WO2021180055A1 - Use of brd4 inhibitor - Google Patents

Abstract

An application of a BRD4 inhibitor in preparation of a drug for treating tumors, in particular, for treating an advanced solid tumor or intractable solid tumor, and a therapeutic regimen and safe and effective dose of the drug for treating the advanced solid tumor or intractable solid tumor. The BRD4 inhibitor can effectively treating an advanced solid tumor or intractable solid tumor, has concrete effects for advanced or intractable breast cancer, colorectal cancer, prostate cancer, gastrointestinal cancer and the like, and has good clinical safety.

Description

Use of a BRD4 inhibitor

This application claims the priority of the prior invention patent application filed with the State Intellectual Property Office of China on March 9, 2020, with the application number 202010156960.2 and the invention title "Use of a BDR4 inhibitor". The full text of this earlier application is incorporated into this application by reference.

Technical field

The invention belongs to the field of medicine, and specifically relates to the application of a BRD4 inhibitor in the preparation of drugs for treating tumors, especially advanced solid tumors or refractory solid tumors, and a method for treating tumors with the inhibitor.

Background technique

Tumor disease is one of the most common diseases in modern times and a common cause of unnatural death. Tumors include benign tumors and malignant tumors. The risk of malignant tumors is very high, and most of the malignant tumors are life-threatening. At present, the methods of treatment of malignant tumors include surgical resection and drug chemotherapy. The existing treatment methods are difficult to produce good results for patients with various advanced tumors including triple-negative breast cancer (TNBC) and colorectal cancer (CRC). Curative effect.

According to the latest data, in 2018, there were about 18 million new cancer cases worldwide, of which breast cancer accounted for 11.6%, ranking second in the world; nearly 9.6 million deaths due to cancer, and breast cancer accounting for 6.6%, ranking sixth in the world . In China, the incidence of breast cancer is 45.29/100000, and the number of cases ranks fifth in the country. At the same time, breast cancer has become the most common type of malignant tumor among women in my country. About 16,000 women in China die from breast cancer each year. Breast cancer has become the fifth cause of death from malignant tumors among women in my country. Triple negative breast cancer (TNBC) refers to breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER-2). In all breast cancer cases, approximately Accounted for 15%. Triple negative breast cancer has a higher incidence in certain ethnic groups such as Asians and African Americans. TNBC patients have the characteristics of younger, familial, aggressive, recurrent and metastatic. Studies have shown that 87% of TNBC patients have negative expression of androgen receptor (AR), which makes them prone to recurrence and distant metastasis. Compared with other types of breast cancer, TNBC has a significant risk of metastasis within 5 years. Larger, metastatic spread mainly occurs in internal organs, especially lung and brain metastasis. The 5-year disease-free survival (DFS) and overall survival (OS) are also significantly lower than those of non-triple-negative breast cancer. Existing breast cancer targeted drugs are ineffective against triple-negative breast cancer. Compared with other types of breast cancer, triple-negative breast cancer is slightly more sensitive to chemotherapy, but the prognosis of conventional chemotherapy is poor and the 5-year survival rate is extremely low, especially for breast cancer. Patients with susceptibility gene (BRCA) mutations have worse efficacy, and there is a very high unmet medical need in this field.

Colorectal cancer, also known as colorectal cancer, is a heterogeneous malignant tumor that grows in the first six feet of the large intestine (colon) and the last 8-10 inches of the large intestine (rectum). It involves various molecular pathways and genetic/epigenetics. The change triggers the sequential transformation of normal mucosa into adenoma and then into cancer. It is a common malignant tumor in the gastrointestinal tract. The early symptoms are not obvious. As the tumor grows, it shows changes in bowel habits, blood in the stool, diarrhea, alternating diarrhea and constipation, and local abdominal pain; in the late stage, anemia, weight loss and other systemic symptoms symptom. The age of onset of colorectal cancer in my country is mostly between 40 and 60 years old. The average age of onset is 48.3 years old, which is 10 to 15 years earlier than Westerners. It is more common in young patients in my country than in Europe and the United States. Colorectal cancer under 30 years old Patients are not uncommon. Colorectal cancer is very dangerous. Because early detection is not timely, more than 80% of patients in my country are actually in the middle and late stages when they are discovered. Due to the loss of the best treatment opportunity, the 5-year survival rate of patients after diagnosis is very low; In our country, 1 person dies of colorectal cancer every 5 minutes. Colorectal cancer is currently mainly treated with surgery, but the quality of life after surgery has also significantly decreased, including: decreased sexual function and excretory function, increased psychological disorders, and decreased social activities. Domestic surveys show that the survival rate of early colorectal cancer after surgery is more than 90-95%, while the late stage is only 5%. As the incidence of colorectal cancer is increasing year by year, and surgical treatment has reached a bottleneck, it is difficult to make a major breakthrough, especially for patients with limited physical conditions that cannot be treated by surgical resection and patients with recurrence and metastasis after surgery. It is particularly important.

Bromodomain and extra-terminal domain, the bromodomain and extra-terminal domain (BET), is a class of proteins with two bromodomains and one extra-terminal domain, which can recognize acetylated lysines in histones . There are 4 BET family members in the human body: BRD2, BRD3, BRD4 and BRDT. Among them, BRD4 (Bromodomain-containing protein 4) is extremely widely expressed in humans. BRD4 protein combines with RNA polymerase II and forward transcription elongation factor to participate in the transcription process of oncogenes such as MYC, BCL2 and BCL6, and then regulate cell transcription. , Marks the mitosis of mammals, regulates the cell cycle, and plays an important role in the occurrence and development of cancer, fibrosis and inflammation. Highly expressed BRD4 has been detected in leukemia, lymphoma, breast cancer, gastrointestinal tumors and other blood system and solid tumor cancer cells and fibrotic cells. Through targeted inhibition of BRD4, tumor cell apoptosis, that is, proliferation slowed, can be induced to achieve anti-tumor effects. Therefore, BRD4, as a promising anti-tumor target, has received extensive attention and development in recent years. BRD4 inhibitors have become a hot field of drug research for solid tumors, leukemia and fibrosis.

BRD4 inhibitors currently have no drugs on the market at home and abroad, but many pharmaceutical companies have small molecule drugs in the clinical stage. For example, Merck's Birabresib (MK-8628, OTX-015), GlaxoSmithKline (GSK) is developing molibresib (GSK-525762A, GSK-525762, I-BET-762, GSK'762), AstraZeneca (AZ) in Research AZ5153 and so on. According to reports, molibresib can be used to treat solid and blood cancers, including multiple myeloma, non-Hodgkin’s lymphoma, acute myeloid leukemia, small cell lung cancer, castration resistant prostate cancer, colorectal cancer, non-small cell lung cancer, and breast cancer Cancer (including metastatic breast cancer, estrogen receptor positive (ER+) breast cancer, and triple negative breast cancer (TNBC)). Birabresib (MK8628) is undergoing clinical research including acute leukemia (AL) and other hematological malignancies (OHM), triple-negative breast cancer, non-small cell lung cancer, castration-resistant prostate cancer, and pancreatic ductal cancer.

Gastrointestinal toxicity is a prominent toxic reaction in the preclinical toxicological studies of BDR4 inhibitors, but the reaction varies greatly among different species. Among them, rats and dogs have poor tolerance, while human intestinal wall This is not sensitive. It is reported that MK-8628 has strong gastrointestinal toxicity when used in rats, but it is relatively well tolerated by humans in the clinical stage. The situation of AZ's BET inhibitor AZ5153 is similar. Common gastrointestinal adverse reactions include diarrhea, constipation, nausea, vomiting, dysgeusia, and mucositis. Hematological toxicity is also one of the main clinical adverse effects of BDR4 inhibitors. Studies have found that BET inhibitors can inhibit miR17-92, thereby significantly up-regulating BIM protein, directly or indirectly activating BAX and BAK proteins, and then controlling internal mitochondrial cell apoptosis The process causes the death of normal hematopoietic cells. Compound OTX015 and MK-8628 have been observed in clinical studies with blood system adverse reactions such as thrombocytopenia, anemia, and neutropenia.

Compound A is a small molecule inhibitor of BRD4, which can significantly inhibit the activity of BRD4 protein and is expected to be used in the treatment of tumors. Its structure is shown in the following formula (I):

WO2019056950A1 discloses the preparation method and activity of compound A, and WO2020192637A1 further discloses the crystal form and use of compound A. Compound A currently does not have sufficient pharmacodynamic and safety evaluation data, so the important risks and exact efficacy of its clinical application are still unclear. The present invention explores the effectiveness and safety of compound A in the treatment of tumors, in order to provide a reference for clinical medication.

Summary of the invention

Based on the above-mentioned defects in the prior art, in the first aspect, the purpose of the present invention is to provide the use of compound A or a pharmaceutically acceptable salt thereof in the preparation of drugs for the treatment of tumors, wherein the tumors are preferably advanced solid tumors or refractory solids Tumor, the structure of the compound A is shown in the following formula (I):

In some embodiments, the solid tumor in the advanced solid tumor or refractory solid tumor is gastrointestinal tumor, colorectal cancer, breast cancer, or prostate cancer.

In some embodiments, the solid tumors in the advanced solid tumors or refractory solid tumors are colorectal cancer with high infiltration of tumor-associated macrophages, triple-negative breast cancer, and androgen-independent prostate cancer.

In some embodiments, the aforementioned subject suffering from the tumor is a human.

In some embodiments, the drug contains a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof. The therapeutically effective amount is preferably about 0.001 to about 1000 mg.

In some embodiments, the administration mode of Compound A or a pharmaceutically acceptable salt thereof is not particularly limited, and the representative administration mode may be oral administration, injection administration, topical administration or in vitro administration. Correspondingly, the medicine uses conventional auxiliary materials and techniques to prepare clinically accepted preparations, such as oral preparations, injection preparations, topical administration preparations, topical preparations, and the like. The drug is in a single-dose dosage form or a multiple-dose dosage form. The dosage form contains about 0.001 mg to about 1000 mg of compound A or a pharmaceutically acceptable salt thereof (calculated as compound A), preferably about 1 mg to about 500 mg, or about 5 mg to about 400 mg, or about 10 mg to about 300 mg, or About 10 mg to about 250 mg, or about 10 mg to about 200 mg, or about 20 mg to about 200 mg, more preferably about 10 mg to about 160 mg, or about 20 mg to about 160 mg, or about 80 mg to about 160 mg; for example, about 10 mg, about 20 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, more preferably about 10 mg, about 20 mg, about 40 mg, about 80 mg, about 120 mg, or about 160 mg, still more preferably about 20 mg, about 40 mg, about 80 mg, about 120 mg, or about 160 mg, or further more preferably about 10 mg or about 40 mg.

In some embodiments, Compound A or a pharmaceutically acceptable salt thereof may be used in combination with one or more of other targeted drugs or chemotherapeutic drugs. The other targeted drugs or chemotherapeutics refer to targeted drugs or chemotherapeutics that are clinically used to treat tumor-related diseases.

In a second aspect, the object of the present invention is to provide a method for treating tumors, the method comprising administering to a subject or patient a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, wherein the tumor is an advanced entity Tumor or refractory solid tumor.

In some embodiments, the solid tumor in the advanced solid tumor or refractory solid tumor is gastrointestinal tumor, breast cancer, colorectal cancer or prostate cancer.

In some embodiments, the solid tumors in the advanced solid tumors or refractory solid tumors are colorectal cancer with high infiltration of tumor-associated macrophages, triple-negative breast cancer, and androgen-independent prostate cancer.

In some embodiments, the subject or patient is a human.

A suitable dosage range of Compound A or a pharmaceutically acceptable salt thereof is from about 0.001 mg/kg to about 1000 mg/kg per day; preferably, from about 0.01 mg/kg to about 100 mg/kg. Preferably, the daily dose of Compound A or its pharmaceutically acceptable salt is about 0.001 mg to about 1000 mg. It is administered in a single dose or in divided doses.

In some embodiments, the above-mentioned administration is oral administration, injection administration, topical administration or in vitro administration; or compound A or a pharmaceutically acceptable salt thereof is prepared into a clinically accepted preparation, and the preparation includes oral preparation, injection preparation , Local administration preparations, topical preparations.

In some embodiments, Compound A or a pharmaceutically acceptable salt thereof (calculated as Compound A) is administered in a daily dose of about 0.001 mg to about 1000 mg, preferably about 1 mg to about 500 mg, or about 5 mg to about 400 mg, or about 10 mg to about 300 mg, or about 10 mg to about 250 mg, or about 10 mg to about 200 mg, or about 20 mg to about 200 mg; more preferably about 10 mg to about 160 mg, or about 20 mg to about 160 mg, or about 80 mg to about 160 mg; for example, about 10mg, about 20mg, about 40mg, about 60mg, about 80mg, about 100mg, about 120mg, about 140mg, about 160mg, more preferably about 10mg, about 20mg, about 40mg, about 80mg, about 120mg or about 160mg, and more preferably about 20 mg, about 40 mg, about 80 mg, about 120 mg, or about 160 mg, administered in a single dose or divided doses.

In some embodiments, the dosage regimen of Compound A or its pharmaceutically acceptable salt is 28 days per cycle, once a day for 21 days, with 7 days off; preferably, compound A or its pharmaceutically acceptable salt is administered every time The pharmaceutically acceptable salt (calculated as compound A) is about 20 mg, about 40 mg, about 80 mg, about 120 mg, and about 160 mg.

In some embodiments, the compound A or a pharmaceutically acceptable salt thereof is used in combination with one or more of other targeted drugs or chemotherapeutic drugs.

In the third aspect, the purpose of the present invention is to provide compound A or a pharmaceutically acceptable salt thereof for the treatment of tumors, preferably advanced solid tumors or refractory solid tumors.

In some embodiments, the solid tumor in the advanced solid tumor or refractory solid tumor is gastrointestinal tumor, colorectal cancer, breast cancer or prostate cancer.

In some embodiments, the solid tumors in the advanced solid tumors or refractory solid tumors are colorectal cancer with high infiltration of tumor-associated macrophages, triple-negative breast cancer, and androgen-independent prostate cancer.

In some embodiments, the subject suffering from the tumor is a human.

In some embodiments, the compound A or a pharmaceutically acceptable salt thereof is prepared into a clinically accepted preparation and then administered. The preparation includes an oral preparation, an injection preparation, a topical administration preparation, and an external preparation.

In some embodiments, Compound A or a pharmaceutically acceptable salt thereof (calculated as Compound A) is administered in a daily dose of about 0.001 mg to about 1000 mg, preferably about 1 mg to about 500 mg, or about 5 mg to about 400 mg, or about 10 mg to about 300 mg, or about 10 mg to about 250 mg, or about 10 mg to about 200 mg, or about 20 mg to about 200 mg; more preferably about 10 mg to about 160 mg, or about 20 mg to about 160 mg, or about 80 mg to about 160 mg; for example, about 10mg, about 20mg, about 40mg, about 60mg, about 80mg, about 100mg, about 120mg, about 140mg, about 160mg, more preferably about 10mg, about 20mg, about 40mg, about 80mg, about 120mg or about 160mg, and more preferably about 20 mg, about 40 mg, about 80 mg, about 120 mg, or about 160 mg, administered in a single dose or divided doses.

In some embodiments, the dosage regimen of Compound A or its pharmaceutically acceptable salt is 28 days per cycle, once a day for 21 days, with 7 days off; preferably, compound A or its pharmaceutically acceptable salt is administered every time The pharmaceutically acceptable salt (calculated as compound A) is about 20 mg, about 40 mg, about 80 mg, about 120 mg, and about 160 mg.

In some embodiments, the compound A or a pharmaceutically acceptable salt thereof can be used in combination with one or more of other targeted drugs or chemotherapeutics.

The dosage of Compound A or its pharmaceutically acceptable salt of the present invention is all calculated as Compound A.

In order to evaluate the efficacy and safety of compound A or its pharmaceutically acceptable salt in the treatment of tumors, the inventor of the present application carried out BDR4 in vitro activity inhibition test, in vivo tumor inhibition test, and clinical phase I study. The results showed that the mechanism of compound A is clear. It can significantly inhibit the activity of BRD4 protein, and it has obvious effect of inhibiting tumor growth in mice with colon cancer, prostate cancer, and triple-negative breast cancer. It is clinically intended to treat advanced solid tumors, including but not limited to gastrointestinal tumors, colorectal cancer, triple-negative breast cancer, prostate cancer, etc. It can be seen that compound A can effectively treat advanced solid tumors, especially gastrointestinal tumors, triple-negative breast cancer, colorectal cancer, and prostate cancer, with good clinical safety.

Term explanation:

"Advanced solid tumor" refers to cancer that has spread outside the origin or organ through local invasion or metastasis. The term "advanced solid tumor" includes locally advanced and metastatic solid tumors.

"Refractory solid tumor" refers to a solid tumor whose condition is still progressing even if anti-tumor therapy is used on the patient. The anti-tumor therapy includes radiotherapy, chemotherapy drug therapy, targeted drug therapy, immune checkpoint drug therapy and the like. In some embodiments, the refractory solid tumor refers to a solid tumor for which standard treatment regimens are ineffective.

Description of the drawings

Figure 1 is a flow chart of the Phase I clinical program.

Detailed ways

The examples listed below are to better illustrate the content of the present invention, but the content of the present invention is not limited to the examples. Those skilled in the art make non-essential improvements and adjustments to the embodiments based on the above-mentioned content of the invention, which still belongs to the protection scope of the present invention.

1. Inhibition test of compound A on BRD4 activity in vitro

Example 1: BRD4 biochemical activity detection

1. Experiment preparation:

1) The experiment uses BRD4-BD1 and BRD4-BD2 proteins from BPS; peptides from ANASPEC; detection reagents from PerkinElmer;

2) Experiments apply TR-FRET experimental principles to screen compounds.

3) Related reference compounds

2. Experimental steps:

1) Prepare the compound board:

The preparation of the compound plate in the experiment is realized by the Echo non-contact nano-upgraded sonic pipetting system:

The compound dilution was completed by the Echo non-contact nano-upgraded sonic pipetting system, which was diluted by 3 times to 10 concentrations: 20000, 6666.67, 2222.22, 740.74, 246.91, 82.305, 27.435, 9.145, 3.048, 1.016nM.

2) Preparation of reaction reagents:

Relevant reagents should be prepared on the day of the experiment:

a) Prepare 1×assay buffer (test buffer solution);

b) Prepare 3× experimental component solution:

1. Take out the reagent and put it on ice to melt naturally for later use;

2. Use 1×assay buffer (test buffer solution) to configure the "solution A" (protein solution), "solution B" (peptide solution), and "solution C" (test reagent solution) used in the experiment, and make each The components form a 3× solution in the experimental reaction system, and the amount of solutions A, B, and C must be sufficient for this round of experiments.

3) Experimental operation steps:

The experiment board is the board containing the gradient concentration of the compound and the corresponding DMSO solution prepared by the Echo non-contact nano-upgraded sonic pipetting system before the experiment:

a) Take out the experimental plate, and add 5μL/well of "solution A" (protein solution) to the second to 23rd columns of the experimental plate, and then add 5ul/well of 1×assay buffer to the first and 24th columns of the experimental plate , Columns 1 and 24 are used as the Min control in the experimental system;

b) Centrifuge at 1000 rpm for 30 seconds;

c) Incubate the plate at 23°C for 20 minutes;

d) After incubating for 20 minutes, add 5 μL/well of "Solution B" (polypeptide solution) to columns 1-24 of the experiment plate;

e) Centrifuge at 1000 rpm for 30 seconds;

f) Incubate the plate at 23°C for 20 minutes;

g) After incubating for 20 minutes, add 5 μL/well of "Solution C" (detection reagent solution) to columns 1-24 of the experiment plate;

h) Centrifuge at 1000 rpm for 30 seconds;

i) Incubate the plate at 23°C for 40 minutes;

j) Place the experiment plate on EnVision and read the plate.

4) Data analysis:

a) Use the corresponding Max control (maximum control) and Min control (minimum control) of each experimental board to convert the Z'value of the experimental board, and ensure that the Z'value of each board is> 0.5;

b) a compound of the control signal by XLFIT5 the IC ₅₀ value _was calculated, and to ensure that it is maintained at less than 3 times the average of the historical data, the results shown in Table 5.

Table 1 BRD4 detection IC ₅₀ test results

5 Conclusion:

Compound A has a significant inhibitory effect on BRD4-BD1 and BRD4-BD2.

2. In vivo drug efficacy test on mouse transplanted tumor model

Example 2: In vivo pharmacodynamic study of compound A in human breast cancer MDA-MB-231_luc cell subcutaneous xenograft tumor model

1. Laboratory animals and group administration

1.1 Experimental animals

Species: Mouse

Strain: BALB/c nude mice

Week age and weight: 6-8 weeks old, weighing 18-22 grams

Sex: female

Supplier: Shanghai Sipuer-Bikai Laboratory Animal Co., Ltd.

1.2 Preparation method of test substance

Table 2 Preparation method of test substance

Note: BID: twice a day.

1.3 Grouping and dosing schedule

Table 3 Groups and dosing schemes of experimental animals for in vivo efficacy

Note: PO: Oral gavage.

2. Experimental methods and procedures

2.1 Cell culture

Human breast cancer MDA-MB-231_luc cells are cultured in a monolayer in vitro, and the culture conditions are RPMI-1640 medium (supplier: Gibco; article number: 22400-089; production lot number: 4868546) with 10% fetal bovine serum, 100U/ml Penicillin and 100μg/ml streptomycin were cultured at 37°C with 5% CO _2. Use pancreatin-EDTA for routine digestion and passage twice a week. When the cells are in the exponential growth phase, the cells are collected, counted, and seeded.

2.2 Tumor cell inoculation

0.2 mL of 10×10 ⁶ MDA-MB-231_luc cells were subcutaneously inoculated on the right back of each nude mouse (PBS:Matrigel=1:1). When the average tumor volume reaches 100-150mm ³ , the drug will be administered in groups.

2.3 Tumor measurement and experimental indicators

The experimental index is to investigate whether the tumor growth is inhibited, delayed or cured. The tumor diameter was measured with vernier calipers twice a week. The calculation formula of tumor volume is: V=0.5a×b ² , a and b represent the long diameter and short diameter of the tumor, respectively.

The anti-tumor efficacy of the compound was evaluated by TGI (%) or the relative tumor growth rate T/C (%).

TGI (%), reflects the tumor growth inhibition rate. Calculation of TGI(%): TGI(%)=[(1-(Average tumor volume at the end of a certain treatment group-average tumor volume at the beginning of the treatment group))/(Average tumor volume at the end of treatment in the solvent control group Volume-The average tumor volume at the start of treatment in the solvent control group)]×100%.

The relative tumor proliferation rate T/C (%) is calculated as follows: T/C%=T _RTV /C _RTV ×100% (T _{RTV: RTV in the} treatment group; C _{RTV: RTV in the} negative control group).

Calculate the relative tumor volume (RTV) according to the results of tumor measurement. The calculation formula is RTV=V _t /V ₀ , where V ₀ is the average tumor volume measured during group administration (ie d _{0 ), V t} It is the average tumor volume at a certain measurement, and the _{data of T RTV and C RTV are} taken on the same day.

2.4 Statistical analysis

Statistical analysis, including the mean and standard error (SEM) of the tumor volume at each time point in each group. The treatment group showed the best treatment effect on the 21st day after the administration at the end of the trial, so statistical analysis was performed based on this data to evaluate the differences between the groups. The comparison between the two groups is analyzed by T-test, and the comparison between three or more groups is analyzed by one-way ANOVA. If the F value is significantly different, the Games-Howell method is used to test. If there is no significant difference in the F value, the Dunnet (2-sided) method is used for analysis. Use SPSS 17.0 for all data analysis. p<0.05 considered a significant difference.

3. Experimental results

After 21 days of administration, the tumor inhibition rate of compound A was TGI=54.85%, T/C=52.99%, compared with the vehicle control group, p=0.200; there was no significant change in animal body weight, and it was well tolerated.

Example 3: In vivo pharmacodynamic study of compound A in human prostate cancer PC-3 cell subcutaneous xenograft tumor model

1. Experimental Design

The preparation method of the test substance is the same as Table 2, and the animal grouping and dosing schedule are the same as Table 3.

2. Experimental animals

Species: Mouse

Strain: BALB/c nude mice

Week age and weight: 6-8 weeks old, weighing 18-22 grams

Sex: male

Supplier: Shanghai Sipuer-Bikai Laboratory Animal Co., Ltd.

3. Experimental methods and procedures

3.1 Cell culture

Human prostate cancer PC-3 cells were cultured in a monolayer in vitro with F-12K medium (supplier: Gibco; article number: 21127-022; production lot number: 1868870) with 10% fetal bovine serum and 100U/mL penicillin Incubate with 100μg/mL streptomycin at 37°C with 5% CO ₂ . Use pancreatin-EDTA for routine digestion and passage twice a week. When the cells are in the exponential growth phase, the cells are collected, counted, and seeded.

3.2 Tumor cell inoculation

0.1 mL of 10×10 ⁶ PC-3 cells were subcutaneously inoculated on the right back of each nude mouse. When the average tumor volume reaches 100-150mm ³ , the drug will be administered in groups.

3.3 Tumor measurement, experimental indicators and statistical analysis are the same as MDA-MB-231 model test.

4. Experimental results

After 21 days of administration, compared with the vehicle control group, the test compound A has a significant tumor-inhibiting effect (T/C=44.63%, TGI=58.4%, p=0.033); the animals have good tolerance.

Example 4: In vivo anti-tumor efficacy study of Compound A in MC38 mouse colon cancer cell animal transplantation tumor model

1. Experimental Design

The preparation method of the test substance is the same as Table 2, and the animal grouping and dosing schedule are shown in Table 4 below.

Table 4 Grouping of experimental animals for in vivo efficacy and dosing plan

2. Experimental animals

Species: Mouse

Strain: C57BL6 mouse

Week age and weight: 6-7 weeks old, weighing 16-20 grams

Sex: female

Supplier: Shanghai Slack Laboratory Animal Co., Ltd.

3. Experimental methods and procedures

3.1 Cell culture

Mouse colon cancer MC38 cells (Heyuan Biotechnology Co., Ltd.) were cultured in a monolayer in vitro with 10% fetal bovine serum in DMEM medium (Gibco, article number: 12100), 37°C and 5% CO _{2 in} an incubator nourish. Use 0.25% pancreatin-EDTA for routine digestion and passage. When the cells are in the exponential growth phase and the saturation is 80% to 90%, the cells are collected, counted, and inoculated.

3.2 Tumor cell inoculation

0.1 mL of 2×10 ⁵ MC38 cells were subcutaneously inoculated on the right back of each mouse. When the average tumor volume reached about 70 mm ³ , randomized administration was performed according to the tumor volume.

3.3 Tumor measurement, experimental indicators and statistical analysis are the same as MDA-MB-231 model test.

4. Experimental conclusion

After administration for 20 days, compared with the vehicle control group, the relative tumor growth rate T/C=33.68% in the 15mg/kg administration group of compound A, the tumor growth inhibition rate TGI=68.81%, p<0.0001; the 25mg/kg administration group Relative tumor proliferation rate T/C=27.59%, TGI=75.21%, p<0.0001; 50mg/kg administration group T/C=10.04%, TGI=93.46%, p<0.0001. Animals in each administration group had significant tumor-inhibiting effects and good tolerance.

3. Toxicological research

Example 5 Single-dose toxicity test in rats

Forty SD rats, half female and half male, were divided into 2 groups, 20 rats in each group, 10 rats/sex/group, and the groups were vehicle control group and compound A group. The single dose of compound A in group A is 100 mg/kg, the single dose volume is 10 mL/kg, and the dose concentration is 10 mg/mL (maximum gavage concentration). The dose is administered twice within 24 hours (8 hours apart). The total dosage is 200 mg/kg. The vehicle control group was given an equal volume of vehicle.

During the test, the animals in the compound A administration group had no symptoms of severe poisoning, and there were no abnormalities in food intake, blood index, coagulation index, and gross anatomical observation. 200mg/kg of compound A may have a reversible toxic effect on the digestive system of SD rats, which can cause animal salivation, watery stools and slightly slower weight gain in females. The serum UREA level of male animals in the compound A administration group decreased. Therefore, under the conditions of this experiment, the maximum tolerated dose (MTD) of compound A administered by oral gavage to SD rats was 200 mg/kg.

Example 6 Dog single-dose toxicity test

Eight Beagle dogs, half female and half male, were randomly divided into 4 groups by sex and weight, with 2 in each group, half female and half male. The groups are: vehicle control group, compound A low-dose group, compound A medium-dose group, and compound A high-dose group. The single doses of the compound low, medium and high dose groups were 6mg/kg, 18mg/kg and 48mg/kg, and they were given twice within 24 hours (8 hours apart), and the total doses were 12mg/kg and 36mg respectively. /kg and 96mg/kg. The vehicle control group was given an equal volume of vehicle.

During the test, the animals in each administration group of Compound A showed no symptoms of severe poisoning, and only transient watery stools appeared after the second administration on the day of administration. The animals in the high-dose group had a decrease in food intake 4 to 5 days after administration. the trend of. The blood indexes WBC and #NEUT of Beagle dogs in each dose group of compound A showed an increasing trend after administration. Therefore, under the experimental conditions, the non-toxic reaction dose (NOAEL) of compound A to Beagle dogs in a single administration is 36 mg/kg; MTD is 96 mg/kg.

Example 7 SD rats repeated administration for 4 weeks and recovery for 4 weeks accompanied by TK toxicity test study

SD rats were divided into 4 groups, each with 46 rats/group (main test 30 rats/group, TK satellite group 16 rats/group), half male and female. The groups were vehicle control group, compound A low-dose group, compound A medium-dose group, and compound A high-dose group. SD rats in the low, medium, and high dose groups of compound A were given 2, 6 and 20 mg/kg of compound A by oral gavage twice a day (8 hours apart) (total doses of 4, 12, and 40 mg/kg, respectively. day) for 4 consecutive weeks, stop the drug and resume for 4 weeks. The vehicle control group was given an equal volume of vehicle.

The results showed that during the administration period, the animals in each group including the vehicle control group had intermittent soft stools, the SD rats in the medium-dose and high-dose groups of Compound A had intermittent salivation, and individual SD rats in the high-dose group still Perianal filth, decreased spontaneous activity, yellow coat, abnormal urine color, and weight loss can be seen. Male animals in the high-dose group had reduced food intake and slow weight gain, and could recover after stopping the drug. Clinicopathological examinations revealed that the test product has a reversible toxic effect on the blood system and coagulation system at medium and high doses. The specific manifestations are increased NEUT and RETIC, decreased LYMPH and/or PLT, prolonged PT, and shortened TT. Histopathological examination revealed that 12-40 mg/kg of compound A had reversible toxic effects on the digestive system, immune system, respiratory system, and skeletal system of female and male SD rats, manifested by the decrease of rectal mucosal goblet cells, spleen and bone marrow lymph nodes Cells decreased, thymus weight/organ coefficient decreased, lung foam macrophages accumulated, and femur and knee joint trabeculae increased. Compound A at 40 mg/kg has a reversible toxic effect on the heart of male SD rats, manifested by monocyte infiltration or myocardial necrosis.

TK results showed that after D1 administration, there was a significant gender difference in the AUC0-24h dose group of compound A. After D1 and D28 administration, the system exposure (AUC0-24h and Cmax) of the other dose groups had no significant gender difference; D1 After administration with D28, the systemic exposure (AUC0-24h and Cmax) of compound A in female animals increased in a dose-proportional manner; the increase in AUC0-24h of compound A in male animals was higher than the increase in dose, and Cmax increased in a dose-proportional manner; It was administered twice a day, and after continuous administration for 4 weeks, there was no accumulation of compound A exposure in the plasma of female and male SD rats.

Therefore, under the test conditions, the MTD is 40 mg/kg/day. At this dose, the AUC0-24h of male and female animals on the 28th day were 23115 and 18017h*nM, respectively. NOAEL is 4mg/kg/day.

Example 8 Beagle dog repeated administration for 28 days, recovery for 28 days with TK toxicity test study

Beagle dogs were randomly divided into 4 groups, 10 per group, half male and half female. The groups were vehicle control group, compound A low-dose group, compound A medium-dose group, and compound A high-dose group. Compound A low, medium, and high dose groups were given 1, 3, and 6 mg/kg of Compound A by oral gavage twice a day (8 hours apart) (total daily doses were 2 mg/kg, 6 mg/kg, and 12 mg/kg, respectively). kg) for 4 consecutive weeks, and 4 weeks of recovery after stopping the drug.

The results showed that during the administration period, 3/10 animals (1 female and 2 male) in the 12 mg/kg compound A dose group were dying, and the cause of death was related to the toxicity of the test product's digestive system, blood system, and immune system. Compound A has a toxic effect on the digestive system of animals after the administration of each dose, which is manifested as vomiting, abnormal stool, loss of appetite, slow or decreased weight gain after administration, and the toxic reaction of animals in the middle and high dose groups is obvious. High-dose compound A can also cause animal hypoactivity, weight loss, tremor, abnormal gait, and abnormal ocular or nasal secretions. High-dose and/or medium-dose Compound A affects Beagle dog hematology indexes (reduction of RETIC, EOS, BASO), coagulation indexes (prolonged APTT, increase of FIB) and blood biochemical indexes (TP, ALB, CREA, UREA decrease, ALT, AST, CHOL, TG, GLU increased), but the effect is reversible. Histopathological examination revealed that 2 mg/kg of compound A had a reversible toxic effect on the immune system (thymus) of Beagle dogs. Compound A at 6-12 mg/kg has reversible toxic effects on the digestive system (pancreas, liver, digestive tract), immune system (thymus/spleen, intestinal-related lymph nodes), blood system (sternal bone marrow) and skin of Beagle dogs. The toxic effects of the reproductive system (testis, epididymis) of Beagle dogs were not fully recovered after the drug was stopped, and extramedullary hematopoiesis could be seen in the spleen in the convalescent animals. Compound A at 12 mg/kg has a reversible toxic effect on the respiratory system (trachea, lung) of Beagle dogs.

TK results showed that after D1 and D28 administration, there was no significant gender difference in the compound A system exposure (AUC0-24h and Cmax) of each dose group; after repeated administration, the compound A exposure (AUC0-24h and Cmax) of each dose group were all No significant accumulation; after the administration of D1 and D28, the increase ratio of AUC0-24h of compound A was higher than the increase in dose, and Cmax both increased in a dose-proportional manner.

Therefore, under the experimental conditions, the lethal dose is 12 mg/kg/day, and the HNSTD (the highest non-seriously toxic dose) is 6 mg/kg/day. At this dose, the 28th day (AUC0-24h) of male and female animals were 9443.7 and 9149.9h*nM, respectively. The lowest dose (LOAEL) at which adverse effects were observed was 2 mg/kg/day.

Four, clinical research

Example 9 Phase I clinical trial study

1. Scheme design

This trial is an open-label, dose-escalation and cohort expansion phase I clinical study for patients with advanced solid tumors. It aims to evaluate the safety, tolerability, pharmacokinetic characteristics and preliminary characteristics of compound A in patients with advanced solid tumors. Anti-tumor activity. Endpoint indicators include:

(1) Safety: the occurrence and frequency of AE, SAE, and DLT; determine the maximum tolerated dose (MTD) (if any) of compound A, phase II clinical recommended dose (RP2D) and dosing schedule.

(2) Pharmacokinetic indicators: including but not limited to AUC _0-last , AUC _0-∞ , C _max , T _max , t _1/2 and CL/F, etc.

(3) Curative effect indicators: total remission rate (ORR), progression-free survival (PFS), disease control rate (DCR), duration of remission (DOR), etc.

This study was conducted in two phases, the first phase (Stage I) was a dose escalation and dose expansion study, and the second phase (Stage II) was a cohort expansion (Extension Cohort) study.

The first stage (Stage I):

Dose escalation studies:

Refer to the HNSTD/MTD dose in the preliminary toxicology test and the in vivo pharmacodynamic test dose to design the clinical research dose. The initial dose of compound A was set at 20 mg/day, and 5 dose groups were initially proposed: 20, 40, 80, 120, and 160 mg. The preset maximum escalating dose is 160 mg/day. This study will follow the "i3+3" dose escalation schedule.

The first cycle of each dose level (31 days), after the subject's single dose on the first day (day 1), if there is no DLT or serious adverse events on the 3rd day of observation, the subject will enter the continuous dosing phase on the 4th day . (The specific dosing frequency will be adjusted according to the patient's tolerance, safety and drug PK parameters).

After completing the first cycle (Cycle 1) of administration, the safety, tolerability, pharmacokinetic characteristics and anti-tumor activity of the subjects were evaluated. If the subject is well tolerated and I agree, the drug will continue to be administered in repeated cycles every 28 days in the subsequent cycle (Cycle 2-Cycle N) until disease progression or intolerable toxicity occurs, or the study is terminated for other reasons.

Dose amplification study:

According to the safety, tolerability and effectiveness data obtained in the dose escalation study, if necessary, the target dose group dose expansion study and the exploratory study of different dosing schemes (such as 21 days of administration and 7 days of withdrawal) can be carried out. Sponsors and investigators will continue to conduct safety evaluations, and based on the available data of previous dose levels, determine the dose level and dosing schedule during dose escalation and dose expansion.

Stage II (Stage II):

After determining the MTD, the preliminary estimated RP2D, and the appropriate dosing regimen in the first phase, a cohort expansion study will be conducted in different indications to observe the safety, tolerability, pharmacokinetic characteristics and resistance of the study drug. Tumor activity.

In the expansion stage, groups of different tumor types may be selected, and each expansion group is expected to enroll 20 to 40 subjects. The sample size and tumor types for the final cohort expansion will be determined after full discussion with the investigator after comprehensive consideration of the first phase and preclinical research data. Moreover, sponsors and investigators may consider ending the enrollment of a cohort early based on the principle of the maximum benefit/risk ratio of the patient.

In the above two phases of Phase I clinical trials, a total of 53-186 patients with malignant advanced solid tumors are planned to be included.

2. Inclusion and exclusion criteria

1) Selection criteria

Subjects eligible to participate in this study must meet the following selection criteria:

1. Age from 18 to 75 (inclusive), regardless of gender.

2. Enrolled subjects must have advanced or metastatic tumors diagnosed by histology or cytology, and have no standard treatment plan, or those who are ineffective or intolerant to the standard treatment plan, or have no conditions to receive standard treatment and meet Requirements for tumor types at the following corresponding stages:

(1) Stage I: Unlimited solid tumor types;

(2) Stage II:

①Arm 1: TNBC

②Arm 2: CRC

③Arm 3: Other possible tumor types

3. There is at least one measurable lesion, and the definition of measurable comes from the RECIST 1.1 standard.

4. ECOG physical status score: 0 to 1 points.

5. The estimated survival time is more than 3 months.

6. The function of major organs meets the following criteria within 7 days before treatment (have not received blood transfusion, EPO, G-CSF or other medical supportive treatment within 14 days before the first administration of the study drug):

7. Women should agree to use contraceptive measures (such as intrauterine device [IUD], contraceptives or condoms) during the study period and within 6 months after the end of the study; serum pregnancy test within 7 days before the study is planned Negative and must be non-lactating subjects; males should agree to subjects who must use contraception during the study period and within 6 months after the end of the study period.

8. Subjects must give informed consent to this study before the experiment, and voluntarily sign a written informed consent form.

2) Exclusion criteria

Subjects can be excluded from any of the following items and not included in the study:

1. Have received chemotherapy, radiotherapy, biological therapy, endocrine therapy, targeted therapy, immunotherapy and other anti-tumor treatments within 4 weeks before using the study drug for the first time, except for the following items: nitrosoureas (such as carmustine) , Lomustine, etc.) or mitomycin C within 6 weeks before the first use of the study drug; oral fluorouracils and small molecule targeted drugs are within 2 weeks before the first use of the study drug or 5 half-lives of the known drug ( Subject to the longer time); Chinese medicine with anti-tumor indications shall be within 2 weeks before the first use of the study drug.

2. Received treatment with other unmarketed clinical investigational drugs within 4 weeks before using the investigational drug for the first time.

3. Received major organ surgery (excluding needle biopsy) or had significant trauma within 4 weeks before using the study drug for the first time.

4. Have received systemic glucocorticoids (prednisone> 10 mg/day or equivalent doses of similar drugs) or other immunosuppressive agents within 14 days before the first use of the study drug; except for the following cases: use of local or eye , Intra-articular, intranasal and inhaled glucocorticoid therapy; short-term use of glucocorticoids for preventive treatment (for example, prevention of contrast agent allergy).

5. Those who have used a strong inhibitor or a strong inducer of CYP3A4 within 14 days before using the study drug for the first time.

6. Have received BET inhibitor treatment in the past.

7. The adverse reactions of previous anti-tumor treatments have not yet recovered to CTCAE 5.0 grade evaluation ≤ 1 (except for the toxicity that the researcher judges no safety risk such as hair loss).

8. Central nervous system metastasis or meningeal metastasis with clinical symptoms, or there is other evidence that the patient's central nervous system metastasis or meningeal metastasis has not been controlled, and the investigator judged it is not suitable for inclusion.

9. Active infections that cannot be controlled (acute or chronic fungal, bacterial, viral or other infections).

10. Have a history of autoimmune diseases, immunodeficiency, including HIV test positive, or have other acquired or congenital immunodeficiency diseases, or have a history of organ transplantation.

11. Active hepatitis B (hepatitis B virus titer>1000 copies/ml or 200IU/ml), allowing preventive antiviral therapy other than interferon; hepatitis C antibody is positive.

12. Have a history of serious cardiovascular disease, including but not limited to:

(1) Severe heart rhythm or conduction abnormalities, such as ventricular arrhythmia that requires clinical intervention, degree II to III atrioventricular block, etc., or other arrhythmias that require antiarrhythmic drugs (excluding anticoagulant drugs) , The coagulation function related to taking anticoagulant drugs must meet the selection criteria);

(2) History of myocardial infarction, angina pectoris, angioplasty, coronary artery graft surgery;

(3) Those with prolonged QT/QTc interval of ECG at baseline (QTcF>480ms);

(4) Baseline echocardiography (ECHO) or multi-gate acquisition (MUGA) technology shows left ventricular ejection fraction (LVEF) ≤50%;

(5) Heart failure, New York College of Cardiology (NYHA) grade II and above;

(6) Poorly controlled hypertension (despite optimal treatment, systolic blood pressure ≥150mmHg and/or diastolic blood pressure ≥95mmHg);

(7) Past or current cardiomyopathy.

13. Unable to swallow the drug orally, or there is a condition that has been judged by the investigator to seriously affect the absorption of the gastrointestinal tract.

14. Patients who have a history of other serious systemic diseases and are judged by the investigator to be unsuitable to participate in clinical trials.

15. Known alcohol or drug dependence.

16. Have a clear history of neurological or mental disorders, including epilepsy or dementia.

17. Women during pregnancy or lactation.

18. The investigator believes that the subject is not suitable for participating in this clinical study for other reasons.

3. Research results

Currently in clinical trials, the number of subjects is small and the treatment period is short, which is not enough to make an accurate estimate of the final results and later plans. Considering the tolerated dose of toxicology test, non-clinical pharmacodynamic dose and the phase I clinical dose of similar compounds, the expected maximum tolerated dose (MTD) of compound A is 120mg/d～160mg/d, and the expected therapeutic dose is 80mg/d. Under the experimental conditions, compound A administered at 80 mg/d, 120 mg/d, and 160 mg/d will have an effective therapeutic effect on patients with advanced solid tumors, especially gastrointestinal tumors, triple-negative breast cancer, colorectal cancer, and prostate cancer. . In the study, if the dose is increased to 160mg/d, the safety and tolerability of the dose group is still good, the investigator and the sponsor can discuss whether to explore a higher dose. The determination of higher doses will be based on the available data at existing dose levels, which cannot be accurately estimated at present.

It can be expected that Compound A will be able to safely and effectively treat advanced solid tumors, especially gastrointestinal tumors, triple negative breast cancer, colorectal cancer, and prostate cancer.

Example 10 Typical case

A patient, male, 62 years old, diagnosed with stage IIIB rectal cancer in 2015, underwent laparoscopic anterior resection and radical resection of rectal cancer, postoperative adjuvant oxaliplatin+5-Fu, postoperative adjuvant pelvic radiotherapy; later experienced oxa Liplatin + capecitabine, FOLFIRI (irinotecan + 5-Fu + leucovorin), simutecan + thalidomide, capecitabine single agent, recombinant anti-EGFR human mouse chimeric monoclonal antibody Injection, regorafenib treatment; in 2017, two radiotherapy for left lung lesions. All cases progressed and the treatment failed. The patient was diagnosed at the time of enrollment: rectal cancer stage IVA.

The patient was given Compound A orally for the first time on 2020-5-13, with a dose of 20 mg. No serious abnormalities were observed for two days, and the administration was continued on 2020-5-15, the dose was 20 mg/day, and the last dose was 2020-7-10, for a total of 2 cycles (28 days per cycle). At the end of the second cycle, an enhanced CT examination was performed, and the comprehensive assessment was SD (stable disease). During the medication, grade 1 hypoalbuminemia, grade 1 anemia, and grade 1 white blood cell count decreased. Hypoproteinemia may not be related to the study drug, and the latter two may be related to the study drug.

The full names and Chinese definitions of English abbreviations in the manual are as follows:

Abbreviations and terms	English full name	Chinese paraphrase
AE	Adverse event	Adverse events
AUC	Area under drug time curve	Area under the drug-time curve
AL	Acute leukemia	Acute leukemia
ALT	Alanine aminotransferase	Alanine aminotransferase
ANC	Absolute neutrophil count	Neutrophil count
APTT	Activeated partial thromboplastin time	Partial thromboplastin time
AR	Androgen receptor	Androgen receptor
AST	Aspartate aminotransferase	Aspartate aminotransferase
BET	Bromodomain and extra-terminal domain	Bromodomain and ultra-terminal structure
BID	Bis in die	Twice a day
BRCA	Breast cancer susceptibility gene	Breast cancer susceptibility gene
BRD4	Bromodomain-containing protein 4	Bromodomain protein 4
CHL	Chinese hamster lung cells	Chinese Hamster Lung Cell
CHO	Chinese Hamster Ovary	Chinese Hamster Egg Cell
CL	Plasma clearance rate	Plasma clearance
Cmax	Peak concentration	Peak concentration
CR	Complete remission	totally relaxed
CRC	Colorectal cancer	Colorectal cancer
DFS	Disease-free survival	Disease-free survival
DLBCL	Diffuse large B-cell lymphoma	Diffuse large B cell lymphoma
DLT	Dose limited toxicity	Dose limiting toxicity
EC50	50%effective concentration	Half effective concentration
ECHO	Echocardiography	Echocardiogram
ER	Estrogen receptor	Estrogen receptor
GPCR	G protein coupled receptor	G protein coupled receptor
HER2	Human epidermal growth factor receptor 2	Human epidermal growth factor receptor
HNSTD	Maximum non serious toxic dose	Highest non-seriously toxic dose
ICF	Informed consent form	Informed consent
INR	International Normalized Ratio	International normalized ratio
IUD	Intrauterine device	Intrauterine device
LOAEL	The lowest dose at which harmful effects are observed	The lowest dose at which harmful effects have been observed
MTD	Maximum torlerate dose	Maximum tolerated dose
MUGA	Multiple uptake gated acquisition scan	Multi-gate circuit detection
NOAEL	No harmful effect dose level	No harmful effect dose level
NYHA	New York Heart Association	New York Heart Association
OHM	Other hematological malignancies	Other hematological malignancies

OS	Overall survival	Overall survival
PD-L1	Programmed cell death-Ligand 1	Programmed death receptor-ligand 1
PR	Partial remission	Partial relief
PD	progression disease	Disease progression
PR	Progesterone acceptor	Progesterone receptor
PT	Prothrombin time	Prothrombin time
RECIST	Response evaluation criteria in solid tumors	Evaluation Criteria for Efficacy of Solid Tumors
RP2D	Recommended phase 2 dose	Phase II clinical trial recommended dose
SAE	Serious adverse event	Serious adverse event
SD	Stable disease	Stable disease
TGI	Tumor growth inhibition	Tumor growth inhibition
T1/2	half life	half life
Tmax	Peak time	Peak time
TNBC	Triple-Negative Breast Cancer	Triple negative breast cancer
ULN	Upper limit of normal	Upper limit of normal
Vdss	Apparent volume of steady state distribution	Steady-state apparent volume of distribution

Claims (14)

1. Application of compound A or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating tumors, the structure of said compound A is shown in the following formula (I):

   

   The tumor is preferably advanced solid tumor or refractory solid tumor, more preferably the solid tumor in the advanced solid tumor or refractory solid tumor is gastrointestinal tumor, colorectal cancer, breast cancer or prostate cancer, further preferably said The solid tumors in advanced solid tumors or refractory solid tumors are colorectal cancer, triple-negative breast cancer, and androgen-independent prostate cancer in which tumor-associated macrophages are highly infiltrated; preferably, the subject suffering from the tumor is a human.
2. The application according to claim 1, wherein the medicine is prepared into clinically accepted preparations, such as oral preparations, injection preparations, topical administration preparations, and topical preparations.
3. The application according to claim 1 or 2, characterized in that: the drug is in a single-dose dosage form or a multi-dose dosage form; the dosage form contains a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof (compound A ), about 0.001 mg to about 1000 mg, preferably about 1 mg to about 500 mg, or about 5 mg to about 400 mg, or about 10 mg to about 300 mg, or about 10 mg to about 250 mg, or about 10 mg to about 200 mg, or about 20 mg -About 200 mg, more preferably about 10 mg to about 160 mg, or about 20 mg to about 160 mg, or about 80 mg to about 160 mg; for example, about 10 mg, about 20 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg , About 160 mg, more preferably about 10 mg, about 20 mg, about 40 mg, about 80 mg, about 120 mg or about 160 mg, still more preferably about 20 mg, about 40 mg, about 80 mg, about 120 mg or about 160 mg, or even more preferably about 10 mg or about 40mg.
4. The application according to any one of claims 1 to 3, wherein the compound A or a pharmaceutically acceptable salt thereof can be used in combination with one or more of other targeted drugs or chemotherapeutic drugs.
5. A method for treating tumors, the method comprising administering to a subject or patient a therapeutically effective amount of compound A or a pharmaceutically acceptable salt thereof, the structure of the compound A is shown in the following formula (I):

   

   The tumor is preferably advanced solid tumor or refractory solid tumor, further preferably the solid tumor in the advanced solid tumor or refractory solid tumor is gastrointestinal tumor, breast cancer, colorectal cancer or prostate cancer, further preferably said The solid tumors in advanced solid tumors or refractory solid tumors are colorectal cancer, triple-negative breast cancer, androgen-independent prostate cancer with high tumor-associated macrophages infiltration; preferably, the subject or patient is a human.
6. The method of claim 5, wherein the administration is oral administration, injection administration, topical administration or in vitro administration; or compound A or a pharmaceutically acceptable salt thereof is made into a clinically acceptable preparation and then administered. The preparations include oral preparations, injection preparations, topical administration preparations, and topical preparations.
7. The method according to any one of claims 5-6, wherein compound A or a pharmaceutically acceptable salt thereof (calculated as compound A) is administered at a daily dose of about 0.001 mg to about 1000 mg, preferably about 1 mg -About 500 mg, or about 5 mg to about 400 mg, or about 10 mg to about 300 mg, or about 10 mg to about 250 mg, or about 10 mg to about 200 mg, or about 20 mg to about 200 mg, more preferably about 10 mg to about 160 mg, or about 20 mg -About 160 mg, or about 80 mg to about 160 mg; for example, about 10 mg, about 20 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, more preferably about 10 mg, about 20 mg, about 40 mg, About 80 mg, about 120 mg, or about 160 mg, and more preferably about 20 mg, about 40 mg, about 80 mg, about 120 mg, or about 160 mg are administered in a single dose or in divided doses.
8. The method according to any one of claims 5-7, wherein the dosage regimen of Compound A or a pharmaceutically acceptable salt thereof is 28 days per cycle, once a day for 21 days, The drug is stopped for 7 days; preferably, about 20 mg, about 40 mg, about 80 mg, about 120 mg, about 160 mg of compound A or a pharmaceutically acceptable salt thereof (calculated as compound A) is administered each time.
9. The method according to any one of claims 5-8, wherein the compound A or a pharmaceutically acceptable salt thereof is used in combination with one or more of other targeted drugs or chemotherapeutic drugs.
10. Compound A or a pharmaceutically acceptable salt thereof is used for the treatment of tumors, the tumors are preferably advanced solid tumors or refractory solid tumors, and more preferably the solid tumors in the advanced solid tumors or refractory solid tumors are gastrointestinal Tumor, colorectal cancer, breast cancer or prostate cancer, further preferably the solid tumor in the advanced solid tumor or refractory solid tumor is colorectal cancer with high infiltration of tumor-associated macrophages, triple-negative breast cancer, androgenic non-androgen Dependent prostate cancer; preferably the subject suffering from the tumor is a human,

    The structure of the compound A is shown in the following formula (I):

    
11. The compound A or a pharmaceutically acceptable salt thereof according to claim 10, wherein the compound A or a pharmaceutically acceptable salt thereof is administered after being prepared into a clinically accepted preparation, and the preparation includes an oral preparation, Injection preparations, topical administration preparations, topical preparations.
12. The compound A or a pharmaceutically acceptable salt thereof according to any one of claims 10-11, wherein the daily dose of compound A or a pharmaceutically acceptable salt thereof (calculated as compound A) is about 0.001 mg to about 1000 mg, preferably about 1 mg to about 500 mg, or about 5 mg to about 400 mg, or about 10 mg to about 300 mg, or about 10 mg to about 250 mg, or about 10 mg to about 200 mg, or about 20 mg to about 200 mg, more preferably About 10 mg to about 160 mg, or about 20 mg to about 160 mg, or about 80 mg to about 160 mg; for example, about 10 mg, about 20 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, more preferably About 10 mg, about 20 mg, about 40 mg, about 80 mg, about 120 mg, or about 160 mg, and more preferably about 20 mg, about 40 mg, about 80 mg, about 120 mg, or about 160 mg are administered in a single dose or divided doses.
13. The compound A or a pharmaceutically acceptable salt thereof according to any one of claims 11-12, wherein the dosage regimen of the compound A or a pharmaceutically acceptable salt thereof is 28 days per cycle, every day It is administered once, administered for 21 days, and stopped for 7 days; preferably, about 20 mg, about 40 mg, about 80 mg, about 120 mg, or about 160 mg of compound A or a pharmaceutically acceptable salt thereof (calculated as compound A) is administered each time.
14. The compound A or a pharmaceutically acceptable salt thereof according to any one of claims 11-13, wherein the compound A or a pharmaceutically acceptable salt thereof is in combination with other targeted drugs or chemotherapeutics. One or more types are used in combination.

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