A composition comprising a heterocyclic shp2 inhibitor and uses thereof

Abstract

The application belongs to the field of pharmaceutical preparations and relates to a composition containing a heterocyclic SHP2 inhibitor and use thereof, in particular to a pharmaceutical composition containing (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous thereof. The pharmaceutical composition provided by the application is completely dissolved, has good stability and good absorption properties, and has a relatively high relative bioavailability.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations and relates to a composition containing a heterocyclic SHP2 inhibitor and its use, specifically to a pharmaceutical composition containing (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidine-4(3H)-one or a pharmaceutically acceptable salt, hydrate, solvate, crystalline or amorphous form thereof. Background Technology

[0002] SHP2 phosphatase is a non-receptor PTP (protein tyrosine phosphatase) encoded by the PTPN11 gene. It comprises two N-terminal SRC (sarcoma gene) homologous domains (SH2), a PTP domain, and a C-terminal tail. X-ray diffraction results indicate that SHP2 interacts with the PTP domain via the N-terminal SH2 domain, blocking ATP entry into the catalytic site; this kinase exists in a self-inhibitory conformation. Some small peptides or proteins that bind to the SH2 domain can activate phosphorylation of this enzyme, promoting cancer development. In cells, SHP2 function is related to downstream receptor tyrosine kinases in the cytoplasm, including RAS-ERK, PI3K-AKT, and JAK-STAT. First, SHP2 binds to RAS and dephosphorylates it, thereby increasing the activity of the effector protein RAF and activating the RAS / ERK / MAPK proliferative signaling pathway. Second, SHP2 participates in the PD-1 / PD-L1 signaling pathway and promotes immune escape. The PD-1 / SHP2 / STAT1 / T-bet signaling axis mediates the immunosuppressive effect of PD-1 on Th1 cells. Therefore, inhibiting PD-1 or SHP2 can restore the immune function of Th1 cells and activate T cells, thereby relieving immunosuppression in the tumor microenvironment.

[0003] SHP2 is associated with the development of a variety of diseases, such as Noonan syndrome, breast cancer, melanoma, gastric cancer, esophageal cancer, lung cancer, colon cancer, head cancer, neuroblastoma, squamous cell carcinoma of the head and neck, anaplastic large cell lymphoma, and glioblastoma.

[0004] Inhibitors targeting the catalytic site of SHP2 generally exhibit poor selectivity and druggability. In recent years, researchers have discovered that inhibiting SHP2 activity through allosteric site modification can improve both activity and selectivity, leading to some progress in drug research. However, the development of superior SHP2 inhibitors remains crucial to obtain drugs with enhanced activity and pharmacokinetic properties for the treatment of SHP2-mediated diseases. Summary of the Invention

[0005] The inventors of this invention have discovered an SHP2 inhibitor, the compound structure of which is shown in Formula (I) below, and its chemical name is (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one (hereinafter referred to as "Compound (I)").

[0006]

[0007] The inventors of this invention have discovered that the compound of formula (I) or its hydrate, solvate or crystals exhibit significant inhibitory activity against SHP2, and are very promising therapeutic agents for SHP2-related diseases.

[0008] To date, no suitable formulations of compounds of formula (I) or their isomers or pharmaceutically acceptable salts have been reported. It is well known in the art that many challenges exist in the formulation of such preparations, such as long-term stability, control of related substances, drug absorption, and bioavailability. These challenges are determined by a variety of factors, including many factors affecting drug absorption, such as drug dissolution or release from the formulation, drug solubility under physiological conditions, and permeability in the gastrointestinal tract.

[0009] Therefore, it is necessary to study compounds of formula (I) or their derivatives in order to provide suitable formulations that meet clinical needs.

[0010] The inventors of this invention discovered in previous studies that the compound of formula (I) is almost insoluble in water, ethanol, and 0.01 mol / L hydrochloric acid solution.

[0011] One object of the present invention is to provide a pharmaceutical composition comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystalline or amorphous form thereof; and (b) a disintegrant, wherein the pharmaceutical composition of the present invention can be completely dissolved in a formulation and has high bioavailability.

[0012] Another object of the present invention is to provide a pharmaceutical preparation containing the pharmaceutical composition of the present invention.

[0013] Another object of the present invention is to provide the use of the pharmaceutical compositions of the present invention in the preparation of medicaments for treating and / or preventing SHP2-mediated diseases.

[0014] To achieve the above objectives, the present invention provides the following technical solution:

[0015] In one aspect, the present invention provides a pharmaceutical composition comprising:

[0016] (a) A compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystalline or amorphous form thereof.

[0017]

[0018] and (b) disintegrants.

[0019] In some specific embodiments, the pharmaceutical composition of the present invention comprises a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof, a disintegrant, a lubricant and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof accounts for about 0.1% to 60% (by weight), preferably about 0.5% to 20% (by weight), and more preferably about 0.5% to 18% (by weight) of the total weight of the pharmaceutical composition.

[0020] In some specific embodiments, the pharmaceutical composition of the present invention comprises a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof, and a disintegrant, wherein the disintegrant is selected from one or more of sodium carboxymethyl starch (CMS-Na), crospovidone, crospovidone carboxymethyl cellulose (CCNa), and low-substituted hydroxypropyl cellulose (L-HPC), preferably one or more of sodium carboxymethyl starch, crospovidone carboxymethyl cellulose, and low-substituted hydroxypropyl cellulose, and more preferably crospovidone carboxymethyl cellulose. It is known in the art that, within a certain range, increasing the disintegrant dosage can promote disintegration and drug dissolution; however, beyond a certain amount, its pro-disintegration effect gradually weakens, and may even hinder drug dissolution. Therefore, it is necessary to study the content and method of use of the disintegrant to achieve the best disintegration effect. In some embodiments, the disintegrant in the pharmaceutical composition provided by the present invention accounts for about 1% to 30% (by weight), preferably 2% to 15% (by weight), and more preferably 2% to 10% (by weight). The inventors of the present invention have found that formulations prepared using the pharmaceutical composition of the present invention containing the above-mentioned disintegrant can significantly improve the dissolution of the compound of formula (I), and the formulations are stable under high humidity, high temperature, light exposure, and accelerated and long-term testing conditions, with few impurities.

[0021] In some embodiments, the pharmaceutical composition provided by the present invention further comprises a binder selected from one or more of alginate, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, carboxymethyl cellulose, powdered cellulose, gelatin, magnesium aluminum sulfate, maltodextrin, povidone, copovidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, methylcellulose, gum arabic, sodium alginate, guar gum, starch, pregelatinized starch, and sucrose. More preferably, the binder is selected from one or more of povidone, hydroxypropyl methylcellulose, and hydroxypropyl cellulose. It is known in the art that an appropriate increase in the amount of binder in the formulation can improve the hardness and flowability of the granules after granulation. However, excessive use can make the granulation process difficult to control, causing the granules to become too wet. Furthermore, it can lead to difficulty in granule disintegration and slow drug dissolution. Therefore, the content of the binder needs to be studied. In some embodiments of the present invention, the binder in the pharmaceutical composition provided by the present invention accounts for about 0% to 30% (weight / weight) of the total weight of the pharmaceutical composition, preferably about 5% to 25% (weight / weight), and more preferably about 5% to 20% (weight / weight).

[0022] In some embodiments, the pharmaceutical composition provided by the present invention further comprises a lubricant, wherein the lubricant is selected from one or more of magnesium stearate, calcium stearate, stearic acid, zinc stearate, sodium stearate fumarate, talc, polyethylene glycol, glyceryl behenate, glyceryl monostearate, glyceryl stearate palmitate, sodium lauryl sulfate, and hydrogenated vegetable oil. More preferably, the lubricant is selected from one or more of magnesium stearate, calcium stearate, zinc stearate, stearic acid, polyethylene glycol, talc, sodium stearate fumarate, and glyceryl behenate. In some embodiments of the present invention, the lubricant in the pharmaceutical composition provided by the present invention accounts for about 0% to 10% (by weight), preferably about 0.5% to 5% (by weight), and more preferably about 1% to 2% (by weight) of the total weight of the pharmaceutical composition.

[0023] In some embodiments, the pharmaceutical compositions provided by the present invention further comprise a filler selected from one or more of microcrystalline cellulose (MCC), powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, mannitol (MAN), starches, lactose (LAC), sugars, celluloses, and inorganic salts. The inventors of the present invention have discovered that pharmaceutical compositions containing reducing sugars such as lactose produce new, unknown impurities under accelerated conditions for 30 days, while formulations using non-reducing sugars such as mannitol as fillers do not produce these impurities under the same accelerated conditions. Furthermore, when the filler in the compositions of the present invention is only water-soluble mannitol, it is highly sensitive to the amount of wetting agent water, the soft material is easily over-wetted, and it fails to completely dissolve in media at pH 1.2 and pH 4.5; when the filler is only microcrystalline cellulose, the compressibility of the particles is poor, and the tablets fail to completely dissolve in a pH 4.5 medium. In some preferred embodiments, the pharmaceutical composition of the present invention comprises two or more fillers, one of which is selected from microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, and pregelatinized starch, and the other fillers are selected from one or more of mannitol, non-reducing sugars, and inorganic salts. More preferably, the other fillers are selected from one or more of starch, dextrin, sorbitol, and mannitol. In some embodiments of the present invention, the fillers in the pharmaceutical composition provided by the present invention account for about 0% to 95% (by weight), preferably about 40% to 90% (by weight), and more preferably about 50% to 85% (by weight) of the total weight of the pharmaceutical composition.

[0024] In some preferred embodiments, the pharmaceutical composition of the present invention comprises a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof, a disintegrant, a binder, a lubricant and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof accounts for about 0.1% to 60% (by weight) of the total weight of the pharmaceutical composition, the disintegrant accounts for about 1% to 30% (by weight) of the total weight of the pharmaceutical composition, the binder accounts for about 0% to 30% (by weight) of the total weight of the pharmaceutical composition, the lubricant accounts for about 0% to 10% (by weight) of the total weight of the pharmaceutical composition, and the filler accounts for about 0% to 95% (by weight) of the total weight of the pharmaceutical composition. More preferably, the pharmaceutical composition of the present invention comprises a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof, a disintegrant, a binder, a lubricant and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof accounts for about 0.5% to 20% (by weight) of the total weight of the pharmaceutical composition, the disintegrant accounts for about 2% to 15% (by weight) of the total weight of the pharmaceutical composition, the binder accounts for about 5% to 25% (by weight) of the total weight of the pharmaceutical composition, the lubricant accounts for about 0.5% to 5% (by weight) of the total weight of the pharmaceutical composition, and the filler accounts for about 40% to 90% (by weight) of the total weight of the pharmaceutical composition. More preferably, the pharmaceutical composition of the present invention comprises a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof, a disintegrant, a binder, a lubricant and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof accounts for about 0.5% to 18% (by weight) of the total weight of the formulation, the disintegrant accounts for about 2% to 10% (by weight) of the total weight of the pharmaceutical composition, the binder accounts for about 5% to 20% (by weight) of the total weight of the pharmaceutical composition, the lubricant accounts for about 1% to 2% (by weight) of the total weight of the pharmaceutical composition, and the filler accounts for about 50% to 85% (by weight) of the total weight of the pharmaceutical composition.

[0025] The present invention also provides a pharmaceutical formulation comprising the pharmaceutical composition of the present invention.

[0026] In some preferred embodiments, the pharmaceutical formulation of the present invention is a tablet, granule, powder, sustained-release formulation, pill, capsule, lozenge, or flat capsule. In one specific embodiment, the sustained-release agent is a sustained-release microgranule. In another specific embodiment, the pharmaceutical formulation of the present invention is a tablet, capsule, or granule.

[0027] In some preferred embodiments, the present invention provides a pharmaceutical formulation comprising the pharmaceutical composition of the present invention, said pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof, a disintegrant, a binder, a lubricant and a filler, wherein, based on the amount of the compound of formula (I), said compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof constitutes about 0.1% to 60% (by weight) of the total weight of the pharmaceutical composition, said disintegrant constitutes about 1% to 30% (by weight) of the total weight of the pharmaceutical composition, said binder constitutes about 0% to 30% (by weight) of the total weight of the pharmaceutical composition, said lubricant constitutes about 0% to 10% (by weight) of the total weight of the pharmaceutical composition, and said filler constitutes about 0% to 95% (by weight) of the total weight of the pharmaceutical composition. More preferably, the pharmaceutical composition of the present invention comprises a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof, a disintegrant, a binder, a lubricant and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof accounts for about 0.5% to 20% (by weight) of the total weight of the pharmaceutical composition, the disintegrant accounts for about 2% to 15% (by weight) of the total weight of the pharmaceutical composition, the binder accounts for about 5% to 25% (by weight) of the total weight of the pharmaceutical composition, the lubricant accounts for about 0.5% to 5% (by weight) of the total weight of the pharmaceutical composition, and the filler accounts for about 40% to 90% (by weight) of the total weight of the pharmaceutical composition. More preferably, the pharmaceutical composition of the present invention comprises a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof, a disintegrant, a binder, a lubricant, and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof accounts for about 0.5% to 18% (by weight) of the total weight of the formulation; the disintegrant accounts for about 2% to 10% (by weight) of the total weight of the pharmaceutical composition; the binder accounts for about 5% to 20% (by weight) of the total weight of the pharmaceutical composition; the lubricant accounts for about 1% to 2% (by weight) of the total weight of the pharmaceutical composition; and the filler accounts for about 50% to 85% (by weight) of the total weight of the pharmaceutical composition.

[0028] Another aspect of the present invention provides a method for preparing the composition of the present invention, comprising mixing the components of the pharmaceutical composition of the present invention.

[0029] In some embodiments, the present invention provides a method for preparing a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof, and a disintegrant, the method comprising: mixing the compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof, and the disintegrant, followed by granulation. In some specific embodiments, in the method for preparing a pharmaceutical formulation according to the present invention, the disintegrant is selected from one or more of sodium carboxymethyl starch, crospovidone, crospovidone carboxymethyl cellulose, or low-substituted hydroxypropyl cellulose.

[0030] In some preferred embodiments, the present invention provides a method for preparing a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a hydrate, a solvate, a crystalline or amorphous form thereof, a filler, and a disintegrant. The method comprises: mixing the compound of formula (I) or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a crystalline or amorphous form thereof, and a filler and a disintegrant, followed by granulation. In some specific embodiments, according to the method for preparing a pharmaceutical formulation of the present invention, the filler is selected from one or more of microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, mannitol, starches, lactose, sugars, celluloses, and inorganic salts; the disintegrant is selected from one or more of sodium carboxymethyl starch, crospovidone, crospovidone carboxymethyl cellulose, or low-substituted hydroxypropyl cellulose.

[0031] In some preferred embodiments, the present invention provides a method for preparing a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a hydrate, a solvate, a crystalline or amorphous form thereof, a lubricant, and a disintegrant. The method comprises: mixing the compound of formula (I) or a pharmaceutically acceptable salt thereof, a hydrate, a solvate, a crystalline or amorphous form thereof, a disintegrant, and a lubricant, followed by granulation. In some specific embodiments, according to the method for preparing a pharmaceutical formulation of the present invention, the lubricant is selected from one or more of magnesium stearate, calcium stearate, zinc stearate, stearic acid, polyethylene glycol, talc, sodium stearate fumarate, and glyceryl behenate; the disintegrant is selected from one or more of sodium carboxymethyl starch, crospovidone, crospovidone carboxymethyl cellulose, or low-substituted hydroxypropyl cellulose.

[0032] In some other preferred embodiments, the present invention provides a method for preparing a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a hydrate, a solvate, a crystal or amorphous form thereof, a filler, a disintegrant, and a lubricant, the method comprising: mixing the compound of formula (I) or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a crystal or amorphous form thereof, and a filler, a disintegrant, and a lubricant, and granulating the mixture. In some specific embodiments, according to the method for preparing the pharmaceutical formulation of the present invention, the filler is selected from one or more of microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, mannitol, starches, lactose, sugars, celluloses, and inorganic salts; the disintegrant is selected from one or more of sodium carboxymethyl starch, crospovidone, crospovidone carboxymethyl cellulose, or low-substituted hydroxypropyl cellulose; the lubricant is selected from one or more of magnesium stearate, calcium stearate, stearic acid, zinc stearate, sodium stearate fumarate, talc, polyethylene glycol, glyceryl behenate, glyceryl monostearate, glyceryl stearate palmitate, sodium lauryl sulfate, and hydrogenated vegetable oil.

[0033] In some other preferred embodiments, the present invention provides a method for preparing a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a hydrate, a solvate, a crystal or amorphous form thereof, a filler, a disintegrant, a lubricant, and a binder, the method comprising: mixing the compound of formula (I) or a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a crystal or amorphous form thereof, and a filler, a disintegrant, a lubricant, and a binder, and granulating the mixture. In some specific embodiments, according to the method for preparing the pharmaceutical formulation of the present invention, the filler is selected from one or more of microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, mannitol, starches, lactose, sugars, celluloses, and inorganic salts; the disintegrant is selected from one or more of sodium carboxymethyl starch, crospovidone, crospovidone carboxymethyl cellulose, or low-substituted hydroxypropyl cellulose; the lubricant is selected from one or more of magnesium stearate, calcium stearate, stearic acid, zinc stearate, sodium stearate fumarate, talc, polyethylene glycol, glyceryl behenate, glyceryl monostearate, glyceryl stearate palmitate, sodium lauryl sulfate, and hydrogenated vegetable oil; and the binder is selected from one or more of povidone, hydroxypropyl methylcellulose, and hydroxypropyl cellulose.

[0034] In some other preferred embodiments, the present invention provides a method for preparing a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a hydrate, a solvate, a crystal or amorphous form thereof, a filler, a disintegrant, a lubricant, a binder, and a wetting agent, the method comprising: mixing the compound of formula (I) or a pharmaceutically acceptable salt thereof, a hydrate, a solvate, a crystal or amorphous form thereof, and a filler, a disintegrant, a lubricant, a binder, and a wetting agent, and granulating the mixture. In some specific embodiments, according to the method for preparing the pharmaceutical formulation of the present invention, the filler is selected from one or more of microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, mannitol, starches, lactose, sugars, celluloses, and inorganic salts; the disintegrant is selected from one or more of sodium carboxymethyl starch, crospovidone, crospovidone carboxymethyl cellulose, or low-substituted hydroxypropyl cellulose; the lubricant is selected from one or more of magnesium stearate, calcium stearate, stearic acid, zinc stearate, sodium stearate fumarate, talc, polyethylene glycol, glyceryl behenate, glyceryl monostearate, glyceryl palmitate stearate, sodium lauryl sulfate, and hydrogenated vegetable oil; the binder is selected from one or more of povidone, hydroxypropyl methylcellulose, and hydroxypropyl cellulose; and the wetting agent is water.

[0035] In some other preferred embodiments, the present invention provides a method for preparing the pharmaceutical formulation of the present invention, the method comprising: thoroughly mixing a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof with a filler, disintegrant, lubricant, binder and wetting agent, granulating, and capsule filling.

[0036] In some other preferred embodiments, the present invention provides a method for preparing the pharmaceutical formulation of the present invention, the method comprising: thoroughly mixing a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form thereof with a filler, disintegrant, lubricant, binder and wetting agent, granulating, and compressing to form tablets.

[0037] In some other preferred embodiments, the present invention provides a method for preparing the pharmaceutical formulation of the present invention, the method comprising: adding fillers, disintegrants, lubricants, binders, and wetting agents to a compound of formula (I) or its pharmaceutically acceptable salts, hydrates, solvates, crystals, or amorphous forms as needed, and then performing granulation by stirring, extrusion granulation, rotary granulation, spray granulation, or direct dry granulation. Alternatively, microgranulation can be used. Furthermore, granulation and pulverization can be performed as needed. Furthermore, tableting or direct capsule filling can be performed as needed; alternatively, disintegrants, fillers, lubricants, binders, antioxidants, colorants, etc., can be selectively added to the above-mentioned granules for tableting or direct capsule filling.

[0038] In some specific embodiments, the present invention provides a method for preparing the pharmaceutical formulation of the present invention, the method comprising:

[0039] (1) Mix the compound of formula (I) or its pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form, disintegrant and optionally binder, filler or lubricant evenly;

[0040] (2) Optionally add an appropriate amount of wetting agent to the mixture in step (1) for wet granulation;

[0041] (3) Dry and granulate the granules from step (2);

[0042] (4) Optionally, the particles, disintegrant, and lubricant from step (3) are mixed evenly;

[0043] (5) Prepare a formulation from the mixture of step (4).

[0044] In some specific embodiments, the present invention provides a method for preparing the pharmaceutical formulation of the present invention, the method comprising:

[0045] (1) The compound of formula (I) or its pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form, disintegrant and optional binder, filler or lubricant is sieved and added to a wet granulator in the prescribed amount and mixed evenly.

[0046] (2) Add an appropriate amount of wetting agent to the mixture in step (1) and perform wet granulation;

[0047] (3) Dry the particles from step (2) in a drying device;

[0048] (4) Optionally, the particles, disintegrant and lubricant from step (3) are added to a changeable drum mixer and mixed evenly.

[0049] (5) Pour the mixture from step (4) into the capsule filling machine to fill the capsules.

[0050] In some specific embodiments, the present invention provides a method for preparing the pharmaceutical formulation of the present invention, the method comprising:

[0051] (1) The compound of formula (I) or its pharmaceutically acceptable salt, hydrate, solvate, crystal or amorphous form, disintegrant and optional binder, filler or lubricant is sieved and added to a wet granulator in the prescribed amount and mixed evenly.

[0052] (2) Add an appropriate amount of wetting agent to the mixture in step (1) and perform wet granulation;

[0053] (3) Dry the particles from step (2) in a drying device;

[0054] (4) Optionally, the particles, disintegrant and lubricant from step (3) are added to a changeable drum mixer and mixed evenly.

[0055] (5) Pour the mixture from step (4) into a tablet press and compress it into tablets.

[0056] Another aspect of the invention provides a method for using the compositions or pharmaceutical preparations of the invention to treat and / or prevent SHP2-mediated diseases, and their use in the preparation of medicaments for treating and / or preventing SHP2-mediated diseases, wherein the SHP2-mediated diseases include, but are not limited to, proliferative diseases, metabolic diseases, or hematologic disorders. In some embodiments, the SHP2-mediated diseases of the invention are cancer.

[0057] In some embodiments, the SHP2-mediated diseases described in this invention include, but are not limited to: acoustic neuroma, adenocarcinoma, adrenal carcinoma, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendothelial sarcoma, angiosarcoma), adnexal cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., breast adenocarcinoma, papillary breast carcinoma, breast cancer, medullary breast carcinoma, triple-negative breast cancer), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchial cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial cancer, ependymoma, endothelial sarcoma (e.g., Kaposi's sarcoma). Sarcoma, multiple idiopathic hemorrhagic sarcomas, endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma), Ewing sarcoma Cancers including sarcoma, ocular cancer (e.g., intraocular melanoma, retinoblastoma), familial eosinophilia, gallbladder cancer, gastric cancer (e.g., gastric adenocarcinoma), gastrointestinal stromal tumors (GIST), head and neck cancers (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), pharyngeal cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic system cancers (e.g., leukemia such as acute lymphoblastic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myeloid leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CML). L-cell and T-cell CLL); lymphomas such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodular marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., "Woldanstrom macroglobulinemia") macroglobulinemia, hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-cell lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodular natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); and mixtures of one or more leukemias / lymphomas as described above;And multiple myeloma (MM), heavy chain diseases (e.g., alpha chain disease, gamma chain disease, μ chain disease), angioblastoma, inflammatory myofibroblastoma, immune cell amyloidosis, renal cell carcinoma (e.g., nephroblastoma, also known as Wilms' tumor), hepatocellular carcinoma (e.g., hepatocellular carcinoma (HCC), malignant hepatocellular carcinoma), lung cancer (e.g., bronchial carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma), leiomyosarcoma (LMS), mastocytosis (e.g., generalized mastocytosis), myelodyplastic syndrome (MDS), mesothelioma, myeloproliferative disorders (MPD) (e.g., polycythemia vera (PV), idiopathic thrombocythemia (ET), idiopathic extramedullary metaplasia (AMM)). This includes myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), eosinophilic leukocytosis (HES), neuroblastoma, neurofibroma (e.g., type 1 or 2 multiple neurofibroma (NF), Schwannoma), neuroendocrine carcinoma (e.g., gastrointestinal pancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma, ovarian clear cell carcinoma, ovarian serous cystadenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary myxoma (IPMN), islet cell tumor), and penile cancer (e.g., Paget's disease of the penis and scrotum). Diseases including pineal gland tumors, primary neuroectodermal tumors (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary duct cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., adnexal cancer), soft tissue sarcomas (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland cancer, sweat gland cancer, synovial tumors, testicular cancer (e.g., seminoma, embryonal testicular carcinoma), thyroid cancer (e.g., papillary thyroid carcinoma, papillary thyroid carcinoma (PTC), medullary thyroid carcinoma), urethral cancer, vaginal cancer, and vulvar cancer (e.g., vulvar Paget's disease), medulloblastoma, adenoid cystic carcinoma, melanoma, and glioblastoma.

[0058] In some preferred embodiments, the present invention provides methods for using the pharmaceutical compositions or pharmaceutical preparations of the present invention to treat SHP2-mediated diseases, and uses in the preparation of medicaments for treating SHP2-mediated diseases, wherein the SHP2-mediated diseases include, but are not limited to: non-small cell lung cancer, breast cancer, esophageal cancer, bladder cancer, lung cancer, hematopoietic system cancers, lymphoma, medulloblastoma, medulloblastoma, rectal adenocarcinoma, colon cancer, gastric cancer, pancreatic cancer, liver cancer, adenoid cystic carcinoma, prostate cancer, lung cancer, head and neck squamous cell carcinoma, brain cancer, hepatocellular carcinoma, melanoma, oligodendroglioma, glioblastoma, testicular cancer, clear cell carcinoma of the ovary, serous cystadenocarcinoma of the ovary, thyroid cancer, multiple myeloma (AML), renal cell carcinoma, mantle cell lymphoma, triple-negative breast cancer, hemoglobinopathies, diabetes, and obesity.

[0059] In the pharmaceutical compositions or formulations of the present invention, any form or amorphous form of the compound of formula (I) can be used, and the resulting formulation exhibits very good stability. In other embodiments, the crystalline form of the compound of formula (I) is used to prepare the pharmaceutical compositions of the present invention.

[0060] The pharmaceutical compositions or pharmaceutical preparations provided by this invention can be completely dissolved in vitro, have few impurities, good stability, and can simplify storage and transportation conditions.

[0061] Terminology Explanation

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0063] The "salt" of this invention can be any salt, particularly a pharmaceutically acceptable salt. In this document, "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt formed by the compound described in this invention with the "acid" or "acidic reagent" of this invention. The "acid" or "acidic reagent" of this invention may be selected from hydrochloric acid, hydrobromic acid, phosphoric acid, aminosulfonic acid, nitric acid, p-toluenesulfonic acid, benzenesulfonic acid, p-aminobenzenesulfonic acid, sulfuric acid, acetic acid, oxalic acid, phenylacetic acid, propionic acid, malonic acid, trifluoroacetic acid, succinic acid, glycolic acid, stearic acid, ascorbic acid, dihydroxynaphthyl acid, hydroxymaleic acid, glutamic acid, benzoic acid, salicylic acid, 2-acetoxybenzoic acid, fumaric acid, ethanedisulfonic acid, oxalic acid, hydroxyethanesulfonic acid, citric acid, D-gluconic acid, lactic acid, L-malic acid, succinic acid, L-tartaric acid, fumaric acid, α-ketoglutarate, hippuric acid, maleic acid, D-tartaric acid, methanesulfonic acid, or analogs thereof. The pharmaceutically acceptable salts of the compounds of this invention can be synthesized from the compounds of this invention containing acidic or basic moieties using conventional chemical methods. Typically, salts of basic compounds can be prepared by stoichiometry or by reacting a free base with a stoichiometric or excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents. Similarly, salts of acidic compounds can be formed by reacting with a suitable inorganic or organic base.

[0064] The term "solvent" refers to the form of the compounds of this invention that form solid or liquid complexes by coordination with solvent molecules, including hydrates, ethanol compounds, acetonitrile compounds, etc.

[0065] The term "crystallization" refers to the various solid forms formed by the compounds described in this invention, including crystalline and amorphous forms. In the compounds of this invention, "hydrogen," "carbon," and "oxygen" include all their isotopes. Isotopes should be understood to include those atoms having the same number of atoms but different mass numbers. For example, isotopes of hydrogen include protium, tritium, and deuterium, and isotopes of carbon include... 13 C and 14 C, oxygen isotopes include 16 O and 18 O etc.

[0066] The term “API” refers to (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidine-4(3H)-one. Attached Figure Description

[0067] Figure 1 The dissolution profiles of the formulations in Examples 3-12 of this invention are shown.

[0068] Figure 2 These are the dissolution curves of the formulations in Examples 13-21 of this invention. Detailed Implementation

[0069] The following representative embodiments are provided to better illustrate the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, all materials used in the following embodiments are commercially available.

[0070] Example 1: Preparation of (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one

[0071]

[0072] Step 1: Preparation of (R)-N-((R)-1'-(4-amino-5-bromo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3H-spiro[benzofuran-2,4'-piperidin]-3-yl)-2-methylpropane-2-sulfinamide

[0073]

[0074] In a 50L glass reactor, N,N-dimethylformamide (DMF, 9.45 kg) was added and stirred at room temperature. Then, (R)-2-methyl-N-((R)-3H-spiro[benzofuran-2,4'-piperidin]-3-yl)propane-2-sulfinamide (1.0 kg, 3.24 mol) and 6-amino-5-bromo-3-methylpyrimidine-2,4(1H,3H)-dione (0.71 kg, 3.23 mol) were added and stirred until homogeneous. 1,8-diazabicycloundec-7-ene (DBU, 2.47 kg, 14.6 mol) was slowly added to the reactor. Benzotriazole-1-tris(trimethylamino)-hexafluorophosphate (BOP reagent, 2.16 kg, 4.89 mol) was divided into four portions, each 0.4–0.6 kg, and added to the reaction solution every 10–15 minutes. After the reaction was complete, ethyl acetate (15.0 kg) was added to a 100 L glass reactor at room temperature, and stirring was started. The reaction solution was then added to the stirred ethyl acetate, followed by purified water (50.0 kg), and stirring was continued for 10 min. The organic phase was separated, concentrated under reduced pressure, and dried to obtain 0.93 kg of the title compound, with a yield of 56.2%.

[0075] Step 2: Preparation of (R)-N-((R)-1'-(4-amino-1-methyl-6-oxo-5-((2-(trifluoromethyl)pyridin-3-yl)thio)-1,6-dihydropyrimidin-2-yl)-3H-spiro[benzofuran-2,4'-piperidin]-3-yl)-2-methylpropane-2-sulfinamide

[0076]

[0077] 1,4-Dioxane (19.0 kg) was added to a 50 L glass reactor. Stirring was started, and (R)-N-((R)-1'-(4-amino-5-bromo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3H-spiro[benzofuran-2,4'-piperidin]-3-yl)-2-methylpropane-2-sulfinamide (0.92 kg, 1.8 mol), sodium 2-(trifluoromethyl)piperidin-3-thiolate (0.51 kg, 2.52 mol), and acetic acid (0.11 kg, 1.8 mol) were added. The temperature was raised and maintained at 60–70 °C. The reaction solution was filtered and concentrated under reduced pressure to obtain 1.07 kg of the crude title compound, with a yield of 97.5%.

[0078] Step 3: Preparation of crude (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one

[0079]

[0080] Weigh 30.0 kg of dichloromethane into a 50 L glass reactor, start stirring, and add (R)-N-((R)-1'-(4-amino-1-methyl-6-oxo-5-((2-(trifluoromethyl)pyridin-3-yl)thio)-1,6-dihydropyrimidin-2-yl)-3H-spiro[benzofuran-2,4'-piperidin]-3-yl)-2-methylpropane-2-sulfinamide (1.07 kg). Cool down, maintaining the reaction solution temperature at 15–25 °C, and add 2.63 L of hydrogen chloride-dioxane solution dropwise. Stir for 2–3 hours. Centrifuge.

[0081] Then, at 20–30°C, slowly add NaOH aqueous solution (0.3 kg sodium hydroxide, 0.6 kg purified water) to adjust the pH of the system to 8–9, and continue stirring for 2–4 hours. Centrifuge the material, wash with purified water, and transfer the collected free alkali solid to a 50 L glass reactor. Add 3.37 kg of purified water / anhydrous ethanol (20:1) solution and slurry. Centrifuge the material, and wash the solid sequentially with purified water and anhydrous ethanol. Dry to obtain 0.51 kg of crude title compound, yield 57.5%.

[0082] Step 4: Preparation of the refined product (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one

[0083]

[0084] Dimethyl sulfoxide (3.37 kg) was added to a 50 L glass reactor, and stirring was started. Then, the crude product obtained in the above reaction (0.51 kg) was added, and the temperature was raised to 75–85 °C with stirring until dissolved. The mixture was kept at this temperature for 0.5–1.0 hours, and anhydrous ethanol (1.58 kg) was added dropwise to precipitate crystals. The heating was then turned off, and the mixture was allowed to cool naturally to about 60 °C. Anhydrous ethanol (2.04 kg) was then added dropwise, and the reaction temperature was controlled at 40–60 °C. After the addition was complete, the mixture was allowed to cool naturally to room temperature (20–30 °C), and stirring was continued for 1–2 hours to precipitate crystals. The mixture was filtered, and the solid was washed with anhydrous ethanol (0.76 kg) and dried to obtain 0.37 kg of solid, with a yield of 72.5%. 1 H NMR (400MHz, DMSO-d6) δ8.40 (d, J=4.0Hz, 1H), 7.52-7.50 (m, 1H), 7.43 (d, J= 8.5Hz,1H),7.33(d,J=7.5Hz,1H),7.14(t,J=7.5Hz,1H),6.87(t,J=7.5Hz,1 H),6.77(d,J=8.0Hz,1H),4.12(s,1H),3.63-3.53(m,2H),3.31-3.24(m,5H) ,2.09-2.03(m,1H),1.92-1.89(m,2H),1.86-1.82(m,2H),1.77-1.74(m,1H). ESI-MS m / z: 505.2 [M+H] + .

[0085] Example 2-21

[0086] Tables 1 to 3 show Examples 2-21, which are formulations made using the compound of formula (I) (abbreviated as API) prepared in Example 1 of the present invention and various disintegrants, binders, fillers, wetting agents, lubricants, etc.

[0087] Table 1

[0088]

[0089] Table 2

[0090]

[0091] Table 3

[0092]

[0093]

[0094] " / " indicates that no symbol was added.

[0095] The preparation methods of Examples 2-21 are as follows:

[0096] (1) Pretreatment of excipients and weighing of raw and excipient materials: API, lactose, mannitol, microcrystalline cellulose, pregelatinized starch, croscarmellose sodium, croscarmellose sodium, low-substituted hydroxypropyl cellulose, croscarmellose, magnesium stearate and sodium stearate fumarate were weighed according to the prescription amounts of Examples 2-21 shown in Tables 1-3, passed through a 60-mesh sieve, and shaken to mix for premixing;

[0097] (2) Add an appropriate amount of wetting agent to the mixture in step (1) and perform wet granulation;

[0098] (3) Dry the particles from step (2) in an oven at 60°C, and then granulate the dried particles by passing them through a sieve.

[0099] (4) Add the particles from step (3) and the amounts of crosslinked sodium carboxymethyl cellulose, magnesium stearate, and sodium stearate from Examples 2-21 shown in Tables 1-3 to a changeable container mixer and mix them evenly.

[0100] (5) Pour the mixture from step (4) into a tablet press and compress it into tablets.

[0101] Comparative Example 1

[0102] The compound represented by the following formula (compound A) was prepared according to the method disclosed in compound 45 of WO2018 / 172984 (PCT / IB2018 / 051973), and identified by proton NMR and mass spectrometry.

[0103]

[0104] Experimental Example 1: Dissolution Test of Examples 3-21

[0105] Dissolution test method:

[0106] Column: Octadecylsilane-bonded silica gel as packing material (Waters Symmetry C18, 4.6×100mm, 3.5μm or equivalent column);

[0107] Mobile phase: 0.1% formic acid aqueous solution - acetonitrile (76:24);

[0108] Detection wavelength: 285 nm, column temperature: 30 ℃, flow rate: 1.0 mL / min, injection volume: 50 μL;

[0109] Dissolution medium: pH 4.5 acetate buffer (sodium acetate trihydrate + glacial acetic acid);

[0110] Dissolution conditions: Paddle jet, 50 rpm; Medium volume: 900 mL; Sample volume: 5 mL. See dissolution rate figures. Figure 1-2 Experimental results show that most of the formulations prepared in Examples 3-21 were fully dissolved in the dissolution medium of pH 4.5 acetate buffer.

[0111] Experimental Example 2: Stability

[0112] The formulations obtained in Examples 16 and 21 above were placed under accelerated conditions (40℃±2℃, 75%RH±5%RH), high humidity conditions (75%±5%, 90%±5%), and high temperature conditions (40℃, 60℃) and tested. The test results showed that the formulations of the present invention were stable, had few impurities, and no significant changes were observed in various indicators.

[0113] The experimental results above show that the (R)-6-amino-2-(3-amino-3H-spiro[benzofuran-2,4'-piperidin]-1'-yl)-3-methyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidine-4(3H)-one formulation containing a disintegrant of the present invention dissolves completely, has good stability, and meets the requirements for clinical use; in addition, the prepared compound of formula (I) has good absorption properties and relatively high bioavailability.

[0114] Experiment Example 3: Cell Proliferation Inhibition Experiment

[0115] 1. Experimental Materials

[0116] Test compounds: Compounds of formula (I) of the present invention and compound A prepared in the comparative example, each compound was prepared in 20 mM solution using DMSO. The concentrations of the compounds acting on NCI-H358 cells were 100 μM, 25 μM, 6.25 μM, 1.56 μM, 0.391 μM, 0.098 μM, 0.024 μM, 0.006 μM, 0.0015 μM, and 0.00038 μM, respectively.

[0117] Human non-small cell lung cancer cells NCI-H358 were purchased from the American Type Culture Collection (ATCC).

[0118] Reagents: CCK-8 proliferation inhibition assay kit, purchased from Jiangsu Kaiji Biotechnology Co., Ltd., China. Instruments: CKX41 inverted microscope, purchased from Olympus, Japan; multi-functional plate reader, purchased from Molecular Devices, USA; cell incubator, purchased from Thermo Fisher Scientific, USA.

[0119] 2. Experimental Methods

[0120] 2.1 Cell Culture:

[0121] Cell thawing: Remove the NCI-H358 cell cryovials from the liquid nitrogen container and place them in a 37°C water bath. Gently shake to thaw as quickly as possible. After thawing, remove the cryovials, sterilize with alcohol swabs, unscrew the caps, aspirate the cell suspension into centrifuge tubes, add 1 mL of serum-containing complete culture medium, mix well, and centrifuge at 1000 rpm for 5 min. Discard the supernatant, add complete culture medium, and repeatedly pipette until the cells are completely dispersed and resuspended. Seed the cells at an appropriate concentration in culture dishes. Incubate at 37°C in a CO2 incubator with 5% CO2 and 95% humidified air.

[0122] Cell passage: When cells reach approximately 80-90% confluence, discard the original culture medium (1640 medium + 10% FBS + 1% penicillin-streptomycin + 1mM sodium pyruvate). Add 1 mL of PBS to wash away any remaining medium, then discard the PBS. Add 1 mL of trypsin digestion solution and digest for 1-2 min. Under a microscope, observe that the pseudopodia of the cells have retracted and become rounded, but the cells have not yet detached in sheets. At this point, discard the trypsin and terminate the digestion with 1-2 mL of complete culture medium. Gently pipette and collect the cell suspension. Centrifuge at 1000 rpm for 5 min. Remove the supernatant, resuspend the cells in complete culture medium, and seed them into culture dishes at the desired density. Incubate in a CO2 incubator at 37°C, 5% CO2, and 95% humidified air. Change the culture medium or passage the cells every 2-3 days depending on cell growth.

[0123] 2.2 Experimental Procedure:

[0124] After passage, NCI-H358 cells were resuspended in fresh culture medium (1640 medium + 3% FBS + 1% penicillin and streptomycin + 1mM sodium pyruvate). Cell counts were then performed at a concentration of 1.5 x 10⁻⁶ cells / mL. 4 Seeds were generated at a density of 100 μL / mL into 96-well cell culture plates, with 100 μL added to each well (equivalent to 1.5 x 10⁻⁶ cells / mL). 3 (cells / well). After 24 hours, 100 μL of fresh medium containing different concentrations (2×) of the drug was added to the original medium. The final concentrations of the compound were 100 μM, 25 μM, 6.25 μM, 1.56 μM, 0.391 μM, 0.098 μM, 0.024 μM, 0.006 μM, 0.0015 μM, and 0.00038 μM, with two replicates for each concentration group. After incubation for 168 hours, the medium in the wells was aspirated and dried as much as possible. 100 μL of medium containing CCK-8 was added (CCK-8:medium = 1:10). After incubation for a certain period, the 96-well plate was removed from the incubator and equilibrated at room temperature for 5 minutes. The absorbance (OD value) at 450 nm was measured using a multi-functional plate reader, and the cell proliferation inhibition rate was calculated. The calculation formula is: Inhibition (%) = 100 - (OD) 实验孔 -OD空白孔 ) / (OD 溶剂对照孔 -OD 空白孔 *100, based on different drug concentrations and their corresponding inhibition rates, IC50 was performed using GraghPad 5.0 software. 50 Curve plotting, data analysis, and final IC calculation. 50 Values. The experimental results are shown in Table 4.

[0125] Table 4

[0126]

[0127] As can be seen from the above experiments, the compound of formula (I) of the present invention exhibits good inhibitory activity against NCI-H358 cells and is very promising as a therapeutic agent for non-small cell lung cancer.

[0128] Experiment Example 4: Pharmacokinetic Experiment

[0129] 1. Experimental Materials

[0130] Compound: The compound of formula (I) of this invention. The drug solvent is Captisol / 50mM sodium acetate, pH 4.6 (10% / 90%, w / v%). Oral administration is prepared as a 0.5 mg / mL clear solution; intravenous administration is prepared as a 0.1 mg / mL clear solution.

[0131] Animals: Male BALB / c mice, SPF grade, purchased from Shanghai Xipu-Bikai Laboratory Animal Co., Ltd.; 18-20g. An acclimatization period of 2-3 days was given before the experiment.

[0132] Instruments: AB (Alberts & Arpels) API 4500 triple quadrupole liquid chromatography-mass spectrometry system, equipped with an electrospray ionization source (ESI), LC-30AD dual pump; SIL-30AC autosampler; CTO-30AC column oven; DGU-20A3R degasser; AnalystQSA01.01 chromatography workstation; Milli-Q ultrapure water system (Millipore Inc.); Qilinbeier Vortex-5 shaker; HITACHI CF16R X II benchtop high-speed refrigerated centrifuge.

[0133] 2. Experimental Methods

[0134] (1) Three mice per group. The compound of formula (I) of the present invention was administered by gavage (IG) at a dose of 10 mg / kg and by intravenous administration (IV) at a dose of 1 mg / kg.

[0135] (2) Blood was collected from the orbital venous plexus at 5 min, 15 min, 30 min, 1 h, 2 h, 6 h, 10 h and 24 h after gavage and intravenous administration and placed in heparinized EP tubes (0.6 mL). After centrifugation at 8000 rpm / min for 5 min, the supernatant plasma was collected, frozen at -20℃ and analyzed by LC-MS / MS.

[0136] (3) Based on the blood drug concentration data obtained in the above steps, a blood drug concentration-time curve was plotted, and the pharmacokinetic parameters were calculated using WinNonlin software. The experimental results are shown in Table 5.

[0137] Table 5

[0138]

[0139] The pharmacokinetics of compound A in the comparative example were determined using the method described in Experimental Example 4. The results showed that the bioavailability (F) of compound A was 51.2%, which was significantly lower than that of the compound of formula (I) of the present invention.

[0140] Experimental results show that the compound of the present invention has a good half-life T. 1 / 2 It has good area under the curve (AUC) and bioavailability (F), indicating good oral absorption and exposure, making it suitable for drug development.

[0141] Although the present invention has been described in detail above, those skilled in the art will understand that various modifications and changes can be made to the invention without departing from its spirit and scope. The scope of the invention is not limited to the detailed description above, but should be attributed to the claims.

Claims

1. A pharmaceutical composition comprising: (a) A compound of formula (I) or a pharmaceutically acceptable salt thereof, ； and (b) disintegrants.

2. The pharmaceutical composition according to claim 1, wherein the disintegrant is one or more selected from sodium carboxymethyl starch, crospovidone, crospovidone carboxymethyl cellulose sodium, and low-substituted hydroxypropyl cellulose.

3. The pharmaceutical composition according to claim 1 or 2, further comprising a binder.

4. The pharmaceutical composition according to claim 3, wherein the binder is selected from one or more of povidone, hydroxypropyl methylcellulose and hydroxypropyl cellulose.

5. The pharmaceutical composition according to claim 1 or 2, further comprising a lubricant.

6. The pharmaceutical composition according to claim 3, further comprising a lubricant.

7. The pharmaceutical composition according to claim 4, further comprising a lubricant.

8. The pharmaceutical composition of claim 5, wherein the lubricant is selected from one or more of magnesium stearate, calcium stearate, stearic acid, zinc stearate, sodium stearate fumarate, talc, polyethylene glycol, glyceryl behenate, glyceryl monostearate, glyceryl palmitate stearate, sodium lauryl sulfate, and hydrogenated vegetable oil.

9. The pharmaceutical composition according to claim 6 or 7, wherein the lubricant is selected from one or more of magnesium stearate, calcium stearate, stearic acid, zinc stearate, sodium stearate fumarate, talc, polyethylene glycol, glyceryl behenate, glyceryl monostearate, glyceryl palmitate stearate, sodium lauryl sulfate, and hydrogenated vegetable oil.

10. The pharmaceutical composition according to claim 1 or 2, further comprising a filler.

11. The pharmaceutical composition of claim 3, further comprising a filler.

12. The pharmaceutical composition of claim 4, further comprising a filler.

13. The pharmaceutical composition of claim 5, further comprising a filler.

14. The pharmaceutical composition according to any one of claims 6-8, further comprising a filler.

15. The pharmaceutical composition of claim 9, further comprising a filler.

16. The pharmaceutical composition of claim 10, wherein the filler is selected from one or more of microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, mannitol, starches, lactose, sugars, celluloses, and inorganic salts.

17. The pharmaceutical composition according to any one of claims 11-13, wherein the filler is selected from one or more of microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, mannitol, starches, lactose, sugars, celluloses and inorganic salts.

18. The pharmaceutical composition of claim 14, wherein the filler is selected from one or more of microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, mannitol, starches, lactose, sugars, celluloses, and inorganic salts.

19. The pharmaceutical composition of claim 15, wherein the filler is selected from one or more of microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, mannitol, starches, lactose, sugars, celluloses, and inorganic salts.

20. The pharmaceutical composition according to claim 1 or 2, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a binder, a lubricant, and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt thereof constitutes about 0.1% to 60% (by weight) of the total weight of the pharmaceutical composition, the disintegrant constitutes about 1% to 30% (by weight) of the total weight of the pharmaceutical composition, the binder constitutes about 0% to 30% (by weight) of the total weight of the pharmaceutical composition, the lubricant constitutes about 0% to 10% (by weight) of the total weight of the pharmaceutical composition, and the filler constitutes about 0% to 95% (by weight) of the total weight of the pharmaceutical composition.

21. The pharmaceutical composition of claim 3, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a binder, a lubricant, and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt thereof constitutes about 0.1% to 60% (by weight) of the total weight of the pharmaceutical composition, the disintegrant constitutes about 1% to 30% (by weight) of the total weight of the pharmaceutical composition, the binder constitutes about 0% to 30% (by weight) of the total weight of the pharmaceutical composition, the lubricant constitutes about 0% to 10% (by weight) of the total weight of the pharmaceutical composition, and the filler constitutes about 0% to 95% (by weight) of the total weight of the pharmaceutical composition.

22. The pharmaceutical composition of claim 4, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a binder, a lubricant, and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt thereof constitutes about 0.1% to 60% (by weight) of the total weight of the pharmaceutical composition, the disintegrant constitutes about 1% to 30% (by weight) of the total weight of the pharmaceutical composition, the binder constitutes about 0% to 30% (by weight) of the total weight of the pharmaceutical composition, the lubricant constitutes about 0% to 10% (by weight) of the total weight of the pharmaceutical composition, and the filler constitutes about 0% to 95% (by weight) of the total weight of the pharmaceutical composition.

23. The pharmaceutical composition of claim 5, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a binder, a lubricant, and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt thereof constitutes about 0.1% to 60% (by weight) of the total weight of the pharmaceutical composition, the disintegrant constitutes about 1% to 30% (by weight) of the total weight of the pharmaceutical composition, the binder constitutes about 0% to 30% (by weight) of the total weight of the pharmaceutical composition, the lubricant constitutes about 0% to 10% (by weight) of the total weight of the pharmaceutical composition, and the filler constitutes about 0% to 95% (by weight) of the total weight of the pharmaceutical composition.

24. A pharmaceutical composition according to any one of claims 6-8, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a binder, a lubricant, and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt thereof constitutes about 0.1% to 60% (by weight) of the total weight of the pharmaceutical composition, the disintegrant constitutes about 1% to 30% (by weight) of the total weight of the pharmaceutical composition, the binder constitutes about 0% to 30% (by weight) of the total weight of the pharmaceutical composition, the lubricant constitutes about 0% to 10% (by weight) of the total weight of the pharmaceutical composition, and the filler constitutes about 0% to 95% (by weight) of the total weight of the pharmaceutical composition.

25. The pharmaceutical composition of claim 9, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a binder, a lubricant, and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt thereof constitutes about 0.1% to 60% (by weight) of the total weight of the pharmaceutical composition, the disintegrant constitutes about 1% to 30% (by weight) of the total weight of the pharmaceutical composition, the binder constitutes about 0% to 30% (by weight) of the total weight of the pharmaceutical composition, the lubricant constitutes about 0% to 10% (by weight) of the total weight of the pharmaceutical composition, and the filler constitutes about 0% to 95% (by weight) of the total weight of the pharmaceutical composition.

26. The pharmaceutical composition of claim 10, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a binder, a lubricant, and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt thereof constitutes about 0.1% to 60% (by weight) of the total weight of the pharmaceutical composition, the disintegrant constitutes about 1% to 30% (by weight) of the total weight of the pharmaceutical composition, the binder constitutes about 0% to 30% (by weight) of the total weight of the pharmaceutical composition, the lubricant constitutes about 0% to 10% (by weight) of the total weight of the pharmaceutical composition, and the filler constitutes about 0% to 95% (by weight) of the total weight of the pharmaceutical composition.

27. A pharmaceutical composition according to any one of claims 11-13, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a binder, a lubricant, and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt thereof constitutes about 0.1% to 60% (by weight) of the total weight of the pharmaceutical composition, the disintegrant constitutes about 1% to 30% (by weight) of the total weight of the pharmaceutical composition, the binder constitutes about 0% to 30% (by weight) of the total weight of the pharmaceutical composition, the lubricant constitutes about 0% to 10% (by weight) of the total weight of the pharmaceutical composition, and the filler constitutes about 0% to 95% (by weight) of the total weight of the pharmaceutical composition.

28. The pharmaceutical composition of claim 14, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a binder, a lubricant, and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt thereof constitutes about 0.1% to 60% (by weight) of the total weight of the pharmaceutical composition, the disintegrant constitutes about 1% to 30% (by weight) of the total weight of the pharmaceutical composition, the binder constitutes about 0% to 30% (by weight) of the total weight of the pharmaceutical composition, the lubricant constitutes about 0% to 10% (by weight) of the total weight of the pharmaceutical composition, and the filler constitutes about 0% to 95% (by weight) of the total weight of the pharmaceutical composition.

29. The pharmaceutical composition according to claim 15 or 16, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a binder, a lubricant, and a filler, wherein, based on the amount of the compound of formula (I), the compound of formula (I) or a pharmaceutically acceptable salt thereof constitutes about 0.1% to 60% (by weight) of the total weight of the pharmaceutical composition, the disintegrant constitutes about 1% to 30% (by weight) of the total weight of the pharmaceutical composition, the binder constitutes about 0% to 30% (by weight) of the total weight of the pharmaceutical composition, the lubricant constitutes about 0% to 10% (by weight) of the total weight of the pharmaceutical composition, and the filler constitutes about 0% to 95% (by weight) of the total weight of the pharmaceutical composition.

30. A pharmaceutical preparation comprising the pharmaceutical composition of any one of claims 1-29.

31. The pharmaceutical preparation according to claim 30, wherein the pharmaceutical preparation is a tablet, granule, powder, sustained-release preparation, pill, capsule, lozenge, or flat capsule.

32. A method for preparing a pharmaceutical formulation according to claim 30 or 31, the method comprising: (1) Mix the compound of formula (I) or its pharmaceutically acceptable salt, disintegrant and optional binder, filler and lubricant evenly; ； (2) Optionally, add an appropriate amount of wetting agent to the mixture in step (1) for wet granulation; (3) Dry the particles from step (2); (4) Optionally, mix the particles, filler and lubricant from step (3) evenly; (5) Prepare a formulation from the mixture of step (4).

33. Use of the pharmaceutical composition of any one of claims 1-29 or the pharmaceutical preparation of claim 30 or 31 in the preparation of a medicament for the treatment and / or prevention of non-small cell lung cancer.

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