A protac compound with rorγt receptor targeted degradation and uses thereof

Abstract

The application discloses a PROTAC compound with RORgamma t receptor targeted degradation and purposes thereof, which is a compound with a structure shown in a general formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutical composition thereof. e is a ligand capable of combining with an E3 ubiquitin ligase; the L is a linking group covalently combining at least one R e and at least one R W ; the R w is a target protein RORgamma t binding ligand; the application is based on the target point of RORgamma t and the PROTAC technology, and a series of RORgamma t-PROTACs are designed and synthesized for the first time. The mechanism is that the target protein ligand and the E3 ubiquitin ligase ligand are combined with POI and E3 ligase respectively, so as to form a ternary complex of "POI-PROTAC-E3 ligase". Then, the POI is marked with a ubiquitination label, and is degraded by a proteasome. The advantages of RORgamma t-PROTACs mainly include targeting of "undruggable proteins", small dosage, low toxicity, and difficulty in drug resistance. W -L-R e (I)

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a PROTAC compound with RORγt receptor-targeted degradation activity 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 compound with RORγt receptor targeted degradation activity 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 compound 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] Preferably, R1 is selected from -CH2-cyclopropane.

[0008] As a specific implementation scheme, 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 bond, -(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.

[0009] Preferably, the L is selected from one of the following structures: , , , , , , .

[0010] As a specific implementation plan, the R w Choose one of the following structures: ; Where X is -H, halogen, -OH or C 1-5Alkyl; Y is -CH2- or C(O); Z is -H2 or -CH3.

[0011] Preferably, the R w Choose one of the following structures: .

[0012] As a preferred embodiment, the PROTAC compound is selected from the following compounds: , , , , , , , , , , , , , , , , , , , , , , , .

[0013] In a second aspect, the present invention provides a pharmaceutical composition comprising the aforementioned PROTAC compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0014] 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.

[0015] Thirdly, the present invention provides the use of the said PROTAC compound or a pharmaceutically acceptable salt thereof, or the said pharmaceutical composition, in the preparation of a medicament for the prevention or treatment of RORγt receptor-mediated diseases.

[0016] As a specific implementation, the PROTAC compound is used to degrade the RORγt receptor, wherein the RORγt receptor-mediated disease is an autoimmune disease or a tumor.

[0017] The present invention also provides pharmaceutically acceptable salts, solvates, precursor compounds or polymorphs of the said PROTAC compounds (RORγt-PROTACs).

[0018] As a specific implementation plan, the pharmaceutically acceptable salt is an inorganic salt, an organic salt, or an amino acid salt.

[0019] 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 aforementioned PROTAC compounds. Specifically, compounds 1-24 can be synthesized using the following four technical solutions: (1) Technical Solution 1:

[0021] 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 benzyl mercaptan, tetra(triphenylphosphine)palladium, 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene, N,N -Diisopropylethylamine, under nitrogen protection, stirred at 100 °C, yields intermediate b.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Preparation of intermediate f 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, and then 4-(hexafluoro-2-hydroxyisopropyl)aniline was added and stirred at room temperature to obtain intermediate f.

[0026] Preparation of intermediates g1-g4 Dissolve intermediate f 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 g1-g4.

[0027] Preparation of final products 1-4 Intermediates g1-g4 were dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring at room temperature. After extraction and evaporation to dryness, dimethyl sulfoxide, triethylamine, and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione were added and stirred at 90 °C to obtain final products 1-4.

[0028] Preparation of intermediates h3-h4 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 g3-g4 were added separately and stirred at room temperature to obtain intermediates h3-h4.

[0029] Preparation of final product 5-6 Intermediates h3-h4 were dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring at room temperature. After extraction and evaporation to dryness, dimethyl sulfoxide, triethylamine, and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione were added and stirred at 90 °C to obtain final products 5-6.

[0030] (2) Technical Solution Two:

[0031] ; 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-based ligands, as shown in the above structure. The RORγt-PROTACs can be synthesized through the following process, including the following steps: Preparation of intermediate j Intermediate i 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 j.

[0032] Preparation of intermediate k Intermediate j, 4-methylthiazole, palladium acetate, and potassium acetate were dissolved in DMA and stirred at 90 °C under N2 protection to obtain intermediate k.

[0033] Preparation of intermediate l Intermediate k was dissolved in dichloromethane, and trifluoroacetic acid was added dropwise. The mixture was stirred at room temperature to obtain intermediate l.

[0034] Preparation of intermediate m 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 l 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 m.

[0035] Preparation of intermediate n 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 minutes, and then intermediate m was added and stirred at room temperature to obtain intermediate n.

[0036] Preparation of intermediate o Intermediate n is dissolved in dichloromethane, and trifluoroacetic acid is added dropwise. The mixture is stirred at room temperature to obtain intermediate o.

[0037] Preparation of intermediates p1-p4 Intermediates g1-g4 were dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring at room temperature. After extraction and evaporation to dryness, triethylamine and bromoacetyl bromide were added, and the mixture was stirred at room temperature to obtain intermediates p1-p4.

[0038] Preparation of intermediate q3-q4 Intermediates h3-h4 were dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring at room temperature. After extraction and evaporation to dryness, triethylamine and bromoacetyl bromide were added, and the mixture was stirred at room temperature to obtain intermediates q3-q4.

[0039] Preparation of final product 7-12 Intermediate O was dissolved in acetonitrile, and triethylamine and intermediates p1-p4 and q3-q4 were added separately. The mixture was stirred at 50 °C to obtain the final product 7-12.

[0040] (3) Technical Solution Three: ; 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 r Intermediate d was dissolved in anhydrous tetrahydrofuran, and bis(trimethylsilylamine) lithium was slowly added dropwise under N2 protection at -78 °C. After stirring for 15 min, tert-butyl bromoacetate was added and stirred at -78 °C to obtain intermediate r.

[0041] Preparation of intermediate s Intermediate r was dissolved in dichloromethane, and trifluoroacetic acid was added. The mixture was stirred at room temperature to obtain intermediate s.

[0042] Preparation of intermediates t1-t4 Intermediate S was dissolved in dichloromethane, and 2-(7-azobenzotriazole)- I,I,I,I -Tetramethylurea hexafluorophosphate, N,N -Diisopropylethylamine was stirred at room temperature for 15 min, and then Linkers of different lengths, such as [2-(2-aminoethoxy)ethyl]carbamate tert-butyl ester, were added and stirred at room temperature to obtain intermediates t1-t4.

[0043] Preparation of intermediates u1-u4 Intermediates t1-t4 were dissolved in tetrahydrofuran, lithium hydroxide was added, and the mixture was stirred at room temperature to obtain intermediates u1-u4.

[0044] Preparation of intermediates v1-v4 Intermediates u1-u4 were 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, and then 4-(hexafluoro-2-hydroxyisopropyl)aniline was added and stirred at room temperature to obtain intermediates v1-v4.

[0045] Preparation of final product 13-16 Intermediates v1-v4 were dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring at room temperature. After extraction and evaporation to dryness, dimethyl sulfoxide, 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 13-16.

[0046] Preparation of intermediates w3-w4 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 v3-v4 were added separately and stirred at room temperature to obtain intermediates w3-w4.

[0047] Preparation of final product 17-18 Intermediates w3-w4 were dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring at room temperature. After extraction and evaporation to dryness, dimethyl sulfoxide, 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 17-18.

[0048] (4) Technical Solution Four:

[0049]

[0050] The linker of the RORγt-PROTACs is selected from flexible PEG chains n = 1, 2, 3, 4; the E3 ligase ligand is selected from the preparation method of pomalidomide and can be synthesized through the following process, including the following steps: Preparation of intermediates x1-x4 Dissolve intermediate C in N,N Add potassium carbonate, linkers of different lengths such as [2-(2-aminoethoxy)ethyl]carbamate to dimethylformamide, stir at 60 °C to obtain intermediates x1-x4.

[0051] Preparation of intermediates y1-y4 Intermediates x1-x4 were dissolved in tetrahydrofuran, and lithium hydroxide was added. The mixture was stirred at room temperature to obtain a series of crude products, which were then 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, and then 4-(hexafluoro-2-hydroxyisopropyl)aniline was added and stirred at room temperature to obtain intermediates y1-y4.

[0052] Preparation of final products 19-22 Intermediates y1-y4 were dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring at room temperature. After extraction and evaporation to dryness, dimethyl sulfoxide, 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 19-22.

[0053] Preparation of intermediate z3-z4 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 y3-y4 were added separately and stirred at room temperature to obtain intermediates z3-z4.

[0054] Preparation of final product 23-24 Intermediates Z3-Z4 were dissolved in dichloromethane, and trifluoroacetic acid was added dropwise while stirring at room temperature. After extraction and evaporation to dryness, dimethyl sulfoxide, 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 23-24.

[0055] Beneficial Effects: Compared with existing technologies, this invention, based on the target RORγt and PROTAC technology, designs and synthesizes a series of RORγt-PROTACs for the first time. Their mechanism of action involves two ligands—the target protein ligand and the E3 ubiquitin ligase ligand—binding to POI and E3 ligase, respectively, forming a ternary complex of "POI-PROTAC-E3 ligase." Subsequently, POI is tagged with ubiquitination and then degraded by the proteasome. The advantages of RORγt-PROTACs mainly include targeting "undruggable proteins," low dosage, low toxicity, and low susceptibility to drug resistance. Confirming the structure, evaluating the bioactivity, and analyzing the structure-activity relationship of the designed and synthesized RORγt-PROTACs to find RORγt-PROTACs with higher degradation activity is of significant scientific importance. Detailed Implementation

[0056] 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.

[0057] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions or as recommended by the manufacturer.

[0058] The structure of the compound was determined by nuclear magnetic resonance (NMR) or high-resolution 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).

[0059] MS measurements were performed using a Micromass Q-TOF mass spectrometer.

[0060] High-performance liquid chromatography (HPLC) analysis was performed using an Agilent 1260 HPLC system.

[0061] Thin-layer chromatography silica gel plates were used by GF Chemical Co., Ltd., Yantai City, Shandong Province. 254 Silicone sheet.

[0062] 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.

[0063] 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.

[0064] 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).

[0065] 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.

[0066] The following embodiments further describe the present invention, but these embodiments are not intended to limit the scope of protection of the present invention.

[0067] Example 1 Preparation of 2-(4-((cyclopropylmethyl)sulfonyl)phenyl)-N-(4-(2-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethoxy)-1,1,1,3,3,3-hexafluoroprop-2-yl)phenyl)acetamide (compound 1)

[0068] Step 1: Preparation of ethyl 2-(4-(benzylthio)phenyl)acetate (intermediate b)

[0069] Acetyl p-bromophenylacetate (4000 mg, 17.46 mmol, 1.0 equiv), tetra-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 solution was purified by silica gel column chromatography (PE:EA = 20:1–10:1) to give intermediate b, yield: 4020 mg, 80%; yellow oil.

[0070] Step 2: Preparation of 2-(4-(chlorosulfonyl)phenyl)acetic acid ester (intermediate c)

[0071] 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.

[0072] Step 3: Preparation of ethyl 2-(4-(cyclopropylmethyl)sulfonyl)phenyl)acetate (intermediate d)

[0073] 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.

[0074] Step 4: Preparation of 2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetic acid (intermediate e)

[0075] 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.

[0076] Step 5: Preparation of 2-(4-((cyclopropylmethyl)sulfonyl)phenyl)-N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropane-2-yl)phenyl)acetamide (intermediate f)

[0077] Intermediate e (716 mg, 2.90 mmol, 1.0 equiv), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1285 mg, 3.4 mmol, 1.2 equiv), and N,N-diisopropylethylamine (0.49 mL, 3.4 mmol, 1.2 equiv) were dissolved in dichloromethane (40 mL). After stirring at room temperature for 15 min, 4-(hexafluoro-2-hydroxyisopropyl)aniline (730 mg, 2.90 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 dichloromethane (50 mL × 3) and water. 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 f, yield: 710 mg, 55%; brown oil.

[0078] Step 6: Preparation of tert-butyl{2-[2-(2-{4-[(cyclopropylmethyl)sulfonyl]phenyl}acetamido)-4-(1,1,1,3,3,3-hexafluoropropane-2-yl)oxy]ethoxy}ethylcarbamate (intermediate g1)

[0079] Intermediate f (300 mg, 0.66 mmol, 1.0 equiv) was dissolved in [a solution]. N,N Dimethylformamide (25 mL) was added to (2-(2-bromoethoxy)ethyl)carbamate tert-butyl ester (210 mg, 0.79 mmol, 1.2 equiv) and potassium carbonate (181 mg, 1.3 mmol, 2 equiv), and stirred at 60 °C for 6 h. The reaction was monitored by TLC until complete. The mixture was extracted with ethyl acetate (50 mL × 3) and water, and the organic phase was collected. The organic phase was back-extracted with saturated sodium chloride and concentrated under reduced pressure. The mixture was purified by column chromatography (PE:EA = 1:1) to give intermediate g1, yield: 191 mg, 43%; pale yellow transparent oil.

[0080] Step 7: Preparation of 2-(4-((cyclopropylmethyl)sulfonyl)phenyl)-N-(4-(2-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethoxy)-1,1,1,3,3,3-hexafluoroprop-2-yl)phenyl)acetamide (Compound 1)

[0081] Intermediate g1 (191 mg, 0.28 mmol, 1.0 equiv) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (0.43 mL, 5.6 mmol, 20 equiv) was added dropwise. The mixture was stirred overnight at room temperature. The reaction was confirmed to be complete by TLC. The reaction solution was quenched with sodium bicarbonate, and the pH was adjusted to 10 with a saturated sodium hydroxide aqueous solution. The mixture 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, which was used directly in the next reaction step. The crude product (163 mg, 0.28 mmol, 1.0 equiv) was dissolved in dimethyl sulfoxide (25 mL), and triethylamine (0.12 mL, 0.84 mmol, 3.0 equiv) and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (93 mg, 0.34 mmol, 1.2 equiv) were added. The reaction was carried out at 90°C for 6 h. The reaction was monitored by TLC until complete. The product was extracted with ethyl acetate (50 mL × 3) and water, and the organic phase was collected and concentrated under reduced pressure. The product was then back-extracted with saturated sodium chloride, and the organic phase was collected and concentrated under reduced pressure. The product was purified by column chromatography (DCM:MeOH = 20:1) to give target compound 1, yield: 75 mg, 32%; green solid.

[0082] 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 f from Example 1 and potassium carbonate by heating. The subsequent operations were consistent with those in Example 1 to prepare compounds 2, 3, and 4, respectively.

[0083] Example 5

[0084] Step 1: Preparation of tert-butyl (22-(4-(2-(4-(((cyclopropylmethyl)sulfonyl)phenyl)acetamido)phenyl)-23,23,23-trifluoro-8-oxo-22-(trifluoromethyl)-3,6,12,15,18,21-hexaoxa-9-azatrityl)carbamate (compound h3)

[0085] Intermediate G3 (300 mg, 0.39 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (0.60 mL, 7.76 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 the pH was adjusted to 10 with a saturated sodium hydroxide solution. Extraction was performed with dichloromethane (50 mL × 3) and water. The organic phase was collected, concentrated under reduced pressure, and 260 mg of crude product was obtained, which was directly used in the next step. 2-[2-(tert-Butoxycarbonylamino)ethoxy]ethoxyacetic acid (123 mg, 0.47 mmol, 1.2 equiv) and 2-(7-azobenzotriazole)- N,N, I,I -Tetramethylurea hexafluorophosphate (177 mg, 0.47 mmol, 1.2 equiv) N,N Diisopropylethylamine (0.08 mL, 0.47 mmol, 1.2 equiv) was dissolved in dichloromethane (25 mL) and stirred at room temperature for 15 min. The crude product obtained after Boc removal (260 mg, 0.39 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 h3, yield: 234 mg, 66%; yellow transparent oil.

[0086] Step 2: Preparation of 2-(4-((cyclopropylmethyl)sulfonyl)phenyl)-N-(4-(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)-23,23,23-trifluoro-8-oxo-22-(trifluoromethyl)-3,6,12,15,18,21-hexaoxa-9-azatoritriane-22-yl)phenyl)acetamide (Compound 5)

[0087] Intermediate h3 (234 mg, 0.26 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (0.39 mL, 5.12 mmol, 20 equiv) was added dropwise. The mixture was stirred overnight at room temperature. The reaction was confirmed to be complete by TLC. The reaction solution was quenched with sodium bicarbonate, and the pH was adjusted to 10 with a saturated sodium hydroxide aqueous solution. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure to obtain 208 mg of crude product, which was directly used in the next reaction step. The crude product (208 mg, 0.26 mmol, 1.0 equiv) was dissolved in dimethyl sulfoxide (25 mL), and triethylamine (0.11 mL, 0.77 mmol, 3.0 equiv) and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (85 mg, 0.31 mmol, 1.2 equiv) were added. The reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC until complete. The product was extracted with ethyl acetate (50 mL × 3) and water, and the organic phase was collected. The product was then back-extracted with saturated sodium chloride solution, and the organic phase was collected and concentrated under reduced pressure. The product was purified by column chromatography (DCM:MeOH = 20:1) to give the target compound 5, yield: 103 mg, 38%; green solid.

[0088] Example 6

[0089] Example 5 was repeated, except that different starting materials were used to prepare compound 6. Specifically, intermediate h4 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.

[0090] Example 7 Preparation of (2R,4S)-1-((R)-2-(tert-butyl)-13-(4-(2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamyl)phenyl)-14,14,14-trifluoro-5-oxo-13-(trifluoromethyl)-9,12-dioxa-3,6-diazatetradecanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 7)

[0091] Step 1: Preparation ( S 1-(1-(4-bromophenyl)ethyl)carbamate tert-butyl ester (compound j)

[0092] Starting material i (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 allowed to proceed 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 j, yield: 1450 mg, 82%; white solid.

[0093] Step 2: Preparation ( S 1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)tert-butyl carbamate (compound k)

[0094] Intermediate J (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 organic phase was extracted with water using dichloromethane (50 mL × 3), collected, and concentrated under reduced pressure. The solution was purified by column chromatography (PE:EA = 5:1) to give intermediate K, yield: 850 mg, 55%; white solid.

[0095] Step 3: Preparation ( S )-1-(4-(4-methylthiazol-5-yl)phenyl)ethane-1-amine (compound l)

[0096] Intermediate K (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 L, yield: 550 mg, 95%; white solid.

[0097] Step 4: Preparation (2 S 4 R )-4-hydroxy- N -(( S )-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (compound m)

[0098] 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 1 (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 using 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 using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure. The intermediate m was separated by column chromatography (DCM:MeOH = 20:1), yield: 703 mg, 97%; white solid.

[0099] Step 5: 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 n)

[0100] 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 m (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 n, yield: 1033 mg, 89%; white solid.

[0101] Step 6: 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 o)

[0102] Intermediate n (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 completeness 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 o was given in 799 mg (94%) as a white solid.

[0103] Step 7: Preparation of 2-bromo-N-(2-(2-((2-(4-(2-(4-(((cyclopropylmethyl)sulfonyl)phenyl)acetamyl)phenyl)-1,1,1,3,3,3-hexafluoroprop-2-yl)oxy)ethoxy)ethyl)acetamide (intermediate p1)

[0104] Intermediate g1 (200 mg, 0.29 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (0.45 mL, 5.86 mmol, 20 equiv) was added dropwise. The mixture was stirred overnight at room temperature. The reaction was confirmed to be complete by TLC. The reaction mixture was quenched with sodium bicarbonate, and the pH was adjusted to 10 with saturated sodium hydroxide solution. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure to obtain 171 mg of crude product, which was directly used 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 bromoacetyl bromide (50 mg, 0.18 mmol, 1.2 equiv) were added. The mixture was stirred for 1 h at room temperature. The reaction was confirmed to be complete by TLC. Extracted with water using dichloromethane (50 mL × 3), the organic phase was collected and concentrated under reduced pressure. The solution was purified by column chromatography (DCM:MeOH = 50:1) to give intermediate p1, yield: 113 mg, 55%; white solid.

[0105] Step 8: Preparation of (2R,4S)-1-((R)-2-(tert-butyl)-13-(4-(2-(4-((cyclopropylmethyl)sulfonyl)phenyl)acetamyl)phenyl)-14,14,14-trifluoro-5-oxo-13-(trifluoromethyl)-9,12-dioxa-3,6-diazatetradecanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazo-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 7)

[0106] Intermediate p1 (113 mg, 0.16 mmol, 1.0 equiv) was dissolved in acetonitrile (25 mL), and triethylamine (0.24 mL, 3.24 mmol, 20 equiv) and intermediate o were added dropwise. The mixture was stirred at room temperature for 5 h. The reaction was confirmed to be complete by TLC. The reaction solution was quenched with ammonium chloride, and extracted with water in dichloromethane (50 mL × 3). The organic phase was collected, concentrated under reduced pressure, and purified by column chromatography (DCM:MeOH = 20:1) to give compound 7, yield: 74 mg, 43%; white solid.

[0107] Examples 8-12 Example 7 was repeated, except that different raw materials were used to prepare compounds 8-10. Specifically, p2, p3, p4, h3, and h4 were prepared by operating step 7 of Scheme 2 as described above, while the remaining operations were consistent with the remaining operations in Scheme 2, corresponding to compounds 8, 9, 10, 11, and 12, respectively.

[0108] Example 13 Preparation of 2-(4-((cyclopropylmethyl)sulfonyl)phenyl)-N4-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindene-4-yl)amino)ethoxy)ethyl)-N1-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropyl-2-yl)phenyl)succinamide (Compound 13)

[0109] Step 1: Preparation of 4-(tert-butyl)1-ethyl 2-(4-((cyclopropylmethyl)sulfonyl)phenyl)succinate (compound r)

[0110] Intermediate d (1000 mg, 3.55 mmol, 1.0 equiv) was dissolved in anhydrous tetrahydrofuran. Under N2 protection and at -78 °C, bis(trimethylsilylaminolithium) (7.09 mL, 7.09 mmol, 2 equiv) was added, and the mixture was stirred for 15 min. Then, tert-butyl bromoacetate (0.78 mL, 5.32 mmol, 1.5 equiv) was added, and the mixture was stirred for 2 h. The reaction was confirmed to be complete by TLC. The reaction 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 = 5:1) to give intermediate r, yield: 800 mg, 57%; pale yellow oil.

[0111] Step 2: Preparation of 3-(4-((cyclopropylmethyl)sulfonyl)phenyl)-4-ethoxy-4-oxobutyric acid (compound s)

[0112] Intermediate r (800 mg, 2.02 mmol, 1.0 equiv) was dissolved in dichloromethane, and trifluoroacetic acid (3.0 mL, 40.39 mmol, 20 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 quenched with sodium bicarbonate, and the pH was adjusted to 3 with dilute hydrochloric acid. The mixture was extracted with water in dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure to give intermediate s, yield: 653 mg, 95%; white solid.

[0113] Step 3: Preparation of ethyl 14-(4-((cyclopropylmethyl)sulfonyl)phenyl)-2,2-dimethyl-4,12-dioxo-3,8-dioxa-5,11-diazapentadecan-15-oate (compound t1)

[0114] Intermediate S (210 mg, 0.62 mmol, 1.0 equiv) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (282 mg, 0.74 mmol, 1.2 equiv) were administered. N,N Diisopropylethylamine (0.13 mL, 0.74 mmol, 1.2 equiv) was dissolved in dichloromethane (20 mL), stirred at room temperature for 15 min, and then [2-(2-aminoethoxy)ethyl] tert-butyl carbamate (151 mg, 0.74 mmol, 1.2 equiv) was added. The mixture was stirred at room temperature for 4 h. The reaction was confirmed to be complete by TLC. The reaction solution was extracted with dichloromethane (50 mL × 3) and water, 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 t1, yield: 230 mg, 71%; yellow oil.

[0115] Step 4: Preparation of ethyl 14-(4-((cyclopropylmethyl)sulfonyl)phenyl)-2,2-dimethyl-4,12-dioxo-3,8-dioxa-5,11-diazapentadecan-15-oate (compound u1)

[0116] Intermediate t1 (230 mg, 0.44 mmol, 1.0 equiv) was dissolved in a tetrahydrofuran:water (1:1) solvent, and lithium hydroxide (55 mg, 1.31 mmol, 3 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 concentrated under reduced pressure, and the pH was adjusted to 3 with 1 N hydrochloric acid. The solution was extracted with dichloromethane (50 mL × 3) and water. The organic phase was collected and concentrated under reduced pressure to give intermediate u1, yield: 206 mg, 95%; white solid.

[0117] Step 5: Preparation of tert-butyl(2-(2-(3-(4-((cyclopropylmethyl)sulfonyl)phenyl)-4-((4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropyl-2-yl)phenyl)amino)-4-oxobutamido)ethoxy)ethyl)carbamate (compound v1)

[0118] Intermediate u1 (206 mg, 0.42 mmol, 1.0 equiv) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (189 mg, 0.50 mmol, 1.2 equiv) were administered. N,N Diisopropylethylamine (0.086 mL, 0.50 mmol, 1.2 equiv) was dissolved in dichloromethane (20 mL), stirred at room temperature for 15 min, and then 4-(hexafluoro-2-hydroxyisopropyl)aniline (107 mg, 0.42 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 dichloromethane (50 mL × 3) and water, 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 = 40:1) to give intermediate v1, yield: 145 mg, 47%; yellow oil.

[0119] Step 6: Preparation of 2-(4-((cyclopropylmethyl)sulfonyl)phenyl)-N4-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindene-4-yl)amino)ethoxy)ethyl)N1-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropyl-2-yl)phenyl)succinamide (Compound 13)

[0120] Intermediate v1 (145 mg, 0.20 mmol, 1.0 equiv) was dissolved in dichloromethane (15 mL), and trifluoroacetic acid (0.30 mL, 3.92 mmol, 20 equiv) was added dropwise. The mixture was stirred overnight at room temperature. The reaction was confirmed to be complete by TLC. The reaction solution was quenched with sodium bicarbonate, and the pH was adjusted to 10 with a saturated sodium hydroxide aqueous solution. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure to obtain 120 mg of crude product, which was directly used in the next reaction step. The crude product (120 mg, 0.19 mmol, 1.0 equiv) was dissolved in dimethyl sulfoxide (25 mL), and triethylamine (0.078 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. The reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC until complete. The product was extracted with ethyl acetate (50 mL × 3) and water, and the organic phase was collected. The product was then back-extracted with saturated sodium chloride solution, and the organic phase was collected and concentrated under reduced pressure. The product was purified by column chromatography (DCM:MeOH = 20:1) to give the target compound 13, yield: 35 mg, 21%; green solid.

[0121] Examples 14-16 Example 13 was repeated, except that different raw materials were used to prepare compounds 14-16. Specifically, tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate, 1,1-dimethyl ethyl 13-amino-5,8,11-trioxa-2-azatridecanoic acid, and 1,1-dimethyl 16-amino-5,8,11,14-tetraoxa-2-azahexadecanoic acid were reacted with intermediate s from step 2 of Example 13 above, while the remaining operations were kept the same to prepare compounds 14, 15, and 16, respectively.

[0122] Example 17

[0123] Step 1: Preparation of tert-butyl(24-(4-((cyclopropylmethyl)sulfonyl)phenyl)-25-((4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropyl-2-yl)phenyl)amino)-8,22,25-trioxo-3,6,12,15,18-pentaoxa-9,21-diazapentadecyl)carbamate (compound w3)

[0124] Intermediate V3 (200 mg, 0.24 mmol, 1.0 equiv) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (0.37 mL, 4.84 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 the pH was adjusted to 10 with a saturated sodium hydroxide solution. Extraction was performed with dichloromethane (50 mL × 3) and water. The organic phase was collected, concentrated under reduced pressure, and 170 mg of crude product was obtained, which was directly used in the next step. 2-[2-(tert-Butoxycarbonylamino)ethoxy]ethoxyacetic acid (74 mg, 0.28 mmol, 1.2 equiv) and 2-(7-azobenzotriazole)- N,N, I,I -Tetramethylurea hexafluorophosphate (107 mg, 0.28 mmol, 1.2 equiv), N,N Diisopropylethylamine (0.05 mL, 0.28 mmol, 1.2 equiv) was dissolved in dichloromethane (20 mL) and stirred at room temperature for 15 min. The crude product obtained after Boc removal (170 mg, 0.24 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 w3, yield: 124 mg, 0.53%; yellow transparent oil.

[0125] Step 2: Preparation of (Compound 17)

[0126] Intermediate w3 (124 mg, 0.13 mmol, 1.0 equiv) was dissolved in dichloromethane (15 mL), and trifluoroacetic acid (0.20 mL, 2.55 mmol, 20 equiv) was added dropwise. The mixture was stirred overnight at room temperature. The reaction was confirmed to be complete by TLC. The reaction solution was quenched with sodium bicarbonate, and the pH was adjusted to 10 with saturated sodium hydroxide solution. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure to obtain 106 mg of crude product, which was directly used in the next reaction step. The crude product (106 mg, 0.12 mmol, 1.0 equiv) was dissolved in dimethyl sulfoxide (25 mL), and triethylamine (0.051 mL, 0.36 mmol, 3.0 equiv) and 2-(2,6-dioxadiazin-3-yl)-4-fluoroisoindoline-1,3-dione (40 mg, 0.14 mmol, 1.2 equiv) were added. The reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC until complete. The product was extracted with ethyl acetate (50 mL × 3) and water, and the organic phase was collected. The product was then back-extracted with saturated sodium chloride solution, and the organic phase was collected and concentrated under reduced pressure. The product was purified by column chromatography (DCM:MeOH = 20:1) to give the target compound 17, yield: 36 mg, 26%; green solid.

[0127] Example 18

[0128] Example 17 was repeated, except that different starting materials were used to prepare compound 18. Specifically, intermediate v4 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 17.

[0129] Example 19 Preparation of 2-(4-(N-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindene-4-yl)amino)ethoxy)ethyl)aminosulfonyl)phenyl)-N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropyl-2-yl)phenyl)acetamide (Compound 19)

[0130] Step 1: Preparation of ethyl acetate 2-(4-(N-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)aminosulfonyl)phenyl)acetate (compound x1)

[0131] Intermediate c (300 mg, 1.15 mmol, 1.0 equiv) was dissolved in... N,N Dimethylformamide (25 mL) was added to tert-butyl [2-(2-aminoethoxy)ethyl]carbamate (351 mg, 1.72 mmol, 1.5 equiv) and potassium carbonate (317 mg, 2.29 mmol, 2 equiv), and stirred at 50 °C for 3 h. The reaction was monitored by TLC until complete. The organic phase was extracted with ethyl acetate (50 mL × 3) and collected. The organic phase was back-extracted with saturated sodium chloride and concentrated under reduced pressure. The product was purified by column chromatography (DCM:MeOH = 40:1) to give intermediate x1, yield: 315 mg, 68.85%; pale yellow transparent oil.

[0132] Step 2: Preparation of tert-butyl(2-(2-((4-(2-((4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropyl-2-yl)phenyl)amino)-2-oxoethyl)phenyl)sulfonamide)ethoxy)ethyl)carbamate (compound y1)

[0133] Intermediate x1 (315 mg, 0.73 mmol, 1.0 equiv) was dissolved in a tetrahydrofuran:water (1:1) solvent, and lithium hydroxide (92 mg, 2.2 mmol, 3 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 concentrated under reduced pressure, and the pH was adjusted to 3 with 1 N hydrochloric acid. The solution was extracted with dichloromethane (50 mL × 3) and water. The organic phase was collected and concentrated under reduced pressure to obtain 280 mg of crude product, which was directly used in the next step of the reaction. The crude product (280 mg, 0.7 mmol, 1.0 equiv) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (318 mg, 0.84 mmol, 1.2 equiv) were then added. N,N Diisopropylethylamine (0.15 mL, 0.84 mmol, 1.2 equiv) was dissolved in dichloromethane (20 mL), stirred at room temperature for 15 min, and then 4-(hexafluoro-2-hydroxyisopropyl)aniline (181 mg, 0.7 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 dichloromethane (50 mL × 3) and water, and the organic phase was collected, concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (DCM:MeOH = 50:1) to obtain intermediate y1, yield: 273 mg, 61%; yellow oil.

[0134] Step 3: Preparation of 2-(4-(N-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindene-4-yl)amino)ethoxy)ethyl)aminosulfonyl)phenyl)-N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropyl-2-yl)phenyl)acetamide (Compound 19)

[0135] Intermediate Y1 (273 mg, 0.43 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (0.65 mL, 8.49 mmol, 20 equiv) was added dropwise. The mixture was stirred overnight at room temperature. The reaction was confirmed to be complete by TLC. The reaction solution was quenched with sodium bicarbonate, and the pH was adjusted to 10 with saturated sodium hydroxide solution. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure to obtain 219 mg of crude product, which was directly used in the next reaction step. The crude product (219 mg, 0.4 mmol, 1.0 equiv) was dissolved in dimethyl sulfoxide (25 mL), and triethylamine (0.17 mL, 1.2 mmol, 3.0 equiv) and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (134 mg, 0.48 mmol, 1.2 equiv) were added. The reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC until complete. The product was extracted with ethyl acetate (50 mL × 3) and water, and the organic phase was collected. The product was then back-extracted with saturated sodium chloride solution, and the organic phase was collected and concentrated under reduced pressure. The product was purified by column chromatography (DCM:MeOH = 40:1) to give the target compound 19, yield: 71 mg, 22%; green solid.

[0136] Examples 20-22 Example 19 was repeated, except that different raw materials were used to prepare compounds 20-22. Specifically, tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate, 1,1-dimethyl ethyl 13-amino-5,8,11-trioxa-2-azatridecanoic acid, and 1,1-dimethyl 16-amino-5,8,11,14-tetraoxa-2-azahexadecanoic acid were reacted with intermediate c from step 1 of Example 19 above, while the remaining operations were kept the same to prepare compounds 20, 21, and 22, respectively.

[0137] Example 23

[0138] Step 1: Preparation of tert-butyl(20-((4-(2-((4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropyl-2-yl)phenyl)amino)-2-oxoethyl)phenyl)sulfonamido)-8-oxo-3,6,12,15,18-pentaoxa-9-azadocosyl)carbamate (compound z3)

[0139] Intermediate Y3 (240 mg, 0.33 mmol, 1.0 equiv) was dissolved in dichloromethane (25 mL), and trifluoroacetic acid (0.50 mL, 6.56 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 the pH was adjusted to 10 with a saturated sodium hydroxide aqueous solution. Extraction was performed with dichloromethane (50 mL × 3) and water. The organic phase was collected, concentrated under reduced pressure, and 197 mg of crude product was obtained, which was directly used in the next step. 2-[2-(tert-Butoxycarbonylamino)ethoxy]ethoxyacetic acid (99 mg, 0.37 mmol, 1.2 equiv) and 2-(7-azobenzotriazole)- N,N, I,I -Tetramethylurea hexafluorophosphate (143 mg, 0.37 mmol, 1.2 equiv) N,N - Diisopropylethylamine (0.065 mL, 0.37 mmol, 1.2 equiv) was dissolved in dichloromethane (25 mL) and stirred at room temperature for 15 min. The crude product obtained after Boc removal (197 mg, 0.31 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 z3, yield: 172 mg, 63%; yellow transparent oil.

[0140] Step 2: Preparation of 2-(4-(N-(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindene-4-yl)amino)-8-oxo-3,6,12,15,18-pentaoxa-9-azadocosano-20-yl)aminosulfonyl)phenyl)-N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropyl-2-yl)phenyl)acetamide (Compound 23)

[0141] Intermediate Z3 (172 mg, 0.20 mmol, 1.0 equiv) was dissolved in dichloromethane (15 mL), and trifluoroacetic acid (0.30 mL, 3.93 mmol, 20 equiv) was added dropwise. The mixture was stirred overnight at room temperature. The reaction was confirmed to be complete by TLC. The reaction solution was quenched with sodium bicarbonate, and the pH was adjusted to 10 with saturated sodium hydroxide solution. The mixture was extracted with water using dichloromethane (50 mL × 3), and the organic phase was collected and concentrated under reduced pressure to obtain 145 mg of crude product, which was directly used in the next reaction step. The crude product (145 mg, 0.19 mmol, 1.0 equiv) was dissolved in dimethyl sulfoxide (25 mL), and triethylamine (0.078 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. The reaction was carried out at 90 °C for 6 h. The reaction was monitored by TLC until complete. The product was extracted with ethyl acetate (50 mL × 3) and water, and the organic phase was collected. The product was then back-extracted with saturated sodium chloride solution, and the organic phase was collected and concentrated under reduced pressure. The product was purified by column chromatography (DCM:MeOH = 20:1) to give the target compound 23, yield: 54 mg, 28%; green solid.

[0142] Example 24

[0143] Example 23 was repeated, except that different starting materials were used to prepare compound 24. Specifically, intermediate z4 was reacted with trifluoroacetic acid, 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid, and 2-(7-azobenzotriazole)- from Example 17 above. I,I,I,I -Tetramethylurea hexafluorophosphate, N,N The product was prepared by reacting diisopropylethylamine, with subsequent operations consistent with those in Example 23.

[0144] Experimental Example 25 (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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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 strains for subsequent experiments.

[0149] 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.

[0150] (2) Degradation activity assay of compound RORγt 1) 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.

[0151] 2) 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.

[0152] 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.

[0153] 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.

[0154] 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 ℃.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] Table 1. Chemical structure of the target product ; ; ; ; ; ; .

[0160] Table 2. 1H NMR and high-resolution data of the target compound .

[0161] Table 3. Activity data of the target compound .

[0162] 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 compound 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 compound or a pharmaceutically acceptable salt thereof according to claim 1, 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 bond, -(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 compound 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 compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The R w Choose 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 compound 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 compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The PROTAC compound is selected from the following compounds: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 8. A pharmaceutical composition comprising the PROTAC compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

9. The use of the PROTAC compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of 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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