Preparation and application of a proteolysis-targeted chimera targeting anti-apoptotic Bcl-2

Abstract

The present invention discloses the preparation and application of a proteolysis-targeted chimera targeting anti-apoptotic Bcl-2, and belongs to the field of biomedical technology. The present invention prepares a heterocyclic compound, an isomer thereof or a pharmaceutically acceptable salt thereof, and the heterocyclic compound has a structure shown in Formula I. The compound provided by the present invention can degrade the anti-apoptotic Bcl-2 protein in tumor cells MDA-MB-231 through the ubiquitin-proteasome system. The present invention can be used as a strategy for developing Bcl-2 protein degraders, and provides a powerful tool for biomedical research.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the preparation and application of a protein hydrolysis targeting chimera targeting anti-apoptotic Bcl-2. Background Art

[0002] Bcl-2 family proteins play a vital role in the process of cell apoptosis. Among them, the anti-apoptotic protein Bcl-2, as a core member of the family, prevents the occurrence of cell apoptosis by interacting with pro-apoptotic proteins and inhibiting their activity. However, in malignant tumor cells, the abnormal expression of Bcl-2 protein often leads to an imbalance of apoptosis, further worsening the cancer. The development of existing Bcl-2 inhibitors is severely limited by factors such as drug resistance and adverse reactions. Therefore, targeted degradation of Bcl-2 protein provides a promising strategy for regulating tumor cell apoptosis.

[0003] Proteolysis targeting chimeras (PROTACs) technology is the most advanced research direction in the field of targeted protein degradation. PROTAC molecules can use the cell's inherent ubiquitin-proteasome system to degrade intracellular proteins. PROTAC molecules can bind to the target protein and E3 ubiquitin ligase at the same time to form an E3 ligase-PROTAC-target protein ternary complex. The formation of this ternary complex will shorten the distance between the target protein and the E3 ligase, allowing the E3 ligase to mediate the ubiquitin-binding enzyme E2 to ubiquitinate the target protein. The labeled protein is then recognized and degraded by the proteasome. PROTAC plays an important role in regulating the expression level of pathogenic proteins and thus controlling their functions in various diseases, bringing unprecedented opportunities for drug development.

[0004] Existing Bcl-2 protein PROTAC molecules have poor effects, such as PROTAC Bcl2degrader-1 with CAS number 2378801-85-3, which has poor effects on DCs of Bcl-2. 50 The value is 3 μM; in the article "Proteolysis Targeting Chimeras for the Selective Degradation of Mcl-1 / Bcl-2 Derived from Nonselective Target Binding Ligands", its PROTAC is for DC of Bcl-2 50The value is 3.0 μM. In the synthesis process of PROTAC molecules, the composition of each part needs to be carefully selected. In the target protein ligand part, its biological activity and selectivity need to be considered; in the E3 ubiquitin ligase ligand part, its selectivity and molecular weight need to be considered; in the link chain part, the water solubility, biological activity and ease of synthesis of the molecule need to be considered; in the synthesis process, not only is the large number of optional structures a challenge, but another challenge is that when the three are finally combined, the actual effect cannot be simply superimposed and can only be verified through experiments. It can be seen that the engineering of developing new Bcl-2 protein PROTAC molecules is full of uncertainty, so the development of more effective PROTACs targeting Bcl-2 proteins is still a topic that needs to be answered urgently. Summary of the invention

[0005] In view of the above prior art, the purpose of the present invention is to provide a preparation and application of a proteolysis-targeted chimera targeting anti-apoptotic Bcl-2.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a heterocyclic compound, an isomer thereof or a pharmaceutically acceptable salt thereof, wherein the heterocyclic compound has a structure shown in Formula I:

[0008]

[0009] Formula I;

[0010] Wherein, n is 0, 1, 2, 3, 4 or 5;

[0011] R1 is selected from Formula II or Formula III,

[0012]

[0013] Formula II;

[0014]

[0015] Formula III.

[0016] Furthermore, the heterocyclic compound is selected from any of the following compounds:

[0017] .

[0018] The second aspect of the present invention provides a pharmaceutical preparation comprising the heterocyclic compound, its isomer or a pharmaceutically acceptable salt thereof, and at least one other pharmacologically active substance.

[0019] The third aspect of the present invention provides a pharmaceutical composition comprising the heterocyclic compound, its isomer or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, excipients and diluents.

[0020] The fourth aspect of the present invention provides a method for preparing the heterocyclic compound, its isomer or a pharmaceutically acceptable salt thereof. When R1 is selected from Formula II, the preparation of the heterocyclic compound represented by Formula I comprises the following steps:

[0021] (1) adding methylsulfonyl chloride to a mixture of raw material 1 and triethylamine, and after the reaction is completed, extracting the organic phase, washing and drying it, filtering it, concentrating it, and separating and purifying it to obtain the first product;

[0022] The mass volume ratio of raw material 1, triethylamine and methanesulfonyl chloride is (1-5) g: (5-15) mL: (1-5) mL;

[0023] (2) dissolving the first product in dimethylformamide, adding 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione and potassium carbonate in sequence, heating to reflux, extracting the organic phase, washing and drying, filtering and concentrating, separating and purifying, and obtaining the second product;

[0024] The mass ratio of the first product, 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione and potassium carbonate is (0.5-5):(0.5-5):(0.5-5);

[0025] (3) adding sodium ascorbate solution and copper sulfate solution to the mixture of the Bcl-2 inhibitor and the second product; heating and stirring, removing the solvent by rotary evaporation, dissolving the residue in ammonium hydroxide solution, extracting the organic phase, and drying; filtering and evaporating the solvent, separating and purifying to obtain a heterocyclic compound;

[0026] The mass volume ratio of Bcl-2 inhibitor, second product, sodium ascorbate solution and copper sulfate solution is (50-200) mg: (50-100) mg: (0.5-5) mL: (0.5-5) mL;

[0027] The mixed solvent, in volume ratio, is tert-butyl alcohol: water = 2 L: 1 L;

[0028] The first raw material is 2-azidoethanol-1-ol or 2-(2-azidoethoxy)ethoxyethane-1-ol; the first product is 2-azidoethyl methanesulfonate or 2-(2-azidoethoxy)ethoxyethyl methanesulfonate; the second product is 4-(2-(2-azidooxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione or 4-(2-(2-(-2-azidoethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione.

[0029] Further, when R1 is selected from Formula III, the preparation of the heterocyclic compound represented by Formula I comprises the following steps:

[0030] (1) Dissolve the raw material 1 in tetrahydrofuran, add sodium chloride, sodium iodoacetate and water, evaporate, and wash with water; after acidification, add sodium chloride to saturation, extract, dry, and evaporate to obtain the first product;

[0031] The mass ratio of raw material 1 and sodium iodoacetate is (1-5) g: (1-5) g;

[0032] (2) adding thionyl chloride dropwise to a tetrahydrofuran solution of the first product, heating to reflux, and rotary evaporating to obtain 2-(2-azidooxy)ethoxy)acetyl chloride; at room temperature, sequentially adding 2-(2-azidooxy)ethoxy)acetyl chloride and triethylamine to a tetrahydrofuran suspension of 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, heating to reflux, cooling to room temperature, and rotary evaporating, dissolving the residue in dichloromethane, washing, collecting the organic phase, drying overnight, distilling off the solvent, and separating and purifying to obtain the second product;

[0033] The mass ratio of thionyl chloride, the first product, triethylamine and 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione is (1-20) g: (1-5) g: (0.01-1) g: (0.01-1) g;

[0034] (3) dissolving the Bcl-2 inhibitor and the second product in a mixed solvent to obtain a suspension; adding sodium L-ascorbate and copper sulfate, heating and stirring, removing the solvent by rotary evaporation, dissolving the residue in NH4OH solution, extracting, drying, removing the solvent by evaporation, separating and purifying to obtain a heterocyclic compound;

[0035] The mass ratio of Bcl-2 inhibitor, second product, sodium L-ascorbate and copper sulfate is (1-5) mg: (1-5) mg: (1-5) mg: (1-5) mg;

[0036] The mixed solvent, in volume ratio, is tert-butyl alcohol: water = 2 L: 1 L;

[0037] The raw material 1 is 2-azidoethanol-1-ol or 2-(2-azidoethoxy)ethoxyethane-1-ol; the first product is 2-(2-azidoethoxy)ethoxyacetic acid or 2-(3-(2-azidoethoxy)-2-methoxypropoxy)acetic acid; the second product is 2-(2-(2-azidooxy)ethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide or 2-(2-(2-azidooxy)ethoxy)methoxy)N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide.

[0038] The fifth aspect of the present invention provides the use of the heterocyclic compound, its isomer or pharmaceutically acceptable salt thereof, the pharmaceutical preparation, and the pharmaceutical composition in the preparation of drugs for treating tumors, inflammation and immune-related diseases.

[0039] Beneficial effects of the present invention:

[0040] The compound PROTAC prepared by the present invention can induce the formation of the Bcl-2-PROTAC-E3 ligase ternary complex, and the E3 ligase can mediate the ubiquitination of the Bcl-2 protein by the ubiquitin-binding enzyme E2, which is then recognized and degraded by the proteasome in the cell. PROTAC is released and participates in the next degradation cycle. Experiments show that the compound provided by the present invention has the effect of degrading Bcl-2 protein in MDA-MB-231 cells, and the compound PROTAC77 has the best degradation activity, inducing DC degradation of Bcl-2 in MDA-MB-231 cells that highly express Bcl-2 protein. 50 The value is 0.206 μM. The present invention can be used as a strategy for developing Bcl-2 protein degraders and provides a powerful tool for biomedical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The synthetic routes of PROTAC67 and PROTAC77. Reagents and conditions: (a) TEA, DMF, rt, 1 h, 0℃, MsCl, rt, 2 h; (b) K2CO3, DMF, 80℃, 10 h; (c) t-BuOH / H2O, CuSO4, sodium L-ascorbate, 50℃, 2 h.

[0042] Figure 2 This is the NMR spectrum of compound 1A.

[0043] Figure 3 This is the NMR spectrum of compound 2A.

[0044] Figure 4 This is the NMR spectrum of compound PROTAC67.

[0045] Figure 5 This is the NMR spectrum of compound 1B.

[0046] Figure 6 This is the NMR spectrum of compound 2B.

[0047] Figure 7 This is the NMR spectrum of compound PROTAC77.

[0048] Figure 8 The synthetic routes of PROTAC49 and PROTAC79. Reagents and conditions: (d) NaH, THF, 0℃, 30min, sodium iodoacetate, rt, 32 h; (e) SOCl2, triethylamine, 70℃, 6 h; (f) t-BuOH / H2O, CuSO4, sodium ascorbate, 50℃, 2 h.

[0049] Fig. 9 This is the NMR spectrum of compound 1G.

[0050] Fig.10 This is the NMR spectrum of compound 2G.

[0051] Fig.11 This is the NMR spectrum of compound PROTAC49.

[0052] Fig.12 This is the NMR spectrum of compound 1H.

[0053] Fig.13 This is the NMR spectrum of compound 2H.

[0054] Fig.14 This is the NMR spectrum of compound PROTAC79.

[0055] Fig.15 This is a Western blot image of Bcl-2 protein level in MDA-MB-231 cells.

[0056] Fig.16 The relative expression level of Bcl-2 protein in MDA-MB-231 cells.

[0057] Fig.17 The diagrams are the structures of Compound 1A, Compound 2A, Compound 1B, Compound 2B, Compound 1G, Compound 2G, Compound 1H, and Compound 2H.

[0058] Fig.18 The structural diagrams of PROTAC67, PROTAC77, PROTAC49 and PROTAC79. DETAILED DESCRIPTION

[0059] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0060] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, and are commensurate with a reasonable benefit / risk ratio.

[0061] The pharmaceutically acceptable salts of the present invention refer to salts of the compounds of the present invention, prepared from compounds with specific substituents found in the present invention and relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and the like; also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups, and thus can be converted into either base or acid addition salts.

[0062] The isomers described in the present invention refer to compounds of the present invention that may exist in specific geometric or stereoisomer forms, including cis- and trans-isomers.

[0063] The pharmacologically active substances of the present invention are selected from any one or more of the following:

[0064] Hormones, hormone analogs and anti-hormones (such as tamoxifen, toremifene, aminoglutethimide, cyproterone acetate, bicalutamide, raloxifene, fulvestrant, flutamide, nilutamide, fludrocortisone, fluoxymesterone, finasteride, buserelin acetate, megestrol acetate, medroxyprogesterone, octreotide), LHRH agonists and antagonists (such as luprolide, goserelin acetate), aromatase inhibitors (such as anastrozole, letrozole, vorozole, exemestane, atamestane, riazole, etc.), etc.

[0065] Suitable formulations for administering the compounds of the invention will be apparent to those of ordinary skill in the art and include, for example, tablets, inhalants, solutions, suppositories, lozenges, syrups, emulsions, dispersible powders, cachets, elixirs, pills, capsules or lozenges; wherein solutions such as injection (subcutaneous, intravenous, intramuscular) solutions, infusion (injection) solutions, etc. The content of one or more pharmaceutically active compounds should range from 0.1 to 90 wt.%, preferably 0.5 to 50 wt.%, of the composition as a whole, i.e., an amount sufficient to achieve the dosage range specified below. If necessary, the specified dose can be given several times a day. Its delivery method can be administered to the body by known means. For example, systemic delivery by vein or local injection into the tissue of interest. It can be optionally administered via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It is understood by those skilled in the art that the actual dose to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0066] Researchers, veterinarians, doctors or other medical personnel in the field can learn the range of therapeutically effective amounts that can be used based on clinical trials or other methods known in the art.

[0067] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0068] The small molecule PROTAC prepared by the present invention comprises three parts: a Bcl-2 inhibitor bound to the Bcl-2 protein, a connecting chain, and pomalidomide or thalidomide, a ligand of the E3 ligase. Small molecule PROTAC can induce the formation of the Bcl-2-PROTAC-E3 ligase ternary complex. The E3 ligase mediates the ubiquitin-binding enzyme E2 to catalyze the transfer of ubiquitin (Ub) to Bcl-2 and form a ubiquitin chain, which is eventually recognized by the proteasome. Bcl-2 further dissociates from the E3 ligase, and the ubiquitinated target protein is recognized and degraded by the proteasome. In this process, the PROTAC molecule acts as a catalyst to promote the degradation of Bcl-2. Once Bcl-2 is recognized and degraded by the proteasome, the PROTAC molecule will be released and continue to participate in the next degradation process.

[0069] The experimental materials used in the examples of the present invention that are not specifically described are all conventional experimental materials in the art, and can be purchased through commercial channels or synthesized according to the literature. The Chinese name of the Bcl-2 inhibitor used in the present invention is pent-4-yn-2-yl (4-(1-(4-bromobenzoyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)carbamate, and the synthesis method is in accordance with the literature "Novel Bcl-2 Inhibitors Selectively Disrupt the Autophagy-Speciffc Bcl-2−Beclin 1 Protein−Protein Interaction" "DOI: 10.1021 / acsmedchemlett.2c00309", the CAS number of pomalidomide is 19171-19-8, and the CAS number of 4-hydroxythalidomide is 5054-59-1.

[0070] Example 1: Preparation of compound PROTAC67

[0071] The synthetic route of compound PROTAC67 is as follows Figure 1 As shown, where n=0.

[0072] Synthesis of compound 1A

[0073] Dissolve 2-azidoethanol-1-ol (1.6 g) in 10 mL dimethylformamide (DMF), add triethylamine (16 mL), stir at room temperature for 1 h, put in an ice bath, add methylsulfonyl chloride (1.9 mL) dropwise, and stir at room temperature for 2 h. TLC detection (volume ratio of the developing solvent is DCM: MeOH = 30:1). After the reaction is complete, add 10 mL of saturated NH4Cl solution to quench the reaction, extract with ethyl acetate (EA) (extract 3 times, each time using 80 mL of EA), take the combined organic phase, wash twice with saturated ammonium chloride solution, then wash twice with saturated brine, then dry with anhydrous sodium sulfate, filter and concentrate, and separate and purify by silica gel column chromatography (eluent by volume ratio, DCM: MeOH = 150:1), and the product is a white powder solid, namely compound 1A, Chinese name 2-azidoethyl methanesulfonate, mass 2.36 g, yield 74.9%. The product was tested by NMR, the results are shown in Figure 2 The structural formula of compound 1A is shown in Fig.17 .

[0074] Synthesis of compound 2A

[0075] 2-Azidoethyl methanesulfonate (500 mg) was dissolved in 10 mL dimethylformamide (DMF), and 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindole-1,3-dione (553.44 mg) and potassium carbonate (557.84 mg) were added in sequence. The mixture was heated to reflux at 80°C for 10 h and detected by TLC. After the reaction was complete, distilled water and ethyl acetate (EA) were added for extraction. The organic phase was washed with water and saturated brine and then dried over anhydrous sodium sulfate. The mixture was filtered and concentrated, and separated and purified by silica gel column chromatography (eluent by volume ratio, DCM: MeOH = 150: 1) to obtain a white solid powder, namely compound 2A, whose Chinese name is 4-(2-(2-azidooxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione, with a mass of 531 mg and a yield of 76.6%. The product was tested by NMR, and the results are shown in Figure 3 The structural formula of compound 2A is shown in Fig.17 .

[0076] Synthesis of compound PROTAC67

[0077] Bcl-2 inhibitor (159.13 mg) and compound 2A (85.82 mg) were dissolved in 4 mL of mixed solvent (volume ratio of tert-butyl alcohol: water = 2L:1L in the mixed solvent). Under light-shielding conditions, 0.1M sodium ascorbate solution (4 mL) and 0.1M copper sulfate solution (1.02 mL) were added to the reaction solution in sequence. After heating and stirring at 50°C for 6 hours, the reaction was complete after TLC detection. The mixture was cooled to room temperature and the solvent was removed by rotary evaporation. The residue was dissolved in 10mL of 2M ammonium hydroxide solution and then extracted with dichloromethane (extracted 3 times, each time with a volume of 40mL of dichloromethane). The organic phases were combined and dried overnight with anhydrous magnesium sulfate. The anhydrous magnesium sulfate was removed by filtration and the solvent was removed by evaporation at 45°C. The crude product was separated and purified by silica gel column chromatography (eluent by volume ratio, DCM: MeOH = 15: 1) to obtain a yellow powdery solid product, namely compound PROTAC67, with a mass of 134 mg and a yield of 61.3%. The product was tested by NMR, and the results are shown in Figure 4 The structural formula of compound PROTAC67 is shown in Fig.18 .

[0078] Example 2: Preparation of compound PROTAC77

[0079] The synthetic route of compound PROTAC77 is as follows Figure 1 As shown, where n=2.

[0080] Synthesis of compound 1B

[0081] The synthesis of compound 1B was carried out in the same manner as the synthesis of compound 1A in Example 1, except that 2-(2-azidoethoxy)ethoxyethane-1-ol (2 g) was used instead of 2-azidoethanol-1-ol (1.6 g). The final product, compound 1B, whose Chinese name is 2-(2-azidoethoxy)ethoxyethyl methanesulfonate, was obtained with a mass of 1.9 g and a yield of 65.7%. The product was tested by NMR, and the results are shown in Figure 5 The structural formula of compound 1B is shown in Fig.17 .

[0082] Synthesis of compound 2B

[0083] The synthesis of compound 2B was carried out in the same manner as the synthesis of compound 2A in Example 1, except that compound 1B (500 mg) was used instead of compound 1A. The final product, compound 2B, whose Chinese name is 4-(2-(2-(-2-azidoethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione, was obtained with a mass of 614 mg and a yield of 72.1%. The product was subjected to NMR testing, and the results are shown in Figure 6The structural formula of compound 2B is shown in Fig.17 .

[0084] Synthesis of compound PROTAC77

[0085] The synthesis of compound PROTAC77 was carried out in the same manner as the synthesis of compound PROTAC67 in Example 1, except that compound 2B (230 mg) was used instead of compound 2A. The final product, compound PROTAC77, was obtained with a mass of 239 mg and a yield of 57.3%. The product was subjected to NMR testing, and the results are shown in Figure 7 The structural formula of compound PROTAC77 is shown in Fig.18 .

[0086] Example 3: Preparation of compound PROTAC49

[0087] The synthetic route of compound PROTAC49 is as follows Figure 8 As shown, where n=1.

[0088] Synthesis of Compound 1G

[0089] 2-(2-Azidoethoxy)ethane-1-ol (1 g) was dissolved in 10 mL of dry tetrahydrofuran, and sodium chloride (304.74 mg) was added in batches under an ice-water bath. After stirring for 30 minutes, sodium iodoacetate (1.59 g) was added to the reaction solution, and stirred at room temperature for 36 h. 10 mL of water was added, and THF in the solution was removed by rotary evaporation. 30 mL of water was added to the residue, and the solution was washed with dichloromethane (3 times, each time with a volume of 40 mL of dichloromethane). 1 N HCl solution was used to acidify (pH = 1), sodium chloride was added to saturation, and dichloromethane was used for extraction (3 times, each time with a volume of 40 mL of dichloromethane). The organic phases were combined and dried over anhydrous magnesium sulfate overnight. The anhydrous magnesium sulfate was removed by filtration, and the solvent was removed by rotary evaporation at 45 ° C to obtain the product as a yellow oily liquid, namely compound 1G, whose Chinese name is 2-(2-azidoethoxy)ethoxyacetic acid, with a mass of 0.7 g and a yield of 48.6%. The product was tested by NMR, and the results are shown in Fig. 9 The structural formula of compound 1G is shown in Fig.17 .

[0090] Synthesis of compound 2G

[0091] Thionyl chloride (2.5 g) was added dropwise to a tetrahydrofuran (10 mL) solution of compound 1G (200 mg). After heating under reflux at 70°C for 3 hours, excess thionyl chloride and solvent were removed by rotary evaporation to obtain yellow oily liquid 2-(2-azidooxy)ethoxy)acetyl chloride (this oily intermediate did not need further purification and was immediately used for the next reaction). At room temperature, 2-(2-azidooxy)ethoxy)acetyl chloride and triethylamine (106.99 mg) were added to a tetrahydrofuran (10 mL) suspension of 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (95.33 mg) in sequence, and then the reaction solution was moved to 70°C and heated under reflux for 6 hours, then cooled to room temperature, and tetrahydrofuran was removed by rotary evaporation at 70°C. The residue was dissolved in 120 mL of dichloromethane, and washed three times with 50 mL of water and 50 mL of saturated sodium chloride solution, respectively. The organic phase was collected and dried over anhydrous magnesium sulfate overnight. The anhydrous magnesium sulfate was removed by filtration, and the solvent was evaporated at 45°C to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (eluent by volume ratio, DCM: MeOH = 15:1) to obtain a white powdery solid product, namely compound 2G, whose Chinese name is 2-(2-(2-azidooxy)ethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide, with a mass of 296 mg. The yield was 59.58%. The product was tested by NMR, and the results are shown in Fig.10 The structural formula of compound 2G is shown in Fig.17 .

[0092] Synthesis of compound PROTAC49

[0093] Bcl-2 inhibitor (86 mg) and compound 2G (60.04 mg) were dissolved in 3.9 mL of a mixed solvent (the volume ratio in the mixed solvent was tert-butyl alcohol: water = 2L: 1L) to obtain a suspension. Under light-proof conditions, sodium L-ascorbate (63 mg) and copper sulfate (25 mg, 0.158 mmol) were dissolved in 1 mL of water, respectively, and added to the above suspension in sequence. After heating and stirring at 50°C for 2 hours, the reaction was complete after TLC detection, and then cooled to room temperature. The solvent was removed by rotary evaporation at 45°C, and the residue was dissolved in 10 mL of 2M NH4OH solution, and then extracted with dichloromethane (extracted 3 times, each time using a volume of 40 mL of dichloromethane), the organic phases were combined, and dried overnight with anhydrous magnesium sulfate. The anhydrous magnesium sulfate was removed by filtration, and the solvent was removed by rotary evaporation at 45°C to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (eluent by volume ratio, DCM: MeOH = 15:1) to obtain a yellow powdery solid product, namely compound PROTAC49, with a mass of 86 mg and a yield of 65.29%. The product was tested by NMR, and the results are shown in Fig.11The structural formula of compound PROTAC49 is shown in Fig.18 .

[0094] Example 4: Preparation of compound PROTAC79

[0095] The synthetic route of compound PROTAC79 is as follows Figure 8 As shown, where n=2.

[0096] Synthesis of compound 1H

[0097] The synthesis of compound 1H was carried out in the same steps as the synthesis of compound 1G in Example 3, except that 2-(3-(2-azidoethoxy)-2-methoxypropoxy)acetic acid (200 mg) was used instead of 2-(2-azidoethoxy)ethane-1-ol (1 g). The final product, compound 1H, whose Chinese name is 2-(3-(2-azidoethoxy)-2-methoxypropoxy)acetic acid, has a mass of 1.9 g and a yield of 65.7%. The product was tested by NMR, and the results are shown in Fig.12 The structural formula of compound 1H is shown in Fig.17 .

[0098] Synthesis of compound 2H

[0099] The synthesis of compound 2H was carried out in the same manner as the synthesis of compound 2G in Example 3, except that compound 1H (200 mg) was used instead of compound 1G. The final product, compound 2H, whose Chinese name is 2-(2-(2-azidooxy)ethoxy)methoxy)N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)acetamide, was obtained with a mass of 289 mg and a yield of 69.0%. The product was subjected to NMR testing, and the results are shown in Fig.13 The structural formula of compound 2H is shown in Fig.17 .

[0100] Synthesis of compound PROTAC79

[0101] The synthesis of compound PROTAC79 was carried out in the same manner as the synthesis of compound PROTAC49 in Example 3, except that compound 2H (66.00 mg) was used instead of compound 2G. The final product, compound PROTAC79, was obtained with a mass of 87 mg and a yield of 63.2%. The product was subjected to NMR testing, and the results are shown in Fig.14 The structural formula of compound PROTAC79 is shown in Fig.18 .

[0102] Example 5: Western blot experiment

[0103] After treating MDA-MB-231 cells that highly express Bcl-2 with PROTAC67, PROTAC77, PROTAC49 and PROTAC79 prepared in Examples 1-4 at different concentrations, the level of intracellular Bcl-2 protein was determined by Western blot experiment, the steps are as follows:

[0104] Extract cell protein and perform protein quantification: cells were lysed with RIPA cell lysis buffer (with protease inhibitors), proteins were collected, and quantified with BCA protein quantification kit (Shanghai Biotech Co., Ltd.) to the same concentration.

[0105] Protein denaturation: Denature the protein by placing it in a 100°C water bath for 8 minutes.

[0106] Load sample and run gel: The sample volume per well is generally 20 μL. The electrophoresis voltage of the upper concentrated gel is 75 V, and the electrophoresis voltage of the lower separation gel is 120 V. Stop the electrophoresis when the 15 KD protein marker reaches about 1 cm from the end of the gel.

[0107] Transfer: Use a wet transfer apparatus for transfer. The PVDF membrane should be soaked in methanol in advance. The constant current is 250 mA and the transfer is performed for 90 minutes. Blocking: Place the PVDF membrane in BSA blocking solution for 1 h (shaking slowly on a shaker). Incubate with primary antibody: Dilute the primary antibody (Bcl2 Polyclonal antibody, Wuhan Tri-Eagle Biotechnology Co., Ltd.) according to the instructions. After adding the primary antibody, incubate overnight at 4°C.

[0108] Washing: Wash 3 times with TBST washing solution, 10 minutes each time. Incubate with secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG (H+L), Shanghai Bio-Tech Biotechnology Co., Ltd.): After adding the secondary antibody, incubate on a shaker at room temperature for 1 h. Washing: Wash 3 times with TBST washing solution, 10 minutes each time. Exposure: Mix equal volumes of ECL developer A and B for exposure.

[0109] MDA-MB-231 cells that highly express Bcl-2 protein were randomly divided into five groups and treated with PROTAC67, PROTAC77, PROTAC49 and PROTAC79, respectively, so that the concentrations of PROTAC in the culture medium were 0.1μM, 0.5μM, 1μM, 5μM and 10μM, respectively. The untreated group was the control group, and the internal standard was GAPDH. Western blot analysis was performed after 24 hours.

[0110] The results are as follows Fig.15 , Fig.16As shown in the figure, the Bcl-2 protein level decreased in a dose-dependent manner, among which compound PROTAC77 had the best degradation activity. PROTAC79, PROTAC49, PROTAC77, and PROTAC67 induced Bcl-2 degradation in MDA-MB-231 cells that highly expressed Bcl-2 protein. 50 The values ​​(drug concentrations that lead to 50% protein degradation) were 0.520 μM, 11.22 μM, 0.206 μM and 1.64 μM, respectively. The above results show that the present invention successfully established a Bcl-2 protein degrader based on the ubiquitin-proteasome system.

[0111] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A heterocyclic compound or a pharmaceutically acceptable salt thereof, characterized in that: The heterocyclic compound has a structure shown in Formula I: Formula I; Wherein, n is 0, 1, 2, 3, 4 or 5; R1 is selected from Formula II or Formula III, Formula II; Formula III.

2. The heterocyclic compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The heterocyclic compound is selected from any of the following compounds: 。 3. A pharmaceutical preparation, characterized in that The invention comprises the heterocyclic compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, and at least one other pharmacologically active substance.

4. A pharmaceutical composition, characterized in that The invention comprises the heterocyclic compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, and one or more pharmaceutically acceptable carriers, excipients and diluents.

5. The method for preparing the heterocyclic compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: When R1 is selected from Formula II, the preparation of the heterocyclic compound represented by Formula I comprises the following steps: (1) adding methylsulfonyl chloride to a mixture of raw material 1 and triethylamine, and after the reaction is completed, extracting the organic phase, washing and drying it, filtering it, concentrating it, and separating and purifying it to obtain the first product; The mass volume ratio of raw material 1, triethylamine and methanesulfonyl chloride is (1-5) g: (5-15) mL: (1-5) mL; (2) dissolving the first product in dimethylformamide, adding 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione and potassium carbonate in sequence, heating to reflux, extracting the organic phase, washing and drying, filtering and concentrating, separating and purifying, and obtaining the second product; The mass ratio of the first product, 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione and potassium carbonate is (0.5-5):(0.5-5):(0.5-5); (3) adding sodium ascorbate solution and copper sulfate solution to the mixture of the Bcl-2 inhibitor and the second product; heating and stirring, removing the solvent by rotary evaporation, dissolving the residue in ammonium hydroxide solution, extracting the organic phase, and drying; filtering and evaporating the solvent, separating and purifying to obtain a heterocyclic compound; The mass volume ratio of Bcl-2 inhibitor, second product, sodium ascorbate solution and copper sulfate solution is (50-200) mg: (50-100) mg: (0.5-5) mL: (0.5-5) mL; In step (3), the solvent used in the mixture is a mixed solvent, and the mixed solvent, by volume ratio, is tert-butyl alcohol: water = 2L:1L; The first raw material is 2-azidoethanol-1-ol or 2-(2-azidoethoxy)ethoxyethane-1-ol; the first product is 2-azidoethyl methanesulfonate or 2-(2-azidoethoxy)ethoxyethyl methanesulfonate; the second product is 4-(2-(2-azidooxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione or 4-(2-(2-(-2-azidoethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione.

6. The method for preparing a heterocyclic compound or a pharmaceutically acceptable salt thereof according to claim 5, characterized in that: When R1 is selected from formula III, the preparation of the heterocyclic compound represented by formula I comprises the following steps: (1) Dissolve the raw material 1 in tetrahydrofuran, add sodium chloride, sodium iodoacetate and water, rotary evaporate, and wash with water; after acidification, add sodium chloride to saturation, extract, dry, and rotary evaporate to obtain the first product; The mass ratio of raw material 1 and sodium iodoacetate is (1-5) g: (1-5) g; (2) adding thionyl chloride dropwise to a tetrahydrofuran solution of the first product, heating to reflux, and rotary evaporating to obtain 2-(2-azidooxy)ethoxy)acetyl chloride; at room temperature, sequentially adding 2-(2-azidooxy)ethoxy)acetyl chloride and triethylamine to a tetrahydrofuran suspension of 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, heating to reflux, cooling to room temperature, and rotary evaporating, dissolving the residue in dichloromethane, washing, collecting the organic phase, drying overnight, distilling off the solvent, and separating and purifying to obtain the second product; The mass ratio of thionyl chloride, the first product, triethylamine and 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione is (1-20) g: (1-5) g: (0.01-1) g: (0.01-1) g; (3) dissolving the Bcl-2 inhibitor and the second product in a mixed solvent to obtain a suspension; adding sodium L-ascorbate and copper sulfate, heating and stirring, and removing the solvent by rotary evaporation; dissolving the residue in NH4OH solution, extracting, drying, and removing the solvent by evaporation, and separating and purifying to obtain a heterocyclic compound; The mass ratio of Bcl-2 inhibitor, second product, sodium L-ascorbate and copper sulfate is (1-5) mg: (1-5) mg: (1-5) mg: (1-5) mg; The mixed solvent, in volume ratio, is tert-butyl alcohol: water = 2 L: 1 L; The raw material 1 is 2-azidoethanol-1-ol or 2-(2-azidoethoxy)ethoxyethane-1-ol; the first product is 2-(2-azidoethoxy)ethoxyacetic acid or 2-(3-(2-azidoethoxy)-2-methoxypropoxy)acetic acid; the second product is 2-(2-(2-azidooxy)ethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide or 2-(2-(2-azidooxy)ethoxy)methoxy)N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)acetamide.

7. Use of the heterocyclic compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, the pharmaceutical preparation according to claim 3, or the pharmaceutical composition according to claim 4 in the preparation of drugs for treating tumors, inflammation and immune-related diseases.

Images