阅读说明：本技术 小分子化合物qw24在制备治疗结直肠癌疾病药物中的应用 (Application of small molecule compound QW24 in preparation of medicine for treating colorectal cancer diseases ) 是由 易正芳 王金花 谢玖清 邢雅婧 仇文卫 刘明耀 于 2019-08-30 设计创作，主要内容包括：本发明公开了一种如式(I)所示的小分子化合物QW24及其在制备治疗结直肠癌疾病药物中的应用,该化合物能通过自噬-溶酶体蛋白降解途径显著下调BMI-1的蛋白质水平,降低结直肠癌肿瘤干细胞的自我更新能力,进而抑制肿瘤细胞的增殖和迁移。在小鼠皮下荷瘤模型中,式(I)所示的小分子化合物QW24显著抑制肿瘤的生长,以及在结直肠癌肝转移动物模型中,能显著抑制肿瘤的转移,并且给药过程中未显示明显的毒性,具有治疗结直肠癌的应用前景。(The invention discloses a small molecular compound QW24 shown in formula (I) and application thereof in preparing a medicine for treating colorectal cancer, wherein the compound can remarkably reduce the protein level of BMI-1 through an autophagy-lysosome protein degradation way, reduce the self-renewal capacity of colorectal cancer tumor stem cells, and further inhibit the proliferation and migration of the tumor cells. In a mouse subcutaneous tumor-bearing model, the small molecular compound QW24 shown in the formula (I) can obviously inhibit the growth of tumors, and in a colorectal cancer liver metastasis animal model, can obviously inhibit the metastasis of the tumors, does not show obvious toxicity in the administration process, and has an application prospect in treating colorectal cancer.)

1. A QW24 small molecule compound or a hydrate or a pharmaceutically acceptable salt thereof, which is characterized in that the structure is shown as formula (I);

2. a pharmaceutical composition comprising a QW24 compound represented by formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. Use of the QW24 small molecule compound or a hydrate or a pharmaceutically acceptable salt thereof according to claim 1, or the pharmaceutical composition according to claim 2, in the preparation of a medicament for the prevention and/or treatment of colorectal cancer diseases.

4. The use according to claim 3, wherein the QW24 compound of formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition is for inhibiting the proliferation, growth, metastasis, migration and promoting apoptosis of colorectal cancer in vitro and/or in vivo.

5. The use of claim 4, wherein the colorectal cancer comprises colorectal cancer cell lines HCT116, HT29, HCT8, HCT15, LS174T, LoVo and CT 26.

6. The use according to claim 3, wherein the QW24 compound of formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition significantly reduces the self-renewal capacity of colorectal cancer tumor stem cells, reduces the number of rectal cancer tumor stem cells.

7. The use according to claim 6, wherein the QW24 compound of formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition inhibits the self-renewal ability of colorectal cancer cell line HCT116 with stem cell properties, reducing the number of HCT 116.

8. The use according to claim 4, wherein the QW24 compound of formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition is for inhibiting liver metastasis of CT26 in colorectal cancer cells in vitro and/or in vivo.

9. The use of claim 3, wherein the QW24 compound of formula (I) or a hydrate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition down-regulates the protein level of BMI-1.

10. The use of claim 9, wherein the QW24 compound of formula (I), or a hydrate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition degrades BMI-1 protein via the autophagy-lysosomal pathway.

11. Use of the QW24 compound or a hydrate or a pharmaceutically acceptable salt thereof according to claim 1, or the pharmaceutical composition according to claim 2, for the preparation of a medicament for the prevention and/or treatment of a malignant tumor disease associated with BMI-1 overexpression.

12. The use of claim 11, wherein the malignancy comprises colon cancer, glioma, prostate cancer, breast cancer, lung cancer, liver cancer, leukemia.

Technical Field

The invention relates to the technical field of medicines, in particular to application of a small molecular compound QW24 in preparation of medicines for treating colorectal cancer diseases.

Background

Colorectal cancer (Colorectal cancer) is the cancer with the third grade of incidence and fatality worldwide, accounting for 10% of all cancer cases, and with the fatality reaching 50%. In 2012 140 million new cases and 694000 people died from colorectal cancer, with more common cases in developed countries, accounting for approximately 65% of the total number of cases. The incidence and mortality of colorectal cancer in China are increasing day by day, which seriously endangers the health of people and also becomes a hotspot for the research of colorectal cancer.

The main treatment method of the colorectal cancer is different along with the development of the colorectal cancer at different stages, the colorectal cancer is mainly treated by means of surgical excision when tumors at the I stage and the II stage of the colorectal cancer are only positioned on the intestinal wall, the tumors are widely spread and metastasized when the cancer is developed to the III stage and the IV stage, the surgical excision therapy is difficult to cure, and the radiotherapy or chemotherapy mode must be combined. The survival rate of colorectal cancer patients gradually decreases as the cancer progresses, and the five-year survival rate at stage iv is only lower than 5%. There is some difficulty in surgical resection at the late stage of cancer, and chemotherapy or radiotherapy is the main treatment means. However, patients with advanced colorectal cancer have serious recurrence problems, survivors after primary cure still have the risk of forming secondary primary tumors in the colon and rectum, and the survival rate of the patients is still low.

Tumor stem cells (CSCs) have been shown to be more resistant to traditional Cancer therapies than non-tumor stem cells, recurrence of colorectal Cancer is closely associated with colorectal Cancer tumor stem cells, and clinical data indicate that if chemotherapeutic drugs can target tumor stem cells, survival rates of patients can be significantly improved. Therefore, tumor stem cells have been the focus of research on cancer treatment. BMI-1 is a key regulatory factor for maintaining the self-renewal capacity of tumor stem cells, can maintain the pluripotency of the tumor stem cells, promote cell proliferation, inhibit cell aging and apoptosis, and further cause drug resistance and relapse of cancer. BMI-1 is highly expressed in colorectal cancer patients and is closely associated with low survival rates of patients. Thus, targeting BMI-1 is considered a new approach to the treatment of colorectal cancer.

Therefore, by combining the latest basic research results of colorectal cancer formation mechanism, the novel small molecular drugs for treating colorectal cancer, especially the drugs with the independent intellectual property rights in China, are developed, the molecular mechanism of the effect is clarified, the foundation is laid for the subsequent research and development of new drugs, and the novel small molecular drugs have important theoretical significance and wide application prospect.

Disclosure of Invention

The invention provides a QW24 small molecule compound or a hydrate or a pharmaceutically acceptable salt thereof, which has a structure shown in a formula (I):

QW24 shown in formula (I) is a small molecular compound with the molecular formula of C₂₇H₂₈N₂O₄And the molecular weight is 444.52.

The invention also provides a pharmaceutical composition, which comprises the QW24 small molecule compound shown as the formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The invention also provides application of the QW24 small-molecule compound shown in the formula (I) or a hydrate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in preparing a medicament for treating colorectal cancer diseases.

In the application of the invention, QW24 shown in the formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition has the following functions in vitro and in vivo: protein levels of BMI-1 were significantly down-regulated by the autophagy-lysosomal protein degradation pathway without affecting their mRNA levels. Has obvious proliferation inhibiting effect on colorectal cancer cell strain and little damage to normal cells. In colorectal cancer cell strains with stem cell-like properties, the self-renewal capacity of colorectal cancer tumor stem cells is remarkably reduced, and further, the proliferation and migration of tumor cells are inhibited. In a mouse subcutaneous tumor-bearing model, the growth of tumors is remarkably inhibited, in a colorectal cancer liver metastasis animal model, the metastasis of the tumors is remarkably inhibited, and no obvious toxicity is shown in the administration process.

In the application of the invention, QW24 shown in formula (I) or hydrate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition significantly reduces the expression of BMI-1 protein.

In the application, QW24 shown in the formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition is used for inhibiting the proliferation, growth, metastasis and migration of colorectal cancer in vitro and/or in vivo and promoting the apoptosis of the colorectal cancer.

Wherein the colorectal cancer comprises colorectal cancer cell strains HCT116, HT29, HCT8, HCT15, LS174T, LoVo and CT 26.

In the application of the invention, QW24 shown in formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition can significantly reduce the self-renewal capacity of colorectal cancer tumor stem cells and reduce the number of rectal cancer tumor stem cells.

In the application of the invention, QW24 shown in formula (I) or a hydrate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition inhibits the self-renewal capacity of colorectal cancer cell strain HCT116 with stem cell property, and reduces the number of HCT 116.

In the application of the invention, QW24 shown in formula (I) or a hydrate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition can inhibit the migration of colorectal cancer cells in vitro and influence the cell morphology.

In the application of the invention, QW24 shown in the formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition can inhibit liver metastasis of colorectal cancer cell CT26 in vitro and/or in vivo.

In the application of the invention, QW24 shown in formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition can down-regulate the protein level of BMI-1 without influencing the mRNA level of the BMI-1 in vitro and/or in vivo and reduce the stability of the BMI-1 protein.

In the application of the invention, QW24 shown in formula (I) or a hydrate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition degrades BMI-1 protein through an autophagy-lysosome pathway.

In the application of the invention, the hydrate, the pharmaceutically acceptable salt or the pharmaceutically acceptable carrier of the QW24 compound shown in the formula (I) and the like also have the same inhibiting effect as the QW24 shown in the formula (I).

In the application of the invention, the QW24 compound shown in the formula (I) can be used alone or in combination with other medicines.

The invention also provides a method for down regulating the expression of the BMI-1 in vitro and/or in vivo by the QW24 compound shown in the formula (I) or the hydrate or the pharmaceutically acceptable salt thereof or the pharmaceutical composition. QW24 as shown in formula (I) can down-regulate the expression of BMI-1 in a concentration-dependent manner.

The invention also provides a method for inhibiting the self-renewal of colorectal cancer tumor stem cells by the QW24 compound shown in the formula (I) or the hydrate or the pharmaceutically acceptable salt thereof or the pharmaceutical composition.

QW24 shown in formula (I) or a hydrate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition significantly inhibits the frequency of colorectal cancer stem-like cell HCT116 sphere formation, i.e., inhibits the ability of tumor initiating cells (tumor stem cells) to self-renew.

The invention provides a method for inhibiting colorectal cancer cell metastasis in vitro and/or in vivo by QW24 shown in formula (I) or a hydrate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition. QW24 shown in formula (I) or its hydrate or pharmaceutically acceptable salt, or pharmaceutical composition can inhibit in vitro migration of colorectal cancer cell lines HCT116, HCT8 and CT26 and liver metastasis of CT26 cells.

The invention provides a method for degrading a BMI-1 protein through an autophagy-lysosome pathway by QW24 shown in a formula (I) or a hydrate or pharmaceutically acceptable salt thereof or a pharmaceutical composition. When the QW24 shown in the formula (I) or the hydrate or the pharmaceutically acceptable salt thereof or the pharmaceutical composition is used for treating HCT116 cells, obvious autophagosome formation is realized, and the expression level of an LC3 II band is increased by Westernblotting detection.

The invention provides an application of a QW24 compound shown in formula (I) or a hydrate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition in preparing a medicament for preventing and/or treating malignant tumor diseases related to BMI-1 overexpression.

The malignant tumor is a BMI-1 over-expression malignant tumor, and comprises colon cancer, glioma, prostatic cancer, breast cancer, lung cancer, liver cancer, leukemia and the like.

The invention has the beneficial effects that: the invention obtains a small molecular compound QW24 shown in formula (I) through an SRB (Sulforhodamine B) colorimetric method and protein immunoblotting screening, wherein the compound can strongly inhibit the proliferation of colorectal cancer cells in vitro and in vivo, and can significantly reduce the protein level of BMI-1 through an autophagy-lysosome protein degradation pathway. In colorectal cancer cell strains with stem cell-like properties, the self-renewal capacity of colorectal cancer tumor stem cells is remarkably reduced, and further, the proliferation and migration of tumor cells are inhibited. In an animal model of colorectal cancer hepatic metastasis, tumor metastasis is remarkably inhibited, and no obvious toxicity is shown during administration. Colorectal cancer metastasis is an important cause of late death of patients, and the invention makes a new breakthrough in colorectal cancer treatment and provides a potential novel medicine for colorectal cancer treatment.

Drawings

FIG. 1 shows that QW24 of formula (I) inhibits proliferation of various colorectal cancer cell lines and down-regulates BMI-1 expression. FIG. 1(A) shows the inhibitory effect of QW24 of formula (I) at different concentrations (cell proliferation activity (%) in the ordinate) in colorectal cancer cell lines of HCT116, HT29, HCT8, HCT15, LS174T, LoVo and CT 26; FIG. 1(B) shows the IC50 of QW24 of formula (I) in different colorectal cancer cell lines; FIG. 1(C) shows the IC50 of QW24 of formula (I) in a normal cell line; fig. 1(D) shows that the QW24 shown in formula (I) has a significant difference in IC50 (half maximal inhibitory concentration (μ M) of QW24 on the ordinate) between colorectal and normal cells; FIG. 1(E) shows that QW24 shown in formula (I) has better inhibitory effect on HCT116 of colorectal cancer cells with dryness than positive compound PTC209, wherein PTC209 is a published BMI-1 inhibitor (cell proliferation activity (%) is ordinate); FIG. 1(F) shows that QW24 of formula (I) has a more pronounced down-regulation of BMI-1 than does positive compound PTC 209; FIG. 1(G) shows the effect of QW24 of formula (I) on the levels of BMI-1 and the protein Ub-H2A downstream of BMI-1 in different colorectal cancer cells.

FIG. 2 shows that QW24 of formula (I) inhibits the self-renewal ability of colorectal cancer tumor stem cells and reduces the number of colorectal cancer tumor stem cells. FIG. 2(A, B, C) shows that QW24 of formula (I) reduces the number and size of HCT116 cell sphere formation; FIG. 2(D) shows an in vitro limiting dilution assay; fig. 2(E) shows the ability of QW24 shown in formula (I) to inhibit self-renewal of colorectal cancer tumor stem cells.

FIG. 3 shows that QW24 of formula (I) inhibits migration of colorectal cancer cells in vitro and affects cell morphology. FIG. 3(A) shows that QW24 of formula (I) inhibits migration of HCT116, HCT8 and CT26 cells of colorectal cancer; FIG. 3 (B) shows that QW24 shown in formula (I) affects cell morphology.

FIG. 4 shows QW24 of formula (I) inhibiting tumor growth and down-regulating BMI-1 protein expression levels in vivo in a subcutaneous tumor-bearing model of colorectal cancer. Figure 4(a, B, C) shows the effect of QW24 and positive compound PTC209 shown in formula (I) in inhibiting HCT116 tumor growth in a colorectal carcinoma subcutaneous tumor-bearing model with QW24 effect more pronounced than PTC 209; FIG. 4(D) shows that QW24 and positive compound PTC209 of formula (I) have no significant effect on mouse body weight; FIG. 4(E) shows that QW24 and positive compound PTC209, represented by formula (I), down-regulate the protein expression level of BMI-1 in tumors; FIG. 4(F) shows the inhibitory effect of QW24 of formula (I) on tumor proliferation and intratumoral BMI-1 in tissue sections.

FIG. 5 shows QW24 of formula (I) inhibits liver metastasis from colorectal cancer and prolongs survival of mice in vivo. Fig. 5(a) shows that QW24 and positive compound PTC209 shown in formula (I) inhibited liver metastasis of colorectal cancer cell CT26 in a mouse model, and QW24 effect was more significant than PTC 209; FIG. 5(B) is a statistical plot showing that QW24 and positive compound PTC209 of formula (I) inhibit liver metastasis of colorectal cancer cell CT26 in a mouse model; FIG. 5(C) shows that QW24 and positive compound PTC209, represented by formula (I), have no significant effect on mouse body weight; FIG. 5(D) shows that QW24 of formula (I) is effective in extending survival of mice and is superior to PTC-209; FIG. 5(E) shows the inhibitory effect of QW24 and positive compound PTC209 of formula (I) on tumors metastasized to the liver and the corresponding statistical plot; FIG. 5(F) shows the effect of QW24 and positive compound PTC209 in inhibiting liver nodule formation shown in formula (I) and the corresponding statistical plot.

FIG. 6 shows that QW24 of formula (I) degrades BMI-1 protein mainly through the lysosomal-autophagy pathway. FIG. 6 (A, B) shows that QW24 of formula (I) has no significant effect on BMI-1mRNA levels; FIG. 6(C) shows that QW24 represented by formula (I) decreases the stability of BMI-1 protein; FIG. 6(D) shows that QW24 of formula (I) causes degradation of BMI-1 protein by inducing the autophagy-lysosomal pathway in cells; fig. 6(E, F, G, H) shows that QW24 shown in formula (I) significantly induced the onset of autophagy.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, but the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art, except for the contents specifically mentioned below, and the present invention is not particularly limited.