AR-CBD PROTAC compound, preparation method thereof and application of AR-CBD PROTAC compound in treatment of breast cancer

Abstract

The invention discloses an AR-CBD PROTAC compound as well as a preparation method and application thereof in treating breast cancer, and relates to the technical field of biological medicines. The preparation principle of the AR-CBD PROTAC compound provided by the invention is that an AR-CBD inhibitor D2 carboxylic acid derivative and an E3 ligand amino derivative with different linkers are respectively subjected to one-step amidation reaction to prepare the AR-CBD PROTAC compound. The AR-CBD PROTAC compound provided by the invention has the advantages that the activity of resisting MCF-7 cells and mutant strains of the MCF-7 cells is obvious, the AR degradation activity is obvious, and the AR-CBD PROTAC compound can be used for preparing the anti-breast cancer medicine.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to AR-CBD PROTAC compounds, their preparation methods, and their applications in the treatment of breast cancer. Background Art

[0002] Breast cancer is the most common cancer among women globally, and estrogen receptor-positive (ER+) breast cancer accounts for approximately 70% of all breast cancers. Targeting estrogen receptor alpha (ERα), endocrine therapy is mainly used clinically, that is, by reducing estrogen levels or blocking the estrogen receptor (ER) signaling pathway to exert an anti-ERα+ breast cancer effect. In the past 30 years, "endocrine therapy" for treating ER+ breast cancer has included: 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; selective estrogen receptor degraders that induce ER degradation as complete antagonists, such as fulvestrant. However, long-term use of the above drugs based on endocrine therapy is prone to primary or secondary drug resistance problems, which has become a major challenge in the treatment of ERα+ breast cancer. Therefore, it is particularly important to develop new anti-drug-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] Research has shown that in ER+ breast cancer, which accounts for approximately 70% of all breast cancers, almost all ER+ breast cancers express androgen receptor (AR). Clinically, selective AR modulators (SARMs) without masculinizing side effects have been applied to the treatment of breast cancer. Research indicates that AR has a therapeutic effect at multiple stages of breast cancer; it may regulate target gene transcription and promote cancer cell proliferation by using a mechanism similar to that of ER and coactivators. Clinical studies have shown that the AR antagonist, prucalopride, inhibits AR-positive breast cancer cells, and has little effect on the proliferation of AR-negative PC3 and DU145 cells. AR antagonists also have a therapeutic effect on breast cancer. For example, the AR antagonist, bicalutamide, had good efficacy in a clinical trial of 26 postmenopausal women (ER-, PR-, AR+) with metastatic breast cancer, with a clinical benefit rate of 19% and good drug tolerance. Therefore, the therapy of targeting AR to treat ER+ breast cancer is expected to overcome the drug resistance caused by the existing endocrine therapy for breast cancer targeting ER, bringing new hope to most breast cancer patients.

[0004] AR is a ligand-regulated nuclear transcription factor encoded by the NR3C4 gene and belongs to the steroid hormone receptor superfamily. It consists of an N-terminal Domain (NTD), a Ligand-Binding Domain (LBD), a Hinge region, and a DNA Binding Domain (DBD). Among them, the LBD is composed of three different functional regions: the Ligand Binding Pocket (LBP), the Activation Function 2 (AF-2) site, and the Binding Function 3 (BF-3) site. AR antagonists mainly competitively bind to the AR LBP with testosterone or dihydrotestosterone (DHT), thereby blocking the androgen signaling pathway and the transcriptional activation of downstream genes. So far, AR antagonists have gone through the development of first-generation and second-generation drugs. Due to the relatively low affinity and easy emergence of drug resistance of the first-generation antagonists, second-generation AR antagonists represented by enzalutamide and apalutamide have emerged one after another, which have higher binding affinity and a significant improvement in activity.

[0005] Currently, drugs that antagonize the androgen receptor in clinical practice are still mainly selective androgen receptor modulators (SARMs) or selective androgen receptor degraders (SARDs). However, under the selective pressure of long-term endocrine therapy, a series of complex adaptive changes occur in breast cancer cells, leading to acquired endocrine resistance. AR antagonists, including enzalutamide, inevitably induce point mutations in the AR LBP, such as T877A, W741C / T877A, and F876L / T877A, resulting in their agonist activity and the rapid emergence of drug resistance. Therefore, it is urgent to explore other key binding sites of AR, supplement new drug development and treatment strategies, and develop new targeted treatment methods that can effectively alleviate the occurrence and development of endocrine-resistant breast cancer.

[0006] Different from the androgen receptor LBP, as one of the strategies for developing drugs that block protein-protein interaction (PPI), the androgen receptor AF-2 domain is another important target for inhibiting AR transcriptional activity. That is, after androgens bind to AR LBP, the LBD undergoes a conformational change and generates a hydrophobic groove on the surface. The hydrophobic groove is composed of helices H3, H4, and H12. It can form specific protein-protein interactions (PPI) with coactivators, such as the LXXLL motif (L = Leucine, X represents any amino acid) in steroid receptor coactivators (SRCs), and then recruit a series of coactivators with histone modification or chromatin recombination, such as p300 / CBP histone acetyltransferase, which plays a key role in the process of AR-regulated gene transcription. The amino-terminal-carboxyl-terminal (N / C) interaction between the FXXLF motif of AR NTD and AF-2 is also particularly important for the transcriptional activity of full-length AR. Therefore, targeting the coactivator binding domain (CBD) of AF-2 can directly block the interaction between AR and coactivators, thus avoiding the drug resistance generated by classical ligand-binding pocket mutations, as well as resisting endocrine resistance caused by bypass activation (such as the EGFR pathway) or coactivator overexpression. Since any mutation in the binding pocket between AR and coactivators will hinder the binding of AR to coactivators, this target has a lower probability of adaptive mutations compared to LBP and good clinical application prospects. Since the first AR AF-2 targeting peptide that uses the NR-box motif (F / W)XXL(F / W) to mimic the coactivator LXXLL motif was reported, small molecule inhibitors (Coactivator Binding Inhibitors, CBI) targeting the AR coactivation site have gradually attracted wide attention. So far, a series of AR CBIs with different structural skeletons have been reported and can effectively inhibit the transcriptional activity of AR. These results indicate that targeting the androgen receptor coactivator binding site can not only be used for the treatment of breast cancer but also has the potential to combat drug resistance.

[0007] Although small molecule inhibitors of the androgen receptor, including CBIs, have shown significant inhibitory effects, studies have shown that there are still a variety of co-regulatory factors in the body that participate in the regulation of androgen receptor activity. There are also studies showing that there is androgen receptor-independent activation caused by additional signaling pathways, leading to the emergence of clinical drug resistance. This makes the emergence of new drug strategies even more important. Therefore, the targeted protein degradation technology (TPD), by specifically degrading target proteins (especially mutant proteins), is expected to overcome the deficiencies of existing small molecule inhibitor drugs. In recent years, a class of technologies for inducing protein degradation has been developed, and its main mechanism of action is to hijack the target protein while catalyzing the ubiquitination of the protein, and then be degraded by the proteasome or lysosome. Such technologies mainly include: proteolysis targeting chimeras (PROTACs), hydrophobic tagging (HyT), and molecular glues, etc.

[0008] The mode of action of PROTAC technology is completely different from that of small molecule inhibitors. This technology mainly exerts its therapeutic effect by constructing a heterobifunctional molecule containing a ligand of the protein of interest (POI ligand), a linker, and a ligand of the E3 ubiquitin ligase, inducing the spatial proximity of the target protein and the E3 ligase, so that the former is ubiquitinated and degraded. Different from the mode of action of traditional small molecule drugs, PROTAC technology adopts an "event-driven" mode. It can regulate protein levels by inducing the degradation of target proteins, without directly inhibiting the activity of target proteins and continuous high-intensity binding. The targeted protein degradation strategy can effectively solve the problem of "undruggable" targets. PROTAC technology has a wide range of applications in the fields of pharmacy and biology. Different from the post-transcriptional regulation of siRNA, the degradation of proteins by PROTAC technology occurs after gene translation, which is very helpful for studying the biological functions of different proteins, and its distribution in the human body and targeted effects on different tissues also give the possibility of drug development for this technology. This technology has great advantages compared with traditional small molecule inhibitors. For example, it has lower requirements for binding affinity, which enables it to target undruggable proteins and overcome target drug resistance mutations; it also has characteristics such as 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. Summary of the Invention

[0009] The present invention provides an AR-CBD PROTAC compound, a preparation method thereof, and an application thereof in the treatment of breast cancer. The AR-CBD PROTAC compound provided by the present invention has significant anti-breast cancer activity and can be developed as a new anti-breast cancer drug, having broad application prospects. It is specifically achieved through the following technologies.

[0010] In the first aspect of the present invention, there is provided an AR-CBD PROTAC compound, the general chemical structure formula of which is: ; wherein, the Linker is selected from , , , , , , , or , and the right end of the Linker is connected to the E3 ligand; The E3 ligand is selected from or .

[0011] Furthermore, the AR-CBD PROTAC compound is selected from any one of the compounds shown in Table 1 below.

[0012] Table 1

[0013]

[0014]

[0015] In the second aspect of the present invention, there is provided a preparation method of any one of the above AR-CBD PROTAC compounds, which is prepared by carrying out a condensation reaction on an E3 ligase ligand derivative containing an amino linker and a D2 carboxylic acid derivative containing a carboxyl linker under the action of a condensing agent and a base; The general chemical structure formula of the E3 ligase ligand derivative is: , or , wherein, n = 1, 2, 3; The Linker is selected from , , , , , , , or ; The general chemical structure formula of the D2 carboxylic acid derivative is: .

[0016] Furthermore, the molar ratio of the D2 carboxylic acid derivative, the E3 ligase ligand derivative, HATU, and DIPEA in the feeding is 1:1.1:1.1:3.

[0017] In the third aspect of the present invention, there is provided a pharmaceutically or physiologically acceptable salt of the AR-CBD PROTAC compound described in any one of the above.

[0018] In the fourth aspect of the present invention, there is provided the AR-CBD PROTAC compound described in any one of the above, or the AR-CBD PROTAC compound prepared by the preparation method described in any one of the above, or the pharmaceutically or physiologically acceptable salt of the above AR-CBD PROTAC compound, for use in the preparation of a medicament for treating breast cancer.

[0019] In the fifth aspect of the present invention, there is provided a medicament for treating breast cancer, comprising the AR-CBD PROTAC compound described in any one of the above, or the pharmaceutically or physiologically acceptable salt of the above AR-CBD PROTAC compound.

[0020] Furthermore, the medicament for treating breast cancer further comprises at least 1 pharmaceutically acceptable carrier or excipient.

[0021] Compared with the prior art, the advantages of the present invention are as follows: The AR-CBD PROTAC compounds provided by the present invention have good anti-breast cancer activity, and the inhibitory activity of some compounds reaches the nmol level; they can be developed as new anti-breast cancer drugs and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the synthesis route of the AR-CBD PROTAC compound.

[0023] Figure 2 It is the degradation activity of compound W8 on AR protein in MCF-7 cells.

[0024] Figure 3 It shows that the degradation of AR protein by compound W8 in MCF-7 cells is time-dependent. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0026] In some embodiments of the present invention, the AR-CBD PROTAC compound is obtained by subjecting an AR-CBD inhibitor D2 carboxylic acid derivative and an E3 ligase ligand (pomalidomide or VHL) to an amide condensation reaction under the action of a condensing agent HATU and a base DIPEA. Three synthetic routes of the AR-CBD PROTAC compound are as Figure 1 shown.

[0027] Specifically, the preparation method of the AR-CBD PROTAC compound is as follows: S1. 3-Isobutoxy-4-nitrobenzoate (Compound 3) is synthesized through the reaction shown in the following formula (1) and used as the raw material for the next reaction.

[0028]

[0029] Further, step S1 may specifically be: First, take a 100 mL round-bottom flask, weigh commercially available methyl 3-hydroxy-4-nitrobenzoate (Compound 1, 5.00 g, 25.36 mmol), put in a magnetic stir bar, and completely dissolve it with DMF (N,N-dimethylformamide, 20 mL); weigh anhydrous potassium carbonate (7.00 g, 50.65 mmol) and pour it into the flask, measure isobutyl iodide (Compound 2, 9.30 g, 50.54 mmol) and add it to the reaction flask; place a reflux condenser at the reaction flask mouth, and the reaction temperature is 80 °C. Monitor by TLC. After the reaction is completed, take the reaction flask out of the furnace and let it cool to room temperature. Add dilute hydrochloric acid (25 mL) to quench the reaction under stirring; extract with EA (ethyl acetate, 3×60 mL) to collect the organic phase, wash twice with saturated sodium chloride, and then spin dry. After separation and purification by column chromatography (the dosage of the eluent petroleum ether and ethyl acetate is PE:EA = 50:1), a yellow-green solid compound (Compound 3, 3.10 g, 12.24 mmol) is obtained with a yield of 48.3%.

[0030] S2. Methyl 4-amino-3-isobutoxybenzoate (Compound 4) is synthesized through the reaction shown in the following formula (2) and used as the raw material for the next reaction.

[0031]

[0032] Further, step S2 can be specifically selected as follows: The raw material obtained from reaction (1) (compound 3, 2.50 g, 9.9 mmol) was dissolved in methanol (30 mL) in a 100 mL round-bottom flask, and Raney nickel suspension (4 mL, 0.24 mmol) was added, and the reaction was carried out at room temperature for 4 h under hydrogen circulation. Monitored by TLC. After the reaction was completed, water was added, filtered, and the filtrate was extracted with EA (3×50 mL), and the organic phase was washed with saturated brine and then rotary evaporated to dryness. The crude product obtained by rotary evaporation was separated and purified by column chromatography (PE:EA = 10:1) to obtain a yellowish-white solid compound (compound 4, 1.36 g, 6.09 mmol), and the yield was 62.1%.

[0033] S3. 3-Isobutoxy-4-nitrobenzoic acid (compound 5) was synthesized through the reaction shown in the following formula (3) and used as the raw material for the next reaction.

[0034]

[0035] Further, step S3 can be specifically selected as follows: The raw material obtained from reaction (1) (compound 3, 2.00 g, 7.9 mmol) was dissolved in methanol (8 mL), and then lithium hydroxide dissolved in water (2 mL) was added to the reaction flask. Monitored by TLC. After the reaction was completed, 1 mol / L dilute hydrochloric acid (3 mL) was added to adjust the pH value of the reaction solution to <3, and the product precipitated. After suction filtration and drying, light yellow solid compound 5 (1.76 g, 7.32 mmol) was obtained, and the yield was 94.2%.

[0036] S4. D2 (compound 6) was synthesized through the reaction shown in the following formula (4) and used as the raw material for the next reaction.

[0037]

[0038] Further, step S4 can specifically be selected as follows: Dissolve the raw material obtained from reaction (3) (compound 5, 1.10 g, 4.6 mmol) in DCM (6 mL) in a 100 mL single-necked round-bottom flask, then add Mukaiyama reagent (2-chloro-1-methylpyridinium iodide, 1.20 g, 4.7 mmol) and triethylamine (2.00 g, 19.8 mmol). Subsequently, stir the reaction system under reflux at 50 °C. After 30 min, add compound 4 (511 mg, 2.3 mmol), and continue the reflux reaction. Monitor by TLC. After the reaction is completed, add sodium bicarbonate (5 mL) to neutralize the reaction solution, then extract with DCM (dichloromethane, 3 × 20 mL). The organic phase is washed with saturated brine and dried by rotary evaporation to obtain the crude product. After separation by column chromatography (eluent ratio PE:EA = 10:1), the purified compound D2 (compound 6) is obtained as a pale yellow solid (0.67 g, 1.51 mmol), with a yield of 73.7%.

[0039] S5. Synthesize 3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamido)benzoic acid (compound 7) through the reaction shown in the following formula (5), and use it as the raw material for the next reaction.

[0040]

[0041] Further, step S5 can specifically be selected as follows: Dissolve the raw material obtained from reaction (4) (compound 6, 0.67 g, 1.5 mmol) in methanol (4 mL), and then dissolve lithium hydroxide in water (2 mL) and add it to the reaction flask. Monitor by TLC. After the reaction is completed, add 1 mol / L dilute hydrochloric acid to adjust the pH value of the reaction solution to < 3, and the product precipitates. Filter and dry to obtain the crude product of compound 7 as a yellow solid (0.63 g, 1.46 mmol), with a yield of 82.7%.

[0042] S6. Synthesize tert-butyl (4-bromobenzyl)carbamate (compound 9) through the reaction shown in the following formula （6） and use it as the raw material for the next reaction.

[0043]

[0044] Further, step S6 can specifically be selected as follows: Take a 100 mL single-necked flask, and add the mixed solvent EtOAc / H2O (V / V = 1 / 1) (20 mL). Subsequently, add successively ( S)-(-)-1-(4-bromophenyl)ethylamine (Compound 8, 6.0 g, 29.99 mmol) and NaHCO3 (2.77 g, 32.99 mmol) were slowly added with (Boc)2O (di-tert-butyl dicarbonate, 7.20 g, 32.99 mmol) under an ice bath. After the addition was completed, the ice bath was removed and the mixture was stirred at room temperature for 3 h. The reaction was monitored by TLC. After the starting compound 8 completely disappeared, the reaction system was extracted with EtOAc, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to obtain the crude product, which was purified by flash chromatography with the mobile phase of petroleum ether / ethyl acetate (V / V = 15:1) to give the white solid product (Compound 9, 8.73 g, 29.09 mmol).

[0045] S7. (4-(4-Methylthiazol-5-yl)benzyl)carbamic acid tert-butyl ester (Compound 11) was synthesized through the reaction shown by the following formula （7） and used as the starting material for the next reaction.

[0046]

[0047] Furthermore, step S7 can be specifically selected as follows: The starting material prepared in reaction (6) (Compound 9, 8.50 g, 28.31 mmol), commercially available 4-methylthiazole (Compound 10, 5.62 g, 56.63 mmol), palladium acetate (0.32 g, 1.42 mmol), and potassium acetate (11.12 g, 113.26 mmol) were placed in a 50 mL round-bottom flask. After evacuation, argon was introduced and the cycle was repeated three times. Subsequently, the solvent dimethylacetamide (DMAC) was added and the reaction was carried out at 150 °C overnight. The reaction was monitored by TLC. After the starting compound 9 completely disappeared, the reaction was quenched with water and extracted with ethyl acetate. After concentration of the organic phase, it was separated and purified by silica gel column chromatography with the mobile phase of petroleum ether / ethyl acetate (V / V = 6:1) to obtain the white solid product (Compound 11, 7.30 g, 22.93 mmol).

[0048] S8. (2S,4R)-4-Hydroxy-2-(((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylic acid tert-butyl ester (Compound 12) was synthesized through the reaction shown by the following formula (8) and used as the starting material for the next reaction.

[0049]

[0050] Furthermore, step S8 can be specifically selected as follows: A 100 mL single-necked flask was taken and DCM (20 mL) was added, and then the reaction （7）The obtained raw material (Compound 11, 7.00 g, 21.98 mmol) was slowly added dropwise with TFA (trifluoroacetic acid, 10.03 g, 87.93 mmol) under an ice bath. After the addition was completed, stirring was continued for 1 h. The reaction was monitored by TLC. After the raw material disappeared, DCM and TFA in the system were removed by rotary evaporation, and the residue was redissolved in DCM. The pH was adjusted to alkaline with ammonia water, and then extracted with DCM. After most of the DCM was removed by rotary evaporation, an appropriate amount of anhydrous DCM (30 mL) was added. Then, DIPEA (N,N-diisopropylethylamine, 11.41 g, 87.93 mmol), HATU (2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 9.20 g, 24.18 mmol), and Boc-Hyp-OH (Boc-L-hydroxyproline, 5.59 g, 24.18 mmol) were added in sequence. After the addition was completed, stirring was carried out at room temperature for 3 h. Monitored by TLC, after the raw material completely disappeared, extraction was carried out with DCM, and after drying by rotary evaporation, column chromatography purification was carried out with petroleum ether / ethyl acetate (V / V = 3 / 1) as the mobile phase to obtain a white solid product (Compound 12, 8.02 g, 18.59 mmol).

[0051] S9. tert-Butyl ((S)-1-((2S,4R)-4-hydroxy-2-(((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (Boc-VHL) was synthesized through the reaction shown in formula (9) below and used as the raw material for the next reaction.

[0052]

[0053] Furthermore, step S9 may specifically be: Take a 50 mL single-necked flask and add DCM (50 mL), and then add the reaction （8）The obtained raw material (Compound 12, 7.80 g, 18.07 mmol) was slowly added dropwise with TFA (8.24 g, 72.30 mmol) under an ice bath. After the addition was complete, stirring was continued for 1 h. The reaction was monitored by TLC. After the raw material disappeared, DCM and TFA in the system were removed by rotary evaporation, and the residue was redissolved in an appropriate amount of DCM. The pH was adjusted to alkaline with ammonia water, and then extracted with DCM. After drying, most of the DCM was removed by rotary evaporation, and an appropriate amount of anhydrous DCM (30 mL) was added. Then, DIPEA (0.09 g, 72.30 mmol), HATU (7.56 g, 19.88 mmol), and Boc-Tle-OH (4.60 g, 19.88 mmol) were added in sequence. After the addition was complete, stirring was carried out at room temperature for 3 h. Monitored by TLC, after the raw material completely disappeared, extraction was carried out with DCM, and after rotary evaporation to dryness, column chromatography purification was carried out with the mobile phase of dichloromethane / methanol (V / V = 30:1) to obtain the white solid product Boc-VHL (8.06 g, 14.79 mmol).

[0054] S10. (2S,4R)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (VHL) was synthesized through the reaction shown in the following formula (10) and used as the raw material for the next reaction.

[0055]

[0056] Further, step S10 can be specifically selected as follows: Take a 50 mL single-necked flask and add DCM, then add the raw material Boc-VHL (400 mg, 0.73 mmol) obtained from reaction (9). Under an ice bath, slowly add dropwise TFA (335 mg, 2.94 mmol). After the addition is complete, continue stirring for 0.5 h. The reaction was monitored by TLC. After the raw material disappeared, DCM and TFA in the system were removed by rotary evaporation, and the residue was redissolved in an appropriate amount of DCM. The pH was adjusted to alkaline with ammonia water, and then extracted with DCM. After drying, DCM was removed by rotary evaporation to obtain the white solid VHL (7.55 g, 14.02 mmol).

[0057] S11. Pomalidomide amino derivatives (15a-b) containing Linkers with different alkyl chain lengths were synthesized through the reaction shown in the following formula (11) and used as the raw material for the next reaction.

[0058]

[0059] Further, step S11 can specifically be selected as follows: Take a 50 mL round-bottom flask, weigh the above-obtained product (compound 13, 1.0 eguiv.) and the tert-butoxycarbonyl amino compound 14a or 14b with a p-toluenesulfonyl group (1.2 eguiv.), dissolve them in 3 mL of anhydrous DMF, then add K2CO3 (2.0 eguiv.), react at 50 °C for 12 h, and monitor the completion of the reaction by TLC. Then extract with EA (3 × 25 mL), wash with saturated NaCl solution (30 mL), dry the organic layer with anhydrous sodium sulfate, remove the solvent under reduced pressure to obtain the crude product, and purify it by silica gel column chromatography with the mobile phase of petroleum ether / ethyl acetate = 1:1 to obtain the corresponding pale yellow solid products 15a or 15b with a yield of 70-90%.

[0060] S12. Synthesize pomalidomide amino derivatives (17a and 17b) containing Linkers with different ether chain lengths through the reaction shown in the following formula (12), and use them as raw materials for the next reaction.

[0061]

[0062] Further, step S12 can specifically be selected as follows: Take a 50 mL round-bottom flask, weigh the above-obtained product (compound 13, 1.0 eguiv.) and the tert-butoxycarbonyl amino compound 16a or 16b with a p-toluenesulfonyl group (1.2 eguiv.), dissolve them in 3 mL of anhydrous DMF, then add K2CO3 (2.0 eguiv.), react at 50 °C for 12 h, and monitor the completion of the reaction by TLC. Then extract with EA (3 × 25 mL), wash with saturated NaCl solution (30 mL), dry the organic layer with anhydrous sodium sulfate, remove the solvent under reduced pressure to obtain the crude product, and purify it by silica gel column chromatography with the mobile phase of petroleum ether / ethyl acetate = 1:1 to obtain the corresponding pale yellow solid products 17a or 17b with a yield of 70-90%.

[0063] S13. Synthesize VHL amino derivatives (19a-e) containing Linkers with different rigid chains through the reactions shown in the following formulas (13), (14), and (15), and use them as raw materials for the next reaction.

[0064]

[0065] For the above reaction formula (13), further, step S13 can specifically be selected as follows: Take a 50 mL round-bottom flask, weigh the VHL (1.0 eguiv.) obtained from reaction formula (10), 18a-d (1.1 eguiv.), HATU (1.1 eguiv.), and DIPEA (4.0 eguiv.), place them in a 25 mL round-bottom flask, add anhydrous DCM, stir overnight at room temperature. After monitoring by TLC that the raw material VHL has completely disappeared, extract with DCM, dry with anhydrous sodium sulfate, concentrate, and then purify by column chromatography. The mobile phase is dichloromethane / methanol (V / V = 15:1), and the corresponding pale yellow solid product 19a-d is obtained with a yield of 80 - 95%.

[0066]

[0067] For the above reaction formula (14), further, step S13 can specifically be selected as follows: Take a 50 mL round-bottom flask, weigh the VHL (300 mg, 0.67 mmol) obtained from reaction formula (10), dissolve it in MeCN (6 mL), successively add tert-butyl 6-bromoacetate (Compound 20, 158 mg, 0.81 mmol), KI (11.1 mg, 0.67 mmol), and K2CO3 (185 mg, 1.34 mmol). After adding the materials, place the reaction in an oil bath and heat to 85 °C, stir overnight. Monitor the reaction by TLC. After the raw materials disappear, extract the reaction with DCM, dry, and then rotary evaporate the organic phase. Concentrate and then purify by column chromatography. The mobile phase is dichloromethane / methanol (V / V = 15:1) to obtain a white solid product (Compound 21, 329 mg, 0.59 mmol) with a yield of 88%.

[0068]

[0069] For the above reaction formula (15), further, step S13 can specifically be selected as follows: Take a 50 mL round-bottom flask, weigh 21 (300 mg, 0.67 mmol) of the compound obtained from reaction formula (14). Under ice bath conditions, slowly add dropwise TFA (289 mg, 2.01 mmol). After the addition is complete, continue stirring for 0.5 h. Monitor the reaction by TLC. After the raw materials disappear, rotary evaporate to remove DCM and TFA in the system, and redissolve the residue in an appropriate amount of DCM. Adjust the pH to alkaline with ammonia water, extract with DCM, dry, and then rotary evaporate to remove DCM. Subsequently, add HATU (281 mg, 0.74 mmol), DIPEA (348 mg, 2.68 mmol), and compound 22 (262 mg, 0.74 mmol) in sequence, add anhydrous DCM, stir overnight at room temperature, monitor by TLC. After the reaction is completed, extract with DCM, dry with anhydrous sodium sulfate, concentrate, and then purify by column chromatography. The mobile phase is dichloromethane / methanol (V / V = 15:1) to obtain a pale yellow solid product (compound 19e, 406 mg, 0.55 mmol), and the yield is 82%.

[0070] S14. Synthesize a series of (A) AR-CBD PROTACs (W1-W2) through the reaction shown in the following formula (16).

[0071]

[0072] Further, step S14 can specifically be selected as follows: Take a 50 mL round-bottom flask, weigh 15a-b (1.0 eguiv.) of the compound obtained from reaction formula (11). Under ice bath conditions, slowly add dropwise TFA (3.0 eguiv.). After the addition is complete, continue stirring for 0.5 h. Monitor the reaction by TLC. After the raw materials disappear, rotary evaporate to remove DCM and TFA in the system, and redissolve the residue in an appropriate amount of DCM. Adjust the pH to alkaline with ammonia water, extract with DCM, dry, and then rotary evaporate to remove DCM. Subsequently, add compound 7 (1.1 eguiv.), HATU (1.1 eguiv.), and DIPEA (4.0 eguiv.) in sequence, add anhydrous DMF, stir overnight at room temperature, monitor by TLC. After the reaction is completed, extract with EA, dry with anhydrous sodium sulfate, concentrate, and then purify by column chromatography. The mobile phase is petroleum ether / ethyl acetate = 1:1 to obtain the corresponding yellow solid products W1-W2, and the yield is 70-80%.

[0073] S15. Synthesize a series of (B) AR-CBD PROTACs (W3–W4) through the reaction shown in the following formula (17).

[0074]

[0075] Further, step S15 can specifically be selected as follows: Take a 50 mL round-bottom flask, weigh the compound 17a or 17b obtained from reaction formula (12) (1.0 eguiv.), under ice bath conditions, slowly add dropwise TFA (3.0 eguiv.). After the addition is complete, continue stirring for 0.5 h. Monitor the reaction by TLC. After the raw materials disappear, rotary evaporate to remove DCM and TFA in the system, and redissolve the residue in an appropriate amount of DCM. Adjust the pH to alkaline with ammonia water, extract with DCM, dry, and then rotary evaporate to remove DCM. Subsequently, add compound 7 (1.1 eguiv.), HATU (1.1 eguiv.), and DIPEA (4.0 eguiv.) in sequence, add anhydrous DMF, stir overnight at room temperature, monitor by TLC. After the reaction is completed, extract with EA, dry with anhydrous sodium sulfate, concentrate, and then purify by column chromatography. The mobile phase is petroleum ether / ethyl acetate = 1:1, and the corresponding yellow solid products W3 - W4 are obtained with a yield of 70 - 80%.

[0076] S16. A series of C) AR-CBD PROTACs (W5–W9) are synthesized through the reaction shown in the following formula (18).

[0077]

[0078] Further, step S6 can specifically be selected as follows: Take a 50 mL round-bottom flask, weigh 19a - e obtained from reaction formulas (13), (14), and (15) (1.0 eguiv.), under ice bath conditions, slowly add dropwise TFA (3.0 eguiv.). After the addition is complete, continue stirring for 0.5 h. Monitor the reaction by TLC. After the raw materials disappear, rotary evaporate to remove DCM and TFA in the system, and redissolve the residue in an appropriate amount of DCM. Adjust the pH to alkaline with ammonia water, extract with DCM, dry, and then rotary evaporate to remove DCM. Subsequently, add compound 7 (1.1 eguiv.), HATU (1.1 eguiv.), and DIPEA (4.0 eguiv.) in sequence, add anhydrous DCM, stir overnight at room temperature, monitor by TLC. After the reaction is completed, extract with DCM, dry with anhydrous sodium sulfate, concentrate, and then purify by column chromatography. The mobile phase is dichloromethane / methanol (V / V = 15:1), and white solid products W5 - W9 are obtained with a yield of 70 - 82%.

[0079] Example 1: Preparation of N-(3-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)propyl)-3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamido)benzamide (Product W1).

[0080] Weigh compound 15a (100 mg, 0.23 mmol). Under ice bath conditions, slowly add dropwise TFA (79 mg, 0.69 mmol). After the addition is complete, continue stirring for 0.5 h. Monitor the reaction by TLC. After the raw material disappears, rotary evaporate to remove DCM and TFA in the system, and redissolve the residue in an appropriate amount of DCM. Adjust the pH to alkaline with ammonia water, extract with DCM, dry, and then rotary evaporate to remove DCM. Subsequently, add compound 7 (108 mg, 0.25 mmol), HATU (95 mg, 0.25 mmol), and DIPEA (119 mg, 0.92 mmol) in sequence, add anhydrous DMF, stir overnight at room temperature, monitor by TLC. After the reaction is completed, extract with EA, dry with anhydrous sodium sulfate, concentrate, and then purify by column chromatography. The mobile phase is petroleum ether / ethyl acetate = 1:1 to obtain the yellow solid product W1 (128 mg, 0.17 mmol), with a yield of 75%.

[0081] 1 H NMR (600 MHz, Chloroform- d ) δ 9.42 (s, 1H), 8.76 (s, 1H), 8.47 (d, J = 8.3 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.82 (dt, J = 6.5, 2.9 Hz, 1H), 7.71(dd, J = 8.4, 7.3 Hz, 1H), 7.67 (d, J = 1.7 Hz, 1H), 7.54 (d, J = 1.8 Hz, 1H), 7.49(d, J = 7.2 Hz, 1H), 7.43 (ddd, J = 15.5, 8.3, 1.7 Hz, 2H), 4.93 (dd, J = 12.5, 5.3Hz, 1H), 4.40 (dt, J = 10.4, 5.1 Hz, 1H), 4.26 – 4.20 (m, 1H), 3.96 (dd, J= 6.4, 3.2 Hz, 2H), 3.93 – 3.91 (m, 2H), 3.68 – 3.62 (m, 1H), 2.81 – 2.77 (m, 1H), 2.74 – 2.62 (m, 2H), 2.25 – 2.13 (m, 5H), 2.06 (ddt, J = 9.8, 4.5, 2.6 Hz, 1H), 1.10 – 1.03 (m, 12H).

[0082] Example 2: Preparation of N-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)butyl)-3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamide)benzamide (Product W2).

[0083] The preparation method of Product W2 in this example is basically the same as that in Example 1; the difference is that starting compound 15a is replaced with starting compound 15b to obtain yellow solid Product W2 with a yield of 78%.

[0084] 1 H NMR (400 MHz, DMSO- d 6) δ 11.14 (s, 1H), 9.84 (s, 1H), 8.65 (s, 1H), 7.93 (d, J = 8.7 Hz, 1H), 7.85 – 7.76 (m, 3H), 7.62 – 7.50 (m, 4H), 7.43 (dd, J = 7.3, 3.1 Hz, 1H), 5.11 – 5.05 (m, 1H), 4.24 (d, J = 6.3 Hz, 2H), 4.02 (d, J = 6.5 Hz, 2H), 3.87 (d, J = 6.4 Hz, 2H), 3.36 (d, J = 6.3 Hz, 3H), 2.64 – 2.47 (m, 3H), 2.08 – 1.99 (m, 3H), 1.85 – 1.80 (m, 2H), 1.74 (t, J = 7.6 Hz, 2H), 0.98 (dd, J = 6.8, 2.6 Hz, 12H).

[0085] Preparation of N-(2-(2-((2-(2-(2-6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethoxy)ethyl)-3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamide)benzamide (Product W3).

[0086] Weigh compound 17a (106 mg, 0.23 mmol). Under ice bath conditions, slowly add dropwise TFA (79 mg, 0.69 mmol). After the addition is complete, continue stirring for 0.5 h. Monitor the reaction by TLC. After the raw material disappears, rotary evaporate to remove DCM and TFA in the system, and redissolve the residue in an appropriate amount of DCM. Adjust the pH to alkaline with ammonia water, extract with DCM, dry, and then rotary evaporate to remove DCM. Subsequently, add compound 7 (108 mg, 0.25 mmol), HATU (95 mg, 0.25 mmol), and DIPEA (119 mg, 0.92 mmol) in sequence, add anhydrous DMF, stir overnight at room temperature, monitor by TLC. After the reaction is completed, extract with EA, dry with anhydrous sodium sulfate, concentrate, and then purify by column chromatography. The mobile phase is petroleum ether / ethyl acetate = 1:1 to obtain the yellow solid product W3 (128 mg, 0.16 mmol), and the yield is 72%.

[0087] 1 H NMR (600 MHz, Chloroform- d ) δ 8.72 (s, 1H), 8.48 (d, J = 8.3 Hz,1H), 7.91 (d, J = 8.3 Hz, 1H), 7.68 (dd, J = 8.4, 7.2 Hz, 1H), 7.64 (d, J = 1.7 Hz,1H), 7.57 (d, J = 1.8 Hz, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.43 (dd, J = 8.4, 1.7 Hz,1H), 7.38 (dd, J = 8.3, 1.7 Hz, 1H), 7.22 (d, J = 8.3 Hz, 2H), 5.02 – 4.97 (m,1H), 4.33 (dd, J = 5.5, 3.0 Hz, 2H), 3.98 – 3.94 (m, 4H), 3.91 (d, J= 6.4 Hz, 2H), 3.80 (t, J = 4.9 Hz, 2H), 3.71 (ddd, J = 20.0, 9.3, 4.7 Hz, 2H), 2.85 – 2.77 (m, 3H), 2.17 (ddd, J = 13.2, 6.5, 3.4 Hz, 2H), 2.11 (td, J = 5.4, 2.5 Hz, 1H), 1.06 (dd, J = 6.7, 3.3 Hz, 12H).

[0088] Example 4: Preparation of N-(2-(2-(2-((2-(2-(2-6-oxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)ethoxy)ethoxy)ethyl)-3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamide)benzamide (Product W4).

[0089] The preparation method of Product W4 in this example is basically the same as that in Example 3; the difference is that starting material compound 17a is replaced with starting material compound 17b to obtain yellow solid Product W4 with a yield of 75%.

[0090] 1 H NMR (600 MHz, Chloroform- d ) δ 8.76 (s, 1H), 8.56 (d, J = 8.4 Hz, 1H), 8.48 (s, 1H), 7.96 (d, J = 8.3 Hz, 1H), 7.69 – 7.64 (m, 2H), 7.58 (d, J = 1.9 Hz, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.43 (dt, J = 8.4, 2.0 Hz, 2H), 7.25 (d, J = 8.5 Hz, 1H), 4.96 (dd, J = 12.3, 5.4 Hz, 1H), 4.36 – 4.32 (m, 2H), 4.02 – 3.93 (m, 6H), 3.85 – 3.82 (m, 2H), 3.74 – 3.72 (m, 4H), 2.91 – 2.74 (m, 3H), 2.22 (ddp, J= 10.0, 6.6, 3.3 Hz, 2H), 2.15 (m, 1H), 1.11 (dd, J = 8.8, 6.7 Hz, 12H).

[0091] Example 5: Preparation of (2S,4R)-4-hydroxy-1-((S)-2-(4-(4-(3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamido)benzoyl)piperazin-1-yl)benzamido)-3,3-dimethylbutanoyl)-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Product W5).

[0092] Weigh compound 19a (168 mg, 0.23 mmol). Under ice bath conditions, slowly add dropwise TFA (79 mg, 0.69 mmol). After the addition is complete, continue stirring for 0.5 h. Monitor the reaction by TLC. After the raw material disappears, rotary evaporate to remove DCM and TFA in the system, and redissolve the residue in an appropriate amount of DCM. Adjust the pH to alkaline with ammonia water, extract with DCM, dry, and then rotary evaporate to remove DCM. Subsequently, add compound 7 (108 mg, 0.25 mmol), HATU (95 mg, 0.25 mmol), DIPEA (119 mg, 0.92 mmol) in sequence, add anhydrous DMF, stir overnight at room temperature, monitor by TLC. After the reaction is completed, extract with DCM, dry with anhydrous sodium sulfate, concentrate, and then purify by column chromatography. The mobile phase is dichloromethane / methanol (V / V = 15:1) to obtain the white solid product W5 (188 mg, 0.18 mmol), and the yield is 78%.

[0093] 1 1H NMR (400 MHz, Chloroform- d ) δ 8.74 (s, 1H), 8.58 (d, J = 8.1 Hz,1H), 7.96 (d, J = 8.3 Hz, 1H), 7.74 – 7.66 (m, 3H), 7.56 (d, J = 7.6 Hz, 1H),7.44 – 7.38 (m, 5H), 7.16 – 7.08 (m, 2H), 6.89 (d, J = 8.5 Hz, 2H), 6.72 (d, J =8.6 Hz, 1H), 5.11 (t, J = 7.1 Hz, 1H), 4.75 (t, J= 7.7 Hz, 2H), 4.55 (s, 1H),4.26 – 4.18 (m, 1H), 3.96 (dd, J = 17.9, 6.4 Hz, 4H), 3.66 (dd, J = 11.2, 3.4 Hz,4H), 3.35 (s, 4H), 2.55 (s, 3H), 2.21 (qd, J = 6.8, 3.1 Hz, 3H), 2.05 (dd, J =13.6, 8.3 Hz, 3H), 1.52 (d, J = 6.9 Hz, 3H), 1.18 – 1.06 (m, 21H).

[0094] Example 6: Preparation of N-((S)-1-(((2S,4R)-4-hydroxy-2-((((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-4-(4-(3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamido)benzoyl)piperazin-1-yl)piperidine-1-carboxamide (Product W6).

[0095] The preparation method of Product W6 in this example is basically the same as that in Example 5; the difference is that starting compound 19a is replaced with starting compound 19b to obtain a white solid product W6 with a yield of 70%.

[0096] 1 H NMR (400 MHz, Chloroform- d ) δ 8.70 (d, J = 7.1 Hz, 2H), 8.53 (dd, J =8.2, 1.8 Hz, 1H), 7.95 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 1.7 Hz, 1H), 7.41 (dd, J =5.9, 3.0 Hz, 5H), 7.11 – 7.01 (m, 2H), 5.13 – 5.05 (m, 1H), 4.93 (td, J = 8.3,5.6 Hz, 1H), 4.55 (d, J = 13.2 Hz, 1H), 4.45 (d, J= 6.0 Hz, 1H), 4.20 – 4.12 (m,1H), 3.97 (d, J = 6.4 Hz, 2H), 3.92 (dd, J = 6.5, 2.7 Hz, 2H), 3.80 (s, 1H), 3.50(s, 1H), 3.39 (d, J = 11.1 Hz, 1H), 3.24 – 3.06 (m, 2H), 2.65 (d, J = 8.8 Hz,4H), 2.55 (d, J = 1.5 Hz, 3H), 2.20 (dq, J = 13.1, 6.3 Hz, 4H), 1.91 (s, 2H),1.48 (d, J = 6.9 Hz, 3H), 1.10 (t, J = 6.9 Hz, 12H), 1.01 (d, J = 8.0 Hz, 9H).

[0097] Example 7: Preparation of (2S,4R)-4-hydroxy-1-((S)-2-(4-(3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamido)benzamido)cyclohexane-1-carboxamido)-3,3-dimethylbutanoyl)-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (W7).

[0098] The preparation method of the product W7 in this example is basically the same as that in Example 5; the difference is that the starting compound 19a is replaced with the starting compound 19c to obtain the white solid product W7 with a yield of 76%.

[0099] 1 H NMR (400 MHz, Chloroform-d) δ 8.75 (s, 1H), 8.69 (s, 1H), 8.53 (d, J = 8.3 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.59 (d, J = 1.6 Hz, 1H), 7.53 (d, J =1.7 Hz, 1H), 7.48 – 7.42 (m, 2H), 7.37 (dd, J = 5.5, 2.0 Hz, 5H), 6.74 (d,J = 7.9 Hz, 1H), 6.47 (d, J = 8.8 Hz, 1H), 5.11 (t, J = 7.2 Hz, 1H), 4.75 (t, J = 8.0Hz, 1H), 4.65 (d, J = 8.8 Hz, 1H), 4.51 (s, 1H), 4.28 – 4.21 (m, 1H), 4.11 (d, J = 11.3 Hz, 1H), 3.92 (dd, J = 14.2, 6.5 Hz, 4H), 3.66 (dd, J = 11.4, 3.5 Hz, 1H), 2.37 (dt, J = 10.9, 7.6 Hz, 3H), 2.17 (dt, J = 13.3, 6.7 Hz, 3H), 1.92 – 1.67 (m, 9H), 1.50 (d, J = 6.9 Hz, 3H), 1.27 (s, 4H), 1.10 – 1.03 (m, 21H).

[0100] Example 8: Preparation of N-((S)-1-(((2S,4R)-4-hydroxy-2-((((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-1-(((1-(3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamido)phenyl)benzoyl)piperidin-4-yl)methyl)piperidine-4-carboxamide W8.

[0101] The preparation method of the product W8 in this example is basically the same as that in Example 5; the difference is that the starting compound 19a is replaced with the starting compound 19d to obtain a white solid product W8 with a yield of 82%.

[0102] 11H NMR (400 MHz, Chloroform-d) δ 8.71 (d, J = 8.7 Hz, 2H), 8.51 (d, J= 8.2 Hz, 1H), 7.94 (d, J = 8.3 Hz, 1H), 7.66 (d, J = 1.7 Hz, 1H), 7.41 (t, J= 5.7 Hz, 5H), 7.08 – 7.01 (m, 2H), 5.07 (q, J = 7.3 Hz, 1H), 4.90 (t, J =8.4 Hz, 1H), 4.46 (s, 1H), 3.97 (d, J = 6.5 Hz, 3H), 3.91 (d, J = 6.4 Hz,2H), 3.69 (d, J = 4.4 Hz, 2H), 3.59 – 3.40 (m, 8H), 3.23 (s, 1H), 3.15 (s,1H), 2.54 (s, 3H), 2.20 (dt, J = 11.6, 6.1 Hz, 4H), 1.61 (s, 4H), 1.48 (d, J= 6.9 Hz, 3H), 1.32 (dd, J = 6.7, 2.9 Hz, 2H), 1.08 (d, J = 7.0 Hz, 12H),1.02 (s, 9H).

[0103] Example 9: Preparation of (2S,4R)-4-hydroxy-1-((S)-2-((2-(9-(3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamido)phenyl)-3,9-diazaspiro[5.5]undec-3-yl)-2-oxoethyl)amino)-3,3-dimethylbutanoyl)-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Product W9).

[0104] The preparation method of Product W9 in this example is basically the same as that in Example 5; the difference is that starting material compound 19a is replaced with starting material compound 19e to obtain white solid Product W9 with a yield of 80%.

[0105] 11H NMR (400 MHz, Methanol-d4) δ 8.89 (s, 1H), 8.08 (d, J = 8.1 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 1.7 Hz, 1H), 7.58 (dd, J = 8.3, 1.6 Hz, 1H), 7.45 (dd, J = 7.5, 3.6 Hz, 5H), 7.12 (d, J = 1.7 Hz, 1H), 7.04 (dd, J = 8.0, 1.7 Hz, 1H), 4.66 – 4.57 (m, 3H), 4.49 – 4.41 (m, 1H), 4.01 (d, J = 6.4 Hz, 2H), 3.91 (d, J = 6.5 Hz, 2H), 3.87 (s, 1H), 3.76 (dd, J = 11.1, 4.1 Hz, 1H), 3.20 (d, J = 13.1 Hz, 1H), 2.89 (d, J = 11.2 Hz, 1H), 2.69 (d, J = 7.0 Hz, 2H), 2.57 (s, 3H), 2.49 (s, 3H), 2.17 (ddq, J = 20.4, 13.5, 6.3 Hz, 5H), 2.04 – 1.84 (m, 8H), 1.52 (d, J = 7.0 Hz, 3H), 1.07 (q, J = 4.2 Hz, 21H).

[0106] The target compounds W1-W9 of the present invention synthesized in Examples 1-9 above respectively, and their chemical structures are shown in Table 1 above.

[0107] Test Example 1: In vitro anti-breast cancer activity of AR-CBD PROTACs compounds.

[0108] CCK-8 (Cell Counting Kit-8) is a detection reagent based on WST-8 that is widely used in the detection of cell proliferation and cytotoxicity. WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-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 methyl sulfate, it is reduced by dehydrogenases in mitochondria to a highly water-soluble orange-yellow formazan product. The more and faster the cells proliferate, 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 live cells. Therefore, this property can be used to directly analyze cell proliferation and toxicity.

[0109] The anti-breast cancer cell proliferation activity of AR-CBD PROTACs compounds was tested by the CCK-8 method, and the specific steps are as follows: (1)Prepare cell suspension: When the growth density of breast cancer cells (MCF-7, MCF-7 EGFR , MCF-7 D538G , MCF-7 Y537S )reaches 80%-90%, digest the cells with 0.25% Trypsin-EDTA, add fresh MEM medium, mix the cell suspension evenly, count using a cell counting chamber, and dilute the cells to a single cell suspension of 5×10 4 cells / mL.

[0110] (2)Plate: Inoculate 100 μL of the single cell suspension into a microplate (tissue culture grade, 96 wells, flat bottom).

[0111] (3)Pre-culture: Incubate the cells in a 37°C, 5% CO2 incubator for about 24 hours.

[0112] (4)Add drugs: Aspirate the original medium, and sequentially add 200 μL of medium containing the test drugs at different concentrations (100 μM - 0.01 μM) to each well of the culture plate (set 3 replicates for each concentration, and set a blank group at the same time), and place it in the incubator for 96 h.

[0113] (5)Harvest the plate: Prepare the CCK-8 working solution according to the ratio of adding 10 μL of CCK-8 reagent to every 100 μL of medium, add 100 μL of the CCK-8 working solution to each well, and place the culture plate in the incubator for 2 - 3 h.

[0114] (6)Measure the plate: Measure the absorbance (OD) at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the IC of the compound against breast cancer cells50 .

[0115] The above experimental results show that: Most of the synthesized AR-CBD PROTACs compounds have good anti-breast cancer activity. Most of these compounds have inhibitory activity against three MCF-7 mutant strains. Among them, the inhibitory activity of some compounds, W6 (IC 50 = 180 - 4250 nM), W7 (IC 50 = 60 - 2820 nM), W8 (IC 50 = 150 - 1520 nM), W9 (IC 50 = 540 - 6710 nM) is better than that of the positive control drug 4-OHT (4-hydroxytamoxifen, IC 50 = 560 - 17980 nM).

[0116] Test Example 2: Degradation activity of AR-CBD PROTACs compounds on AR protein in vitro. In this test example, the product W8 was used as a representative of AR-CBD PROTACs compounds, and the degradation activity of AR-CBD PROTACs compounds on ERα protein was detected by immunoblot analysis. The specific steps are as follows:

[0117] (1) Treat MCF-7 cells with different concentrations of ERα PROTAC compounds for 24 h, and obtain whole cell lysates with RIPA buffer and protein loading buffer.

[0118] (2) Analyze the protein concentration of the samples with a BCA protein assay kit (KR0008), and adjust the volume of the samples for electrophoresis according to the standard protein curve.

[0119] (3) Separate the proteins with a 7.5% or 10% SDS-PAGE gel and transfer them to a PVDE membrane with a thickness of 0.45 μM (Millipore, 000027346).

[0120] (4) Block the membrane with 5% bovine serum albumin (BSA, KR9048-466-8) or 5% non-fat milk at room temperature for 2 h. Incubate the primary antibody at 4°C for more than 12 h and the secondary antibody on a shaker at room temperature for more than 1 h.

[0121] (5) Detect the proteins using ultrasensitive enhanced chemiluminescence (ECL, meilunbio, MAO186-2) reagent.

[0122] The test results are shown in Figure 2 、 3As shown. The test results indicate that product W8 has obvious degradation activity on AR protein in MCF-7 cells. In addition, product W8 can induce AR degradation in a time- and concentration-dependent manner. At 12 h and a concentration of only 1 µM, it can significantly reduce the protein level of Hsp90.

[0123] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple variations all fall within the protection scope of the present invention.

Claims

1. An AR-CBD PROTAC compound, characterized in that, The general chemical structure formula is: ； Among them, the Linker is selected from , , , , , , , or , and the right end of the Linker is connected to the E3 ligand; The E3 ligand is selected from or .

2. The AR-CBD PROTAC compound according to claim 1, wherein Any one selected from the following compounds: N-(3-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)propyl)-3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamido)benzamide, N-(2-(2-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)ethoxy)ethyl)-3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamide)benzamide, N-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)butyl)-3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamide)benzamide, N-(2-(2-(2-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)ethoxy)ethoxy)ethyl)-3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamide)benzamide, (2S,4R)-4-hydroxy-1-((S)-2-(4-(4-(3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamido)benzoyl)piperazin-1-yl)benzamido)-3,3-dimethylbutanoyl)-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, N-((S)-1-((2S,4R)-4-hydroxy-2-(((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-4-(4-(3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamide)benzoyl)piperazin-1-yl)piperidine-1-carboxamide, (2S,4R)-4-hydroxy-1-((S)-2-(4-(3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamido)benzamido)cyclohexanecarboxamido)-3,3-dimethylbutanoyl)-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, (2S,4R)-4-hydroxy-1-((S)-2-((2-(9-(3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamido)benzoyl)-3,9-diazaspiro[5.5]undec-3-yl)-2-oxoethyl)amino)-3,3-dimethylbutanoyl)-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, N-((S)-1-((2S,4R)-4-hydroxy-2-(((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-1-((1-(3-isobutoxy-4-(3-isobutoxy-4-nitrobenzamido)benzoyl)piperidin-4-yl)methyl)piperidine-4-carboxamide.

3. A method for preparing an AR-CBD PROTAC compound, characterized in that, The AR-CBD PROTAC compound is the AR-CBD PROTAC compound according to any one of claims 1 or 2; The AR-CBD PROTAC compound is prepared by condensing a derivative of an E3 ligase ligand containing an amino linker and a derivative of D2 carboxylic acid containing a carboxyl linker under the action of a condensing agent and a base; The general chemical structure formula of the E3 ligase ligand derivative is as follows: , or , where n = 1, 2, 3; The Linker is selected from , , , , , , , or ; The general chemical structure formula of the D2 carboxylic acid derivative is as follows: .

4. The preparation method of the AR-CBD PROTAC compound according to claim 3, characterized in that, The molar ratio of the D2 carboxylic acid derivative, the E3 ligase ligand derivative, HATU, and DIPEA is 1:1.1:1.1:

3.

5. A pharmaceutically or physiologically acceptable salt of the AR-CBD PROTAC compound according to claim 1 or 2.

6. Use of the AR-CBD PROTAC compound according to claim 1 or 2, or the AR-CBD PROTAC compound prepared by the preparation method according to claim 3 or 4, or a pharmaceutically or physiologically acceptable salt of the AR-CBD PROTAC compound according to claim 5, in the preparation of a medicament for treating breast cancer.

7. A drug for treating breast cancer, characterized in that, Comprising the AR-CBD PROTAC compound according to claim 1 or 2, or a pharmaceutically or physiologically acceptable salt of the AR-CBD PROTAC compound according to claim 5.

8. The product according to claim 7, wherein, It further comprises at least one pharmaceutically acceptable carrier or excipient.

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