add the dissolved solution into toluene (3 mL), and continue stirring until A solid precipitated out, and was centrifuged to obtain a solid, which was dried under vacuum at room temperature.

Weigh a small amount of the obtained solid sample and spread it on a glass slide, heat it to 180°C with a hot plate and keep the temperature for 5 minutes, then cool down naturally to room temperature to obtain a solid. The sample was taken for X-ray powder diffraction, and it was shown as a crystalline solid (crystal form XIII, anhydrous crystal) with good crystallinity. The spectrum is shown in FIG. 5 , and the XRPD diffraction peak data is shown in Table 3. The sample was taken for DSC-TGA test. The DSC graph shows that the sample has two endothermic peaks at 194.07°C and 200.51°C. The TGA graph shows that the sample has a weight loss of 0.216% between room temperature and 180°C, see Figure 6.

Table 3: XRPD diffraction peak data table of crystal form XIII of Example 3

Peak position (2θ)°	Relative Strength%	Peak position (2θ)°	Relative Strength%	Peak position (2θ)°	Relative Strength%
8.926	6.7	18.196	21.5	25.727	7.6
9.516	8.2	19.073	33.4	26.294	12.1
14.291	20.7	20.809	100.0	26.885	11.9
15.816	92.8	22.135	6.4	30.754	8.2
16.593	9.2	22.462	6.2	33.664	5.6
17.848	35.6	24.388	19.2	the	the

Note: Select peaks with relative peak intensity >5.0% to list in the table.

Comparative example 1-2

A certain amount of compound sample represented by formula (A) was weighed, added with 0.4 mL of binary mixed solvent, and stirred at room temperature for 7 days or 50°C for 1 day, but no solid was obtained.

Table 4: Experimental conditions and results of comparative examples 1-2

Test Example 1: Research on Hygroscopicity of Different Crystal Forms of Compound of Formula (A)

The crystalline form I (Example 1) and the crystalline form V (Example 2) of the compound of formula (A) were placed in the DVS sample chamber for testing. The samples after DVS were taken for X-ray powder diffraction.

Experimental results:

Table 5: DVS test results of different crystal forms

Embodiment and initial crystal form	△W(RH=95%)	ΔW(RH=0%)	After DVS crystal form
Example 1/Form I	+0.13%	0%	Crystal form unchanged
Example 2/Form V	+0.06%	-0.08%	Crystal form unchanged

The data show that: Form I and Form V have no hygroscopicity.

Test Example 2: Solid Stability Experiment of Different Crystal Forms of the Compound of Formula (A)

An appropriate amount of formula (A) compound crystalline form I (Example 1) and crystalline form V (Example 2) samples were placed in a weighing bottle, at high temperature (60°C) and high humidity (25°C/92.5%RH) , Light (25°C/4500lux) and accelerated (40°C/75%RH) conditions were left open for 7 days and 15 days respectively, and samples were taken for X-ray powder diffraction to investigate the formula (A) compound crystal form I ( Example 1) and the stability of Form V (Example 2) under different conditions.

Table 6: Results of solid stability experiments of different crystal forms

The data show that both the crystal form I of Example 1 and the crystal form V of Example 2 can maintain stable crystal forms under high temperature, high humidity, light and accelerated conditions, and can also maintain chemical stability.

Test Example 3: Stability experiment of different crystal forms of the compound of formula (A) in different pH solutions

Investigate the stability of formula (A) compound crystal form I (embodiment 1) and crystal form V (embodiment 2) in the buffer medium of pH 2-7.

Table 7: Stability test results of different crystal forms in different pH solutions

The data show that both the crystal form I of Example 1 and the crystal form V of Example 2 can maintain stable crystal forms in solutions with different pHs.

Test Example 4: Stability experiment of different crystal forms of the compound of formula (A) in biosolvent media

The stability of crystal form I (Example 1) and Form V (Example 2) of the compound of formula (A) in pure water and three biosolvent media (FaSSIF, FeSSIF and FaSSGF) was investigated.

Table 8: Stability test results of different crystal forms in biosolvent media

The data show that the crystalline form I of Example 1 and the crystalline form V of Example 2 can maintain stable crystal forms in water and different biosolvent media.

Test Example 1: The inhibitory effect test of the compound of the present application on CDK9, CDK1, CDK2, CDK4, CDK5, CDK6 and CDK7

1. Purpose of the experiment

Detect the inhibitory effect of the compound on CDK1/2/4/5/6/7/9 kinase, and draw the effective IC ₅₀ value.

2. CDKs detection family

CDK1/CDK2/CDK4/CDK5/CDK6/CDK7/CDK9

Table 9: Information on Kinases, Substrates and ATP in In Vitro Assays

3. Detection process

3.1 Compound dilution

The compound of formula (A) was diluted with DMSO to 11 concentrations, 3-fold dilution, wherein the highest concentration of the reference compound staurosporine was 1 μM, and the highest concentration of the test compound was 10 μM.

3.2 Enzyme reaction

Compounds (50 nL) dissolved in DMSO were transferred to enzyme reaction plates using sonication (Echo). Add 5 μL of CDK enzyme dilution to the enzyme reaction plate, centrifuge and incubate at room temperature for 10 minutes. Take 5 μL of the substrate premix and add it to the plate. The final concentrations of the substrate and ATP in each well are shown in Table 1. After centrifugation, react at 30°C for 120 minutes.

3.3 Termination reaction and signal detection

Add 10 μL of stop solution to each well, incubate at room temperature for 120 minutes after centrifugation, and then incubate overnight at 4°C. The signal value was read using the HTRF program on the Envision instrument, and data analysis was performed, and the IC ₅₀ (inhibitory concentration at 50% of the maximum effect) value was expressed in nM. The results are shown in Table 10.

Table 10: Inhibitory effects of compounds represented by formula (A) on CDK1, 2, 4, 5, 6, 7 and 9

compound	CDK1	CDK2	CDK4	CDK5	CDK6	CDK7	CDK9
Compound A	93.21	563.61	784.82	283.52	2795.36	4443.59	6.92

The above test shows that the compound represented by formula (A) has selective and effective inhibitory effect on CDK9 inhibition.

Test Example 2: In vitro inhibitory effect of the compound represented by formula (A) on the proliferation of different tumor cell lines

1. Purpose of the test

The in vitro inhibitory effect of the compound represented by formula (A) on the proliferation of various tumor cell lines was investigated.

2. Test principle

The trade name of MTT is thiazolium blue, which is a tetrazolium salt of a dye that can accept hydrogen atoms. Amber dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to insoluble blue-purple crystals and deposit them in cells, while dead cells have no such function. Dimethyl sulfoxide can dissolve the blue-purple complex in the cells, and its light absorption value is measured at a wavelength of 490-550nm with an enzyme-linked immunosorbent detector, which can indirectly reflect the number of cells. Within a certain cell number range, the amount of MTT crystal formation is proportional to the cell number. The drug to be tested was diluted to different concentrations in sequence, and added to a 96-well plate. After a certain period of drug action, the OD value was measured. The OD value can reflect the number of living cells, and the IC ₅₀ value was calculated with SPSS19.0.

3. Test equipment

Model 371 _CO2 Incubator: Thermo Corporation

IX70-142 Inverted Fluorescent Microscope: Olympus

HFsafe-1500 biological safety cabinet: Shanghai Lishen Scientific Instrument Co., Ltd.

Varloskan flash microplate reader: Thermo company

Precision electronic balance: Mettler AL204

TD6 centrifuge: Changsha Xiangrui Centrifuge Co., Ltd.

4. Test material:

4.1 Cells and media

Note: Culture medium manufacturer: Gibco.

4.2 Test material

name	Specification	Manufacturer
fetal bovine serum	500mL/bottle	Cellmo
PBS	2L/bag	Solarbio
DMSO	500mL/bottle	recovery
MTT	5g/bottle	Amresco
0.25% Trypsin-EDTA	500mL/bottle	Gibco
MEM NEAA	100mL/bottle	Gibco
sodium pyruvate	100mL/bottle	Gibco
Puromycin	1mL	Gibco

4.3 Reagent preparation

5mg/mL MTT working solution: Weigh 0.5g of MTT and dissolve in 100mL PBS, filter and sterilize with a 0.22μm microporous membrane, and store in a 4°C refrigerator (use within two weeks) or -20°C for long-term storage.

5. Test method

5.1 Planking

Suspension cells: Count after resuspension by centrifugation. After making a certain density of cell suspension with complete medium, pipette and blow evenly to inoculate in a 96-well plate, 100 μL per well, and then culture in a CO ₂ incubator, and use it for detection after 24 hours of attachment.

5.2 Drug preparation

Weigh an appropriate amount of the compound represented by formula (A) and add the calculated amount of DMSO to dissolve, aliquot and store at -20°C; the prepared concentration is 10mM.

5.3 Dosing

Dilute the 10mM concentration stock solution of the compound represented by formula (A) into DMSO solutions of different concentrations (8 concentrations) of the compound (3-fold dilution, 20×final concentration), and take the DMSO solutions (10 μL) of the compounds of each concentration respectively, and use The cell culture medium (90 μL) was diluted to make a working solution (2×final concentration), and the working solution (100 μL) of compounds of various concentrations was added to a 96-well plate inoculated with cells (1× final concentration, the highest final concentration was 1000nM), each drug concentration was set up with 3 replicate wells, and corresponding blank wells (medium only) and normal wells (drug concentration 0) were set up, and the culture was continued in a _CO2 incubator.

6. Detection

Take out the 96-well plate, and observe the degree of cell overgrowth under a microscope. It was detected by MTT method.

MTT method: Add 20 μL of MTT to each well, incubate in the incubator for about 4 hours, discard the liquid in the well, add 150 μL DMSO to each well, place in a shaker for 5-10 minutes, and detect with a microplate reader at a wavelength of 550 nm.

Use the following formula to calculate the inhibition rate of cell growth: Inhibition rate (%) = (OD value of normal well - OD value of administration well) / (OD value of normal well - OD value of blank well) × 100%

7. Data analysis

Use SPSS19.0 statistical software to calculate the IC ₅₀ value of the drug.

See Table 11 for the cell proliferation inhibition results of the compound represented by formula (A).

Table 11: Cell proliferation inhibition test data of compounds represented by formula (A)

cell type	IC50 (nM)
MVs 4-11	34
Hep3B-Luc	71
SMMC7721-Luc	119
PLC/PRF/5	292
DU145	85.5
MDA-MB-231	64.4
RPMI-8826	96.8
MM.1S	22.3
SU-DHL-4	11.2
Jeko-1	21.6

Experimental conclusion: According to the data in the table, the compound represented by formula (A) has a strong inhibitory effect on various tumor cell lines.

Test Example 3: Investigation of hERG Inhibitory Activity in Vitro

1. Purpose of the test:

Rapidly activating human delayed rectifier outward potassium current (IKr), mainly mediated by hERG ion channels, is involved in human cardiomyocyte repolarization. Blocking this current by drugs is the main reason for the clinical appearance of QT prolongation syndrome, even acute arrhythmia and even sudden death. Using the whole-cell patch clamp technique, the blocking effect of the compound represented by formula (A) on hERG channel was detected on the CHO-K1 cell line stably expressing hERG channel, and the half maximal inhibitory concentration IC ₅₀ of the compound was determined as a comprehensive As part of the cardiac safety assessment, a preliminary evaluation of its in vitro safety screening for cardiotoxicity was carried out.

2. Test method:

This test includes the following aspects:

●Using manual patch clamp technique to record hERG current on CHO-K1 cell line stably expressing hERG channel;

Calculate the inhibition rate of each concentration according to the hERG tail current;

●Each compound was tested at 5 concentrations, and the IC ₅₀ value was calculated;

●Test 3 cells for each concentration;

• A positive control drug.

hERG currents were recorded using the whole-cell patch clamp technique. Take the cell suspension and add it to the cell tank, and place it on the stage of the upright microscope. After the cells adhered to the wall, they were perfused with extracellular fluid at a flow rate of 1–2 mL/min. The glass microelectrode is drawn in two steps by a microelectrode pulling instrument, and its water resistance value is 2-5MΩ. After establishing whole-cell recordings, the clamping potential was maintained at -80mV. Depolarization to +60mV and repolarization to -50mV elicited hERG tail currents when voltage stimulation was given. All recordings were performed after the current was stabilized. The administration of extracellular perfusion starts at a low concentration, and each concentration lasts for 5-10 minutes until the current is stable, and then the next concentration is given. The half maximal inhibitory concentration ( _IC50 ) of the test compound was obtained by the best fit of the Logistic equation.

Amitriptyline is one of the most widely used drugs for blocking hERG current, so it was used as a positive control drug in this study.

3. The results are shown in Table 12:

Table 12: IC ₅₀ values of compounds represented by formula (A) on hERG current recorded on CHO-K1 stable cell line

In the above test, the IC ₅₀ result of the active control drug amitriptyline on hERG current inhibition is consistent with the historical results of the test party and also with the results reported in the literature, indicating that the results of this test are credible. The above test results show that the compound shown in the measured formula (A) can not reach the _half -inhibition of hERG current at the highest test concentration, so the IC cannot be measured, indicating that within the detection concentration range of this test, the compound shown in the formula (A) The compound has no obvious inhibitory effect on the hERG channel, which can reflect to a certain extent that the compound represented by the formula (A) has low or no cardiotoxicity, and has positive significance for drug safety evaluation.

Test Example 4: In vivo investigation of anti-tumor activity of drugs - in vivo pharmacodynamics of the compound represented by formula (A) in human acute myeloid leukemia MV 4-11 cell subcutaneous xenograft tumor model

Cell culture: IMDM medium containing 10% fetal bovine serum (FBS), 37°C, 5% CO ₂ .

NODSCID mice, female, 6-8 weeks old, weighing about 18-22 grams, were subcutaneously inoculated with 0.1 mL (1×10 ⁸ ) of MV 4-11 cells on the right back of each mouse. When the average tumor volume reached 150 cubic millimeters, administration began, and the dosage and method of administration were shown in the table below. The tumor volume was measured twice a week, and the volume was measured in cubic millimeters. When the average tumor volume of the solvent group grew to more than 800 cubic millimeters, the administration was terminated, and the difference in average tumor volume between the test compound group and the solvent group was compared. The antitumor efficacy of compounds was evaluated by TGI (%). TGI (%) reflects tumor growth inhibition rate.

Solvent: DMSO:HP-β-CD (0.5g/mL):water ratio is 2%:20%:78% (v/v/v).

Calculation of TGI (%): TGI (%)=[1-(average tumor volume at the end of certain treatment group administration-average tumor volume at the beginning of this treatment group)/(average tumor volume at the end of solvent control group administration-solvent The average tumor volume at the beginning of the control group)]×100%. The results are shown in Table 13.

Table 13: In vivo tumor inhibition test data

group	number of animals	Method of administration	Dosage	Days of administration	TGI(%)
Solvent group	5	qd, p.o.	--	16	--
Compound A	5	qd, p.o.	5mg/kg	16	62.9

Test Conclusions:

The compound represented by the formula (A) exhibits good drug efficacy in vivo in the human acute myeloid leukemia MV 4-11 cell subcutaneous xenograft tumor model, and has a significant tumor inhibitory effect.

Test Example 5: In vivo investigation of the antitumor activity of the drug - the in vivo drug effect of the compound represented by formula (A) in the subcutaneous xenograft tumor model of human promyelocytic acute leukemia HL-60 cells.

Cell culture: IMDM medium containing 20% fetal bovine serum (FBS), 37°C, 5% CO ₂ ;

NU/NU mice, female, 6-8 weeks old, weighing about 18-22 grams, each mouse was subcutaneously inoculated with 0.1 mL of HL-60 cell suspension (containing about 1×10 ⁷ cells) in the armpit of the right forelimb. When the average tumor volume reached 150 cubic millimeters, the drug was started, and the dosage and method of administration were shown in the table below. The tumor volume was measured 2-3 times a week, and the volume was measured in cubic millimeters. When the average tumor volume in the solvent group grew to more than 800 cubic millimeters, the administration was terminated, and the difference in average tumor volume between the test compound group and the solvent group was compared. The antitumor efficacy of compounds was evaluated by TGI (%). TGI (%) reflects tumor growth inhibition rate.

Solvent: DMSO:HP-β-CD (0.5g/mL):water ratio is 2%:20%:78% (v/v/v).

Calculation of TGI (%): TGI (%)=[1-(average tumor volume at the end of certain treatment group administration-average tumor volume at the beginning of this treatment group)/(average tumor volume at the end of solvent control group administration-solvent The average tumor volume at the beginning of the control group)]×100%. The results are shown in Table 14.

Table 14: In vivo tumor inhibition test data

group	number of animals	Method of administration	Dosage	Days of administration	TGI(%)
Solvent group	5	qd, p.o.	--	9	--
Compound A	5	qd, p.o.	5mg/kg	9	58.0

Test Conclusions:

The compound represented by formula (A) exhibits good drug efficacy in vivo in the subcutaneous xenograft tumor model of human promyelocytic acute leukemia HL-60 cells. Nine days after the start of administration, the compound represented by formula (A) has a significant tumor-inhibiting effect.

Test Example 6: Investigation of Drug Toxicity to Mice

Experimental animals: ICR mice (5 weeks), male and female

Solvent: Solvent: DMSO:HP-β-CD (0.5g/mL):water ratio is 2%:20%:78% (v/v/v).

Test method: ICR mice were divided into 7 groups according to weight balance, with 5 males and 5 mice in each group. The method of administration is intragastric administration, once a day, for 7 consecutive days, and the dosage and results are shown in the table below.

test results:

Test conclusion: With the increasing dose, the control drug BAY1251152 exhibited dose-dependent toxicity, and the number of animal deaths increased; Compound A of the present application did not appear to die in experimental animals at the same dose as the control drug. It can be seen that the compound A of the present application A was significantly better tolerated than the control drug BAY1251152 in both female and male animals.

While the foregoing application has been described in some detail, by way of illustration and example, for purposes of clarity of understanding, it should be apparent from the teachings of the present application that one of ordinary skill in the art can, without departing from the spirit or scope of the appended claims, It is subject to certain changes and modifications.

Claims (13)

1. A compound represented by formula (A) in solid form,

   
2. A compound represented by formula (A) in crystalline form.
3. The compound represented by formula (A) in crystalline form according to claim 2, wherein the crystalline form is a solvent-free and anhydrous crystalline form or a hydrate crystalline form, preferably a solvent-free and anhydrous crystalline form.
4. The crystal form I of the compound shown in formula (A) is characterized in that, using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.3±0.2°, 19.4±0.2°, 20.1 ±0.2°;

   Alternatively, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.3±0.2°, 19.4±0.2°, 20.1±0.2°, 23.0±0.2°;

   Alternatively, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.3±0.2°, 19.4±0.2°, 20.1±0.2°, 21.5±0.2°, 23.0±0.2°;

   Alternatively, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.3±0.2°, 19.4±0.2°, 20.1±0.2°, 23.0±0.2°, 26.1±0.2°;

   Alternatively, it has an X-ray powder diffraction pattern substantially as shown in FIG. 1 .
5. The crystal form I according to claim 4, whose differential scanning calorimetry curve has an endothermic peak at 204.33±5°C;

   Alternatively, it has a DSC profile substantially as shown in FIG. 2 .
6. The crystal form I according to claim 4, whose thermogravimetric analysis curve has a weight loss of 0.565%±0.2% between room temperature and 180°C±5°C;

   Alternatively, it has a TGA profile substantially as shown in FIG. 2 .
7. The crystal form V of the compound shown in formula (A) is characterized in that, using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 11.3 ± 0.2 °, 18.3 ± 0.2 °, 22.8 ±0.2°;

   Alternatively, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 11.3±0.2°, 16.2±0.2°, 18.3±0.2°, 21.0±0.2°, 22.8±0.2°;

   Alternatively, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 11.3±0.2°, 16.2±0.2°, 18.3±0.2°, 21.0±0.2°, 22.8±0.2°, 23.5±0.2°;

   Alternatively, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.4±0.2°, 11.3±0.2°, 16.2±0.2°, 18.3±0.2°, 19.7±0.2°, 21.0±0.2°, 22.8 ±0.2°, 23.5±0.2°;

   Alternatively, it has an X-ray powder diffraction pattern substantially as shown in FIG. 3 .
8. The crystal form V according to claim 7, characterized in that its differential scanning calorimetry curve has an endothermic peak at 185.87±5°C;

   Alternatively, it has a DSC profile substantially as shown in FIG. 4 .
9. The crystal form V according to claim 7, characterized in that its thermogravimetric analysis curve has a weight loss of 0.926%±0.2% between room temperature and 170±5°C;

   Alternatively, it has a TGA profile substantially as shown in FIG. 4 .
10. The crystal form XIII of the compound represented by formula (A) is characterized in that, using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 15.8±0.2°, 19.1±0.2°, 20.8 ±0.2°;

    Alternatively, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 14.3±0.2°, 15.8±0.2°, 17.8±0.2°, 19.1±0.2°, 20.8±0.2°;

    Alternatively, it has an X-ray powder diffraction pattern substantially as shown in FIG. 5 .
11. A crystalline composition comprising the crystalline form I of any one of claims 4-6, the crystalline form V of any one of claims 7-9, the crystalline form XIII of claim 10, or Two or more of these.
12. A pharmaceutical composition comprising the compound of formula (A) in solid form according to claim 1, or the compound of formula (A) in crystalline form according to claim 2 or 3, or the crystalline compound of claim 11 combination.
13. According to the compound shown in the formula (A) of the solid form according to claim 1, the compound shown in the formula (A) of the crystal form described in claim 2 or 3, the formula (A) described in any one of claims 4 to 10 ), the crystalline form of the compound shown in ), the crystalline composition of claim 11, or the use of the pharmaceutical composition of claim 12 in the preparation of medicines for treating CDK9-mediated diseases; preferably, the CDK9 Mediated diseases include cell proliferative or inflammatory diseases.

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