Crystal form of heteroaryl derivative PARP inhibitor and application thereof

Abstract

The invention relates to a crystal form of a compound N-cyclopropyl-5-(4-((7-ethyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-3-yl) methyl) piperazine-1-yl) pyridinecarboxamide, a preparation method of the crystal form and application of the crystal form in preparation of related medicines. # imgabs0 #

Description

A crystal form of a heteroaryl derivative PARP inhibitor and its use Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to multiple crystal forms of a small molecule compound having PARP-1 inhibitory activity, and uses thereof in preparing medicines for treating related diseases. Background Art

[0002] Approximately 5% of breast cancer cases are associated with germline mutations in the BRCA1 / 2 genes (3% for BRCA1 and 2% for BRCA2). Breast cancers caused by BRCA1 mutations are mostly triple-negative (70%), while BRCA2 mutations are more likely to cause estrogen receptor-positive (ER-positive) breast cancer (70%). BRCA1 / 2 genes are tumor suppressor genes that play a crucial role in DNA damage repair and normal cell growth. Mutations in these genes can inhibit normal DNA repair after damage, leading to homologous recombination deficiency (HRD). This occurs when BRCA function is lost or other homologous recombination-related genes are mutated or lost, preventing double-strand breaks from being repaired through homologous recombination (HRR), ultimately leading to cancer. Poly(ADP-ribose) polymerase (PARP) is a DNA repair enzyme that plays a key role in the DNA repair pathway. PARP is activated when DNA breaks occur. As a molecular sensor of DNA damage, PARP recognizes and binds to DNA breaks, activating and catalyzing the poly(ADP-ribosylation) of the receptor protein, thus participating in the DNA repair process. PARP plays a key role in DNA single-strand base excision and repair. In HRD tumor cells, double-strand DNA repair is impossible, and PARP inhibitors block single-strand repair, resulting in a "synthetic lethality" effect that leads to cell death. PARP inhibitors act as a "trapping" for PARP proteins, trapping them bound to damaged DNA. This directly prevents other DNA repair proteins from binding, ultimately leading to cell death. Several PARP inhibitors have been successfully developed, such as olaparib, rucaparib, and niraparib. However, adverse reactions limit their use in combination with chemotherapeutic agents. This may be due to the lack of selectivity of marketed PARP inhibitors for the PARP family. These side effects include intestinal toxicity caused by tankyrase inhibition and hematotoxicity caused by PARP-2 inhibition. Therefore, it is of great clinical significance to develop highly selective PARP-1 inhibitors and reduce the related toxic side effects of non-selective PARP inhibitors.

[0003] When used to treat humans, it is important that a crystalline form of a therapeutic agent, such as N-cyclopropyl-5-(4-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)piperazin-1-yl)picolinamide, retain its polymorphic and chemical stability, solubility, and other physicochemical properties over time and across different manufacturing batches of the agent. If these physicochemical properties vary over time and between batches, administration of a therapeutically effective dose becomes problematic and can result in toxic side effects or ineffective treatment, particularly if a particular polymorph decomposes into less active, inactive, or toxic compounds prior to use. Therefore, it is very important to select a crystalline agent form that is stable, reproducible, and possesses physicochemical properties that are favorable for its use as a therapeutic agent.

[0004] Summary of the Invention

[0005] The present invention relates to a compound represented by formula (I), N-cyclopropyl-5-(4-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)piperazin-1-yl)picolinamide, including crystalline forms of the compound and a method for preparing the compound, as well as pharmaceutical compositions and medical applications thereof. The compounds provided by the present invention have high selectivity, good activity, and low toxic and side effects. The various crystalline forms have excellent properties such as high purity, good solubility, stable physical and chemical properties, resistance to high temperatures, high humidity, and strong light, and low hygroscopicity.

[0006] A crystalline form of a compound of formula (I):

[0007] In certain specific embodiments, the crystalline form is crystalline form B, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 19.31°±0.2°, 20.37°±0.2°, 22.23°±0.2°, 22.90°±0.2°, 23.70°±0.2°, and 27.18°±0.2°.

[0008] In certain specific embodiments, the crystalline form is crystalline form B, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 11.12°±0.2°, 15.96°±0.2°, 16.93°±0.2°, 19.31°±0.2°, 20.37°±0.2°, 22.23°±0.2°, 22.90°±0.2°, 23.70°±0.2°, 25.45°±0.2°, 26.52°±0.2°, 27.18°±0.2°, 29.14°±0.2°, and 32.78°±0.2°.

[0009] In certain specific embodiments, the crystalline form is Form B, and its X-ray powder diffraction pattern using Cu-Kα radiation is substantially as shown in FIG3 .

[0010] In certain specific embodiments, the differential scanning calorimetry analysis curve and the thermogravimetric analysis curve of Form B are shown in Figures 1 and 2, respectively.

[0011] In certain specific embodiments, the crystalline form is crystalline form E, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 10.76°±0.2°, 15.03°±0.2°, 17.79°±0.2°, and 19.28°±0.2°.

[0012] In certain specific embodiments, the crystalline form is crystalline form E, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 3.63°±0.2°, 7.18°±0.2°, 10.76°±0.2°, 15.03°±0.2°, 17.47°±0.2°, 17.79°±0.2°, 19.28°±0.2°, 21.33°±0.2°, 23.76°±0.2°, and 27.19°±0.2°.

[0013] In certain specific embodiments, the crystalline form is crystalline form E, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 3.63°±0.2°, 7.18°±0.2°, 7.80°±0.2°, 10.27°±0.2°, 10.76°±0.2°, 15.03°±0.2°, 17.27°±0.2°, 17. 47°±0.2°, 17.79°±0.2°, 19.28°±0.2°, 20.09°±0.2°, 20.63°±0.2°, 21.33°±0.2°, 22.41°±0.2°, 23.76°±0.2°, 24.02°±0.2°, 25.89°±0.2°, 27.19°±0.2°, 27.67°±0.2°.

[0014] In certain embodiments, Form E has an X-ray powder diffraction pattern substantially as shown in FIG6 using Cu-Kα radiation.

[0015] In certain specific embodiments, the differential scanning calorimetry analysis curve and the thermogravimetric analysis curve of Form E are shown in Figures 4 and 5, respectively.

[0016] In certain specific embodiments, the crystalline form is crystalline form F, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 9.66°±0.2°, 10.70°±0.2°, 14.24°±0.2°, 19.26°±0.2°, 21.11°±0.2°, and 22.10°±0.2°.

[0017] In certain specific embodiments, the crystalline form is crystalline form F, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 9.66°±0.2°, 10.70°±0.2°, 14.24°±0.2°, 17.34°±0.2°, 19.26°±0.2°, 21.11°±0.2°, 22.10°±0.2°, and 24.77±0.2°.

[0018] In certain specific embodiments, the crystalline form is Form F, which has an X-ray powder diffraction pattern substantially as shown in FIG9 using Cu-Kα radiation.

[0019] In certain specific embodiments, the crystalline form is Form F, and its differential scanning calorimetry analysis curve and thermogravimetric analysis curve are shown in Figures 7 and 8, respectively.

[0020] In certain specific embodiments, the crystalline form is crystalline form G, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 7.81°±0.2°, 10.30°±0.2°, 11.70°±0.2°, 19.44°±0.2°, and 20.66°±0.2°.

[0021] In certain specific embodiments, the crystalline form is crystalline form G, wherein, using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ positions: 7.81°±0.2°, 9.47°±0.2°, 10.30°±0.2°, 11.70°±0.2°, 12.37°±0.2°, 19.44°±0.2°, 19.75°±0.2°, 20.03°±0.2°, 20.41°±0.2°, 20.66°±0.2°, 22.49°±0.2°, and 26.77°±0.2°.

[0022] In certain specific embodiments, the crystalline form is crystalline form G, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 7.81°±0.2°, 9.47°±0.2°, 10.30°±0.2°, 11.70°±0.2°, 12.37°±0.2°, 17.27°±0.2°, 19.44°±0.2°, 19.75°±0 .2°, 20.03°±0.2°, 20.41°±0.2°, 20.66°±0.2°, 21.18°±0.2°, 21.60°±0.2°, 22.49°±0.2°, 26.38°±0.2°, 26.77°±0.2°, 27.50°±0.2°, 28.96°±0.2°, 33.69°±0.2°, and 37.22°±0.2°.

[0023] In certain specific embodiments, the crystalline form is Form G, which has an X-ray powder diffraction pattern substantially as shown in FIG12 using Cu-Kα radiation.

[0024] In certain specific embodiments, the crystalline form is Form G, and its differential scanning calorimetry analysis curve and thermogravimetric analysis curve are shown in Figures 10 and 11, respectively.

[0025] In certain specific embodiments, the crystalline form is crystalline form H, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 5.11°±0.2°, 8.73°±0.2°, 10.39°±0.2°, 15.92°±0.2°, 16.99°±0.2°, 17.34°±0.2°, 18.07°±0.2°, 21.09°±0.2°, 23.46°±0.2°, 24.79°±0.2°, 25.49°±0.2°, and 26.33°±0.2°.

[0026] In certain specific embodiments, the crystalline form is Form H, which has an X-ray powder diffraction pattern substantially as shown in FIG15 using Cu-Kα radiation.

[0027] In certain specific embodiments, the differential scanning calorimetry analysis curve and the thermogravimetric analysis curve of Form H are shown in FIG13 and FIG14 , respectively.

[0028] Optionally, the present invention provides a crystalline form of a hydrate of Compound I, optionally, the water content of the hydrate is 0.1-4, 0.1-3, 0.1-2 or 0.1-1; optionally, the water content of the hydrate is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0.

[0029] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of any of the aforementioned crystalline forms and a pharmaceutically acceptable carrier and / or excipient, preferably 1-600 mg of the therapeutically effective amount calculated as the free base. The pharmaceutical composition may be in the form of a unit dosage form (a unit dosage form is also referred to as a "dose strength").

[0030] The present invention also provides use of the crystalline form or composition described in any one of the aforementioned embodiments in the preparation of a drug for treating / preventing a PARP-mediated disease. Furthermore, the PARP-mediated disease is a tumor.

[0031] The present invention also provides a method for treating a disease in a mammal, comprising administering to a subject a therapeutically effective amount of a crystalline form or a composition thereof as described in any of the aforementioned schemes, wherein the disease is preferably a tumor, and the therapeutically effective amount is preferably 1-600 mg calculated as the free base. In some embodiments, the mammal of the present invention includes a human.

[0032] As used herein, an "effective amount" or "therapeutically effective amount" refers to administering a sufficient amount of a crystalline form disclosed herein to alleviate, to some extent, one or more symptoms of the disease or condition being treated. In some embodiments, the result is a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a crystalline form disclosed herein required to provide a clinically significant reduction in disease symptoms. Examples of therapeutically effective amounts are based on the free base and include, but are not limited to, 1-600 mg, 1-500 mg, 1-400 mg, 1-300 mg, 1-250 mg, 1-200 mg, 1-150 mg, 1-125 mg, 1-100 mg, 1-80 mg, 1-60 mg, 1-50 mg, 1-40 mg, 1-25 mg, 1-20 mg, 5-300 mg, 5-250 mg, 5-200 mg, 5-150 mg, 5-125 mg, 5-100 mg, 5-90 mg, 5-70 mg, 5-8 0mg, 5-60mg, 5-50mg, 5-40mg, 5-30mg, 5-25mg, 5-20mg, 10-600mg, 10-500mg, 10-450mg, 10-400mg, 10-300mg, 10-250mg, 10-200mg, 10-150mg, 10-125mg, 10-100mg, 10-90mg, 10-80mg, 10-70mg, 10-60mg, 10-50mg, 10-40mg, 10-30mg, 10-20mg; 20-600mg, 20-500mg, 20-400mg, 20-350mg, 20-300mg, 20-250mg, 20-200mg, 20-150mg , 20-125mg, 20-100mg, 20-90mg, 20-80mg, 20-70mg, 20-60mg, 20-50mg, 20-40mg, 20-30 mg; 50-600mg, 50-500mg, 50-400mg, 50-300mg, 50-250mg, 50-200mg, 50-150mg, 50-125 mg, 50-100mg; 100-600mg, 100-500mg, 100-400mg, 100-300mg, 100-250mg, 100-200mg;

[0033] In some embodiments, the pharmaceutical composition or formulation of the present invention contains the above-mentioned therapeutically effective amount of the crystalline form of the present invention;

[0034] The present invention relates to a pharmaceutical composition or pharmaceutical preparation, comprising a therapeutically effective amount of the crystalline form of the present invention and a carrier and / or excipient. The pharmaceutical composition can be in the form of a unit dosage form (the amount of the main drug in the unit dosage form is also referred to as a "preparation specification"). In some embodiments, the pharmaceutical composition includes, but is not limited to, 1 mg, 1.25 mg, 2.5 mg, 5 mg, 10 mg, 12.5 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 125 mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 2 75mg, 300mg, 325mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg.

[0035] A method for treating a disease in a mammal, comprising administering to a subject a therapeutically effective amount of a crystalline form of the present invention, and a pharmaceutically acceptable carrier and / or excipient, wherein the therapeutically effective amount is preferably 1-600 mg calculated as the free base, and the disease is preferably a tumor, particularly a brain tumor.

[0036] A method for treating a disease in a mammal, the method comprising administering a crystalline form of the present invention, and a pharmaceutically acceptable carrier and / or excipient, calculated as a free base, to a subject at a daily dose of 1-600 mg / day, the daily dose being a single dose or divided doses. In some embodiments, the daily dose includes but is not limited to 10-600 mg / day, 20-600 mg / day, 25-600 mg / day, 50-600 mg / day, 75-600 mg / day, 100-600 mg / day, 200-600 mg / day, 10-600 mg / day, 20-600 mg / day, 25-600 mg / day, 50-600 mg / day, 75-600 mg / day, In some embodiments, the daily dose includes but is not limited to 1 mg / day, 5 mg / day, 10 mg / day, 20 mg / day, 25 mg / day, 50 mg / day, 75 mg / day, 100 mg / day, 125 mg / day, 150 mg / day, 200 mg / day, 400 mg / day, and 600 mg / day.

[0037] The present invention relates to a kit, which may include a crystalline form in a single-dose or multi-dose form. The kit contains the crystalline form of the present invention, and the amount of the crystalline form of the present invention is the same as that in the above-mentioned pharmaceutical composition.

[0038] The amounts of the crystalline forms according to the invention in the present invention are in each case calculated as the free base.

[0039] "Preparation specifications" refers to the weight of the main drug contained in each vial, tablet or other unit preparation.

[0040] The crystalline form of the present invention is present in an amount of about 5% to about 100% by weight of the drug substance; in certain embodiments, in an amount of about 10% to about 100% by weight of the drug substance; in certain embodiments, in an amount of about 15% to about 100% by weight of the drug substance; in certain embodiments, in an amount of about 20% to about 100% by weight of the drug substance. In some embodiments, the drug substance is present at about 25% to about 100% by weight; in some embodiments, the drug substance is present at about 30% to about 100% by weight; in some embodiments, the drug substance is present at about 35% to about 100% by weight; in some embodiments, the drug substance is present at about 40% to about 100% by weight; in some embodiments, the drug substance is present at about 45% to about 100% by weight; in some embodiments, the drug substance is present at about 50% to about 100% by weight; in some embodiments, the drug substance is present at about 55% to about 100% by weight; in some embodiments, the drug substance is present at about 60% to about 100% by weight; in some embodiments, the drug substance is present at about 65% to about 100% by weight. In certain embodiments, the drug substance is present in an amount of about 70% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present in an amount of about 75% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present in an amount of about 80% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present in an amount of about 85% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present in an amount of about 90% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present in an amount of about 95% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present in an amount of about 98% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present in an amount of about 99% to about 100% by weight of the drug substance; and in certain embodiments, substantially all of the drug substance is present as substantially pure crystals.

[0041] The crystalline form of the present invention can be prepared by the following preparation method:

[0042] 1. Volatilization experiment: The clarified sample solution is exposed to evaporate at different temperatures until the solvent is dry.

[0043] 2. Slurry experiment: A supersaturated solution of the sample (with insoluble solids present) is stirred at a certain temperature in different solvent systems.

[0044] 3. Antisolvent test: Dissolve the sample in a good solvent, add an antisolvent (poor solvent), stir the precipitated solid for a short time and filter it immediately.

[0045] 4. Cooling crystallization experiment: dissolve a certain amount of sample in the corresponding solvent at high temperature, and then stir and crystallize directly at room temperature or low temperature.

[0046] 5. Polymer template experiment: Add different types of polymer materials to the sample clear solution and leave it open at room temperature to evaporate until the solvent is dry.

[0047] 6. Thermal method experiment: The sample is treated according to certain thermal method crystallization conditions and cooled to room temperature.

[0048] 7. Water vapor diffusion experiment: Place the sample in a certain humidity environment at room temperature.

[0049] The terms "good solvent" and "poor solvent" in the present invention are relative. In a pair of solvents, the one with higher solubility is a good solvent, and the one with lower solubility is a poor solvent. The solvents used in the above-mentioned preparation methods, unless otherwise specified, may be a single solvent or a combination of two or more.

[0050] The X-ray powder diffraction, DSC pattern, and TGA pattern disclosed in the present invention, and those substantially the same also fall within the scope of the present invention.

[0051] Unless stated otherwise, the terms used in the specification and claims have the following meanings.

[0052] “IC 50 ” refers to the half-maximal inhibitory concentration, which is the concentration at which half of the maximum inhibitory effect is achieved.

[0053] “Ether solvent” refers to a chain compound or a cyclic compound containing an ether bond -O- and having 2 to 10 carbon atoms. Specific examples include, but are not limited to, tetrahydrofuran, diethyl ether, propylene glycol methyl ether, methyl tert-butyl ether, isopropyl ether, or 1,4-dioxane.

[0054] "Alcohol solvent" refers to one or more "hydroxyl" substituted "C 1-6 The group is derived from one or more hydrogen atoms on the "alkyl" group, the "hydroxyl" and "C 1-6 "Alkyl" is as defined above, and specific examples include but are not limited to methanol, ethanol, isopropanol, n-propanol, isopentanol or trifluoroethanol.

[0055] “Ester solvent” refers to a combination of a lower organic acid containing 1 to 4 carbon atoms and a lower alcohol containing 1 to 6 carbon atoms, and specific examples include but are not limited to ethyl acetate, isopropyl acetate or butyl acetate.

[0056] "Ketone solvents" refer to compounds in which a carbonyl group (-C(O)-) is connected to two hydrocarbon groups. Depending on the hydrocarbon groups in the molecule, ketones can be divided into aliphatic ketones, alicyclic ketones, aromatic ketones, saturated ketones and unsaturated ketones. Specific examples include but are not limited to: acetone, acetophenone, and 4-methyl-2-pentanone.

[0057] "Nitrile solvents" refer to solvents in which one or more "cyano" groups replace "C 1-6 The group derived from one or more hydrogen atoms on the "alkyl" group, the "cyano" and "C 1-6 "Alkyl" is as defined above, and specific examples include but are not limited to acetonitrile or propionitrile.

[0058] "Halogenated hydrocarbon solvents" refer to solvents in which one or more halogen atoms replace "C 1-6 The group derived from one or more hydrogen atoms on the "alkyl", the "halogen atom" and "C 1-6 "Alkyl" is as defined above, and specific examples include but are not limited to: dichloromethane, 1,2-dichloroethane, chloroform or carbon tetrachloride.

[0059] As used herein, "the crystal of the present invention", "the crystal form of the present invention", "the crystal form of the present invention" and the like can be used interchangeably.

[0060] The "room temperature" mentioned in the present invention generally refers to 4-30°C, preferably 20±5°C.

[0061] The crystal structure of the present invention can be analyzed using various analytical techniques known to those skilled in the art, including but not limited to, X-ray powder diffraction (XRD), differential scanning calorimetry (DSC) and / or thermogravimetric analysis (TGA), also known as thermogravimetry (TG).

[0062] As used herein, "2θ or 2θ angle" refers to the peak position expressed in degrees (°) based on the setup of an X-ray diffraction experiment, and is typically the unit of the abscissa in a diffraction pattern. If the incident beam forms an angle θ with a certain lattice plane and the reflection is diffracted, the experimental setup requires recording the reflected beam in 2θ angles. It should be understood that the specific 2θ value of a specific crystal form mentioned herein is intended to represent the 2θ value (expressed in degrees) measured using the X-ray diffraction experimental conditions described herein, and the error range of the 2θ may be ±0.3, ±0.2, or ±0.1.

[0063] It is understood that the numerical values ​​described and protected by the present invention are approximate values. Variations in the numerical values ​​may be due to equipment calibration, equipment errors, crystal purity, crystal size, sample size and other factors.

[0064] It is understood that the crystal form of the present invention is not limited to the characteristic spectra that are exactly the same as the characteristic spectra described in the drawings disclosed in the present invention, such as XRD, DSC, and TGA. Any crystal form having characteristic spectra that are substantially the same or essentially the same as those described in the drawings falls within the scope of the present invention.

[0065] It is understood that, as is well known in the art of differential scanning calorimetry (DSC), the melting peak height of a DSC curve depends on many factors related to sample preparation and instrument geometry, while the peak position is relatively insensitive to experimental details. Therefore, in some embodiments, the crystalline compound of the present invention has a DSC pattern with characteristic peak positions, has substantially the same properties as the DSC pattern provided in the accompanying drawings of the present invention, and has a measurement error tolerance of within ±5°C, generally required to be within ±3°C.

[0066] "Carrier" refers to a system that does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the administered compound, and can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug and deliver the drug to the target organ. Non-limiting examples include microcapsules and microspheres, nanoparticles, liposomes, etc.

[0067] "Excipient" refers to a substance that is not itself a therapeutic agent and is used as a diluent, adjuvant, binder, and / or vehicle that is added to a pharmaceutical composition to improve its handling or storage properties or to allow or facilitate the formation of a compound or pharmaceutical composition into a unit dosage form for administration. As known to those skilled in the art, pharmaceutical excipients can provide various functions and can be described as wetting agents, buffers, suspending agents, lubricants, emulsifiers, disintegrants, absorbents, preservatives, surfactants, colorants, flavoring agents, and sweeteners. Examples of pharmaceutical excipients include, but are not limited to: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, hydroxypropyl methylcellulose, hydroxypropyl cellulose, microcrystalline cellulose, and cross-linked carboxymethylcellulose (e.g., sodium cross-linked carboxymethylcellulose); (4) tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other nontoxic compatible substances used in pharmaceutical preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG1 is a differential scanning calorimetry analysis curve of Form B of the compound represented by formula (I).

[0069] FIG2 is a thermogravimetric analysis spectrum of Form B of the compound represented by formula (I).

[0070] FIG3 is an X-ray powder diffraction pattern of Form B of the compound represented by formula (I).

[0071] FIG4 is a differential scanning calorimetry analysis curve of Form E of the compound represented by formula (I).

[0072] FIG5 is a thermogravimetric analysis spectrum of Form E of the compound represented by formula (I).

[0073] FIG6 is an X-ray powder diffraction pattern of Form E of the compound represented by formula (I).

[0074] FIG7 is a differential scanning calorimetry analysis curve of Form F of the compound represented by formula (I).

[0075] FIG8 is a thermogravimetric analysis spectrum of Form F of the compound represented by formula (I).

[0076] FIG9 is an X-ray powder diffraction pattern of Form F of the compound represented by formula (I).

[0077] FIG10 is a differential scanning calorimetry analysis curve of Form G of the compound represented by formula (I).

[0078] FIG11 is a thermogravimetric analysis spectrum of Form G of the compound represented by formula (I).

[0079] FIG12 is an X-ray powder diffraction pattern of Form G of the compound represented by formula (I).

[0080] FIG13 is a differential scanning calorimetry analysis curve of Form H of the compound represented by formula (I).

[0081] FIG14 is a thermogravimetric analysis spectrum of Form H of the compound represented by formula (I).

[0082] FIG15 is an X-ray powder diffraction pattern of Form H of the compound represented by formula (I).

[0083] FIG16 is a tumor growth curve of the mouse MDA-MB-436 subcutaneous transplanted tumor model.

[0084] FIG17 is a curve showing changes in animal body weight in a mouse MDA-MB-436 subcutaneous transplanted tumor model. DETAILED DESCRIPTION

[0085] The technical solutions of the present invention are described in detail below with reference to the accompanying drawings and embodiments, but the protection scope of the present invention includes but is not limited to them.

[0086] The structures of the compounds were determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). NMR shifts (δ) were expressed in 10-6 The unit of ppm is given. NMR measurements were performed using a Bruker Avance III 400 and Bruker Avance 300 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS).

[0087] MS was measured using Agilent 6120B (ESI) and Agilent 6120B (APCI).

[0088] HPLC analysis was performed using a LC-20AT (Shimadzu) high pressure liquid chromatograph (Kromasil 100-5-C18, 4.6 mm×250 mm).

[0089] XRD analysis was performed using a Bruker D8 Advance Diffractometer. X-ray powder diffraction analysis was performed as follows.

[0090] Table 1 XRD test parameters

[0091] The TGA and DSC images were collected on a TA 5500 thermogravimetric analyzer and a TA 2500 differential scanning calorimeter, respectively. The test parameters are shown in the following table.

[0092] Table 2 DSC and TGA test parameters

[0093] The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from companies such as Titan Technology, Anage Chemical, Shanghai Demer, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, and Bailingwei Technology.

[0094] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0095] Unless otherwise specified in the examples, the room temperature is 20°C to 30°C.

[0096] The following describes in detail the implementation process of the present invention and the beneficial effects produced by specific embodiments, which is intended to help readers better understand the essence and characteristics of the present invention and is not intended to limit the scope of implementation of this case.

[0097] DMAc: N,N-dimethylacetamide

[0098] NMP: N-methylpyrrolidone

[0099] MTBE: Methyl tert-butyl ether

[0100] Example 1: Preparation of Compound I

[0101] first step:

[0102] Ethyl 6-methyl-5-nitronicotinate (10 g, 47.6 mmol) and selenium dioxide (21.14 g, 190.5 mmol) were dissolved in 1,4-dioxane (100 ml) and refluxed at 100° C. overnight. After the reaction, the mixture was filtered through a funnel padded with diatomaceous earth, the diatomaceous earth was washed with ethyl acetate, and the filtrate was concentrated. The residue was separated and purified by silica gel column chromatography (eluent ratio: ethyl acetate: petroleum ether (v / v) = 0% to 40%) to obtain compound 1A (10.104 g, 94.7%) as a yellow oil.

[0103] LCMS (ESI) m / z = 225.1 [M+1] +

[0104] Step 2:

[0105] Sodium hydride (2.695 g, 112.3 mmol) was dissolved in anhydrous tetrahydrofuran (100 ml), stirred at 0°C, and triethyl 2-butyl acrylate (28.3 g, 112.3 mmol) was added dropwise. After the addition was completed, the mixture was stirred at 0°C for 20 min, heated to 40°C and stirred for 10 min, and transferred to a dry ice ethanol bath. Compound 1A (10.48 g, 46.8 mmol) was dissolved in anhydrous tetrahydrofuran (100 ml) and added dropwise to the reaction flask, and the dry ice was kept. The mixture was stirred in an ethanol bath for 1 h. After the reaction was completed, saturated ammonium chloride solution (100 ml) was added to quench the reaction, and ethyl acetate (200 ml) was added for extraction. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (200 ml × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (eluent ratio: ethyl acetate: petroleum ether (v / v) = 0-10%) to give compound 1B (11.57 g, 76.8%), a mixture of two isomers, as a yellow oil.

[0106] LC-MS (ESI) m / z = 323.1 [M+1] +

[0107] Step 3:

[0108] Compound 1B (11.57 g, 35.9 mmol) was dissolved in ethanol (50 ml), and 10% palladium-carbon catalyst (1 g) was added. The mixture was replaced with hydrogen three times and stirred at room temperature overnight. The mixture was filtered through a funnel padded with diatomaceous earth, and the diatomaceous earth was washed with anhydrous ethanol. The filtrate was concentrated, and 4M hydrochloric acid-dioxane solution (60 ml) was added to the residue. The mixture was stirred at room temperature for 1 h and concentrated. Ethyl acetate (50 ml) was added to the residue, stirred, and filtered. The filter cake was washed with ethyl acetate and dried to give compound 1C (4.28 g, 42.0%) as a white solid.

[0109] 1 H NMR(400MHz,DMSO-d6)δ10.39(s,1H),8.62(d,1H),7.75(s,1H),4.38–4.29(m,2H),3.24(dd,1H), 2.97(dd,1H),2.62–2.53(m,1H),1.83–1.64(m,1H),1.55–1.35(m,1H),1.33(dd,3H),0.94(t,3H).

[0110] Step 4:

[0111] Compound 1C (4.28 g, 17.3 mmol) and 2,3-dichloro-5,6-dicyanobenzoquinone (4.309 g, 19.0 mmol) were dissolved in dioxane (86 ml) and refluxed at 100° C. for 3.5 h. After the reaction, saturated sodium bicarbonate solution (40 ml) and ethyl acetate (120 ml) were added. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (120 ml × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (eluent ratio: ethyl acetate: petroleum ether = 0-50%) to obtain compound 1D (3.375 g, 79.5%) as a light yellow solid.

[0112] LC-MS (ESI) m / z = 247.1 [M+1] +

[0113] Step 5:

[0114] Compound 1D (3.375 g, 13.72 mmol) was dissolved in anhydrous tetrahydrofuran (150 ml) and stirred at -78°C. Lithium aluminum hydride (1.564 g, 41.16 mmol) was added portionwise, and the mixture was stirred at -78°C for 20 min. The mixture was then heated to -40°C and stirred for 20 min. After the reaction, 1 M hydrochloric acid was added to adjust the pH of the system to neutral. The solvent was removed by distillation under reduced pressure. 100 ml of methanol / dichloromethane (1:10) was added to the resulting residue to dissolve the residue. The mixture was ultrasonically shaken for 10 min, filtered, and the filtrate was collected. The filter cake was redissolved in 100 ml of methanol / dichloromethane (1:10). This process was repeated eight times. The filtrates were combined and concentrated to obtain compound 1E (2.8 g, 100%) as a light yellow solid.

[0115] 1 H NMR (400MHz, DMSO) δ11.86(s,1H),8.37(d,1H),7.72(d,1H),7.62(d,1H),5.44(t,1H),4.61(d,2H),2.57–2.51(m,2H),1.18(t,3H).

[0116] Step 6:

[0117] 1E (100 mg, 0.49 mmol) was added to dichloromethane (2.5 mL), and DMF (1 mL) was added to dissolve the mixture. Thionyl chloride (350 mg, 2.94 mmol) was added dropwise at 0°C and reacted at room temperature for 1 hour. LCMS showed that the reaction of the raw material was complete and the product was generated. The product was directly dried to obtain compound 1F (109 mg, crude product) for the next reaction.

[0118] LC-MS(ESI):m / z=223.1, 225.1[M+H] +

[0119] Step 7:

[0120] 5-Bromopyridine-2-carboxylic acid methyl ester (2.16 g, 10 mmol) and N-Boc-piperazine (2.03 g, 11 mmol) were dissolved in 1,4-dioxane (100 mL), and Cs2CO3 (6.5 g, 20 mmol) and RuPhos-Pd-G3 (253 mg, 0.3 mmol) were added. The reaction was carried out at 100°C under nitrogen protection overnight. The reaction was stopped after LCMS detection was completed, and the mixture was cooled to room temperature. The filtrate was collected by filtration, and the residue was washed with ethyl acetate (20 mL × 3). The filtrate was concentrated, and a small amount of anhydrous ethanol was added. The mixture was heated to dissolve, and a large amount of petroleum ether was added. After cooling, the precipitated crystals were collected to obtain compound 2 (2.37 g, 73.4%) as a light yellow solid.

[0121] LC-MS (ESI): m / z = 321.1 [M+H] + .

[0122] Step 8:

[0123] Compound 2 (400 mg, 1.24 mmol) was dissolved in THF (10 mL) and H2O (1 mL), and LiOH (30 mg, 1.24 mmol) was added. The reaction was stirred at room temperature for 2 h, and the solvent was distilled off under reduced pressure. The mixture was diluted with water and extracted with ethyl acetate (20 mL×3). The organic phases were combined, dried over anhydrous Na2SO4, filtered and dried, and DMF (10 mL) was added to the resulting solid. HATU (565 mg, 1.49 mmol) was added with stirring and stirred at room temperature. After the solid was completely dissolved, DIEPA (2 mL) was added, and finally, excess cyclopropylamine was added. The mixture was stirred at room temperature overnight. After the reaction was completed as monitored by LCMS, 50 mL of ethyl acetate was added to the system, washed with water (50 mL×4), the organic phase was collected, dried over anhydrous sodium sulfate, filtered and evaporated to dryness, and separated by silica gel chromatography (PE:EA (v / v) = 1:0 to 1:1) to give compound 3 (309 mg, 71.5%) as a light yellow solid.

[0124] LC-MS (ESI): m / z = 347.2 [M+H] + .

[0125] Step 9:

[0126] 3 (309 mg, 0.89 mmol) was dissolved in methanol (5 mL), and dioxane hydrochloride (5 mL, 4 M) solution was added. The mixture was reacted at room temperature for two hours and then dried to obtain compound 4 (200 mg, crude product).

[0127] LC-MS (ESI): m / z = 247.1 [M+H] + .

[0128] Step 10:

[0129] 1F (100 mg, 0.44 mmol) and compound 4 (200 mg, 0.81 mmol) were dissolved in anhydrous acetonitrile (10 mL), and potassium iodide (8 mg, 0.05 mmol) and DIPEA (0.5 mL) were added. After nitrogen replacement, the reaction was carried out at 80°C for 8 hours. LCMS detection showed that the reaction of the raw materials was complete and product was generated. The system was concentrated, and a saturated sodium bicarbonate solution (20 mL) was added. The mixture was extracted with a mixed solution of DCM:MeOH (v / v) = 10:1 (10 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and passed through a column (DCM:MeOH (v / v) = 1:0 to 10:1) to obtain compound I (76 mg, 38.1%).

[0130] 1 H NMR(400MHz,DMSO-d6)δ11.84(s,1H),8.40(d,1H),8.32(d,1H),8.23(d,1H),7.83(d,1 H),7.75(s,1H),7.63(d,1H),7.39(dd,1H),3.65(s,2H),3.35–3.31(m,4H,overlapped with solvent DMSO peak),2.90–2.80(m,1H),2.59–2.52(m,6H, overlapped with solvent DMSO peak),1.19(t,3H),0.66(dd,2H),0.63(q,2H).

[0131] LC-MS (ESI): m / z = 433.2 [M+H] + .

[0132] Preparation of crystal forms

[0133] Example 2: Preparation of Form B

[0134] 50 mg of the compound of Formula I was added to 1 ml of CHCl₃, suspended and stirred at room temperature for 3 days, filtered, and dried to obtain Form B. The differential scanning calorimetry curve, thermogravimetric analysis, and X-ray powder diffraction (XRD) pattern of Form B of Compound I are shown in Figures 1-3. Specific peak values ​​are shown in Table 3.

[0135] Table 3

[0136] Example 3: Preparation of Form E

[0137] 50 mg of the compound of Formula I was added to 1 ml of DMAc and slowly cooled from room temperature to obtain Form E. The sample was slowly cooled to 5°C, clarified, and then stored at -20°C for 11 days to precipitate a solid. The solid was then air-dried at room temperature for 1 day and then vacuum-dried in a vacuum drying oven at room temperature for 2 days to obtain Form E. The differential scanning calorimetry, thermogravimetric analysis, and X-ray powder diffraction (XRD) patterns of Form E of Compound I are shown in Figures 4-6. Specific peaks are shown in Table 4.

[0138] Table 4

[0139] Example 4: Preparation of Form F

[0140] 50 mg of the compound of Formula I was added to 1 mL of THF / H₂O (2:1, v / v), and 5 mL of 1,4-dioxane was added dropwise. The mixture was allowed to evaporate slowly at room temperature. The differential scanning calorimetry, thermogravimetric analysis, and X-ray powder diffraction (XRD) patterns of Form F of Compound I are shown in Figures 7-9. Specific peaks are shown in Table 5.

[0141] Table 5

[0142] Example 5: Preparation of Form G

[0143] 50 mg of the compound of Formula I was added to 1 ml of MeOH, stirred at room temperature for 3 days, and then filtered and dried to obtain Form G. The differential scanning calorimetry (DSC) curve, thermogravimetric analysis (TGA) pattern, and X-ray powder diffraction (XRD) pattern of Form G of Compound I are shown in Figures 10-12. Specific peak values ​​are shown in Table 6. Comprehensive analysis, including TGA, confirmed that Form G is an anhydrous crystalline form.

[0144] Table 6

[0145] Example 6: Preparation of Form H

[0146] 50 mg of the compound of Formula I was added to NMP until the solution became clear. MTBE was then added dropwise until solid precipitated. The mixture was stirred for 1-2 hours, filtered, and dried at room temperature for 32 hours, followed by vacuum drying at 50°C for 6 hours to obtain Form H. The differential scanning calorimetry (DSC) curve, thermogravimetric analysis (TGA) pattern, and X-ray powder diffraction (XRD) pattern of Form H of Compound I are shown in Figures 13-15. Specific peak values ​​are shown in Table 7.

[0147] Table 7

[0148] Biological test cases

[0149] 1. PARP1 enzyme activity test

[0150] PARP1 chemiluminescence detection kit was purchased from BPS Bioscience. The histone solution in the kit was diluted 5 times with 1X PBS, 25 μL of histone dilution was taken to the microplate, and incubated at 4°C overnight. After the incubation, the plate was washed 3 times with PBST (0.05% Tween-20), 100 μL of blocking solution was taken to the microplate, and incubated at 25°C for 90 minutes; after the incubation, the plate was washed 3 times with PBST. 2.5 μL of compounds of different concentrations diluted in the test buffer and 12.5 μL of substrate mixed solution (1.25 μL 10X PARP test buffer; 1.25 μL 10X PARP test mixture; 2.5 μL Activated DNA, 7.5 μL double distilled water) were added to the microplate. The PARP1 enzyme was diluted to 2 ng / μL, 10 μL was taken to the microplate, and the reaction system was incubated at 25°C for 60 minutes;

[0151] After incubation, wash the plate three times with PBST. Dilute Streptavidin-HRP 50-fold with blocking buffer, then transfer 25 μL to the microplate and incubate at 25°C for 30 minutes. After incubation, wash the plate three times with PBST. Mix ELISA ECL substrate A and substrate B at a 1:1 (v / v) ratio, transfer 50 μL to the microplate, and read the chemiluminescence value.

[0152] According to the formula [(1-(RLU sample -RLU min ) / (RLU max -RLU min )) × 100%], where RLUsample is the reading of the compound well, RLUmax is the reading of the solvent control well, and RLUmin is the reading of the control well without PARP1 enzyme. GraphPad Prism software was used to fit the curve using four parameters (log (inhibitor) vs. response--Variable slope) and calculate the IC 50 value.

[0153] Table 8

[0154] 2. PARP2, PARP5A, PARP5B, PARP6, PARP7, PARP14 and PARP15 enzyme activity test

[0155] PARP2, PARP5A, PARP5B, PARP6, PARP7, PARP14 and PARP15 chemiluminescent detection kits were purchased from BPS Bioscience. The histone solution in the kit was diluted 5-fold with 1X PBS, and 25 μL of the histone dilution solution was added to a microplate and incubated at 4°C overnight. After the incubation, the plate was washed 3 times with PBST (0.05% Tween-20), 100 μL of blocking solution was added to the microplate, and incubated at 25°C for 90 minutes; after the incubation, the plate was washed 3 times with PBST. 2.5 μL of compound 1 diluted in test buffer and 5 μL of substrate mixed solution were added to the microplate. 5 μL of diluted PARP enzyme was added to the microplate, and the reaction system was incubated at 25°C for 60 minutes.

[0156] After incubation, wash the plate three times with PBST. Dilute Streptavidin-HRP 50-fold with blocking buffer, then transfer 25 μL to the microplate and incubate at 25°C for 30 minutes. After incubation, wash the plate three times with PBST. Mix ELISA ECL substrate A and substrate B at a 1:1 (v / v) ratio, transfer 25 μL to the microplate, and read the chemiluminescence value.

[0157] According to the formula [(1-(RLU sample -RLU min ) / (RLU max -RLU min ))×100%] to calculate the inhibition rate, where RLU sample Read the compound wells, RLU max Reading value for solvent control well, RLU min The values ​​of the control wells without PARP1 enzyme were read and the IC was calculated using GraphPad Prism software using a four-parameter curve fitting (log (inhibitor) vs. response--Variable slope). 50 value.

[0158] Test results: The compounds of the present invention have a weak inhibitory effect on PARP2 enzyme activity in vitro, and their corresponding IC 50 The value is 27.47nM; the compound has a very weak inhibitory effect on the enzyme activities of PARP5A, PARP5B, PARP6, PARP7, PARP14 and PARP15 in vitro, and the corresponding IC 50 The values ​​are all greater than 500nM. The specific test results are shown in the following table.

[0159] Table 9

[0160] The compounds of the present invention have good PARP1 inhibition selectivity.

[0161] 3. MDA-MB-436 cell activity test experiment

[0162] Human breast tumor MDA-MB-436 cells were purchased from ATCC and cultured in Leibovitz's L-15 medium (supplemented with 10 μg / mL insulin, 16 μg / mL glutathione, 10% fetal bovine serum, and 1% double-antibody) at 37°C in a CO2-free incubator. On the first day, cells in the exponential growth phase were harvested and resuspended in culture medium to a cell count of 4,000 cells / 135 μL. 135 μL of this cell suspension was added to each well of a 96-well cell culture plate and incubated overnight. On the second day, various concentrations of compounds were added and the cells were incubated in an incubator for 7 days. Following the incubation period, 75 μL of pre-thawed and room temperature CTG solution was added to each well according to the instructions of the CellTiter-Glo kit (Promega, G7573). The cells were mixed using a microplate shaker for 2 minutes and then incubated at room temperature for 10 minutes before fluorescence signal was measured using an Envision 2104 plate reader (PerkinElmer). The inhibition rate was calculated using the formula [(1–(RLU compound –RLU blank ) / (RLU control –RLU blank ))×100%], where RLU compound The readings for the drug-treated groups, RLU control is the average value of the solvent control group, RLU blank The IC values ​​were calculated using GraphPad Prism software. 50 value.

[0163] Test results: The compound of the present invention has a significant inhibitory effect on breast tumor cells MDA-MB-436.

[0164] Table 10

[0165] 4. Mouse MDA-MB-436 subcutaneous transplant tumor model

[0166] Human breast cancer MDA-MB-436 cells were placed in Leibovitz's L-15 medium (supplemented with 10 μg / mL insulin, 16 μg / mL glutathione, 10% fetal bovine serum, and 1% double-antibody) and cultured at 37°C. Twice a week, routine digestion and passage were performed with trypsin. When the cell saturation reached 80%-90% and the number reached the required level, the cells were harvested, counted, and inoculated. 0.2 mL (10×10 6MDA-MB-436 cells (with Matrigel, volume ratio of 1:1) were subcutaneously inoculated into the right back of BALB / c nude mice (provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd.). The average tumor volume reached approximately 180 mm 3 The grouping and dosing began at 1:00 pm (recorded as Day 0). The vehicle group was given 5% DMSO, 30% PEG400, and 65% 20% sulfobutyl-β-cyclodextrin solution, and the drug group was given the compound (Day 0-Day 10: 1 mg / kg; Day 11-Day 28: 0.1 mg / kg). The dosing frequency was once a day for a 29-day dosing cycle, with a 14-day drug-free observation period. After grouping, tumor diameter was measured twice a week with a vernier caliper. The tumor volume was calculated as follows: V = 0.5 × a × b 2 , a and b represent the major and minor diameters of the tumor, respectively. The tumor inhibitory efficacy of the compound was evaluated using TGI (%) = [1 – (average tumor volume of a treatment group at the end of dosing – average tumor volume of that treatment group at the start of dosing) / (average tumor volume of the solvent control group at the end of treatment – ​​average tumor volume of the solvent control group at the start of treatment)] × 100%. Tumor growth curves and animal body weight change curves are shown in Figures 16 and 17, respectively.

[0167] Test results: After 28 days of administration, the compound achieved a TGI of 119%; after discontinuation of the drug, tumors in animals receiving the compound did not regenerate. There was no significant weight loss in animals receiving the compound. Together, these results demonstrate that the compound exhibits excellent efficacy in inhibiting tumor growth and inducing tumor regression, with good tolerability.

[0168] 5. Pharmacokinetic test in rats

[0169] 1.1 Experimental Animals: Male SD rats, approximately 220 g, 6 to 8 weeks old, 6 rats per compound, purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0170] 1.2 Experimental Design: On the day of the experiment, six SD rats were randomly divided into groups according to body weight. They were fasted but not watered for 12-14 hours before administration and fed 4 hours after administration.

[0171] Table 11

[0172] Note: Intravenous administration solvent: 10% DMA + 10% Solutol + 80% Saline; Oral administration solvent: 5% DMSO + 30% PEG400 + 65% (20% SBE-CD)

[0173] (DMA: dimethylacetamide; Solutol: polyethylene glycol-15-hydroxystearate; Saline: normal saline; DMSO: dimethyl sulfoxide; SBE-CD: β-cyclodextrin)

[0174] Before and after dosing, blood (0.15 mL) was collected via the orbital cavity under isoflurane anesthesia. The blood was placed in an EDTAK2 centrifuge tube and centrifuged at 5000 rpm at 4°C for 10 minutes to collect plasma. Blood was collected from both the intravenous and oral gavage groups at 0, 5, 15, 30 minutes, and 1, 2, 4, 6, 8, and 24 hours. All samples were stored at -80°C prior to analysis and quantitative analysis was performed using LC-MS / MS. The test results for some examples are shown below.

[0175] Table 12

[0176] -:not applicable.

[0177] Conclusion: The compound has good pharmacokinetic characteristics in rats.

[0178] Crystal form test example

[0179] Stability testing

[0180] Samples were tested at 92.5% RH, 40°C, 60°C, etc., and the purity (expressed as a percentage) was detected by HPLC. The experimental results are shown in Table 15.

[0181] The preparation method of the test solution and the HPLC purity test conditions are shown in Tables 13 and 14;

[0182] Table 13 Preparation method of test solution

[0183] Table 14 HPLC purity test conditions

[0184] Table 15 Chemical stability of Form G under different conditions (purity determined by HPLC)

[0185] Note: RRT stands for relative retention time;

[0186] Conclusion: Form G has good chemical stability.

Claims

1. A crystalline form of a compound of formula (I):

2. The crystal form according to claim 1, wherein The crystal form is crystal form B, and using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ positions: 19.31°±0.2°, 20.37°±0.2°, 22.23°±0.2°, 22.90°±0.2°, 23.70°±0.2°, and 27.18°±0.2°.

3. The crystalline form according to claim 2, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ positions: 11.12°±0.2°, 15.96°±0.2°, 16.93°±0.2°, 19.31°±0.2°, 20.37°±0.2°, 22.23°±0.2°, 22.90°±0.2°, 23.70°±0.2°, 25.45°±0.2°, 26.52°±0.2°, 27.18°±0.2°, 29.14°±0.2°, and 32.78°±0.2°.

4. The crystalline form according to claim 2 or 3, whose X-ray powder diffraction pattern is substantially as shown in Figure 3.

5. The crystal form according to claim 1, wherein The crystalline form is crystalline form E, and using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ positions: 3.63°±0.2°, 7.18°±0.2°, 10.76°±0.2°, 15.03°±0.2°, 17.47°±0.2°, 17.79°±0.2°, 19.28°±0.2°, 21.33°±0.2°, 23.76°±0.2°, and 27.19°±0.2°.

6. The crystalline form according to claim 5, using Cu-Kα radiation, has an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2θ positions: 3.63°±0.2°, 7.18°±0.2°, 7.80°±0.2°, 10.27°±0.2°, 10.76°±0.2°, 15.03°±0.2°, 17.27°±0.2°, 17.47 °±0.2°, 17.79°±0.2°, 19.28°±0.2°, 20.09°±0.2°, 20.63°±0.2°, 21.33°±0.2°, 22.41°±0.2°, 23.76°±0.2°, 24.02°±0.2°, 25.89°±0.2°, 27.19°±0.2°, 27.67°±0.2°.

7. The crystalline form according to claim 5 or 6, using Cu-Kα radiation, has an X-ray powder diffraction pattern substantially as shown in FIG6.

8. The crystal form according to claim 1, wherein The crystal form is crystal form F, and using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ positions: 9.66°±0.2°, 10.70°±0.2°, 14.24°±0.2°, 17.34°±0.2°, 19.26°±0.2°, 21.11°±0.2°, 22.10°±0.2°, and 24.77±0.2°.

9. The crystalline form according to claim 8, using Cu-Kα radiation, has an X-ray powder diffraction pattern substantially as shown in FIG9.

10. The crystal form according to claim 1, wherein The crystalline form is crystalline form G, and using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ positions: 7.81°±0.2°, 9.47°±0.2°, 10.30°±0.2°, 11.70°±0.2°, 12.37°±0.2°, 19.44°±0.2°, 19.75°±0.2°, 20.03°±0.2°, 20.41°±0.2°, 20.66°±0.2°, 22.49°±0.2°, and 26.77°±0.2°.

11. The crystalline form according to claim 10, using Cu-Kα radiation, has an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2θ positions: 7.81°±0.2°, 9.47°±0.2°, 10.30°±0.2°, 11.70°±0.2°, 12.37°±0.2°, 17.27°±0.2°, 19.44°±0.2°, 19.75°±0.2° °, 20.03°±0.2°, 20.41°±0.2°, 20.66°±0.2°, 21.18°±0.2°, 21.60°±0.2°, 22.49°±0.2°, 26.38°±0.2°, 26.77°±0.2°, 27.50°±0.2°, 28.96°±0.2°, 33.69°±0.2°, 37.22°±0.2°.

12. The crystalline form according to claim 10 or 11, using Cu-Kα radiation, has an X-ray powder diffraction pattern substantially as shown in Figure 12.

13. The crystal form according to claim 10 or 11, wherein the differential scanning calorimetry analysis curve and the thermogravimetric analysis curve are shown in FIG10 and FIG11 respectively.

14. The crystal form according to claim 1, wherein The crystalline form is crystalline form H, and using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ positions: 5.11°±0.2°, 8.73°±0.2°, 10.39°±0.2°, 15.92°±0.2°, 16.99°±0.2°, 17.34°±0.2°, 18.07°±0.2°, 21.09°±0.2°, 23.46°±0.2°, 24.79°±0.2°, 25.49°±0.2°, and 26.33°±0.2°.

15. The crystalline form according to claim 14, which has an X-ray powder diffraction pattern substantially as shown in Figure 15 using Cu-Kα radiation.

16. A pharmaceutical composition, wherein The pharmaceutical composition contains a therapeutically effective amount of the crystalline form according to any one of claims 1 to 15, and a pharmaceutically acceptable carrier and / or excipient. Preferably, the therapeutically effective amount is 1 to 600 mg calculated as the free base.

17. Use of the crystalline form according to any one of claims 1 to 15, or the pharmaceutical composition according to claim 16, in the preparation of a medicament for treating tumors.

18. A method for treating a disease in a mammal, the method comprising administering to a subject a therapeutically effective amount of the crystalline form according to any one of claims 1 to 15, wherein the therapeutically effective amount is preferably 1 to 600 mg calculated as the free base, and the disease is preferably a tumor.

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