WO2021089005A1 - Use of fgfr inhibitor - Google Patents

Abstract

Provided is the use of an FGFR inhibitor. More specifically, provided are an FGFR inhibitor for treating FGFR-related tumors or a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising same, a method for treating FGFR-related tumors by means of using a drug comprising same, and the use thereof in the preparation of a drug for treating FGFR-related tumors. In vitro and in vivo test results show that compound A has the effect of inhibiting the activity of digestive or urinary system tumors related to abnormal FGFR expression, can be used to develop a drug for treating diseases involving digestive or urinary system tumors, and has important clinical application values.

Description

Use of a FGFR inhibitor

Cross-references to related applications

This application claims the priority of the Chinese patent application number 201911086889.9 filed on November 8, 2019 and the Chinese patent application number 202010321669.6 filed on April 22, 2020. The entire contents of the above two patent applications are hereby incorporated by reference.

Technical field

The invention belongs to the field of medicine. More specifically, the present invention relates to an FGFR inhibitor or a pharmaceutically acceptable salt thereof for the treatment of FGFR-related tumors, a pharmaceutical composition containing the same, a method for treating FGFR-related tumors using a drug containing the same, and the preparation thereof Use in drugs for treating FGFR-related tumors.

Background technique

Fibroblast growth factor receptor (FGFR) is a receptor for fibroblast growth factor (FGF) signal transduction, and its family consists of four members (FGFR1-4). Through FGFR, FGF plays an important role in many physiological regulation processes such as cell proliferation, cell differentiation, cell migration and angiogenesis. A lot of research evidence shows that abnormal FGF signaling pathway (high expression, gene amplification, gene mutation, chromosome recombination, etc.) is directly related to many pathological processes such as tumor cell proliferation, migration, invasion and angiogenesis, and FGFR is in many tumors such as Non-small cell lung cancer, breast cancer, gastric cancer, bladder cancer, urothelial cancer, endometrial cancer, liver cancer, prostate cancer, cervical cancer, colon cancer, esophageal cancer, myeloma, etc. all show overexpression or overactivation. For example, studies have found that FGFR2 fusion mutations occur in about 10-20% of patients with intrahepatic cholangiocarcinoma; FGFR3 gene changes in bladder cancer have a strong correlation with low-grade pathological and clinical low-stage cancers, more than 70% The pathological low-grade non-invasive urothelial papilloma contains FGFR3 mutation; and high expression of FGFR1, FGFR2 and FGFR3 are found in gastric cancer tissues and gastric cancer cells. Therefore, FGFR has become an important therapeutic target, attracting a wide range of research and development interest.

According to the mechanism of action, inhibitors that target the kinase domain of the FGFR membrane can be divided into ATP-competitive inhibitors, non-ATP-competitive reversible inhibitors, and irreversible inhibitors. Among them, non-ATP-competitive reversible inhibitors and irreversible inhibitors affect kinases. The inhibitory activity is not affected by the high ATP concentration in the cell and in the body. In recent years, some FGFR-targeted drugs for the treatment of the above-mentioned tumor diseases have entered the clinical trial stage. For example, Balversa (Erdafitinib) has become the world's first pandemic drug for metastatic urothelial cancer approved by the FDA. FGFR reversible inhibitor targeted drugs. In addition, other FGFR1-4 inhibitors (for example, BGJ-398, Debio-1347 and TAS-120, etc.) are in different clinical trials for solid tumors such as urothelial cell carcinoma and liver cancer. The structural formulas of the above drugs are as follows:

However, studies have found that pan-FGFR (pan-FGFR) reversible inhibitors such as BGJ-398 will inevitably develop drug resistance after 4-6 months of use, resulting in reduced efficacy, while irreversible inhibitors such as TAS-120 Patients who are resistant to BGJ-398 are still effective, showing the unique advantages of irreversible inhibitors. But so far, no FGFR1-4 irreversible inhibitor has been approved for marketing at home and abroad.

Primary liver cancer (referred to as liver cancer) is a relatively common malignant tumor and the fourth leading cause of cancer deaths in the world, posing a serious threat to human life and health. Liver cancer mainly includes hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and combined hepatocellular cholangiocarcinoma (c-CC-CC). Liver cancer has no obvious clinical symptoms in the early stage, and is often found in the middle and late stages, with high malignancy, high recurrence rate, poor treatment effect, and poor prognosis. The clinical treatment of liver cancer mainly includes surgical treatment, transcatheter arterial chemoembolization (TACE), radiotherapy and chemotherapy. However, there is still a risk of cancer in the residual liver tissue of the patient after surgery, and the 5-year risk of recurrence exceeds 70%. Sorafenib is currently the only molecularly targeted drug approved for the treatment of advanced liver cancer, but its effect on liver cancer patients with liver function Child-Pugh grade B is still poor.

Urothelial cancer (UCC) is a common malignant tumor worldwide, and it is also one of the most common clinical malignancies in urology in my country. Among them, 90%-95% of urothelial cancers are bladder cancer. Urothelial cell carcinoma is mostly non-muscular invasive at the first diagnosis, but it has a high recurrence rate; and with the increase in the number of recurrences, the malignancy of the tumor will increase and turn into a muscular invasive tumor. Patients with metastatic urothelial cancer with FGFR gene alterations have a poor prognosis and a low response rate to treatment. There are significant clinical needs that are far from being met in this type of patients. For decades, the standard of treatment for urothelial cancer has been based on Cisplatin (Cisplatin) chemotherapy. The curative effect of second-line chemotherapy drugs Vinflunine or taxanes is slightly improved, the historical objective response rate (ORR) is only about 10%, and the median overall survival (overall survival, OS) is 7-9 months. For advanced or metastatic urothelial cancer, the overall response rate of PD-1/PD-L1 checkpoint inhibitors approved in recent years is only 15% to 20%, and the median OS is about 10 months. Therefore, many patients Did not benefit.

Gastric cancer is also one of the most common malignant tumors in the world, with a relatively poor prognosis and a serious threat to human health. Due to the lack of a mature early screening system and the atypical symptoms of early gastric cancer and the low detection rate, most patients are already in the advanced stage when they are diagnosed. Today's treatment measures for gastric cancer mainly include surgical treatment, systemic application of chemical drugs, radiotherapy and molecular targeted drug therapy. Targeted therapy is a drug treatment aimed at specific tumor targets. However, due to the strong heterogeneity of gastric cancer and other reasons, clinical studies on targeted therapy of gastric cancer have few successes and many failures.

Cholangiocarcinoma (CCA) is a malignant tumor of epithelial cells with different characteristics of cholangiocarcinoma. The incidence has increased by nearly 20% in the past 10 years, accounting for about 3% of gastrointestinal tumors and 10-15% of hepatobiliary malignancies. According to anatomical location, cholangiocarcinoma can be divided into intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma (eCCA), the latter is further divided into hilar cholangiocarcinoma (pCCA) and distal cholangiocarcinoma (distal cholangiocarcinoma) , DCCA). Radical surgery is the only cure, but there are no characteristic clinical manifestations in the early stage of the disease. About two-thirds of patients have lost the opportunity of surgery at the first diagnosis, and the 5-year survival rate is about 10%. Even if surgically removed, the recurrence rate of cholangiocarcinoma 1 year after surgery is still as high as 50%. For unresectable locally advanced metastatic biliary tumors (including intrahepatic and extrahepatic cholangiocarcinoma, gallbladder cancer, ampullary carcinoma, etc.), the standard first-line chemotherapy regimen (gemcitabine combined with cisplatin) only brings a median survival of 11.7 months , The prognosis is extremely poor. However, FGFR inhibitors have shown promise in clinical trials of cholangiocarcinoma. For example, the FGFR inhibitor BGJ-398 has obtained excellent phase II trial results in advanced cholangiocarcinoma with FGFR gene fusion, mutation, and amplification, and objective remission The rate and disease control rate in FGFR gene fusion patients reached 18.8% and 83.3%.

Intrahepatic cholangiocarcinoma refers to malignant tumors of bile duct epithelium located in the liver at level 2 and above. It is also called intrahepatic cholangiocarcinoma. It belongs to a type of primary liver cancer (accounting for 10% to 15%). Can be attributed to a type of cholangiocarcinoma (about 10%). Intrahepatic cholangiocarcinoma is highly malignant, has strong invasion and lymph node metastasis characteristics, and is difficult to diagnose early. The prognosis of the patient is poor. The overall 5-year survival rate is less than 10%, and the median survival time after surgical resection is 36 months. At present, the only cure for intrahepatic cholangiocarcinoma is early detection and surgical resection, but the recurrence rate after resection is also high. As for patients with locally advanced unresectable, recurrence and metastasis, there is a lack of effective measures that can significantly improve the prognosis. Intrahepatic cholangiocarcinoma is insensitive to traditional chemotherapy, radiotherapy and recent tumor immunotherapy. There is no standard treatment for cholangiocarcinoma patients who have failed chemotherapy with the first-line gemcitabine. It is reported in the literature to accept various programs such as 5-fluorouracil (5-FU) and irinotecan, 5-FU and oxaliplatin, 5-FU and cisplatin, 5-FU or capecitabine and sunitinib In patients with second-line chemotherapy, the median disease progression-free survival (PFS) and overall survival (OS) were 3.2 and 6.7 months, respectively. There was no PFS or OS between the regimens. Significant differences. There have been many reports on the molecular mechanism of intrahepatic cholangiocarcinoma. Nakamura et al. conducted a comprehensive genomic analysis of 260 cholangiocarcinoma patients and found that 40% of the cases showed FGFR gene changes, and FGFR2 was highly expressed in intrahepatic cholangiocarcinoma. Using next-generation sequencing technology, it was also found that 13%-50% of patients with intrahepatic cholangiocarcinoma carried FGFR2 gene fusions, of which 13.6% of patients had FGFR2-BICC1 and FGFR2-AHCYL1 gene fusions. These findings provide new opportunities for the treatment of intrahepatic cholangiocarcinoma.

At present, several FGFR inhibitors are in different stages of clinical research on cholangiocarcinoma, such as BGJ-398 (reversible selective inhibitor), ARQ087 (ATP competitive inhibitor), TAS-120 (irreversible pan-FGFR inhibitor) Et al. The first FGFR inhibitor reported in cholangiocarcinoma was BGJ-398. The study reported that 3 patients with FGFR2 fusion-positive intrahepatic cholangiocarcinoma who received BGJ-398 treatment developed clinically acquired FGFR inhibitor resistance. Further studies found polyclonal secondary mutations in the kinase domain of FGFR2, including 3 cases The FGFR2V564F gene mutation exists in all patients. TAS-120 is a highly selective and irreversible pan-FGFR inhibitor. Studies have shown that TAS-120 has clinical effects on BGJ-398-resistant intrahepatic cholangiocarcinoma patients, and can inhibit a variety of secondary FGFR2 mutations. However, there is no literature that discloses the cell-level inhibitory activity data of TAS-120. Currently, no targeted drugs for the treatment of intrahepatic cholangiocarcinoma have been approved for marketing.

WO2019034076A1 discloses FGFR inhibitors and their medical uses, including compound A (Example 2). This patent application discloses the evaluation results of compound A on FGFR wild-type kinase in vitro inhibitory activity, the evaluation results on mutant kinase in vitro inhibitory activity and the pharmacokinetic evaluation results in mice, but it does not disclose which diseases Compound A can be used for. the treatment.

Summary of the invention

[Object of the present invention]

An object of the present invention is to provide a use of Fibroblast Growth Factor Receptor (FGFR) inhibitor compound A or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of FGFR-related tumors.

Another object of the present invention is to provide an FGFR inhibitor compound A or a pharmaceutically acceptable salt thereof, which is used for the treatment of FGFR-related tumors.

Another object of the present invention is to provide a method for treating FGFR-related tumors, the method comprising administering to a subject or patient a drug containing a therapeutically effective amount of an FGFR inhibitor compound A or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of FGFR-related tumors, which comprises an FGFR inhibitor compound A or a pharmaceutically acceptable salt thereof and optionally a pharmaceutically acceptable carrier.

[Technical Solution of the Invention]

A technical problem to be solved by the present invention is to provide a small molecule compound with excellent FGFR1-4 wild-type and mutant kinase inhibitory activity, and a pharmaceutical composition, use and treatment method thereof.

Another technical problem to be solved by the present invention is to provide a small molecule with excellent FGFR1-4 wild-type and mutant kinase inhibitory activity and excellent in vitro/in vivo anti-FGFR-related tumors (especially digestive or urinary system tumors) activity Compound and its pharmaceutical composition, use and treatment method.

Another technical problem to be solved by the present invention is to provide an excellent FGFR1-4 wild-type and mutant kinase inhibitory activity and excellent in vitro/in vivo anti-FGFR-related tumor (especially digestive or urinary system tumors) activity, and has Small molecule compounds with good safety and their pharmaceutical compositions, uses and treatment methods.

Another technical problem to be solved by the present invention is to provide an excellent FGFR1-4 wild-type and mutant kinase inhibitory activity and excellent in vitro/in vivo anti-FGFR-related tumor (especially digestive or urinary system tumors) activity, and has Small molecule compounds with excellent plasma stability and safety, and pharmaceutical compositions, uses and treatment methods thereof.

In order to solve the above technical problems, the inventor of the present application conducted further experiments on the basis of the prior art WO2019034076A1, and found that compound A has good inhibitory activity on both wild-type and mutant FGFR1-4, and can significantly inhibit FGFR1 -4 Proliferation of digestive or urinary system tumor cells and xenograft tumor models related to abnormal expression, and has high plasma stability and safety, which suggests that compound A can be used to develop treatments for FGFR-related digestive or urinary system tumor diseases drug. The results of the above studies have led to the completion of the technical solution of the present invention.

Specifically, in the first aspect of the present invention, there is provided the use of an FGFR inhibitor compound A or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of FGFR-related tumors, wherein the compound A has The structure is as follows:

In one embodiment, the FGFR-related tumor is one or more of digestive or urinary system tumors.

In one embodiment, the FGFR-related tumor is any one of gastric cancer, liver cancer, urothelial cancer, cholangiocarcinoma, or any combination thereof.

In one embodiment, the FGFR-related tumor is gastric cancer. In a preferred embodiment, the gastric cancer is gastric cancer with FGFR2 gene amplification.

In one embodiment, the FGFR-related tumor is liver cancer. In a preferred embodiment, the liver cancer is a liver cancer with high FGFR4/FGF19 expression. In a preferred embodiment, the liver cancer is a liver cancer with high FGFR3 expression. In a preferred embodiment, the liver cancer is any one of hepatocellular carcinoma, intrahepatic cholangiocarcinoma, mixed liver cancer, or any combination thereof.

In one embodiment, the FGFR-related tumor is urothelial carcinoma. In a preferred embodiment, the urothelial cancer is bladder cancer. In a preferred embodiment, the bladder cancer is a bladder cancer with high FGFR3 expression and FGFR3-TACC3 fusion.

In one embodiment, the FGFR-related tumor is cholangiocarcinoma. In a preferred embodiment, the cholangiocarcinoma is a cholangiocarcinoma with high FGFR2 expression. In a preferred embodiment, the cholangiocarcinoma is any one of intrahepatic cholangiocarcinoma, hilar cholangiocarcinoma, distal cholangiocarcinoma, or any combination thereof. In a more preferred embodiment, the cholangiocarcinoma is intrahepatic cholangiocarcinoma.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is the only active ingredient in the drug.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is used in combination with one or more other targeted drugs or chemotherapeutics.

In one embodiment, the drug is formulated into a clinically accepted formulation. In a preferred embodiment, the preparation is an oral preparation, an injection preparation or a topical preparation.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.001 mg/kg to about 1000 mg/kg. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.01 mg/kg to about 100 mg/kg. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.02 mg/kg to about 50 mg/kg. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.03 mg/kg to about 20 mg/kg.

In one embodiment, the medicament contains a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the therapeutically effective amount is 0.001-1000 mg. In a preferred embodiment, the therapeutically effective amount is 0.01-100 mg. In a preferred embodiment, the therapeutically effective amount is 0.1-50 mg. In a preferred embodiment, the therapeutically effective amount is 0.5-30 mg.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a single dose or in divided doses.

In one embodiment, the drug is administered orally, by injection, topical, or in vitro. In a preferred embodiment, the drug is administered orally or by injection.

In the second aspect of the present invention, there is provided an FGFR inhibitor compound A or a pharmaceutically acceptable salt thereof for the treatment of FGFR-related tumors, wherein the compound A has the following structure:

In one embodiment, the FGFR-related tumor is one or more of digestive or urinary system tumors.

In one embodiment, the FGFR-related tumor is any one of gastric cancer, liver cancer, urothelial cancer, cholangiocarcinoma, or any combination thereof.

In one embodiment, the FGFR-related tumor is gastric cancer. In a preferred embodiment, the gastric cancer is gastric cancer with FGFR2 gene amplification.

In one embodiment, the FGFR-related tumor is liver cancer. In a preferred embodiment, the liver cancer is a liver cancer with high FGFR4/FGF19 expression. In a preferred embodiment, the liver cancer is a liver cancer with high FGFR3 expression. In a preferred embodiment, the liver cancer is any one of hepatocellular carcinoma, intrahepatic cholangiocarcinoma, mixed liver cancer, or any combination thereof.

In one embodiment, the FGFR-related tumor is urothelial carcinoma. In a preferred embodiment, the urothelial cancer is bladder cancer. In a preferred embodiment, the bladder cancer is a bladder cancer with high FGFR3 expression and FGFR3-TACC3 fusion.

In one embodiment, the FGFR-related tumor is cholangiocarcinoma. In a preferred embodiment, the cholangiocarcinoma is a cholangiocarcinoma with high FGFR2 expression. In a preferred embodiment, the cholangiocarcinoma is any one of intrahepatic cholangiocarcinoma, hilar cholangiocarcinoma, distal cholangiocarcinoma, or any combination thereof. In a more preferred embodiment, the cholangiocarcinoma is intrahepatic cholangiocarcinoma.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is used as the sole active ingredient.

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

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is formulated into a clinically acceptable preparation. In a preferred embodiment, the preparation is an oral preparation, an injection preparation or a topical preparation.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.001 mg/kg to about 1000 mg/kg. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.01 mg/kg to about 100 mg/kg. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.02 mg/kg to about 50 mg/kg. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.03 mg/kg to about 20 mg/kg.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is formulated in a medicament in a therapeutically effective amount. In a preferred embodiment, the therapeutically effective amount is 0.001-1000 mg. In a preferred embodiment, the therapeutically effective amount is 0.01-100 mg. In a preferred embodiment, the therapeutically effective amount is 0.1-50 mg. In a preferred embodiment, the therapeutically effective amount is 0.5-30 mg.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a single dose or in divided doses.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered orally, by injection, topical, or in vitro. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered by oral administration or injection.

In the third aspect of the present invention, there is provided a method of treating FGFR-related tumors, the method comprising administering to a subject or patient a therapeutically effective amount of an FGFR inhibitor compound A or a pharmaceutically acceptable salt thereof Drug, wherein the compound A has the following structure:

In one embodiment, the FGFR-related tumor is one or more of digestive or urinary system tumors.

In one embodiment, the FGFR-related tumor is any one of gastric cancer, liver cancer, urothelial cancer, cholangiocarcinoma, or any combination thereof.

In one embodiment, the FGFR-related tumor is gastric cancer. In a preferred embodiment, the gastric cancer is gastric cancer with FGFR2 gene amplification.

In one embodiment, the FGFR-related tumor is liver cancer. In a preferred embodiment, the liver cancer is a liver cancer with high FGFR4/FGF19 expression. In a preferred embodiment, the liver cancer is a liver cancer with high FGFR3 expression. In a preferred embodiment, the liver cancer is any one of hepatocellular carcinoma, intrahepatic cholangiocarcinoma, mixed liver cancer, or any combination thereof.

In one embodiment, the FGFR-related tumor is urothelial carcinoma. In a preferred embodiment, the urothelial cancer is bladder cancer. In a preferred embodiment, the bladder cancer is a bladder cancer with high FGFR3 expression and FGFR3-TACC3 fusion.

In one embodiment, the FGFR-related tumor is cholangiocarcinoma. In a preferred embodiment, the cholangiocarcinoma is a cholangiocarcinoma with high FGFR2 expression. In a preferred embodiment, the cholangiocarcinoma is any one of intrahepatic cholangiocarcinoma, hilar cholangiocarcinoma, distal cholangiocarcinoma, or any combination thereof. In a more preferred embodiment, the cholangiocarcinoma is intrahepatic cholangiocarcinoma.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered as the sole active ingredient.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in combination with one or more other targeted drugs or chemotherapeutic drugs.

In one embodiment, the drug is formulated into a clinically accepted formulation. In a preferred embodiment, the preparation is an oral preparation, an injection preparation or a topical preparation.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.001 mg/kg to about 1000 mg/kg. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.01 mg/kg to about 100 mg/kg. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.02 mg/kg to about 50 mg/kg. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.03 mg/kg to about 20 mg/kg.

In one embodiment, the therapeutically effective amount is 0.001-1000 mg. In a preferred embodiment, the therapeutically effective amount is 0.01-100 mg. In a preferred embodiment, the therapeutically effective amount is 0.1-50 mg. In a preferred embodiment, the therapeutically effective amount is 0.5-30 mg.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a single dose or in divided doses.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered orally, by injection, topical, or in vitro. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered by oral administration or injection.

In the fourth aspect of the present invention, there is provided a pharmaceutical composition for the treatment of FGFR-related tumors, which comprises an FGFR inhibitor compound A or a pharmaceutically acceptable salt thereof and optionally a pharmaceutically acceptable , Wherein the compound A has the following structure:

In one embodiment, the FGFR-related tumor is one or more of digestive or urinary system tumors.

In one embodiment, the FGFR-related tumor is any one of gastric cancer, liver cancer, urothelial cancer, cholangiocarcinoma, or any combination thereof.

In one embodiment, the FGFR-related tumor is gastric cancer. In a preferred embodiment, the gastric cancer is gastric cancer with FGFR2 gene amplification.

In one embodiment, the FGFR-related tumor is liver cancer. In a preferred embodiment, the liver cancer is a liver cancer with high FGFR4/FGF19 expression. In a preferred embodiment, the liver cancer is a liver cancer with high FGFR3 expression. In a preferred embodiment, the liver cancer is any one of hepatocellular carcinoma, intrahepatic cholangiocarcinoma, mixed liver cancer, or any combination thereof.

In one embodiment, the FGFR-related tumor is urothelial carcinoma. In a preferred embodiment, the urothelial cancer is bladder cancer. In a preferred embodiment, the bladder cancer is a bladder cancer with high FGFR3 expression and FGFR3-TACC3 fusion.

In one embodiment, the FGFR-related tumor is cholangiocarcinoma. In a preferred embodiment, the cholangiocarcinoma is a cholangiocarcinoma with high FGFR2 expression. In a preferred embodiment, the cholangiocarcinoma is any one of intrahepatic cholangiocarcinoma, hilar cholangiocarcinoma, distal cholangiocarcinoma, or any combination thereof. In a more preferred embodiment, the cholangiocarcinoma is intrahepatic cholangiocarcinoma.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is the only active ingredient in the pharmaceutical composition.

In one embodiment, the pharmaceutical composition also contains one or more other targeted drugs or chemotherapeutic drugs as active ingredients.

In one embodiment, the pharmaceutical composition is formulated into a clinically accepted formulation. In a preferred embodiment, the preparation is an oral preparation, an injection preparation or a topical preparation.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.001 mg/kg to about 1000 mg/kg. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.01 mg/kg to about 100 mg/kg. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.02 mg/kg to about 50 mg/kg. In a preferred embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a daily dosage range from about 0.03 mg/kg to about 20 mg/kg.

In one embodiment, the pharmaceutical composition contains a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the therapeutically effective amount is 0.001-1000 mg. In a preferred embodiment, the therapeutically effective amount is 0.01-100 mg. In a preferred embodiment, the therapeutically effective amount is 0.1-50 mg. In a preferred embodiment, the therapeutically effective amount is 0.5-30 mg.

In one embodiment, the compound A or a pharmaceutically acceptable salt thereof is administered in a single dose or in divided doses.

In one embodiment, the pharmaceutical composition is administered by oral administration, injection administration, topical administration or in vitro administration. In a preferred embodiment, the pharmaceutical composition is administered by oral administration or injection.

The above embodiments represent exemplary embodiments of the present invention, but the present invention is not limited to the above embodiments. In addition, the various technical features in the above embodiments of the present invention can be combined with each other to form one or more new technical solutions. These new technical solutions also fall within the scope of the present invention, as long as such new technical solutions are What is technically feasible is fine.

[Beneficial effects of the present invention]

In order to prove that the compound A of the present invention is an effective FGFR1-4 inhibitor against FGFR-related tumors, especially tumors of the digestive or urinary system, the present invention evaluated the in vitro kinase inhibitory activity of compound A against FGFR1-4 wild-type and mutant types and compared with The proliferation inhibitory activity of FGFR-related exemplary digestive or urinary system tumors (including gastric cancer, liver cancer, bladder cancer, and cholangiocarcinoma) models, and the inhibitory effect of compound A on tumor growth was further evaluated for several tumor xenograft models.

In vitro kinase activity test and cell test results show that compound A has good in vitro kinase inhibitory activity on FGFR1-4 wild type and mutant type; on human gastric cancer cells (SNU-16) with FGFR2 gene amplification, FGFR3 high expression and FGFR3 -TACC3 fusion human bladder cancer cells (RT112/84), FGFR4/FGF19 high-expressing human liver cancer cells (Hep3B), FGFR2 high-expressing human cholangiocarcinoma cells HuCCT1 and human intrahepatic cholangiocarcinoma cells RBE have good inhibition of proliferation Effect; It has a significant inhibitory effect on human gastric cancer SNU-16 cell subcutaneous xenograft tumor model, human bladder cancer RT112/84 cell subcutaneous xenograft tumor model and human liver cancer LI-03-0332 subcutaneous xenograft model.

In addition, in order to investigate the pharmaceutical properties of compound A, the present invention measured the plasma stability of compound A in the human and mouse plasma stability test, and the structure was similar to the reference compound (Example 8 in WO2019034076A1 (S configuration)) A comparison was made. The results of plasma stability show that compound A has better stability in both mouse and human plasma.

Furthermore, it is known that, relative to reversible inhibitors, irreversible inhibitors enhance their affinity with the target through covalent bonding with the target protein, which is the fundamental reason why irreversible inhibitors exhibit high biological activity. However, if off-target, the affinity-enhancing effect of irreversible inhibitors will also occur on off-target sites, resulting in enhanced toxic and side effects [see Yang Bo et al. Research Progress on Small Molecule Covalent Inhibitors, Acta Pharmaceutical Sciences, 2014 , 49(2):158-165]. Out of concerns that irreversible inhibitors may bring greater toxic and side effects, the present invention detects the off-target effects of compound A on a variety of important non-target kinases, and the results show that compound A has low off-target effects.

Furthermore, in order to evaluate the safety of compound A in vivo, the present invention conducted safety pharmacology tests and acute toxicity tests on dogs and mice. The results show that compound A has no significant effect on the detection indicators, and the safety is good.

In summary, the inventors of the present application have found through experiments that compound A has the activity of selectively inhibiting FGFR1-4, has good inhibitory activity on both wild-type and mutant FGFR1-4, and can significantly inhibit the abnormality of FGFR1-4. Expression related to the proliferation of digestive or urinary system tumor cells and xenograft tumor models, and has high plasma stability and safety. It can be seen that compound A has the activity of inhibiting digestive or urinary system tumors related to abnormal expression of FGFR, can be used to develop drugs for the treatment of digestive or urinary system tumor diseases, and has important clinical application value.

[Detailed description of the invention]

Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A specific term or phrase should not be considered uncertain or unclear without a special definition, but should be understood in its ordinary meaning.

"Compound A" refers to a compound with the following structure as an FGFR inhibitor:

For its synthesis method, please refer to Example 2 of WO2019034076A1.

"Pharmaceutically acceptable salt" refers to a conventional non-toxic salt formed by the reaction of Compound A of the present invention with an inorganic acid, organic acid, inorganic base or organic base. The salt is within the scope of reliable medical judgment and is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, and reasonable benefits/risks Commensurate. A pharmaceutically acceptable base addition salt or acid addition salt can be obtained by contacting compound A with a sufficient amount of non-toxic acid or base in a pure solution or a suitable inert solvent.

"Pharmaceutically acceptable carrier" refers to a carrier suitable for formulating a pharmaceutical composition of Compound A or a pharmaceutically acceptable salt thereof or a clinically accepted preparation thereof. The carrier is within the scope of reliable medical judgment and is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, and reasonable benefits/risks Commensurate. "FGFR" refers to fibroblast growth factor receptor, which is a receptor for fibroblast growth factor signaling, and its family consists of four members (FGFR1-4). Through FGFR, FGF plays an important role in many physiological regulation processes such as cell proliferation, cell differentiation, cell migration and angiogenesis.

"FGFR-related tumors" refer to tumors whose occurrence or development is closely related to the expression and activation of any one or any combination of FGFR1-4, including but not limited to gastric cancer, bladder cancer, urothelial cancer, liver cancer, Cholangiocarcinoma (eg intrahepatic cholangiocarcinoma).

"Targeted drug" refers to a targeted drug that is clinically used to treat tumor-related diseases. It is endowed with a targeting ability to enable the active ingredient or its carrier to target a specific target lesion site, and accumulate or release the active ingredient at that site , To form a relatively high concentration, thereby improving the curative effect while inhibiting side effects and reducing damage to normal tissues and cells.

"Chemotherapeutic drugs" refer to drugs that can act on different links in the growth and reproduction of tumor cells, inhibit or kill tumor cells, and are clinically used to treat tumor-related diseases. It is currently one of the main methods of treating tumors.

"Clinically accepted preparations" are within the scope of reliable medical judgment, suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic reactions or other problems or complications, and A formulation with a reasonable benefit/risk ratio commensurate. The clinically accepted preparations include oral preparations, injection preparations, and topical preparations.

According to the present invention, the dosage and frequency of administration of the compound A or its pharmaceutically acceptable salt can be performed by conventional methods such as modeling, dose escalation studies, or clinical trials, and by considering the characteristics and severity of the disease to be treated. The degree, age, general condition and weight of the patient, as well as the specific compound administered, its pharmacokinetic properties, and the route of administration are determined. A suitable daily dosage range of the compound A or a pharmaceutically acceptable salt thereof is from about 0.001 mg/kg to about 1000 mg/kg; preferably, from about 0.01 mg/kg to about 100 mg/kg; further Preferably, from about 0.02mg/kg to about 50mg/kg; still more preferably, from about 0.03mg/kg to about 20mg/kg (wherein, "kg" refers to the administration object (ie, the subject Or the weight of the patient). Preferably, the daily dose of compound A or its pharmaceutically acceptable salt is 0.001 mg-1000 mg, and more preferably, the daily dose of compound A or its pharmaceutically acceptable salt is 0.01-1000 mg. 100mg; More preferably, the daily dose of Compound A or its pharmaceutically acceptable salt is 0.1-50 mg; More preferably, the daily dose of Compound A or its pharmaceutically acceptable salt is 0.5-30mg; More preferably, the daily dosage of Compound A or its pharmaceutically acceptable salt is 0.1mg, 0.5mg, 1mg, 2mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 36mg, 40mg, 45mg, 50mg, 55mg, 60mg, 70mg, 72mg, 100mg, 200mg, 500mg, 800mg, 1000mg, given in single or divided doses.

According to the present invention, the compound A or a pharmaceutically acceptable salt thereof is included in the drug or pharmaceutical composition in a therapeutically effective amount. The therapeutically effective amount is preferably 0.001-1000 mg, more preferably 0.01-100 mg, still more preferably 0.1-50 mg, still more preferably 0.5-30 mg, administered in a single dose or in divided doses.

According to the present invention, the therapeutically effective amount, administration dose or administration dose of the compound A or a pharmaceutically acceptable salt thereof is calculated as compound A.

According to the present invention, subjects or patients suffering from FGFR-related tumors can be human and non-human mammals such as mice, rats, guinea pigs, cats, dogs, cows, horses, sheep, pigs, monkeys, etc., more preferably humans. .

[Other embodiments of the invention]

The present invention also relates to the following embodiments:

1. The use of a fibroblast growth factor receptor (FGFR) inhibitor in the preparation of a medicine for treating FGFR-related tumors.

2. The use according to scheme 1, characterized in that the tumors include gastric cancer, liver cancer, and urothelial cancer.

3. The use according to scheme 2, characterized in that the liver cancer is hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and mixed liver cancer.

4. The use according to scheme 2, characterized in that the urothelial cancer is bladder cancer.

5. The use according to any one of schemes 1 to 4, characterized in that the inhibitor can be used in combination with one or more of targeted drugs and chemotherapeutic drugs.

6. The use according to any one of schemes 1 to 4, characterized in that the medicine is made into a clinically accepted preparation, and the preparation is preferably an oral preparation, an injection preparation, or an external preparation.

7. The use according to any one of schemes 1 to 4, characterized in that the daily dose of the inhibitor ranges from about 0.001 mg/kg to about 1000 mg/kg; preferably from about 0.01 mg/kg To about 100 mg/kg; can be administered in a single dose or in divided doses.

8. The use according to any one of the schemes 1 to 4, characterized in that the drug contains a therapeutically effective amount of the inhibitor, the therapeutically effective amount is preferably 0.001-1000 mg; it can be administered in a single dose or in divided doses.

Further, the present invention also relates to the following embodiments:

1. The use of an FGFR inhibitor compound A or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of FGFR-related tumors.

2. The use according to scheme 1, characterized in that the tumor is cholangiocarcinoma.

3. The use according to scheme 2, characterized in that the cholangiocarcinoma includes intrahepatic cholangiocarcinoma, hilar cholangiocarcinoma and distal cholangiocarcinoma; preferably intrahepatic cholangiocarcinoma.

4. The use according to scheme 1 or 2, characterized in that the medicine also contains one or more other targeted medicines and chemotherapeutics.

5. The use according to scheme 1 or 2, characterized in that the medicine is made into a clinically accepted preparation, and the preparation is preferably an oral preparation, an injection preparation, or an external preparation.

6. The use according to scheme 1 or 2, characterized in that the medicine contains a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, and the therapeutically effective dose is preferably 0.001 mg-1000 mg, more preferably 0.01-100 mg, more preferably 0.1-50 mg, still more preferably 0.5-30 mg; it can be administered in a single dose or in divided doses.

7. A method for treating FGFR-related tumor diseases, characterized in that a drug containing a therapeutically effective dose of Compound A or a pharmaceutically acceptable salt thereof is administered to a subject or patient.

8. The method according to scheme 7, wherein the tumor is cholangiocarcinoma.

9. The method according to claim 8, wherein the cholangiocarcinoma includes intrahepatic cholangiocarcinoma, hilar cholangiocarcinoma, and distal cholangiocarcinoma; preferably, it is intrahepatic cholangiocarcinoma.

10. The method according to any one of the schemes 7-9, characterized in that the administration may be oral administration, injection administration, topical administration or in vitro administration, preferably oral administration or injection administration.

Detailed ways

In order to further understand the present invention, specific embodiments of the present invention will be described in detail below in conjunction with examples. However, it should be understood that the purpose of these descriptions is only to further illustrate the features and advantages of the present invention, and does not constitute any limitation to the aspects of the present invention.

Example 1: Evaluation of FGFR1-4 wild-type kinase inhibitory activity in vitro

The ³³ P-ATP membrane filtration experiment was used to determine the inhibitory effect of test compound A on each wild-type FGFR kinase.

Buffer conditions: 20mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) (pH 7.5), 10mM MgCl ₂ , 1mM EGTA, 0.02% (v/v%) benzze 35 (Brij35) ), 0.02 mg/mL BSA, 0.1 mM Na ₃ VO ₄ , 2 mM DTT, 1% (v/v%) DMSO.

Test procedure: At room temperature, the test compound was dissolved in DMSO to prepare a 10 mM solution for later use. Add the substrate (see Table 1) to the newly prepared reaction buffer, add the specific kinase (see Table 2) to the substrate solution, and mix gently. The sonic technology (Echo550) was used to transfer the compound dissolved in DMSO to the kinase reaction mixture. The initial concentration of the test compound in the reaction mixture was 10 μM, and a total of 10 concentrations were diluted by 4 times. After 15 minutes of incubation, ³³ P-ATP (illuminance 0.01μCi/μL final) was added to the reaction system to start the reaction. Incubate the kinase reaction at room temperature for 120 minutes; transfer the reaction to P81 ion exchange paper (Whatman#3698-915). The filter membrane was extensively washed with 0.75% (v/v%) phosphoric acid, and the radioactive phosphorylated substrate remaining on the filter paper was measured. The kinase activity data is expressed as the ratio of the kinase activity of the test well (containing the test compound) and the blank well (only containing DMSO). The IC ₅₀ value is obtained by curve fitting with Prism4 software (GraphPad). The experimental results are shown in Table 3.

Table 1: Information about the substrate in the in vitro test

Table 2: Related information of kinases and ATP in in vitro tests

Table 3: Activity of test compounds against FGFR1-4 wild-type kinase (IC ₅₀ , nM)

Compound	FGFR1	FGFR2	FGFR3	FGFR4
TAS-120	0.784	0.397	1.340	4.650
Compound A	0.155	0.085	0.790	0.292

Note: TAS-120 was purchased from Glpbio, catalog number GC191561.

Conclusion: The compound A of the present invention can significantly inhibit the activity of FGFR1-4 wild-type kinase, and the effect is better than that of the reference compound TAS-120.

Example 2: Human and mouse plasma stability test

Test method: Thaw the frozen plasma for 10-20 minutes. After the plasma is completely thawed, place it in a centrifuge and centrifuge at 3220×g (centrifugal force) for 5 minutes to remove suspended solids and sediments. Measure the plasma pH value, and adjust the pH to the range of 7.40±0.10 with 1% (v/v%) phosphoric acid solution or 1M sodium hydroxide solution. Prepare 96-well incubation plates, named T0 (0min), T10 (10min), T30 (30min), T60 (60min), T120 (120min). Add 198 μL of mouse or human blank plasma to the corresponding incubation plate, and then add 2 μL of the working solution of the test compound (DMSO solution) to the corresponding incubation plate (two parallel wells are prepared for each sample). Incubate all samples in a 37°C water bath. The final incubation concentration of the test compound was 2 μM, and the final organic phase content was 1.0% (v/v%). At the end of each incubation time point, take out the corresponding incubation plate, and add 400 μL of acetonitrile to each corresponding sample well to precipitate the protein. After sealing and shaking all sample plates, centrifuge at 3220×g for 20 minutes. Take 50μL of supernatant and add 100μL of ultrapure water to dilute, mix well and analyze by LC/MS/MS method.

The experimental results are shown in Table 4.

Table 4: Results of plasma stability test

Conclusion: Compared with the reference compound in the table (Example 8 (S configuration) in WO2019034076A1), compound A has better stability in both mouse and human plasma.

Example 3: Evaluation of in vitro inhibitory activity of mutant kinases

The ³³ P-ATP membrane filtration experiment was used to determine the inhibitory effect of test compound A on each mutant FGFR kinase.

Buffer conditions: 20 mM Hepes (pH 7.5), 10 mM MgCl ₂ , 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na ₃ VO ₄ , 2 mM DTT, 1% DMSO.

Test procedure: Add the substrate (see Table 5) to the newly prepared reaction buffer, add the specific kinase (see Table 6) to the substrate solution, and mix gently. The sonic technology (Echo550) was used to transfer the compound dissolved in DMSO to the kinase reaction mixture. The initial concentration of the test compound in the reaction mixture was 10 μM, and a total of 10 concentrations were diluted by 4 times. After 15 minutes of incubation, ³³ P-ATP (illuminance 0.01μCi/μL final) was added to the reaction system to start the reaction. Incubate the kinase reaction at room temperature for 120 minutes, transfer the reaction to P81 ion exchange paper (Whatman#3698-915), and wash the filter membrane extensively with 0.75% phosphoric acid; measure the residual radioactive phosphorylated substrate on the filter paper. The kinase activity data is expressed as the ratio of the kinase activity of the test well (containing the test compound) and the blank well (only containing DMSO), and the IC ₅₀ value is obtained by curve fitting with Prism4 software (GraphPad). The experimental results are shown in Table 7.

Table 5: Information about the substrate

Table 6: Information about kinases and ATP

Table 7: Activity of test compounds on FGFR mutant enzyme (IC ₅₀ , nM)

Kinase	TAS-120	BGJ-398	Compound A
FGFR1(V561M)	605	1310	26.8
FGFR2(E565G)	1.37	7.24	0.12
FGFR2(N549H)	2.36	29.8	0.23
FGFR2(V564F)	255	6520	42.1
FGFR3(K650M)	1.99	27	0.17
FGFR3(V555M)	22.8	888	24.7
FGFR4(N535K)	8020	10200	17.5
FGFR4(V550M)	93.9	3960	4.24

Note: BGJ-398 was purchased from Glpbio, catalog number GC10055.

Conclusion: The compound A of the present invention can significantly inhibit the activity of FGFR1-4 mutant kinase, and the effect is better than that of the reference compounds TAS-120 and BGJ-398.

Example 4: Evaluation of the proliferation inhibitory activity of human gastric cancer cells, bladder cancer cells and liver cancer cells

CellTiter-Glo ^TM live cell detection kit was used to determine the effects of test compound A on human gastric cancer cells (SNU-16) with FGFR2 gene amplification, human bladder cancer cells with high FGFR3 expression and FGFR3-TACC3 fusion (RT112/84) and Inhibition of proliferation of human liver cancer cells (Hep3B) with high FGFR4/FGF19 expression. Among them, the medium of SNU-16 is PRMI-1640 medium (Invitrogen, catalog number 11875093) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution, Hep3B cells and RT112/84 cells. The medium is an EMEM medium (ATCC, catalog number: 30-2003) supplemented with a final concentration of 10% fetal bovine serum and a 1% penicillin/streptomycin solution.

Test procedure: Digest the SNU-16, RT112/84 and Hep3B cells that have reached 80% cell fusion with trypsin, centrifuge and resuspend the count, and use the culture medium to make 100,000, 20,000 and 70,000 cells/mL SNU-16, RT112/84 and Hep3B cell suspension were added to a 96-well cell culture plate (90 μL/well) and placed in _{a cell culture incubator containing 5% CO 2} at 37°C. After the cells were cultured for 24 hours, the reference compound Epirubicin and the test compound A were dissolved in DMSO into a mother liquor with a concentration of 30 mM. Use SNU-16, RT112/84 and Hep3B medium to further dilute the diluted compound stock solution, and transfer the diluted mixture to the corresponding cell plate. The final concentration of the test compound is 30μM (as IC50 test The initial concentration), 5 times decreasing dilution 9 concentrations, 9 concentrations are: 30μM, 6μM, 1.2μM, 0.24μM, 0.048μM, 0.0096μM, 0.0019μM, 0.0004μM and 0.00008μM, mix and centrifuge, set Cultured in a cell incubator containing 5% CO ₂ at 37°C for 3 days. Take out the 96-well cell culture plate, add CellTiterGlo (CTG, Chemiluminescence Cell Activity Detection Kit) reagent (100 μL/well), mix and centrifuge, and incubate at room temperature for 10 minutes. Use Envision multi-marker analyzer to read. Calculate the inhibition rate based on the original data. The calculation formula of the inhibition rate is: Inhibition rate %=(DMSO control-well to be tested)/(DMSO control-medium control)×100. Data analysis was performed using statistical software XLFit calculated IC _50. The experimental results are shown in Table 8.

Table 8: The inhibitory effect of compound A on the proliferation of SNU-16, RT112/84 and Hep3B cells (IC ₅₀ , nM)

Compound	SNU-16	RT112/84	Hep3B
Compound A	5.0	2.3	5.1
Epirubicin	274.6	75.9	451.9

Note: Epirubicin was purchased from TOCRIS, article number 3260, batch number 2A7193516.

Conclusion: Compound A has a significant inhibitory effect on the proliferation of tested SNU-16, RT112/84 and Hep3B cells. It can be seen that compound A has a significant inhibitory effect on the proliferation of tumor cells with abnormal FGFR expression, and the effect is better than that of the reference compound. Spectrum anticancer drug Epirubicin.

Example 5: Evaluation of inhibitory activity on the proliferation of human cholangiocarcinoma cells

The MTT method was used to determine the inhibitory effect of test compound A on the proliferation of human cholangiocarcinoma cells HuCCT1 with high FGFR2 expression and human intrahepatic cholangiocarcinoma cells RBE. The cell culture medium used was PRMI-1640 medium (Gibco, lot number: 8119264) supplemented with fetal bovine serum at a final concentration of 10%.

Test procedure: Inoculate a certain number of cells in logarithmic growth phase in a 96-well plate (100μL/well), and add 100μL of culture medium containing different concentration gradients of compound A or the control drug TAS-120 to each well after 24 hours of attachment. There are 3 multiple holes for each drug concentration, and corresponding blank holes (only medium) and normal holes (drug concentration is 0). After 72 hours of drug action, add MTT working solution (5mg/mL, 20μL per well), act at 37°C for 4 hours, shake the plate to remove the supernatant, add 150μL of DMSO (analytical purity); Wipe the plate clean, and detect the optical density (OD) at 550nm with a microplate reader.

Use the following formula to calculate the inhibition rate of cell growth:

Inhibition rate (%) = ( _normal OD value-OD value _{dosing hole} ) / ( _normal OD value-OD value _{blank hole} ) × 100%

The inhibition rate at each concentration was calculated by SPSS19.0 medicament half maximal inhibitory concentration IC _50. The experimental results are shown in Table 9.

Table 9: Inhibitory effect of compound A on the proliferation of HuCCT1 and RBE cells (IC ₅₀ , μM)

Compound	HuCCT1	RBE
Compound A	1.451±0.464	1.068±0.130
TAS-120	＞20	＞200

Conclusion: Compound A has a good inhibitory effect on the proliferation of HuCCT1 and RBE cells tested, while TAS-120 has almost no inhibitory effect on the proliferation of HuCCT1 and RBE cells, with significant differences.

Example 6: In vivo pharmacodynamic evaluation of human gastric cancer SNU-16 cell subcutaneous xenograft tumor BALB/c nude mouse model

The BALB/c nude mouse model of human gastric cancer SNU-16 xenograft tumors amplified by FGFR2 gene was used to determine the inhibitory effect of compound A on human gastric cancer.

Cell culture: SNU-16 cells were cultured in vitro under RPMI-1640 medium containing 10% fetal bovine serum and 2mM L-glutamine, cultured in an incubator containing 5% CO ₂ at 37°C. Use pancreatin-EDTA for routine digestion and passage twice a week. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected, counted, and the test is carried out according to the following steps.

Test procedure: 0.2 mL of ^{a cell suspension containing 5×10 6} SNU-16 cells and base glue (PBS: Matrigel = 1:1 (v/v)) was subcutaneously inoculated on the right side of each mouse Back. On the 13th day after inoculation, when the average tumor volume reached 144 mm ³ , they were divided into groups by stratified randomization, with 6 mice in each group. The reference compound TAS-120 and the test compound A are dissolved in an aqueous solution containing 0.5% (w/v) methyl cellulose (MC) and 0.5% (v/v) Tween 80 into an appropriate solution, and administered at 10 mL/kg Volumetric gavage was given to mice for 28 consecutive days. 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 is expressed by the tumor growth inhibition rate (abbreviated as tumor inhibition rate) TGI (%). 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-average tumor volume at the start of treatment in the solvent control group)]×100%.

The experimental results are shown in Table 10.

Table 10: Effect of Compound A on SNU-16 Xenograft Tumor Model Tumor Volume (Mean±SEM, mm ³ , n=6)

Note: The comparison between two groups is analyzed by T-test, the comparison between three or more groups is analyzed by one-way ANOVA, and Prism is used for data analysis.

Conclusion: Compound A administered once a day (QD) at doses of 10, 20 and 40 mg/kg, all have significant anti-tumor effect, and the anti-tumor effect has a dose-dependent trend. After 28 days of administration, the tumor inhibition rate (TGI) was 63%, 86%, and 98%, respectively, and the p values were all less than 0.05 compared with the solvent control group. At the same dose, the anti-tumor effect of compound A is better than that of TAS-120, and at a dose of 40 mg/kg, the therapeutic effect of the two is significantly different, which is statistically significant (p<0.05).

Example 7: In vivo pharmacodynamic evaluation of human bladder cancer RT112/84 cell subcutaneous xenograft tumor BALB/c nude mouse model

Using the human bladder cancer cell RT112/84 xenograft BALB/c nude mouse model with high FGFR3 expression and FGFR3-TACC3 fusion, the inhibitory effect of compound A on human bladder cancer was determined.

Cell culture: RT112/84 cells were cultured in a monolayer in vitro, and the culture conditions were EMEM medium (Gibco, catalog number 11140076) containing 10% fetal bovine serum, 1% NEAA (non-essential amino acids), and 2mM L-glutamine (Gibco, catalog number 11140076). Cultivation in an incubator containing 5% CO _2. Use pancreatin-EDTA for routine digestion and passage twice a week. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected, counted, and the test is carried out according to the following steps.

Test procedure: 0.2 mL of ^{a cell suspension (PBS: Matrigel=1:1 (v/v)) containing 10×10 6} RT112/84 cells and base glue was subcutaneously inoculated on the right back of each mouse. When the average tumor volume reached 188 mm ³ , they were divided into groups by stratified randomization, with 8 mice in each group. The control compound TAS-120 and the test compound A were dissolved into an appropriate solution with an aqueous solution containing 0.5% (w/v) MC and 0.5% (v/v) Tween 80, and were administered to mice by gavage at a dose volume of 10 mL/kg , Continuous administration for 20 days. 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 is expressed by the tumor growth inhibition rate TGI (%). 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-average tumor volume at the start of treatment in the solvent control group)]×100%.

The experimental results are shown in Table 11.

Table 11: Effect of compound A on tumor volume of RT112/84 xenograft tumor model (mean ± SEM, mm ³ , n=8)

Conclusion: Compound A administered once a day (QD) at doses of 5, 10 and 20 mg/kg, all have significant anti-tumor effects. After 20 days of administration, the tumor inhibition rate (TGI) was 67%, 79%, and 83%, respectively, and the p values were all less than 0.05 compared with the solvent control group. At a dose of 20 mg/kg, compound A and TAS-120 have comparable anti-tumor effects.

Example 8: In vivo pharmacodynamic evaluation of human liver cancer LI-03-0332 subcutaneous xenograft model

A subcutaneous xenograft tumor model constructed by nude mice after inoculation with LI-03-0332 human liver cancer tissue with high expression of FGFR3 was used to determine the inhibitory effect of compound A on human liver cancer.

Tumor tissue: The LI-03-0332 model of human liver cancer was originally established from clinical samples resected by surgery. The collection and use of specimens strictly abide by national, hospital and company-related ethical laws and regulations. The passage naming rule is: the tumor sample is inoculated into nude mice as the P0 generation, and the subsequent passage is the P1 generation, and so on, the recovered sample is named FP, and the tumor tissue used in this experiment is the FP9 generation.

Test procedure: The LI-03-0332 tumor tissue was cut into 20-30mm ³ pieces after removing the necrotic tissue. After adding base glue, the liver cancer tumor tissue was subcutaneously inoculated on the right back of each mouse, a total of 149 mice were inoculated Mice. On the 22nd day after inoculation, when the measured average tumor volume reached 135mm ³ , random stratified grouping was used according to tumor volume and animal body weight. There were 6 mice in the solvent control group and 8 mice in each of the remaining groups. The control compound TAS-120 and the test compound A were dissolved into an appropriate solution with an aqueous solution containing 0.5% (w/v) MC and 0.5% (v/v) Tween 80, and were administered to mice by gavage at a dose volume of 10 mL/kg , Continuous administration for 21 days. 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 is expressed by the tumor growth inhibition rate TGI (%). 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-average tumor volume at the start of treatment in the solvent control group)]×100%.

The experimental results are shown in Table 12.

Table 12: Effect of compound A on tumor volume (mean ± SEM, mm ³ ) of LI-03-0332 xenograft tumor model

Note: The solvent control group contains 6 animals, and the other groups contain 8 animals.

Conclusion: Compound A administered once a day (QD) at doses of 5, 10, and 20 mg/kg, all have significant anti-tumor effects. After 21 days of administration, the tumor inhibition rate (TGI) was 81%, 79%, and 87%, respectively, and the p-values were all less than 0.0001 compared with the solvent control group. At a dose of 20 mg/kg, compound A and TAS-120 have comparable anti-tumor effects.

Example 9: Evaluation of off-target effects of important kinases

Using Eurofins' Enzyme Profiling Services technology platform, the inhibitory effect of compound A on the activity of a variety of non-target kinases was analyzed. The detection of the compound for each kinase uses the Eurofins standard Kinase Profiler ^TM experimental procedure, which follows the relevant standard operating procedures. Protein kinase (except ATM(h) and DNA-PK(h)) tests are carried out by the amount of radiation, while lipid kinase, ATM(h), ATR/ATRIP(h) and DNA-PK(h) are tested by

Perform testing.

Test procedure: Take an appropriate amount of 50 times working solution of test compound A (100% DMSO dissolved) into the test hole (the final concentration of compound A is 0.1μM), then add the mixed solution of kinase and substrate; add the selected The concentration of ATP solution starts to react; the mixture of kinase and substrate and compound do not need to be pre-incubated before ATP is added.

The experimental results are shown in Table 13.

Table 13: Evaluation results of off-target effect

Conclusion: At a concentration of 0.1 μM, compound A has no obvious inhibitory effect on more than 50 kinases tested.

Example 10: Safety pharmacology evaluation

The Functional Observation Combination Test (FOB) was used to evaluate the effect of single intragastric administration of test compound A on the central nervous system function of SD rats. Divide 20 male and female rats into 4 groups, each of which has 5 male and female rats. Orally administered a vehicle (containing 0.5% (weight/volume, w/v) methylcellulose and 0.2% (w/v) Tween 80). Aqueous solution) or compound A at doses of 0.5, 1.2 and 3 mg/kg. The administration volume for all animals was 5 mL/kg. In this experiment, animals were observed for mortality and weighed. FOB observations were performed before the test, 2 hours after the administration, 8 hours after the administration, and 24 hours after the administration. Observation indicators include motor function, behavior change, coordination function, sensory/motor reflex and body temperature. As a result, no changes related to the test article were seen at each observation time point. Under the test conditions, there was no effect of test compound A on the central nervous system of rats.

Using telemetry, the effects of oral administration of test compound A on the cardiovascular parameters of awake beagle dogs were evaluated. Female and male dogs were orally administered a control formulation (vehicle: an aqueous solution containing 0.5% (w/v) methylcellulose and 0.2% (w/v) Tween 80), and the test compound at a dose of 0.3, 0.6 or 2 mg/kg A. The survival rate of the animals is observed every day. Cage-side observations were conducted twice a day on non-dosing days, and detailed clinical observations were performed before and after each dosing. The electrocardiogram, heart rate and blood pressure of all experimental animals were continuously recorded at least 2 hours before each administration to about 24 hours after administration. Print the 2 time points before administration (at least 30 minutes apart) and 6 time points after administration (0.5 hour, 1 hour, 2 hours, 4 hours, 12 hours and 24 hours after administration) for each administration An ECG waveform with a length of about 30 seconds. The results showed that under the test conditions, beagle dogs were given a single oral gavage of 0.3, 0.6 and 2 mg/kg of test compound A. Within 24 hours after administration, there were no cardiovascular indicators related to the test product. Variety.

A single oral gavage of the test compound A was given to SD rats to evaluate its effect on respiratory function. Divide 20 female and male rats into 4 groups, each group has 5 male and female, and the control formulation (vehicle: containing 0.5% (w/v) methyl cellulose and 0.2% (w/v) Tween 80 aqueous solution) was administered orally. Or 0.5, 1.2 or 3 mg/kg of test compound A. The administration volume for all animals was 5 mL/kg. Before administration, about 15 minutes of respiratory data (tidal volume, respiratory rate, ventilation volume per minute) were collected as the baseline of respiratory parameters before grouping. The collection time points on the day of administration were before the administration, 2 hours after the administration, 8 hours after the administration, and 24 hours after the administration, and collected data for approximately 15 minutes. The results showed that 24 hours after a single oral administration, the test article had no effect on the rat's respiratory rate, tidal volume and minute ventilation.

The experimental results are shown in Table 14.

Table 14: Results of safety pharmacology test

Conclusion: Compound A has no effect on the central nervous system and respiratory system of SD rats and the cardiovascular system of Beagle dogs, suggesting that compound A has high safety.

Example 11: Acute toxicity test

1. Toxicity test of single intragastric administration in rats

Test method: 40 rats were randomly divided into 4 groups, 10 rats in each group, half male and half male, and compound A and the control solvent were administered by oral gavage. The dose of compound A was 10, 40 or 400 mg/kg, respectively. The solvent is an aqueous solution containing 0.5% (w/v) MC and 0.2% (w/v) Tween 80 to detect the maximum tolerated dose of compound A. The administration volume is 10 mL/kg, and the observation period is 14 days.

Test results: Only in the 400mg/kg dose group, abnormal yellow stools and a slight increase in total bilirubin were observed. There were no deaths, clinical symptoms, gross lesions, weight changes, food intake, and clinical tests (hematology, blood coagulation, serum biochemistry, urinalysis) related to the test product in the female and male animals in the other dose groups compared with the solvent control group. difference. Therefore, the maximum tolerated dose (MTD) for a single administration under the experimental conditions is 400 mg/kg.

2. Toxicity test of single intragastric administration in dogs

Test method: 8 beagle dogs were randomly divided into 4 groups, 2 dogs in each group, half male and half male, and compound A and the control solvent were given by oral gavage. The dose of compound A was 15, 50 or 250 mg/ kg, the solvent is an aqueous solution containing 0.5% (w/v) MC and 0.2% (w/v) Tween 80. The administration volume is 5mL/kg. The animals were fasted overnight before dosing. The observation period is 14 days. Both males and females undergo necropsy on the 21st day.

Test results: A single dose of 15, 50, or 250 mg/kg of compound A was administered to beagle dogs. There were no mortality, clinical symptoms, body weight, food intake, hematology, blood coagulation, serum biochemistry, urine Fluid analysis parameters, gross pathological changes, and histopathological adverse reactions. Therefore, it is believed that the maximum tolerable dose (MTD) of male and female beagle dogs under this test condition is 250 mg/kg.

Conclusion: Compound A has a high tolerable dose in toxicity test of single intragastric administration and has good safety.

The various aspects of the present invention have been exemplified by the above-mentioned embodiments. Obviously, the above-mentioned embodiments are only given for the purpose of clear illustration, and are not intended to limit the scope of the present invention in any way. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above examples. It is unnecessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from this are still within the scope of protection created by the present invention.

Claims (10)

1. Use of an FGFR inhibitor compound A or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating FGFR-related tumors, wherein the compound A has the following structure:

   
2. The use according to claim 1, wherein the FGFR-related tumor is one or more of digestive or urinary system tumors; preferably any one of gastric cancer, liver cancer, urothelial cancer, cholangiocarcinoma, or any of them combination.
3. The use according to claim 2, wherein the liver cancer is any one or any combination of hepatocellular carcinoma, intrahepatic cholangiocarcinoma, mixed liver cancer; the urothelial cancer is bladder cancer; the bile duct The cancer is any one or any combination of intrahepatic cholangiocarcinoma, hilar cholangiocarcinoma, and distal cholangiocarcinoma, and is preferably intrahepatic cholangiocarcinoma.
4. The use according to any one of claims 1 to 3, wherein the compound A or a pharmaceutically acceptable salt thereof is the only active ingredient in the drug, or is combined with one or more other targets Combination of drugs or chemotherapy drugs.
5. The use according to any one of claims 1 to 3, wherein the drug is prepared into a clinically accepted preparation, preferably an oral preparation, an injection preparation or an external preparation.
6. The use according to any one of claims 1-3, wherein the compound A or a pharmaceutically acceptable salt thereof is from about 0.001 mg/kg to about 1000 mg/kg, preferably from about 0.01 mg/kg to It is administered in a daily dosage range of about 100 mg/kg, more preferably from about 0.02 mg/kg to about 50 mg/kg, and still more preferably from about 0.03 mg/kg to about 20 mg/kg.
7. The use according to any one of claims 1 to 3, wherein the medicament contains a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof.
8. The use according to claim 7, wherein the therapeutically effective amount is 0.001-1000 mg, preferably 0.01-100 mg, more preferably 0.1-50 mg, still more preferably 0.5-30 mg.
9. The use according to any one of claims 1-3, wherein the compound A or a pharmaceutically acceptable salt thereof is administered in a single dose or in divided doses.
10. The use according to any one of claims 1 to 3, wherein the drug is administered by oral administration, injection administration, topical administration or in vitro administration, preferably by oral administration or injection administration.