Diagnosis and treatment integrated ERalpha PROTACs compound for detecting and degrading ERalpha as well as preparation method and application of diagnosis and treatment integrated ERalpha PROTACs compound

Abstract

The invention discloses a diagnosis and treatment integrated ERalpha PROTACs compound for detecting and degrading ERalpha as well as a preparation method and application thereof, and belongs to the technical field of medicines. The structure of the diagnosis and treatment integrated PROTAC compound is shown as a general formula (I), and OBHSA containing carboxyl linkers and WUN29654 amino derivatives with different linkers are used as raw materials to be subjected to a one-step amidation reaction to prepare the ERalpha PROTAC compound. Results of cytotoxicity, immunoblotting analysis and optical characteristics show that the diagnosis and treatment integrated PROTAC compound has remarkable activity of resisting MCF-7 cells and mutant strains thereof, ERalpha protein degradation activity and obvious fluorescence emission signals at 400-500 nm, and can be used for preparing anti-breast cancer drugs and / or breast cancer diagnosis drugs. # imgabs0 #

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, and 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-resistant 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 the first-line treatment for patients with ERα+ breast cancer, mainly exerts its anti-breast cancer effect by reducing estrogen levels or blocking the ER signaling pathway. In the past 30 years, "endocrine therapy" for the treatment of ER+ breast cancer includes aromatase inhibitors that inhibit the production of endogenous estrogen, such as letrozole; selective estrogen receptor modulators that bind to ER and regulate its activity, such as tamoxifen; and selective estrogen receptor degraders that induce ER degradation as complete antagonists, such as fulvestrant. However, long-term use of endocrine therapy often leads to primary or secondary drug resistance. In recent years, significant progress has been made in the molecular pharmacology of ERα and the exploration of the mechanism of endocrine resistance that affects the key pathways of ER action. There is solid evidence that ERα still plays an important role in advanced breast cancer, promotes disease development, and continues to be a potential therapeutic target. The successful application of fulvestrant in clinical practice further proves that the strategy of degrading ERα protein provides a practical and effective way to overcome endocrine therapy resistance.

[0004] Proteolysis targeted chimera (PROTAC) technology is an emerging drug development strategy 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 removing pathogenic proteins and playing a role in treating diseases. This technology has great advantages over traditional small molecule inhibitors, such as its low binding affinity requirements, 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. So far, more than 10 PROTACs have entered clinical trials, such as ARV-110, ARV-471, DT2216, etc.

[0005] Despite the success of PROTACs, it is still difficult to detect and track protein degradation in real time. Currently, most methods for evaluating protein degradation activity rely on Western Blot. However, more convenient visualization tools will help develop more effective analytical methods and help to better understand the mechanism 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 function of detecting and degrading ERα to visualize the protein degradation process and improve diagnostic and therapeutic effects will be a potential targeted treatment strategy for breast cancer. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a PROTACs compound that has the function of detecting and degrading ERα, that is, an integrated diagnosis and treatment ERαPROTACs compound, which has significant anti-breast cancer activity and fluorescence properties, and can realize the visualization of ERα protein degradation. The purpose of the present invention is also to provide the application of the integrated diagnosis and treatment ERαPROTACs compound, which can be developed as a new anti-cervical cancer drug and has broad application prospects. The purpose of the present invention is also to provide a method for preparing the integrated diagnosis and treatment ERαPROTACs compound.

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

[0008] In the first aspect, a diagnostic and therapeutic integrated 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 tests that the above-mentioned ERαPROTACs compounds can degrade ERα in a concentration-dependent manner and have significant anti-proliferative activity against breast cancer cells, and can be used to prepare anti-breast cancer drugs.

[0012] Preferably, the integrated diagnosis and treatment 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 above-mentioned integrated diagnosis and treatment ERαPROTACs compounds or pharmacologically or physiologically acceptable salts thereof. The drug may also contain pharmaceutically acceptable excipients, which can be prepared according to existing 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 diagnosis and treatment ERαPROTACs compound of general formula (I) is provided, comprising the following steps: a WUN29654 derivative containing an amino linker and an OBHSA derivative containing a carboxyl linker undergo an amide condensation reaction in an organic solvent under the action of a condensing agent and a base to obtain the PROTACs compound. The synthetic routes of some ERαPROTACs compounds are as follows:

[0020]

[0021] Synthetic route of ERαPROTACs compounds for integrated diagnosis and treatment

[0022] Preferably, in the method for preparing the diagnostic and therapeutic integrated ERαPROTACs compound represented by the general formula (I), the condensing agent comprises one or more of HATU, HBTU, PyBOP, DCC, etc.; the base comprises DIPEA, Et 3N、Na 2 CO 3 、NaHCO 3 The organic solvent includes one or more of DMF, DCM, DMC, 2-MeTHF, etc. The molar ratio of OBHSA derivative, WUN29654 derivative, condensation agent and base is 1.0-1.8:1:1.1-3.0:3.0-6.0.

[0023] Advantages and beneficial effects of the present invention: The integrated diagnosis and treatment ERαPROTACs compounds of the present invention have good anti-breast cancer activity and fluorescence properties, and can realize the visualization of ERα protein degradation, and the inhibitory activity of some compounds reaches the sub-micromolar level. The integrated diagnosis and treatment ERαPROTACs compounds of the present invention can be developed as new anti-breast cancer drugs, and have broad application prospects in improving both diagnostic and therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 :The degradation activity of WF-1, 3, 4, and 5 on ERα protein in MCF-7 cells was detected by immunoblotting analysis.

[0025] Figure 2 :The degradation activity of WF-2 on ERα protein in MCF-7 cells was detected by immunoblotting analysis.

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

[0027] Figure 4 : Microscope 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 methods of the present invention and are not intended to limit the remainder of the present invention in any way.

[0029] The preparation of the diagnostic and therapeutic integrated ERαPROTACs 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) is synthesized by the reaction shown in the following formula (1) and used as the raw material for the next step reaction.

[0031]

[0032] First, a 100 mL round-bottom flask was used to weigh 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), and 25 mL of anhydrous acetonitrile was added. Anhydrous triethylamine (0.7 mg, 6.94 mmol) was slowly added dropwise. The reaction was continued at room temperature for 12 h. After TLC monitoring, the reaction was complete. After the reaction was completed, the acetonitrile and triethylamine were removed by concentration under reduced pressure. Ethyl acetate was added to dissolve the mixture, 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) successively. The organic layer was dried over anhydrous sodium sulfate, filtered and dried to obtain a crude product, which was purified by column chromatography with a mobile phase of petroleum ether / ethyl acetate (V / V=30 / 1) to obtain a white solid compound 3 with a yield of 86%.

[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, and the reaction system was placed in an ice bath and stirred for 3 h. After TLC monitoring, when the raw material completely disappeared, the reaction system was extracted with DCM, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, and spin-dried to obtain a crude product, which 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, 25 mL of DCM was added, and BBr was added at -20 °C. 3 (2.6 mL, 27.33 mmol) was reacted for 12 h, and 10 mL of water was added to quench the reaction, extracted with ethyl acetate (3 × 20 mL), and saturated NaHCO 3The organic layer was dried over anhydrous sodium sulfate and desolventized to obtain a crude product, which was purified by 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 anhydrous and oxygen-free air was evacuated. Diisobutylaluminum hydride (DIBAL-H, 8 mL, 7.93 mmol) was added at -78°C and reacted for 12 h, and then 2 M HCl was added to quench the reaction. 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 crude product was removed by desolvation under reduced pressure and purified by silica gel column with 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 step reaction.

[0043]

[0044] 4-Methoxyaniline 7 (0.5 g, 4.06 mmol) was dissolved in DCM, 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 obtain 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 obtain 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 obtain 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 step reaction.

[0046]

[0047] First, different commercially available compounds 13a-c (1.0 eguiv.) were dissolved in DCM and reacted with (Boc) in the presence of TEA (0.1 eguiv.). 2 O (1.2 equiv.) was reacted in an ice bath to give compounds 14a-c. Compounds 14a-c (1.0 equiv.) were reacted with p-toluenesulfonyl chloride (1.2 equiv.), DMAP (0.2 equiv.), and TEA (2.0 equiv.) in DCM at room temperature to give 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 raw material for the next step.

[0049]

[0050] First, commercially available compound 13d (1.0 eguiv.) was dissolved in DCM and reacted with (Boc) in the presence of TEA (0.1 eguiv.). 2 O (1.2 equiv.) was reacted in an ice bath to obtain compound 14d, which was then extracted with DCM (3×25 mL), washed with saturated NaCl solution (30 mL), and the organic layer was dried over anhydrous sodium sulfate and desolventized to obtain a crude product, which was directly used for 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 obtain compound 15d. It was then extracted with DCM (3×25 mL), washed with saturated NaCl solution (30 mL), and the organic layer was dried over anhydrous sodium sulfate and desolventized to obtain a crude product, which was purified by silica gel column with a mobile phase of petroleum ether / ethyl acetate = 3:1, and the yield was 78%.

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

[0052]

[0053] First, take a 100 mL round-bottom flask, weigh commercially available 3-hydroxy-1,8-naphthalene dicarboxylic anhydride 16 (1.21 g, 5.65 mmol) and 3-amino-2,6-piperidindione 17 (0.72 g, 5.65 mmol), dissolve them in 10 mL of anhydrous DMF, and then add K 2 CO 3(1.56 g, 11.3 mmol), the reaction was continued at room temperature for 12 h, and the reaction was complete after TLC monitoring. It was then extracted with EA (3×25 mL), washed with saturated NaCl solution (30 mL), and the organic layer was dried over anhydrous sodium sulfate, desolvated under reduced pressure to obtain a crude product, which was purified by silica gel column with a mobile phase of petroleum ether / ethyl acetate = 1:1 to obtain 1.32 g of yellow solid product 18, with a yield of 72%.

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

[0055]

[0056] Take a 50 mL round-bottom flask, weigh the above-obtained product 18 (1.0 eguiv.) and the tert-butyloxycarbonylamino compound 15a-c (1.2 eguiv.) with a p-toluenesulfonyl group, dissolve them in 3 mL of anhydrous DMF, and then add K 2 CO 3 (2.0 eguiv.), reacted at 50°C for 12 h, and the reaction was complete after TLC monitoring. It was then extracted with EA (3×25 mL), washed with saturated NaCl solution (30 mL), and the organic layer was dried over anhydrous sodium sulfate, desolvated under reduced pressure to obtain a crude product, which was purified by silica gel column with a mobile phase of petroleum ether / ethyl acetate = 1:1 to obtain light yellow solid products 19a-c with a yield of 40-60%.

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

[0058]

[0059] Take a 50 mL round-bottom flask, weigh the above-obtained product 18 (0.30 g, 0.93 mmol) and the tert-butyloxycarbonylamino compound 15d (0.40 g, 1.12 mmol) with a p-toluenesulfonyl group, dissolve them in 3 mL of anhydrous DMF, and then add K 2 CO 3 (0.25 g, 1.86 mmol), reacted at 50°C for 12 h, and the reaction was complete after TLC monitoring. It was then extracted with EA (3×25 mL), washed with saturated NaCl solution (30 mL), and the organic layer was dried over anhydrous sodium sulfate, desolvated under reduced pressure to obtain a crude product, which was purified by silica gel column with a mobile phase of petroleum ether / ethyl acetate = 1:1 to obtain a light yellow solid product 19d with a yield of 55%.

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

[0061]

[0062] Take a 25 mL round-bottom flask, weigh the above product 19a-c (1.0 eguiv.), dissolve it in 1 mL of anhydrous DCM, and add 0.5 mL of CF under ice bath. 3 COOH, and then reacted at room temperature for 30 minutes. The reaction was monitored by TLC to be complete. After that, the product 20a-c was obtained by spin drying with a yield of 100%, which was directly used as the raw material for the next step.

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

[0064]

[0065] Take a 25 mL round-bottom flask, weigh the above product 19d (1.0 eguiv.), dissolve it in 1 mL of anhydrous DCM, and add 0.5 mL of CF under ice bath. 3 COOH, then reacted at room temperature for 30 min, and the reaction was complete after TLC monitoring. Then it was spin-dried to obtain a light yellow solid product 20d with a yield of 100%, which was directly used as the raw material for the next step reaction.

[0066] 13. Compound 22a-b was synthesized by the following reaction and used as the raw material for the next step.

[0067]

[0068] Take a 50 mL round-bottom flask, weigh the above product 12 (1.0 eguiv.) and commercially available tert-butyl bromoacetate or tert-butyl 6-bromohexanoate 21a-b (1.2 eguiv.), dissolve them in 3 mL of anhydrous DMF, and then add K 2 CO 3 (2.0 eguiv.), KI (0.1 eguiv.), reacted at room temperature for 12 h, and the reaction was complete after TLC monitoring. It was then extracted with EA (3×25 mL), washed with saturated NaCl solution (30 mL), and the organic layer was dried over anhydrous sodium sulfate, desolvated under reduced pressure to obtain a crude product, which was purified by silica gel column with a mobile phase of petroleum ether / ethyl acetate = 5:1 to obtain a light yellow solid product 22a-b with a yield of 70-80%.

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

[0070]

[0071] Take a 25 mL round-bottom flask, weigh the above obtained product 22a-b (1.0 eguiv.) and the compound 6 (1.0 eguiv.) obtained by the above formula (4), pump out the water and oxygen, add a small amount of anhydrous THF, react at 90°C for 12 hours, and monitor the reaction completion by TLC. Then extract with EA (3×25 mL), wash with saturated NaCl solution (30 mL), dry the organic layer with anhydrous sodium sulfate, desolventize under reduced pressure to obtain a crude product, and purify with a silica gel column, the mobile phase is petroleum ether / ethyl acetate = 3:1, and obtain a light yellow solid product 23a-b with a yield of 40-55%.

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

[0073]

[0074] Take a 25mL round-bottom flask, weigh the above product 23a-b (1.0 eguiv.), dissolve it in 1mL anhydrous DCM, add 0.3mL CF3COOH under ice bath, react at room temperature for 30min, monitor the reaction completion by TLC, and then spin dry to obtain white solid product 24a-b with a yield of 100%, which is directly used as the raw material for the next step 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: Weigh compound 24a (0.10 g, 0.17 mmol) and 20a (0.0 61g, 0.15mmol), HATU (0.086g, 0.23mmol), DIPEA (0.099mL, 0.6mmol) were placed in a 25mL round-bottom flask, anhydrous DMF was added, and the mixture was stirred overnight at room temperature. After the raw material 20a disappeared completely as monitored by TLC, it was extracted with EA, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography. The mobile phase ratio was dichloromethane / methanol (V / V=10:1) to obtain a white solid product WF-1 (0.096g, 0.10mmol) with 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 TLC monitoring, the raw material 20d completely disappeared, and then extracted with EA, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography. The mobile phase ratio was dichloromethane / methanol (V / V=10:1) to obtain a white solid product WF-4 (0.084 g, 0.08 mmol) with 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 TLC monitoring of the complete disappearance of the raw material 20a, it was extracted with EA, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography. The mobile phase ratio was dichloromethane / methanol (V / V=10:1) to obtain a white solid product WF-1 (0.096 g, 0.10 mmol) with a yield of 58%.

[0084] 1 H NMR (600 MHz, DMSO-d 6)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 to obtain a white solid product WF-2 with a yield of 83%.

[0088] 1 H NMR (600 MHz, DMSO-d 6)δ10.97(s,1H),9.63(s,1H),9.58(s,1H),8.31(dt,J=8.3,4.0Hz,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 to obtain a light yellow solid product WF-3 with a yield of 42%.

[0091] 1 H NMR (600 MHz, DMSO-d 6)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=8.6Hz,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] 1 H NMR (600 MHz, DMSO-d 6)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 TLC monitoring, the raw material 20d completely disappeared, and then extracted with EA, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography. The mobile phase ratio was dichloromethane / methanol (V / V=10:1) to obtain a white solid product WF-4 (0.084 g, 0.08 mmol) with a yield of 50%.

[0098] 1 H NMR (600 MHz, DMSO-d 6 )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 WST-8-based widely used cell proliferation and cytotoxicity detection reagent. WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazolyl monosodium salt) is a compound similar to MTT (chemical name: 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide). Under the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate, it is reduced by dehydrogenase in mitochondria to a highly water-soluble orange-yellow formazan product (Formazan). The more cells proliferate and the faster the speed, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of the color is proportional to the number of living cells, so this characteristic can be used to directly analyze cell proliferation and toxicity.

[0106] The anti-breast cancer cancer cell proliferation activity of the integrated diagnostic and therapeutic ERαPROTACs compound was tested by CCK-8 method. 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: at 37°C, 5% CO 2 Incubate the cells in an incubator for about 24 hours.

[0110] 4) Adding drugs: Aspirate the original culture medium, add 200 μL of culture medium containing different concentrations (100 μM-0.01 μM) of the drug to be tested to each well of the culture plate in turn (set up 3 replicate wells for each concentration, and set up a blank group at the same time), and incubate in an incubator for 96 hours.

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

[0112] 7) Test plate: Use an ELISA reader to measure the absorbance (OD) at 450nm and calculate the IC of the compound against breast cancer cells 50 , the results are shown in Table 3.

[0113] Table 3. Antiproliferative activity of ERαPROTACs (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 the three MCF-7 mutants. For example, compound WF-2 has inhibitory activity 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 with RIPA buffer and protein loading buffer. The protein concentration of the sample was analyzed with 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 with 7.5% or 10% SDS-PAGE gel and transferred to a PVDE membrane with a thickness of 0.45 μM (Millipore, 000027346). The membrane was blocked with 5% bovine serum albumin (BSA, KR9048-466-8) or 5% skim milk at room temperature for 2 hours, the primary antibody was incubated at 4°C for more than 12 hours, and the secondary antibody was 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 ERαPROTACs for integrated diagnosis and treatment

[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] The MEM medium containing cells (MCF-7) was placed in a cell culture incubator at 37°C. After the cells recovered, they were transferred to a confocal dish and cultured for 24 hours. After 24 hours, different concentrations of WF-2 were added to incubate the cells. After 30 minutes, the cells were rinsed 3 times with PBS buffer, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. After 10 minutes, PBS was added to wash the cells to wash away the free drugs and observe the imaging results. The instrument uses a LECA-LCS-SP8 confocal laser scanning microscope for imaging.

[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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

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

2. The integrated diagnosis and treatment ERαPROTACs compound according to claim 1, characterized in that: Selected from the following compounds: 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, 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, 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, 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, 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.

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

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

5. Use of the integrated diagnosis and treatment ERαPROTACs compound or a pharmacologically or physiologically acceptable salt thereof according to claim 1 or 2 in the preparation of a breast cancer diagnostic drug.

6. A drug, characterized in that: Comprising one or more of the diagnostic and therapeutic ERαPROTACs compounds or pharmacologically or physiologically acceptable salts thereof as described in claim 1 or 2; 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 ERαPROTACs compound according to claim 1 or 2, characterized in that: The method comprises the following steps: a WUN29654 derivative containing an amino linker and an OBHSA derivative containing a carboxyl linker undergo an amide condensation reaction in an organic solvent under the action of a condensing agent and a base to obtain the integrated diagnosis and treatment ERαPROTAC compound; The WUN29654 derivative containing a hydroxyl linker is The structure of the OBHSA derivative containing a carboxyl linker is Among them, 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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