CN115710248A - Novel selective estrogen receptor down-regulator compounds, methods of preparation and uses - Google Patents

Abstract

The invention discloses a selective estrogen receptor down-regulator compound, a preparation method and application thereof, and the compound has the following structure.

Description

Novel selective estrogen receptor down-regulator compounds, methods of preparation and uses

Technical Field

The invention relates to chemical drugs, a preparation method and application, in particular to a selective estrogen receptor down-regulator compound, a preparation method and application.

Background

Estrogens have roles in protein synthesis, coagulation, lipid balance, fluid balance, melanin, gastrointestinal function, pulmonary function, cognition, immune response, and heart disease. Estrogen receptors ("ERs") are ligand-activated transcriptional regulators that mediate the induction of a variety of biological effects through interaction with endogenous estrogens, including 17 β -estradiol and estrones. ER has been found to have two subtypes, era and ER β, and both receptors are involved in the regulation and development of the female reproductive tract. ER and estrogen regulate biological processes through several different pathways. The classical pathway involves the binding of ligand-activated ER to specific DNA sequence motifs known as Estrogen Response Elements (ERE). ER can also participate in non-classical pathways such as ERE-independent gene transcription through protein-protein interactions with other transcription factors, non-genomic pathways with rapid action, and ligand-independent pathways involving activation through other signaling pathways. This ER signaling is critical not only for the development and maintenance of female reproductive organs, but also for bone metabolism and bone mass, lipid metabolism, cardiovascular protection, and central nervous system conduction. Studies in this area have identified the great complexity of estrogen and ER activity. The goal of drug development has been to create new compounds that modulate estrogenic activity, either by acting as antagonists or agonists or by acting as partial antagonists or partial agonists. One goal has been to identify full anti-estrogens (full antagonists) that have the effect of stopping all estrogenic activity in the body. Fulvestrant is an example of a complete estrogen receptor antagonist with no agonist activity, which is a selective estrogen receptor down-regulator (SERD). Fulvestrant is disclosed by Imperial Chemical Industries (ICI) in U.S. patent No.4,659,516 and sold by Astra Zeneca under the trade name fastodex. It is indicated for the treatment of hormone receptor positive metastatic breast cancer in postmenopausal women with disease progression after anti-estrogen treatment. Fulvestrant has limited water solubility and can only be injected intramuscularly (TM), and cannot be administered orally. Patient compliance is poor and clinical use is limited. Another class of anti-estrogens is Selective Estrogen Receptor Modulators (SERMs) which act as antagonists or agonists in a gene-or tissue-specific manner. The goal of SERM treatment is to identify drugs with a mixed spectrum that provide advantageous targeted antiestrogenic activity and avoid adverse off-target effects or exhibit the attendant advantageous estrogenic side effects (J Med Chem 2020, 63, 15094-15114.). An example of a SERM is tamoxifen, originally sold under the trade name Nolvadex by AstraZeneca. Tamoxifen is also disclosed by ICI in U.S. patent No.4,659,516. (see also U.S. patent nos. 6,774,122 and 7,456,160). Tamoxifen is a prodrug that is metabolized into 4-hydroxy tamoxifen and N-desmethyl-4-hydroxy tamoxifen (which has high binding affinity for the estrogen receptor). Tamoxifen is indicated to prevent further breast cancer after breast cancer treatment and to treat lymph node positive breast cancer in women after mastectomy and radiotherapy. Tamoxifen may affect bone health. Tamoxifen may cause osteopenia in premenopausal women (bonethinning), while it may be beneficial for bone health in postmenopausal women. Serious side effects have been noted, including an increased risk of uterine cancer in postmenopausal women and "tumor outbreaks" (tumor flares) in women with breast cancer that has spread to the bone. In addition to these side effects, some women who initially respond to tamoxifen experience acquired resistance over time, and in some cases, ER-positive breast cancer becomes not only tamoxifen resistant, but tamoxifen becomes an agonist inducing tumor proliferation. Three-line treatment of breast cancer includes steroidal and non-steroidal Aromatase Inhibitors (AI) which block estrogen production and thus ER-dependent growth. These drugs, which include letrozole, anastrozole and exemestane, carry the risk of removing all estrogen from women after menopause, thereby increasing the risk of osteopenia, osteoporosis and bone fractures (J Med Chem 2019, 62, 11301-11323.)

Various SERDs, SERMs, and AIs have been disclosed. SERM raloxifene was disclosed by Eli Lilly in 1981 (U.S. Pat. Nos. 4,418,068;5,478,847, 5,393,763 and 5,457,117) for the prevention of breast cancer and the treatment of osteoporosis. In june 2011, aragon pharmaceuticals disclosed benzopyran derivatives and acobifene analogs for the treatment of tamoxifen resistant breast cancer (see W02011/156518, us patent nos. 8,455,534 and 8,299,112). Genentech discloses a series of tetrahydropyrido [3,4-b ] indol-1-yl compounds with estrogen receptor modulating activity in US2016/0175289 and three compounds (one of which GDN-0810) for use in a combination therapy for estrogen receptor modulation in US 2015/0258080. AstraZeneca is currently developing AZD9496, a novel oral selective estrogen receptor down-regulator for estrogen receptor positive breast cancer patients (WO 2014/191726). In view of the often disastrous effects of estrogen regulatory disorders, including cancer, tumors and particularly breast cancer, there remains a strong need to produce new drugs with significant anti-estrogen efficacy without unacceptable side effects.

Disclosure of Invention

The purpose of the invention is as follows: it is an object of the present invention to provide novel selective estrogen receptor down-regulator compounds (SERDs).

Another object of the present invention is to provide a preparation method and use of the selective estrogen receptor down-regulator compound.

The technical scheme is as follows: a selective estrogen receptor down-regulator compound having the structure:

wherein:

m is 0, 1, 2, 3 or 4;

n is 0, 1, 2, 3 or 4;

x is selected from-O-, -CH ₂ -、-S-、-NR ₁₇ 、-CHF-、-CF ₂ -or a cycloalkyl group;

y is selected from-C (O) -, -CF ₂ -, cycloalkyl, -CH ₂ -or-CHF-;

ring A is selected from phenyl, biphenylyl, naphthyl, quinolinyl, 5-or 6-membered monocyclic heteroaryl, cycloalkyl, or 7-, 8-, 9-, or 10-membered bicyclic heterocyclyl;

ring B is selected from phenyl, heteroaryl, thiophenyl, 5-or 6-membered monocyclic heteroaryl, cycloalkyl or 7-, 8-, 9-or 10-membered bicyclic heterocyclyl;

R ₁ selected from-CH = CHCOOR ₁₇ 、-NR ₁₇ (CO)COOR ₁₇ 、-COOR ₁₇ cycloalkyl-COOR ₁₇ 、-C ₂ -C ₆ alkenylene-COOR ₁₇ 、-C ₂ -C ₆ alkynylene-COOR ₁₇ 、-CH＝CHC(O)R ₁₆ 、-NR ₁₇ (CO)C(O)R ₁₆ 、-C(O)R ₁₆ -cycloalkyl-C (O) R ₁₆ 、-C ₂ -C ₆ alkenylene-C (O) R ₁₆ and-C ₂ -C ₆ alkynylene-C (O) R ₁₆ ；

R ₂ And R ₃ Is selected from-OR ₁₅ 、-SR ₁₅ 、-N(R ₁₅ ) ₂ Hydrogen, aryl, heteroaryl, halogen, -CN, -NO ₂ Haloalkyl, cycloalkyl, thiol, nitroso, C ₁ -C ₆ Alkyl, hydroxy, -O (C) ₁ -C ₆ Alkyl), -O (C) ₁ -C ₆ Fluoroalkyl group), -SF ₅ 、-B(OH) ₂ 、-B(OR ₁₅ ) ₂ 、-C(O)OR ₁₅ 、-C(O)R ₁₆ 、-C(S)R ₁₆ 、-OSO ₂ OR ₁₅ 、-OSO ₂ R ₁₆ 、-NHSO ₂ OR ₁₅ 、-NHSO ₂ R ₁₆ -N (alkyl) SO ₂ OR ₁₅ -N (alkyl) SO ₂ R ₁₆ 、-OP(O)(OR ₁₅ ) ₂ 、-OP(O)(R ₁₆ ) ₂ 、-P(O)(OR ₁₅ ) ₃ 、-P(O)(R ₁₆ ) ₃ 、-P(O)OR ₁₅ 、-P(O)R ₁₆ 、-SO ₂ R ₁₆ 、-SO ₂ OR ₁₅ Alkyne, alkene, arylalkyl, aryloxy, heteroarylalkyl or C ₁ -C ₆ A fluoroalkyl group;

R ₁₅ selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl;

R ₁₆ is selected from-N (R) ₁₅ ) ₂ 、-SR ₁₅ OR-OR ₁₅ ；

R ₁₇ Selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, -C (O) R ₁₅ 、-C(S)R ₁₅ 、-C(O)R ₁₆ 、-C(S)R ₁₆ Or a heteroaryl group.

Further, said R ₁ Selected from-COOH, -NH (CO) COOH or-CH = CHCOOH. The ring A is phenyl, biphenylyl, naphthyl or quinolyl. Ring B is phenyl or 5-or 6-membered monocyclic heteroaryl.

Further, the selective estrogen receptor down-regulator compound is characterized by being any one of the following compounds:

a pharmaceutical composition comprising a therapeutically effective amount of one or more selective estrogen receptor down-regulator compounds of claim 1 or 2 and a pharmaceutically acceptable carrier.

The selective estrogen receptor down-regulator compound is used for preparing a medicine for treating estrogen related disorder diseases.

Further, the estrogen-related disorder is breast cancer, ovarian cancer, endometrial cancer, prostate cancer, lung cancer, cancer that has metastasized to bone, or bone loss.

Further, the breast cancer is hormone receptor positive metastatic breast cancer, tamoxifen resistant breast cancer or triple negative breast cancer. The selective estrogen receptor down-regulator compound is characterized in that the synthetic route is as follows:

scheme 1:

scheme 2:

scheme 3:

the structure of the final product of the above synthetic route is as follows:

wherein R is ₁ 、R ₂ 、R ₃ M and n are defined as the technical scheme, and z is 0, 1, 2, 3 or 4.

Ring A is selected from phenyl, biphenylyl, naphthyl, quinolinyl, 5-or 6-membered monocyclic heteroaryl, cycloalkyl, or 7-, 8-, 9-, or 10-membered bicyclic heterocyclyl;

ring B is selected from phenyl, heteroaryl, thiophenyl, 5-or 6-membered monocyclic heteroaryl, cycloalkyl or 7-, 8-, 9-or 10-membered bicyclic heteroaryl

A cyclic group;

R ₁ selected from-CH = CHCOOR ₁₇ 、-NR ₁₇ (CO)COOR ₁₇ 、-COOR ₁₇ cycloalkyl-COOR ₁₇ 、-C ₂ -C ₆ alkenylene-COOR ₁₇ 、-C ₂ -C ₆ alkynylene-COOR ₁₇ 、-CH＝CHC(O)R ₁₆ 、-NR ₁₇ (CO)C(O)R ₁₆ 、-C(O)R ₁₆ -cycloalkyl-C (O) R ₁₆ 、-C ₂ -C ₆ alkenylene-C (O) R ₁₆ and-C ₂ -C ₆ alkynylene-C (O) R ₁₆ ；

R ₂ And R ₃ Is selected from-OR ₁₅ 、-SR ₁₅ 、-N(R ₁₅ ) ₂ Hydrogen, aryl, heteroaryl, halogen, -CN, -NO ₂ Haloalkyl, cycloalkyl, thiol, nitroso, C ₁ -C ₆ Alkyl, hydroxy, -O (C) ₁ -C ₆ Alkyl), -O (C) ₁ -C ₆ Fluoroalkyl group), -SF ₅ 、-B(OH) ₂ 、-B(OR ₁₅ ) ₂ 、-C(O)OR ₁₅ 、-C(O)R ₁₆ 、-C(S)R ₁₆ 、-OSO ₂ OR ₁₅ 、-OSO ₂ R ₁₆ 、-NHSO ₂ OR ₁₅ 、-NHSO ₂ R ₁₆ -N (alkyl) SO ₂ OR ₁₅ -N (alkyl) SO ₂ R ₁₆ 、-OP(O)(OR ₁₅ ) ₂ 、-OP(O)(R ₁₆ ) ₂ 、-P(O)(OR ₁₅ ) ₃ 、-P(O)(R ₁₆ ) ₃ 、-P(O)OR ₁₅ 、-P(O)R ₁₆ 、-SO ₂ R ₁₆ 、-SO ₂ OR ₁₅ Alkyne, alkene, arylalkyl, aryloxy, heteroarylalkyl or C ₁ -C ₆ A fluoroalkyl group.

Has the advantages that: the compound improves the pharmacokinetic property, is an orally taken estrogen receptor down-regulator, realizes the nanomolar level of activity, and has very obvious effect on preventing tumors such as breast cancer and the like.

Drawings

FIG. 1 is a Western blot analysis;

FIG. 2 is a photograph of an immunofluorescence assay;

FIG. 3 shows a bar graph of the effect of compounds 13 and 19 on fulvestrant and tamoxifen and control on the activity of ERE luciferase transcription in MCF-7 cells, the y-axis is the relative ERE luciferase transcription activity, the x-axis is compounds 13 and 19 on fulvestrant and tamoxifen and control;

FIG. 4 shows a graph of the induction of block of the G0/G1 phase of the MCF-7 cell cycle by compounds 13 and 19, the y-axis being the number of MCF-7 cells and the x-axis being the range of the cell cycle, after 24h of treatment with 13 and 19, MCF-7 cells were collected, stained with PI, analyzed by flow cytometry, compared to control DMSO, ^* p＜0.05， ^** p＜0.01；

FIG. 5 is a bar graph showing the effect of compounds 13 and 19 on the ER signaling pathway, the y-axis being the expression of GREB1, PGR, TFF1 genes in MCF-7 cells, the x-axis being the regional range of cell cycles of treatment with different concentrations of compound, N =3, ns > 0.05, ^* p＜0.05， ^** p＜0.01， ^*** p＜0.001；

FIG. 6 shows a graph of compound 19 in vivo PK studies, with the concentration of compound 19 in the blood of the mice on the y-axis, x-

The axis is the time corresponding to the complete PK of compound 19 (including 3mg/kg i.v. and 30mg/kg oral) which showed the best antiproliferative profile in the in vitro assay;

figure 7 shows a physical plot and histogram of the mice transplant tumor volume and mouse body weight for compound 13 and 19 versus fulvestrant and tamoxifen and the control group, and a plot of the mouse tumor volume and tumor-bearing mouse weight over time for the oral compound 13 and 19 versus fulvestrant and tamoxifen groups;

7A is a real object image of the breast cancer tumor of the mouse after 30 days of administration, y-axes of the images 7B and 7C are the transplanted tumor volume and the tumor weight of the mouse, x-axes are the corresponding compound 13 and 19 groups, fulvestrant and tamoxifen groups and a control group, y-axes of the images 7D and 7E are the transplanted tumor volume and the tumor-bearing mouse weight, x-axes are time, the long diameter (a) and the short diameter (B) of the tumor are measured by vernier calipers, and the tumor volume is calculated according to the following formula: v =1/2 × a × b ² When the mean tumor volume reaches 100mm ³ On the left and right, mice were randomly divided into 5 groups (n =6 per group), designated: control group, tamoxifen, fulvestrant, compound 13 and compound 19, tamoxifen mice mouthTaking 30mg/kg/d of tamoxifen, and taking 1930mg/kg/d of compound orally for 19 groups of mice; compound 13 mice were orally administered at 30mg/kg/d, and fulvestrant was intraperitoneally injected at 10mg/kg/d. Mice body weights and tumor volumes were recorded every 5 days during a 30 day treatment period;

FIG. 8 shows HE-stained sections of mice heart, liver, spleen, kidney and rat uterus for effects of compounds 13 and 19 with fulvestrant and tamoxifen and control groups for histological examination of heart, liver, spleen, kidney and rat uterus, dissecting tissues, paraffin embedding, sectioning (6 μm thick), sections stained with hematoxylin and eosin (H & E);

FIG. 9 shows a graph of Ki67, ER and PR expression in mouse tumor tissue for compound 13 and 19 versus fulvestrant and tamoxifen and controls, 9A is an immunohistochemistry plot, 9B, 9C and 9D plots on the y-axis are Ki67, ER and PR expression indices in mouse tumor tissue, on the x-axis are compound 13 and 19 versus fulvestrant and tamoxifen and controls, ^* p＜0.05， ^** p＜0.01；

FIG. 10 shows H & E staining patterns and cube plots of the effects of compounds 13 and 19 on endometrium of female rat pups with estrogen and tamoxifen and control groups, 10A is the H & E staining pattern, 10B is the ratio of uterine wet weight to body weight of female rat pups on y-axis, and compounds 13 and 19 on estrogen and tamoxifen and control groups on x-axis;

FIGS. 11, 12 show graphs of binding assays of compounds 13 and 19 to ER, and FIG. 11 is RMSD of compounds 13 and 19 to ER receptors as a function of time simulated; FIG. 12 is RMSF data for amino acids in the ligand binding domain of compounds 13 and 19 with ER, the docking position of compounds 13 and 19 with ER;

FIGS. 13, 14 show a docking scheme of proteins with compounds 13 and 19, evaluating the interaction between compounds 13 and 19 and the ER ligand binding domain (PDB ID:1R 5K), compounds 13 and 19 can interact with key amino acids Glu353 and Arg394 to form stable H bonds in the binding pocket using SchrodingerMaestro 2019 docking analysis, the dimethyl-substituted phenyl ring of compound 13 and the dimethyl-and fluorine-substituted phenyl ring of compound 19 are accommodated in the hydrophobic pocket formed by Leu346, met388, phe404, met421 and Leu 525.

Detailed Description

Example 1

The following table shows the classes of intermediates synthesized in scheme 1

Intermediate 1: 3-chloro-6-fluorobenzo [ b ] thiophene-2-carbonyl chloride

This intermediate was synthesized from step 1 in 15mL SOCl ₂ To the solution was added (E) -3- (4-fluorophenyl) acrylic acid (2g, 12mmol), and to the reaction mixture was added several drops of pyridine. The reaction mixture was heated at reflux for 3 days as most of the starting material was converted to product. After completion of the reaction, the reaction mixture was evaporated to remove SOCl ₂ The crude product 1 was used as such without further purification.

Intermediate 2: 3-chloro-6-fluoro-N-methoxy-N-methylbenzo [ b ] thiophene-2-carboxamide

This intermediate was synthesized in step 2 by adding N, O-dimethylhydroxylamine hydrochloride (1.5g, 24mmol) and Et in an ice bath ₃ N (5mL, 36mmol) to a solution of intermediate 1 (crude 2 g) in DCM (15 mL). The reaction was carried out at 0 ℃ and then heated to room temperature for 5 hours. After the reaction was completed, the reaction was quenched with ice water and extracted with ethyl acetate. The organic extract was washed with brine and dried over anhydrous sodium sulfate. The organic extracts were evaporated under reduced pressure and purified by flash chromatography using (10-40% ethyl acetate in petroleum ether) as the mobile phase to give 1.5g (46%) of a yellow solid. ¹ H NMR(500MHz，CDCl ₃ )δ＝7.90(dd，J＝8.9，5.0，1H)，7.49(dd，J＝8.4，2.2，1H)，7.24(m，1H)，3.72(s，3H)，3.40(s，3H).13C NMR(126MHz，CDCl ₃ )δ163.14，161.29(d，J＝249.1Hz)，139.41(d，J＝10.5Hz)，132.55，125.71，124.72(d，J＝9.5Hz)，124.46，114.76(d，J＝24.7Hz)，108.45(d，J＝25.8Hz)，61.98，33.48.

Intermediates 1 to 3: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (phenyl) methanone

The intermediate is synthesized by steps 1-3 in the reaction route 1, compound intermediates 1-2 (200mg, 0.73mmol) are accurately weighed and placed in a 50mL eggplant-shaped bottle, 1mol/L phenyl magnesium bromide 1.46mL is added, and stirring is carried out for 6h under the protection of nitrogen. After the reaction was complete, quench the reaction by adding a small amount of water and remove the gum by adding a small amount of dilute hydrochloric acid. The reaction liquid was extracted with ethyl acetate, the organic phase was collected, and a certain amount of anhydrous sodium sulfate was added thereto and allowed to stand for 2 hours. The product was isolated by dry column packing, dry loading and silica gel column chromatography (PE: EA = 19: 1). Pale yellow powder, yield 86.6%. ¹ H NMR(500MHz，CDCl ₃ )δ7.93(dd，J＝9.0，5.1Hz，1H)，7.92-7.89(m，2H)，7.68-7.61(m，1H)，7.57-7.49(m，3H)，7.29(td，J＝8.8，2.3Hz，1H).

Intermediate 1-4-a: methyl (E) 3- (4- ((2-benzoyl-6-fluorobenzo [ b ]]Thiophen-3-yl) oxy) phenyl) acrylate this intermediate was synthesized by steps 1-4 of scheme 1 by weighing intermediate 1-3 (150mg, 0.52mmol) in a 50mL eggplant-shaped flask, adding 2mL DMF to dissolve the reaction, adding 1.5 equivalents of 4-bromo-3-fluorophenol (148mg, 0.78mmol) and 3 equivalents of cesium carbonate (508.2mg, 1.56mmol), raising the reaction temperature to 80 deg.C and refluxing for 12h. After the reaction was completed, the reaction was cooled to room temperature, and the reaction solution was extracted with ethyl acetate. The organic phase was collected, washed with saturated brine, and then a certain amount of anhydrous sodium sulfate was added to the organic phase and allowed to stand for 2 hours. Dry column filling, dry sample loading, silica gel columnThe product was isolated by chromatography (PE: EA = 4: 1). A pale yellow solid was obtained in yield: 95.3 percent. ¹ H NMR(500MHz，CDCl ₃ )δ7.70(m，2H)，7.64-7.50(m，3H)，7.39(t，J＝7.8Hz，2H)，7.28-7.23(m，1H)，7.16(td，J＝8.8，2.2Hz，1H)，6.37(dd，J＝9.5，2.8Hz，1H)，6.31(ddd，J＝8.9，2.8，0.9Hz，1H).

The following intermediates were prepared by a procedure analogous to steps 1-4 using the appropriate starting materials:

intermediate 1-4-b: (3- (4-bromo-2-methylphenoxy) -6-fluorobenzo [ b ] thiophen-2-yl) (phenyl) methanone

¹ H NMR(500MHz，Chloroform-d)δ7.72(dd，J＝8.2，1.3Hz，2H)，7.55(dd，J＝8.5，2.3Hz，1H)，7.52-7.43(m，2H)，7.34(dd，J＝8.2，7.5Hz，2H)，7.20-7.16(m，1H)，7.10(td，J＝8.8，2.3Hz，1H)，7.03(m，1H)，6.33(d，J＝8.7Hz，1H)，2.01(s，3H).

Intermediates 1-4-c: (3- (4-bromo-3-methylphenoxy) -6-fluorobenzo [ b ] thiophen-2-yl) (phenyl) methanone

¹ H NMR(500MHz，Chloroform-d)δ7.69(dd，J＝8.3，1.3Hz，2H)，7.62-7.47(m，3H)，7.41-7.30(m，2H)，7.21(d，J＝8.7Hz，1H)，7.13(td，J＝8.8，2.3Hz，1H)，6.46-6.41(m，1H)，6.29(dd，J＝8.8，3.0Hz，1H)，2.19(s，3H).

Intermediates 1-4-d: intermediate 1-4-b: (3- (4-bromo-biphenyloxy) -6-fluorobenzo [ b ] thiophen-2-yl) (phenyl) methanone this intermediate was the crude product and was directly subjected to the next reaction.

Intermediate 1-5-a: methyl (E) -3- (4- ((2-benzoyl-6-fluorobenzo [ b ] thiophen-3-yl) oxy) -2-fluorobenzene) acrylate

The intermediate is synthesized by steps 1-5 in the reaction route 1, weighing a compound 1-4-a (100mg, 0.22mmol) and placing the compound in a 50mL glass pressure resistant tube, adding 2mL DMF for dissolving, then adding 5 equivalents of methyl acrylate (0.1mL, 1.1mmol) and,2 equivalents of triethylamine (0.06ml, 0.06mmol) and 0.1 times equivalent of triphenylphosphine palladium chloride are used as catalysts, the reaction temperature is raised to 130 ℃, and the reaction is heated for 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature, and the reaction solution was extracted with ethyl acetate. The organic phase was collected, washed with saturated brine, and then a certain amount of anhydrous sodium sulfate was added to the organic phase and allowed to stand for 2 hours. Dry packing, dry loading, and silica gel column chromatography to isolate the product (PE: EA = 4: 1). After isolation, a pale yellow oil was obtained in 41.3% yield. ¹ H NMR(500MHz，CDCl ₃ )δ7.75-7.70(m，2H)，，7.67(d，J＝16.2Hz，1H)，7.63(dd，J＝8.9，5.0Hz，1H)，7.60(dd，J＝8.4，2.2Hz，1H)，7.57-7.53(m，1H)，7.39(t，J＝7.8Hz，1H)，7.32-7.27(m，1H)，7.18(td，J＝8.8，2.2Hz，1H)，6.44(dd，J＝8.7，2.4Hz，1H)，6.39(d，J＝16.2Hz，1H)，6.35(dd，J＝11.5，2.5Hz，1H)，3.81(s，1H).

The following intermediates were prepared by a procedure analogous to steps 1-5 using the appropriate starting material, methyl acrylate:

intermediate 1-5-b: methyl (E) -3- (4- ((2-benzoyl-6-fluorobenzo [ b ] thiophen-3-yl) oxy) -3-methylphenyl) acrylate

¹ H NMR(500MHz，Chloroform-d)δ7.75-7.66(m，2H)，7.59-7.54(m，2H)，7.50-7.45(m，2H)，7.33(dd，J＝8.4，7.3Hz，2H)，7.22(d，J＝2.1Hz，1H)，7.11(m，2H)，6.45(d，J＝8.5Hz，1H)，6.26(d，J＝16.0Hz，1H)，3.78(s，3H)，2.04(s，3H).

Intermediates 1-5-c: methyl (E) -3- (4- ((2-benzoyl-6-fluorobenzo [ b ] thiophen-3-yl) oxy) -2-methylphenyl) acrylate

¹ H NMR(500MHz，Chloroform-d)δ7.81(d，J＝15.9Hz，1H)，7.73-7.66(m，2H)，7.60(dd，J＝8.9，5.1Hz，1H)，7.56(dd，J＝8.5，2.2Hz，1H)，7.53-7.48(m，1H)，7.38-7.33(m，2H)，7.30(d，J＝8.6Hz，1H)，7.13(td，J＝8.8，2.3Hz，1H)，6.42(dd，J＝8.6，2.7Hz，1H)，6.38(d，J＝2.7Hz，1H)，6.20(d，J＝15.9Hz，1H)，3.78(s，3H)，2.24(s，3H).

Intermediates 1-5-d: methyl (E) -3- (4 '- ((2-benzoyl-6-fluorobenzo [ b ] thiophen-3-yl) oxy) -1,1' -biphenyl ] -4-yl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ7.74(dd，J＝11.5，10.3Hz，3H)，7.67(dd，J＝8.9，5.1Hz，1H)，7.59(dd，J＝11.4，5.2Hz，3H)，7.55-7.50(m，3H)，7.40-7.34(m，4H)，7.16(td，J＝8.8，2.2Hz，1H)，6.69(d，J＝8.7Hz，2H)，6.48(d，J＝16.0Hz，1H)，3.84(s，3H).

The following table shows the intermediate types synthesized in scheme 2

Intermediate 2-3-a: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (3-fluorophenyl) methanone

The intermediate is synthesized in step 2-3 of reaction route 2, 1-bromo-3-fluorobenzene (761mg, 3.65mmol) is accurately weighed and placed in a 50mL eggplant-shaped bottle, magnesium powder with the same equivalent, 2mL anhydrous tetrahydrofuran and iodine are added, nitrogen is used for protection, and the temperature is raised to 80 ℃ for reaction. After the reaction was observed to fade, the reaction was carried out for 2h. Then accurately weighed intermediate 2 (200mg, 0.73mmol) is added, and stirred for 6h under the condition of nitrogen protection at normal temperature. After the reaction was complete, quench the reaction by adding a small amount of water and remove the gum by adding a small amount of dilute hydrochloric acid. The reaction liquid was extracted with ethyl acetate, and the organic phase was collected and added with a certain amount of anhydrous sodium sulfate and allowed to stand for 2 hours. The product was isolated by silica gel column chromatography (PE: EA = 19: 1). A pale yellow solid was isolated in 87.7% yield. ¹ H NMR(500MHz，CDCl ₃ )δ7.97(dd，J＝8.9，5.0Hz，1H)，7.69(d，J＝7.7Hz，1H)，7.62-7.56(m，2H)，7.55-7.48(m，1H)，7.39-7.29(m，2H).

The following intermediates were prepared by a procedure analogous to step 2-3 using the appropriate starting materials:

intermediate 2-3-b: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (4-fluoro-2-methylphenyl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ7.64-7.57(m，3H)，7.37(dd，J＝8.4，5.8Hz，1H)，7.30(d，J＝8.8Hz，2H)，7.15(td，J＝8.8，2.2Hz，1H)，6.83-6.75(m，2H)，6.52(d，J＝8.8Hz，2H)，6.31(d，J＝16.0Hz，1H)，3.82(s，3H)，2.14(s，3H).

Intermediate 2-3-c: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (2-ethylphenyl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ7.94(dd，J＝9.0，5.1Hz，1H)，7.55(dd，J＝8.3，2.2Hz，1H)，7.49(td，J＝7.7，1.2Hz，1H)，7.43-7.37(m，2H)，7.31(d，J＝7.6Hz，1H)，7.26(dd，J＝8.9，2.3Hz，1H)，2.77(q，J＝7.5Hz，2H)，1.24(t，J＝7.6Hz，3H).

Intermediate 2-3-d: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (2-isopropylphenyl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ7.93(dd，J＝9.0，5.1Hz，1H)，7.57-7.47(m，3H)，7.37-7.33(m，1H)，7.32-7.29(m，1H)，7.28-7.24(m，1H)，3.31-2.98(m，1H)，1.28(s，3H)，1.27(s，3H).

Intermediate 2-3-e: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (3,5-dimethylphenyl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ7.96(dd，J＝8.9，5.0Hz，1H)，7.57(dd，J＝8.3，2.1Hz，1H)，7.52(s，2H)，7.33-7.29(m，1H)，7.22(s，1H)，2.42(s，6H).

Intermediate 2-3-f: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (4-methoxyphenyl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ7.97(dd，J＝8.9，5.0Hz，1H)，7.58(dd，J＝8.3，2.2Hz，1H)，7.50(s，1H)，7.38(d，J＝8.7Hz，1H)，7.31(td，J＝8.8，2.3Hz，1H)，7.17(d，J＝9.2Hz，1H)，2.46(s，3H).

Intermediate 2-3-g: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (4- (trifluoromethoxy) phenyl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ8.01-7.93(m，3H)，7.57(dd，J＝8.3，2.2Hz，1H)，7.36(d，J＝8.1Hz，2H)，7.31(td，J＝8.8，2.3Hz，1H).

Intermediate 2-3-h: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (2,4-dimethylphenyl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ7.93(dd，J＝9.0，5.0Hz，1H)，7.54(dd，J＝8.3，2.2Hz，1H)，7.39(d，J＝7.8Hz，1H)，7.30-7.24(m，3H)，7.15(s，1H)，7.10(d，J＝7.8Hz，1H)，2.42(s，6H).

Intermediate 2-3-i: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (phenyl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ7.93(dd，J＝9.0，5.1Hz，1H)，7.92-7.89(m，2H)，7.68-7.61(m，1H)，7.57-7.49(m，3H)，7.29(td，J＝8.8，2.3Hz，1H).

Intermediate 2-3-j: benzo [ b ] thiophen-4-yl (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ8.15(d，J＝8.1Hz，1H)，7.95(dd，J＝9.0，5.0Hz，1H)，7.91(d，J＝5.5Hz，1H)，7.83(dd，J＝7.4，0.8Hz，1H)，7.67(d，J＝5.5Hz，1H)，7.58(dd，J＝8.3，2.2Hz，1H)，7.46(t，J＝7.7Hz，1H)，7.33-7.29(m，1H).

Intermediate 2-3-k: benzofuran-5-yl (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ8.22(d，J＝1.6Hz，1H)，7.98-7.91(m，2H)，7.75(d，J＝2.2Hz，1H)，7.63(d，J＝8.6Hz，1H)，7.58(dd，J＝8.3，2.3Hz，1H)，7.31(td，J＝8.9，2.3Hz，1H)，6.90(dd，J＝2.2，0.8Hz，1H).

Intermediate 2-3-1: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (naphthalen-2-yl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ8.44(s，1H)，8.03-7.93(m，5H)，7.70-7.65(m，1H)，7.63-7.57(m，2H)，7.32(td，J＝8.8，2.3Hz，1H).

Intermediate 2-3-m: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (naphthalen-1-yl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ8.21-8.16(m，1H)，8.08(d，J＝8.2Hz，1H)，7.97(dd，J＝6.5，2.8Hz，1H)，7.92(dd，J＝9.0，5.0Hz，1H)，7.79-7.75(m，1H)，7.61-7.55(m，4H)，7.28-7.25(m，1H).

Intermediate 2-3-n: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (isoquinolin-7-yl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ9.41(s，1H)，8.67(d，J＝5.7Hz，1H)，8.37(s，1H)，8.15(d，J＝8.5Hz，1H)，8.10-8.06(m，1H)，7.98(dd，J＝8.9，5.0Hz，1H)，7.79(d，J＝5.7Hz，1H)，7.60(dd，J＝8.2，2.1Hz，1H)，7.33(td，J＝8.8，2.1Hz，1H).

2-4-a intermediate: methyl (E) -3- (4- ((6-fluoro-2- (3-fluorobenzoyl) benzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

The intermediate is synthesized by the steps 2-4 in the reaction scheme 2, the intermediate 2-3-a (100.0mg, 0.31mmol) is weighed and placed in a 50mL eggplant-shaped bottle, 2mL DMF is added to dissolve reactants, 2 equivalents of trans-p-coumaric acid methyl ester (110.4mg, 0.62mmol) and 3 equivalents of cesium carbonate (302.3mg, 0.93mmol) are added, the reaction temperature is raised to 80 ℃, and the reflux is carried out for 12 hours. After the reaction was completed, the reaction was cooled to room temperature, and the reaction solution was extracted with ethyl acetate. The organic phase was collected, washed with saturated brine, and then a certain amount of anhydrous sodium sulfate was added to the organic phase and allowed to stand for 2 hours. The product was isolated by dry column packing, dry loading and silica gel column chromatography (PE: EA = 9: 1). A yellow oil was isolated in 45.6% yield. ¹ H NMR(500MHz，CDCl ₃ )δ7.64-7.57(m，3H)，7.52-7.49(m，1H)，7.43-7.38(m，1H)，7.36-7.30(m，3H)，7.24-7.19(m，1H)，7.19-7.13(m，1H)，6.68-6.61(m，2H)，6.30(d，J＝16.0Hz，1H)，3.81(s，3H).

The following intermediates were prepared by a procedure analogous to steps 2-4 using the appropriate starting materials:

intermediate 2-4-b: methyl (E) -3- (4- ((6-fluoro-2- (4-fluoro-2-methylbenzoyl) benzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ7.64-7.57(m，3H)，7.37(dd，J＝8.4，5.8Hz，1H)，7.30(d，J＝8.8Hz，2H)，7.15(td，J＝8.8，2.2Hz，1H)，6.83-6.75(m，2H)，6.52(d，J＝8.8Hz，2H)，6.31(d，J＝16.0Hz，1H)，3.82(s，3H)，2.14(s，3H).

Intermediate 2-4-c: methyl (E) -3- (4- ((2- (2-ethylbenzoyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ7.62-7.53(m，3H)，7.34-7.29(m，2H)，7.28-7.25(m，2H)，7.16-7.07(m，3H)，6.54-6.42(m，2H)，6.33-6.24(m，1H)，3.81(s，3H)，1.28(t，J＝7.1Hz，2H)，1.04(t，J＝7.6Hz，3H).

Intermediate 2-4-d: methyl (E) -3- (4- ((6-fluoro-2- (2-isopropylbenzoyl) benzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ7.62-7.54(m，2H)，7.51(dd，J＝8.9，5.0Hz，1H)，7.33(t，J＝7.6Hz，1H)，7.28-7.25(m，3H)，7.23(d，J＝7.5Hz，1H)，7.10(td，J＝8.8，2.2Hz，1H)，7.03(t，J＝7.4Hz，1H)，6.48(d，J＝8.7Hz，2H)，6.29(d，J＝16.0Hz，1H)，3.80(s，3H)，3.09-2.83(m，1H)，1.08(s，3H)，1.07(s，3H).

Intermediates 2-4-e: methyl (E) -3- (4- ((2- (3,5-dimethylbenzoyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ7.64-7.56(m，3H)，7.34(d，J＝8.7Hz，2H)，7.17-7.11(m，2H)，6.67(d，J＝8.7Hz，2H)，6.31(d，J＝16.0Hz，1H)，3.81(s，3H)，2.24(s，6H).

Intermediate 2-4-f: methyl (E) -3- (4- ((6-fluoro-2- (4 v methoxybenzoyl) benzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ7.82-7.77(m，2H)，7.64-7.55(m，3H)，7.31(t，J＝5.7Hz，2H)，7.16(td，J＝8.8，2.3Hz，1H)，6.90-6.86(m，2H)，6.72-6.66(m，2H)，6.29(d，J＝16.0Hz，1H)，3.88(s，3H)，3.80(s，3H).

Intermediate 2-4-g: methyl (E) -3- (4- ((6-fluoro-2- (4- (trifluoromethoxy) benzoyl) benzo 1b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ7.76-7.71(m，2H)，7.65(dd，J＝8.9，5.0Hz，1H)，7.62-7.55(m，2H)，7.30(s，2H)，7.21-7.14(m，3H)，6.58(d，J＝8.8Hz，2H)，6.29(d，J＝16.0Hz，1H)，3.81(s，3H).

Intermediate 2-4-h: methyl (E) -3- (4- ((2- (2,4-dimethylbenzoyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ7.64-7.56(m，3H)，7.26(s，3H)，7.14(td，J＝8.8，2.2Hz，1H)，6.91(d，J＝7.8Hz，1H)，6.88(s，1H)，6.53-6.48(m，2H)，6.30(d，J＝16.0Hz，1H)，3.82(s，3H)，2.32(s，3H)，2.11(s，3H).

Intermediates 2-4-i: methyl (E) -3- (4- ((2-benzoyl-6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ7.72(dd，J＝8.3，1.3Hz，2H)，7.66-7.54(m，3H)，7.56-7.49(m，1H)，7.40-7.33(m，2H)，7.28(s，2H)，7.16(td，J＝8.8，2.3Hz，1H)，6.61(d，J＝8.8Hz，2H)，6.29(d，J＝16.0Hz，1H).3.81(s，3H).

Intermediate 2-4-j: methyl (E) -3- (4- ((2- (benzo [ b ] thiophene-4-carbonyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ7.99(d，J＝8.0Hz，1H)，7.70-7.65(m，2H)，7.61(dd，J＝8.3，1.5Hz，1H)，7.44(dd，J＝15.3，5.6Hz，3H)，7.32(t，J＝7.7Hz，1H)，7.22-7.13(m，1H)，7.09(d，J＝8.6Hz，2H)，6.42(d，J＝8.6Hz，2H)，6.18(d，J＝16.0Hz，1H)，3.81(s，3H).

Intermediate 2-4-k: methyl (E) -3- (4- ((2- (benzofuran-5-carbonyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ8.04(d，J＝1.5Hz，1H)，7.74-7.70(m，2H)，7.64(dd，J＝8.9，5.1Hz，1H)，7.60(dd，J＝8.5，2.1Hz，1H)，7.56(d，J＝15.9Hz，1H)，7.47(d，J＝8.6Hz，1H)，7.25(d，J＝8.7Hz，2H)，7.17(td，J＝8.8，2.2Hz，1H)，6.77(d，J＝1.4Hz，1H)，6.59(d，J＝8.8Hz，2H)，6.26(d，J＝16.0Hz，1H)，3.80(s，3H).

Intermediate 2-4-1: methyl (E) -3- (4- ((2- (2-naphthyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ8.28(s，1H)，7.87(d，J＝8.2Hz，1H)，7.83-7.77(m，2H)，7.74(dd，J＝8.5，1.5Hz，1H)，7.67-7.59(m，3H)，7.56-7.51(m，2H)，7.21-7.14(m，3H)，6.53(d，J＝8.7Hz，2H)，6.23(d，J＝16.0Hz，1H)，3.80(s，3H).

Intermediate 2-4-m: methyl (E) -3- (4- ((2- (1-naphthyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ7.88(dd，J＝14.1，8.3Hz，2H)，7.78(d，J＝8.2Hz，1H)，7.61(d，J＝6.8Hz，2H)，7.58(dd，J＝8.9，5.1Hz，1H)，7.44-7.33(m，4H)，7.13(td，J＝8.8，2.1Hz，1H)，7.01(d，J＝8.7Hz，2H)，6.28(d，J＝8.6Hz，2H)，6.14(d，J＝16.0Hz，1H)，3.81(s，3H).

Intermediate 2-4-n: methyl (E) -3- (4- ((6-fluoro-2- (quinoline-7-carbonyl) benzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，DMSO)δ9.38(s，1H)，8.54(d，J＝5.7Hz，1H)，8.37(s，1H)，8.18-8.10(m，2H)，7.83(dd，J＝8.5，1.6Hz，1H)，7.77(d，J＝5.7Hz，1H)，7.61-7.56(m，1H)，7.51(d，J＝16.0Hz，1H)，7.45(d，J＝8.8Hz，2H)，7.36(td，J＝9.0，2.3Hz，1H)，6.61(d，J＝8.8Hz，2H)，6.43(d，J＝16.0Hz，1H)，3.70(s，3H).

The following table shows the classes of intermediates synthesized in scheme 3

Intermediate 3-3: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (4-fluoro-2,6-dimethylphenyl) methanol

The intermediate is synthesized by the step 3-3 in the reaction route 3, the intermediate 2 (200mg, 0.73mmol) is accurately weighed and placed in a 50mL eggplant-shaped bottle, tetrahydrofuran is added for dissolution, 1.3 equivalent of 1mol/L diisobutylaluminum hydride (0.95ml, 0.95mmol) is added, and the mixture is stirred in ice bath. After the reaction is finished, adding saturated sodium tartrate aqueous solution, and stirring until the solution is clear. The reaction liquid was extracted with ethyl acetate, the organic phase was collected, and a certain amount of anhydrous sodium sulfate was added thereto and allowed to stand for 2 hours. The product was isolated by dry column packing, dry loading and silica gel column chromatography (PE: EA = 19: 1). A white solid was isolated in 71.6% yield. ¹ H NMR(500MHz，CDCl ₃ )δ7.72(dd，J＝8.8，5.0Hz，1H)，7.46(dd，J＝8.5，2.2Hz，1H)，7.20(td，J＝8.9，2.3Hz，1H)，6.79(d，J＝9.3Hz，2H)，6.57(s，1H)，2.43(s，6H).

Intermediate 3-4-a: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (4-fluoro-2,6-dimethylphenyl) methanol

The intermediate is synthesized by the steps 3-4 in the reaction route 3, 2-bromo-5-fluoro-1,3-dimethylbenzene (741.0mg, 3.65mmol) is accurately weighed and placed in a 50mL eggplant-shaped bottle, the same equivalent of magnesium powder and 2mL anhydrous tetrahydro-2Furan and iodine are reacted under the protection of nitrogen and the temperature is raised to 80 ℃. After the reaction was observed to fade, the reaction was carried out for 2h. Then accurately weighed compound intermediate 3-3 (200mg, 0.73mmol) is added, and stirred for 6h under the protection of nitrogen at normal temperature. After the reaction was complete, quench the reaction by adding a small amount of water and remove the gum by adding a small amount of dilute hydrochloric acid. The reaction liquid was extracted with ethyl acetate, the organic phase was collected, and a certain amount of anhydrous sodium sulfate was added thereto and allowed to stand for 2 hours. The product was isolated by silica gel column chromatography (PE: EA = 4: 1). A pale yellow oil was isolated in 77.4% yield. ¹ H NMR(500MHz，CDCl ₃ )δ7.72(dd，J＝8.8，5.0Hz，1H)，7.46(dd，J＝8.5，2.2Hz，1H)，7.20(td，J＝8.9，2.3Hz，1H)，6.79(d，J＝9.3Hz，2H)，6.57(s，1H)，2.43(s，6H).

The following intermediates were prepared by a procedure analogous to steps 3-4 using the appropriate starting materials:

intermediate 3-4-b: (4-chloro-2,6-dimethylphenyl) (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) methanol

¹ H NMR(500MHz，CDCl ₃ )δ7.72(dd，J＝8.9，5.0Hz，1H)，7.46(dd，J＝8.5，2.3Hz，1H)，7.20(td，J＝8.9，2.3Hz，1H)，7.08(s，2H)，6.56(s，1H)，2.42(s，6H).

Intermediate 3-4-c: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (2,6-dimethylphenyl) methanol

¹ H NMR(500MHz，CDCl ₃ )δ7.72(dd，J＝8.9，5.0Hz，1H)，7.45(dd，J＝8.5，2.3Hz，1H)，7.22-7.16(m，2H)，7.08(d，J＝7.6Hz，2H)，6.63(s，1H)，2.45(s，6H).

Intermediate 3-5-a: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (4-fluoro-2,6-dimethylphenyl) methanone

The intermediate is synthesized by the steps 3-5 in the reaction route 3, the compound intermediate 3-4-a (200mg, 0.59mmol) is weighed and placed in a 50mL eggplant-shaped bottle, dichloromethane is added for dissolution, 5 equivalents of PCC is added, and the mixture is stirred until the reaction is finished. The reaction liquid was extracted with ethyl acetate, the organic phase was collected, and a certain amount of anhydrous sodium sulfate was added thereto and allowed to stand for 2 hours. The product was isolated by dry column packing, dry loading and silica gel column chromatography (PE: EA = 19: 1). A pale yellow solid was isolated in 94.4% yield. ¹ H NMR(500MHz，CDCl ₃ )δ7.97-7.90(m，1H)，7.56(d，J＝8.1Hz，1H)，7.28-7.24(m，1H)，6.84(d，J＝9.4Hz，2H)，2.24(s，6H).

The following intermediates were prepared by a procedure analogous to steps 3-5 using the appropriate starting materials:

intermediate 3-5-b: (4-chloro-2,6-dimethylphenyl) (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ7.94(dd，J＝9.0，5.0Hz，1H)，7.56(dd，J＝8.3，2.1Hz，1H)，7.30-7.25(m，3H)，7.14(s，2H)，2.22(s，6H).

Intermediate 3-5-c: (3-chloro-6-fluorobenzo [ b ] thiophen-2-yl) (2,6-dimethylphenyl) methanone

¹ H NMR(500MHz，CDCl ₃ )δ7.93(dd，J＝9.0，5.0Hz，1H)，7.55(dd，J＝8.3，2.2Hz，1H)，7.30(d，J＝7.7Hz，1H)，7.26(dd，J＝8.8，2.3Hz，1H)，7.12(d，J＝7.7Hz，2H)，2.24(s，6H).

Intermediate 3-6-a: methyl (E) -3- (4- ((6-fluoro-2- (4-fluoro-2,6-dimethylbenzoyl) benzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

The intermediate is synthesized by the steps 3-6 in the reaction scheme 3, the intermediate 3-5-a (200.0mg, 0.58mmol) is weighed and placed in a 50mL eggplant-shaped bottle, 2mLDMF is added to dissolve the reactant, 2 equivalents of trans-p-coumaric acid methyl ester (206.5mg, 1.16mmol) and 3 equivalents of cesium carbonate (565.6mg, 1.74mmol) are added) The reaction temperature was raised to 80 ℃ and refluxed for 12h. After the reaction was completed, the reaction was cooled to room temperature, and the reaction solution was extracted with ethyl acetate. After the organic phase is collected, the organic phase is washed by saturated saline solution, and then a certain amount of anhydrous sodium sulfate is added into the organic phase to be kept stand for 2 hours. The product was isolated by dry column packing, dry loading and silica gel column chromatography (PE: EA = 9: 1). A yellow oil was isolated in 44.7% yield. ¹ H NMR(500MHz，CDCl ₃ )δ7.64-7.58(m，2H)，7.50(dd，J＝8.9，5.0Hz，1H)，7.31(d，J＝8.7Hz，2H)，7.12(td，J＝8.9，2.3Hz，1H)，6.56(d，J＝9.5Hz，2H)，6.50(d，J＝8.7Hz，2H)，6.32(d，J＝16.0Hz，1H)，3.82(s，3H)，2.10(s，6H).

The following intermediates were prepared by a procedure analogous to steps 3-6 using the appropriate starting materials:

intermediate 3-6-b: methyl (E) -3- (4- ((2- (4-chloro-2,6-dimethylbenzoyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ7.61(dd，J＝16.9，8.9Hz，2H)，7.52(dd，J＝8.9，5.1Hz，1H)，7.32(d，J＝8.7Hz，2H)，7.13(td，J＝8.8，2.1Hz，1H)，6.83(s，2H)，6.47(d，J＝8.6Hz，2H)，6.33(d，J＝16.0Hz，1H)，3.82(s，3H)，2.08(s，6H).

Intermediate 3-6-c: methyl (E) -3- (4- ((2- (2,6-dimethylbenzoyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylate

¹ H NMR(500MHz，CDCl ₃ )δ7.63-7.58(m，2H)，7.49(dd，J＝8.9，5.1Hz，1H)，7.28-7.26(m，2H)，7.11(td，J＝8.9，2.1Hz，1H)，7.06(t，J＝7.7Hz，1H)，6.85(d，J＝7.6Hz，2H)，6.47(d，J＝8.6Hz，2H)，6.31(d，J＝16.0Hz，1H)，3.82(s，3H)，2.11(s，6H).

Example 1: synthesis procedures for representative Compounds

Compound 1: (E) -3- (4- ((2-benzoyl-6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

The compound is synthesized in the step 7, the intermediate 2-5-i (100mg, 0.24mmol) is weighed and placed in a 50mL eggplant-shaped bottle, 4mL tetrahydrofuran is added to dissolve and stir evenly, 5 times of equivalent of sodium hydroxide and 4mL water are added, and stirring is carried out for 12h. After the reaction is finished, the pH is adjusted to 1 with 1mol/L dilute hydrochloric acid. The reaction liquid was extracted with ethyl acetate, the organic phase was collected, and a certain amount of anhydrous sodium sulfate was added thereto and allowed to stand for 2 hours. The product was isolated by dry column chromatography on silica gel (DCM: meOH = 9: 1). A pale yellow solid was isolated in 86.6% yield and 99.61% purity. ¹ H NMR(500MHz，MeOD)δ7.79(dd，J＝8.8，2.3Hz，1H)，7.71-7.59(m，3H)，7.57-7.51(m，2H)，7.39(d，J＝8.3Hz，4H)，7.23(td，J＝9.0，2.3Hz，1H)，6.63(d，J＝8.8Hz，2H)，6.32(d，J＝16.0Hz，1H). ¹³ C NMR(126MHz，CDCl ₃ )δ192.92，173.01，166.88(d，J＝249.1Hz)，163.45，150.78，147.63，144.61(d，J＝11.2Hz)，141.80，136.43，133.37，133.32，132.44，131.83，131.62(d，J＝3.6Hz)，131.61，128.69(d，J＝9.8Hz)，121.03，119.57，118.46(d，J＝25.4Hz)，113.17(d，J＝26.6Hz).ESI-MS(-)[m/z]：417.39[M-H] ^-

Compounds 2 through 21 were prepared by a procedure similar to step 7 using the appropriate starting materials. These compounds were characterized as follows:

compounds 2 to 5 are synthesized by scheme 1

Compound 2: (E) -3- (4- ((2-benzoyl-6-fluorobenzo [ b ] thiophen-3-yl) oxy) -2-fluorobenzene) acrylic acid:

136.06，132.58，129.85(d，J＝4.4Hz)，129.14，128.47，127.98，127.83(d，J＝3.6Hz)，124.56(d，J＝9.9Hz)，119.43，117.08(d，J＝12.0Hz)，114.73(d，J＝25.5Hz)，111.56，109.29(d，J＝26.3Hz)，103.60(d，J＝26.8Hz).ESI-MS(-)[m/z]：435.45[M-H] ^- .

compound 3: (E) -3- (4- ((2-benzoyl-6-fluorobenzo [ b ] thiophen-3-yl) oxy) -3-methylphenyl) acrylic acid

The compound is obtained by reacting an intermediate 1-5-b. ¹ H NMR(500MHz，MeOD)δ7.80(dd，J＝8.8，2.2Hz，1H)，7.71-

3.7Hz)，127.01，126.90，124.76(d，J＝9.8Hz)，116.64，114.47(d，J＝25.4Hz)，113.40，109.26(d，J＝26.3Hz)，14.63.ESI-MS(-)[m/z]：431.46[M-H] ^-

Compound 4: (E) -3- (4- ((2-benzoyl-6-fluorobenzo [ b ] thiophen-3-yl) oxy) -2-methylphenyl) acrylic acid

137.87，132.40，129.49，128.51，128.03，127.88，127.74，127.36(d，J＝3.6Hz)，124.76(d，J＝9.8Hz)，117.89，117.34，114.48(d，J＝25.4Hz)，113.26，109.19(d，J＝26.3Hz)，18.28.ESI-MS(-)[m/z]：431.48[M-H] ^-

Compound 5: (E) -3- (4 '- (2-benzoyl-6-fluorobenzo [ b ] thiophen-3-yl) oxy) - (1,1' -biphenyl ] -4-yl) acrylic acid

128.04，127.14，125.66，119.52，116.44，115.56(d，J＝25.0Hz)，110.62(d，J＝26.3Hz).ESI-MS(-)[m/z]：493.58[M-H] ^-

Compounds 6 to 18 were synthesized from scheme 2:

compound 6: (E) -3- (4- ((6-fluoro-2- (3-fluorobenzoyl) benzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

139.70(d，J＝6.7Hz)，129.99，129.78(d，J＝7.7Hz)，129.21，128.98，127.41(d，J＝3.5Hz)，125.07(d，J＝9.6Hz)，124.68(d，J＝3.0Hz)，119.58(d，J＝21.3Hz)，116.21，115.87，115.75(d，J＝23.0Hz)，115.08(d，J＝25.0Hz)，109.54(d，J＝25.7Hz).ESI-MS(m/z)：435.45[M-H] ^-

Compound 7: (E) -3- (4- ((2- (2-ethylbenzoyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

8.9Hz)，135.17，130.81(d，J＝9.5Hz)，130.29，129.71(d，J＝4.4Hz)，129.65(d，J＝1.3Hz)，125.74(d，J＝9.9Hz)，118.60，117.59(d，J＝21.7Hz)，115.71，115.70(d，J＝25.2Hz)，112.64(d，J＝21.7Hz)，110.74(d，J＝26.4Hz)，19.24.ESI-MS(-)[m/z]：449.48[M-H] ^-

Compound 8: (E) -3- (4- ((6-fluoro-2- (4-fluoro-2-methylbenzoyl) benzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

130.81(d，J＝9.5Hz)，130.29，129.71(d，J＝4.4Hz)，129.65(d，J＝1.3Hz)，125.74(d，J＝9.9Hz)，118.60，117.59(d，J＝21.7Hz)，115.71，115.70(d，J＝25.2Hz)，112.64(d，J＝21.7Hz)，110.74(d，J＝26.4Hz)，19.24.ESI-MS(-)[m/z]：449.48[M-H] ^-

Compound 9: (E) -3- (4- ((6-fluoro-2- (3-fluorobenzoyl) benzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

163.62(d，J＝83.3Hz)，161.63(d，J＝79.8Hz)，159.71，146.90，145.77，140.67(d，J＝10.7Hz)，139.70(d，J＝6.7Hz)，129.99，129.78(d，J＝7.7Hz)，129.21，128.98，127.41(d，J＝3.5Hz)，125.07(d，J＝9.6Hz)，124.68(d，J＝3.0Hz)，119.58(d，J＝21.3Hz)，116.21，115.87，115.75(d，J＝23.0Hz)，115.08(d，J＝25.0Hz)，109.54(d，J＝25.7Hz).ESI-MS(-)[m/z]：459.55[M-H] ^-

Compound 10: (E) -3- (4- ((2- (3,5-dimethylbenzoyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

134.54，130.43，129.65，129.31，128.36，126.59，125.62(d，J＝10.0Hz)，118.67，116.53，115.44(d，J＝25.3Hz)，110.62(d，J＝26.4Hz)，20.91.ESI-MS(-)[m/z]：445.53[M-H] ^-

Compound 11: (E) -3- (4- ((6-fluoro-2- (4-methoxybenzoyl) benzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

(126MHz，DMSO)δ186.67，168.04，163.81，162.35(d，J＝247.2Hz)，159.33，145.73，143.39，139.94(d，J＝11.4Hz)，132.01，130.44，130.12，129.62，129.46，128.13(d，J＝3.6Hz)，125.17(d，J＝9.9Hz)，118.58，116.29，115.46(d，J＝25.1Hz)，114.14，110.53(d，J＝26.4Hz).ESI-MS(-)[m/z]：447.47[M-H] ^-

Compound 12: (E) -3- (4- ((6-fluoro-2- (4- (trifluoromethoxy) benzoyl) benzo [ b ] thiophen-3-yl) oxy) phenyl)

MHz，DMSO)δ187.42，168.08，162.73(d，J＝248.4Hz)，159.12，151.48，147.23，143.18，140.66(d，J＝11.5Hz)，136.94，131.29，130.33，129.70，129.50，127.90(d，J＝3.5Hz)，125.64(d，J＝9.8Hz)，120.99，120.35(d，J＝257.4Hz)，118.83，115.99，115.71(d，J＝25.4Hz)，110.66(d，J＝26.5Hz).ESI-MS(-)[m/z]：501.47[M-H] ^-

Compound 13: (E) -3- (4- ((2- (2,4-dimethylbenzoyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

131.62，130.18，129.84，129.69(d，J＝3.5Hz)，129.55，128.59，126.22，125.60(d，J＝10.0Hz)，118.49，115.87，115.61(d，J＝25.1Hz)，110.66(d，J＝26.1Hz)，21.39，19.32.ESI-MS(-)[m/z]：445.48[M-H] ^-

Compound 14: (E) -3- (4- ((2- (1-naphthoyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

133.48，131.90，131.57，129.72(d，J＝3.5Hz)，129.56，128.47，128.06，127.17，126.74，126.22，125.15(d，J＝9.7Hz)，124.82，124.31，115.86，115.33，114.98(d，J＝24.9Hz)，114.44，109.61(d，J＝25.7Hz).ESI-MS(m/z)：467.48[M-H] ^-

Compound 15: (E) -3- (4- ((2- (2-naphthoyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

Hz)，159.11，146.64，143.36，140.42(d，J＝11.4Hz)，135.29，135.06，132.14(d，J＝4.9Hz)，131.87，131.20，130.39，129.69(d，J＝3.1Hz)，129.39，129.13(d，J＝7.9Hz)，128.49，128.21(d，J＝3.6Hz)，128.16，127.35，125.57(d，J＝9.7Hz)，124.68，118.57，116.35，115.55(d，J＝25.0Hz)，110.65(d，J＝26.3Hz).ESI-MS(m/z)：467.48[M-H] ^-

Compound 16: (E) -3- (4- ((2- (benzofuran-5-formyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

159.20，157.13，148.09，146.32，143.16，140.19(d，J＝11.2Hz)，133.08，130.34，129.66，129.48，128.15(d，J＝3.6Hz)，127.50，125.86，125.37(d，J＝10.0Hz)，123.97，118.83，116.22，115.53(d，J＝25.1Hz)，111.74，110.58(d，J＝26.4Hz)，107.75.ESI-MS(m/z)：457.45[M-H] ^-

Compound 17: (E) -3- (4- ((2- (benzo [ b ] thiophen-4-formyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

143.20，140.76，140.53(d，J＝11.5Hz)，136.99，132.88，130.35，130.13，129.67，129.51，128.23(d，J＝3.5Hz)，127.26，126.72，125.56(d，J＝10.0Hz)，123.73，122.82，118.58，115.93，115.59(d，J＝25.2Hz)，110.62(d，J＝26.3Hz).ESI-MS(m/z)：473.42[M-H] ^-

Compound 18: (E) -3- (4- ((6-fluoro-2- (isoquinoline-7-formyl) benzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

Hz)，159.16，152.79，147.43，144.08，143.90，140.79(d，J＝11.5Hz)，139.28，134.66，130.55，129.48(d，J＝2.6Hz)，129.35，128.57，128.32，128.12(d，J＝3.5Hz)，126.02，125.76(d，J＝10.0Hz)，121.51，117.21，116.20，115.70(d，J＝25.1Hz)，110.73(d，J＝26.4Hz).ESI-MS(m/z)：468.47[M-H] ^-

Compounds 19 to 21 Synthesis from scheme 3

Compound 19: (E) -3- (4- ((6-fluoro-2- (4-fluoro-2,6-dimethylbenzoyl) benzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

＝10.8Hz)，136.77(d，J＝8.6Hz)，135.76，129.62，129.46，128.90，125.43(d，J＝9.6Hz)，116.15，115.24，115.08，115.04，114.16(d，J＝21.4Hz)，109.85(d，J＝25.6Hz)，19.32(d，J＝1.5Hz).ESI-MS(-)[m/z]：463.50[M-H] ^-

Compound 20: (E) -3- (4- ((2- (4-chloro-2,6-dimethylbenzoyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

The compound is obtained by reacting an intermediate 3-6-b. ¹ H NMR(500MHz，DMSO)δ12.32(s，1H)，8.14(dd，J＝9.1，2.3Hz，1H)，7.55-7.48(m，4H)，7.34(td，J＝9.0，2.4Hz，1H)，6.99(s，2H)，6.57(d，J＝8.7Hz，2H)，6.39(d，J＝

Compound 21: (E) -3- (4- ((2- (2,6-dimethylbenzoyl) -6-fluorobenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid

141.00(d，J＝11.5Hz)，139.97，133.54，130.14，129.62，129.50，129.37，127.72，126.05(d，J＝10.1Hz)，118.44，115.80(d，J＝25.6Hz)，115.57，111.07，110.90，110.60，19.20.ESI-MS(-)[m/z]：445.52[M-H] ^-

And (3) performance test:

(1) Survival of MCF-7 cells or Ishikawa cells

The MTT method detects the cell viability. MCF-7 the medium was changed to phenol red free RPMI-1640 and activated charcoal treated FBS before each experiment to remove estrogen for 2 days. (1000-2000 MCF-7 cells per well in 96-well plates) 1000-2000 MCF-7 cells per well were seeded in 96-well plates and cultured overnight to allow the cells to adhere. All compounds were dissolved in DMSO, controls were treated with 0.1% DMSO, and cells were incubated for 96-120 h after treatment. 5% MTT (5 mg/mL, phosphate Buffered Saline (PBS)) reagent was added to the cells, and after about 4h of incubation, the culture medium was discarded, and 200. Mu.L DMSO was added (absorbance was measured at 490nm after shaking for about 10min on a shaker) and shaken for about 10min. Finally, the absorbance was measured at 490 nm. Calculating the growth inhibition rate of the compound by an MTT method to be (Ac-As)/(Ac-Ab) x 100%, wherein Ac is the absorbance of a control hole; the absorbance of the sample well is As; ab is blank well absorbance, the activity of ishikawa cells is detected by an MTT method, 3000-4000 ishikawa cells are inoculated into a 96-well plate, and the cells are cultured overnight to be attached to the wall. All compounds were dissolved in DMSO, controls were treated with 0.1% DMSO, 20 μ M compounds for 72h, cells were added with 5% mtt (5 mg/mL, phosphate Buffered Saline (PBS)) reagent, after about 4h of incubation, the culture was discarded, 200 μ l DMSO was added, (absorbance was measured at 490nm after shaking about 10min on a shaker) and shaken for about 10min. Finally, the absorbance was measured at 490 nm. Calculating the growth inhibition rate of the compound by an MTT method to be (Ac-As)/(Ac-Ab) x 100%, wherein Ac is the absorbance of a control hole; the absorbance of the sample well is As; ab is the absorbance of the blank well, and the results are shown in Table 1.

TABLE 1 antiproliferative Activity of fluorinated SERD Compounds in MCF-7 and Ishikawa cells

Western blotting:

MCF-7 cells were cultured for 2 days with phenol red free medium and activated charcoal treated FBS prior to the experiment. When cells were cultured to 80% density in 6-well plates, control and test compounds were added for 24 hours of treatment. The cells were then washed with pre-cooled PBS and lysed by adding cell lysate to extract intracellular proteins (then, the cells were washed in cold PBS and resuspended in cell radioimmunoprecipitation assay (RIPA) lysis buffer to prepare a cell extract). Loading according to the calculated protein concentration. The electrophoresis conditions were 20mA and 120min. The film transfer condition is 100V,90min, and the operation must be carried out in an ice bath environment. After 2 hours of blocking with 5% skim milk at room temperature, wash 3 times with PBST for 10 minutes each. The primary antibody was then diluted in primary antibody diluent at the appropriate ratio (5% bovine serum albumin was prepared from PBST, ER α for each specific antibody was diluted 1: 1000, and 5% bovine serum albumin was used as diluent) and incubated overnight at 4 ℃. Primary antibody was recovered and secondary antibody was incubated on a shaker for 2h and then washed 3 times with PBST (secondary antibody incubated 2h at room temperature and then on a shaker, membrane washed 3 times with PBST). Finally, the ECL luminescence solution is uniformly dropped on the surface of the membrane (the ECL luminescence solution and ECL chemiluminescence solution are uniformly mixed and spread on the surface of the membrane), and the strip is subjected to imaging analysis by a gel imager. The results are shown in FIG. 1.

(2) Immunofluorescence detection

MCF-7 cells were cultured 48h before the experiment with de-activin conditions (FBS without phenol Red 1640 and charcoal treatment). Cells were in 24 wells (4X 10 per well) ⁴ Individual cells) were cultured overnight in a plate. After attachment, the cells are treated with the test compound and incubated for 24h. All samples were fixed and then closed. After incubation overnight at 4 ℃ with the corresponding diluted antibody, a secondary antibody conjugated to a fluorophore was added. Then, the cells were washed three times with PBS. Nuclei were stained with DAPI, followed by immunofluorescence detection using a fluorescence microscope (Leica, dim 8). The results are shown in FIG. 2.

MCF-7 cell Estrogen Response Element (ERE) luciferase assay:

MCF-7 cells were cultured 48h before the experiment with de-activin conditions (FBS without phenol Red 1640 and charcoal treatment). The cells were cultured at 1.5X 10 ⁴ The density of cells/well was seeded in 96-well plates and co-transfected with 100ng of ERE-luciferase plasmid (3X ERE TATA luc waters a gift from Donald McDonnell (Addge plasma #11354 http:// n2t. Net/addge: 11354. Lipofectamine8000 transfection reagent (Beyotime) was used to transfect 24h in RPIM 1640 medium according to the instructions. After 24h the cells were treated with the corresponding compounds 24h, after 24h the luciferase activity was determined with the luciferase assay system (Beyotime). The results are shown in FIG. 3.

(3) Cell cycle arrest

MCF-7 cells were cultured 48h before the experiment with de-activin conditions (FBS without phenol Red 1640 and charcoal treatment). The cells were cultured at 5X 10 ⁵ The density of cells/well was seeded in 6-well plates, treated with the corresponding compounds for 24h, followed by collection and treatment of cells according to the Kjeldahl cell cycle assay kit instructions, followed by detection using flow cytometry. The results are shown in FIG. 4.

(4) qPCR detection

Extraction of RNA using Total RNA extraction kit (Vazyme)

IIIlst Strand cDNA Synthesis Supermix for qPCR (gDNA digesterter plus) (YEASEN) recording to the manufacturing's primers) quantitative real-time PCR was performed on the 7500RT-PCR system using

qPCR A qPCR was performed with a SYBR Green Master Mix (YEASEN). The relative difference of gene expression is calculated by a delta Ct method by taking beta-actin as a normalization control. The following primers were used (Biotech Co., ltd.):

β-actin(forward)：5’-TCAAGATCATTGCTCCTCCTGA-3’

β-actin(reverse)：5’-CTCGTCATACTCCTGCTTGCTG-3’

TFF1(forward)：5’-TTGTGGTTTTCCTGGTGTCA-3’

TFF1(reverse)：5’-CCGAGCTCTGGGACTAATCA-3’

GREB1(forward)：5’-ATGGGAAATTCTTACGCTGGAC-3’

GREB1(reverse)：5’-CACTCGGCTACCACCTTCT-3’

PR(forward)：5’-AGCCAGAGCCCACAATACAG-3’

PR(reverse)：5’-GACCTTACAGCTCCCACAGG-3’

the results are shown in FIG. 5

(5) Pharmacokinetic experiments on target Compounds

6 SPF stagesFemale ICR mice, 6-8 weeks old, 18-22g in weight, purchased from Beijing Wintonlihua laboratory animal technology Co., ltd, animal license number: SCXK (Jing) 2021-0006; the experimental animals were received and acclimatized for one week, followed by pharmacokinetic studies. The temperature of the animal room is 20-26 ℃; humidity is 40-70%; alternating illumination and darkness within 12 h; freely drinking water and taking food; animals were fasted for 12h before the start of the experiment. A single dose single cycle dosing regimen was used in this study, with 6 healthy animals selected and divided into 2 groups, scored as two groups 1, 2. The dosing regimen is shown in table 2. Following dosing, blood was collected from the jugular vein at specific collection time points as shown in table 3 below. Sampling requirement and processing: collecting blood, placing in a heparin sodium anticoagulation tube under wet ice environment, standing for more than 15min at 6000r/min, centrifuging for 3min, separating blood plasma, and storing at-20 deg.C. Determining the concentration of the sample at each time point for each animal using LC-MS/MS; for samples with high concentrations above the limit of detection, it is necessary to dilute the sample with blank plasma to a quantitative range of concentrations and then perform the assay. Microsoft Excel2010 spreadsheet software was selected for data management, and WinNolin 8.2 software was used to calculate pharmacokinetic parameters. Absolute bioavailability calculation formula: f (%) = (Dose) _jv ×AUC _oral(0-∞) )/(Dose _oral ×AUC _iv(0-∞) ) X 100%. Calculation of pharmacokinetic parameters: and (4) according to the blood concentration-time data of each tested animal measured in the test, obtaining the main pharmacokinetic parameters of the tested animal. Comprising T _max (time to peak), C _max (peak concentration), t _1/2 (elimination half-life period), AUC (area under blood concentration-time curve), and the like.

TABLE 2 dosing regimen

Following dosing, blood was collected from the jugular vein at the specific collection time points shown in tables 5-7 below.

TABLE 3 blood sample Collection design

The results are shown in tables 4 and 5 and FIG. 6

Table 4 in vivo pharmacokinetic data for compound 19.

TABLE 5 plasma concentrations following oral or intravenous administration of Compound 19 ^*

^* Compound 19 was gavaged at 3mg/kg (intravenous) and 30mg/kg (oral) in PEG400/CMC = 1: 9 water. Data are mean plasma concentrations of three mice at different time points. ND: no data

(6) Establishment of nude mouse subcutaneous transplantation tumor model and determination of mouse weight and tumor volume related indexes

Female nude mice and female immature SD rats were purchased from dichroa febrifuga cave laboratory animals ltd. These animals were maintained under specific pathogen-free conditions. All animals were treated strictly according to the guidelines for laboratory animal care and use and the principles of vertebrate utilization and care, and all animal work was approved by the animal Care Committee of the institute of Biotechnology, beijing. Animal experiments were performed according to the regulations of "guide to conservation and use of laboratory animals" issued by the department of science and technology of the people's republic of China.

Tumor inoculation nude mice were injected subcutaneously with MCF-7 cells (1X 106) suspended in PBS. In the experiment, nude mice were injected with 0.15mg/ml/day of estrogen subcutaneously. The tumor length (a) and length (b) were measured with a vernier caliper and the tumor volume was calculated according to the following formula: v =1/2 × a × b2. When the average tumor volume reachesTo 100mm ³ On the left and right, mice were randomly divided into 4 groups (n =6 per group), designated: control, tamoxifen, 19 and 13 groups. The tamoxifen group mice were injected with 30mg/kg/d tamoxifen. 19 groups of mice were injected with compound 1930mg/kg/d; the gavage compound 13 30mg/kg/d in 13 groups of mice. Mouse body weight and tumor volume were recorded every 5 days during a 30 day treatment period. Thereafter, all mice were sacrificed and tumor, heart, liver, spleen and kidney were collected. The results are shown in FIG. 7.

(7) HE stained section

Tumors, heart, liver, spleen, kidney and rat uterus were selected for histological examination. Dissected tissue, paraffin embedded, and sectioned (6 μm thick). Sections were stained with hematoxylin and eosin (H & E).

1) And putting the prepared tissue paraffin section into an electric heating constant-temperature oven, and baking for 3 hours at 60 ℃.

2) The dried paraffin sections were dewaxed in xylene I and xylene II for 5min, respectively, and then soaked in 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol and 70% ethanol for 1min, respectively, and washed with distilled water for 2min.

3) Staining with hematoxylin for 5min, differentiating with hydrochloric acid ethanol for 30s, and washing with clear water.

4) Staining with eosin dye solution for 1min, and washing off residual dye solution.

5) 70% ethanol for 30s;80% ethanol for 30s; twice with 90% ethanol for 3min each time. Absolute ethyl alcohol is added twice, each time is 5 minutes, dimethylbenzene is transparent, the mixture is sealed by neutral glue, and the observation is carried out under a microscope. The results are shown in FIG. 8.

(8) Immunohistochemistry

Tumor tissues were fixed in 4% formalin for 24 hours, paraffin-embedded, and 6 μm sections were cut and mounted on plus slides. The sections were dewaxed in xylene and then hydrated by exposure to graded alcohol (100%, 95%,70%,50%, water). The slides were immersed in the antigen retrieval solution and heated for 10 minutes, cooled to room temperature, infiltrated in TBST for 15 minutes, rinsed with water, and then incubated with 3% hydrogen peroxide for 10 minutes to quench endogenous peroxidase activity to block non-specific binding, and the slides were incubated with blocking solution for 30 minutes. Incubation with Ki67 primary antibody (1: 500 dilution), ER primary antibody (1: 250 dilution) or PR primary antibody (1: 100 dilution), respectively, was carried out overnight at 4 ℃. Negative controls replaced primary antibody with blocking solution. After washing the primary antibody with TBST, peroxidase-labeled polymer coupled with goat anti-rabbit immunoglobulin was added for 30 minutes. For signal detection, slides were incubated with Elite abc-chromogenic reagent and diaminobenzidine mixture for 30 minutes and up to 5 minutes. Slides were counterstained with hematoxylin, dehydrated in graded alcohol, and viewed under the microscope. The results are shown in FIG. 9.

(9) Uterine wet weight determination

30 female immature SD rats with 3 weeks of age and the weight of about 100g are adaptively bred for 7d before the experiment. Control, 0.1mg/kg estradiol, tamoxifen, compound 19, compound 13 were orally administered 1 time for 2 consecutive days. The uteri of each group of rats were collected the first day after the end of the experiment. Peripheral adipose tissues were removed, rat uterus was washed with PBS 2-3 times, drained, stored, and weighed. Recording the wet weight of the uterus and calculating the uterus coefficient. Uterus coefficient = wet weight of uterus (g)/weight of body (g) × 100, and the results are shown in fig. 10.

(10) Molecular docking

The crystal structure of docking analysis and molecular dynamics simulation ER alpha (PDB ID:1R 5K) was obtained from the PDB website; the protein was prepared from the protein preparation module in schrodinger Maestro 2019. Water is removed from the protein and polar hydrogen is added. Meanwhile, the protonation state of all residues is considered to be that His is protonated, and other residues such as Asp, arg, glu and Lys are pretreated to be in a deprotonation state. Generating a receptor grid by using a receptor grid generating method; the resulting binding site is the binding pocket for ER α, and comprises several key amino acid residues. The three-dimensional structure of the ligand was constructed with Chemdraw software and optimized with Schrodinger software. The ligand is then docked to the rigid protein using a docking module. The docking results were analyzed. Molecular dynamics simulations (100 ns) were performed using Desmond in Schrodinger Maestro 2019. The model is solvated by the system builder in orthogonal boxes that can transfer the water molecules of the interaction potential (TIP 3P) with an edge distance of

Sodium ions were added to neutralize the system. Calculations were performed using OPLS4 force field. Subsequently, a molecular dynamics simulation of 100ns was performed for each system in the NPT system. The temperature and pressure were kept constant with a langevin thermostat and a langevin gas pressure regulator, respectively. The resulting RMSD, RMSF, hydrogen bonding and hydrophobic interactions were analyzed throughout the traces by using the simulated interaction map module in schrodinger Maestro 2019, the results of which are shown in fig. 11-14.

FIG. 1 is a Western blot analysis; wherein the A, B, C graph is a western blot analysis showing estrogen receptor downregulation of compounds 1-21 at a concentration of 10 μmol/L compared to the known compounds fulvestrant and tamoxifen. Figure 1D is a western blot analysis showing estrogen receptor downregulation of compounds 13 and 19 compared to the known compound fulvestrant at different concentrations. The experiment proves that the compounds 13 and 19 can degrade estrogen receptors at 10 mu mol/L, are equivalent to the positive drug fulvestrant, and show good target protein degradation effect.

FIG. 2 is a diagram of immunofluorescence analysis; compounds 13 and 19 are shown to have an estrogen receptor down-regulated immunofluorescence assay at a concentration of 10 μmol/L compared to the known compound fulvestrant. Compounds 13 and 19 were shown to degrade estrogen receptors in vitro, comparable to the positive drug fulvestrant.

FIG. 3 shows a bar graph of the effect of compounds 13 and 19 on fulvestrant and tamoxifen and control on the activity of ERE luciferase transcription in MCF-7 cells, the y-axis is the relative ERE luciferase transcription activity, the x-axis is compounds 13 and 19 on fulvestrant and tamoxifen and control; this experiment shows that compounds 13 and 19 can inhibit estrogen receptor signaling pathway transduction and inhibit tumor proliferation.

FIG. 5 shows that compounds 13 and 19 can inhibit estrogen signaling pathway transduction and inhibit breast cancer cell proliferation.

The experiment in figure 6 shows that compound 19 has very excellent pharmacokinetic properties and good in vivo metabolic properties.

The experiment in figure 7 proves that the compounds 13 and 19 have better effect than the positive medicaments fulvestrant and tamoxifen in the in vivo animal tumor-bearing experiment and have the property of becoming candidate medicaments;

figure 8 demonstrates that compounds 13 and 19 are less toxic and have less short-term toxicity to heart, liver, spleen and kidney.

Fig. 9 shows that compounds 13 and 19 can down-regulate Ki67, ER and PR receptors in vivo, and have better in vivo effect.

The experiment of figure 10 shows that compounds 13 and 19 do not have an agonistic effect on the endometrium, are full antagonists, and can prevent the onset of endometrial cancer.

The experiments in fig. 11 and 12 show that the compounds 13 and 19 can be well docked with estrogen receptors, and the compounds 13 and 19 are proved to act on the estrogen receptors from the perspective of molecular simulation.

FIGS. 13 and 14 show that compounds 13 and 19 can interface well with estrogen receptors and can form effects with key amino acids of estrogen receptors.

Claims (10)

1. A selective estrogen receptor down-regulator compound having the structure:

wherein:

m is 0, 1, 2, 3 or 4;

n is 0, 1, 2, 3 or 4;

x is selected from-O-, -CH ₂ -、-S-、-NR ₁₇ 、-CHF-、-CF ₂ -or a cycloalkyl group;

y is selected from-C (O) -, -CF ₂ -, cycloalkyl, -CH ₂ -or-CHF-;

ring A is selected from phenyl, biphenylyl, naphthyl, quinolinyl, 5-or 6-membered monocyclic heteroaryl, cycloalkyl, or 7-, 8-, 9-, or 10-membered bicyclic heterocyclyl;

ring B is selected from phenyl, heteroaryl, thiophenyl, 5-or 6-membered monocyclic heteroaryl, cycloalkyl or 7-, 8-, 9-or 10-membered bicyclic heterocyclyl;

R ₁ selected from-CH = CHCOOR ₁₇ 、-NR ₁₇ (CO)COOR ₁₇ 、-COOR ₁₇ cycloalkyl-COOR ₁₇ 、-C ₂ -C ₆ alkenylene-COOR ₁₇ 、-C ₂ -C ₆ alkynylene-COOR ₁₇ 、-CH＝CHC(O)R ₁₆ 、-NR ₁₇ (CO)C(O)R ₁₆ 、-C(O)R ₁₆ -cycloalkyl-C (O) R ₁₆ 、-C ₂ -C ₆ alkenylene-C (O) R ₁₆ and-C ₂ -C ₆ alkynylene-C (O) R ₁₆ ；

R ₂ And R ₃ Is selected from-OR ₁₅ 、-SR ₁₅ 、-N(R ₁₅ ) ₂ Hydrogen, aryl, heteroaryl, halogen, -CN, -NO ₂ Haloalkyl, cycloalkyl, thiol, nitroso, C ₁ -C ₆ Alkyl, hydroxy, -O (C) ₁ -C ₆ Alkyl), -O (C) ₁ -C ₆ Fluoroalkyl group), -SF ₅ 、-B(OH) ₂ 、-B(OR ₁₅ ) ₂ 、-C(O)OR ₁₅ 、-C(O)R ₁₆ 、-C(S)R ₁₆ 、-OSO ₂ OR ₁₅ 、-OSO ₂ R ₁₆ 、-NHSO ₂ OR ₁₅ 、-NHSO ₂ R ₁₆ -N (alkyl) SO ₂ OR ₁₅ -N (alkyl) SO ₂ R ₁₆ 、-OP(O)(OR ₁₅ ) ₂ 、-OP(O)(R ₁₆ ) ₂ 、-P(O)(OR ₁₅ ) ₃ 、-P(O)(R ₁₆ ) ₃ 、-P(O)OR ₁₅ 、-P(O)R ₁₆ 、-SO ₂ R ₁₆ 、-SO ₂ OR ₁₅ Alkyne, alkene, arylalkyl, aryloxy, heteroarylalkyl or C ₁ -C ₆ A fluoroalkyl group;

R ₁₅ selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl;

R ₁₆ is selected from-N (R) ₁₅ ) ₂ 、-SR ₁₅ OR-OR ₁₅ ；

R ₁₇ Selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, -C (O) R ₁₅ 、-C(S)R ₁₅ 、-C(O)R ₁₆ 、-C(S)R ₁₆ Or a heteroaryl group.

2. The selective estrogen receptor down-regulator compound of claim 1, wherein R is ₁ Selected from-COOH, -NH (CO) COOH or-CH = CHCOOH.

3. The selective estrogen receptor down-regulator compound of claim 1, wherein ring a is phenyl, biphenyl, naphthyl or quinolyl.

4. The selective estrogen receptor down-regulator compound of claim 1, wherein ring B is phenyl or 5-or 6-membered monocyclic heteroaryl.

5. A selective estrogen receptor down-regulator compound according to any one of claims 1 to 4 being any one of:

6. a pharmaceutical composition comprising a therapeutically effective amount of one or more selective estrogen receptor down-regulator compounds of any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. Use of a selective estrogen receptor down-regulator compound of any one of claims 1 to 5 in the manufacture of a medicament for the treatment of an estrogen-related disorder.

8. The use according to claim 7, wherein the estrogen-related disorder is breast cancer, ovarian cancer, endometrial cancer, prostate cancer, lung cancer, cancer that has metastasized to bone, or bone loss.

9. The use according to claim 7, wherein the breast cancer is hormone receptor positive metastatic breast cancer, tamoxifen resistant breast cancer, or triple negative breast cancer.

10. A process for the preparation of a selective estrogen receptor down-regulator compound according to any one of claims 1 to 5 by the following synthetic route:

scheme 1:

scheme 2:

scheme 3:

wherein R is ₁ 、R ₂ 、R ₃ M, n are as defined in claim 1, z is 0, 1, 2, 3 or 4;

ring A is selected from phenyl, biphenylyl, naphthyl, quinolinyl, 5-or 6-membered monocyclic heteroaryl, cycloalkyl, or 7-, 8-, 9-, or 10-membered bicyclic heterocyclyl;

ring B is selected from phenyl, heteroaryl, thiophenyl, 5-or 6-membered monocyclic heteroaryl, cycloalkyl or 7-, 8-, 9-or 10-membered bicyclic heterocyclyl;

R ₁ selected from-CH = CHCOOR ₁₇ 、-NR ₁₇ (CO)COOR ₁₇ 、-COOR ₁₇ cycloalkyl-COOR ₁₇ 、-C ₂ -C ₆ alkenylene-COOR ₁₇ 、-C ₂ -C ₆ alkynylene-COOR ₁₇ 、-CH＝CHC(O)R ₁₆ 、-NR ₁₇ (CO)C(O)R ₁₆ 、-C(O)R ₁₆ -cycloalkyl-C (O) R ₁₆ 、-C ₂ -C ₆ alkenylene-C (O) R ₁₆ and-C ₂ -C ₆ alkynylene-C (O) R ₁₆ ；

R ₂ And R ₃ Is selected from-OR ₁₅ 、-SR ₁₅ 、-N(R ₁₅ ) ₂ Hydrogen, aryl, heteroaryl, halogen, -CN, -NO ₂ Haloalkyl, cycloalkyl, thiol, nitroso, C ₁ -C ₆ Alkyl, hydroxy, -O (C) ₁ -C ₆ Alkyl), -O (C) ₁ -C ₆ Fluoroalkyl group), -SF ₅ 、-B(OH) ₂ 、-B(OR ₁₅ ) ₂ 、-C(O)OR ₁₅ 、-C(O)R ₁₆ 、-C(S)R ₁₆ 、-OSO ₂ OR ₁₅ 、-OSO ₂ R ₁₆ 、-NHSO ₂ OR ₁₅ 、-NHSO ₂ R ₁₆ -N (alkyl) SO ₂ OR ₁₅ -N (alkyl) SO ₂ R ₁₆ 、-OP(O)(OR ₁₅ ) ₂ 、-OP(O)(R ₁₆ ) ₂ 、-P(O)(OR ₁₅ ) ₃ 、-P(O)(R ₁₆ ) ₃ 、-P(O)OR ₁₅ 、-P(O)R ₁₆ 、-SO ₂ R ₁₆ 、-SO ₂ OR ₁₅ Alkyne, alkene, arylalkyl, aryloxy, heteroarylalkyl or C ₁ -C ₆ A fluoroalkyl group.

Images