CN105753753A - Compound for adjusting activity of estrogen-related receptor and medical application of compound - Google Patents

Abstract

The invention discloses a compound with formula I and its pharmaceutically acceptable salt and its application. The compound and its pharmaceutically acceptable salt and precursor molecule can be used to prepare estrogen-related receptor (Estrogen-related receptor, ERR) activity, drugs for the prevention and treatment of breast cancer, prostate cancer, gastric cancer, colon cancer, ovarian cancer, cervical cancer and diseases caused by metabolic abnormalities.

Description

A class of compounds that regulate the activity of estrogen-related receptors and their medical use

technical field

The present invention relates to a class of compounds used to regulate the activity of estrogen-related receptors and their medical use.

Background technique

Orphan nuclear receptors (ONRs), a member of the transcription factor superfamily, are a class of nuclear receptors that can produce biological functions without binding to ligands, and can directly participate in the regulation of various functions. Gene. ERR (estogen-related receptors, NR3B) is an important ONR superfamily, and ERRs belong to the third nuclear receptor superfamily, mainly including three subtypes, namely ERRα (NR3B1), ERRβ (NR3B2) and ERRγ (NR3B3 ). The reason why ERRs are called "estrogen-related receptors" is that ERRs not only participate in the complex signal transduction system of estrogen, but also are closely related to diseases caused by estrogen. At the same time, ERRs share high sequence homology with estrogen receptor α (ERα), which can be activated by non-hormonal signals.

ERR mainly includes three functional domains: N-terminal domain (N-terminal domain, NTD), DNA-binding domain (DBD) and ligand-binding domain (ligand-binding domain, LBD), LBD also includes activation function 2 (AF2) motif. NTD has a low degree of conservation and contains activation function region 1 (activation function, AF1), which is mainly involved in post-transcriptional covalent modification, such as phosphorylation and ubiquitination site modification. The ERR receptor DBD region contains two zinc finger structures, which mainly recognize and bind special sequences in the regulatory region of target gene DNA. The LBD at the C-terminus of ERR is highly conserved and contains a ligand-binding pocket site for receptor dimerization, which can co-activate with coactivators such as peroxisome proliferator-activated receptor γ (PPARγ) Sub-1α (PPARγcoactivator1α, PGC-1α), PGC-1β interacts with co-repressor factors to regulate the transcriptional activity of ERR (relevant literature: Bianco S, et.al, J Steroid Biochem Mol Biol.2012, 130(3- 5):180-185; Giguère V, et.al, Cold Spring Harb Symp Quant Biol.2011,76:57-61; Deblois G, et.al, Nat Rev Cancer.2013,13(1):27-36 ).

ERRα is widely expressed in tissues, such as heart, intestinal tract, brain, spinal cord, brown fat, bone, kidney and endometrial cancer cells. ERRα is mainly localized in the nucleus among the many cells it expresses, based on specificity Environmental, nutritional or metabolic signals are involved in many physiological processes, especially the physiological processes regulated by ERα, and affect the signaling pathway of ERα. ERRβ is mainly related to biological development, mainly expressed in liver, stomach, skeletal muscle, heart, kidney and other parts. ERRγ is mainly expressed in the spinal cord, central nervous system, liver and breast tissue (related literature: Stein RA, et.al, Endocr Relat Cancer.2006, 13Suppl 1:S25-32; Ariazi EA, et.al, Curr Top Med Chem. 2006,6(3):203-215; Ranhotra HS, J Recept Signal Transduct Res.2010,30(4):193-205; Ariazi EA, et.al, Cancer Res.2002,62(22):6510- 6518).

Studies have found that the precise regulation of certain specific genes by ERRα plays an important role in the energy metabolism of the body. The metabolic processes that ERRα participates in the body mainly include: glucose metabolism, lipid metabolism, mitochondrial oxidative metabolism, and adaptive energy metabolism. These functions of ERRα affect the development of tissues and organs, cell aging, and the occurrence and development of tumors. ERRα is considered to be a potential therapeutic target for metabolic diseases such as diabetes and obesity. Compound 29, an inverse agonist of ERRα, down-regulates blood glucose and triglyceride levels in various diabetic and obese rat models, reduces body weight, and improves insulin resistance (relevant literature: Patch RJ, et.al, J Med Chem, 2011Feb 10; 54(3):788-808).

In recent years, studies have revealed that ERRα is closely related to estrogen-dependent tumors such as breast cancer, endometrial cancer, and cervical cancer, and more studies have shown that many non-estrogen-dependent tumors such as prostate cancer, gastric adenocarcinoma, and colorectal cancer The occurrence and development of cancer and clinical prognosis are also related to the expression of ERRα. Clinical studies have confirmed that ERRα is significantly up-regulated in tumors such as breast cancer, colon cancer, ovarian cancer, and prostate cancer, and is an independent risk factor for poor prognosis. In the malignant and invasive breast cancer cell line MDA-MB-231, down-regulation of ERRα inhibited the migration ability of MDA-MB-231, while re-expression of ERRα restored the migration activity of MDA-MB-231, indicating that ERRα plays an important role in breast cancer cells. play an important role in migration. Moreover, ERRα can combine with HIF-1α in the form of protein-protein interaction, and co-regulate the transcription of HIF-1α target genes in tumor cells under hypoxic/nomoxic conditions. Ao et al. found that ERRα can interact with HIF-1α and promote the transcription induced by HIF-1α in the study of the correlation between ERRα’s participation in hypoxic transcriptional response and the growth of solid tumors, which is extremely important for the function of HIF-1α. By upregulating/downregulating ERRα expression or using ERRα inhibitors, the transcriptional activity of hypoxic genes can be effectively inhibited, thereby reducing angiogenesis and growth of solid tumors in vivo. These studies have shown that ERRα may be a potential therapeutic target for various tumors (related literature: Suzuki T., et.al, Cancer research.2004, 64(13): 4670-4676; Stein R.A., et.al, Cancer Res .2008,68(21):8805-8812; Boudjadi S.et.al, Am JPathol.2013,183(1):266-276; Ao A,et.al,Proc Natl Acad Sci U S A.2008,105 (22):7821-7826). Moreover, the ERRα inverse agonist XCT790 inhibited the proliferation and angiogenesis of various tumor cells. The ERRα inverse agonist SR16388 effectively inhibited the growth of prostate cancer in a tumor-bearing nude mouse model. These studies show that modulators targeting ERRα may be used as clinically effective anti-tumor drugs (related literature: Zhang L., et.al, Eur J Pharmacol.2015,769:167-176; WuF., et.al , Chem Biol Interact.2009,181(2):236-242; Wang J., et.al, Cell Prolif.2010,43(2):103-113; Duellman S., et.al, Biochem Pharmacol.2010 , 80(6):819-826.).

Studies have shown that GSK5182, an inverse agonist targeting ERRγ, can effectively reduce blood sugar in diabetic mouse models (db/db mice and DIO mice) to normal levels by inhibiting glycogen production, indicating that ERRγ regulators are expected to be used for Treatment of metabolic diseases such as diabetes and obesity (related literature: Kim D., et.al, Diabetes. 2013, 62(9): 3093-3102).

In summary, estrogen-related receptors can be used as new therapeutic targets for tumors and metabolic diseases, and small molecule modulators based on estrogen-related receptor opening are very likely to become therapeutic targets for related diseases (tumor and diabetes, etc.) new drug molecules.

Contents of the invention

The technical problem to be solved by the present invention is to provide a compound used as an estrogen-related receptor modulator.

The technical scheme that solves the above-mentioned technical problem is as follows:

Compounds in formula I or pharmaceutically acceptable salts or stereoisomers thereof:

The present invention also provides a medicine composed of the above compounds.

A drug for treating tumors or metabolic diseases, comprising two parts, one part is the compound of formula I or its pharmaceutically acceptable salt and prodrug molecule, and the other part is a pharmaceutically acceptable carrier.

Another technical problem to be solved in the present invention is to provide the application of the above compounds.

Another object of the present invention is the application of the compound or its pharmaceutically acceptable salts and prodrug molecules as modulators of estrogen-related receptors in the treatment of tumors or metabolic diseases.

The above compound and its pharmaceutically acceptable salt or stereoisomer are used as estrogen-related receptor modulators in the preparation of medicines for treating tumor diseases.

Preferably, the tumor disease is any one of the following diseases: (1) breast cancer; (2) prostate cancer; (3) gastric cancer; (4) colon cancer; (5) ovarian cancer; (6) cervical cancer ; (7) endometrial cancer.

Preferably, the metabolic disease is any one of the following diseases: (1) type 2 diabetes; (2) osteoporosis; (3) obesity; (4) hyperlipidemia; Atherosclerosis and its complications; (6) fatty liver; (7) vascular stenosis; (8) hyperglycemia; (9) insulin resistance; (10) hypercholesterolemia.

The present invention adopts the above technical scheme. Compared with the prior art, the compound can become a new drug molecule for treating related diseases (tumor, diabetes, etc.), and has broad application prospects.

Description of drawings

Fig. 1 Effect of compounds of the present invention on ERRα transcriptional activation at cellular level (reporter gene experiment).

Fig. 2 Effect of compounds of the present invention on ERRβ transcriptional activation at cell level (reporter gene experiment).

Fig. 3 Effect of compounds of the present invention on ERRγ transcriptional activation at the cellular level (reporter gene experiment).

Fig. 4 Competitive binding experiment of compounds of the present invention on molecular level ERRα (time-resolved fluorescence resonance energy transfer technique, TR-FRET).

Fig. 5 Effects of compounds of the present invention on the migration function of breast cancer cell MDA-MB-231.

Fig. 6 Inhibitory effect of compounds of the present invention on human-derived breast cancer cell MDA-MB-231 xenograft nude mouse model.

detailed description

In the following, the present invention will be described in detail and specifically through specific examples, so as to better understand the present invention, but the following examples do not limit the scope of the present invention.

Various biological and pharmacological evaluations using the compounds of the present invention will be described in detail below.

Example 1

Compounds were tested for transcriptional regulatory activity on ERR using a reporter gene.

This example illustrates that the compounds involved in the present invention can effectively inhibit the expression of reporter genes regulated by ERRs in human embryonic kidney cells HEK-293, indicating that the compounds involved in the present invention can effectively regulate the function of ERRs. Reporter gene testing techniques are familiar to those skilled in the art.

We used the GAL4 fusion receptor activation method to test the transcriptional regulation activity of compounds on ERRs. The GAL4 fusion receptor activation method is based on the principle of mammalian cell single hybridization, that is, the receptor LBD and the yeast transcription factor DBD are constructed into a fusion protein expression plasmid, and the receptor LBD acts as the activation domain (Activation domain, AD). When the GAL4 fusion receptor plasmid is co-transfected with the reporter gene plasmid driven by the GAL4 DNA response element (UAS), under the action of the receptor agonist, the GAL4 fusion receptor will bind to 5×UAS and drive the reporter gene. Express. Under the action of receptor inverse agonists or antagonists, the combination of GAL4 fusion receptor and 5×UAS is reduced, which will inhibit the expression of reporter gene.

In the experiment of the present invention, we transiently co-transfected the Gal4-ERRs plasmid with a plasmid expressing 5×UAS and coupled to a firefly luciferase reporter gene (using the β-gal expression plasmid as an internal reference) into mammalian HEK-293 In the cells, add different concentrations of compounds to act for 24 hours, and detect the activity changes of luciferase and β-gal.

1. Transfection:

Inoculate HEK-293 cells in a 6cm culture dish, 6.0×10 ⁴ /dish, and the culture medium is 10% FBSDMEM, so that the growth state is good. After culturing at 37°C in 5% CO ₂ for 24 hours, 6×10 ³ cells per well were seeded in 96-well Costar cell culture plates and cultured overnight. When the cell density reaches 70%, take 50mL of serum-free medium (DMEM) in a 1.5mL centrifuge tube, and then add 2mL of transfection reagent 3000Transfection Reagent, flick several times by hand, mix and place at room temperature for 5min. According to the recombinant plasmid and 3000Transfection Reagent was mixed at a ratio of 1:5, and 20pmol of recombinant plasmids (pGal4-ERRs LBD, p5×UAS-Luc and pβ-gal) were added to DMEM and mixed gently. Dilute the recombinant plasmid in serum-free DMEM and then dilute it with DMEM The mixture of 3000 Transfection Reagent was flicked and mixed, and left at room temperature for 20 minutes to form a DNA/lipofectamine complex. Add the incubated DNA/lipofectamine complex dropwise to the culture dish containing cells and culture medium, gently shake the cell culture dish back and forth to increase the cell transfection efficiency, and put the culture dish at 37°C, 5% CO ₂ Cultivate in the incubator for 6 hours, and replace with fresh culture medium (to reduce the cytotoxicity of liposomes). Cells were digested again and seeded in 96-well Costar cell culture plates at 1×10 ⁴ /well.

2. Add compound and positive drug XCT790:

Eight hours after transfection, the compounds were added to the 96-well plate. Dilute the positive control XCT790 and the compound to 10× target concentration and add it to a 96-well Costar cell culture plate with a volume of 10 μl. Continue to culture for 24 hours in 3 duplicate wells at each concentration. After 24 hours of culture, discard the culture medium in the culture plate and add cells 100 μl of the lysate, 50 μl each was used to measure the activity of firefly luciferase and β-galactosidase with an MD multifunctional microplate reader, and the relative luciferase activity (firefly luciferase activity/β-galactosidase Activity ratio) to calculate the transcriptional regulatory activity of the compound on ERRs.

The experimental results show that the compounds involved in the present invention can dose-dependently inhibit the transcriptional activation of ERRα (IC50=1.92±0.10 μM), which is consistent with the trend of the positive control XCT790 (IC50=0.32±0.02 μM). The results are shown in FIG. 1 .

Similarly, the compound also has a certain inhibitory activity on the transcriptional activation of ERRβ and ERRγ, and the inhibition rates of ERRβ and ERRγ at 20 μM reached 32.93% and 51.37%, respectively, but IC50>20μM, the results are shown in Figure 2 and Figure 3.

Example 2

The time-resolved fluorescence resonance energy transfer detection technology (Time-Resolved Fluorescence Resonance Energy Transfer assay, TR-FRET assay) was used to test the competitive binding activity of the compounds of the present invention to ERRα at the molecular level.

This example illustrates that the compounds involved in the present invention can effectively inhibit the combination of ERRα and PGC1α, indicating that the compounds involved in the present invention competitively bind to ERRα (molecular level). TR-FRET is a technique familiar to those skilled in the art.

FRET (Fluorescence Resonance Energy Transfer) is fluorescence resonance energy transfer, which is based on the energy transfer of two fluorophores (donor and acceptor), when the two fluorophores are close to each other. Interactions between biomacromolecules can be measured with fluorescent labels and energy transfer between the two. When the two groups are close enough, the excitation source (such as flash lamp or laser) excites the fluorescent donor, which can cause energy transfer to the acceptor, so that the acceptor emits fluorescence at a specific wavelength.

Compared with FRET, TR-FRET is commonly used in drug screening. We use LanthaScreen ^TM Estrogen Related Receptor alpha TR-FRET Coactivator Assay to analyze the interaction between the LBD of ERRα and the coactivator PGC-1α. When the LBD of ERRα interacts with it After the inverse agonist is combined, the conformation changes, the binding force with the coactivator PGC-1α is weakened, and the signal at 520nm is weakened, so it can be used to screen compounds that compete with ERRα.

The specific operation is as follows:

Add 30μl of 1M DTT to 5.97ml of TR-FRET coregulator buffer G;

Add DMSO to TR-FRET co-regulator buffer G as a pair (the final concentration of DMSO is 2%), and add 10 μl of the above solution to a 384-well experimental plate;

Dilute the compound or XCT790 step by step with DMSO in a certain proportion, and dilute to 12 concentration gradients;

Dilute the above compound or XCT790 with TR-FRET coregulator buffer G;

After mixing, transfer the compounds from the previous step to a 384-well assay plate.

Prepare 4×ERRα-LBD buffer with pre-cooled TR-FRET co-regulator buffer G;

Add the 4×ERRα-LBD buffer prepared above to the 384-well experimental plate

Prepare 2 μM fluorescein-labeled PGC1α (4X) and 20 nM Tb anti-GST antibody (4X) with TR-FRET coregulator buffer G at room temperature;

Add the antibody solution prepared above into the 384-well experimental plate;

After gently shaking and mixing, incubate the 384-well experimental plate at room temperature for 8 hours in the dark;

It was detected at wavelengths of 520nm and 495nm, and the competitive binding activity of the compound to ERRα was calculated by the emission intensity ratio (520/495).

The experimental results show that the compounds involved in the present invention can dose-dependently antagonize the combination of ERRα and PGC1α, indicating that the compound can competitively bind ERRα (IC50=0.82±0.11 μM), which is consistent with the trend of the positive control XCT790 (IC50=0.22±0.01 μM), the results are shown in Figure 4.

Example 3

The CCK-8 cell proliferation assay was used to determine the inhibitory effect of the compound on the proliferation of various tumor cell lines.

This example illustrates that the compounds involved in the present invention can effectively inhibit the proliferation of various tumor cell lines, indicating that the compounds involved in the present invention exhibit effective anti-tumor activity in vitro. The CCK-8 cell proliferation assay is a technique familiar to those skilled in the art.

1. Tumor cell culture and seeding 96-well cell culture plate:

Take out the tumor cells in the logarithmic growth phase in good condition, observe the cell morphology, if the cells have been 80% confluent, start to passage. Wash and discard the old culture medium in the petri dish, wash with sterile 1mL PBS for 3-5 times, add 1mL of warmed trypsin, shake the dish horizontally to make the trypsin evenly distributed and fully digested. Put the cells in the incubator for 1-2 minutes, take out the cells, and observe the digestion of the cells under an inverted microscope. When the gap between the cells becomes larger and the protrusions of the cells retract and become round, immediately add 10% FBS culture medium to stop the digestion, and use a pipette gun Gently pipette the cells, repeat gently pipetting the digested cells to detach and disperse, form a uniform cell suspension, and adjust the cell density. Inoculate the cell suspension into a 96-well plate with a cell number of 5× ¹⁰³ /well, 200 μL per well, and culture in a 37°C 5% CO ₂ incubator. After the cells adhere to the wall, discard the original 10% FBS culture solution, wash with sterile PBS for 3-5 times, then replace with 0.1% FBS culture solution, starve and culture for 24 hours, so that the cells are in a homogeneous state in the G0/G1 phase .

2. Compounds interfere with tumor cells

The outermost circle of the 96-well plate is easy to evaporate, so the cells are only inoculated in the middle area of the 96-well plate, and 100 μL of sterilized double-distilled water is added to each well around the circle to prevent evaporation. During the intervention, the original 0.1% FBS culture solution was discarded and replaced with fresh 10% FBS DMEM culture solution, and different concentrations of compounds (using DMSO as solvent) were added, the final concentration of DMSO was 0.5%, and the effect of the compounds on tumor cell proliferation was detected. The effect was detected after 48 hours of culture.

3. Detection

After the specified intervention time, the old culture solution in the 96-well plate was discarded, new culture solution was added, and 10 μL of CCK-8 reagent was added to each well, and placed in a 5% CO2 incubator at 37°C for 2 hours. The absorbance value (A450) was detected at a single wavelength of 450nm using a 9602 enzyme label analyzer. For each concentration, 6 replicate wells were set, and the average value was taken, and the experiment was repeated 3 times. Calculation formula of inhibition rate: inhibition rate (%)=(OD compound-ODbasic)/(ODcontrol-ODbasic)×100.

Experimental results show that the compounds involved in the present invention can dose-dependently inhibit the proliferation of various tumor cells (cell lines such as breast cancer, prostate cancer, and lung cancer), especially for ER (-) breast cancer cell MDA-MB-231. Proliferation inhibitory effect is particularly significant (IC50=2.17±0.19μM), the results are shown in Table 1

Cell line IC50(μM) MDA-MB-231[ER(-)] 2.17±0.19 MDA-MB-435[ER(-)] 3.98±0.56 T47D[ER(+)] 13.72±1.41 MCF-7[ER(+)] 9.86±1.26 HELA >15 PC3 6.48±0.77 LNCap >15 A549 4.13±0.64

Table 1 Compounds of the present invention inhibit the proliferation of various tumor cells

Example 4

Transwell tumor cell migration assay was used to determine the effect of compounds on the migration function of breast cancer cell MDA-MB-231.

This example illustrates that the compounds involved in the present invention can effectively inhibit the migration of breast cancer cells MDA-MB-231, indicating that the compounds involved in the present invention exhibit effective anti-migration activity of breast cancer cells in vitro. Transwell tumor cell migration assay is a technique familiar to those skilled in the art.

1. Prepare the Transwell chamber:

Matrigel 1:8 (50mg/L) dilution was used to coat the upper chamber surface of the bottom membrane of the Transwell chamber to coat the basement membrane, and air-dried at 4°C. After air-drying, hydrate the basement membrane, suck out the residual liquid in the culture plate, add 50uL serum-free DMEM culture solution containing 10g/L BSA to each well, and incubate in a 37°C incubator for 30min.

2. Prepare cell suspension:

1) Cells in good growth state were taken, and after the cells were subcultured and adhered to the wall, they were replaced with 1% FBS DMEM cell culture medium for starvation culture for 12-24 hours to remove the influence of serum on cell migration.

2) Digest the cells, centrifuge to discard the culture medium after the digestion is terminated, wash with PBS 1-2 times, and resuspend with 1% FBS DMEM cell culture medium. Adjust the cell density to 1-10×10 ⁵ , and try to ensure that the control group and the treatment group have the same cell density.

3. Cell inoculation:

1) Take 200 μL of cell suspension and add it to the Transwell chamber, and add 1, 5, 10, 20 μM XCT790 to the upper chamber of the Transwell

2) Add 500 μL of medium containing 10ng/ml VEGF to the lower chamber of the 24-well plate (pay special attention when seeding the plate, once air bubbles appear, lift the small chamber to remove the air bubbles, and then put the small chamber into the culture plate)

3) Incubate the 24-well plate in a 37° C., 5% CO ₂ incubator for 6, 12, and 24 hours.

4. Dyeing:

1) Discard the culture medium in the well, and fix with 90% alcohol at room temperature for 30 minutes.

2) Staining: 0.1% crystal violet was used for staining. First prepare and stain with 0.1% crystal violet, then stain with 0.1% crystal violet at room temperature for 10 minutes, rinse with clean water, and gently wipe off the upper layer of non-migrated cells with a cotton swab.

5. Cell counting:

Use a Leica DC 300F upright microscope to observe and take pictures (100×), turn the Transwell chamber upside down, and you can clearly see the cells attached to the upper and lower chambers of the bottom membrane of the chamber. Randomly select 5 fields of view to count the number of cells. The formula for calculating the inhibition rate is: inhibition rate (%)=(cell count of the control group−cell count of the experimental group)/cell count of the control group×100%.

The experimental results show that after MDA-MB-231 breast cancer cells are treated with different concentrations of the compounds (0, 1, 5, 10 μM) of the present invention for 24 hours, the positive control group, that is, the 10% FBS intervention group, significantly promotes MDA-MB-231 migration. The migration inhibition rate of 1μM compound was 19.69%; the migration inhibition rate of 5μM group was 55.87%; the migration inhibition rate of 10μM compound was 72.29%. The results of the migration experiment showed that the greater the concentration of the compound, the more obvious the inhibition on the migration of MDA-MB-231 cells, the results are shown in Figure 5.

Example 5

The nude mouse xenograft model was used to determine the effect of the compound on the breast cancer cell MDA-MB-231 tumor-bearing nude mouse tumor.

This example illustrates that the compounds involved in the present invention can effectively inhibit the growth of subcutaneous breast cancer in nude mice, and at the same dose (30mg/kg), the tumor inhibitory effect is close to that of cyclophosphamide, indicating that the compounds involved in the present invention show Potent in vivo anti-breast cancer activity. The xenograft tumor model in nude mice is a technique familiar to those skilled in the art.

1. Preparation of cell suspension

Breast cancer MDA-MB-231 cells in the logarithmic growth phase were collected, digested, resuspended with sterile PBS, and centrifuged at 1000 rpm for 5 min. Then repeat the above operation twice to enrich MDA-MB-231 cells to remove dead cells and improve seeding efficiency and tumor formation rate. After centrifugation, the cells were resuspended with sterile PBS to prepare a single cell suspension of 5×10 ⁷ /mL for inoculation in the right axilla of nude mice.

2. Inoculation of nude mice

20 female nude mice, 18-20g in total, were prepared for inoculation after one week of adaptive culture in the animal room. Fully mix the MDA-MB-231 cells with adjusted cell concentration to disperse the cells evenly, so that each mouse can be inoculated with the same amount of cells. Then use a 1 mL sterile syringe to draw 1 mL of 5×10 ⁷ /mL MDA-MB-231 single cell homogeneous suspension, empty the air in the needle, and make the piston point at the integer reading position. After fixing the nude mice, turn the bevel of the syringe needle up, and flick the syringe tube with your fingers to make the cells mix again. Accurately puncture the needle of the syringe into the right axillary skin of the nude mice, insert the needle 1 cm into the injection site, and then inject 0.1 mL of MDA-MB-231 single cell homogeneous suspension. After subcutaneous injection of cells, pull out the needle and immediately press the needle hole for 30 seconds.

3. Intervention and observation of tumor growth

After the inoculation, the nude mice were fed routinely and the tumor growth in the right axilla of the nude mice was recorded daily. About the first week after tumor inoculation, a raised nodule can be seen in the right armpit of nude mice, indicating successful inoculation. Continue to record the growth of tumors in nude mice after successful inoculation.

When the tumors in the nude mice grew to 80-100 mm ³ , the nude mice were randomly divided into groups. Ten rats in each group, three groups in total, one group is the blank control group, one group is the cyclophosphamide group, and one group is the experimental group. The blank control group was given medicine twice a week, each time being a corresponding volume of normal saline; the cyclophosphamide group was given medicine twice a week, each 30mg/kg intraperitoneal injection; A 30mg/kg compound was injected intraperitoneally. After the administration, the tumor changes in nude mice in each group were observed more closely, and the size of the tumor changes were measured. According to the formula: tumor volume=length×width×height×π/6, the change of tumor volume was calculated.

4. Dissecting the mouse

After three weeks of administration, the nude mice were killed, the tumor site of the nude mice was wiped with alcohol, and the nude mice were carefully dissected with dissecting forceps and scissors. Lin's fixative container.

The experimental results showed that on the 30th day, the tumor volume of the 30mg/kg compound nude mice group was only 56.3% of that of the model group, indicating that the compound, as a specific inverse agonist of ERRs, could significantly inhibit the growth of MDA-MB-231 cells in nude mice The growth of breast cancer xenograft tumors, the results are shown in Figure 6.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as limiting the patent scope of the present invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent for the present invention should be based on the appended rights.

Claims (5)

1. the compound in formula I or its pharmaceutically acceptable salt

2. treating a medicine for tumor or metabolic disease, including two parts, a part is claim 1 institute The compound stated or its pharmaceutically acceptable salt and prodrugs, another part is pharmaceutically acceptable load Body.

3. medicine as claimed in claim 2 answering in the medicine of preparation treatment tumor or metabolic disease With.

Apply the most as claimed in claim 3, it is characterised in that described tumor disease is breast carcinoma, prostate Cancer, gastric cancer, colon cancer, ovarian cancer, cervical cancer or carcinoma of endometrium.

Apply the most as claimed in claim 3, it is characterised in that described metabolic disease be type Ⅱdiabetes mellitus, Osteoporosis, obesity, hyperlipidemia, atherosclerosis and complication thereof, fatty liver, angiostenosis, Hyperglycemia, insulin resistant or hypercholesterolemia.