Werner syndrome degradation agents, methods of making and using the same

Abstract

The application discloses a new bifunctional small molecule (I) with WRN degradation activity and a preparation method and application thereof. The compound can significantly inhibit tumor cell proliferation at a micromolar concentration level, has a double function of targeted inhibition and degradation, and has an application prospect of improving the drug resistance and toxicity of an anti-tumor drug.

Description

Technical Field

[0001] This invention relates to a novel bifunctional small molecule, its preparation method, and its applications, and more particularly to a bifunctional small molecule with WRN protein degradation activity, its preparation method, and its applications. Background Technology

[0002] In tumor cells with mismatch repair-deficient (dMMR), damage to microsatellite regions cannot be corrected in time, leading to the deletion or amplification of repetitive sequences—a phenomenon known as microsatellite instability (MSI). MSI is commonly found in sporadic non-hereditary tumors such as colorectal cancer, endometrial cancer, gastric cancer, and ovarian cancer. Currently, immune checkpoint inhibitors have been shown to be effective against several tumors carrying MSI-H (high microsatellite instability). However, the response rate of MSI-H tumors to immune checkpoint inhibitors is limited, and the dosage of immunosuppressants is limited by toxicity.

[0003] Studies have found that dMMR / MSI tumor cells undergo synthetic lethality after the WRN gene is knocked out or the WRN protein is depleted, leading to cell death. WRN (Werner helicase) belongs to the RecQ helicase family, and its gene is located on chromosome 8p11-12, consisting of 34 exons. The WRN protein is composed of 1432 amino acids and has five domains: a 3'→5' exonuclease domain at the N-terminus, an ATPase domain, a DNA-binding RQC (RecQ C-terminus) domain, a HRDC (helicase and ribonuclease DC-terminus) domain mediating protein-protein interactions, and a nuclear localization signaling domain at the C-terminus. Among these, the ATPase and RQC domains are the core of WRN's helical function and are key regions involved in its synthetic lethality.

[0004] Currently, Novartis' small molecule inhibitor HRO-761 and Vividion Therapeutics' covalent small molecule inhibitor VVD-133214 are in Phase I clinical trials, both specifically targeting the helicase domain of WRN. However, long-term use of WRN small molecule inhibitors can lead to drug resistance, ultimately reducing the therapeutic effect. Summary of the Invention

[0005] Purpose of the invention: The first objective of this invention is to provide a novel bifunctional small molecule; the second objective is to provide a method for preparing the compound; and the third objective is to provide a pharmaceutical application of the compound and its pharmaceutical composition.

[0006] Technical solution: The present invention relates to compounds represented by general formula (I) or pharmaceutically acceptable salts thereof.

[0007]

[0008] in:

[0009] R 1 Selected from

[0010] R 2 for R 3 Selected from H, OCH3, OH, CN, or halogens, R 4 Selected from H, F, Cl, Br, I or substituted C1-C4 alkyl groups, R 5 Selected from H, F, Cl, Br, I, SF3, C(O)H or substituted C1-C4 alkyl, substituted C1-C4 alkenyl, substituted C1-C4 alkynyl, substituted C3-C5 cycloalkyl or OCF3, R 6 The substituent is selected from H, F, Cl, Br, I, OH, CN, substituted C1-C4 alkyl, substituted C3-C5 cycloalkyl, or substituted C1-C4 alkoxy, wherein the substituent is selected from at least one H, F, Cl, Br, or I; X is selected from CR. 7 Or N, R 7 Selected from H, F, Cl, Br, or I;

[0011] A is selected from

[0012] n is an integer selected from 1 to 11 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11), m is an integer selected from 1 to 3 (1, 2, 3), and R 8 Selected from H, F, Cl, Br, I, or OH; Y, Z, and K are independently selected from CH, N, or CR. 9 R 9 Selected from H, F, Cl, Br, I, OCH3, OH, CN, CF3 or OCF3.

[0013] Preferred, R 1 Selected from

[0014] Preferred, R 2 Selected from

[0015] Preferably, A is selected from

[0016] n is an integer selected from 1 to 9 (1, 2, 3, 4, 5, 6, 7, 8, 9), and m is an integer selected from 1 to 3 (1, 2, 3).

[0017] Preferably, the compound described in this invention is selected from any one of the following compounds:

[0018]

[0019]

[0020]

[0021]

[0022] Protein degradation-targeting chimeras (PROTACs) are hybrid bifunctional compounds that recruit both target proteins and their degrading enzymes, specifically degrading the target proteins via the ubiquitin-proteasome pathway. This invention utilizes PROTAC technology to develop degraders targeting WRN, providing a new option for WRN-targeting drug development and potentially addressing issues such as drug resistance and toxicity associated with WRN inhibitors.

[0023] Preferably, the pharmaceutically acceptable salt is an acid addition salt formed by the compound and any one of the following acids: hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, malic acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or ferulic acid.

[0024] "Pharmaceutically acceptable salt" refers to the salt of the compounds of this invention, prepared by reacting a compound with a specific substituent discovered in this invention and a relatively non-toxic acid or base. When the compounds of this invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the free 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 amine, or magnesium salts or similar salts. When the compounds of this invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the free 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 (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfuric acid (forming sulfates or bisulfates), 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, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Acids such as citric acid, tartaric acid, and methanesulfonic acid; organic acid salts also include salts of organic acids such as amino acids (e.g., arginine), glucuronic acid, etc. Certain specific compounds of the present invention contain basic and acidic functional groups, thus allowing them to be converted into any base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and then the parent compound is separated, thereby regenerating the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubilities in polar solvents.

[0025] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of both. Non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0026] The method for preparing the compound or its pharmaceutically acceptable salt described in this invention is selected from any of the following methods:

[0027] Method 1: When A is When compound I-1 is subjected to acylation, iodination, substitution, coupling, deprotection and substitution, the compound IA is obtained.

[0028]

[0029] Compound I-3 is prepared from compounds I-1 and I-2 via an acylation reaction. The base used is triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium hydrogen hydride, potassium tert-butoxide, sodium methoxide, or sodium ethoxide, preferably N,N-diisopropylethylamine. The reaction solvent used is one or a mixture of any two of dichloromethane, ethyl acetate, acetone, tetrahydrofuran, dichloromethane, acetonitrile, toluene, ethylene glycol dimethyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, or dimethyl sulfoxide, preferably dichloromethane. The reaction temperature is 0–100°C, preferably 0–30°C.

[0030] Compound I-4 is prepared from compound I-3 via a substitution reaction, wherein the iodine salt used is potassium iodide or sodium iodide, preferably potassium iodide; the reaction solvent used is one or a mixture of any two of dichloromethane, ethyl acetate, acetone, tetrahydrofuran, dichloromethane, acetonitrile, toluene, ethylene glycol dimethyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide, preferably acetone; the reaction temperature is 0–100°C, preferably 50–80°C.

[0031] Compound I-6 is prepared from compounds I-4 and I-5, using a base such as triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium hydrogen hydride, potassium tert-butoxide, sodium methoxide, or sodium ethoxide, preferably N,N-diisopropylethylamine; the reaction solvent is one or a mixture of any two of ethyl acetate, acetone, tetrahydrofuran, dichloromethane, acetonitrile, toluene, ethylene glycol dimethyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, or dimethyl sulfoxide, preferably dioxane; the reaction temperature is 0–100°C, preferably 20–30°C.

[0032] Compound I-8 is prepared by a Suzuki coupling reaction of compound I-6 and organoboron reagent I-7. The solvent used is selected from one or a mixture of any two of the following: tetrahydrofuran, acetonitrile, N-methylpyrrolidone, N,N-dimethylacetamide, dimethyl sulfoxide, N,N-dimethylformamide, 1,4-dioxane, or water, preferably a 3:1 mixture of 1,4-dioxane and water. The base used is selected from potassium bicarbonate, potassium carbonate, sodium bicarbonate, sodium carbonate, potassium phosphate, sodium acetate, triethylamine, or N,N-diisopropylamine. The catalyst used is selected from tetra(triphenylphosphine)palladium (Pd(PPh3)4), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (Pd(dppf)Cl2), palladium acetate (Pd(OAc)2), tris(dibenzylacetone)palladium (Pd2(dba)3), or bis(triphenylphosphine)palladium dichloride (Pd(PPh3)2Cl2), preferably Pd(dppf)Cl2; the reaction temperature is 25℃~150℃, preferably 60~100℃.

[0033] Compound I-9 is prepared from compound I-8 by acid deprotection, wherein the acid used is hydrogen chloride, trifluoroacetic acid, trifluoromethanesulfonic acid, sulfuric acid, hydrobromic acid or phosphoric acid, preferably hydrogen chloride; the reaction solvent used is one or a mixture of any two of ethyl acetate, tetrahydrofuran, dioxane, methanol, ethanol or diethyl ether, preferably dioxane; the reaction temperature is 0 to 60°C, preferably 20 to 30°C.

[0034] Compound IA is prepared from compounds I-9 and I-10. The base used is triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium hydrogen hydride, potassium tert-butoxide, sodium methoxide, or sodium ethoxide, preferably N,N-diisopropylethylamine. The reaction solvent is one or a mixture of any two of ethyl acetate, acetone, tetrahydrofuran, dichloromethane, acetonitrile, toluene, ethylene glycol dimethyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, or dimethyl sulfoxide, preferably N,N-dimethylformamide. The reaction temperature is 0–150°C, preferably 25–80°C.

[0035] Method 2: When A is When compound I-9 and compound I-11 condense, the compound IB is obtained.

[0036]

[0037] Compound IB is prepared from compounds I-9 and I-11. The base used is triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, sodium hydroxide, sodium hydroxide, sodium hydroxide, potassium tert-butoxide, sodium methoxide, or sodium ethoxide, preferably N,N-diisopropylethylamine. The condensing agent used is N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI). The reaction mixture comprises 1-hydroxybenzotriazole (HOBt), propyl phosphoric anhydride (T3P), and trichlorotriazine, preferably N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU); the reaction solvent is one or a mixture of any two of ethyl acetate, acetone, tetrahydrofuran, dichloromethane, acetonitrile, toluene, ethylene glycol dimethyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, or dimethyl sulfoxide, preferably N,N-dimethylformamide; the reaction temperature is 0–60°C, preferably 0–25°C.

[0038] Method 3: When A is When compound I-9 condenses with compound I-12, the compound IC is obtained.

[0039]

[0040] Compound IB is prepared from compounds I-9 and I-12. The base used is triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, sodium hydroxide, sodium hydroxide, sodium hydroxide, potassium tert-butoxide, sodium methoxide, or sodium ethoxide, preferably N,N-diisopropylethylamine. The condensing agent used is N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (HATU), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI). The reaction mixture comprises 1-hydroxybenzotriazole (HOBt), propyl phosphoric anhydride (T3P), and trichlorotriazine, preferably N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU); the reaction solvent is one or a mixture of any two of ethyl acetate, acetone, tetrahydrofuran, dichloromethane, acetonitrile, toluene, ethylene glycol dimethyl ether, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, or dimethyl sulfoxide, preferably N,N-dimethylformamide; the reaction temperature is 0–60°C, preferably 0–25°C.

[0041] Among them, R 1 R 2 The definitions of n are as described above;

[0042] The compounds IA, IB, and IC obtained by the above method are salted with pharmaceutically acceptable acids to obtain the pharmaceutically acceptable salts.

[0043] The pharmaceutical compositions of the present invention comprise the compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0044] The pharmaceutically acceptable carrier can be an excipient widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods to allow the active ingredient to dissolve at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipient can be an inert filler, or it may provide a function such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The pharmaceutical excipient may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0045] The pharmaceutical compositions of the present invention can be prepared using any method known to those skilled in the art, based on the disclosure. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0046] The pharmaceutical compositions of this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0047] The compounds described in this invention, or their pharmaceutically acceptable salts or pharmaceutical compositions thereof, are used in the preparation of drugs for WRN protein degrading agents.

[0048] Preferably, the drug is a drug for treating tumors.

[0049] Further preferably, the drug is a drug for treating colorectal cancer, gastric cancer, prostate cancer, endometrial cancer, adrenocortical cancer, uterine cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer, or ovarian cancer.

[0050] The hydrates, solvates, or crystals of the compounds of general formula (I) of this invention, and their applications in the preparation of antitumor drugs, are also within the scope of protection of this invention.

[0051] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0052] The compounds designed in this invention can significantly inhibit tumor cell proliferation, reaching micromolar concentration levels, and have dual functions of targeted inhibition and degradation, thereby improving the drug resistance and toxicity of antitumor drugs. Detailed Implementation

[0053] The technical solution of the present invention will be further described below with reference to the embodiments.

[0054] Example 1: Synthesis of N-(2-chloro-4-(trifluoromethyl)phenyl)2-(2-(3,6-dihydro-2H-pyran-4-yl)6-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)-2-oxoethyl)piperazin-1-yl)5-ethyl-7-oxo[1,2,4]triazol[1,5-a]pyrimidin-4(7H)-yl)acetamide (IA-1)

[0055] (1) Synthesis of 2-bromo-N-(2-chloro-4-(trifluoromethyl)phenyl)acetamide (I-3)

[0056] 2-Chloro-4-(trifluoromethyl)aniline (5.00 g, 25.57 mmol) and triethylamine (2.60 g, 25.57 mmol) were dissolved in DCM (50 mL). Bromoacetyl bromide (5.40 g, 26.84 mmol) was slowly added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 6 hours. TLC (PE:EA = 4:1) showed that the starting material had reacted completely, and the reaction was stopped. Approximately 200 mL of water was added, and the mixture was extracted three times with 50 mL of DCM. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was subjected to column chromatography (PE:EA = 10:1) to give 6.96 g of a white solid, with a yield of 86.0%. 1 H NMR(300MHz, DMSO-d6)δ(ppm):10.18(s,1H),8.07(d,J=8.4Hz,1H),7.91(s,1H),7.73(d,J=8.1Hz,1H),4.24(s,2H).MS(ESI+)m / z:316.1[M+H] + .

[0057] (2) Synthesis of 2-iodo-N-(2-chloro-4-(trifluoromethyl)phenyl)acetamide (I-4)

[0058] Compound I-3 (10.00 g, 31.60 mmol) was dissolved in acetone (136 mL), and KI (5.76 g, 34.70 mmol) was added. The mixture was refluxed for 0.5 hours. TLC (PE:EA = 8:1) showed that the starting material had reacted completely, and the reaction was stopped. The reaction solution was cooled and filtered, and the filtrate was evaporated to dryness to give 11.71 g of a yellow solid, with a yield of 101.9%. MS (ESI+) m / z: 363.9 [M+H] + .

[0059] (3) Synthesis of 4-(2-bromo-4-(2-((2-chloro-4-(trifluoromethyl)phenyl)amine)-2-oxoethyl)-5-ethyl-7-oxo-4,7-2H-[1,2,4]triazol[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester (I-6)

[0060] 1.50 g (3.51 mmol) of 4-(2-bromo-5-ethyl-7-oxo-4,7-2H-[1,2,4]triazol[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester (I-4) was dissolved in 1,4-dioxane (15 mL), and DIEA (1.36 g, 10.53 mmol) was added. The mixture was stirred overnight at room temperature. TLC (PE:EA = 1:1) showed that the starting material had reacted completely, and the reaction was stopped. Approximately 60 mL of water was added, and the mixture was extracted three times with 20 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was purified by column chromatography to give 1.32 g of a pale yellow intermediate, with a yield of 56.7%. MS (ESI+) m / z: 662.10 [M+H] + .

[0061] (4) Synthesis of 4-(4-(2-(((2-chloro-4-(trifluoromethyl)phenyl)amine)-2-oxoethyl)-2-(3,6-dihydro-2H-pyran-4-yl)-ethyl-7-oxo-4,7-2H-[1,2,4]triazol[1,5-a]pyrimidin-6-yl)piperazine-1-carboxylic acid tert-butyl ester (I-8)

[0062] Compound I-6 (1.32 g, 1.99 mmol), pinacol ester of 3,6-dihydro-2H-pyran-4-boronic acid (0.63 g, 2.98 mmol), Pd(dppf)Cl2 (0.15 g, 0.20 mmol), and K3PO4 (1.27 g, 6.00 mmol) were dissolved in a mixed solvent (10 mL) of 1,4-dioxane and water (1,4-dioxane:water = 4:1). The mixture was reacted under N2 protection at 60 °C for 3 hours. TLC (DCM:MeOH = 20:1) showed complete reaction of the starting material, and the reaction was stopped. Approximately 50 mL of water was added, and the mixture was extracted three times with 15 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by column chromatography to give 0.976 g of a pale yellow solid, with a yield of 77.1%. 1H NMR (400MHz, DMSO-d6) δ (ppm): 10.35 (s, 1), 8.06 (d, J = 4.8 Hz, 1H), 7.96 (d, J = 2.4H z,1H),7.72(dd,J=4.8Hz,2.4Hz,1H),6.83(s,1H),5.31(s,1H),4.25(q,J=4.8Hz, 2H),3.93(m,2H),3.80(t,J=5.6Hz,2H),3.38(m,2H),2.96(m,2H),2.65(d,J=10.8 Hz,2H),2.52(m,2H),1.43(s,9H),1.17(t,J=7.6Hz,3H).MS(ESI+)m / z:666.0[M+H] + .

[0063] (5) Synthesis of N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-6-(piperazin-1-yl)-[1,2,4]triazol[1,5-a]pyrimidin-4(7H)-yl)acetamide (I-9)

[0064] Compound I-8 (0.98 g, 1.47 mmol) was dissolved in DCM (10 mL). A 4 M HCl solution of Dioxane (20 mL) was slowly added dropwise to the system under ice bath conditions, and the reaction was allowed to proceed overnight at room temperature. TLC (DCM:MeOH = 15:1) showed complete reaction of the starting material, and the reaction was stopped. The solution was evaporated to dryness to give 0.67 g of a white solid, yield 75.9%. MS (ESI+) m / z: 566.2 [M+H] + .

[0065] (6) Synthesis of N-(2-chloro-4-(trifluoromethyl)phenyl)2-(2-(3,6-dihydro-2H-pyran-4-yl)6-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)-2-oxoethyl)piperazin-1-yl)5-ethyl-7-oxo[1,2,4]triazol[1,5-a]pyrimidin-4(7H)-yl)acetamide (IA-1)

[0066] Compound I-9 (0.15 g, 0.27 mmol) and 2-bromo-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)acetamide (0.13 g, 0.32 mmol) were dissolved in DMF (1.5 mL), and DIEA (0.17 g, 1.33 mmol) was added. The reaction was carried out overnight at room temperature. TLC (DCM:MeOH = 10:1) showed that the starting material had reacted completely, and the reaction was stopped. Approximately 15 mL of water was added, and the mixture was extracted three times with 5 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to give a crude product. The crude product was purified by column chromatography to give 0.14 g of a yellow solid, with a yield of 62.0%. 1 H NMR(300MHz,DMSO-d6)δ(ppm):11.15(s,1),10.83(s,1),10.35(s,1),8.76(d,J=8.4Hz,1H) ,8.07(m,1H),7.97(s,1H),7.86(t,J=7.9Hz,1H),7.71(d,J=8.5Hz,1H),7.61(d,J=7.3Hz,1 H),6.82(s,1H),5.31(s,2H),5.17(m,1H),4.25(s,2H),3.81(m,4H),3.26(m,2H),2.96(m,6 H),2.65(m,5H),2.45(m,2H),2.11(s,1H),1.17(t,J=7.2Hz,3H).MS(ESI+)m / z:879.3[M+H] + .

[0067] Example 2: Synthesis of 4-(4-(4-(2-((2-chloro-4-(trifluoromethyl)phenyl)amine)-2-oxoethyl)-2-(3,6-dihydro-2H-pyran-4-yl)-ethyl-7-oxo-4,7-dihydro-[1,2,4]triazol[1,5-a]pyrimidin-6-yl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)butyramide (IA-2)

[0068] Compound I-9 (0.15 g, 0.27 mmol) and 4-bromo-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)butyramide (0.17 g, 0.40 mmol) were dissolved in DMF (2 mL), and DIEA (0.14 g, 1.06 mmol) was added. The mixture was heated to 60 °C and reacted for 4 h. TLC (DCM:MeOH = 10:1) showed that the starting material had basically reacted to completion, and the reaction was stopped. Approximately 15 mL of water was added, and the mixture was extracted three times with 5 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was subjected to column chromatography to obtain 0.15 g of a yellow solid, with a yield of 61.2%. 1 H NMR (400MHz, DMSO-d6) δ (ppm): 11.16 (s, 1H), 10.41 (s, 1H), 9.80 (s, 1H), 8.46 (t, J = 8.4Hz, 1H), 8.05 (t, J = 8.0Hz, 1H) ,7.96(d,J=2.1Hz,1H),7.85(t,J=8.4Hz,1H),7.72(dd,J=8.8,2.1Hz,1H),7.63(s,J=8.0Hz,1H),6.83(s,1H),5.32( s,2H),5.15(q,J=12.8,5.4Hz,1H),4.25(d,J=2.9Hz,2H),3.80(t,J=5.5Hz,2H),3.67(m,2H),3.37(m,6H),2.90(m,4 H),2.70(m,3H),2.56(m,3H),2.52(m,1H),2.07(m,1H),1.92(m,2H),1.16(t,J=7.5Hz,3H).MS(ESI+)m / z:907.3[M+H] + .

[0069] Example 3: Synthesis of 6-(4-(4-(2-((2-chloro-4-(trifluoromethyl)phenyl)amine)-2-oxoethyl)-2-(3,6-dihydro-2H-pyran-4-yl)-ethyl-7-oxo-4,7-dihydro-[1,2,4]triazol[1,5-a]pyrimidin-6-yl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)hexanoamide (IA-3)

[0070] Compound I-9 (0.15 g, 0.27 mmol) and 6-bromo-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)hexamethylenetetramine (0.14 g, 0.32 mmol) were dissolved in DMF (1 mL), and DIEA (0.171 g, 1.33 mmol) was added. The mixture was heated to 80 °C and reacted for 6 h. TLC (DCM:MeOH = 10:1) showed that the starting material had basically reacted to completion, and the reaction was stopped. Approximately 10 mL of water was added, and the mixture was extracted three times with 4 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was subjected to column chromatography to obtain 0.15 g of a yellow solid, with a yield of 62.1%. 1 H NMR (400MHz, DMSO-d6) δ (ppm): 11.15 (s, 1H), 10.41 (s, 1H), 9.72 (s, 1H), 8.47 (t, J = 8.8Hz, 1H), 8.05 (t, J = 7.4Hz, 1H), 7.9 6(d,J=2.1Hz,1H),7.84(t,J=7.9Hz,1H),7.72(dd,J=8.7,2.1Hz,1H),7.62(d,J=7.3Hz,1H),6.83(s,1H),5.32(s,2H),5.1 5(q,J=5.4Hz,1H),4.25(d,J=2.9Hz,2H),3.80(t,J=5.5Hz,2H),3.73(m,2H),3.36(m,6H),2.95(m,3H),2.91(m,2H),2.79 (m,2H),2.68(m,2H),2.54(m,2H),2.07(m,1H),1.66(m,4H),1.37(m,2H),1.16(t,J=7.4Hz,3H).MS(ESI+)m / z:935.3[M+H] + .

[0071] Example 4: Synthesis of 8-(4-(4-(2-((2-chloro-4-(trifluoromethyl)phenyl)amine)-2-oxoethyl)-2-(3,6-dihydro-2H-pyran-4-yl)-ethyl-7-oxo-4,7-dihydro-[1,2,4]triazol[1,5-a]pyrimidin-6-yl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)octamide (IA-4)

[0072] Compound I-9 (0.15 g, 0.27 mmol) and 8-bromo-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)octamide (0.15 g, 0.32 mmol) were dissolved in DMF (1.5 mL), and DIEA (0.17 g, 1.33 mmol) was added. The mixture was heated to 80 °C and reacted for 6 h. TLC (DCM:MeOH = 10:1) showed that the starting material had basically reacted to completion, and the reaction was stopped. Approximately 15 mL of water was added, and the mixture was extracted three times with 5 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was subjected to column chromatography to obtain 0.15 g of a pale yellow solid, with a yield of 56.8%. 1 HNMR (400MHz, DMSO-d6) δ (ppm): 11.17 (s, 1H), 10.44 (s, 1H), 9.72 (s, 1H), 8.47 (t, J = 8.4Hz, 1H), 8.05 (t, J = 7.5Hz ,1H),7.97(d,J=2.1Hz,1H),7.83(t,J=8.0Hz,1H),7.72(dd,J=8.8,2.2Hz,1H),7.62(d,J=7.3Hz,1H),6.83(s,1H ),5.33(s,2H),5.15(q,J=5.4Hz,1H),4.25(d,J=2.9Hz,2H),3.80(m,4H),3.38(m,6H),2.90(m,6H),2.63(m,2H), 2.54(m,1H),2.46(m,2H),2.05(m,1H),1.65(m,4H),1.35(m,6H),1.16(t,J=7.5Hz,3H).MS(ESI+)m / z:963.3[M+H] + .

[0073] Example 5: Synthesis of 10-(4-(4-(2-((2-chloro-4-(trifluoromethyl)phenyl)amine)-2-oxoethyl)-2-(3,6-dihydro-2H-pyran-4-yl)-ethyl-7-oxo-4,7-dihydro-[1,2,4]triazol[1,5-a]pyrimidin-6-yl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)decanoamide (IA-5)

[0074] Compound I-9 (0.15 g, 0.27 mmol) and 10-bromo-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)decanoamide (0.16 g, 0.32 mmol) were dissolved in DMF (1.5 mL), and DIEA (0.17 g, 1.33 mmol) was added. The mixture was heated to 80 °C and reacted for 6 h. TLC (DCM:MeOH = 10:1) showed that the starting material had basically reacted to completion, and the reaction was stopped. Approximately 15 mL of water was added, and the mixture was extracted three times with 5 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was subjected to column chromatography to obtain 0.138 g of a pale yellow solid, with a yield of 52.5%. 1 H NMR (400MHz, DMSO-d6) δ (ppm): 11.15 (s, 1H), 10.36 (s, 1H), 9.69 (s, 1H), 8.48 (dd, J = 8.4, 5.4Hz, 1H), 8.06 (t, J = 7.2Hz, 1H), 7.9 6(d,J=2.1Hz,1H),7.82(t,J=8.4,1H),7.71(dd,J=8.7,2.2Hz,1H),7.60(d,J=7.3Hz,1H),6.81(m,1H),5.30(s,2H),5.14(dd,J =12.8,5.4Hz,1H),4.24(d,J=2.9Hz,2H),3.79(t,J=5.4Hz,2H),3.57(d,J=11.3Hz,2H),3.36(m,6H),2.90(m,5H),2.61(m,3H), 2.54(m,1H),2.46(m,2H),2.06(m,1H),1.63(m,2H),1.45(m,2H),1.30(m,10H),1.16(t,J=7.4Hz,3H).MS(ESI+)m / z:991.3[M+H] + .

[0075] Example 6: Synthesis of 2-(4-(4-(2-((2-chloro-4-(trifluoromethyl)phenyl)amino)-2-oxoethyl)-2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-4,7-dihydro-[1,2,4]triazol[1,5-a]pyrimidin-6-yl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)butyramide (IA-6)

[0076] Compound I-9 (0.15 g, 0.27 mmol) and 2-bromo-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)acetamide (0.13 g, 0.32 mmol) were dissolved in DMF (1.5 mL), and DIEA (0.171 g, 1.33 mmol) was added. The reaction was carried out at room temperature for 6 h. TLC (DCM:MeOH = 10:1) showed that the starting material had basically reacted to completion, and the reaction was stopped. Approximately 15 mL of water was added, and the mixture was extracted three times with 5 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was subjected to column chromatography to obtain 0.042 g of a pale yellow solid, with a yield of 17.2%. 1 H NMR (400MHz, CDCl3) δ (ppm): 11.14 (s, 1H), 10.53 (s, 1H), 10.38 (s, 1H), 8.32 (d, J = 1.8Hz, 1H), 8.04 (d, J = 8.4Hz ,1H),8.02(d,J=8.4Hz,1H),7.97(d,J=2.1Hz,1H),7.90(d,J=8.2Hz,1H),7.72(dd,J=8.9,2.1Hz,1H),6.83(m,1 H),5.32(s,2H),5.14(m,1H),4.25(m,2H),3.80(t,J=4.2Hz,2H),3.74(m,2H),3.29(m,1H),2.90(m,5H),2.56( m,4H),2.43(m,1H),2.06(m,1H),1.29(m,1H),1.21(m,2H),1.18(m,3H),0.84(m,1H).MS(ESI+)m / z:879.3[M+H] + .

[0077] Example 7: Synthesis of 4-(4-(4-(2-((2-chloro-4-(trifluoromethyl)phenyl)amino)-2-oxoethyl)-2-(3,6-dihydro-2H-pyran-4-yl)-5-ethyl-7-oxo-4,7-dihydro-[1,2,4]triazol[1,5-a]pyrimidin-6-yl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)butyramide (IA-7)

[0078] Compound I-9 (0.15 g, 0.27 mmol) and 4-bromo-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)butyramide (0.13 g, 0.32 mmol) were dissolved in DMF (1.5 mL), and DIEA (0.17 g, 1.33 mmol) was added. The mixture was heated to 80 °C and reacted for 6 h. TLC (DCM:MeOH = 10:1) showed that the starting material had basically reacted to completion, and the reaction was stopped. Approximately 15 mL of water was added, and the mixture was extracted three times with 5 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was subjected to column chromatography to obtain 0.052 g of a pale yellow solid, with a yield of 20.8%. 1 H NMR (400MHz, CDCl3) δ (ppm): 11.13 (s, 1H), 10.74 (s, 1H), 10.41 (s, 1H), 8.29 (s, 1H), 8 .05(d,J=8.6Hz,1H),7.92(m,3H),7.72(d,J=8.7Hz,1H),6.83(s,1H),5.31(m,2H),5.1 2(m,1H),4.25(m,2H),3.80(t,J=5.5Hz,2H),3.64(m,2H),2.90(m,5H),2.64(m,5H),2 .05(m,1H),1.89(m,2H),1.23(m,2H),1.16(t,J=7.4Hz,3H).MS(ESI+)m / z:907.3[M+H] + .

[0079] Example 8: Synthesis of N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-6-(4-((2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amine)acetyl)piperazin-1-yl)-5-ethyl-7-oxo-[1,2,4]triazol[1,5-a]pyrimidin-4(7H)-yl)acetamide (IB-1)

[0080] Compound (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)glycine (0.097 g, 0.29 mmol) was dissolved in DMF (2 mL), and HATU (0.11 g, 0.29 mmol) and DIEA (0.21 g, 1.59 mmol) were added. After stirring at room temperature for half an hour, compound I-9 (0.15 g, 0.27 mmol) was added to the reaction system, and the reaction was carried out at room temperature for 6 hours. TLC (DCM:MeOH = 15:1) showed that the starting material had basically reacted completely, and the reaction was stopped. About 15 mL of water was added, and the mixture was extracted three times with 5 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was subjected to column chromatography to obtain 0.15 g of a yellow solid, with a yield of 65.3%. 1 H NMR (400MHz, DMSO-d6) δ (ppm): 11.15 (s, 1H), 10.36 (s, 1H), 8.07 (t, J = 7.8Hz, 1H), 7.97 (d, J = 2.1Hz, 1H), 7.72 (m ,1H),7.61(t,J=7.8Hz,1H),7.10(m,2H),6.83(m,1H),5.33(s,2H),5.08(m,1H),4.40(m,2H),4.24(d,J=4Hz,2H) ,4.16(d,J=16.8Hz,1H),3.95(d,J=12.3Hz,1H),3.80(t,J=5.5Hz,2H),3.54(m,1H),3.44(s,1H),3.24(m,2H),3 .00(m,2H),2.88(m,2H),2.75(m,2H),2.60(m,2H),2.05(m,1H),1.20(t,J=7.4Hz,3H).MS(ESI+)m / z:879.3[M+H] + .

[0081] Example 9: Synthesis of N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-6-(4-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amine)butyryl)piperazin-1-yl)-5-ethyl-7-oxo-[1,2,4]triazol[1,5-a]pyrimidin-4(7H)-yl)acetamide (IB-2)

[0082] Compound 4-((2-(2,6-dioxopiperidin-3-yl)-dioxoisoindoline-4-yl)amine)butyric acid (0.070 g, 0.19 mmol) was dissolved in DMF (2 mL), and HATU (0.072 g, 0.19 mmol) and DIEA (0.15 g, 1.17 mmol) were added. After stirring at room temperature for half an hour, compound I-9 (0.11 g, 0.19 mmol) was added to the reaction system, and the reaction was carried out at room temperature for 6 hours. TLC (DCM:MeOH = 15:1) showed that the starting material had basically reacted completely, and the reaction was stopped. About 15 mL of water was added, and the mixture was extracted three times with 5 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was subjected to column chromatography to obtain 0.11 g of a yellow solid, with a yield of 65.0%. 1 H NMR (400MHz, CDCl3) δ (ppm): 9.00 (s, 1H), 8.46 (d, J = 8.7Hz, 1H), 8.22 (m, 1H), 7.63 (d, J = 2.0Hz, 1H), 7.53 (m, 2H), 7.0 9(d,J=7.1Hz,1H),7.00(d,J=8.6Hz,1H),6.95(m,1H),6.33(m,1H),5.10(m,2H),4.92(m,1H),4.64(d,J=12.8Hz,1H), 4.36(q,J=2.8Hz,2H),3.91(t,J=5.4Hz,2H),3.82(m,1H),3.64(m,2H),3.40(q,J=6.6Hz,2H),3.25(m,1H),3.12(m,2 H),2.82(m,4H),2.71(m,4H),2.49(m,2H),2.13(m,1H),2.04(m,2H),1.29(t,J=7.1Hz,3H).MS(ESI+)m / z:907.3[M+H] + .

[0083] Example 10: Synthesis of N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-6-(4-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amine)hexanoyl)piperazin-1-yl)-5-ethyl-7-oxo-[1,2,4]triazol[1,5-a]pyrimidin-4(7H)-yl)acetamide (IB-3)

[0084] Compound 6-((2-(2,6-dioxopiperidin-3-yl)-dioxoisoindoline-4-yl)amine)hexanoic acid (0.12 g, 0.32 mmol) was dissolved in DMF (2 mL), and HATU (0.12 g, 0.32 mmol) and DIEA (0.21 g, 1.59 mmol) were added. After stirring at room temperature for half an hour, compound IA-1-9 (0.15 g, 0.27 mmol) was added to the reaction system, and the reaction was carried out at room temperature for 6 hours. TLC (DCM:MeOH = 15:1) showed that the starting material had basically reacted completely, and the reaction was stopped. About 15 mL of water was added, and the mixture was extracted three times with 5 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was subjected to column chromatography to obtain a yellow solid of 0.22 g, with a yield of 87.6%. 1 H NMR (400MHz, CDCl3) δ (ppm): 9.02 (s, 1H), 8.47 (d, J = 8.7Hz, 1H), 8.17 (s, 1H), 7.63 (d, J = 2.0Hz, 1H), 7.51 (m, 2H), 7.07 (d, J = 7 .0Hz,1H),6.96(m,1H),6.89(d,J=8.5Hz,1H),6.23(t,J=5.6Hz,1H),5.11(d,J=6.1Hz,2H),4.90(m,1H),4.64(d,J=12.3Hz,1H ),4.36(d,J=2.9Hz,2H),3.91(t,J=5.4Hz,2H),3.84(d,J=13.0Hz,1H),3.71(m,1H),3.59(m,1H),3.27(m,3H),3.15(m,2H),2 .83(m,4H),2.71(m,4H),2.39(m,2H),2.13(m,1H),1.74(m,4H),1.49(m,2H),1.30(t,J=7.5Hz,3H).MS(ESI+)m / z:935.3[M+H] + .

[0085] Example 11: Synthesis of N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-6-(4-(8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amine)octanoyl)piperazin-1-yl)-5-ethyl-7-oxo-[1,2,4]triazol[1,5-a]pyrimidin-4(7H)-yl)acetamide (IB-4)

[0086] Compound 8-((2-(2,6-dioxopiperidin-3-yl)-dioxoisoindoline-4-yl)amine)octanoic acid (0.13 g, 0.32 mmol) was dissolved in DMF (2 mL), and HATU (0.12 g, 0.32 mmol) and DIEA (0.21 g, 1.59 mmol) were added. After stirring at room temperature for half an hour, compound I-9 (0.150 g, 0.265 mmol) was added to the reaction system, and the reaction was carried out at room temperature for 6 hours. TLC (DCM:MeOH = 15:1) showed that the starting material had basically reacted completely, and the reaction was stopped. About 15 mL of water was added, and the mixture was extracted three times with 5 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by column chromatography to give 0.13 g of a yellow solid, with a yield of 49.8%. 1 H NMR (400MHz, CDCl3) δ (ppm): 9.03 (s, 1H), 8.46 (d, J = 8.7Hz, 1H), 8.21 (s, 1H), 7.63 (d, J = 2.0Hz, 1H), 7.51 (m, 2H), 7 .06(d,J=7.0Hz,1H),6.95(m,1H),6.88(d,J=8.5Hz,1H),6.22(m,1H),5.11(m,2H),4.89(m,1H),4.64(d,J=12.5Hz ,1H),4.36(q,J=2.8Hz,2H),3.82(m,3H),3.70(t,J=11.3Hz,1H),3.59(t,J=10.9Hz,1H),3.26(m,3H),3.14(m,2H) ,2.79(m,8H),2.39(m,2H),2.12(m,1H),1.66(m,4H),1.39(m,6H),1.30(t,J=7.3Hz,3H).MS(ESI+)m / z:963.3[M+H] + .

[0087] Example 12: Synthesis of N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-6-(4-(11-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amine)undecanoyl)piperazin-1-yl)-5-ethyl-7-oxo-[1,2,4]triazol[1,5-a]pyrimidin-4(7H)-yl)acetamide (IB-5)

[0088] Compound 11-((2-(2,6-dioxopiperidin-3-yl)-dioxoisoindoline-4-yl)amine)undecanoic acid (0.14 g, 0.32 mmol) was dissolved in DMF (2 mL), and HATU (0.12 g, 0.32 mmol) and DIEA (0.21 g, 1.59 mmol) were added. After stirring at room temperature for half an hour, compound I-9 (0.15 g, 0.27 mmol) was added to the reaction system, and the reaction was stopped at room temperature for 6 hours. TLC (DCM:MeOH = 15:1) showed that the starting material had basically reacted completely, and the reaction was stopped. About 15 mL of water was added, and the mixture was extracted three times with 5 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was subjected to column chromatography to obtain 0.18 g of a yellow solid, with a yield of 66.8%. 1 H NMR (400MHz, CDCl3) δ (ppm): 9.03 (s, 1H), 8.46 (d, J = 8.7Hz, 1H), 8.25 (d, J = 7.3Hz, 1H), 7.63 (d, J = 2.0Hz, 1H), 7.50 (m, 2H), 7. 06(d,J=7.1Hz,1H),6.96(m,1H),6.87(d,J=8.6Hz,1H),6.22(m,1H),5.11(d,J=5.7Hz,2H),4.90(m,1H),4.64(d,J=12.7Hz,1H ),4.35(q,J=2.8Hz,2H),3.90(t,J=5.4Hz,2H),3.85(d,J=12.7Hz,1H),3.70(t,J=11.3Hz,1H),3.59(t,J=11.5Hz,1H),3.25( m,3H),3.14(m,2H),2.73(m,8H),2.36(m,2H),2.12(m,1H),1.66(m,4H),1.40(m,2H),1.32(m,3H).MS(ESI+)m / z:1005.3[M+H] + .

[0089] Example 13: Synthesis of N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-6-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)piperidin-4-acyl)piperazin-1-yl)-5-ethyl-7-oxo-[1,2,4]triazol[1,5-a]pyrimidin-4(7H)-yl)acetamide (IC-1)

[0090] Compound 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)piperidin-4-carboxylic acid (0.12 g, 0.32 mmol) was dissolved in DMF (2 mL), and HATU (0.12 g, 0.32 mmol) and DIEA (0.21 g, 1.59 mmol) were added. After stirring at room temperature for half an hour, compound I-9 (0.15 g, 0.27 mmol) was added to the reaction system, and the reaction was carried out at room temperature for 6 hours. TLC (DCM:MeOH = 15:1) showed that the starting material had basically reacted completely, and the reaction was stopped. About 15 mL of water was added, and the mixture was extracted three times with 5 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was subjected to column chromatography to obtain a yellow solid of 0.23 g, with a yield of 92.2%. 1 H NMR (400MHz, CDCl3) δ (ppm): 9.03 (s, 1H), 8.47 (d, J = 8.7Hz, 1H), 8.20 (m, 1H), 7.63 (d, J = 2.0Hz, 1 H),7.53(m,2H),7.35(d,J=7.3Hz,1H),7.18(d,J=8.3Hz,1H),6.95(m,1H),5.13(m,2H),5.95(m, 1H),4.66(m,1H),4.36(m,2H),3.91(t,J=5.3Hz,3H),3.78(m,3H),3.60(m,1H),3.32(m,1H),3.0 9(m,4H),2.79(m,9H),2.10(m,3H),1.87(m,2H),1.32(d,J=7.0Hz,3H).MS(ESI+)m / z:933.3[M+H] + .

[0091] Example 14: Synthesis of N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(2-(3,6-dihydro-2H-pyran-4-yl)-6-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-acyl)piperazin-1-yl)-5-ethyl-7-oxo-[1,2,4]triazol[1,5-a]pyrimidin-4(7H)-yl)acetamide (IC-2)

[0092] Compound 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-carboxylic acid (0.12 g, 0.32 mmol) was dissolved in DMF (2 mL), and HATU (0.12 g, 0.32 mmol) and DIEA (0.21 g, 1.59 mmol) were added. After stirring at room temperature for half an hour, compound I-9 (0.15 g, 0.27 mmol) was added to the reaction system, and the reaction was carried out at room temperature for 6 hours. TLC (DCM:MeOH = 15:1) showed that the starting material had basically reacted to the end, and the reaction was stopped. About 15 mL of water was added, and the mixture was extracted three times with 5 mL of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was subjected to column chromatography to obtain a yellow solid of 0.21 g, with a yield of 85.3%. 1 H NMR (400MHz, CDCl3) δ (ppm): 9.03 (s, 1H), 8.42 (m, 2H), 7.63 (m, 2H), 7.51 (dd, J = 8.8, 2.1Hz, 1H), 7.25(d,J=2.3Hz,1H),7.03(dd,J=8.6,2.3Hz,1H),6.93(m,1H),5.13(m,2H),4.93(m,1H),4.63(d ,J=12.8Hz,1H),4.34(m,2H),3.95(m,5H),3.73(m,1H),3.58(m,1H),3.33(t,J=11.9Hz,1H),3.0 8(m,4H),2.79(m,9H),2.12(m,1H),1.95(m,4H),1.30(t,J=7.6Hz,3H).MS(ESI+)m / z:933.3[M+H] + .

[0093] Example 15: Evaluation of the inhibitory activity of the compound on tumor cell proliferation

[0094] 1. Experimental Methods

[0095] (1) Human colon adenocarcinoma cell line (SW48) cultured to the logarithmic growth phase was plated into 96-well plates at a pre-specified density in a medium containing fetal bovine serum;

[0096] (2) Cells were treated with a compound or medium (DMSO) 24 hours later, and day 0 plates were collected for analysis;

[0097] (3) After the drug was applied, the 96-well plate was placed in a constant temperature incubator at 37°C and 5% CO2. After 5 days, 20 μL of 1.0% MTT thiazolyl blue solution was added to each well.

[0098] (4) Continue to place in a constant temperature incubator. After 4 hours, use a suction device to remove the supernatant culture medium, add 150 μL DMSO to each well, and place on a decolorizing shaker to mix until the crystals dissolve.

[0099] (5) Measure the absorbance at 570 nm using a multi-functional microplate reader and calculate the IC50 using the modified Kohl's method. 50 value:

[0100] lgIC 50 =Xm-I[P-(3-Pm-Pn) / 4].

[0101] 2. Experimental Results

[0102] The specific results are shown in Table 1. IC 50 <1μM (denoted as: A); IC 50 =1~10μM (denoted as: B); IC 50 >10μM (denoted as: C).

[0103] Table 1. Results of the inhibitory activity of the compounds on SW48 cell line.

[0104] Number <![CDATA[IC 50 ]]> Number <![CDATA[IC 50 ]]> I-A-1 C I-B-1 C I-A-2 B I-B-2 C I-A-3 C I-B-3 C I-A-4 B I-B-4 C I-A-5 C I-B-5 C I-A-6 B I-C-1 C I-A-7 B I-C-2 C

[0105] Table 1 shows that the compounds of the present invention exhibit good inhibitory activity against the human colon adenocarcinoma SW48 cell line, inhibiting IC50. 50 The value reached the micromolar concentration level.

Claims

1. A compound of general formula (I) or a pharmaceutically acceptable salt thereof, ， in: R 1 Selected from ; R 2 for ; A is selected from n is selected from 3 or 7.

2. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound is selected from any one of the following compounds: 。 3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The pharmaceutically acceptable salt is an acid addition salt formed by the compound and any one of the following acids: hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, malic acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or ferulic acid.

4. A method for preparing the compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Compound IA is obtained by acylation, iodination, substitution, coupling, deprotection, and substitution of compound I-1; ； Among them, R 1 R 2 The definitions of n are as described in claim 1; The compound IA obtained by the above method is salted with a pharmaceutically acceptable acid to obtain the pharmaceutically acceptable salt.

5. A pharmaceutical composition, characterized in that, It comprises the compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

6. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 5, in the preparation of a medicament for a WRN protein degrader.

7. The application according to claim 6, characterized in that, The drug mentioned is a drug for treating tumors.

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