阅读说明：本技术 一种化合物在癌症治疗药物中的应用 (Application of compound in cancer treatment medicine ) 是由 周维英 李小丽 周端方 宋燚 张欢 龙良源 于 2020-08-31 设计创作，主要内容包括：本发明涉及生物医药领域,具体涉及一种化合物在癌症治疗药物中的应用。本方案的化合物Zinc-09具有较好的抗癌活性以及SGK3(血清和糖皮质激素诱导的蛋白激酶3)靶向性,可靶向性抑制SGK3的下游通路,对SGK3介导的癌细胞的发生发展产生抑制作用。本方案的Zinc-09可以解决由于乳腺癌的高异质性造成的现有药物不能满足靶向精准治疗的需求的问题。Zinc-09及其衍生物可被开发为靶向SGK3的抑制剂从而发挥其抗癌作用。(The invention relates to the field of biological medicines, in particular to application of a compound in a cancer treatment medicine. The compound Zinc-09 has good anticancer activity and SGK3 (protein kinase 3 induced by serum and glucocorticoid) targeting property, can inhibit the downstream pathway of SGK3 in a targeted manner, and has an inhibiting effect on the generation and development of SGK 3-mediated cancer cells. The Zinc-09 of the scheme can solve the problem that the existing medicine cannot meet the requirement of targeted precise treatment due to high heterogeneity of the breast cancer. Zinc-09 and derivatives thereof can be developed into SGK3 targeted inhibitors to exert anticancer effects.)

1. The use of a compound for the manufacture of a medicament for the treatment of cancer, wherein: the structural formula of the compound is shown as a formula (I);

2. use of a compound according to claim 1 for the manufacture of a medicament for the treatment of cancer, wherein: the cancer is estrogen receptor positive breast cancer.

3. Use of a compound according to claim 2 for the manufacture of a medicament for the treatment of cancer, wherein: the breast cancer is a breast cancer with high SGK3 expression.

4. Use of a compound according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment of cancer, wherein: the drug is an inhibitor of protein expression of SGK3 or p-SGK 3.

5. Use of a compound according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment of cancer, wherein: the drug is an inhibitor of SGK3 protein phosphorylation.

6. Use of a compound according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment of cancer, wherein: the drug is a G1 phase blocker of breast cancer cells.

7. Use of a compound according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment of cancer, wherein: the medicine is an autophagy promoter for breast cancer cells.

8. Use of a compound according to claim 1 for the manufacture of a medicament for the treatment of cancer, wherein: IC of the compound for inhibiting MCF-7 cell activity in vitro 72h after MCF-7 cells are treated by the compound₅₀The value was 263.0. mu.M.

9. Use of a compound according to claim 1 for the manufacture of a medicament for the treatment of cancer, wherein: IC of said compound for inhibiting activity of ZR-75-1 cell in vitro 72h after treating ZR-75-1 cell with said compound₅₀The value was 114.7. mu.M.

10. Use of a compound according to claim 1 for the manufacture of a medicament for the treatment of cancer, wherein: IC of the compound in vitro inhibiting activity of T47D cells 72h after treatment of T47D cells with the compound₅₀The value was 131.6. mu.M.

Technical Field

The invention relates to the field of biological medicines, in particular to application of a compound in a cancer treatment medicine.

Background

Breast cancer has become one of the most common tumors that are seriously threatening the health or even life of women, and its resulting mortality has been the first in female tumor death. The occurrence and development process of breast cancer and the mechanism thereof are extremely complex. With the continuous and deep understanding of the biological behavior of breast cancer and the transformation and updating of the treatment concept, the treatment of breast cancer enters the comprehensive treatment era of surgery, chemotherapy and radiotherapy. Among them, drug chemotherapy is still one of the main means for clinical treatment of breast cancer.

Targeted therapy is an emerging, more efficient therapeutic approach than global chemotherapy. For example, Gefitinib (Gefitinib), Erlotinib (Erlotinib) and Trastuzumab (Trastuzumab) based on receptor tyrosine kinase family members such as Epidermal Growth Factor Receptor (EGFR) and human epidermal growth factor receptor 2(HER2) targeted inhibitors, and the like, as well as dual inhibitors of EGFR and HER2 such as lapatinib. The molecular targeting drugs mainly target specific cells, genes or receptors, have the advantages of strong specificity, high efficiency, small side effect, improvement of the overall survival time of patients and the like, are widely used in clinic, and obtain exciting results. However, due to the high heterogeneity of breast cancer, the requirement of clinical precise treatment cannot be met, and the existing drug resistance problem provides a new challenge for the targeted treatment of breast cancer. Therefore, the development of new targeting anti-breast cancer drugs aiming at different molecular phenotypes is urgently needed to meet the requirement of clinical accurate treatment of breast cancer.

Disclosure of Invention

The invention aims to provide application of a compound in a cancer treatment drug so as to solve the problem that the existing drug cannot meet the requirement of targeted precise treatment due to high heterogeneity of breast cancer.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of a compound in preparing a cancer treatment drug is characterized in that the structural formula of the compound is shown as a formula (I);

the principle and the advantages of the scheme are as follows: the compound of the scheme is abbreviated as Zinc-09, and the systematic name is 6- [2- (3,4-dimethoxyphenyl) vinyl]-5-nitro-2,4(1H,3H) -pyrimidinidione (6- [2- (3,4-dimethoxyphenyl) ethenyl)]-5-nitro-2,4(1H,3H) -pyrimidinedione) of formula C₁₄H₁₃N₃O₆. By retarding the cell cycle to the G1 phase so as to inhibit cell proliferation and promoting autophagy so as to inhibit the occurrence and development of tumors, Zinc-09 has the function and value of inhibiting the occurrence and development of tumors. And the Zinc-09 has the function of targeted inhibition of serum and glucocorticoid-induced protein kinase 3(SGK3), and can inhibit SGK 3-mediated cancer cell generation and development related pathways. Among them, SGK3 is a signal molecule involved in signal transduction of various cells, and has been found to play an important role in the intracellular phosphorylation cascade, and its expression can promote proliferation, invasion and metastasis of tumor cells, and plays an important role in the processes of cell growth, differentiation, survival, substance transport, etc. SGK3 is regulated by estrogen, promoting estrogen-mediated survival of tumor cells. Therefore, the SGK3 inhibitor (Zinc-09) has the potential effect of inhibiting the breast cancer of women. In the prior art, no research on the anti-breast cancer effect of a small molecule inhibitor of SGK3 exists.

When the inventor carries out research and development of anti-cancer drugs, in order to select candidate compounds which can have SGK3 targeting, the inventor carries out computer-assisted screening on 200 ten thousand compounds in a library to obtain more than ten thousand candidate compounds. In the computer-aided screening stage, because no existing SGK3 single crystal structure exists, the inventor performs homology modeling to obtain a model of the SGK3 single crystal structure. Then, 200 ten thousand compounds in the library are subjected to matching screening by using a model of the SGK3 single crystal structure, and ten thousand candidate compounds are obtained. The inventors then performed a high throughput screening of these candidate compounds. Through comprehensive research on the cancer cell inhibition activity, action targets and the like of the candidate compounds, the compound Zinc-09 is finally found to have better activity of inhibiting cancer cell proliferation. And other candidate compounds are eliminated due to the problems of poor anticancer activity or poor targeting.

The efficacy proves that the Zinc-09 has better anticancer activity and SGK3 targeting property. In estrogen receptor positive breast cancer, an overexpressed estrogen receptor promotes downstream SGK3 overexpression, and further influences biological behaviors such as proliferation and migration of cancer cells, apoptosis of cells and the like. The Zinc-09 can inhibit the expression of SGK3 and p-SGK3 proteins, further inhibit a downstream pathway of SGK3 and generate an inhibiting effect on the generation and development of cancer cells. In addition, Western blot results show that Zinc-09 can play an anti-cancer role by down-regulating the expression of G1 stage-related proteins CyclinD1 and P-CyclinD1 and up-regulating the expression of P21 so as to block the cell cycle process in the G1 stage. The Zinc-09 acts on cancer cells at different times, the expression of autophagy marker proteins LC3 II/LC 3I is obviously increased along with the prolonging of the acting time, and the expression of autophagy inhibiting genes mTOR and p-mTOR proteins is obviously reduced. It is shown that Zinc-09 exerts the purpose of anti-breast cancer by inducing autophagy and cycle arrest.

In conclusion, the compound (Zinc-09) has the beneficial effects that: the Zinc-09 can be used for inhibiting the expression of SGK3 and p-SGK3 proteins in a targeted manner, so that the proliferation and development of cancer cells are inhibited. The Zinc-09 can meet the requirement of targeted and accurate treatment in clinical treatment and has specific treatment effect on SGK 3-related pathway-mediated cancer. Zinc-09 can inhibit cancer cells by promoting multiple pathways such as G1 phase arrest and autophagy. And the toxicological experiment result shows that Zinc-09 has low toxicity to normal cells and high safety.

Further, the cancer is an estrogen receptor positive type breast cancer.

In estrogen receptor positive breast cancer (ER + breast cancer), a large amount of Estrogen Receptor (ER) is expressed, and the ER starts the overexpression of downstream serum and glucocorticoid-induced protein kinase 3(SGK3), so that the biological behaviors of proliferation, migration, apoptosis and the like of breast cancer cells are influenced. In estrogen receptor positive breast cancers, breast cancer cells receive growth signals from estrogen, which is the most common type of breast cancer. The compound (Zinc-09) of the scheme can inhibit the expression of SGK3 and p-SGK3 proteins, inhibit the generation and development of ER-mediated tumor cells in estrogen receptor positive breast cancer, and further play a role in treating the breast cancer.

Further, the breast cancer is a breast cancer with high expression of SGK 3.

The expression of SGK3 protein is regulated by estrogen, and promotes the survival of tumor cells mediated by estrogen. In some breast cancers, there is a case where the SGK3 protein is highly expressed, i.e., the SGK3 protein is expressed in an amount exceeding the normal level. The compound (Zinc-09) has an inhibition effect on the expression of SGK3 protein, can also inhibit the phosphorylation of SGK3 protein, has a potential effect on inhibiting female breast cancer, and can be used for treating the breast cancer with high SGK3 expression. Zinc-09 is a small molecule inhibitor against SGK 3.

Further, the drug is an inhibitor of protein expression of SGK3 or p-SGK 3.

Serum and glucocorticoid-induced protein kinase 3(SGK3) is a newly discovered downstream signaling molecule of PI3K in recent years, which is widely expressed in mammalian tissues and cells, and participates in regulating cell proliferation, survival, migration, substance transmembrane transport, and the like by phosphorylating downstream target proteins. SGK3 is a Ser/Thr protein kinase that is a member of the SGK family. The study of this protein kinase and its function has been poorly understood in the past. SGK3 is another key molecule in PI3K/Akt/mTOR signaling pathway, has high homology with another important kinase family protein kinase B (PKB/AKT) in the signaling pathway, can recognize the same substrate sequence and phosphorylate some same substrates, such as: GSK3 beta (glycogenolytic kinase beta), BAD (Bcl-2associated treated promoter), FOXO3a (forkhead transcription factor 3a), PRAS40(Proline-rich AKT substrate of 40kDa) and TSC2(Tuberous research factor 2), etc., are closely related to the occurrence of various human diseases including tumors.

SGK3 is related to expression of breast cancer Estrogen (ER) receptors, has the function of mediating INPP4B dependent PI3K signaling in breast cancer, the expression of SGK3 in invasive breast cancer is higher than that of normal breast tissue, and the over-expression of SGK3 can influence malignant biological behaviors such as breast cancer cell proliferation, apoptosis, invasion and migration. Therefore, the SGK3 inhibitor has the potential effect of inhibiting female breast cancer, but no research on the anti-breast cancer effect of the SGK3 small-molecule inhibitor exists at present.

The compound (Zinc-09) can inhibit the expression of SGK3 protein, and can be used as an SGK3 small molecule inhibitor to be applied to the treatment of breast cancer (see example 3 for details).

Further, the drug is an inhibitor of protein phosphorylation of SGK 3.

The compound (Zinc-09) of the scheme not only has an inhibition effect on the expression of SGK3 protein, but also inhibits the phosphorylation of SGK3 protein, namely inhibits the activation of SGK3 protein (see example 3 for details).

Further, the drug is a blocker of the G1 phase of breast cancer cells.

Cell cycle regulation is closely related to the occurrence and development of breast cancer, and a complete cell cycle comprises three detection points: G1/S checkpoint, G2/M checkpoint and spindle assembly checkpoint, which can complete the cell cycle without being stimulated by an external signal once the cell passes through the G1/S checkpoint, therefore the G1/S phase checkpoint is considered to be the most important cell cycle checkpoint and disruption of cell cycle progression also often occurs at the G1 phase. Cyclin D1 and CDK4 are key proteins for regulating G1 phase, cyclin D1 is combined with CDK4 protein in the middle and later stages of G1, the activity of CDK4 protein is activated and maintained, the protein has a promoting effect on cell cycle, P21 is a cell cycle inhibitor, and the protein participates in cell cycle regulation and control by mainly inhibiting the activity of CDKs complex, and blocks cells from being transited from the G1 phase to the S phase. Western blot results show that Zinc-09 can play an anti-breast cancer role by down-regulating the expression of G1 stage-associated proteins CyclinD1 and P-CyclinD1 and up-regulating the expression of P21, thereby blocking the cell cycle progression in the G1 stage (see example 4 for details).

Further, the medicament is an autophagy promoter for breast cancer cells.

Autophagy (also called type ii programmed cell death) is a process in which cells degrade damaged organelles and macromolecular substances by lysosomes under the control of autophagy-related genes. A great deal of research in recent years shows that autophagy is closely related to tumors, and can regulate the formation, proliferation, metastasis, energy metabolism and other aspects of tumors. The autophagy plays a dual role in the occurrence and development of the breast cancer, on one hand, the stimulation of external conditions such as hunger, hypoxia, high temperature and the like or the damage of organelles, the accumulation of cytoplasmic components and other internal environment changes can induce the autophagy of cells, and the autophagy can effectively isolate and eliminate the damaged organelles and misfolded proteins and has positive significance for the growth, development and maintenance of homeostasis of the cells; on the other hand, when autophagy is too strong or the duration is too long, pathological behaviors such as cell metabolic disorder and excessive apoptosis can be caused if the autophagy exceeds the regulation and control capability of cells. Therefore, the goal of treating breast cancer can be achieved by promoting autophagy which induces cell death. The microtubule-associated protein Light Chain (LC)3, the mammalian target of rapamycin (mTOR) and the like play an important role in the process, and our results show that the Zinc-09 acts on the ZR-75-1 cells of the breast cancer at different times, the autophagy marker protein LC3 II/LC 3I is obviously increased along with the prolonging of the acting time, and the expression of autophagy inhibiting genes mTOR and P-mTOR protein is obviously reduced. The SGK3 inhibitor candidate drug Zinc-09 is shown to play the aim of resisting breast cancer by inducing autophagy. (see example 4 for details).

Further, the compound inhibits IC of MCF-7 cell activity in vitro 72h after MCF-7 cells are treated by the compound₅₀The value was 263.0. mu.M.

IC of the compound for inhibiting MCF-7 cell activity in vitro 72h after MCF-7 cells are treated by the compound₅₀The value was 263.0. mu.M. The Zinc-09 has a strong in-vitro inhibition effect on MCF-7 cells (ER + breast cancer cells), and a certain anticancer activity is suggested.

Further, the compounds inhibit IC of ZR-75-1 cell activity in vitro 72h after treating ZR-75-1 cells with the compounds₅₀The value was 114.7. mu.M.

IC of said compound for inhibiting activity of ZR-75-1 cell in vitro 72h after treating ZR-75-1 cell with said compound₅₀The value was 114.7. mu.M. Zinc-09 has strong in-vitro inhibition effect on ZR-75-1 cells (ER + breast cancer cells), and the fact that the Zinc-09 has anti-cancer activity is suggested.

Further, the compounds inhibit IC of T47D cell activity in vitro 72h after treatment of T47D cells with the compounds₅₀The value was 131.6. mu.M.

IC of said compounds for inhibiting ZR-75-1 cell activity in vitro 72h after T47D cells were treated with said compounds₅₀The value was 131.6. mu.M. The Zinc-09 has strong in-vitro inhibition effect on T47D cells (ER + breast cancer cells), and the in-vitro inhibition effect is suggested to have anticancer activity.

In addition, the inhibition rate of the compound on MCF-10A cell (normal mammary epithelial cell) activity was comparable to that of carboplatin 48h after treating MCF-10A cells with 200. mu.M of the compound Zinc-09 described in this protocol. The Zinc-09 is suggested to have low toxicity to normal cells and high safety.

Drawings

FIG. 1 shows the inhibition rate of Zinc-07 on MCF-7 cells in example 1 of the present invention.

FIG. 2 shows the inhibition rate of Zinc-08 of example 1 of the present invention on MCF-7 cells.

FIG. 3 shows the inhibition rate of Zinc-09 of example 1 of the present invention on MCF-7 cells.

FIG. 4 shows the inhibition rate of Zinc-13 on MCF-7 cells according to example 1 of the present invention.

FIG. 5 shows the inhibition rate of Zinc-09 on MCF-7 cells at different concentrations and different treatment times of example 1 of the present invention.

FIG. 6 shows the inhibition rate of Zinc-09 on ZR-75-1 cells at different concentrations and different treatment times of example 1 of the present invention.

FIG. 7 shows the inhibition rate of Zinc-09 on T47D cells at different concentrations and different treatment times of example 1 of the present invention.

FIG. 8 shows the results of in vitro cytotoxicity evaluation of Zinc-09 of example 2 of the present invention against normal mammary epithelial cell MCF-10A.

FIG. 9 shows the expression of SGK3 protein in 6 breast cancer cell lines of example 3 of the present invention.

FIG. 10 shows the protein expression of Zinc-09 of example 3 of the present invention after acting on ER + breast cancer cells ZR-75-1.

FIG. 11 shows the expression of SGK3 protein in T47D-tet-on-SGK3 cell line under induction of DOX in example 3 of the present invention.

FIG. 12 shows the inhibition of the expression level of SGK3 protein by Zinc-09 of example 3 of the present invention.

FIG. 13 shows the expression of the G1 phase-related protein by Zinc-09 of example 4 of the present invention.

FIG. 14 shows the expression of autophagy-related proteins by Zinc-09 of example 4 of the present invention.

Detailed Description

In examples 1-4, compounds Zinc-01, Zinc-02, Zinc-03, Zinc-04, Zinc-05, Zinc-06, Zinc-07, Zinc-08, Zinc-09, Zinc-10, Zinc-11, Zinc-12, Zinc-13, Zinc-14 are available from ChemBridge; the breast cancer cell strains MCF-7, ZR-75-1 and T47D, and normal breast epithelial cells MCF-10A are purchased from ATCC; DMEM and 1640 medium were purchased from Hyclone; thiazole blue (MTT) was purchased from bio-engineering (shanghai) gmbh; antibodies β -actin, Cyclin D1, P-Cyclin D1, P21, LC3, m-TOR, P-mTOR were purchased from Cell Signaling technology.