An integrated diagnostic and therapeutic ERα PROTACs compound for detecting and degrading ERα, and its preparation method and application

Abstract

The present invention discloses an integrated diagnostic and therapeutic ERαPROTACs compound for detecting and degrading ERα, and its preparation method and application, which belong to the field of medical technology. The structure of the integrated diagnostic and therapeutic PROTAC compound of the present invention is shown in general formula (I), and the OBHSA containing a carboxyl linker is respectively reacted with a WUN29654 amino derivative with a different linker as a raw material to prepare the ERαPROTAC compound by a one-step amide reaction. Cytotoxicity, immunoblotting analysis and optical properties showed that the integrated diagnostic and therapeutic PROTAC compound of the present invention has significant anti-MCF-7 cell and mutant strain activity, ERα protein degradation activity, and a significant fluorescence emission signal at 400-500nm, which can be used to prepare anti-breast cancer drugs and / or breast cancer diagnostic drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to an integrated diagnostic and therapeutic ERαPROTACs compound for detecting and degrading ERα, as well as a preparation method and application thereof. Background Art

[0002] Breast cancer is the most common cancer in women worldwide, of which estrogen receptor-positive (ER+) breast cancer accounts for about 70% of all breast cancers. With estrogen receptor α (ERα) as the target, endocrine therapy is mainly used clinically, that is, to exert anti-ERα+ breast cancer effects by reducing estrogen levels or blocking the estrogen receptor (ER) signaling pathway. However, long-term use of endocrine therapy can easily lead to primary or secondary drug resistance, which has become a major challenge in the treatment of ERα+ breast cancer. Therefore, it is particularly important to find new anti-resistance breast cancer drugs with diverse structures and functions, high efficiency and low toxicity, so as to solve clinical drug resistance and improve the treatment effect of ERα+ breast cancer.

[0003] Endocrine therapy, as a first-line treatment option for patients with ERα+ breast cancer, primarily exerts its anti-breast cancer effects by reducing estrogen levels or blocking ER signaling pathways. Over the past 30 years, "endocrine therapy" for ER+ breast cancer has included aromatase inhibitors, such as letrozole, which inhibit endogenous estrogen production; selective estrogen receptor modulators, such as tamoxifen, which bind to and modulate ER activity; and selective estrogen receptor degraders, such as fulvestrant, which act as full antagonists and induce ER degradation. However, long-term use of endocrine therapy often leads to the development of primary or secondary resistance. Recent advances have been made in understanding the molecular pharmacology of ERα and the mechanisms that influence its action and promote endocrine resistance. Conclusive evidence indicates that ERα continues to play a crucial role in advanced breast cancer, driving disease progression and remaining a potential therapeutic target. The successful clinical application of fulvestrant further demonstrates that strategies to degrade ERα protein offer a promising approach to overcoming resistance to endocrine therapy.

[0004] Proteolysis targeting chimera (PROTAC) technology is an emerging drug development strategy developed based on the ubiquitin-proteasome system. This technology constructs a heterologous bifunctional molecule formed by coupling a protein of interest ligand (POI ligand), a linker (Linker) and an E3 ubiquitin ligase ligand (E3 ligand), inducing the target protein and the E3 ligase to be close to each other in space, so that the former is ubiquitinated and degraded, thereby clearing the pathogenic protein and playing a role in treating the disease. This technology has great advantages over traditional small molecule inhibitors. For example, it has low requirements for binding affinity, which enables it to target undruggable proteins and overcome target resistance mutations. In addition, it also has the characteristics of small dosage, high activity, high selectivity and low toxicity. To date, more than 10 PROTACs have entered clinical trials, such as ARV-110, ARV-471, DT2216, etc.

[0005] Despite the success of PROTACs, real-time detection and tracking of protein degradation remains difficult. Currently, most methods for assessing protein degradation activity rely on Western Blot. However, more convenient visualization tools will help develop more effective analytical methods and help better understand the mechanisms of protein degradation. Therefore, new strategies need to be developed to visualize the protein degradation process and improve diagnostic and therapeutic effects. In view of this, using PROTAC technology to develop a series of compounds that have the ability to detect and degrade ERα simultaneously for visualizing the protein degradation process and improving diagnostic and therapeutic effects will be a potential targeted treatment strategy for breast cancer. Summary of the Invention

[0006] The present invention aims to overcome the deficiencies of the prior art and provide a PROTAC compound that simultaneously detects and degrades ERα, namely, an integrated diagnostic and therapeutic ERα PROTAC compound. These compounds have significant anti-breast cancer activity and fluorescent properties, enabling visualization of ERα protein degradation. The present invention also aims to provide applications of the integrated diagnostic and therapeutic ERα PROTAC compound, which can be developed as a new anti-cervical cancer drug with broad application prospects. The present invention also aims to provide a method for preparing the integrated diagnostic and therapeutic ERα PROTAC compound.

[0007] In order to achieve the above object, the technical solutions adopted by the present invention are as follows:

[0008] In the first aspect, an integrated diagnostic and therapeutic ERαPROTACs compound represented by general formula (I) or a pharmacologically or physiologically acceptable salt thereof is provided.

[0009]

[0010] Among them, Linker is selected from n=an integer of 1-5, m=an integer of 1-7, and x=an integer of 1-3.

[0011] The present invention found through in vitro anti-breast cancer activity testing that the above-mentioned ERαPROTACs compound can degrade ERα in a concentration-dependent manner, and at the same time has significant anti-proliferative activity against breast cancer cells, and can be used to prepare anti-breast cancer drugs.

[0012] Preferably, the integrated diagnostic and therapeutic ERαPROTACs compound is selected from the compounds shown in Table 1 below:

[0013] Table 1

[0014]

[0015]

[0016]

[0017] In a second aspect, the present invention provides the use of any one of the above-mentioned integrated diagnostic and therapeutic ERαPROTACs compounds or pharmacologically or physiologically acceptable salts thereof in the preparation of anti-breast cancer drugs and / or breast cancer diagnostic drugs.

[0018] In a third aspect, a drug is provided, comprising one or more of the aforementioned integrated diagnostic and therapeutic ERα PROTAC compounds or pharmacologically or physiologically acceptable salts thereof. The drug may further comprise pharmaceutically acceptable excipients and may be prepared according to conventional pharmaceutical techniques. The drug is an anti-breast cancer drug or a breast cancer diagnostic drug.

[0019] In a fourth aspect, a method for preparing an integrated diagnostic and therapeutic ERα PROTAC compound of formula (I) is provided, comprising the following steps: an amino-linker-containing WUN29654 derivative and a carboxyl-linker-containing OBHSA derivative undergo an amide condensation reaction in an organic solvent in the presence of a condensing agent and a base to obtain the PROTAC compound. The synthetic routes for some ERα PROTAC compounds are as follows:

[0020]

[0021] Synthetic routes of integrated diagnostic and therapeutic ERα PROTACs compounds

[0022] Preferably, in the method for preparing the integrated diagnostic and therapeutic ERαPROTACs compound represented by the general formula (I), the condensing agent comprises one or more of HATU, HBTU, PyBOP, and DCC; the base comprises one or more of DIPEA, Et3N, Na2CO3, and NaHCO3; and the organic solvent comprises one or more of DMF, DCM, DMC, and 2-MeTHF. The molar ratio of the OBHSA derivative, WUN29654 derivative, condensing agent, and base is 1.0-1.8:1:1.1-3.0:3.0-6.0.

[0023] Advantages and benefits of the present invention: The integrated diagnostic and therapeutic ERαPROTACs compounds of the present invention exhibit excellent anti-breast cancer activity and fluorescent properties, enabling visualization of ERα protein degradation. Some compounds exhibit submicromolar inhibitory activity. The integrated diagnostic and therapeutic ERαPROTACs compounds of the present invention can be developed as new anti-breast cancer drugs, with broad application prospects for simultaneously improving diagnostic and therapeutic efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Immunoblotting analysis was used to detect the degradation activity of WF-1, 3, 4, and 5 on ERα protein in MCF-7 cells.

[0025] Figure 2 : Immunoblotting analysis was used to detect the degradation activity of WF-2 on ERα protein in MCF-7 cells.

[0026] Figure 3 : Fluorescence emission spectra of ERαPROTACs (WF-1–WF-5).

[0027] Figure 4 : Microscopic observation of WF-2 imaging in cells. DETAILED DESCRIPTION

[0028] The features and advantages of the present invention can be further understood through the following detailed description. The examples provided are merely illustrative of the method of the present invention and are not intended to limit the remainder of the present invention in any way.

[0029] The preparation of the integrated diagnostic and therapeutic ERα PROTACs compound represented by general formula (I) of the present invention specifically comprises the following steps:

[0030] 1. 2-(4-methoxyphenyl)-2-oxoethyl 2-(4-methylphenyl) acetate (3) was synthesized by the reaction shown in the following formula (1) and used as the raw material for the next reaction.

[0031]

[0032] First, commercially available 2-bromo-1-(4-bromophenyl)ethane-1-one 1 (1.59 g, 6.94 mmol) and p-methoxyphenylacetic acid 2 (1.15 g, 6.94 mmol) were weighed into a 100 mL round-bottom flask. 25 mL of anhydrous acetonitrile was added, and anhydrous triethylamine (0.7 mg, 6.94 mmol) was slowly added dropwise. The reaction was continued at room temperature for 12 h. TLC monitored the reaction to be complete. After the reaction was completed, the acetonitrile and triethylamine were removed by concentration under reduced pressure. Ethyl acetate was added for dissolution, and the mixture was washed with dilute hydrochloric acid (2 M, 30 mL), saturated sodium bicarbonate (2×30 mL), and saturated sodium chloride (30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and dried to obtain the crude product, which was purified by column chromatography using petroleum ether / ethyl acetate (V / V=30 / 1) as the mobile phase to obtain compound 3 as a white solid in an 86% yield.

[0033] 2. 3,4-bis(4-methoxyphenyl)furan-2(5H)-one (4) is synthesized by the reaction shown in the following formula (2) and used as the raw material for the next reaction.

[0034]

[0035] The raw material 3 (1.00 g, 3.18 mmol) obtained by reaction (1) was dissolved in anhydrous acetonitrile (6 mL), and commercially available 1,8-diazacyclo[5,4,0]undecene-7 (DBU) (0.97 g, 6.36 mmol) was added in sequence. The reaction system was placed in an ice bath and stirred for 3 h. After TLC monitoring, the raw material disappeared completely. The reaction system was extracted with DCM, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and spin-dried to obtain a crude product. The crude product was purified by column chromatography with a mobile phase of petroleum ether / ethyl acetate (V / V = 10 / 1) to obtain a light yellow solid product 4 (0.58 g, 1.97 mmol) with a yield of 62%.

[0036] 3. 3,4-bis(4-hydroxyphenyl)furan-2(5H)-one (5) is synthesized by the reaction shown in the following formula (3) and used as the raw material for the next reaction.

[0037]

[0038] The raw material 4 (1.26 g, 4.25 mmol) obtained by reaction (2) was placed in a 100 mL round-bottom flask, and 25 mL of DCM was added. BBr3 (2.6 mL, 27.33 mmol) was added at -20°C and reacted for 12 h. Then, 10 mL of water was added to quench the reaction, and the mixture was extracted with ethyl acetate (3×20 mL), washed with saturated NaHCO3 solution (15 mL), and the organic layer was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain the crude product, which was purified on a silica gel column with a mobile phase of petroleum ether / ethyl acetate = 3:1 to obtain 0.93 g of compound 5 with a yield of 82%.

[0039] 4. 4,4'-(furan-3,4-diyl)diphenol (6) is synthesized by the reaction shown in the following formula (4) and used as the raw material for the next reaction.

[0040]

[0041] The raw material 5 (1.12 g, 4.17 mmol) obtained by reaction (3) was placed in a 50 mL round-bottom flask, and the mixture was evacuated to anhydrous and oxygen-free conditions. Diisobutylaluminum hydride (DIBAL-H, 8 mL, 7.93 mmol) was added at -78°C and allowed to react for 12 h. The reaction was then quenched by adding 2 M HCl. The mixture was then extracted with ethyl acetate (3 × 25 mL), washed with saturated NaCl solution (30 mL), and the organic layer was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain the crude product, which was purified on a silica gel column using a mobile phase of petroleum ether / ethyl acetate = 3:1 to obtain 0.44 g of compound 6 with a yield of 42%.

[0042] 5. N-(4-Hydroxyphenyl)-N-(2,2,2-trifluoroethyl)prop-2-ene-1-sulfonamide (12) was synthesized by the following reaction and used as the raw material for the next reaction.

[0043]

[0044] 4-Methoxyaniline 7 (0.5 g, 4.06 mmol) was dissolved in DCM, and DMAP (0.05 g, 0.41 mmol) and trifluoroacetic anhydride 8 (0.90 g, 4.26 mmol) were added at 0°C, and the mixture was stirred at room temperature for 4 h to obtain compound 9. Compound 9 (0.89 g, 4.06 mmol) was dissolved in THF and reduced with borane dimethyl sulfide (0.61 g, 8.12 mmol) at 65 ° C to give compound 10. Compound 10 (1.0 g, 4.87 mmol) was reacted with dichloroethanesulfonyl chloride (0.95 g, 5.84 mmol) and triethylamine (1.48 g, 14.61 mmol) in DCM solvent at room temperature to give compound 11. Compound 11 (1.00 g, 3.39 mmol) was reacted with BBr3 (1.70 g, 6.77 mmol) in DCM solvent at -20 ° C to give compound 12.

[0045] 6. The tert-butyloxycarbonylamino compound 15a-c with a p-toluenesulfonyl group was synthesized by the following reaction and used as the raw material for the next reaction.

[0046]

[0047] First, various commercially available compounds 13a-c (1.0 equiv) were dissolved in DCM and reacted with (Boc)2O (1.2 equiv) in the presence of TEA (0.1 equiv) under ice-cold conditions to yield compounds 14a-c. Compounds 14a-c (1.0 equiv) were then reacted with p-toluenesulfonyl chloride (1.2 equiv), DMAP (0.2 equiv), and TEA (2.0 equiv) in DCM at room temperature to yield compounds 15a-c.

[0048] 7. The tert-butyloxycarbonylamino compound 15d with a p-toluenesulfonyl group was synthesized by the following reaction and used as the starting material for the next reaction.

[0049]

[0050] First, commercially available compound 13d (1.0 equiv.) was dissolved in DCM and reacted with (Boc)2O (1.2 equiv.) in the presence of TEA (0.1 equiv.) under ice-cold conditions to yield compound 14d. The product was then extracted with DCM (3 × 25 mL), washed with saturated NaCl (30 mL), and the organic layer dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to yield the crude product, which was used directly in the next step without further purification. Compound 14d (1.0 equiv.) was reacted with p-toluenesulfonyl chloride (1.2 equiv.), DMAP (0.2 equiv.), and TEA (2.0 equiv.) in DCM at room temperature to yield compound 15d. The product was then extracted with DCM (3 × 25 mL), washed with saturated NaCl (30 mL), and the organic layer dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to yield the crude product, which was purified on a silica gel column using a mobile phase of petroleum ether / ethyl acetate (3:1) in a yield of 78%.

[0051] 8. Compound 18 (WUN29654) was synthesized by the following reaction and used as the raw material for the next reaction.

[0052]

[0053] First, commercially available 3-hydroxy-1,8-naphthalene dicarboxylic anhydride 16 (1.21 g, 5.65 mmol) and 3-amino-2,6-piperidinedione 17 (0.72 g, 5.65 mmol) were weighed into a 100 mL round-bottom flask and dissolved in 10 mL of anhydrous DMF. K₂CO₃ (1.56 g, 11.3 mmol) was then added. The reaction was continued at room temperature for 12 h, and TLC monitored the reaction for completion. The product was then extracted with EA (3 × 25 mL) and washed with saturated NaCl solution (30 mL). The organic layer was dried over anhydrous sodium sulfate and stripped under reduced pressure to afford the crude product. Purification was performed on a silica gel column using a mobile phase of petroleum ether / ethyl acetate (1:1) to yield 1.32 g of the yellow solid product 18, a 72% yield.

[0054] 9. Compounds 19a-c were synthesized by the following reactions and used as starting materials for the next step.

[0055]

[0056] In a 50 mL round-bottom flask, the above-obtained product 18 (1.0 eq. vol.) and the p-toluenesulfonyl-containing tert-butyloxycarbonylamino compounds 15a-c (1.2 eq. vol.) were weighed and dissolved in 3 mL of anhydrous DMF. KCO (2.0 eq. vol.) was added and allowed to react at 50°C for 12 h. TLC monitored the reaction for completion. The mixture was then extracted with EA (3 x 25 mL) and washed with saturated NaCl solution (30 mL). The organic layer was dried over anhydrous sodium sulfate and stripped under reduced pressure to afford the crude product. Purification on a silica gel column using a mobile phase of petroleum ether / ethyl acetate (1:1) afforded the products 19a-c as pale yellow solids in yields of 40-60%.

[0057] 10. Compound 19d was synthesized by the following reaction and used as the starting material for the next reaction.

[0058]

[0059] In a 50 mL round-bottom flask, the above-obtained product 18 (0.30 g, 0.93 mmol) and the p-toluenesulfonyl-containing tert-butyloxycarbonylamino compound 15d (0.40 g, 1.12 mmol) were weighed and dissolved in 3 mL of anhydrous DMF. KCO (0.25 g, 1.86 mmol) was then added and allowed to react at 50°C for 12 h. TLC monitored the reaction for completion. The product was then extracted with EA (3 × 25 mL) and washed with saturated NaCl solution (30 mL). The organic layer was dried over anhydrous sodium sulfate and stripped under reduced pressure to afford the crude product. The product 19d was purified on a silica gel column using a mobile phase of petroleum ether / ethyl acetate (1:1) to afford a pale yellow solid in a 55% yield.

[0060] 11. Compounds 20a-c were synthesized by the following reaction and used as starting materials for the next step.

[0061]

[0062] In a 25 mL round-bottom flask, the above-obtained product 19a-c (1.0 equv.) was weighed and dissolved in 1 mL of anhydrous DCM. 0.5 mL of CF3COOH was added under ice-cooling. The mixture was allowed to react at room temperature for 30 minutes. Completion of the reaction was monitored by TLC. The product 20a-c was then spin-dried to give a pale yellow solid in 100% yield. This was used directly as the starting material for the next reaction.

[0063] 12. Compound 20d was synthesized by the following reaction and used as the starting material for the next reaction.

[0064]

[0065] In a 25 mL round-bottom flask, the above-obtained product 19d (1.0 equv.) was weighed and dissolved in 1 mL of anhydrous DCM. 0.5 mL of CF3COOH was added under ice-cooling. The mixture was allowed to react at room temperature for 30 min. Completion was monitored by TLC. The product 20d was then spin-dried to give a pale yellow solid in 100% yield, which was used directly as the starting material for the next reaction.

[0066] 13. Compounds 22a-b were synthesized by the following reaction and used as starting materials for the next step.

[0067]

[0068] In a 50 mL round-bottom flask, the above-obtained product 12 (1.0 eq. vol.) and commercially available tert-butyl bromoacetate or tert-butyl 6-bromohexanoate 21a-b (1.2 eq. vol.) were weighed and dissolved in 3 mL of anhydrous DMF. KCO (2.0 eq. vol.) and KI (0.1 eq. vol.) were then added. The mixture was allowed to react at room temperature for 12 h, and the reaction was monitored for completion by TLC. The mixture was then extracted with EA (3 x 25 mL) and washed with saturated NaCl solution (30 mL). The organic layer was dried over anhydrous sodium sulfate and stripped under reduced pressure to afford the crude product. Purification on a silica gel column using a mobile phase of petroleum ether / ethyl acetate (5:1) afforded the product 22a-b as a pale yellow solid in a yield of 70-80%.

[0069] 14. Compound 23a-b was synthesized by the following reaction and used as the starting material for the next reaction.

[0070]

[0071] In a 25 mL round-bottom flask, the above-obtained product 22a-b (1.0 equv.) and compound 6 (1.0 equv.) obtained from formula (4) were weighed. After evacuating the anhydrous and oxygen-free solution, a small amount of anhydrous THF was added. The reaction was allowed to proceed at 90°C for 12 h. TLC monitored the reaction for completion. The product was then extracted with EA (3 × 25 mL) and washed with saturated NaCl solution (30 mL). The organic layer was dried over anhydrous sodium sulfate and desolvated under reduced pressure to obtain the crude product. The product 23a-b was purified on a silica gel column using a mobile phase of petroleum ether / ethyl acetate (3:1) to obtain a pale yellow solid product in a yield of 40-55%.

[0072] 15. Compound 24a-b was synthesized by the following reaction and used as the starting material for the next reaction.

[0073]

[0074] In a 25 mL round-bottom flask, the above-obtained product 23a-b (1.0 equv.) was weighed and dissolved in 1 mL of anhydrous DCM. 0.3 mL of CF3COOH was added under ice-cooling. The mixture was allowed to react at room temperature for 30 min. Completion of the reaction was monitored by TLC. The product 24a-b was then spin-dried to give a white solid in 100% yield, which was used directly as the starting material for the next reaction.

[0075] 16. The integrated diagnostic and therapeutic ERαPROTACs compounds (WF1-WF3, WF-5) were synthesized through the following reaction.

[0076]

[0077] Taking the preparation of 2-(4-(((1S,2R,4S)-5,6-bis(4-hydroxyphenyl)-N-(2,2,2-trifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene)-2-sulfonamido)phenoxy)-N-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-5-yl)oxy)butyl)acetamide WF-1 as an example, the steps are as follows: compound 24a (0.10 g, 0.17 mmol) and compound 20a (0.0 61 g, 0.15 mmol), HATU (0.086 g, 0.23 mmol), and DIPEA (0.099 mL, 0.6 mmol) were placed in a 25 mL round-bottom flask, anhydrous DMF was added, and the mixture was stirred overnight at room temperature. After the complete disappearance of the raw material 20a as monitored by TLC, it was extracted with EA, dried over anhydrous sodium sulfate, and concentrated, and then purified by column chromatography with a mobile phase ratio of dichloromethane / methanol (V / V = 10:1) to obtain a white solid product WF-1 (0.096 g, 0.10 mmol) in a yield of 58%.

[0078] The preparation method of the integrated diagnostic and therapeutic ERαPROTACs compounds WF-2, WF-3 and WF-5 is the same as above.

[0079] 17. The integrated diagnostic and therapeutic ERαPROTAC compound (WF-4) was synthesized by the following reaction.

[0080]

[0081] Compound 24a (0.10 g, 0.17 mmol), 20d (0.06 g, 0.15 mmol), HATU (0.086 g, 0.23 mmol), and DIPEA (0.1 mL, 0.60 mmol) were weighed and placed in a 25 mL round-bottom flask. Anhydrous DMF was added and stirred overnight at room temperature. After the complete disappearance of the raw material 20d as monitored by TLC, the product was extracted with EA, dried over anhydrous sodium sulfate, and concentrated. After purification by column chromatography, the mobile phase ratio was dichloromethane / methanol (V / V = 10:1) to give a white solid product WF-4 (0.084 g, 0.08 mmol) in a yield of 50%.

[0082] [Example 1] Preparation of 2-(4-(((1S,2R,4S)-5,6-bis(4-hydroxyphenyl)-N-(2,2,2-trifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene)-2-sulfonamido)phenoxy)-N-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-5-yl)oxy)butyl)acetamide WF-1.

[0083] Compound 24a (0.10 g, 0.17 mmol), 20a (0.061 g, 0.15 mmol), HATU (0.086 g, 0.23 mmol), and DIPEA (0.099 mL, 0.6 mmol) were weighed and placed in a 25 mL round-bottom flask. Anhydrous DMF was added and stirred overnight at room temperature. After complete disappearance of the starting material 20a as monitored by TLC, the product was extracted with EA, dried over anhydrous sodium sulfate, and concentrated. After purification by column chromatography, the mobile phase ratio was dichloromethane / methanol (V / V = 10:1) to give a white solid product WF-1 (0.096 g, 0.10 mmol) in a yield of 58%.

[0084] 1H NMR (600MHz, DMSO-d6) δ10.98(s,1H),9.63(s,1H),9.57(s,1H),8.25(d,J=8.3Hz,1H),8.19(d,J=7.2Hz,1H),8 .12(d,J=4.5Hz,1H),7.95(dd,J=66.3,2.6Hz,1H),7.85(dd,J=5.6,2.6Hz,1H),7.73(dt,J=10.7,7.7Hz,1H),7. 25(dd,J=9.0,3.1Hz,2H),7.06(dd,J=18.1,7.9Hz,4H),6.86(dd,J=9.0,3.1Hz,2H),6.70(d,J=8.2Hz,2H),6.63 (d,J=8.0Hz,2H),5.77(dd,J=11.7,5.4Hz,1H),5.42(s,1H),5.26(d,J=4.1Hz,1H),4.44(t,J=7.8Hz,4H),4.17–

[0085] 4.09(m,2H),3.48(t,J=4.1Hz,1H),3.19(d,J=6.9Hz,2H),2.89(ddd,J=22.1,12.0,5.1Hz,1H),2.5 5(dq,J=15.1,4.6Hz,2H),1.99(p,J=6.1,5.3Hz,1H),1.74(h,J=6.7Hz,2H),1.60(h,J=7.4Hz,2H).

[0086] [Example 2] Preparation of 2-(4-(((1S,2R,4S)-5,6-bis(4-hydroxyphenyl)-N-(2,2,2-trifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene)-2-sulfonamido)phenoxy)-(N-6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-benzo[d]isoquinolin-5-yl)oxy)hexyl)acetamide WF-2.

[0087] The preparation method is the same as that described in Example 1, except that the raw material 20a is replaced by the raw material 20b, and a white solid product WF-2 is obtained with a yield of 83%.

[0088] 1H NMR (600MHz, DMSO-d6) δ10.97(s,1H),9.63(s,1H),9.58(s,1H),8.31(dt,J=8.3,4.0H z,1H),8.23(d,J=7.4Hz,1H),8.05(d,J=2.7Hz,1H),7.92(dd,J=6.8,3.2Hz,1H),7.81 –7.72(m,2H),7.26(d,J=8.7Hz,2H),7.11–7.04(m,4H),6.88–6.80(m,2H),6.70(dd,J =8.6,2.1Hz,2H),6.65(dd,J=8.5,3.5Hz,2H),5.78(dd,J=11.5,5.4Hz,1H),5.42(d,J =1.2Hz,1H),5.27(d,J=3.8Hz,1H),4.50–4.43(m,2H),4.42(d,J=2.2Hz,1H),4.21–4. 09(m,3H),3.57(dt,J=6.6,3.3Hz,1H),3.49(ddd,J=7.4,4.9,1.6Hz,1H),3.09(d,J=9 .4Hz,2H),2.99(t,J=5.2Hz,1H),2.91–2.86(m,1H),2.57(t,J=3.0Hz,1H),2.55–2.52 (m,1H),1.77(dd,J=11.5,5.3Hz,4H),1.41(dt,J=20.7,7.6Hz,4H),1.34–1.29(m,2H).

[0089] [Example 3] Preparation of 2-(4-(((1S,2R,4S)-5,6-bis(4-hydroxyphenyl)-N-(2,2,2-trifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene)-2-sulfonamido)phenoxy)-N-(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-5-yl)oxy)octyl)acetamide WF-3.

[0090] The preparation method is the same as that described in Example 1, except that the raw material 20a is replaced by the raw material 20c, and a light yellow solid product WF-3 is obtained with a yield of 42%.

[0091] 1H NMR (600MHz, DMSO-d6) δ11.01(s,1H),9.67(s,1H),9.62(s,1H),8.36–8.16(m,2H),8.10–7.96(m,3H),7.86–7.80(m,1H),7.29(d,J=8.6H z,2H),7.15–7.08(m,4H),6.89(d,J=8.8Hz,2H),6.76–6.72(m,2H),6.70–6.67(m,2H),5.82(dt,J=11.3,5.3Hz,1H),5.46(d,J=1.3Hz,1H ),5.33–5.30(m,1H),4.54–4.47(m,2H),4.45(s,2H),4.20(q,J=6.1Hz,2H),3.52(dd,J=7.7,4.8Hz,1H),3.13–3.08(m,2H),2.96–2.90(m ,1H),2.58(t,J=4.9Hz,1H),2.07–1.96(m,2H),1.95–1.87(m,2H),1.83–1.77(m,2H),1.44(dt,J=20.1,7.5Hz,4H),1.29(d,J=3.2Hz,2H).

[0092] [Example 4] Preparation of 6-(4-(((1S, 2R, 4S)-5,6-bis(4-hydroxyphenyl)-N-(2,2,2-trifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene)-2-sulfonamido)phenoxy)-N-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-benzo[d]isoquinolin-5-yl)oxy)hexyl)hexanamide WF-5.

[0093] The preparation method is the same as that described in Example 1, except that raw material 20a is replaced by raw material 20c, and raw material 24a is replaced by raw material 24b, to obtain a light yellow solid product WF-5 with a yield of 68%.

[0094] 1H NMR (600MHz, DMSO-d6) δ11.03(s,1H),9.70(s,1H),9.64(s,1H),8.41–8.17(m,2H),8.06–7.93(m,1H),7.90(dt,J=5.3,2.5Hz,1H),7. 82–7.72(m,2H),7.25(d,J=8.6Hz,2H),7.12(ddt,J=18.2,6.6,2.1Hz,4H),6.84–6.79(m,2H),6.75(d,J=8.5Hz,2H),6.69(dd,J=8.7,2 .4Hz,2H),5.83(dt,J=11.3,5.1Hz,1H),5.48(d,J=1.3Hz,1H),5.32(dd,J=4.2,1.4Hz,1H),4.49(dd,J=8.9,4.7Hz,2H),4.20–4.11(m, 2H),3.94–3.86(m,2H),3.51(dd,J=8.1,4.6Hz,1H),3.09–3.02(m,2H),2.99–2.89(m,1H),2.64–2.55(m,2H),2.11–2.02(m,3H),1.98–

[0095] 1.89(m,2H),1.83–1.76(m,2H),1.71–1.65(m,2H),1.57–1.52(m,2H),1.49–1.41(m,4H),1.36(d,J=11.6Hz,4H).

[0096] [Example 5] Preparation of (2-(4-(((1S,2R,4S)-5,6-bis(4-hydroxyphenyl)-N-(2,2,2-trifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene)-2-sulfonamido)phenoxy)-N-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-5-yl)oxy)ethoxy)ethyl)acetamide WF-4.

[0097] Compound 24a (0.10 g, 0.17 mmol), 20d (0.06 g, 0.15 mmol), HATU (0.086 g, 0.23 mmol), and DIPEA (0.1 mL, 0.60 mmol) were weighed and placed in a 25 mL round-bottom flask. Anhydrous DMF was added and stirred overnight at room temperature. After the complete disappearance of the raw material 20d as monitored by TLC, the product was extracted with EA, dried over anhydrous sodium sulfate, and concentrated. After purification by column chromatography, the mobile phase ratio was dichloromethane / methanol (V / V = 10:1) to give a white solid product WF-4 (0.084 g, 0.08 mmol) in a yield of 50%.

[0098] 1 H NMR(600MHz,DMSO-d6)δ11.03(s,1H),9.69(s,1H),9.64(s,1H),8.39–8.24( m,2H),8.16(q,J=4.3,3.3Hz,1H),8.05(dd,J=66.1,2.6Hz,1H),7.95(t,J=3. 2Hz,1H),7.83–7.76(m,1H),7.35–7.26(m,2H),7.17–7.07(m,4H),6.92–6.85 (m,2H),6.78–6.72(m,2H),6.71–6.68(m,2H),5.83(dd,J=11.7,5.9Hz,1H),5 .47(d,J=1.2Hz,1H),5.34–5.28(m,1H),4.57–4.44(m,4H),4.36–4.29(m,2H ),3.87–3.80(m,2H),3.57(q,J=6.0Hz,2H),3.53(td,J=5.7,3.1Hz,1H),3.35 (t,J=5.8Hz,2H),2.93(ddt,J=20.2,17.5,4.9Hz,1H),2.60(dq,J=14.6,5.4, 4.8Hz, 2H), 2.05 (dq, J=10.8, 5.2Hz, 1H), 1.93 (td, J=12.0, 10.1, 6.1Hz, 2H).

[0099] The chemical structures of the target compounds WF-1-WF-5 of the present invention synthesized above are shown in Table 2.

[0100] Table 2. Chemical structures of WF-1-WF-5

[0101]

[0102]

[0103] [Example 6]

[0104] 1. In vitro anti-breast cancer activity of ERαPROTACs compounds integrated with diagnosis and treatment

[0105] CCK-8 (Cell Counting Kit-8) is a widely used cell proliferation and cytotoxicity assay based on WST-8. WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonatophenyl)-2H-tetrazolium monosodium salt) is a compound similar to MTT (chemical name: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Under the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate, it is reduced by mitochondrial dehydrogenases to a highly water-soluble orange-yellow formazan product. The color becomes darker as the number and rate of cell proliferation increase, while the color becomes lighter as the cytotoxicity increases. For the same cell population, the color intensity is proportional to the number of viable cells, making this property a direct indicator of cell proliferation and cytotoxicity analysis.

[0106] The anti-breast cancer cancer cell proliferation activity of the integrated diagnostic and therapeutic ERα PROTACs compound was tested by CCK-8 assay. The specific steps are as follows:

[0107] 1) Preparation of cell suspension: breast cancer cells (MCF-7, MCF-7 EGFR , MCF-7 D538G , MCF-7 Y537S When the growth density reached 80%-90%, the cells were digested with 0.25% Trypsin-EDTA, fresh MEM medium was added, the cell suspension was mixed, and the cells were counted using a cell counting plate. The cells were diluted to 5×10 4 Single cell suspension at 100 cells / mL.

[0108] 2) Plating: 100 μL of single cell suspension was inoculated into a microplate (tissue culture grade, 96-well, flat bottom).

[0109] 3) Pre-culture: Incubate the cells in a 37°C, 5% CO2 incubator for about 24 hours.

[0110] 4) Drug Addition: Aspirate the original culture medium and add 200 μL of culture medium containing different concentrations (100 μM-0.01 μM) of the test drug to each well of the culture plate (set up 3 replicates for each concentration, and also set up a blank group), and incubate in the incubator for 96 hours.

[0111] 6) Plate collection: Prepare CCK-8 working solution at a ratio of 10 μL CCK-8 reagent per 100 μL culture medium. Add 100 μL CCK-8 working solution to each well and incubate the culture plate in an incubator for 2-3 hours.

[0112] 7) Plate Assay: Measure the absorbance (OD) at 450 nm using a microplate reader and calculate the IC of the compound against breast cancer cells 50 , the results are shown in Table 3.

[0113] Table 3. Anti-proliferative activity of ERα PROTACs (WF-1–WF-5) in breast cancer cells

[0114]

[0115] The above experimental results show that the synthesized ERαPROTACs compounds have good anti-breast cancer activity, and most of them have inhibitory activity against three MCF-7 mutants, such as compound WF-2 against MCF-7 EGFR (IC 50 =1.40±0.15μM), for MCF-7 D538G (IC 50 =4.31±2.18μM), for MCF-7 Y537S (IC 50 =9.29±2.7 μM).

[0116] 2. Degradation activity of ERα PROTACs compounds on ERα protein in MCF-7 cells

[0117] The degradation activity of the integrated diagnostic and therapeutic ERαPROTACs compounds on ERα protein was detected by immunoblotting analysis. The specific steps are as follows: MCF-7 cells were treated with different concentrations of ERαPROTAC compounds for 24 hours, and whole cell lysates were obtained using RIPA buffer and protein loading buffer. The protein concentration of the samples was analyzed using a BCA protein analysis kit (KR0008), and the volume of the sample for electrophoresis was adjusted according to the standard protein curve. The proteins were separated using 7.5% or 10% SDS-PAGE gels and transferred to 0.45 μM thick PVDE membranes (Millipore, 000027346). The membranes were blocked with 5% bovine serum albumin (BSA, KR9048-466-8) or 5% skim milk at room temperature for 2 hours, the primary antibodies were incubated at 4°C for more than 12 hours, and the secondary antibodies were incubated at room temperature for more than 1 hour on a shaker. Proteins were detected using ultrasensitive enhanced chemiluminescence (ECL, meilunbio, MAO186-2) reagents.

[0118] The above experimental results ( Figure 1 、 2) showed that WF-1 and 4 had no obvious degradation activity on ERα protein in MCF-7 cells, while WF-2, 3, and 5 had certain degradation activity on ERα protein in MCF-7 cells. Among them, WF-2 had the best degradation ability on ERα protein in MCF-7 cells, and it was in a time-dependent manner.

[0119] [Example 7] Fluorescence properties of integrated diagnostic and therapeutic ERα PROTACs

[0120] Fluorescence performance test The probe (WF-1–WF-5) solution was prepared in 10 mM PBS (pH = 7.4), and the optical properties of the probes were measured using a UV-visible spectrophotometer and a Hitachi Shimadzu UV-2600F-4600 fluorescence spectrophotometer.

[0121] The above experimental results ( Figure 3 ) showed that ERαPROTACs (WF-1–WF-5) compounds all had fluorescence emission signals at 400-500nm.

[0122] [Example 8] Cell imaging capability of WF-2

[0123] MCF-7 cells were placed in a 37°C cell culture incubator in MEM medium. After recovery, the cells were transferred to a confocal microplate and cultured for 24 hours. After 24 hours, the cells were incubated with various concentrations of WF-2. After 30 minutes, the cells were rinsed three times with PBS buffer, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. After 10 minutes, the cells were washed with PBS to remove free drug and imaged. Images were acquired using a LECA-LCS-SP8 confocal laser scanning microscope.

[0124] The above experimental results ( Figure 4 ) showed that WF-2 fluorescence changes in MCF-7 cells were observed in a concentration-dependent manner, and the cell imaging effect was most obvious when the WF-2 concentration was 100 μM, indicating that WF-2 has certain potential for the diagnosis of breast cancer.

[0125] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An integrated diagnostic and therapeutic ERα PROTACs compound, characterized in that: The structure is shown in the general formula (I): （I） Among them, Linker is selected from 、 , n=1-5, m=1-7, x=1-3.

2. The integrated diagnosis and treatment ERα PROTACs compound according to claim 1, characterized in that Selected from the following compounds: 2-(4-(((1 S , 2 R , 4 S )-5,6-bis(4-hydroxyphenyl)-N-(2,2,2-trifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene)-2-sulfonamido)phenoxy)-N-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-5-yl)oxy)butyl)acetamide, 2-(4-(((1 S , 2 R , 4 S )-5,6-bis(4-hydroxyphenyl)-N-(2,2,2-trifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene)-2-sulfonamido)phenoxy)-(N-6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-benzo[d]isoquinolin-5-yl)oxy)hexyl)acetamide, 2-(4-(((1 S , 2 R , 4 S )-5,6-bis(4-hydroxyphenyl)-N-(2,2,2-trifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene)-2-sulfonamido)phenoxy)-N-(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-5-yl)oxy)octyl)acetamide, 2-(4-(((1 S , 2 R , 4 S )-5,6-bis(4-hydroxyphenyl)-N-(2,2,2-trifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene)-2-sulfonamido)phenoxy)-N-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-5-yl)oxy)ethoxy)ethyl)acetamide, 6-(4-(((1 S , 2 R , 4 S )-5,6-bis(4-hydroxyphenyl)-N-(2,2,2-trifluoroethyl)-7-oxabicyclo[2.2.1]hept-5-ene)-2-sulfonamido)phenoxy)-N-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-benzo[d]isoquinolin-5-yl)oxy)hexyl)hexanamide.

3. A pharmacologically or physiologically acceptable salt of the integrated diagnostic and therapeutic ERα PROTACs compound according to claim 1 or 2.

4. Use of the integrated diagnostic and therapeutic ERα PROTACs compound according to claim 1 or 2, or a pharmacologically or physiologically acceptable salt thereof, in the preparation of an anti-breast cancer drug.

5. Use of the integrated diagnostic and therapeutic ERα PROTACs compound according to claim 1 or 2, or a pharmacologically or physiologically acceptable salt thereof, in the preparation of a breast cancer diagnostic drug.

6. A drug, characterized in that: Comprising one or more of the integrated diagnostic and therapeutic ERα PROTACs compound according to claim 1 or 2 or a pharmacologically or physiologically acceptable salt thereof; the drug is an anti-breast cancer drug or a breast cancer diagnostic drug.

7. The drug according to claim 6, characterized in that: Also contains pharmaceutically acceptable excipients.

8. The method for preparing the integrated diagnosis and treatment ERα PROTACs compound according to claim 1 or 2, characterized in that: The method comprises the following steps: performing an amide condensation reaction of a WUN29654 derivative containing an amino linker and an OBHSA derivative containing a carboxyl linker in an organic solvent under the action of a condensing agent and a base to obtain the integrated diagnosis and treatment ERα PROTAC compound; The WUN29654 derivative containing an amino linker is or The structure of the OBHSA derivative containing a carboxyl linker is ; where m=1-5, n = 1-7, x=1-3.

9. The preparation method according to claim 8, characterized in that: The condensing agent includes HATU, HBTU, PyBOP, and DCC; the base includes DIPEA, Et3N, Na2CO3, and NaHCO3.

10. The preparation method according to claim 8, characterized in that: The organic solvent includes DMF, DCM, DMC, and 2-MeTHF.

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