Use of a bis-heterocyclic derivative in the treatment of breast cancer and as an antitumor drug sensitizer

Abstract

The present application relates to the field of biological medicine, and provides application of a double heterocyclic derivative in treatment of breast cancer and as an anti-tumor drug sensitizer; the present application discloses application of a double heterocyclic derivative in inhibition of breast cancer cell proliferation, inhibition of formation, growth and metastasis of breast cancer tumor, and enhancement of chemotherapeutic sensitivity of breast cancer cells and tumor. The present application firstly finds that the compound can inhibit proliferation of breast cancer cells at low concentration without affecting normal cells, and improve chemotherapeutic sensitivity of breast cancer cells. The compound can significantly inhibit formation and growth of breast cancer tumor and enhance chemotherapeutic sensitivity of breast cancer tumor at a concentration without obvious organ toxicity. The present application helps to clarify anticancer activity and chemotherapeutic sensitization of the compound, and provides a pharmaceutical basis for drug development in the field of breast cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and provides the application of a biheterocyclic derivative in the treatment of breast cancer and as a sensitizer for antitumor drugs. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide, and its incidence and mortality rates have been rising steadily in recent years, with a significant trend towards younger patients. As a highly heterogeneous malignant disease, current clinical treatment for breast cancer still primarily relies on surgery, radiotherapy, and chemotherapy. However, in clinical practice, many patients gradually develop resistance to conventional therapies during treatment, leading to decreased treatment effectiveness and disease progression. Therefore, finding novel potential drugs that combine anticancer activity with chemosensitivity enhancement has become an important direction in current breast cancer research. Summary of the Invention

[0003] Purpose of the invention: The purpose of this invention is to provide an application of a biheterocyclic derivative, specifically including inhibiting the proliferation of breast cancer cells, inhibiting the formation, growth and metastasis of breast cancer tumors, and enhancing the chemosensitivity of breast cancer cells and tumors.

[0004] The technical problem this invention aims to solve is to explore the application of this bi-heterocyclic derivative in inhibiting the proliferation of breast cancer cells, inhibiting the formation, growth and metastasis of breast cancer tumors, and enhancing the chemosensitivity of breast cancer cells, thereby providing a pharmaceutical basis for drug development in the field of breast cancer treatment. Technical solution

[0005] The use of 2-[(4,6-dimethyl-2-quinazolinyl)amino]-6-phenyl-4(3H)-pyrimidinone in the preparation of drugs for treating breast cancer, characterized in that the structural formula is shown in BY-8 below: .

[0006] Application of 2-[(4,6-dimethyl-2-quinazolinyl)amino]-6-phenyl-4(3H)-pyrimidinone in the preparation of sensitizers for tumor drugs.

[0007] The drug is formulated from 2-[(4,6-dimethyl-2-quinazolinyl)amino]-6-phenyl-4(3H)-pyrimidinone or a pharmaceutically acceptable salt thereof with pharmaceutically acceptable excipients.

[0008] According to claim 4, the drug dosage form is a gel, soft capsule, oral preparation, injection, or lyophilized powder for injection.

[0009] The breast cancers mentioned include: Luminal A type, Luminal B type, and triple-negative breast cancer.

[0010] The breast cancer has developed resistance to anti-tumor drugs.

[0011] The antitumor drug mentioned is doxorubicin.

[0012] The name of this bicyclic derivative is "2-[(4,6-dimethyl-2-quinazolinyl)amino]-6-phenyl-4(3H)-pyrimidinone", its IPUAC name is "2-[(4,6-dimethylquinazolin-2-yl)amino]-4-phenyl-1H-pyrimidin-6-one", its CAS number is 336176-57-9, its molecular formula is C20H17N5O, its molecular weight is 343.39, and its chemical structure is shown in BY-8. .

[0013] It should be noted that BY-8 has a reversible structure, as detailed below: .

[0014] The breast cancer cells mentioned include human breast cancer cell line MCF-7 cells and its doxorubicin-resistant strain MCF-7-Adr cells, MDA-MB-231 cells and BT-474 cells, with normal cells as controls being breast epithelial cells MCF-10A.

[0015] Furthermore, the cells were seeded into 96-well plates at a density of 2000-3000 cells / well.

[0016] The breast cancer tumors described were tumors formed subcutaneously in nude mice (BALB / c Nude) by MDA-MB-231 cells and doxorubicin-resistant MCF-7-Adr cells.

[0017] BY-8 can inhibit the proliferation of breast cancer cells in vitro and enhance the drug sensitivity of breast cancer cells to doxorubicin. At doses without significant organ toxicity, it inhibits the formation and growth of breast cancer tumors in vivo, reduces the frequency of stem cells in tumors, inhibits the metastasis of breast cancer cells, and enhances the drug sensitivity of breast cancer tumors to doxorubicin. The results of this invention provide a theoretical basis for using BY-8 as an anticancer drug or chemosensitizer to treat breast cancer.

[0018] In studies on the inhibition of breast cancer tumor formation and growth, the usage and dosage of BY-8 were as follows: breast cancer cells were treated with 5 μM BY-8 in complete culture medium for 48 hours, and then administered the treatment at a rate of 10... 7 10 6 10 5 The cell count was used to subcutaneously implant tumors in nude mice.

[0019] In studies on inhibiting breast cancer metastasis, BY-8 was used as follows: 5-week-old nude mice with lung metastasis were injected via the tail vein, once every three days, at a dose of 10 mg / kg each time, for 21 days.

[0020] In a study on improving the drug sensitivity of breast cancer tumors to doxorubicin, BY-8 was administered via tail vein injection into tumor-bearing nude mice at a dose of 10 mg / kg for BY-8 and 2.5 mg / kg for 21 days.

[0021] Beneficial Effects: Breast cancer, especially triple-negative breast cancer, is characterized by its high invasiveness and poor differentiation, with a high risk of distant metastasis. Visceral metastasis is more common than bone metastasis, and brain metastasis is also more frequent. Currently, there is a lack of effective endocrine therapy and molecularly targeted therapy drugs. Commonly used chemotherapy drugs (such as doxorubicin) are prone to drug resistance, leading to treatment ineffectiveness or recurrence. Furthermore, at therapeutic doses, they have a strong killing effect on normal cells. Drug resistance and off-target effects of antitumor drugs are technical challenges that urgently need to be addressed in this field. This invention discovers that BY-8 has certain anti-breast cancer cell activity. More importantly, this invention discovers that BY-8 can improve the drug sensitivity of breast cancer cells to doxorubicin, especially against breast cancer cells that have developed resistance to antitumor drugs, such as… Figure 6 As shown, for doxorubicin-resistant MCF-7-Adr tumors, treatment with doxorubicin showed no significant difference compared to the blank control group, indicating ineffectiveness against this tumor. However, when doxorubicin was used in combination with BY-8, a highly significant difference was observed, demonstrating the sensitizing effect of this invention. Furthermore, as... Figure 2 and Figure 7 As shown, BY-8 at therapeutic doses showed no visible toxicity to normal MCF-10A cells or major organs, demonstrating that the present invention has a tumor-targeting effect; the present invention expands the clinical application of BY-8.

[0022] Furthermore, this invention discloses the application of a biheterocyclic derivative in inhibiting breast cancer cell proliferation, suppressing breast cancer tumor formation, growth, and metastasis, and enhancing the chemosensitivity of breast cancer cells and tumors. This invention is the first to discover that this compound, at low concentrations, can inhibit breast cancer cell proliferation without affecting normal cells and improve the chemosensitivity of breast cancer cells. At concentrations without significant organ toxicity, it can significantly inhibit the formation and growth of breast cancer tumors and enhance the chemosensitivity of breast cancer tumors. This invention helps to elucidate the anticancer activity and chemosensitizing effect of this compound, providing a pharmaceutical basis for drug development in the field of breast cancer treatment. Attached image description: Figure 1The effect of BY-8 on cell proliferation was analyzed using the CCK8 assay. In this study, A represents the effect of BY-8 on the proliferation of normal breast epithelial cells, while B and C represent the effects of BY-8 on the proliferation of breast cancer cells.

[0023] Figure 2 To analyze the IC50 of BY-8 in mammary epithelial cells (A) and breast cancer cells (BD) using the CCK8 assay. 50 .

[0024] Figure 3 To analyze the effect of BY-8 on the doxorubicin sensitivity of breast cancer cells (AC) and doxorubicin-resistant breast cancer cells (D) using the CCK8 assay.

[0025] Figure 4 The effect of BY-8 on the tumor initiation ability of breast cancer cells was analyzed using a limiting dilution method in nude mice xenograft tumors. AB represents images and statistical analysis of tumors formed by MDA-MB-231 cells, and CD represents images and statistical analysis of tumors formed by MCF-7-Adr cells.

[0026] Figure 5 To establish a nude mouse lung metastasis model, the effect of BY-8 on the in vivo metastatic ability of breast cancer cells was analyzed. In the figures, A shows an image of the nude mouse lung with arrows indicating metastatic lesions; B shows the weight change of the nude mice during BY-8 treatment; and C shows an H&E stained scan image of a paraffin-embedded lung section.

[0027] Figure 6 This study analyzed the effect of BY-8 on the sensitivity of breast cancer tumors to doxorubicin in tumor-bearing nude mice treated with BY-8 and / or Adr. AB represents images and statistical analysis of tumors formed by MCF-7 cells after treatment, and CD represents images and statistical analysis of tumors formed by MCF-7-Adr cells.

[0028] Figure 7 Organ toxicity analysis of BY-8 was performed on visceral sections of healthy nude mice after treatment with solvent or BY-8.

[0029] Note: In the attached diagram, doxorubicin is referred to as Adr, and the solvent for BY-8 and Adr is DMSO. Detailed Implementation

[0030] The present invention is further described below through embodiments. However, it should be understood that the following specific embodiments are only used to help those skilled in the art understand the present invention, and are not intended to limit the present invention.

[0031] Example 1: CCK8 assay analysis of the effect of BY-8 on breast cancer cell proliferation: Cells were seeded into 96-well plates. After 24 hours, different concentrations of BY-8 were added. After 48 hours, 100 μL of CCK8 solution diluted 10 times was added to each well, and the cells were incubated for 1 hour. The absorbance at 450 nm was measured, and the viable cell percentage was calculated.

[0032] The results are as follows Figure 1 As shown, at low concentrations (≤25μM), BY-8 can significantly inhibit the proliferation of breast cancer cells, but has no effect on the proliferation of breast epithelial cells.

[0033] Example 2: CCK8 assay analysis of the IC50 of BY-8 in breast epithelial cells and breast cancer cells. 50 : The specific implementation method is the same as in Example 1, using Graphpad Prism 10.1.2 for IC processing. 50 The calculation.

[0034] Example 3: CCK8 assay analysis of the effect of BY-8 on the sensitivity of breast cancer cells to doxorubicin: Cells were seeded into 96-well plates. After 24 hours, different concentrations of Adr and the same concentration of BY-8 were added. After 48 hours, 100 μL of CCK8 solution diluted 10-fold was added to each well, and the cells were incubated for 1 hour. The absorbance at 450 nm was measured, and IC50 analysis was performed using Graphpad Prism 10.1.2. 50 The calculation.

[0035] The results are as follows Figure 3 As shown, BY-8 enhances the doxorubicin sensitivity of breast cancer cells.

[0036] Example 4: Analysis of the effect of BY-8 on the tumor initiation ability of breast cancer cells using nude mouse xenograft tumor assay combined with limiting dilution method: Breast cancer cells were expanded and cultured. After the last passage, when the cells grew to 30%-40%, the medium was replaced with complete medium containing BY-8, and cultured for 48 hours. After trypsin digestion and cell collection, the cells were counted and then subcutaneously introduced into the axilla of nude mice. Tumor formation was observed 14 days later.

[0037] The results are as follows Figure 4 As shown, BY-8 treatment weakened the tumorigenicity of breast cancer cells.

[0038] Example 5: A nude mouse lung metastasis model was established to analyze the effect of BY-8 on the in vivo metastatic ability of breast cancer cells. 1×10 6A nude mouse lung metastasis model was established by injecting cells into nude mice via the tail vein. DMSO or BY-8 was administered via tail vein injection every three days, and the mice were weighed and their weight recorded. After 24 days, the nude mice were sacrificed, and their lungs were harvested and fixed in paraformaldehyde. H&E staining analysis was performed to observe lung nodules.

[0039] The results are as follows Figure 5 As shown, after administration of BY-8, the number of lung nodules in mice was significantly reduced. Recording the mouse body weight revealed no significant difference compared to the solvent control group, suggesting that fluvastatin has the function of inhibiting breast cancer cell metastasis in vivo with low toxicity.

[0040] Example 6: Analysis of the effect of BY-8 on the sensitivity of breast cancer tumors to doxorubicin in tumor-bearing nude mice treated with BY-8 and / or Adr: Tumor-bearing nude mice were treated with BY-8 and / or Adr via tail vein injection every three days. The mice were sacrificed after 24 days, and the tumors were collected and weighed.

[0041] The results are as follows Figure 6 As shown, the tumor weight in nude mice treated with both BY-8 and Adr was significantly smaller than that in mice treated with Adr alone, indicating that BY-8 can improve the doxorubicin sensitivity of breast cancer tumors in vivo.

[0042] Example 7: Analysis of organ toxicity of BY-8 in visceral sections of healthy nude mice after treatment with solvent or BY-8: Healthy nude mice were treated with DMSO or BY-8 via tail vein injection every three days. After 24 days, the mice were sacrificed, and their hearts, livers, spleens, lungs, and kidneys were collected for H&E staining analysis to observe whether organ damage occurred.

[0043] The results are as follows Figure 7 As shown, BY-8 showed no significant organ toxicity at the administered dose.

Claims

1. 2-[(4,6-dimethyl-2-quinazolinyl)amino]-6-phenyl-4(3H)-pyrimidinone in the preparation of drugs for treating breast cancer. The application of 2,2-[(4,6-dimethyl-2-quinazolinyl)amino]-6-phenyl-4(3H)-pyrimidinone in the preparation of sensitizers for antitumor drugs, characterized in that, The tumor is breast cancer, and the antitumor drug is doxorubicin. 3.2-[(4,6-dimethyl-2-quinazolinyl)amino]-6-phenyl-4(3H)-pyrimidinone in combination with doxorubicin in the preparation of drugs for treating breast cancer.

4. The application according to claim 1 or 2, characterized in that, The drug is formulated from 2-[(4,6-dimethyl-2-quinazolinyl)amino]-6-phenyl-4(3H)-pyrimidinone or a pharmaceutically acceptable salt thereof with pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The drug dosage form is a gel, oral preparation, injection, or lyophilized powder for injection.

6. The application according to any one of claims 1-3, characterized in that, The breast cancers mentioned include: Luminal A, Luminal B, or triple-negative breast cancer.

7. The application according to claim 6, characterized in that, The breast cancer has developed resistance to anti-tumor drugs.

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