PROTAC chimera for targeted degradation of ROR [gamma] t receptor and application of PROTAC chimera

Abstract

The invention discloses a PROTAC chimera for targeted degradation of an ROR [gamma] t receptor and application of the PROTAC chimera, the structure of the chimera is RW-L-Re, Re is a ligand capable of being combined with E3 ubiquitin ligase, L is a linking group covalently combined with at least one Re and at least one RW, and Rw is a target protein ROR [gamma] t binding ligand and is selected from one of the following structures. A series of PROTAC chimeras capable of degrading an ROR gamma t receptor in a targeted manner are designed and synthesized for the first time, a ternary complex is formed mainly by combining a target protein ligand and an E3 ubiquitin ligase ligand with POI and E3 ligase respectively, and then the POI is labeled with a ubiquitination tag and is further degraded by proteasome. Experimental results prove that the PROTAC chimera designed by the invention has excellent ROR [gamma] t receptor degradation activity, and can be used for preparing related drugs for treating autoimmune diseases or tumors.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a PROTAC chimera that targets and degrades the RORγt receptor and its uses. Background Technology

[0002] Retinoic acid receptor-related orphan receptors (RORs) belong to the ligand-dependent transcription factor nuclear receptor superfamily, comprising three subtypes: RORα, RORβ, and RORγ. These are encoded by the genes RORA, RORB, and RORRC, respectively. RORγ has two subtypes: RORγ1 and RORγ2 (RORγt). RORγ1 is mainly found in multiple sites, including adipose tissue, muscle, kidneys, and liver. RORγt is primarily highly expressed in lymphoid organs such as the thymus. Studies have shown that RORγt is a specific transcription factor for T helper cell 17 (Th17), inducing Th17 cell differentiation and the production of the pro-inflammatory cytokine interleukin-17 (IL-17), playing a crucial role in autoimmune diseases and tumor development and progression.

[0003] To date, several RORγt small molecule inhibitors have entered clinical trials, with the fastest reaching Phase II. However, these small molecules require high dosages to maintain activity and suffer from drawbacks such as poor selectivity and easy drug resistance. Therefore, there is an urgent need to research and develop new strategies for the treatment of this disease.

[0004] In recent years, novel targeted protein degradation technologies that utilize inherent protein degradation mechanisms to target and degrade specific proteins have attracted significant attention from researchers. Among them, Proteolysis Targeting Chimera (PROTAC) is one of the fastest-growing and most mature technologies in this field, offering significant advantages in improving efficacy and selectivity, reducing toxic side effects, and overcoming drug resistance. It has brought new methods and strategies for treating autoimmune diseases and cancer. Summary of the Invention

[0005] Purpose of the invention: In order to solve the problems existing in the prior art, the present invention provides a PROTAC chimera that targets and degrades RORγt receptor and its uses.

[0006] Technical Solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a PROTAC chimera of general formula (I) or a pharmaceutically acceptable salt thereof: R W -LR e (I) Wherein, the R e It is a ligand capable of binding to E3 ubiquitin ligase; The L is covalently bonded to at least one R. e and at least one R W Linking groups; The R w The target protein RORγt binding ligand is selected from one of the following structures: ; Among them, R1 is selected from C 1-5 Alkoxy, amino, C 1-5 Alkylamino, C 1-5 Alkyl, phenyl, benzyl or -CH2-3-10 membered cycloalkyl.

[0007] As a preferred embodiment, R1 is selected from -CH2-cyclopropane.

[0008] As a specific implementation scheme, L has the following structure:

[0009] Wherein, Q and G are independently selected from -CH2-, -O-, -NH-, -C(O)- or -NHC(O)-; Z1~Z8 are independently selected from the following: -(CO)-, -NH-, -O-, -C(O)-, -S(O)-, -S(O)2-, -NHHC(O)-, -S(O)NH-, C6-C 10 Aromatic rings, C5-C 10 heterocyclic aromatic rings or C3-C 10 Nitrogen-containing heterocycles; n1 to n9 are independent integers between 0 and 12.

[0010] Preferably, the L is selected from one of the following structures: .

[0011] As a specific implementation scheme, Rw is selected from one of the following structures: ; Where X is -H, halogen, -OH or C 1-5 Alkyl; Y is -CH2- or C(O); Z is -H2 or -CH3.

[0012] Preferably, the R w Choose one of the following structures: .

[0013] As a preferred embodiment, the PROTAC chimera is selected from the following compounds: , , .

[0014] In a second aspect, the present invention provides a pharmaceutical composition comprising the aforementioned PROTAC chimera or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

[0015] The pharmaceutical composition may be a solid or liquid pharmaceutical preparation, and the dosage form of the pharmaceutical composition includes, but is not limited to, tablets, capsules, powders, granules, suspensions, or injections.

[0016] Thirdly, the present invention provides the use of the described PROTAC chimera or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof, in the preparation of a medicament for the prevention or treatment of RORγt receptor-mediated diseases.

[0017] As a specific implementation, the drug is used to degrade RORγt, and the RORγt receptor-mediated diseases are autoimmune diseases or tumors.

[0018] The present invention also provides pharmaceutically acceptable salts, solvates, precursor compounds or polymorphs of the said PROTAC chimera.

[0019] Specifically, the pharmaceutically acceptable salt is an inorganic salt, an organic salt, or an amino acid salt; The inorganic salts include: sodium salts, hydrochloride salts, trifluoroacetate salts, sulfates, phosphates, diphosphates, hydrobromide salts, or nitrates; The organic salts include: maleate, acetate, fumarate, tartrate, succinate, lactate, p-toluenesulfonate, salicylate, and oxalate. The amino acid salts include: arginine, ornithine, lysine, leucine, isoleucine, glycine, cystine, cysteine, tyrosine, alanine, phenylalanine, histidine, serine, threonine, methionine, tryptophan, glutamate, aspartate, valine, methionine, proline, or hydroxyproline.

[0020] This invention also provides a method for preparing the PROTAC chimera, and more specifically, compounds 1-18 can be synthesized using the following four technical solutions: (1) Technical Solution 1: ; The linker of the RORγt-PROTACs is selected from flexible PEG chains n = 1, 2, 3, 4; the E3 ligase ligand is selected from CRBN ligands, such as pomalidomide. The RORγt-PROTACs can be synthesized through the following process, including the following steps: Preparation of intermediate b Dissolve raw material a in 1,4-dioxane, and add tetrakis(triphenylphosphine)palladium, 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene, and... N,N -Diisopropylethylamine, under nitrogen protection, stirred at 100 °C, yields intermediate b.

[0021] Preparation of intermediate c Dissolve intermediate b in a mixed solution of acetic acid and water, and slowly add at 0 °C. N -Chlorosuccinimide, stirred at room temperature, yields intermediate c.

[0022] Preparation of intermediate d Intermediate c was dissolved in a mixed solution of tetrahydrofuran and water, and sodium bicarbonate and sodium sulfite were added. The mixture was stirred at 70 °C. After evaporating the reaction solution to dryness, it was dissolved in dimethyl sulfoxide, and bromomethyl(cyclopropane) was added. Under nitrogen protection, the mixture was stirred at 100 °C to obtain intermediate d.

[0023] Preparation of intermediate e Intermediate d is dissolved in methanol, sodium hydroxide is added, and the mixture is stirred at room temperature to obtain intermediate e.

[0024] Preparation of intermediates g1-g4 Dissolve raw material f in N,N Add linkers of different lengths, such as (2-(2-bromoethoxy)ethyl)carbamate, triethylamine, and sodium iodide to dimethylformamide, and stir at 100 °C to obtain intermediates g1-g4.

[0025] Preparation of intermediates h1-h4 Intermediates g1-g4 were dissolved in dichloromethane, and triethylamine was added dropwise. Then p-nitrobenzoyl chloride was slowly added and stirred at room temperature to obtain intermediates h1-h4.

[0026] Preparation of intermediates i1-i4 Intermediates h1-h4 were dissolved in a mixed solution of ethanol and water, respectively. Iron powder and ammonium chloride were added, and the mixture was stirred at room temperature to obtain intermediates i1-i4.

[0027] Preparation of intermediates j1-j4 Intermediate e was dissolved in dichloromethane, and 2-(7-azabenzotriazole)- I,I,I,I-Tetramethylurea hexafluorophosphate and N,N -Diisopropylethylamine was stirred at room temperature for 15 min. Intermediates i1-i4 were added separately and stirred at room temperature to obtain intermediates j1-j4.

[0028] Preparation of final products 1-4 Intermediates J1-J4 were dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring at room temperature. After extraction and evaporation to dryness, triethylamine and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione were added, and the mixture was stirred at 90 °C to obtain final products 1-4.

[0029] Preparation of intermediate k3-k4 Dissolve 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid in dichloromethane, then add 2-(7-azabenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate, DIPEA, stirred at room temperature for 15 min. Intermediates j3-j4 were added separately and stirred at room temperature to obtain intermediates k3-k4.

[0030] Preparation of final product 5-6 Intermediates K3-K4 were dissolved separately in dichloromethane, and trifluoroacetic acid was added dropwise while stirring at room temperature. After extraction and evaporation to dryness, triethylamine and 2-(2,6-dioxopiridine-3-yl)-4-fluoroisoindoline-1,3-dione were added, and the mixture was stirred at 90 °C to obtain the final products 5-6.

[0031] (2) Technical Solution Two: ; The linker of the RORγt-PROTACs is selected from flexible PEG chains n = 1, 2, 3, 4; the E3 ligase ligand is selected from CRBN ligands, such as pomalidomide. The RORγt-PROTACs can be synthesized through the following process, including the following steps: Preparation of intermediate m Raw material l was dissolved in anhydrous tetrahydrofuran solution, and lithium aluminum hydride was slowly added at 0 °C. After reflux and stirring, intermediate m was obtained.

[0032] Preparation of intermediate n Intermediate m was dissolved in dichloromethane, potassium tert-butoxide and 4-dimethylaminopyridine were added, and tert-butyldimethylchlorosilane was slowly added at 0 °C. Then the mixture was stirred at room temperature to obtain intermediate n.

[0033] Preparation of intermediate o Intermediate n was dissolved in dichloromethane, and triethylamine and p-nitrobenzoyl chloride were added. The mixture was stirred at room temperature to obtain intermediate o.

[0034] Preparation of intermediate p Intermediate O was dissolved in a mixed solvent of ethanol and water, and iron powder and ammonium chloride were added. The mixture was stirred at 90 °C to obtain intermediate P.

[0035] Preparation of intermediate q Intermediate e was dissolved in dichloromethane, and 2-(7-azabenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate and N,N - Diisopropylethylamine, stirred at room temperature for 15 min. Add intermediate p, and stir at room temperature to obtain intermediate q.

[0036] Preparation of intermediate r Intermediate q was dissolved in anhydrous tetrahydrofuran solution, tetrabutylammonium fluoride was added, and the mixture was stirred at room temperature to obtain intermediate r.

[0037] Preparation of intermediates s1-s4 Dissolve intermediate r in N,N Add potassium carbonate, tert-butyl (2-(2-bromoethoxy)ethyl)carbamate, and other linkers of different lengths to dimethylformamide, and stir at 60 °C to obtain intermediates s1-s4.

[0038] Preparation of intermediates 7-10 Intermediates s1-s4 were dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring at room temperature. After extraction and evaporation to dryness, triethylamine and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione were added and stirred at 90 °C to obtain the final products 7-10.

[0039] Preparation of intermediate t3-t4 Dissolve 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid in dichloromethane, then add 2-(7-azabenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate, DIPEA, stirred at room temperature for 15 min. Intermediates s3-s4 were added separately and stirred at room temperature to obtain intermediates t3-t4.

[0040] Preparation of final product 11-12 Intermediates t3-t4 were dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring at room temperature. After extraction and evaporation to dryness, triethylamine and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione were added and stirred at 90 °C to obtain the final products 11-12.

[0041] (3) Technical Solution Three: ; The linker of the RORγt-PROTACs is selected from flexible PEG chains (n ​​= 1, 2); the E3 ligase ligand is selected from VHL ligands, as shown in the above structure ab. The corresponding RORγt-PROTACs can be synthesized through the following process, including the following steps: Preparation of intermediates u1-u2 Dissolve intermediate q in N,N Add potassium carbonate, tert-butyl 2-(2-bromoethoxy)acetate, and other linkers of different lengths to dimethylformamide, and stir at 60 °C to obtain intermediates u1-u2.

[0042] Preparation of intermediate w Intermediate v was dissolved in a mixed solution of ethyl acetate and water, and 4-dimethylaminopyridine and di-tert-butyl dicarbonate were added. The mixture was stirred at room temperature to obtain intermediate w.

[0043] Preparation of intermediate x Intermediate w, 4-methylthiazole, palladium acetate, and potassium acetate were dissolved in DMA and stirred at 90 °C under N2 protection to obtain intermediate x.

[0044] Preparation of intermediate y Intermediate x was dissolved in dichloromethane, and trifluoroacetic acid was added dropwise. The mixture was stirred at room temperature to obtain intermediate y.

[0045] Preparation of intermediate z Boc- L Hydroxyproline was dissolved in dichloromethane, and 2-(7-azobenzotriazole)- was added. I,I,I,I -Tetramethylurea hexafluorophosphate, N,N Diisopropylethylamine was stirred at room temperature for 15 min, then intermediate y was added and stirred at room temperature. After extraction and evaporation to dryness, the intermediate was dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring at room temperature to obtain intermediate z.

[0046] Preparation of intermediate aa Will N -Boc- L -Tertiary leucine is dissolved in dichloromethane, and 2-(7-azobenzotriazole)- is added. I,I,I,I -Tetramethylurea hexafluorophosphate, N,N -Diisopropylethylamine was stirred at room temperature for 15 min, and then intermediate z was added and stirred at room temperature to obtain intermediate aa.

[0047] Preparation of intermediate ab The intermediate aa was dissolved in dichloromethane, and trifluoroacetic acid was added dropwise. The reaction was carried out at room temperature to obtain intermediate ab.

[0048] Preparation of final product 13-14 Dissolve u1-u2 separately in methanol, add lithium hydroxide and stir at room temperature. Evaporate MeOH to dryness, adjust pH to 4-5 with 1 N hydrochloric acid to obtain the intermediate. Dissolve the intermediate in dichloromethane, add 2-(7-azobenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate, N,N - Diisopropylethylamine, stirred at room temperature for 15 min. Add intermediate ab to the reaction solution and react at room temperature to obtain the final product 13-14.

[0049] (4) Technical Solution Four: ; The linker of the RORγt-PROTACs is selected from flexible PEG chains n = 1, 2, 3, 4; the E3 ligase ligand is selected from VHL ligands, as shown in the above structure ab. The corresponding RORγt-PROTACs can be synthesized by the following process, including the following steps: Preparation of intermediate ac1-ac4 Different linkers, such as 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetic acid, were dissolved in dichloromethane, and 2-(7-azobenzotriazole)- was added to the reaction solution. I,I,I,I -Tetramethylurea hexafluorophosphate, N,N - Diisopropylethylamine, stirred at room temperature for 15 min. Add intermediate ab to the reaction solution and stir at room temperature to obtain intermediate ac1-ac4.

[0050] Preparation of intermediates ad1-ad4 Intermediates ac1-ac4 were dissolved separately in dichloromethane, and trifluoroacetic acid was added dropwise. The mixture was stirred at room temperature to obtain intermediates ad1-ad4.

[0051] Preparation of final product 15-18 U2 was dissolved in methanol, and LiOH was added with stirring at room temperature to obtain an intermediate. MeOH was evaporated to dryness, the pH was adjusted to 4-5 with 1 N hydrochloric acid, and the mixture was extracted with dichloromethane and water. The organic phase was collected and concentrated under reduced pressure. The intermediate was dissolved in dichloromethane, and 2-(7-azobenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate, N,N -Diisopropylethylamine, stirred at room temperature for 15 min. Intermediates ad1-ad4 were added to the reaction solution and reacted at room temperature to obtain the final product 15-18.

[0052] Beneficial Effects: Compared with existing technologies, this invention is the first to design and synthesize a series of PROTAC chimeras capable of targeting and degrading the RORγt receptor. These chimeras primarily form a ternary complex by binding two ligands—a target protein ligand and an E3 ubiquitin ligase ligand—to POI and E3 ligase, respectively. Subsequently, the POI is tagged with ubiquitination and then degraded by the proteasome. Experimental results confirm that the PROTAC chimeras designed in this invention possess excellent RORγt receptor degradation activity and can be used to prepare drugs for treating autoimmune diseases or tumors. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions or as recommended by the manufacturer.

[0055] The structure of the compound was determined by nuclear magnetic resonance (NMR) or high-resolution MRI (HRMS). NMR measurements were performed using a Bruker's Ascend NMR spectrometer, and chemical shift values ​​were calculated using... δ The unit is ppm, and the coupling constant is expressed as... J The values ​​are expressed in Hz, the solvent used for measurement is deuterated chloroform (CDCl3), and the internal standard is tetramethylsilane (TMS).

[0056] MS measurements were performed using a Micromass Q-TOF mass spectrometer.

[0057] High-performance liquid chromatography (HPLC) analysis was performed using an Agilent 1260 HPLC system.

[0058] Thin-layer chromatography silica gel plates were used by GF Chemical Co., Ltd., Yantai City, Shandong Province. 254 Silicone sheet.

[0059] Silica gel column chromatography generally uses GF from Yantai Xinnuo Chemical Co., Ltd., Shandong Province. 254 200-300 mesh silica gel is used as the carrier.

[0060] The degradation activity of the compound was determined by constructing RORγt overexpressing cells, extracting total cellular protein, and detecting the expression level of RORC protein by Western blotting.

[0061] The cells and plasmids used for overexpression were: HEK-293T human embryonic kidney cells, pLVX-puro plasmid from Clontech (Beijing), pMD2.G plasmid (number 12259) and psP AX2 plasmid (number 12260) from Addgen (all preserved by our project group), and human RORC cDNA plasmid from UBO Biotechnology Co., Ltd. (Chongqing).

[0062] The main reagents and antibodies used for overexpression were as follows: plasmid extraction kit and gel extraction kit were purchased from OME-GA (USA); 10×Cutsmart buffer (Catalog No. B7204S), restriction endonucleases NotI (Catalog No. R0189V), EcoRI (Catalog No. R3101V), and T4 DNA ligase were purchased from NEB Biotechnology Co., Ltd. (USA); DMEM cell culture medium and fetal bovine serum (FBS) were purchased from Shanghai Adamas Reagent Co., Ltd.; gene amplification-related reagents 10×PCR buffer, DNA polymerase, and dNTPs were purchased from Nanjing Novizan Biotechnology Co., Ltd.; transfection reagent lipo8000 was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; RORC polyclonal antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd.; β-actin and secondary antibodies for HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG were obtained from Zhongshan Jinqiao Co., Ltd.

[0063] The following embodiments further describe the present invention, but these embodiments are not intended to limit the scope of protection of the present invention.

[0064] Example 1 4-(2-(4-(cyclopropylmethyl)sulfonyl)phenyl)acetamido)- N -(2-(2-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)ethoxy)ethyl)- N Preparation of phenylbenzamide (compound 1)

[0065] Step 1: Preparation of ethyl 2-(4-(benzylthio)phenyl)acetate (intermediate b)

[0066] Acetyl p-bromophenylacetate (4000 mg, 17.46 mmol, 1.0 equiv), tetrakis(triphenylphosphine)palladium (302 mg, 0.26 mmol, 0.015 equiv), and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (151 mg, 0.26 mmol, 0.015 equiv) were administered. N,NDiisopropylethylamine (4.55 mL, 26.19 mmol, 1.5 equiv) and benzyl mercaptan (1.64 mL, 13.97 mmol, 0.8 equiv) were dissolved in 1,4-dioxane (150 mL). The reactants were reacted at 100 °C for 5 h under nitrogen protection, and the reaction was confirmed to be complete by TLC. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The purified intermediate (PE:EA = 20:1–10:1) was obtained by silica gel column chromatography, yielding 4020 mg (80%); a yellow oil.

[0067] Step 2: Preparation of ethyl acetate 2-(4-(chlorosulfonyl)phenyl)acetate (intermediate c)

[0068] Intermediate b (4020 mg, 14.04 mmol, 1.0 equiv) was dissolved in a mixed solvent of acetic acid (120 mL) and water (30 mL). The solution was then dissolved in an ice bath. N 1-Chlorosilicate (7467 mg, 56.15 mmol, 4.0 equiv) was slowly added to the reaction solution. The reaction was allowed to proceed at room temperature for 2 h. The reaction was confirmed to be complete by TLC. The reaction was quenched with sodium bicarbonate, extracted with dichloromethane (50 mL × 3) and water, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 10:1) to give intermediate c, yield: 3900 mg, 98%; colorless transparent oil.

[0069] Step 3: Preparation of ethyl 2-(4-(cyclopropylmethyl)sulfonyl)phenyl)acetate (intermediate d)

[0070] Intermediate c (3900 mg, 13.82 mmol, 1.0 equiv) was dissolved in tetrahydrofuran (60 mL) and water (20 mL). Sodium bicarbonate (2322 mg, 27.65 mmol, 2.0 equiv) and sodium sulfite (2090 mg, 16.59 mmol, 1.2 equiv) were added to the reaction mixture, and the mixture was heated to 70 °C and stirred for 2 h. The reaction was confirmed to be complete by TLC. The reaction mixture was concentrated under reduced pressure to obtain the intermediate, which was dissolved in dimethyl sulfoxide (50 mL). (Bromomethyl)cyclopropane (4 mL, 41.47 mmol, 3.0 equiv) was added, and the mixture was stirred at 100 °C for 3 h under nitrogen protection. The reaction was confirmed to be complete by TLC. The reaction mixture was extracted with dichloromethane (50 mL × 3) and water, and the organic phase was collected and concentrated under reduced pressure. The crude product was then back-extracted with ethyl acetate (50 mL × 3) and water, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 10:1) to obtain intermediate d, yield: 807 mg, 21%; brown oil.

[0071] Step 4: Preparation of 2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetic acid (intermediate e)

[0072] Intermediate d (807 mg, 2.90 mmol, 1.0 equiv) was dissolved in methanol (15 mL), and a 1 N aqueous sodium hydroxide solution was added dropwise to the reaction solution. The reaction was allowed to proceed at room temperature for 2 h. After the reaction was confirmed to be complete by TLC, the reaction solution was poured into water, and the pH was adjusted to approximately 4 with 1 N dilute hydrochloric acid. The mixture was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give intermediate d in 716 mg (97%) as a brown oil.

[0073] Step 5: Preparation of tert-butyl(2-(2-(phenylamino)ethoxy)ethyl)carbamate (intermediate g1)

[0074] (2-(2-bromoethoxy)ethyl)carbamate tert-butyl ester (300 mg, 1.12 mmol, 1.0 equiv), aniline (125 mg, 1.34 mmol, 1.2 equiv), triethylamine (792 mg, 7.84 mmol, 7.0 equiv), and sodium iodide (16 mg, 0.11 mmol, 0.1 equiv) were dissolved in DMF and reacted at 100 °C for 6 h. The reaction was confirmed to be complete by TLC. The reaction solution was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. Then, it was back-extracted with water using ethyl acetate (50 mL × 3), and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. This crude product was purified by column chromatography (PE:EA = 10:1) to give intermediate g1, yield: 102 mg, 32%; brown oil.

[0075] Step 6: Preparation of tert-butyl(2-(2-(4-nitro- N -Phenylacetamide)ethoxy)ethyl)carbamate (intermediate h1)

[0076] Intermediate g1 (102 mg, 0.36 mmol, 1.0 equiv) and triethylamine (74 mg, 0.73 mmol, 2 equiv) were dissolved in dichloromethane. 4-Nitrobenzoyl chloride (82 mg, 0.44 mmol, 1.2 equiv) was added to the reaction solution, and the reaction was carried out at room temperature for 1 h. The reaction was confirmed to be complete by TLC. The reaction solution was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure to obtain a crude product. This crude product was purified by column chromatography (PE:EA = 3:1) to give intermediate h1, yield: 132 mg, 84%; brown oil.

[0077] Step 7: Preparation of tert-butyl(2-(2-(4-amino- N -Phenylacetamide)ethoxy)ethyl)carbamate (intermediate i1)

[0078] Intermediate h1 (132 mg, 0.30 mmol, 1.0 equiv), iron powder (86 mg, 1.54 mmol, 5.0 equiv), and ammonium chloride (33 mg, 0.61 mmol, 2.0 equiv) were dissolved in EtOH / H2O (4:1) and reacted at 90 °C for 1 h. The reaction was confirmed to be complete by TLC. The mixture was filtered, and the filtrate was concentrated and extracted with water using dichloromethane (50 mL × 3). The organic phase was collected, concentrated under reduced pressure, and the crude product was obtained. The crude product was purified by column chromatography (PE:EA = 1:1) to obtain intermediate i1, yield: 119 mg, 97%; brown oil.

[0079] Step 8: Preparation of tert-butyl(2-(2-(4-(2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamido)- N -Phenylacetamide)ethoxy)ethyl)carbamate (intermediate j1)

[0080] Intermediate e (74 mg, 0.29 mmol, 1.0 equiv), 2-(7-azobenzotriazole)- I,I,I, N' -Tetramethylurea hexafluorophosphate (132 mg, 0.35 mmol, 1.2 equiv) N,N Diisopropylethylamine (0.06 mL, 0.35 mmol, 1.2 equiv) was dissolved in dichloromethane (25 mL). After stirring at room temperature for 15 min, intermediate i1 (119 mg, 0.29 mmol, 1.0 equiv) was added. The mixture was stirred at room temperature for 2 h. The reaction was confirmed to be complete by TLC. The reaction solution was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (EA:PE = 3:1) to obtain intermediate j1, yield: 113 mg, 61%; brown oil.

[0081] Step 9: Preparation of 4-(2-(4-(cyclopropylmethyl)sulfonyl)phenyl)acetamido)- N -(2-(2-((2-(2-(2-6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)- N -Phenylenamide (Compound 1)

[0082] Intermediate J1 (113 mg, 0.20 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (0.30 mL, 4.00 mmol, 20 equiv) was added dropwise with stirring at room temperature overnight. The reaction was confirmed to be complete by TLC. The reaction mixture was quenched with sodium bicarbonate, extracted with dichloromethane (50 mL × 3) and water, and the organic phase was collected and concentrated under reduced pressure to obtain the crude product, which was directly used in the next reaction. The crude product (100 mg, 0.19 mmol, 1.0 equiv) was dissolved in... N,N In dimethylformamide (25 mL), triethylamine (0.08 mL, 0.57 mmol, 3.0 equiv) and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (63 mg, 0.23 mmol, 1.2 equiv) were added, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC until complete. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The mixture was then back-extracted with ethyl acetate, and the organic phase was collected and concentrated under reduced pressure. The mixture was purified by column chromatography (DCM:MeOH = 20:1) to give target compound 1 in 45 mg (42%) as a green solid.

[0083] Examples 2-4 Example 1 was repeated, except that different raw materials were used to prepare compounds 2-4. Specifically, (2-(2-(2-bromoethoxy)ethoxy)ethyl)carbamate tert-butyl ester, (2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethyl)carbamate tert-butyl ester, and (14-bromo-3,6,9,12-tetraoxatetradecyl)carbamate tert-butyl ester were reacted with raw material a from Example 1, triethylamine, and sodium iodide under heating. Subsequent operations were consistent with those in Example 1 to prepare compounds 2, 3, and 4, respectively.

[0084] Example 5

[0085] Step 1: Preparation of tert-butyl 1-(4-(2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamido)phenyl)-1,15-dioxo-2-phenyl-5,8,11,17,20-pentaoxa-2,14-diazadocosa-22-yl)carbamate (compound k3)

[0086] Intermediate J3 (100 mg, 0.15 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (0.24 mL, 3.12 mmol, 20 equiv) was added dropwise with stirring at room temperature overnight. The reaction was monitored by TLC until complete. The reaction solution was quenched with sodium bicarbonate, and extracted with dichloromethane (50 mL × 3) and water. The organic phase was collected, concentrated under reduced pressure, and 93 mg of crude product was obtained, which was directly used in the next step. 2-[2-(tert-Butoxycarbonylamino)ethoxy]ethoxyacetic acid (45 mg, 0.17 mmol, 1.2 equiv) and 2-(7-azobenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate (64 mg, 0.17 mmol, 1.2 equiv) N,N Diisopropylethylamine (0.03 mL, 0.17 mmol, 1.2 equiv) was dissolved in dichloromethane (25 mL × 3) and stirred at room temperature for 15 min. The crude product obtained after Boc removal (93 mg, 0.14 mmol, 1.0 equiv) was added to the reaction solution and stirred at room temperature for 2 h. The reaction was confirmed to be complete by TLC. The reaction solution was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected, concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM:MeOH = 20:1) to obtain intermediate K3, yield: 94 mg, 66%; a colorless, transparent oil.

[0087] Step 2: Preparation of 4-(2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamyl)-N-(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)-8-oxo-3,6,12,15,18-penta-9-azaindoline-20-yl)-N-phenylbenzamide (Compound 5)

[0088] Intermediate K3 (150 mg, 0.17 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (0.26 mL, 3.45 mmol, 20 equiv) was added dropwise with stirring at room temperature overnight. The reaction was monitored by TLC until complete. The reaction solution was quenched with sodium bicarbonate, and extracted with dichloromethane (50 mL × 3) and water. The organic phase was collected, concentrated under reduced pressure, and yielded 122 mg of crude product, which was directly used in the next reaction. The crude product (122 mg, 0.16 mmol, 1.0 equiv) was dissolved in... N,NIn dimethylformamide (25 mL), triethylamine (0.07 mL, 0.48 mmol, 3.0 equiv) and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (53 mg, 0.19 mmol, 1.2 equiv) were added, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC until complete. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The mixture was then back-extracted with ethyl acetate, and the organic phase was collected and concentrated under reduced pressure. The mixture was purified by column chromatography (DCM:MeOH = 20:1) to give target compound 5, yield: 76 mg, 44%; green solid.

[0089] Example 6

[0090] Example 5 was repeated, except that different starting materials were used to prepare compound 6. Specifically, intermediate j4 was reacted with trifluoroacetic acid, 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid, and 2-(7-azobenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate, N,N The product was prepared by reacting diisopropylethylamine, and the subsequent operations were consistent with those in Example 5.

[0091] Example 7 4-(2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamido)- N -(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)phenyl)- N Preparation of ethylbenzamide (compound 7)

[0092] Step 1: Preparation of 2-ethylaminophenol (intermediate m)

[0093] In a 100 mL round-bottom flask, using anhydrous THF as solvent, add o-acetaminophen (3000 mg, 19.9 mmol, 1.0 equiv), cool to 0 °C, and slowly add lithium aluminum hydride (1509 mg, 39.7 mmol, 2.0 equiv). Invert the flask to 80 °C and react for 5 h. TLC was used to confirm the completeness of the reaction. The reaction was quenched with saturated ammonium chloride, filtered through a vacuum funnel, and the filtrate was collected. The filtrate was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The product was purified by column chromatography (PE:EA = 5:1) to obtain intermediate m, yield: 2395 mg, 88%; brown solid.

[0094] Step 2: Preparation of 2-((tert-butyldimethylsiloxy)- N -Ethylaniline (intermediate n)

[0095] Intermediate m (2395 mg, 17.4 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), potassium tert-butoxide (1955 mg, 17.4 mmol, 1.0 equiv) and 4-dimethylaminopyridine (212 mg, 1.74 mmol, 0.1 equiv) were added, and the mixture was cooled to 0 °C. Tert-butyldimethylchlorosilane (3132 mg, 20.8 mmol, 1.2 equiv) was slowly added, and the mixture was allowed to react at room temperature for 2 h. The reaction was monitored by TLC until complete. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The solution was purified by column chromatography (PE:EA = 50:1) to give intermediate n, yield: 3930 mg, 90%; white solid.

[0096] Step 3: Preparation N -(2-((tert-butyldimethylsiloxy)phenyl)- N 1-Ethyl-4-nitrobenzamide (intermediate o)

[0097] Intermediate n (3930 mg, 15.6 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and triethylamine (6.3 mL, 46.8 mmol, 3.0 equiv) and 4-nitrobenzyl chloride (4343 mg, 23.4 mmol, 1.5 equiv) were added. The reaction was carried out at room temperature for 1 h. The reaction was confirmed to be complete by TLC. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The mixture was purified by column chromatography (PE:EA = 20:1) to give intermediate o, yield: 5366 mg, 86%; white solid.

[0098] Step 4: Preparation of 4-amino- N -(2-((tert-butyldimethylsilyl)oxy)phenyl)- N -Ethylbenzamide (intermediate p)

[0099] Intermediate O (3930 mg, 15.6 mmol, 1.0 equiv) was dissolved in a mixture of ethanol and water (4:1), iron powder (4360 mg, 46.8 mmol, 5.0 equiv) and ammonium chloride (1669 mg, 31.2 mmol, 2.0 equiv) were added, and the reaction was carried out at room temperature for 1 h. The reaction was confirmed to be complete by TLC. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The purified intermediate P was obtained by column chromatography (PE:EA = 3:1), yielding 5252 mg, 91%; white solid.

[0100] Step 5: Preparation N -(2-((tert-butyldimethylsiloxy)phenyl)-4-(2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamido)- N -Ethylbenzamide (intermediate q)

[0101] Intermediate e (3605 mg, 14.2 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and 2-(7-azobenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate (6475 mg, 17.0 mmol, 1.2 equiv). N,N - Diisopropylethylamine (2.9 mL, 17.0 mmol, 1.2 equiv), stirred for 15 min. Intermediate p (5252 mg, 14.2 mmol, 1.0 equiv) was added. The reaction was checked by TLC to confirm completion. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The mixture was purified by column chromatography (PE:EA = 1:1) to give intermediate q, yield: 4818 mg, 56%; white solid.

[0102] Step 6: Preparation of 4-(2-(4-(cyclopropylmethyl)sulfonyl)phenyl)acetamido)- N -Ethyl- N 2-Hydroxyphenyl)benzamide (intermediate r)

[0103] Intermediate q (4818 mg, 7.9 mmol, 1.0 equiv) was dissolved in anhydrous tetrahydrofuran (25 mL), and tetrabutylammonium fluoride (15.8 mL, 15.8 mmol, 2.0 equiv) in 1.0 mol / L in THF was added. The mixture was stirred at room temperature for 1 h. The reaction was monitored by TLC until complete. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The mixture was purified by column chromatography (EA:PE = 5:1) to give intermediate r, yield: 3860 mg, 98%; white solid.

[0104] Step 7: Preparation of (2-(2-(2-(4-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamido)- N -Ethylbenzamide)phenoxy)ethoxy)ethyl)tert-butyl carbamate (intermediate S1)

[0105] Intermediate r (100 mg, 0.20 mmol, 1.0 equiv) was dissolved in... N,N Dimethylformamide (25 mL) was added to (2-(2-bromoethoxy)ethyl)carbamate tert-butyl ester (65 mg, 0.24 mmol, 1.2 equiv) and potassium carbonate (56 mg, 0.40 mmol, 2 equiv), and stirred at 60 °C for 6 h. The reaction was monitored by TLC until complete. The organic phase was extracted with water using dichloromethane (50 mL × 3) and collected. The organic phase was back-extracted with saturated sodium chloride and concentrated under reduced pressure. The intermediate s1 was purified by column chromatography (EA:PE = 5:1) to obtain a colorless, transparent oil, yielding 97 mg (72%).

[0106] Step 8: Preparation of (2-(2-(2-(4-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamido)- N -Ethylbenzamide)phenoxy)ethoxy)ethyl)tert-butyl carbamate (compound 7)

[0107] Intermediate S1 (112 mg, 0.16 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (0.24 mL, 3.24 mmol, 20 equiv) was added dropwise with stirring at room temperature overnight. The reaction was confirmed to be complete by TLC. The reaction solution was quenched with sodium bicarbonate, and the mixture was extracted with water in dichloromethane (50 mL × 3). The organic phase was collected, concentrated under reduced pressure, and the crude product (97 mg, yield: 98%, colorless transparent oil) was used directly in the next reaction. The crude product (97 mg, 0.15 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and triethylamine (0.06 mL, 0.45 mmol, 3.0 equiv) and 2-(2,6-dioxadiazin-3-yl)-4-fluoroisoindoline-1,3-dione (50 mg, 0.18 mmol, 1.2 equiv) were added. The reaction was carried out at 90 °C for 6 h. The reaction was confirmed to be complete by TLC. The product was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The product was then back-extracted with ethyl acetate, and the organic phase was collected and concentrated under reduced pressure. The product was purified by column chromatography (DCM:MeOH = 20:1) to give compound 7, yield: 58 mg, 43%; green solid.

[0108] Examples 8-10 Example 7 was repeated, except that different raw materials were used to prepare compounds 8-10. Specifically, (2-(2-(2-bromoethoxy)ethoxy)ethyl)carbamate tert-butyl ester, (2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethyl)carbamate tert-butyl ester, and (14-bromo-3,6,9,12-tetraoxatetradecyl)carbamate tert-butyl ester were reacted with intermediate q from Example 2 and potassium carbonate by heating. The remaining operations were consistent with those in Example 2 to prepare compounds 8, 9, and 10, respectively.

[0109] Example 11

[0110] Step 1: Preparation of (20-(2-(4-(2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamido)- N 1-Ethylbenzamido)phenoxy)-8-oxo-3,6,12,15,18-pentaoxa-9-azacarbonyl)tert-butyl carbamate (intermediate t3)

[0111] Intermediate S3 (120 mg, 0.16 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (0.25 mL, 3.13 mmol, 20 equiv) was added dropwise with stirring at room temperature overnight. The reaction was monitored by TLC until complete. The reaction solution was quenched with sodium bicarbonate, and extracted with dichloromethane (50 mL × 3) and water. The organic phase was collected, concentrated under reduced pressure, and 100 mg of crude product was obtained, which was directly used in the next step of the reaction. 2-[2-(tert-Butoxycarbonylamino)ethoxy]ethoxyacetic acid (45 mg, 0.19 mmol, 1.2 equiv) and 2-(7-azobenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate (69 mg, 0.19 mmol, 1.2 equiv) N,N Diisopropylethylamine (0.04 mL, 0.19 mmol, 1.2 equiv) was dissolved in dichloromethane (25 mL × 3) and stirred at room temperature for 15 min. The crude product obtained after Boc removal (100 mg, 0.15 mmol, 1.0 equiv) was added to the reaction solution and stirred at room temperature for 2 h. The reaction was confirmed to be complete by TLC. The reaction solution was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected, concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM:MeOH = 20:1) to give intermediate t3, yield: 80 mg, 54%; colorless transparent oil.

[0112] Step 2: Preparation of 4-(2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamido)- N -(2-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)-8-oxo-3,6,12,15,18-pentaoxa-9-azaindoline-20-yl)oxy)phenyl)- N -Ethylbenzamide (Compound 11)

[0113] Intermediate t3 (80 mg, 0.10 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (0.15 mL, 2.00 mmol, 20 equiv) was added dropwise with stirring at room temperature overnight. The reaction was monitored by TLC until complete. The reaction mixture was quenched with sodium bicarbonate, extracted with dichloromethane (50 mL × 3) and water, and the organic phase was collected and concentrated under reduced pressure to give 70 mg of crude product, which was directly used in the next reaction. The crude product (70 mg, 0.09 mmol, 1.0 equiv) was dissolved in... N,NIn dimethylformamide (25 mL), triethylamine (0.04 mL, 0.27 mmol, 3.0 equiv) and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (31 mg, 0.11 mmol, 1.2 equiv) were added, and the reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC until complete. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The mixture was then back-extracted with ethyl acetate, and the organic phase was collected and concentrated under reduced pressure. The mixture was purified by column chromatography (DCM:MeOH = 20:1) to give target compound 11, yield: 30 mg, 29%; green solid.

[0114] Example 12

[0115] Example 11 was repeated, except that different starting materials were used to prepare compound 12. Specifically, intermediate t4 was reacted with trifluoroacetic acid, 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid, and 2-(7-azobenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate, N,N The product was prepared by reacting diisopropylethylamine, with subsequent operations consistent with those in Example 11.

[0116] Example 13

[0117] Step 1: Preparation of tert-butyl 2-(2-(2-(4-(2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamido)- N -Ethylbenzamide)phenoxy)ethoxy)acetate (compound u1)

[0118] Intermediate q (400 mg, 0.81 mmol, 1.0 equiv) was dissolved in N,N-dimethylformamide, and 2-(2-bromoethoxy)tert-butyl acetate (213 mg, 0.81 mmol, 1.1 equiv) and potassium carbonate (134 mg, 0.97 mmol, 1.2 equiv) were added. The mixture was stirred at 60 °C for 6 h. The reaction was monitored by TLC until complete. The reaction solution was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The organic phase was then back-extracted with water using ethyl acetate (50 mL × 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The solution was purified by column chromatography (PE:EA = 1:1) to give intermediate u1, yield: 452 mg, 85%; a colorless, transparent oil.

[0119] Step 2: Preparation ( S1-(1-(4-bromophenyl)ethyl)carbamate tert-butyl ester (compound w)

[0120] Starting material v (1000 mg, 5.0 mmol, 1.0 equiv) and sodium bicarbonate (312 mg, 3.72 mmol, 0.74 equiv) were dissolved in ethyl acetate:water (1:1). Di-tert-butyl dicarbonate (1314 mg, 6.0 mmol, 1.2 equiv) was added under ice bath conditions, and the reaction was carried out for 2 h. The reaction was monitored by TLC until complete. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The solution was purified by column chromatography (PE:EA = 20:1) to give intermediate w, yield: 1450 mg, 82%; white solid.

[0121] Step 3: Preparation ( S 1-(1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)tert-butyl carbamate (compound x)

[0122] Intermediate w (1450 mg, 4.80 mmol, 1.0 equiv), 4-methylthiazole (962 mg, 9.70 mmol, 2.0 equiv), palladium acetate (11 mg, 0.04 mmol, 0.01 equiv), and potassium acetate (952 mg, 9.70 mmol, 2.0 equiv) were dissolved in dimethylacetamide. The reaction was carried out at 90 °C for 4 h under nitrogen protection. The reaction was confirmed to be complete by TLC. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The mixture was purified by column chromatography (PE:EA = 5:1) to give intermediate x, yield: 850 mg, 55%; white solid.

[0123] Step 4: Preparation ( S )-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethane-1-amine (compound y)

[0124] Intermediate x (850 mg, 2.64 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (4.04 mL, 52.80 mmol, 20.0 equiv) was added dropwise. The mixture was stirred at room temperature for 1 h. The reaction was monitored by TLC until complete. The reaction was quenched with sodium bicarbonate. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The mixture was purified by column chromatography (DCM:MeOH = 20:1) to give intermediate y, yield: 550 mg, 95%; white solid.

[0125] Step 5: Preparation (2) S 4 R )-4-hydroxy- N -(( S )-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (compound z)

[0126] Boc- L 2-Hydroxyproline (582 mg, 2.52 mmol, 1.0 equiv), 2-(7-Azobenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate (1149 mg, 3.02 mmol, 1.2 equiv). N,N - Diisopropylethylamine (0.52 mL, 3.02 mmol, 1.2 equiv). After stirring at room temperature for 15 min, intermediate y (550 mg, 2.52 mmol, 1.0 equiv) was added, and the reaction was allowed to proceed at room temperature for 2 h. The reaction was confirmed to be complete by TLC. The mixture was extracted with water in dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The intermediate was purified by column chromatography (EA:PE = 5:1) to give 932 mg, 85%; white solid. This was used directly in the next reaction. The intermediate (932 mg, 2.15 mmol, 1.0 equiv) was dissolved in dichloromethane, and trifluoroacetic acid (3.3 mL, 43.00 mmol, 20.0 equiv) was added dropwise. The reaction was allowed to proceed overnight at room temperature. The reaction was confirmed to be complete by TLC. The reaction was quenched with sodium bicarbonate. The mixture was extracted with water in dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The intermediate z was separated by column chromatography (DCM:MeOH = 20:1), yield: 703 mg, 97%; white solid.

[0127] Step Six: Preparation (( S )-1-((2 S 4 R)-4-hydroxy-2-((( S 1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)carbamoyl)pyrrolidone-1-yl)-3,3-dimethyl-1-oxobut-2-yl)tert-butyl carbamate (compound aa)

[0128] Will N -Boc -L -Tertiary leucine (490 mg, 2.12 mmol, 1.0 equiv), 2-(7-azobenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate (966 mg, 2.54 mmol, 1.2 equiv). N,N -Diisopropylethylamine (0.44 mL, 2.54 mmol, 1.2 equiv). After stirring at room temperature for 15 min, intermediate z (703 mg, 2.12 mmol, 1.0 equiv) was added, and the reaction was allowed to proceed at room temperature for 2 h. The reaction was confirmed to be complete by TLC. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The solution was purified by column chromatography (EA:PE = 5:1) to give intermediate aa, yield: 1033 mg, 89%; white solid.

[0129] Step 7: Preparation (2) S ,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (compound ab)

[0130] The intermediate aa (1033 mg, 1.90 mmol, 1.0 equiv) was dissolved in dichloromethane, and trifluoroacetic acid (2.9 mL, 38.00 mmol, 20.0 equiv) was added dropwise. The reaction was allowed to proceed overnight at room temperature. TLC was used to confirm the completion of the reaction. The reaction was quenched with sodium bicarbonate. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. Intermediate ab was given in 799 mg (94%) as a white solid.

[0131] Step 8: Preparation (2) S 4 R )-1-(( S )-2-(2-(2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamido)- N-Ethylbenzamido)phenoxy)ethoxy)acetamido)-3,3-dimethylbutyryl)-4-hydroxy- N -(( S )-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 13)

[0132] Intermediate u1 (100 mg, 0.15 mmol, 1.0 equiv) was dissolved in methanol (10 mL), and lithium hydroxide (24 mg, 0.15 mmol, 4.0 equiv) was added. The mixture was stirred at room temperature for 3 h. The reaction was monitored by TLC until complete. The methanol was evaporated to dryness, the pH was adjusted to 4-5 with 1 N hydrochloric acid, and the mixture was extracted with dichloromethane (50 mL × 3) and water. The organic phase was collected and concentrated under reduced pressure to give the intermediate, 84 mg, 93%; a colorless, transparent oil. This intermediate (84 mg, 0.14 mmol, 1.0 equiv) was dissolved in dichloromethane (10 mL), and 2-(7-azobenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate (63 mg, 0.17 mmol, 1.2 equiv). N,N - Diisopropylethylamine (0.03 mL, 0.17 mmol, 1.2 equiv) was stirred at room temperature for 15 min, and intermediate ab (62 mg, 0.14 mmol, 1.0 equiv) was added. The reaction was allowed to proceed at room temperature for 2 h. The reaction was confirmed to be complete by TLC. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The solution was purified by column chromatography (DCM:MeOH = 20:1) to give target compound 13, yield: 50 mg, 35%; white solid.

[0133] Example 14

[0134] Example 13 was repeated, except that different raw materials were used to prepare compound 14. Specifically, 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid was reacted with intermediate q from step one of Example 13 and potassium carbonate by heating, while keeping the other operations the same.

[0135] Example 15

[0136] Step 1: Preparation of (2-(2-((() S )-1-((2 S 4 R )-4-hydroxy-2-((( S)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)carbamoyl)pyrrolidone-1-yl)-3,3-dimethyl-1-oxobut-2-yl)amino)-2-oxoethoxy)ethyl)tert-butyl carbamate (compound ac1)

[0137] 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetic acid (100 mg, 0.45 mmol, 1.0 equiv) was dissolved in dichloromethane (10 mL), and 2-(7-azobenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate (205 mg, 0.54 mmol, 1.2 equiv). N,N - Diisopropylethylamine (0.09 mL, 0.54 mmol, 1.2 equiv) was stirred at room temperature for 15 min. Intermediate ab (178 mg, 0.45 mmol, 1.0 equiv) was added to the reaction mixture, and the reaction was carried out at room temperature for 2 h. The reaction was confirmed to be complete by TLC. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The solution was purified by column chromatography (DCM:MeOH = 20:1) to give intermediate ac1, yield: 138 mg, 46%; a colorless, transparent oil.

[0138] Step 2: Preparation of (2-(2-((( S )-1-((2 S 4 R )-4-hydroxy-2-((( S )-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)carbamoyl)pyrrolidone-1-yl)-3,3-dimethyl-1-oxobut-2-yl)amino)-2-oxoethoxy)ethyl)tert-butyl carbamate (compound ad1)

[0139] Intermediate ac1 was dissolved in dichloromethane, and trifluoroacetic acid was added dropwise. The reaction was carried out at room temperature for 2 h. TLC analysis confirmed the reaction was complete. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The crude intermediate ad1 was used directly in the next reaction, yield: 95 mg, 86%; a colorless, transparent oil.

[0140] Step 3: Preparation (2) S 4 R )-1-(( S )-2-(tert-butyl)-17-(2-(4-(2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamido)-N (-ethylbenzamido)phenoxy)-4,10-dioxo-6,12,15-trioxa-3,9-diazaheptadecyl)-4-hydroxy- N -((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 15)

[0141] Intermediate U2 (100 mg, 0.15 mmol, 1.0 equiv) was dissolved in methanol (10 mL), and lithium hydroxide (24 mg, 0.15 mmol, 4.0 equiv) was added. The mixture was stirred at room temperature for 3 h. The reaction was monitored by TLC until complete. The methanol was evaporated to dryness, the pH was adjusted to 4-5 with 1 N hydrochloric acid, and the mixture was extracted with water using dichloromethane (50 mL × 3). The organic phase was collected, concentrated under reduced pressure, and the intermediate was directly used in the next reaction. 80 mg, 91%; colorless, transparent oil. Intermediate U2 (80 mg, 0.13 mmol, 1.0 equiv) was dissolved in dichloromethane (10 mL), and 2-(7-azobenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate (62 mg, 0.16 mmol, 1.2 equiv). N,N - Diisopropylethylamine (0.03 mL, 0.16 mmol, 1.2 equiv) was stirred at room temperature for 15 min. Intermediate ad1 (76 mg, 0.13 mmol, 1.0 equiv) was added to the reaction mixture, and the reaction was carried out at room temperature for 2 h. The reaction was confirmed to be complete by TLC. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The solution was purified by column chromatography (DCM:MeOH = 20:1) to give target compound 15, yield: 45 mg, 30%; white solid.

[0142] Examples 16-18 Example 15 was repeated, except that different starting materials were used to prepare compounds 16-18. Specifically, 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid, 2-[2-[2-[2-(Boc-amino)ethoxy]ethoxy]ethoxy]acetic acid, N -tert-butyloxycarbonyl-tetraethylene glycol-acetic acid was prepared by heating the intermediate ab from step 2 of Example 15 above and potassium carbonate, while keeping the rest of the operation consistent.

[0143] Experimental Example 19 (1) Construction of RORγt overexpressing cells Step 1: Primer design and synthesis. Specificity was verified using NCBI Primer-BLAST to avoid non-specific amplification, and then the primers were synthesized.

[0144] Step 2: Amplification of the RORC gene. The PCR reaction system (50 μL) consisted of 5 μL of 10×PCR buffer, 1 μL each of forward and reverse primers (10 μM), 4 μL of dNTPs, 1 μL of template plasmid (100 ng / mL), and sterile, enzyme-free water to make up the difference. The PCR amplification program was: 95℃ for 3 min, 98℃ for 10 s, 60℃ for 15 s, and 68℃ for 3 min for 30 cycles; followed by 68℃ for 10 min. The PCR products were then detected by 1% agarose gel electrophoresis, and the target band was recovered.

[0145] Step 3: Construction of recombinant lentiviral overexpression plasmid. Prepare the pLVX digestion reaction system: EcoRI 2 μL, NotI 2 μL, 10×NEB Buffer 15 μL, pLVX-puro vector 4 µg. After mixing, add ultrapure water, centrifuge briefly at low speed, and seal the microcentrifuge tube with sealing film. Detect the digestion products by 1% agarose gel electrophoresis and recover the target band. Ligate the digested pLVX vector plasmid with the RORC target gene fragment. Reaction system (20 μL): RORC target gene fragment 2 μL, digested pLVX 20 ng, T4 ligase 2 μL, 10×T4 buffer 2 μL, sterile enzyme-free water to a final volume of 20 μL, incubate at 22 ℃ in a metal bath for 1 h. Remove 100 μL of competent DH-5α cells stored at -80 ℃ and place on ice. Add the ligation product to the competent cells and mix well. Insert the microcentrifuge tubes into ice and incubate for 30 min. Preheat the water bath to 42 °C. Seal the microcentrifuge tubes with sealing film, insert them onto a float, and subject them to heat shock treatment in a 42 °C water bath for 90 s. Immediately afterward, place them into ice for 2 min. Add 800 μL of antibiotic-free LB broth to the heat-shocked competent DH-5α cells, and incubate in a bacterial shaker at 37 °C and 220 rpm for 1 h. Centrifuge for 5 min. Discard the supernatant, resuspend the cells in 20 μL of LB broth, and incubate overnight at 37 °C.

[0146] Step 4: Prepare virus particles using HEK293T cells. HEK293T cell passage and plating: Digest a plate of well-grown HEK293T cells, resuspend the cells, and plate them into 6 cm cell culture dishes, with approximately 7 × 10⁶ cells per dish. 5Once the cell density reached 70%, the transfection system was prepared as follows: psPAX2 1.5 µg, PMD2.G 1.5 µg, DMEM (without penicillin, streptomycin, and serum) 300 μL, target plasmid 1.5 µg, and lipo8000 8 μL. These components were mixed in a centrifuge tube, then evenly added to a HEK293T centrifuge tube, gently shaken to mix, and placed in an incubator. After 6 h, the medium was changed. After 48 h, the supernatant was collected into a centrifuge tube and centrifuged at 1000 rpm and 4 °C for 5 min. Single cells were filtered out of the viral solution, aliquoted, and stored at -80 °C for later use.

[0147] Step 5: Viral infection of HEK293T cells. HEK293T cells in the logarithmic growth phase were digested, resuspended, and passaged into 6 cm cell culture dishes, with approximately 3 × 10⁶ cells per dish. 5 Cells were screened for puromycin for 24 hours. The cell status was observed. If the infection efficiency was low and many cells died, the cell culture medium containing puromycin was replaced. If the cells reached 90% confluence, they were passaged. The passaged cells were then screened again using cell culture medium containing puromycin to select stable lines for subsequent experiments.

[0148] Step 6: RORC expression level detection. Total cellular protein was extracted, and RORC protein expression levels were detected by Western blotting. Image Lab was used for grayscale value statistical analysis and quantitative analysis.

[0149] (2) Degradation activity assay of compound RORγt Cell Culture and Compound Treatment HEK293T cells in logarithmic growth phase were seeded into 6-well plates at a density of 5 × 10⁶ cells per well. 6 To ensure that the confluence of the cells is 70% to 80% on the second day, add 2 mL of complete culture medium and incubate overnight at 37 ℃ in a 5% CO2 incubator.

[0150] Extraction of total cellular protein a. Cell collection: Set the centrifuge to 5000 rpm and 4 °C, place the cells on ice, discard the culture medium, and wash twice with ice-cold PBS. b. Cell lysis: Add cell lysis buffer to the cell pellet. Before use, add PMSF and protease inhibitor to the RIPA lysis buffer. Mix the cell pellet thoroughly by pipetting with a pipette tip, and incubate on ice for 1 h, vortexing every 15 min. Set the centrifuge to 12000 rpm and 4 °C, centrifuge for 20 min, and carefully aspirate the supernatant into a new centrifuge tube.

[0151] 3) BCA method for measuring protein concentration Thaw the protein sample on ice. Dilute 5 mg / mL BSA standard protein (included in the BCA kit) with PBS buffer to different concentrations: 0 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL. In a clean 96-well plate, add 20 μL of the standard protein at each well. Add 2 μL of the test compound to each well, along with 18 μL of PBS diluent. Perform two replicates per sample.

[0152] Absorbance measurement and protein concentration calculation: Remove the 96-well plate after reaction and place it in a microplate reader. Open the cap, vibrate at medium speed for 30 seconds, and measure the absorbance at 562 nm. Analyze the results using Excel software. First, take the average of two replicates. Plot a standard curve with the absorbance of the BSA standard protein on the x-axis and the concentration of the BSA standard protein on the y-axis. Calculate the formula based on the curve, and substitute the absorbance value of the protein to be tested into the formula to calculate the protein concentration of the lysate.

[0153] 4) Prepare the protein sample for loading. Depending on the protein concentration, add different volumes of 6× loading buffer, mix well, and centrifuge briefly. Seal the microcentrifuge tubes and place them in a preheated 100 ℃ metal bath for denaturation for 5 min, then on ice for 2 min, followed by brief centrifugation. Store at -80 ℃.

[0154] 5) SDS-PAGE electrophoresis and membrane transfer Add the processed protein samples sequentially to the gel wells, along with the protein marker. Add 1× electrophoresis buffer, turn on the power, and use a constant voltage of 80 V for the stacking gel stage. After the bromophenol blue enters the separating gel, adjust the voltage to 120 V and continue electrophoresis until the bromophenol blue reaches the bottom of the gel (approximately 90 min). Turn off the power. Pre-cool the transfer buffer and pour it into the transfer tank. First, cut a 0.45 µm PVDF membrane to match the gel size. Activate the PVDF membrane by soaking it in methanol. Remove the gel casting plate from the electrophoresis tank, carefully pry it open with a peeler, remove the gel, and discard the stacking gel. Reserve the separating gel for later use. Place the sponge, filter paper, activated PVDF membrane, and separating gel into the transfer clamp in sequence. Remove any air bubbles between the PVDF membrane and the separating gel. Then add the filter paper and sponge, and gently remove any remaining air bubbles. Close the clamp and place the sandwich into the transfer tank. Connect the membrane side of the transfer clamp to the positive terminal of the power supply, and the separating gel side to the negative terminal. Perform the transfer at a constant voltage of 90 V for 2 hours. After connecting the transfer tank, place it in an ice box filled with an ice-water complex to cool the transfer tank.

[0155] 6) Blocking and antibody incubation Place the PVDF membrane in an incubation chamber, add 5% skim milk (prepared with TBST), and incubate on a shaker at room temperature for 1 h to block non-specific binding sites on the membrane. Primary antibody incubation: Discard the blocking solution, add 1:1000 diluted RORC primary antibody and GAPDH primary antibody, and incubate overnight on a shaker at 4°C. Wash the membrane: Discard the primary antibody solution, and wash the membrane three times with 1×TBST for 10 min each time to remove unbound primary antibody. Secondary antibody incubation: Add 1:5000 diluted HRP-labeled secondary antibody, and incubate on a shaker at room temperature for 1 h. Wash the membrane: Discard the secondary antibody solution, and wash the membrane three times with 1×TBST for 10 min each time.

[0156] 7) Chemiluminescence color development and imaging Following the ECL chemiluminescence reagent kit instructions, mix reagent A and reagent B in a 1:1 ratio to prepare the chemiluminescence solution, using immediately after preparation. Start the imaging system, adjust the exposure time, acquire images, and save images with clear bands.

[0157] 8) Results Analysis Image Lab software was used to analyze the grayscale values ​​of the WB bands, and the grayscale values ​​of the RORC protein and internal control protein bands in each well were measured. The relative protein content was calculated as follows: the ratio of the RORC grayscale value of the solvent control group (0 μM compound) to the grayscale value of the internal control was 1. The ratio of (RORC grayscale value / internal control grayscale value) of each compound concentration group to the control group was the relative protein content.

[0158] Table 1. Chemical structure of the target product

[0159] Table 2. 1H NMR and high-resolution data of the target compound .

[0160] Table 3. Activity data of the target compound .

[0161] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A PROTAC chimera of general formula (I) or a pharmaceutically acceptable salt thereof: R W -L-R e （I） in, The R e It is a ligand capable of binding to E3 ubiquitin ligase; The L is covalently bonded to at least one R. e and at least one R W Linking groups; The R w The target protein RORγt binding ligand is selected from one of the following structures: ; Among them, R1 is selected from C 1-5 Alkoxy, amino, C 1-5 Alkylamino, C 1-5 Alkyl, phenyl, benzyl or -CH2-3-10 membered cycloalkyl.

2. The PROTAC compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, R1 is selected from -CH2-cyclopropane.

3. The PROTAC chimera according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that... The L has the following structure: ； Wherein, Q and G are independently selected from -CH2-, -O-, -NH-, -C(O)- or -NHC(O)-; Z1~Z8 are independently selected from the following: -(CO)-, -NH-, -O-, -C(O)-, -S(O)-, -S(O)2-, -NHHC(O)-, -S(O)NH-, C6-C 10 Aromatic rings, C5-C 10 heterocyclic aromatic rings or C3-C 10 Nitrogen-containing heterocycles; n1 to n9 are independent integers between 0 and 12.

4. The PROTAC chimera according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The L is selected from one of the following structures: 。 5. The PROTAC chimera according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The Rw is selected from one of the following structures: ； Where X is -H, halogen, -OH or C 1-5 Alkyl; Y is -CH2- or C(O); Z is -H2 or -CH3.

6. The PROTAC chimera according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The R w Choose one of the following structures: 。 7. The PROTAC chimera according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The PROTAC chimera is selected from the following compounds: 、 、 。 8. A pharmaceutical composition comprising the PROTAC chimera as described in any one of claims 1-7 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

9. The use of the PROTAC chimera according to any one of claims 1-7 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 7, in the preparation of a medicament for the prevention or treatment of RORγt receptor-mediated diseases.

10. The application according to claim 9, characterized in that, The diseases mediated by the RORγt receptor are autoimmune diseases or tumors.

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