CN102977018A - Application of indenoindene isoquinoline estrogen receptor beta adjustment agent in resisting breast cancer - Google Patents

Abstract

The present invention relates to an estrogen receptor modulator of formula (I) or a pharmaceutically acceptable salt thereof. Such compounds can combine with estrogen receptor β, effectively produce anti-breast cancer and other effects, and simultaneously produce regulatory effects on ovary and other parts. This type of compound has a strong effect of binding estrogen receptor β in vitro, and can effectively antagonize the combination of estrogen and estrogen receptor β, thereby treating breast cancer caused by excess estrogen, and simultaneously producing Regulatory effect.

Description

Anti-breast cancer application of a class of indenoisoquinoline estrogen receptor beta modulators

technical field

The invention relates to a class of indenoisoquinoline estrogen receptor beta modulators and their anti-breast cancer effects, which simultaneously produce regulatory effects on ovary and other parts.

Background technique

In recent years, breast cancer has become the first common malignant tumor in women, and the research on anti-breast cancer drugs has important academic and practical significance.

Studies have found that the occurrence and development of breast cancer is related to estrogen receptor (ER). ER is a nuclear receptor, including two subtypes of ERα and ERβ. The amino acid sequences of ERα and ERβ share 96% and 53% homology in the DNA binding domain (DBD) and ligand binding domain (LBD), respectively. There is a large amount of ERα distribution in the classic target tissues of estradiol, such as uterus and breast, while the non-classic target tissues of estradiol, such as prostate, testis, and ovary, have low expression or no expression of ERα, while ERβ is high. Express. In addition, the transcriptional activities of ERα and ERβ and their interactions with cofactors also tend to differ.

Like ERα, ERβ can also bind to estrogen and regulate gene expression by binding to the estrogen response element (ERE) on the target gene or interact with other nuclear proteins to change the transcriptional activity of the gene. But when signaling through the AP1 site, ERβ-mediated estrogenic effects are completely opposite to those of ERα.

Much evidence suggests that ERβ is an attractive target for anti-breast cancer therapy. ERβ is a new subtype of ER, which is distributed in both normal breast and breast cancer tissues, with more in the former. It and the biological characteristics of ERα show a similar but relative, synergistic and antagonistic relationship under different conditions, and they jointly participate in and regulate the occurrence and development of breast cancer, and affect the sensitivity and prognosis of hormone therapy. Existence makes the biological characteristics of breast cancer complex and variable. Tamoxifen (Tamoxifen) is the first cancer targeted therapy drug for the treatment of breast cancer. In 1977, as a selective estrogen receptor modulator, it was approved by the FDA for the treatment of postmenopausal advanced breast cancer. However, it has a stimulating effect on uterine cells, and long-term use can easily lead to endometrial hyperplasia or endometrial cancer. In addition, it can also cause side effects such as vasodilation and venous thrombosis. In 1997, Eli Lilly's raloxifene was approved by the FDA for the treatment of osteoporosis. In 2007, the FDA approved its new indication for reducing the risk of breast cancer in postmenopausal women with osteoporosis. But raloxifene has been warned to increase the risk of stroke-related death. Raloxifene is a second-generation selective estrogen receptor modulator that prevents breast cancer without increasing the risk of endometrial cancer. Toremifene is a structural analogue of tamoxifen, which has been approved in the United States and other countries for the treatment of invasive breast cancer. Compared with tamoxifen, it has less potential genotoxic effects, causing secondary uterine The risk of endometrial cancer is less than with tamoxifen. At present, there are few studies on ERβ regulators. Trilostane (Modrenal) is a subtype-selective estrogen receptor regulator. In breast cancer cell MCF-7 cells, Trilostane can increase the binding capacity of estradiol and ERβ. The maximum amount, but does not increase the maximum amount of estradiol binding to ERα, and in MCF-7 and mouse uterine cells, it can increase the expression of ERβ. In the UK, Trilostane has been approved for the treatment of early postmenopausal breast cancer relapsed after endocrine therapy, and is currently being studied for prostate cancer and premenopausal breast cancer. TAS-108 (SR16234) is another subtype-selective estrogen receptor modulator associated with breast cancer, which is an antagonist of ERα and an agonist of ERβ. In vitro reporter assays show that TAS-108 is a full antagonist of ERα in the presence or absence of estradiol; it exhibits partial agonist activity against ERβ. TAS-108 inhibits the growth of tamoxifen-resistant breast cancer cells, DMBA-induced human breast tumors in mice, and estrogen-stimulated MCF-7 xenografts with minimal uterotropic activity. Phase I clinical trials showed that TAS-108 has anti-tumor activity, is well tolerated, and has no effect on endometrial thickness. A phase II trial reached similar conclusions, showing that TAS-108 could treat early-stage breast cancer in postmenopausal women. Phase III clinical trials are under planning. TAS-108 does not increase bone loss, which may be related to it being a partial agonist of ERβ. TAS-108 is a highly anticipated anti-breast cancer drug. The advantages of ERβ regulators are growing day by day, and the study of ERβ regulators has increasingly become an important basis for the study of selective estrogen receptor modulators and the expansion of anti-breast cancer drugs.

Contents of the invention

The purpose of the present invention is to develop the application of a class of estrogen receptor beta regulators in the treatment of breast cancer. The compounds screened in the present invention are a batch of directedly synthesized compounds that can bind to the ERβ target. The high-throughput screening model established by the fluorescence polarization method is used to screen the batch of compounds in vitro, and to search for lead compounds and candidate drugs through functional verification. Preliminary pharmacodynamic and mechanism studies were carried out on the active compounds obtained from the screening, and a class of indenoisoquinoline-like ERα modulators was found to provide candidate drugs for anti-breast cancer treatment, and at the same time produce regulatory effects on the ovary and other parts. This type of compound was synthesized by the Medicinal Chemistry Laboratory of China Pharmaceutical University. Among them, the synthesis method of the test compounds 001672 and 001626 can be obtained from the patent Xiang Hua, Wang Tianlin, Xiao Hong, You Qidong, Yao Yao, Li Xiaobo, Liao Qingjiang. Indenoisoquine Indenoisoquinolinone derivatives, their preparation method and their medical use. ZL 20091 0233991.7 and Hua Xiang, Tianlin Wang, Hong Xiao, Qidong You, Yao Yao, Xiaobo Li, QingjiangLiao. Indenoisoquinolinone derivatives, manufacturing method and medical use thereof. WO2011047515A1. get.

The technical scheme of the invention is: establishing a screening model for ERβ receptor targeting regulators, performing primary screening, secondary screening, and structure-activity relationship analysis to obtain a class of candidate drugs with anti-breast cancer effects. Specific steps are as follows:

Step 1: Establish a high-throughput screening model for ERβ modulators.

Step 2: Validate the model with positive drugs.

Step 3: Use the ERβ modulator high-throughput screening model to perform primary screening and rescreening of the test compound, draw the binding curve of the test compound to antagonize estradiol and ERβ, and determine the IC ₅₀ value.

The present invention provides the medical use of the above-mentioned compound or its pharmaceutically acceptable salt and its pharmaceutical composition, especially in the prevention, delay or treatment of diseases mediated by ERβ, especially the use in anti-breast cancer drugs, and at the same time in The ovaries and other parts produce regulatory effects. The effect of the above-mentioned directedly synthesized compounds in the treatment of breast cancer belongs to the protection scope of the present invention.

Description of drawings:

Figure 1: The inhibition curve of positive drug 4-OH-Tamoxifen antagonizing the binding of estradiol to ERβ.

Figure 2: Inhibition curve of test compound 001672 antagonizing the binding of estradiol to ERβ.

Figure 3: The inhibition curve of the test compound 001626 antagonizing the binding of estradiol to ERβ.

Figure 4: Inhibition curve of test compound 000356 antagonizing the binding of estradiol to ERβ.

Detailed ways

The specific embodiment of the present invention is described below in conjunction with accompanying drawing:

1. Test of the activity of the compound to be tested to antagonize the binding of estradiol to ERβ

1) Experimental materials

Estrogen Receptor-alpha (ERβ) Human Recombinant (Invitrogen, USA), FluormoneTMES2 (Invitrogen, USA), ES2Screening Buffer (Invitrogen, USA), Estradiol is the standard product stored in our laboratory, domestic analytically pure, Tamoxifen is the medicinal chemistry of China Pharmaceutical University Standards, 384-well black microplates (Corning, USA), pipette tips (Axygen, USA) provided by the laboratory.

2) Experimental steps

●Precisely weigh each compound to be tested, add DMSO solvent to form a mother solution, and then use ES2Screening Buffer to prepare a solution of the compound to be tested to the required concentration, and the initial screening concentration is about 1×10 ^-3 mol/L.

●Preparation of 2X Fluormone ^TM ES2/ERβ Complex by solution: use ES2 Screening Buffer to prepare 2X Fluormone TM ES2 and ERβ mixed solution so that the final concentration of Fluormone ^TM ES2 is 9nM, and the concentration of ERβ is 30nM.

● Competitive binding experiment: first add 50 μl of compound to each well of a 384-well black microwell plate, and then add 50 μl of 2X Fluormone ^TM ES2 and ERβ mixed solution to each well. At the same time, add 50 μl estradiol solution (1nM), 50 μl 2X Fluormone ^TM ES2 and ERβ mixed solution as 100% competitive binding control, add 50 μl Buffer, 50 μl 2X Fluormone ^TM ES2 and ERβ mixed solution as 0% competitive binding control and add 100 μl Buffer as a blank control. Avoid light operation. Incubate at room temperature (20-25°C) for 90min.

●Use a microplate reader to read the polarization value of each well.

2. Data processing

1) According to the formula, calculate the inhibition rate of positive drug and test compound to antagonize the combination of estradiol and ERβ

2) Draw the positive drug 4-OH-Tamoxifen to antagonize the binding curve between estradiol and ERβ and determine the IC ₅₀ value, see Figure 1.

3) Use the millibias value and the logarithmic concentration value to plot, draw the binding curve of the compound to be tested to antagonize the binding of estradiol to ERβ and determine the IC ₅₀ value, see Figure 2-4.

Re-screening test results

The IC ₅₀ of the positive drug 4-OH-Tamoxifen measured by the in vitro estrogen receptor β modulator screening model is 0.024nM. The compounds obtained from the screening can be summarized into one category according to the structure of the mother nucleus. The structural formula is consistent with the in vitro estrogen receptor β activity Screen _IC50 as follows:

I: 6-aryl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione

Claims (3)

1. A compound of the following formula or a pharmaceutically acceptable salt thereof:

in: R ₁ and R ₂ independently represent H, -OH, C ₁ -C ₄ alkoxy or C ₁ -C ₆ alkoxycarbonyl; R ₃ and R ₄ independently represent CO(CH ₂ ) ₂ CH ₃ , CO(CH ₂ ) ₃ CH ₃ or C ₁ -C ₆ alkyl; or R ₃ and R ₄ and the nitrogen atom connected to it are combined to form piperidine Pyridyl, 2-methylpiperidinyl, homopiperidinyl, morpholinyl, pyrrolidinyl, 3-methylpyrrolidinyl, 3,3-dimethylpyrrolidinyl, 3,4-dimethyl Pyrrolidinyl, piperazinyl, N-methylpiperazinyl, N-ethylpiperazinyl, N-phenylpiperazinyl or N-benzylpiperazinyl; X represents O, S, NH, _CH2 or -CO-; m = 0 or 1; n=2 or 3. 2. Use of the compound of formula (I) claimed in claim 1 or a pharmaceutically acceptable salt thereof for the preparation of an estrogen receptor beta modulator, and at the same time produce a regulatory effect on the ovary and other parts. 3. The use according to claim 2, as an estrogen receptor beta modulator for the treatment of breast cancer, and simultaneously produces a regulatory effect on the ovary and other positions.

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