WO2014180304A1

WO2014180304A1 - Use of j1-001 compound as anti-cancer drug

Info

Publication number: WO2014180304A1
Application number: PCT/CN2014/076888
Authority: WO
Inventors: 刘天罡
Original assignee: 武汉臻智生物科技有限公司
Priority date: 2013-05-07
Filing date: 2014-05-06
Publication date: 2014-11-13
Also published as: CN104138369B; CN104138369A

Abstract

Provided in the present invention is the use of a J1-001 compound as an anti-cancer drug, including the use of the compound shown in formula I or pharmaceutical acceptable salts thereof in the preparation of a drug for inhibiting the proliferation of tumour cells.

Description

Use of Jl-001 compound as an anticancer drug

Priority information

This application requests a patent application filed on May 7, 2013 from the State Intellectual Property Office of China, with a patent application number of 2013 10163640.X, and a patent application number of 2013 10298008.6 submitted to the State Intellectual Property Office of China on July 16, 2013. Priority and interest in the patent application, which is incorporated herein in its entirety by reference. Technical field

The present invention relates to the field of biomedicine, and in particular to the use of a J1-001 compound as an anticancer drug. Background technique

Currently, cancer has become one of the leading causes of human death. Clinically, chemotherapy, radiation therapy and biological immunotherapy are used to kill tumor cells, but it is impossible to cure tumors fundamentally. Most of them are because the drug resistance of tumor cells seriously affects the clinical chemotherapy effect (according to the American Cancer Society estimates, 90%) The above tumor patients die from varying degrees of tumor cell resistance), and due to the presence of cancer stem cells, most cancer patients are prone to recurrence of cancer or tumor cell migration. Therefore, the search for a highly effective, low-toxic, drug-resistant tumor cell sensitive drug that inhibits the proliferation of tumor stem cells has a very positive effect on the treatment of cancer. Summary of the invention

The present invention is directed to solving at least some of the above technical problems or at least providing a useful commercial choice.

To this end, the object of the present invention is to propose the use of a J1-001 compound for inhibiting proliferation of tumor cells and/or tumor stem cells, which can be used for the treatment of cancer, in the preparation of a medicament.

The invention is

Formula I

Or a pharmaceutically acceptable salt thereof (herein, a compound of the formula I and a pharmaceutically acceptable salt thereof are collectively referred to as a J1-001 compound) can be used alone for inhibiting malignant cells, drug-resistant malignant cells and/or Or proliferation of cancer stem cells.

In a first aspect of the invention, the invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for inhibiting proliferation of tumor cells and/or tumor stem cells,

Formula I.

It should be noted that cancer stem cells maintain the vitality of tumor cell population through self-renewal and immortalization; the movement and migration ability of cancer stem cells make tumor cell metastasis possible; tumor stem cells can be dormant for a long time and have various tolerances. Drug molecules are not sensitive to the external physical and chemical factors that kill tumor cells, so tumors often recur after a period of time after conventional tumor treatment methods eliminate most common tumor cells. Thus, there is a need to develop a drug that inhibits the proliferation of cancer stem cells. According to an embodiment of the present invention, the inventors have found that the compound of the formula I or a pharmaceutically acceptable salt thereof can be used alone to inhibit the proliferation of cancer stem cells. The inventors have unexpectedly found that breast cancer cell MCF-7 stem cells are mixed and cultured in vitro with a J1-001 compound, especially a J1-001 sodium salt. The MTT colorimetric method was used to calculate the inhibition rate of J1-001 sodium salt on tumor stem cell proliferation and IC _5Q (half inhibition rate). The inventors found that J1-001 sodium salt inhibited breast cancer cell MCF-7 stem cells. Thus, it can be concluded that the J1-001 sodium salt has a good inhibitory effect on the tumor stem cells, especially the breast cancer cells MCF-7 stem cells.

According to an embodiment of the present invention, the inventors have found that the compound of the formula I or a pharmaceutically acceptable salt thereof can be used alone for inhibiting the proliferation of malignant tumor cells and drug-resistant malignant cells. The inventors have unexpectedly discovered that the J1-001 compound can effectively inhibit the proliferation of malignant tumor cells and drug-resistant malignant tumor cells by mixing J1-001 compound with malignant tumor cells and drug-resistant malignant tumor cells. Thus, the compound of the formula I or a pharmaceutically acceptable salt thereof can be used for the treatment of malignant tumor cells and drug-resistant malignant tumor cells, thereby providing a novel drug for treating such diseases.

The term "pharmaceutically acceptable salt" as used herein, means a salt form commonly used in the field of medicinal chemistry, that is, substantially non-toxic and capable of providing the desired pharmacokinetic properties, orally, absorbing, distributing, metabolizing or The salt form of excretion. According to an embodiment of the present invention, the "pharmaceutically acceptable salt" may be a common acid addition salt or a base addition salt such as a potassium salt or a sodium salt.

According to the above embodiment of the invention, the invention may also have the following additional technical features:

According to an embodiment of the invention, the tumor cells are malignant tumor cells and drug resistant malignant tumor cells. According to an embodiment of the present invention, the malignant tumor cell is at least one selected from the group consisting of a breast cancer cell, a liver cancer cell, a lung cancer cell, a gastric cancer cell, and an ovarian cancer cell. The inventors discovered that the proliferation of malignant tumor cells can be inhibited by studying the use of the J1-001 compound. Therefore, further studies have found that the proliferation of breast cancer cells, liver cancer cells, lung cancer cells, gastric cancer cells and ovarian cancer cells Has an inhibitory effect. According to a specific embodiment of the present invention, the inventors will be MCF-7 (human breast cancer cells), Bel-7402 (human liver cancer cells), HepG2 (human liver cancer cells), NCI-H460 (human lung cancer cells), SGC-7901 ( Human gastric cancer cells and HO-8910 (human ovarian cancer cells) were mixed and cultured in vitro with J1-001 compound, especially its sodium salt. Using the MTT colorimetric method to calculate the inhibition rate of J1-001 compound on tumor cell proliferation and IC _5Q (half inhibition rate), the inventors found that J1-001 compound to MCF-7, Bel-7402, HepG2, NCI-H460, SGC-7901 and HO-8910 have different degrees of inhibition, which can be concluded that the J1-001 compound has a good inhibitory effect on the above tumor cells. The inventors have also surprisingly found by analyzing the results of the examples that the J1-001 compound has a significantly stronger inhibitory effect on the proliferation of ovarian cancer cells than paclitaxel, thereby further illustrating the use of the J1-001 compound for the treatment of malignant tumors under these conditions. The superiority.

According to an embodiment of the present invention, the drug resistant malignant tumor cell is selected from the group consisting of Bel-7402 liver cancer resistant cells and

MCF-7/TAX breast cancer resistant cells. By investigating the use of the J1-001 compound, the inventors unexpectedly found that it can inhibit the proliferation of drug-resistant malignant cells. According to a specific embodiment of the present invention, the inventors will have Bel-7402/5-Fu (human liver cancer resistant cells) or MCF-7/TAX (breast cancer resistant cells) and J1-001 compounds, especially J1-001 sodium salts. Mix culture in vitro. The MTT colorimetric method was used to calculate the inhibition rate and IC ₅₀ (half inhibition rate) of J1-001 sodium salt on tumor cell proliferation. The inventors found that J1-001 sodium salt is resistant to human liver cancer resistant cells and human breast cancer resistant cells. It has an inhibitory effect. Thus, it can be concluded that the J1-001 sodium salt has a good inhibitory effect on drug-resistant malignant cells under these conditions.

According to an embodiment of the invention, the cancer stem cell is a breast cancer stem cell selected from the group consisting of MCF-7 (CD24-CD44 ⁺ ). The inventors unexpectedly found that it can inhibit the proliferation of cancer stem cells by studying the use of the J1-001 compound. According to a specific embodiment of the present invention, the inventors mixed MCF-7 (CD24-CD44+) breast cancer stem cells with a J1-001 compound, especially a J1-001 sodium salt, in vitro. Using MTT colorimetric method to calculate the inhibition rate of J1-001 sodium salt on tumor cell proliferation and IC _5Q (half inhibition rate), the inventors found that J1-001 sodium salt against MCF-7 (CD24-CD44 ⁺ ) breast cancer stem cells It has an inhibitory effect. Thus, it can be concluded that the J1-001 sodium salt has a good inhibitory effect on breast cancer stem cell cells under these conditions.

Thus, in a second aspect of the invention, the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof for use in the preparation of a medicament

Formula I.

According to an embodiment of the invention, the pharmaceutically acceptable salt is a sodium or potassium salt.

According to an embodiment of the present invention, the cancer is at least one selected from the group consisting of breast cancer, liver cancer, lung cancer, gastric cancer, and ovarian cancer.

The use of the present formula I or a pharmaceutically acceptable salt thereof for the preparation of an anticancer drug has not been reported in the literature worldwide. According to a specific embodiment of the present invention, the inventors unexpectedly found that Formula I or a pharmaceutically acceptable salt thereof has an effect of inhibiting the proliferation of malignant tumor cells and drug-resistant malignant cells and/or tumor stem cells, and further, the inventors It has been found that Formula I or a pharmaceutically acceptable salt thereof is used as an active ingredient in the treatment of an anticancer drug to prepare an anticancer drug, and it is found to have a remarkable anticancer effect. Thus, Formula I or a pharmaceutically acceptable salt thereof can be used as an active ingredient for the treatment of cancer drugs. According to one embodiment of the invention, the above-described resistance comprising Formula I or a pharmaceutically acceptable salt thereof Cancer drugs can be used to treat or prevent breast cancer, liver cancer, lung cancer, stomach cancer and ovarian cancer. The invention thus provides a novel use of Formula I or a pharmaceutically acceptable salt thereof, thereby providing a new class of anticancer drugs for the treatment of cancer. According to a specific embodiment of the present invention, Formula I or a pharmaceutically acceptable salt thereof can be used as an active ingredient in an anticancer drug, and particularly has a significant inhibitory effect on the proliferation of drug-resistant tumor cells and/or tumor stem cells, and thus can Fundamentally achieve the purpose of preventing and treating cancer.

Further, in a third aspect of the invention, according to an embodiment of the invention, the invention provides a pharmaceutical composition comprising: a compound of formula I or a pharmaceutically acceptable salt thereof (collectively referred to as J1 -001 compound) ) and pharmacy

As described above, the pharmaceutical composition can effectively inhibit the proliferation of tumor cells and/or tumor stem cells, as well as for treating cancer.

Further, according to an embodiment of the present invention, the compound of the formula I may be produced by chemical synthesis or biosynthesis, for example, by microbial fermentation. According to an embodiment of the invention, the pharmaceutically acceptable salt is a sodium or potassium salt.

The term "pharmaceutically acceptable excipient" as used herein may include any of the common excipients that may be used pharmaceutically, such as, but not limited to, binders, fillers, film-coated polymers, plasticizers, and helpers. Flowing agents, disintegrating agents, lubricants, and the like.

According to a particular embodiment of the invention, the pharmaceutical composition may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which may enhance the shelf life or efficacy of the pharmaceutical composition. By the embodiment of the present invention, the manner of delivering the pharmaceutical composition is not subject to any limitation, and various known delivery systems can be employed as long as the J1-001 compound or the J1-001 compound can be effectively administered in combination with other drugs. To prevent, manage, treat or ameliorate a condition or one or more of its symptoms, such as cancer.

According to an embodiment of the present invention, the pharmaceutical composition may be administered in a gastrointestinal dosage form and a parenteral administration form. According to an embodiment of the present invention, the gastrointestinal administration dosage form is a preparation in which the pharmaceutical composition enters the stomach and intestine after oral administration, and functions locally or absorbs at the administration site. According to an embodiment of the present invention, the parenteral administration form is a preparation in which the pharmaceutical composition functions in a local action or is absorbed after administration at a site other than an oral administration route. Thus, it is possible to further ensure that the J1-001 compound or a pharmaceutically acceptable salt thereof acts on malignant tumor cells, drug-resistant malignant tumor cells, and/or cancer stem cells in various forms, and enhances treatment of malignant tumor cells in various ways, The effect of drug resistant malignant cells and/or cancer stem cells.

According to an embodiment of the present invention, the J1-001 compound is an acidic ester-soluble polyether antibiotic, which has an inhibitory effect on Gram-positive and negative bacteria; has strong anti-coccidial activity, and is currently used for prevention and treatment. Laying hens and broilers Coccidiosis; At the same time, the toxicity and triad test results show that the antibiotic is safe and has no side effects. According to an embodiment of the present invention, the manner of obtaining the J1 - 001 compound is not particularly limited. According to a specific embodiment of the present invention, the J1 - 001 compound may be produced by fermentation with a microorganism containing a gene cluster encoding the compound, or may be Produced by chemical synthesis or chemical semi-synthesis. Therefore, preparation of a malignant tumor cell and a drug-resistant malignant tumor cell and/or a tumor stem cell using the J1-001 compound has good industrial applicability. On the other hand, the J1 -001 compound has long been used as an antibiotic for the control of chicken coccidia. At the same time, the antibiotic toxicity and triad test results show that it is safe and has no side effects. Therefore, the J1 - 001 compound has operability and applicability in preparation for pharmaceutical use. From the above, the J1-001 compound has great market prospects for the preparation of a medicament for treating malignant tumors.

Further, according to an embodiment of the present invention, the inventors unexpectedly found that the J1 -001 compound inhibited the proliferation of drug-resistant tumor cells significantly better than paclitaxel. In the field of modern medical technology, existing anticancer drugs have a good effect on the treatment of cancer in a certain period of time, but because of the long cycle of treatment of cancer, long-term use of a certain anticancer drug is prone to drug resistance. Therefore, there is a need to develop more drugs for treating cancer, especially drugs which have a significant inhibitory effect on tumor cells which have developed resistance. The inventors have surprisingly found that the J1-001 compound is effective in inhibiting a variety of human malignant cells, particularly drug-resistant tumor cells, and its effect is superior to various existing chemotherapeutic drugs. Thereby, the superiority of the J1-001 compound as a medicament for treating malignant tumors is further improved.

Further, according to an embodiment of the present invention, the inventors have surprisingly found that the J1-001 compound can inhibit the proliferation of tumor stem cells, and the effect thereof is superior to various existing chemotherapeutic drugs, for example: J1-001 compound for tumor stem cells The inhibition of proliferation is significantly stronger than that of doxorubicin, that is, the J1-001 compound is superior in the treatment of malignant tumor drugs.

Further, according to an embodiment of the present invention, the means for administering the prepared drug is also not particularly limited, such as parenteral administration (for example, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural administration, tumor Internal administration and mucosal administration (eg, intranasal and oral routes). In a specific embodiment, the medicament of the present invention can be administered intramuscularly, intravenously, intratumorally, orally, intranasally, pulmonaryly, or subcutaneously. The J1-001 compound can be administered by any convenient route, for example by infusion or a single rapid intravenous injection, by absorption through an epithelial or mucosal skin lining (eg, oral mucosa, rectum, and intestinal mucosa, etc.), by antibody protein specific treatment. And, and can be administered in conjunction with other biologically active agents. According to an embodiment of the invention, the administration of the J1-001 compound may be systemic or topical administration.

In particular embodiments, it may be desirable to topically administer a therapeutic agent of the invention in a region in need of treatment; this may be accomplished by, for example, but not limited to, local infusion, injection, or by implantation, the implant being porous Or non-porous materials, including membranes and matrices, such as silicone rubber membranes, polymers, fibrous matrices (eg, Tissuel®), or collagen matrices.

The additional aspects and advantages of the invention will be set forth in part in the description which follows. DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from Figure 1 shows the results of detection of IC _5Q of various cell lines under the action of J1 -001 sodium salt;

Figure 2 shows the results of the effect of J1 -001 sodium salt on tumors in mice;

Figure 3 shows the results of the effect of J1 -001 sodium salt on breast cancer cells in mice. Detailed description of the invention

The invention is described below with reference to the specific embodiments, which are intended to be illustrative, and not to limit the invention in any way. And the J1 -001 sodium salt used in the following examples has the following

Example 1 : Inhibition of proliferation of HO-8910 cells by J1-001 sodium salt and paclitaxel

HO-8910 (ovarian cancer cells) in logarithmic growth phase were respectively taken and digested with 0.25%-EDTA to prepare a cell suspension having a concentration of 5×10 ⁴ cells/mL. The cell suspension was seeded in a 96-well cell culture plate at 200 μL per well; the blank group was a group containing 200 μί of cell-free medium per well. The above-mentioned cells-inoculated 96-well cell culture plates were placed in a cell culture incubator overnight to allow the cells to adhere. The sample J1-001 sodium salt and the existing anticancer drug paclitaxel were respectively dissolved in DMSO, and were prepared as a storage solution of 100 mmol/L, and then diluted into a sample solution of different concentrations (final concentration of DMSO less than 0.1%) with the corresponding medium before use. Add 200μί different concentrations of sample solution (0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 4.0, 8.0 μηιοΙ/L) or paclitaxel solution (0.05, 0.1, 0.2, 0.5, 1.0, 2.0, respectively) in different experimental groups. 4.0, 8.0 μιηο1/ί); The control group was a group in which 200 μί medium was added to the adherent cells; the blank group was a group in which no cells were added with 200 μί medium. Then, the 96-well cell culture plate supplemented with different concentrations of the drug solution (0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 4.0, 8.0 μm Ι Ι / L sample solution or paclitaxel solution) was cultured in a cell culture incubator for 48 hours. 20 μί 0.5 mg/mL of thiazolyl blue (MTT) was added to each well of the above-mentioned 96-well cell culture plate taken out, and then cultured in a cell culture incubator. After 2 hours, 96-well cell culture plates co-incubated with MTT were centrifuged at 2000 rpm for 10 minutes in a high speed centrifuge. After centrifugation, the supernatant in a 96-well cell culture plate was removed, and then the formazan blue crystals formed at the bottom of the 96-well plate were dissolved by adding 200 μL of dimethyl sulfoxide (DMSO) to each well. The 96-well cell culture plate with DMSO was shaken on a plate shaker for 5 minutes, and the OD value (reference wavelength was 490 nm) was measured at 570 nm using an enzyme-linked immunosorbent assay to calculate the inhibition rate and IC _5Q. (half the inhibition rate).

Inhibition rate%= ( A-Ao ) I ( Α-Αι ) χ 100%,

Where: Α represents the OD value of the control group; A _Q represents the OD value of the sample group; represents the OD value of the blank group.

Table 1. IC ₅ Q values of J1-001 sodium salt paclitaxel HO-8910

A pair of HO 50910 IC ₅₀ Jl-001 sodium salt 1.43

Paclitaxel 7.41

From the results shown in Table 1, it was found that the J1-001 sodium salt had a more significant inhibitory effect on the proliferation of HO-8910 cells in vitro than the paclitaxel, and the IC _5Q value of the HO-8910 cell line was reduced by 5.18 compared with paclitaxel. Times. It indicated that the sodium salt of J1-001 had a strong inhibitory effect on the proliferation of HO-8910 tumor cells, and its inhibitory effect was stronger than that of paclitaxel. Example 2: Inhibition of proliferation of various cell lines in vitro by J1-001 sodium salt

The experimental method is the same as in Example 1, except that the selected cell lines are MCF-7 (human breast cancer cells), Bel-7402 (human liver cancer cells), HepG2 (human liver cancer cells), NCI-H460 (human lung cancer). Cells), SGC-7901 (human gastric cancer cells) and HO-8910 (human ovarian cancer cells).

From the results shown in Figure 1, it can be seen that the sodium salt of J1-001 has different inhibitory effects on the proliferation of MCF-8, Bel-7402, HepG2, NCI-H460, SGC-7901 and HO-8910 cells in vitro, among them, MCF- 7 is most sensitive to J1-001 sodium salt and exhibits the lowest IC _5Q value. It can be seen that the J1-001 sodium salt has a good inhibitory effect on the proliferation of the above tumor cells. Example 3: Inhibition of J1-001 sodium salt on proliferation of multidrug-resistant tumor cells in vitro

The experimental method was the same as in Example 1, except that the selected cells were Bel-7402 (human liver cancer cells); Bel-7402/5-Fu (human liver cancer resistant cells).

Table 2. J1-001 sodium, taxol and adriamycin of ₅₀ values Bel-7402 and Bel-7402/5-Fu an IC

From the experimental results shown in Table 2, it can be seen that the J1-001 sodium salt has a good inhibitory effect on the cell proliferation of the resistant Bel-7402/5-Fu and the non-resistant tumor cell line Bel-7402, and the non-resistant tumor is resistant. The cell line-inhibiting activity of Bel-7402 is similar to that of paclitaxel in inhibiting cancer cell proliferation, and is superior to doxorubicin in inhibiting cancer cell proliferation; it is worth mentioning that J1-001 sodium salt is resistant to The Bel-7402/5-Fu tumor cell line exhibited the lowest IC _5Q value. The antitumor activity of paclitaxel and doxorubicin against 5-Fu-resistant Bel-7402 liver cancer cell line was significantly decreased. Therefore, the above experiments suggest that J1-001 has a unique advantage in anti-tumor resistance and is a potential drug with potential for development. Example 4: Antitumor effect of J1-001 sodium salt in vivo

The thawed frozen HepG2-branch was quickly thawed at 37 °C, the force B RPMI1640 medium was 10 mL, and the lOOOO min ^{1 was} washed twice, and the RPMI1640 medium containing 10% fetal bovine serum was added, 37 ° C, 5 % C0 ₂ incubator In the medium culture, change the cell culture medium between days, and pass the cells to 70%-80% fullness. After 1 week of culture, collect the cell suspension, centrifuge lOOOrmin- ¹ for 5 min, and adjust the cells to 1 mL per mL with serum-free medium. Contains lx lO ⁷ , spare. BABL/c bare at around 5 weeks of age Mice, 3 days after HepG2 cells were inoculated subcutaneously in the axilla of each 0.2 mL (lmL each containing 2>^<107 HepG2 cells). After the tumor was grown to a certain size, the tumor-bearing nude mice were sacrificed. The tumor-bearing mouse tumors were taken out under aseptic conditions, inoculated, and passed through the body for 3 generations. The tumor tissues were cut into l-3mm ³ small pieces in sterile PBS, inoculated by plugging method, and 30 naked in 6 weeks old. The rat armpit was inoculated subcutaneously with 0.2 mL, and the inoculation was observed. Nude mice with tumor mass uniformity when the tumor volume was as long as 100 mm ³ were randomly divided into two groups: model group and J1-001 group, with 6 rats in each group, and a blank group. For the 21-day course, the blank, model group was given the corresponding volume of dilution. It was administered once every 4 days for 5 times. The activity and death of the nude mice were observed at the time of administration, and the tumor size was measured. The long diameter a and the short diameter b (mm) of the tumor were measured with a vernier caliper, and the tumor was weighed after the animal died. The following are specific groupings and administrations:

Blank group: intraperitoneal administration (i.p.) 0.2mL/10g dilution solution

Model group: intraperitoneal administration (i.p.) 0.2mL/10g dilution solution

J1-001 group: intraperitoneal administration (i.p.) 0.2mL/10g 0.05mg/mL dose is lmg/kg

After the 20th day of administration, the animals were sacrificed after blood was taken (the serum was separated after blood was taken), and the tumor masses of each group were peeled off and weighed, and photographed after being placed.

Tumor inhibition rate (%) = (model group tumor volume - experimental group tumor volume y model group tumor volume χ ΐοο %;

RESULTS: There were 2 deaths in the model group and J1-001 group. At the same time, the results from Fig. 2 showed that the tumor size in the animals after treatment with J1-001 was significantly smaller than that in the model group. The tumor inhibition rate was calculated to be about 50%. This result demonstrates that J1-001 is able to inhibit tumor growth in vivo. Example 5 Inhibition of J1-001 sodium salt and paclitaxel on cell proliferation of breast cancer MCF-7 and its resistant strains

Quickly thaw frozen MCF-7 (human breast cancer cells), MCF-7/TAX (human breast cancer resistant cells) at 37 °C, add 5mL of 1640 culture solution, 1000r « ιηίη ¹ 2 times by centrifugation, add The 1640 medium containing 10% fetal bovine serum was cultured in an incubator at 37 ° C and 5 % CO ₂ , and the cell culture medium was changed overnight. The cells were passaged to 70%-80% of full scale, and after 1 week of culture, The cell suspension was collected, centrifuged for 1 min with ΙΟΟΟι·· min- ¹ , and the cells were adjusted to 50,000 cells per ml with 1640 medium, and used. The MCF-7, MCF-7/TAX cell suspension was inoculated into a 96-well plate, 100 μί per well, and cultured in a 37 ° C, 5 % CO ₂ incubator. After 24 h, the corresponding test drug was added to each well and cultured in an incubator at 37 ° C, 5 % CO ₂ . After 24 h, CCk-8 reagent ΙΟ μί culture solution was added to each well, and the absorbance was measured at 450 nm.

Calculate the inhibition rate according to the following formula:

Inhibition rate =[(Α2-Α3)-(Α1-Α3)]/(Α2-Α3) χ 100%,

among them:

A1...test drug absorbance,

Α2... model group absorbance,

A3... blank group absorbance. Table 3. J1-001 sodium, paclitaxel values ₅₀ MCF-7, MCF-7 / TAX an IC

The IC _{5 of the} test substance against MCF-7. ( μηιοΙ/Ρ IC ₅ Q ( μΓηοΙ/L ) for MCF-7/TAX Jl-001 sodium salt 4.27 10.96

Paclitaxel 42.16 3091

It can be seen from the results in Table 3 that the sodium salt of J1-001 has strong antitumor activity against MCF-7, MCF-7/TAX tumor strains, and its IC _5Q values are similar. Compared to paclitaxel, J1-001 sodium salt exhibited lower IC _5Q for both MCF-7 and MCF-7/TAX. For MCF-7 tumor cell lines, the IC _5Q value of J1-001 sodium salt is about 10 times different from that of paclitaxel; for MCF-7/TAX tumor cell line, the IC _5Q value is about 282 times different from that of paclitaxel, suggesting that J1-001 sodium Salt has a unique advantage in fighting breast cancer. Example 6 Effect of J1-001 sodium salt on anti-breast cancer tumor cells in vivo

Rapidly thaw frozen MCF-7-branch at 37 °C, force B RPMI1640 medium 10 mL, lOOOr · ηώι· ¹ centrifugal washing

2 times, add RPMI1640 medium containing 10% fetal bovine serum, incubate in 37 ° C, 5 % CO ₂ incubator, change the cell culture medium between days, pass the cells to 70%-80% fullness, and culture. after one week, the cell suspension were collected, 1000r * min-l centrifuged 5 min, incubated with serum-free solution containing lml per cell regulation IX 10 ⁷ th, spare.

BABL/c nude mice around 5 weeks old were inoculated subcutaneously with 0.2 mL of MCF-7 cells (2 x 10 ⁷ MCF-7 cells per ml) after 3 days. After the tumor was grown to a certain size, the tumor-bearing nude mice were sacrificed. The tumor-bearing mouse tumors were taken out under aseptic conditions, inoculated, and passed through the body for 3 generations. The tumor tissues were cut into l-3mm ³ small pieces in sterile PBS, inoculated by plugging method, and 70 naked at 6 weeks old. Rats were inoculated subcutaneously with 0.2 mL to observe the inoculation.

Nude mice with tumor mass uniformity when the tumor volume was as long as 100 mm ³ were randomly divided into 5 groups: tumor-bearing saline group, tumor-bearing paclitaxel group (10 mg/kg), and tumor-bearing Jl-001 sodium salt high dose. Group (lmg/kg), tumor-bearing Jl-001 sodium salt low-dose group (0.5mg/kg), each group of 6 rats, and a blank group. For the 21-day course, the blank, model group was given the corresponding volume of dilution. The drug was administered once every 3 days for 5 times, and the animals were treated with blood after 20 days. The following are specific groupings and administrations: Blank group: intraperitoneal administration (ip) 0.2ml/10g dilution solution,

Model group: intraperitoneal administration (i.p.) 0.2ml/10g dilution solution,

Paclitaxel group: intraperitoneal administration (i.p.) 0.2ml/10g 0.5mg/ml dose of 10mg/kg,

Jl-001 sodium salt high dose group: intraperitoneal administration (i.p.) 0.2ml/10g 0.05mg/ml dose is lmg/kg,

Jl-001 sodium salt low dose group: intraperitoneal administration (i.p.) 0.2ml/10g 0.025mg/ml The dose is 0.5mg/kg.

On the 21st day of administration, blood was taken and serum was separated for use. The animals were sacrificed, and each group of tumors was taken and weighed. Test results: As shown in Fig. 3, the tumor volume of the tumor-bearing model group was larger, and the tumor volume of the drug-administered group was decreased, and the high-dose group of paclitaxel and J1-001 was effective. As can be seen from Table 4, compared with the model group, the tumor weight of each administration group was significantly decreased, P < 0.05, and it was confirmed that Jl-001 sodium salt can inhibit tumor growth of breast cancer cells in vivo.

Jl-001 sodium salt high dose group 4 1 0.35 ± 0.05

Jl-001 sodium salt low dose group 5 0.5 0.40 ± 0.19 Example 7 J1-001 sodium salt inhibits the proliferation of breast cancer cells MCF-7 stem cells

The MCF-7 cells in logarithmic growth phase were digested with 0.25%-EDTA trypsin to prepare a cell suspension with a concentration of 1×10 ⁶ cells/mL, centrifuged at 500 g for 10 min, and the culture medium was changed to suspension culture solution. Take 1 mL of MCF-7 cell suspension, connect to a 75 cm ² flask, add 9 mL of fresh suspension medium, and incubate in an incubator. After the cells were proliferated for two days, the cell suspension was centrifuged at 500 g for 10 min, resuspended in fresh suspension culture medium, repeatedly beaten as a single cell suspension, and counted at a cell density of 1×10 ⁶ cells/mL. Cultivate the bottle.

The cell surface molecular markers (CD24 ^{+ /} CD44 ^{+ / )} were identified by flow cytometry to identify tumor stem cell subsets. According to the experimental method of Al-Hajj et al. (refer to: Al-Hajj M, etc., for the induction of tumorigenic breast cancer cells) Prospective identification of tumorigenic breast cancer cells, Proc Natl Acad Sci USA. 2003, 100(7): 3983-8., which is incorporated herein by reference, for the detection of CD24-CD44+ in cells by flow cytometry. The content of cells. MCF-7 cells were cultured in suspension culture for 7 weeks. The cell suspension was digested with trypsin-EDTA and mechanically blown into a single cell suspension. The cell density was adjusted to 1×10 ⁸ cells/mL, and the cells were divided into cells. Control group, CD24 single staining group (CD24), CD44 single staining group (CD44) and double staining group (TEST), according to the following table, add the corresponding antibody solution for labeling, set at 4 ° C for 20 min after avoiding light The cells were washed twice with PBS, the cells were resuspended, and the cells of the CD24 OD44+ phenotype were sorted by an upflow cytometer.

The sorted MCF-7 cells (CD24-CD44+) 500 g were centrifuged for 10 min, and the fresh suspension medium was replaced to adjust the cell density to 5×10 ⁴ /mL; the MCF-7 adherent cells in the logarithmic growth phase were taken. After cellopreservation with 0.25%-EDTA trypsin, a cell suspension having a concentration of 5×10 ⁵ /mL was prepared, and the above two cell suspensions were separately inoculated into a 96-well cell culture plate at 200 μί per well. Before the test, each well cell was divided into a blank group, a control group and a drug-administered group, wherein the blank group was a cell-free group plus a medium, the control group was a medium to which cells were added, and the drug-administered group was a cell. Different concentrations of test drugs were added, and doxorubicin was selected as a control for listed anti-tumor drugs in the test, as shown in Table 5 below.

Table 5. Gradient concentration preparation of each test drug

The 96-well cell culture plate was cultured in a cell culture incubator for 48 hours, and 20 μί 0.5 mg/mL of thiazolyl blue (MTT) was added to each well, and the cells were further cultured in a cell culture incubator for 2 hours, and then 96-well cells were added. The plates were centrifuged at 2000 rpm for 10 minutes in a high speed centrifuge. After centrifugation, the supernatant in the 96-well cell culture plate was removed, and then 200 μl of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan blue crystal formed at the bottom of the 96-well plate, using an enzyme-linked immunosorbent assay. The OD value was measured at a wavelength of 570 nm (reference wavelength was 490 nm), and the inhibition rate was calculated and IC _5Q (half the inhibition rate) was obtained. The results are shown in Table 6.

Inhibition rate%= ( A-A0 ) I ( A-A1 ) l 00%, Where: A represents the OD value of the control group; AO represents the OD value of the sample group; A1 represents the OD value of the blank group.

IC of test drug against MCF-7 (CD24-CD44+) cells:

From the data in Table 6, it can be seen that J1-001 sodium salt showed lower IC ₅ o on breast cancer cell stem cells, indicating that J1-001 has a significant inhibitory effect on tumor stem cell proliferation. Industrial applicability

The use of the J1-001 compound of the present invention as an anticancer drug is specifically: the use of a compound of the formula I or a pharmaceutically acceptable salt thereof for the preparation of a medicament for inhibiting tumor cells and/or tumor stem cells The proliferation, that is, the compound of the formula I or a pharmaceutically acceptable salt thereof can be effectively used for the preparation of a medicament for inhibiting the proliferation of tumor cells and/or tumor stem cells. Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations of those details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples", etc. Particular features, structures, materials or features described in the examples or examples are included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Claims

claims

1. The use of the compound represented by Formula I or its pharmaceutically acceptable salt in the preparation of medicines for inhibiting tumor cells and/or tumors

Formula I.

2. The use according to claim 1, characterized in that the tumor cells are malignant tumor cells and drug-resistant malignant tumor cells.

3. The use according to claim 1, wherein the pharmaceutically acceptable salt is sodium salt or potassium salt.

4. The use according to claim 2, characterized in that the malignant tumor cells are at least one selected from the group consisting of breast cancer cells, liver cancer cells, lung cancer cells, gastric cancer cells and ovarian cancer cells.

5. The use of the compound represented by Formula I or its pharmaceutically acceptable salt in the preparation of medicines for the treatment of cancer.

Formula I

6. The use according to claim 5, characterized in that the pharmaceutically acceptable salt is sodium salt or potassium salt.

7. The use according to claim 5, characterized in that the cancer is at least one selected from the group consisting of breast cancer, liver cancer, lung cancer, gastric cancer and ovarian cancer.

8. A pharmaceutical composition, characterized in that it includes:

The compound represented by formula I or a pharmaceutically acceptable salt thereof;

and pharmaceutical

Formula I,

Optionally, the compound represented by Formula I is prepared by microbial fermentation, chemical synthesis or chemical semi-synthesis.

9. The pharmaceutical composition according to claim 8, characterized in that the pharmaceutical composition is in a gastrointestinal administration dosage form and a parenteral administration dosage form.

10. The pharmaceutical composition according to claim 8, wherein the pharmaceutically acceptable salt is a sodium salt or a potassium salt.