WO2023040914A1 - Pharmaceutical use of cdk4/6 inhibitor - Google Patents

Abstract

Disclosed are a method for treating cancer with a CDK4/6 inhibitor and a corresponding pharmaceutical use. The CDK4/6 inhibitor is 5-fluoro-4-(7'-fluoro-2'-methylspiro[cyclopentane-1, 3'-indole]-5'-yl)-nitrogen-(5-(1-methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine, and said cancer comprises colorectal cancer, liver cancer, or breast cancer. The CDK4/6 inhibitor has a good tumor suppression effect.

Description

A kind of medical application of CDK4/6 inhibitor technical field

The application belongs to the field of medicine and relates to the medical use of a CDK4/6 inhibitor.

Background technique

Breast cancer is one of the most common malignant tumors in women. Global statistics show that about 1.7 million people (accounting for 25% of all female cancer patients) are diagnosed with breast cancer each year, and about 520,000 people (accounting for 15% of all female cancer patients) %) died from the disease. Breast cancer is a systemic disease. In the middle and early stages of the disease, breast cancer cells can enter the circulatory system and metastasize throughout the body. At present, the treatment of breast cancer is still mainly based on surgery, chemoradiotherapy, and endocrine therapy, and the survival rate of patients with advanced breast cancer is low and it is difficult to cure. The pathological types of breast cancer include luminal A type (Luminal A type), luminal B type (Luminal B type), normal breast-like type, human epidermal growth factor receptor 2 (HER-2) overexpression type and basal cell-like type, Among them, basal cell-like breast cancer and triple-negative breast cancer are a type of breast cancer that are negative for estrogen receptor (ER), progesterone receptor (PR), and HER-2, accounting for about all newly diagnosed cases of breast cancer. 15%-20%. Human epidermal growth factor receptor-2 (HER2), overexpression and amplification of HER2 are found in different types of solid tumors (including breast cancer, gastric cancer, etc.). For patients with positive HER2 tests, trastuzumab targeting HER2 combined with chemotherapy and other treatment regimens has a significant effect. For HER2-negative patients with advanced breast cancer who have failed endocrine therapy, chemotherapy is still the main treatment. The "China Breast Cancer Diagnosis and Treatment Standards (2018 Edition)" issued by the National Health and Medical Commission pointed out that chemotherapy drugs commonly used in advanced breast cancer include anthracyclines (epirubicin, doxorubicin, doxorubicin), taxanes, etc. , vinorelbine, capecitabine, gemcitabine, platinum, etc. However, due to severe side effects, the clinical application of chemotherapy drugs is limited, and patients often stop treatment because they cannot tolerate severe adverse reactions. Therefore, it is particularly important to develop therapeutic regimens with better efficacy and lower toxicity.

Colorectal cancer (CRC) is a combination of colon cancer and rectal cancer. It is one of the most common malignant tumors in the world, and its incidence rate varies greatly in different regions of the world. It is highest in America and Oceania, in the middle in Europe, and lower in Asia and Africa. In the United States, the morbidity and mortality of colorectal cancer are at the forefront of all tumors; in China, the morbidity and mortality of colorectal cancer are also increasing , transverse colon, etc. The common histological types of colorectal cancer include adenocarcinoma, adenosquamous carcinoma, spindle cell carcinoma, squamous cell carcinoma, and undifferentiated carcinoma; among them, adenocarcinoma is the most common and can be divided into cribriform comedotype adenocarcinoma, medullary carcinoma , micropapillary carcinoma, mucinous adenocarcinoma, serrated adenocarcinoma, and signet ring cell carcinoma.

Primary liver cancer is the sixth most common cancer (6% of all cancers) and the second leading cause of cancer death (9% of all cancer deaths) worldwide (World Cancer Report (2014) World Health Organization. Chapters 1.1 and 5.6. , ISBN 9283204298). Hepatocellular carcinoma (Hepatocellular Carcinoma, HCC) is the most common type of primary liver cancer. HCC mostly occurs on the basis of hepatitis cirrhosis, and it appears about 10 to 20 years after the original liver damage. Most liver cancers are asymptomatic in the early stage, and most patients have reached locally advanced stage or developed distant metastasis when diagnosed. There is no standard treatment for advanced liver cancer, and clinical treatment is facing severe challenges. The survival time of advanced HCC patients in China is generally 3 to 6 months, and the longest in the world is no more than 1 year, which has a lot to do with the lack of effective systemic treatment for these patients. HCC is resistant to common chemotherapy, and its treatment methods generally include surgery (hepatectomy, liver transplantation, and palliative surgery), non-surgical treatment (local therapy, arterial chemoembolization, chemotherapy, radiotherapy, biological therapy, and molecular targeted therapy) and Other treatments (including participation in clinical research). Many cancers that are also found in the liver are not true liver cancers but arise from secondary liver cancers elsewhere in the body that have spread to the liver (ie, metastases). Typically, the starting site is the gastrointestinal tract because of the liver's proximity to many of these metabolically active, blood-rich organs near blood vessels and lymph nodes (eg, pancreatic cancer, gastric cancer, colon cancer, and carcinoid tumors primarily of the appendix (carcinoid tumor)). Secondary liver cancers can also arise from metastatic breast, ovarian, lung, kidney, and prostate cancers.

Therefore, there is still a need to find a drug with significant efficacy for treating breast cancer, colorectal cancer or liver cancer.

Contents of the invention

In view of the deficiencies of the prior art and actual needs, the present invention provides 5-fluoro-4-(7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indol]-5'-yl A new drug application of )-nitrogen-(5-(1-methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine. 5-fluoro-4-(7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indol]-5'-yl)-nitrogen-(5-(1-methylpiperidine -4-yl)pyridin-2-yl)pyrimidin-2-amine is a highly selective CDK4/6 inhibitor, the inventors unexpectedly found that this compound, compared to other drugs with the same target Breast, colorectal, and liver cancers had unexpectedly better outcomes.

One aspect of the present invention provides a method for treating breast cancer, colorectal cancer or liver cancer, the method comprising administering a therapeutically effective amount of compound (I) or a pharmaceutically acceptable salt thereof to a subject in need, preferably the subject The subject is a mammal, more preferably said subject is a human,

Another aspect of the present invention provides the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating breast cancer, colorectal cancer or liver cancer.

Another aspect of the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer, wherein the cancer is breast cancer, colorectal cancer or liver cancer.

In certain embodiments, the pharmaceutically acceptable salt is a fumarate.

In certain embodiments, the breast cancer is hormone receptor (HR) positive breast cancer; preferably, the hormone receptor (HR) positive breast cancer is estrogen receptor positive (ER ⁺ ) breast cancer , progesterone receptor positive (PR ⁺ ) breast cancer, or ER ⁺ PR ⁺ breast cancer; more preferably, the hormone receptor (HR) positive breast cancer is estrogen receptor positive (ER ⁺ ) and HER2 negative breast cancer, breast cancer that is progesterone receptor positive (PR ⁺ ) and HER2 negative, or breast cancer that is ER ⁺ PR ⁺ and HER2 negative.

Unless otherwise specified, the term "compound of the present invention" refers to the compound of formula (I) and its salt, including the pharmaceutically acceptable salt of the compound and all stereoisomers (including but not limited to diastereoisomers) and enantiomers), tautomers, isotopic compounds, prodrugs, solvates, and hydrates thereof.

The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness of the free acids and bases of the specified compound without adverse biological effects. Examples of pharmaceutically acceptable salts include, but are not limited to: salts formed between compound (I) and any of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, Heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, formic acid Sulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, p-toluenesulfonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid; preferably fumarate.

The prodrugs of the compounds of the present invention are included in the protection scope of the present invention. In general, the prodrugs refer to functional derivatives that are readily converted in vivo to the desired compound. Therefore, the term "administering" in the treatment method provided by the present invention includes administering the compound disclosed in the present invention, or although not explicitly disclosed but can be converted into the compound disclosed in the present invention after administration to the subject to treat the described compound. various diseases. Routine methods for selecting and preparing suitable prodrug derivatives are described, for example, in such books as Design of Prodrugs, H. Bundgaard, Elsevier, 1985.

The compounds of the present invention may contain one or more asymmetric centers and may thereby give rise to diastereoisomers and optical isomers. The present invention includes all possible diastereoisomers and their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers and their pharmaceutically acceptable salts.

The above formula I does not exactly define the stereostructure of a certain position of the compound. The present invention includes all stereoisomers of the compound represented by formula I and pharmaceutically acceptable salts thereof. Furthermore, mixtures of stereoisomers and isolated specific stereoisomers are also included in the present invention. During the synthesis of such compounds, or during the use of racemization or epimerization procedures well known to those of ordinary skill in the art, the products obtained may be mixtures of stereoisomers.

When the compound represented by formula I has tautomers, unless otherwise stated, the present invention includes any possible tautomers and their pharmaceutically acceptable salts, and their mixtures.

When the compound represented by formula I and its pharmaceutically acceptable salts exist in the form of solvates or polymorphs, the present invention includes any possible solvates and polymorphs. The type of solvent forming a solvate is not particularly limited as long as the solvent is pharmacologically acceptable. For example, water, ethanol, propanol, acetone, and the like can be used.

The invention also includes all pharmaceutically acceptable isotopic compounds which are identical to the compounds of the invention except that one or more atoms have the same atomic number but an atomic mass or mass number different from the atomic mass or mass prevailing in nature Atomic substitution of numbers. Examples of isotopes suitable for inclusion in compounds of the invention include, but are not limited to, isotopes of hydrogen (e.g., deuterium ( ^2H ), tritium ( ^3H )); isotopes of carbon (e.g., ^13C and ^14C ); Isotopes (such as ³⁷ Cl); isotopes of iodine (such as ¹²⁵ I); isotopes of nitrogen (such as ¹³ N and ¹⁵ N); isotopes of oxygen (such as ¹⁷ O and ¹⁸ O); isotopes of phosphorus (such as ³² P); Isotopes of sulfur (eg ³⁴ S).

The term "treatment" generally refers to obtaining a desired pharmacological and/or physiological effect. The effect may be therapeutic in terms of partial or complete stabilization or cure of the disease and/or side effects due to the disease. As used herein, "treatment" encompasses any treatment of a disease in a patient, including: (a) inhibiting the symptoms of the disease, ie arresting its development; or (b) relieving the symptoms of the disease, ie causing regression of the disease or symptoms.

The term "effective amount" refers to an amount or dose of a compound of formula (I) or a pharmaceutically acceptable salt thereof and an amount or dose of endocrine therapy that provides an effective response in a patient under diagnosis or treatment.

The term "treatment failure" refers to intolerable side effects, disease progression during treatment, or recurrence after treatment.

The term "subject" is a mammal, preferably a human.

Beneficial effects of the present invention:

1) The compound of the present invention has a significant inhibitory effect on human breast cancer, and compared with the positive control drug Abemaciclib, it shows a better tumor inhibitory effect.

2) The compound of the present invention has a significant inhibitory effect on human colorectal cancer, and compared with the positive control drugs Palbociclib and Abemaciclib, it shows a better tumor inhibitory effect.

3) The compound of the present invention does not show obvious drug toxicity and side effects, has good tolerance, and has good clinical application prospects for treating colorectal cancer.

4) The compound of the present invention has a significant inhibitory effect on human liver cancer, and compared with the positive control drugs Palbociclib and Abemaciclib, it shows a better tumor inhibitory effect.

5) The compound of the present invention does not show obvious drug toxicity and side effects, has good tolerance, and has good clinical application prospects for treating liver cancer.

Description of drawings

Figure 1: Changes in body weight of each administration group in the human-derived colorectal cancer xenograft mouse model.

Figure 2: Changes in body weight of each administration group in the human-derived liver cancer xenograft mouse model.

Detailed ways

The experimental methods in the following examples are conventional methods unless otherwise specified. The chemical raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.

Acronym

Example 1 5-fluoro-4-(7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indol]-5'-yl)-nitrogen-(5-(1-methyl (Piperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine fumarate (Compound A)

First, 5-fluoro-4-(7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indol]-5'-yl)- nitrogen-(5-(1-methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine.

Then, under nitrogen protection, at room temperature, dichloromethane (22.0 volumes) and ethanol (22.0 volumes) were added to the reactor, and 5-fluoro-4-(7'-fluoro-2'-methylspiro[ Cyclopentane-1,3'-indol]-5'-yl)-nitrogen-(5-(1-methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine (2.070kg ), warming up to 30-40 degrees Celsius, stirring until all dissolved, cooling to room temperature, and transferring the solution to a solvent bucket for subsequent use. Under the protection of nitrogen, the above solution was filtered through a microporous filter, and transferred to the reactor, stirred, dichloromethane and ethanol were evaporated at normal pressure, and then the temperature of the reactor was kept at 80 ± 5 degrees Celsius, and the fumaric acid ( 1.0eq) of ethanol solution (12 volumes) was slowly dropped into the reaction kettle through a microporous filter, and kept stirring overnight. Cool down to 20-30 degrees Celsius, continue to stir for at least 1 hour, centrifuge, and collect the filter cake. The filter cake was placed in a vacuum oven and dried overnight to obtain 1.605kg 5-fluoro-4-(7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indole]-5' -yl)-nitrogen-(5-(1-methylpiperidin-4-yl)pyridin-2-yl)pyrimidin-2-amine fumarate (compound A), yield 63.6%.

Embodiment 2 The compounds of the present invention inhibit the proliferation of breast cancer cell lines

Using four breast cancer cell lines T47D (ER ⁺ /PR ⁺ /HER2 ^- ), MCF-7 (ER ⁺ /PR ⁺ /HER2 ^- ), 4T1 (ER ^- /PR ^- /HER2 ^- ), Du4475 (ER ^- / PR ⁻ /HER2 ⁻ ) was used in the proliferation inhibition test of the compound of the present invention. The positive control drug Abemaciclib was prepared by the inventor according to the synthesis method recorded in WO2010075074A1. The cell detection equipment was In Cell Analyzer 2200 (GE Healthcare). Reagents or consumables are listed in the table below:

Table 1 Reagents or consumables

Reagents or consumables	Item No.	factory
DMSO	D2650	Sigma
PBS	20012-027	Gibco
DAPI	D8417	Sigma
formaldehyde	47608	Sigma
Triton ™ X-100	T9284	Sigma
96-well black clear bottom cell plate	6005182	PE

Abemaciclib and Compound A were dissolved in DMSO to prepare a 10 mM stock solution. And put it in a -80°C refrigerator for long-term storage; take 5 μL of 10 mM Abemaciclib and Compound A stock solutions, and dilute them into 60 μM working solutions respectively, starting at 60 μM, and diluting 10 points five times.

Inoculate each type of cells in the logarithmic growth phase at 4000cells/100μl/well into a black transparent bottom 96-well plate and culture overnight at 37°C; take out the 96-well plate, add 20μl of the diluted sample to the 96-well plate, and treat at 37°C for 72h ; After 72 hours, take out the 96-well plate, add neutral formaldehyde fixative solution (formaldehyde:PBS=1:9), 50μl/well, fix at room temperature for 10-30min; wash 2 times with 1x PBS, 100μl/well, 0.2% Triton ^TM -X100 Permeabilize for 5-10 minutes; wash 2 times with 1x PBS, 100 μl/well, stain with DAPI (1:5000 dilution in PBS), 50 μl/well, incubate at room temperature in the dark for 20 minutes; wash 3 times with 1x PBS, 100 μl/well, finally add 1x PBS 100μl/well; scan with In Cell Analyzer to analyze the number of cells in each well. The inhibition rate of each compound at each concentration point was calculated according to the following formula, and the _IC50 value was obtained by curve fitting with the software Graphpad Prism 6.0.

The test results are shown in Table 2. The results show that Compound A has obvious growth inhibitory activity on four breast cancer cells T47D, MCF-7, 4T1, and Du4475. Compared with the positive control drug Abemaciclib, Compound A has higher growth inhibitory activity. .

Table 2 The inhibitory activity of test substance on the proliferation of different breast cancer cell lines

Example 3 Pharmacodynamic experiments of compounds of the present invention in human breast cancer xenograft mouse model

One day before tumor inoculation, 0.18 mg estrogen pellets were implanted in the right abdomen of NOD/SCID mice. Inoculate 2×10 ⁷ human breast cancer MCF-7 cells into mice, and start administration when the average tumor volume reaches about 138 mm ³ .

The test was divided into Compound A 5.0mg/kg, 10.0mg/kg and 50.0mg/kg groups, Abemaciclib25.0mg/kg group, and Vehicle group. The preparations of the above test and reference substances are shown in Table 3. Among them, the positive control drug Abemaciclib was prepared by the inventors according to the synthesis method described in WO2010075074A1.

The preparation of table 3 test sample and reference substance

Ten mice in each group were administered orally orally, once a day, for a total of 28 days. Changes in tumor volume and body weight were observed regularly, and curative effect was evaluated based on relative tumor proliferation rate (T/C) and relative tumor inhibition rate (TGI).

Tumor volume calculation formula: tumor volume (mm ³ )=1/2×(a×b ² ) (where a represents the long diameter and b represents the short diameter).

Relative tumor proliferation rate (T/C), that is, the percentage value of the relative tumor volume or tumor weight between the treatment group and the control group at a certain time point. Calculated as follows:

T/C (%)=T _RTV /C _RTV ×100% (T _RTV : the average RTV of the treatment group; C _RTV : the average RTV of the Vehicle group; RTV=V _t /V ₀ , V ₀ is the tumor volume of the animal when grouping , V _t is the tumor volume of the animal after treatment);

The calculation formula for relative tumor inhibition rate (TGI) is as follows: TGI (%)=(1-T/C)×100%. (T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment group and the control group at a specific time point, respectively).

The results are shown in Table 4, the results show that: compound A (5mg/kg, 10mg/kg, 50mg/kg) treatment group all showed the effect of tumor suppression on the 42nd day, and the relative tumor proliferation rate (T/C) was 54% , 30%, 19%, compound A (10mg/kg, 50mg/kg) treatment group showed better tumor suppression effect than positive drug Abemaciclib (25mg/kg) group.

It can be seen that compound A exhibits obvious tumor suppressive effect in the xenograft mouse model of human breast cancer, and has a good clinical application prospect in the treatment of breast cancer.

The inhibitory activity of table 4 test substance in breast cancer MCF-7 xenograft model

Embodiment 4 The compound of the present invention is to the proliferation inhibitory determination of liver cancer cell line

Using 31 liver cancer cell lines SNU-761, PLC/PRF/5, SK-HEP-1, SNU-475, SNU-739, SNU-368, HCCC9810, SNU-423, SNU-449, SNU-182, SNU- 387, MHCC97L, MHCC97H, HL7702, SNU-398, HLE, SNU-886, MHCC97, Hepa1-6, Huh-1, Huh-7, JHH-7, Hep G2, BRL-3a, BNL CL.2, BEL7405, BRL, HLF, HCCLM3, Li-7, and H22 were used in the proliferation inhibition assay of the compounds of the present invention. The positive control drug Abemaciclib was prepared by the inventor according to the synthesis method recorded in WO2010075074A1. The cell detection equipment was In Cell Analyzer 2200 (GE Healthcare) , the reagents or consumables used in the experiment are shown in Table 5 below:

Table 5 Reagents or consumables

Abemaciclib and Compound A were dissolved in DMSO to prepare a 10 mM stock solution. And put it in a -80°C refrigerator for long-term storage; take 5 μL of 10 mM Abemaciclib and Compound A stock solutions, and dilute them into a 60 μM working solution, starting at 60 μM, three-fold dilution of Abemaciclib by 10 points, and six-fold dilution of Compound A 10 points.

Inoculate each type of cells in the logarithmic growth phase at 4000cells/100μl/well into a black transparent bottom 96-well plate, and culture overnight at 37°C; take out the 96-well plate, add 20μl of the diluted sample to the 96-well plate, and treat at 37°C for 72h Take out the 96-well plate after 72 hours, add neutral formaldehyde fixative solution (formaldehyde:PBS=1:9), fix at room temperature for 10-30min at 50 μl/well; wash twice with 1x PBS, 100 μl/well, permeabilize with 0.2% TritonTM-X100 Chemical treatment for 5-10min; wash 2 times with 1x PBS, 100μl/well, stain with DAPI (1:5000 dilution in PBS), 50μl/well, incubate at room temperature in the dark for 20min; wash 3 times with 1x PBS, 100μl/well, finally add 1x PBS 100μl/well; scan with In Cell Analyzer to analyze the number of cells in each well. The inhibition rate of each compound at each concentration point was calculated according to the following formula, and the IC50 value was obtained by curve fitting with the software Graphpad Prism 6.0.

The test results are shown in Table 6, the results show that: compound A showed proliferation inhibitory activity on 31 kinds of liver cancer cells; among them, compound A had inhibitory activity on 21 kinds of liver cancer cell lines SNU-761, PLC/PRF/5, SK-HEP-1 , SNU-475, SNU-739, SNU-368, HCCC9810, SNU-423, SNU-449, SNU-387, MHCC97L, MHCC97H, SNU-398, HLE, SNU-886, MHCC97, Hepa1-6, Huh-1 , Huh-7, JHH-7, and Li-7 have obvious growth inhibitory activity (IC ₅₀ ≤1μM); compared with the positive control drug Abemaciclib, compound A has inhibitory effect on 20 liver cancer cell lines SNU-761, PLC/PRF/5 , SNU-475, SNU-739, SNU-368, HCCC9810, SNU-423, SNU-449, SNU-387, MHCC97L, MHCC97H, SNU-398, HLE, SNU-886, MHCC97, Hepa1-6, Huh-1 , Huh-7, JHH-7, Li-7 have higher proliferation inhibitory activity.

Table 6 The inhibitory activity of the test substance on the proliferation of different liver cancer cell lines

NA: Not Available

Example 5 Pharmacodynamic experiments of compounds of the present invention in human liver cancer xenograft mouse model

from

Liver cancer xenograft model LI1088 (Crown Biotechnology Co., Ltd.) tumor-bearing mice were harvested from tumor tissues, cut into 2-3mm diameter tumor pieces and inoculated subcutaneously at the right anterior scapula of Balb/c nude mice. 117.61mm ³ , randomly grouped according to tumor size.

The positive control drugs were Palbociclib and Abemaciclib, Palbociclib was prepared by the inventor according to the synthesis method described in WO2003062236A1, and Abemaciclib was prepared by the inventor according to the synthesis method described in WO2010075074A1.

The test is divided into compound A 10.0mg/kg, 25.0mg/kg and 50.0mg/kg groups, Palbociclib 25.0mg/kg group, Abemaciclib 25.0mg/kg group, and Vehicle group. 7.

The preparation of table 7 test sample and reference substance

Eight mice in each group were administered orally orally, once a day, for a total of 28 days. Changes in tumor volume and body weight were observed regularly, and curative effect was evaluated based on relative tumor proliferation rate (T/C) and relative tumor inhibition rate (TGI).

Tumor volume calculation formula: tumor volume (mm ³ )=1/2×(a×b ² ) (where a represents the long diameter and b represents the short diameter).

Relative tumor proliferation rate (T/C), that is, the percentage value of the relative tumor volume or tumor weight between the treatment group and the control group at a certain time point. Calculated as follows:

T/C (%)=TRTV/CRTV×100% (TRTV: the average RTV of the treatment group; CRTV: the average RTV of the Vehicle group; RTV=Vt/V0, V0 is the tumor volume of the animal when grouped, and Vt is the animal's tumor volume after treatment tumor volume);

The calculation formula for relative tumor inhibition rate (TGI) is as follows: TGI (%)=(1-T/C)×100%. (T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment group and the control group at a specific time point, respectively).

The results are shown in Table 8 and Figure 1. The results show that the Compound A (25mg/kg) treatment group and the Compound A (50mg/kg) treatment group showed extremely significant tumor inhibition on the 25th day, and the relative tumor proliferation rate T/C (%) were 42.32% and 22.29% respectively, relative to the Vehicle group, statistically significant difference (respectively P=0.038 and P=0.003); Compound A (10mg/kg) treatment group showed slight The tumor inhibitory effect, the relative tumor proliferation rate T/C (%) was 91.29%, there was no statistical difference relative to Vehicle group (P=0.690); Palbociclib (25mg/kg) and Abemaciclib (25mg/kg) treatment groups were at On day 25, it showed a slight tumor inhibitory effect, and the relative tumor proliferation rates T/C (%) were 83.64% and 77.57%, respectively, and there was no statistical difference compared with the Vehicle group (P=0.802, P=0.578). In summary, the relative tumor proliferation rate (T/C) of the compound A (25mg/kg, 50mg/kg) treatment group on the 25th day was significantly higher than that of the positive drug Palbociclib (25mg/kg) and Abemaciclib (25mg/kg) groups. Better tumor suppressive effect.

Compound A (10mg/kg, 25mg/kg, 50mg/kg), Palbociclib (25mg/kg) and Abemaciclib (25mg/kg) each treatment group had no animal death, the overall body weight of the animal was good, and each administration group had no obvious drug toxicity.

It can be seen that compound A (25mg/kg, 50mg/kg) has shown obvious tumor inhibitory effect in human liver cancer xenograft mouse model, and the compound has no obvious toxic and side effects, and has good clinical efficacy in the treatment of liver cancer. Application prospects.

Table 8 Inhibitory activity of test substance in liver cancer xenograft model

Note: P value is to compare each group with Vehicle group by T test.

Example 6 Determination of the inhibition of the proliferation of colorectal cancer cell lines by the compounds of the present invention

Adopt 14 kinds of colorectal cancer cell lines DLD-1, HCT8, HCT15, Caco-2, LOVO, HCT116, CT26.WT, Colon-26, SW620, SW948, Colo205, Colo320DM, HT29, LS174T for the proliferation of the compound of the present invention In the inhibition assay test, the positive control drug Abemaciclib was prepared by the inventor according to the synthesis method recorded in WO2010075074A1. The cell detection equipment was In Cell Analyzer 2200 (GE Healthcare). The reagents or consumables used in the experiment are shown in the following table:

Table 9 Reagents or consumables

Abemaciclib and Compound A were dissolved in DMSO to prepare a 10 mM stock solution. And put it in a -80°C refrigerator for long-term storage; take 5 μL of 10 mM Abemaciclib and Compound A stock solutions, and dilute them respectively to form a 60 μM working solution. With 60 μM as the initial concentration, Abemaciclib is three-fold diluted 10 points, and Compound A is six-fold diluted 10 points.

Inoculate each type of cells in the logarithmic growth phase at 4000cells/100μl/well into a black transparent bottom 96-well plate, and culture overnight at 37°C; take out the 96-well plate, add 20μl of the diluted sample to the 96-well plate, and treat at 37°C for 72h Take out the 96-well plate after 72 hours, add neutral formaldehyde fixative solution (formaldehyde:PBS=1:9), fix at room temperature for 10-30min at 50 μl/well; wash twice with 1x PBS, 100 μl/well, permeabilize with 0.2% TritonTM-X100 Chemical treatment for 5-10 minutes; wash 2 times with 1x PBS, 100 μl/well, stain with DAPI (1:5000 dilution in PBS), 50 μl/well, incubate at room temperature in the dark for 20 minutes; wash 3 times with 1x PBS, 100 μl/well, finally add 1x PBS 100μl/well; scan with In Cell Analyzer to analyze the number of cells in each well. The inhibition rate of each compound at each concentration point was calculated according to the following formula, and the IC50 value was obtained by curve fitting with the software Graphpad Prism 6.0.

The test results are shown in Table 10. The results show that Compound A exhibited anti-proliferation activity on 14 colorectal cancer cell lines, among which Compound A had inhibitory activity on 11 colorectal cancer cell lines DLD-1, HCT8, HCT15, Caco-2 , LOVO, HCT116, SW620, SW948, Colo205, HT29, LS174T have obvious growth inhibitory activity (IC ₅₀ ≤1μM); compared with the positive control drug Abemaciclib, compound A has the effect on 8 kinds of colorectal cancer cell lines DLD-1, HCT8 , HCT15, LOVO, HCT116, SW948, Colo205, HT29 have higher proliferation inhibitory activity.

Table 10 The inhibitory activity of the test substance on the proliferation of different colorectal cancer cell lines

NA: Not Available

Example 7 Pharmacodynamic experiments of compounds of the present invention in human-derived colorectal cancer xenograft mouse model

5×10 ⁶ human colorectal cancer COLO 205 cells were inoculated into BALA/c nude mice, and the administration began when the average volume of the tumor reached about 142 mm ³ .

The test is divided into compound A 5.0mg/kg, 10.0mg/kg, 25.0mg/kg and 50.0mg/kg groups, positive control drugs are Palbociclib 25.0mg/kg and Abemaciclib 25.0mg/kg groups, and Vehicle group, the above test The preparation of product and reference substance is shown in Table 11. Among them, the positive control drug Palbociclib was prepared by the inventor according to the synthesis method described in WO2003062236A1, and Abemaciclib was prepared by the inventor according to the synthesis method described in WO2010075074A1.

The preparation of table 11 test sample and reference substance

Ten mice in each group were administered orally orally, once a day, for a total of 35 days. Changes in tumor volume and body weight were observed regularly, and curative effect was evaluated based on relative tumor proliferation rate (T/C) and relative tumor inhibition rate (TGI).

Tumor volume calculation formula: tumor volume (mm ³ )=1/2×(a×b ² ) (where a represents the long diameter and b represents the short diameter).

Relative tumor proliferation rate (T/C), that is, the percentage value of the relative tumor volume or tumor weight between the treatment group and the control group at a certain time point. Calculated as follows:

T/C (%)=TRTV/CRTV×100% (TRTV: the average RTV of the treatment group; CRTV: the average RTV of the Vehicle group; RTV=Vt/V0, V0 is the tumor volume of the animal when grouped, and Vt is the animal's tumor volume after treatment tumor volume);

The calculation formula for relative tumor inhibition rate (TGI) is as follows: TGI (%)=(1-T/C)×100%. (T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment group and the control group at a specific time point, respectively).

The results are shown in Table 12 and Fig. 2. The results show that: the compound A (5mg/kg, 10mg/kg, 25mg/kg, 50mg/kg) treatment groups all showed the effect of tumor suppression on the 35th day, and the relative tumor proliferation rate ( T/C) is 57%, 46%, 28%, 8%, relative to Vehicle group, statistically significant difference (p value is respectively 0.003, <0.001, <0.001, <0.001); and compound A (25mg /kg, 50mg/kg) treatment group showed better tumor suppression effect than positive drug Palbociclib (25mg/kg) and Abemaciclib (25mg/kg) group.

During the experiment, the overall body weight of the animals was good, and each administration group showed no obvious drug toxicity, and the test drug showed good tolerance.

It can be seen that compound A (25mg/kg, 50mg/kg) showed obvious tumor inhibitory effect in the human-derived colorectal cancer xenograft mouse model, and the compound had no obvious toxic and side effects, and had a good therapeutic effect. Prospects of clinical application in rectal cancer.

Table 12 Inhibitory activity of test substance in colorectal cancer xenograft model

Note: P value is to compare each group with Vehicle group by T test.

The present invention has been described through the above-mentioned embodiments, but it should be understood that the above-mentioned embodiments are only for the purpose of illustration and description, and are not intended to limit the present invention to the scope of the described embodiments. In addition, those skilled in the art can understand that the present invention is not limited to the above-mentioned embodiments, and more variations and modifications can be made according to the teachings of the present invention, and these variations and modifications all fall within the claimed scope of the present invention. within the range. The protection scope of the present invention is defined by the appended claims and their equivalent scope.

Claims (18)

1. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of breast cancer, colorectal cancer or liver cancer,

   
2. The purposes of the compound of formula (I) or its pharmaceutically acceptable salt in the preparation of the medicine for treating breast cancer,

   
3. The purposes of the compound of formula (I) or its pharmaceutically acceptable salt in the preparation of the medicine for the treatment of colorectal cancer,

   
4. The purposes of the compound of formula (I) or its pharmaceutically acceptable salt in the preparation of the medicine for the treatment of liver cancer,

   
5. The use according to any one of claims 1-4, wherein the pharmaceutically acceptable salt is fumarate.
6. The use according to any one of claims 1-2 or 5, wherein the breast cancer is hormone receptor (HR) positive breast cancer; preferably, the hormone receptor (HR) positive breast cancer is estrogen receptor (HR) positive breast cancer. Hormone receptor positive (ER ⁺ ) breast cancer, progesterone receptor positive (PR ⁺ ) breast cancer, or ER ⁺ PR ⁺ breast cancer; more preferably, the hormone receptor (HR) positive breast cancer is estrogen Receptor-positive (ER ⁺ ) and HER2-negative breast cancer, progesterone receptor-positive (PR ⁺ ) and HER2-negative breast cancer, or ER ⁺ PR ⁺ and HER2-negative breast cancer.
7. A method for treating breast cancer, liver cancer or colorectal cancer, the method comprising administering a therapeutically effective amount of compound (I) or a pharmaceutically acceptable salt thereof to a subject in need,

   
8. A method for treating breast cancer, the method comprising administering a therapeutically effective amount of Compound (I) or a pharmaceutically acceptable salt thereof to a subject in need thereof,

   
9. A method for treating liver cancer, the method comprising administering a therapeutically effective amount of compound (I) or a pharmaceutically acceptable salt thereof to a subject in need thereof,

   
10. A method for treating colorectal cancer, the method comprising administering a therapeutically effective amount of compound (I) or a pharmaceutically acceptable salt thereof to a subject in need thereof,

    
11. The method according to any one of claims 7-10, wherein the pharmaceutically acceptable salt is fumarate.
12. The method according to any one of claims 7, 8 or 11, wherein the breast cancer is hormone receptor (HR) positive breast cancer; preferably, the hormone receptor (HR) positive breast cancer is estrogen receptor (HR) positive breast cancer. Hormone receptor positive (ER ⁺ ) breast cancer, progesterone receptor positive (PR ⁺ ) breast cancer, or ER ⁺ PR ⁺ breast cancer; more preferably, the hormone receptor (HR) positive breast cancer is estrogen Receptor-positive (ER ⁺ ) and HER2-negative breast cancer, progesterone receptor-positive (PR ⁺ ) and HER2-negative breast cancer, or ER ⁺ PR ⁺ and HER2-negative breast cancer.
13. The compound of formula (I) or a pharmaceutically acceptable salt thereof is used for treating cancer, wherein the cancer is breast cancer, colorectal cancer or liver cancer.
14. A compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer, wherein the cancer is breast cancer,

    
15. A compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer, wherein the cancer is colorectal cancer,

    
16. A compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer, wherein the cancer is liver cancer,

    
17. The compound or pharmaceutically acceptable salt thereof for the use according to any one of claims 13-16, wherein the pharmaceutically acceptable salt of the compound of formula (I) is of formula ( I) Fumarate salts of compounds.
18. The compound for use according to any one of claims 13-14 or 17, or a pharmaceutically acceptable salt thereof, wherein the breast cancer is hormone receptor (HR) positive breast cancer; preferably, The hormone receptor (HR) positive breast cancer is estrogen receptor positive (ER ⁺ ) breast cancer, progesterone receptor positive (PR ⁺ ) breast cancer, or ER ⁺ PR ⁺ breast cancer; more preferably, all The hormone receptor (HR) positive breast cancer is estrogen receptor positive (ER ⁺ ) and HER2 negative breast cancer, progesterone receptor positive (PR ⁺ ) and HER2 negative breast cancer, or ER ⁺ PR ⁺ and HER2-negative breast cancer.

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