CN116854667A - Proteolytic targeted chimera for targeted degradation of PD-L1 and application thereof - Google Patents

Abstract

The invention provides a proteolytic targeting chimeric body for targeted degradation of PD-L1 and application thereof, and a PROTAC degradation agent for targeted degradation of PD-L1 protein, which is developed based on a novel PD-L1 protein small molecule inhibitor. According to the invention, the PD-L1 ligand and the ligand of the E3 ligase are coupled through connectors (linker) with different types and different chain lengths, so that the serial PD-L1 targeted PROTAC molecules are successfully prepared, the PD-L1 protein can be effectively targeted, the content of the PD-L1 protein is reduced, meanwhile, the protein has better in vivo activity, the growth of colorectal cancer MC-38 cells of mice can be obviously inhibited, the normal cytotoxicity is lower, and the characteristics of high efficiency and low toxicity are met.

Description

Proteolytic targeted chimera for targeted degradation of PD-L1 and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a proteolytic targeting chimeric body for targeted degradation of PD-L1 and application thereof.

Background

PROTAC (protein-targeting chimeras) is one of the research hotspots in the pharmaceutical field in recent years, and is a brand new technology for inducing targeted protein degradation through ubiquitin-proteinase system (UPS). The PROTAC consists of three special elements: e3 ubiquitin ligase ligand, target protein ligand and Linker (Linker). The E3 ubiquitin ligase ligand is responsible for specifically recruiting E3 ubiquitin ligase, the target protein ligand is used for targeting and capturing target proteins, and the Linker is used for combining the two ligands to form a stable ternary complex. Unlike traditional small protein molecule inhibitors, the mechanism of action of procac is event driven, requiring no long-term target occupation. After finishing ubiquitination marking of one target protein, PROTAC can be separated from the target protein and E3 ubiquitin ligase to continuously mark the next protein. Therefore, in the process of exerting the degradation effect, a small amount of PROTAC molecules can achieve the aim of inducing degradation of a large amount of target proteins. In addition, compared with kinase inhibitors, the PROTAC molecule has lower affinity requirement on target protein, and the PROTAC molecule has wide application prospect on targets which cannot be prepared at present by reasonably designing the molecular structure of the PROTAC. So far, the PROTAC technology has become an important tool for chemical degradation of specific proteins to treat tumors. Such as AR, MDM2, CDK6, CDK9, BRD, BET, ALK, PARP-1, etc., have been developed as PROTAC molecules.

PD-L1 protein is an important immunosuppressive molecule, contributing to tumor immune escape and tumor progression, and overexpression thereof can transmit negative regulation signals, resulting in T cell failure. During the last decade, some research groups have been actively looking for small molecule PD-1/PD-L1 modulators, e.g., the BASEMERIZU (BMS) company reported a series of resorcinol dibenzyl ether-based PD-L1 inhibitors. Although the small molecule inhibitor has a simple structure, few remodelling sites are difficult to break through the patent, and the affinity of the small molecule and the target is lower than that of the antibody, so that off-target phenomenon can occur, thereby reducing the curative effect and increasing the toxicity, and further mechanism exploration and clinical curative effect evaluation are required.

Therefore, it is possible to design PD-1/PD-L1 molecular degrading agents to reduce or down regulate the content of PD-L1 protein by using PROTACs technology, and it is necessary to develop a more effective means for degrading PD-L1, and by degrading PD-L1 by PROTAC, the PD-L1 signal pathway can be more effectively inhibited, so that the method is possible to become a potential cancer treatment method.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention aims to provide a small molecule inhibitor targeting PD-L1 protein and application thereof, and the invention develops a PROTAC degradation agent targeting and degrading PD-L1 based on a novel PD-L1 small molecule inhibitor. Based on the research of PD-L1 small molecule inhibitors in the early stage of a laboratory, a plurality of PD-L1 small molecule inhibitors with excellent activity are found (see patent CN202111475689 of the invention), and the invention provides a PROTAC degradation agent for targeted degradation of PD-L1 based on PD-L1 small molecules and E3 ubiquitin ligase ligand in patent CN202111475689 through a proper linker, and a preparation method and application thereof.

The invention provides a proteolytic targeting chimeric body (PROTAC), which is a compound with a structural general formula shown in formula I:

the dibenzyl ether in formula I represents a ligand moiety that binds to a PD-L1 protein;

in formula I, E3 ligand represents an E3 ubiquitin ligase ligand; linker is a Linker, i.e. a Linker, connecting the PD-L1 protein-bound ligand and the E3 ubiquitin ligase ligand;

wherein: r is R ₁ Hydrogen or halogen substituted in the 2, 3 or 4 position of the benzene ring; r is R ₂ Hydrogen, halogen or methyl substituted in the 2, 4, 5 or 6 position of the benzene ring; r is R ₃ Is hydrogen,

Linker includes: one of saturated fatty chain, unsaturated fatty chain, polyethylene glycol chain, nitrogen-containing five-membered/six-membered heterocyclic ring, substituted benzene ring and amino acid condensed short polypeptide chain;

e3 ligand has any one of the structures shown in the following formulas or an isomer form thereof:

the proteolytic targeting chimera for targeted degradation of PD-L1 is characterized in that the Linker has a structure shown in the following formula:

wherein Z is ₀ 、Z ₁ 、Z ₂ Each independently selected fromAny one of the groups, m0, m1, m2, m3, m4, m5, m6 are each independently selected from any one of integers from 0 to 15;

or the Linker has one of the structures shown in the following formula:

wherein Cx is selected from a 3-8 membered heterocycle containing 1-4 heteroatoms, a 3-8 membered cycloalkyl, a 6-8 membered aryl, or a single bond At least one heteroatom selected from O, S, N; cy is selected from a 3-8 membered heterocycle, a 3-8 membered cycloalkyl, a 6-8 membered aryl or a single bond, said heterocycle containing 1-4 heteroatoms, the heteroatoms being selected from at least one of O, S, N; z is-CH ₂ -NH or-O; m is an integer between 1 and 6, and n is an integer between 0 and 6.

The proteolytic targeting chimera of the target degradable PD-L1, or a tautomer, a meso form, a racemate, an enantiomer, a mixture of one or more diastereoisomers or pharmaceutically acceptable salts thereof. Representative structural formulas of the proteolytic targeting chimeras are selected from one of the following:

the application of the proteolytic targeting chimera for targeted degradation of PD-L1 in preparing antitumor drugs.

Furthermore, the proteolytic targeting chimera is used for preparing antitumor drugs for treating non-small cell lung cancer or breast cancer by taking PD-L1 as a target.

The beneficial effects obtained by the invention are as follows: the invention examines cytotoxicity, binding and ability to degrade PD-L1 and anti-tumor activity in vivo of the synthesized compounds, including three tumor cells: non-small cell lung cancer cell A549, human breast cancer cell MDA-MB-231 and murine colon cancer cell line MC-38. The results show that: several compounds produced stronger PD-L1 degradation to three tumor cell lines, and obtained good anti-tumor activity in mouse model. The invention has important value for discovering new PD-L1 targeted degradation agents.

Drawings

Fig. 1.A: western blot experiment is carried out to determine the PD-L1 expression level of HCC-827 in whole cell lysate in the presence of PROTAC molecule; b: compounds 25i and 25j induce degradation of PD-L1 proteins in tumor cells SK-N-AS, SY5Y, 786-O and MC 38; c: quantitative graphs of degradation activity of compounds 25i and 25j on HCC-827.

FIG. 2 Compounds 25i and 25j induce PD-L1 degradation on the cell membrane and cytoplasm of HCC-827. A: flow cytometry detected the level of HCC-827 cell surface PD-L1 after 24h treatment with 10 μm compound; b: results of the membrane protein extraction kit assay HCC-827 in the presence of 10 μm of compounds 25i and 25j, results of degradation of the membrane and intracellular PD-L1 protein. C: quantification of the cell membrane and cytoplasm after 25i and 25j treatment with PD-L1 protein.

FIG. 3 Compounds 25i and 25j promote T cell mediated tumor killing in HCC-827 and Jurkat co-culture systems.

FIG. 4. Mechanism studies of compounds 25i and 25j targeting degradation of PD-L1. A: effects of Compounds 25i and 25j, the CRBN ligand pomalidomide, PD-L1 ligand A56 on PD-L1 expression in HCC-827 cells; b: HCC-827 cells were pretreated with the proteasome inhibitor MG-132 for 4h,25i and 25j for 24h; c: HCC-827 cells were pretreated with the lysosomal inhibitor Bafilomycin for 4h,25i and 25j for 24h. PD-L1 protein changes were determined using the Westernblot experiment.

Fig. 5 compounds 25i and 25j inhibited tumor growth in the MC38 mouse colon cancer model. A: tumor volume change curves, measured every 2 days; b: mice body weight change profile, measured every 2 days.

Fig. 6. Safety evaluation. A: pathological sections of major tissues (heart, liver, spleen, lung, kidney) of the MC38 mouse colon cancer model, organs were stained with hematoxylin and eosin (H & E); b: serum biochemical analysis.

Fig. 7.A: representative immunohistochemical detection of PD-L1, granzyme B and perforin in tumor tissue. The scale bar is 200 mu m; b: quantitative qPCR analysis of mRNR expression of CXCR3, CXCL9 and CXCL10 in tumors; c: the Westernblot experiment determines PD-L1 levels in mouse tumor tissues.

Fig. 8.A: flow cytometry analysis of cd4+ T cell and cd8+ T cell ratios in lymph nodes of control and dosing groups; b: flow cytometry analyzed the ratio of cd4+ T cells to cd8+ T cells in tumor tissues of control and dosing groups.

Detailed Description

The invention will be further illustrated with reference to specific examples, but the scope of the invention is not limited thereto.

The known starting materials of the present invention may be synthesized using or according to methods known in the art, or may be purchased from Shanghai Taitan technologies, shaohuang chemical technology, shanghai Bi medicine technologies, an Naiji chemistry, myhel chemistry, and the like.

The reaction can be carried out under argon atmosphere or nitrogen atmosphere without any particular explanation in examples. An argon or nitrogen atmosphere means that the reactor flask is connected to a balloon of argon or nitrogen of about 1L volume.

The examples are not particularly described, the reaction temperature at room temperature is 20-30 ℃, the temperature of ice bath condition is 0 ℃, and the reaction time is 12-16 hours.

The reaction progress in the examples was monitored by Thin Layer Chromatography (TLC), the developing reagent used for the reaction, the column chromatography eluent system used for purifying the compound, the developing reagent system for thin layer chromatography and the rapid purification preparation of the liquid C18-bonded phase separation cartridge system comprising: the volume ratio of the solvent is adjusted according to the polarity of the compound.

Without special description in the examples, step 1 refers to a Suzuki coupling reaction in which arylboronic acid or boric acid ester and halogenated aromatic hydrocarbon are subjected to cross coupling; step 2 refers to bromination reaction of aryl benzyl alcohol under the condition of brominating reagent; step 3 refers to a reaction of coupling an arylbenzyl bromide compound with a phenolic hydroxyl compound; step 4 refers to the coupling reaction of phenolic hydroxyl compound and cyano-substituted bromomethylbenzene or chloromethylpyridine; step 5 refers to the substitution reaction of the E3 ligase ligand 2- (2, 6-dioxo-piperidin-3-yl) -4-fluoro-isoindoline-1, 3-dione with an amino-functional linker; step 6 refers to a reaction of removing an amino protecting group by trifluoroacetic acid; step 7 refers to the reductive amination of carbonyl groups with amines; step 8 refers to an acid amine condensation reaction in which a carboxyl compound and an amino compound are amidated; step 9 refers to the oxidation of hydroxyl compounds to aldehyde by dessmartin; step 10 refers to the amidation reaction of an amino compound with an acyl halide compound; step 11 refers to the azide reaction of a haloalkane with sodium azide; step 12 refers to the Click reaction of the azide with the alkyne.

Measuring instrument: nuclear magnetic resonance spectroscopy was performed using a Bruker AV-400Bruker AV-600 type nuclear magnetic resonance apparatus. Mass spectrometry was performed using ZAD-2F and VG300 mass spectrometers. HPLC purity determination method: the purity of the compound was confirmed by HPLC (high performance liquid chromatography) analysis, and it was confirmed that the purity was 95% or more. HPLC analysis was performed using a C18 column (InertSustatin C18, 4.6X105 mm,5 μm), shimadzu LC-20A, UV detection at 254 nm. The mobile gradient phase consisted of B (pure water and 0.1% trifluoroacetic acid) and A (100% methanol) at a flow rate of 1.0mL/min.

Example 1: preparation of 3- { [ (2- { [ (2- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo-isoindol-4-yl ] amino } ethyl) amino ] methyl } -5- ({ [3- (2-fluorophenyl) -2-methylphenyl ] methyl } oxy) phenyl) oxy ] methyl } benzonitrile (13 a) chemical formula 13 a:

chemical reaction procedure for the preparation of compound 13 a:

step 1, intermediate 3: [3- (2-fluorophenyl) -2-methylphenyl ] methanol

3-bromo-2-methylbenzyl alcohol (1.039 g,5.19 mmol), 2-fluorobenzeneboronic acid (0.77 g,5.5 mmol), K ₂ CO ₃ (1.725 g,12.5 mmol) and Pd (PPh) ₃ ) ₄ (0.06 g,0.052 mmol) in 1, 4-dioxane (15 mL) and H ₂ The mixture in O (2 mL) was degassed and heated to 80 ℃. At N ₂ The lower run was continued for 12 hours. Concentrating the dioxane in the reaction solution under reduced pressure, and pouring the obtained mixture into H ₂ O (30 mL) and then the mixture was extracted with ethyl acetate (30 mL. Times.3). The organic phase was washed with brine (30 mL. Times.2), dried over anhydrous Na ₂ SO ₄ Drying and vacuum concentration gave 0.99g of a white solid in 88.2% yield.

Step 2, intermediate 4:3- (bromomethyl) -1- (2-fluorophenyl) -2-toluene

To a solution of intermediate 3 (0.99 g,4.58 mmol) and triphenylphosphine (1.8 g,6.87 mmol) in DCM (15 mL) under ice-bath conditions was slowly added NBS (1.22 g,6.87 g). The reaction was warmed to room temperature and stirred for 12h, tlc indicated the reaction was complete. The reaction was slowly quenched with water and then concentrated under reduced pressure to give a residue. The crude product was purified by silica gel chromatography (eluting with volume ratio petroleum ether/ethyl acetate=40/1) to give 1.05g of a white solid in 82.6% yield.

Step 3, intermediate 6:4- ({ [3- (2-fluorophenyl) -2-methylphenyl ] methyl } oxy) -2-hydroxybenzaldehyde

To a solution of intermediate 4 (1 g,3.59 mmol) and compound 5 (0.5 g,3.62 mmol) in DMF (15 mL) was added potassium carbonate (0.74 g,5.07 mmol) and sodium iodide (0.54 g,3.60 mmol). Heating to 60 ℃, stirring for 3h, and TLC shows that the reaction is complete. Pouring the mixture into H ₂ O (30 mL) and then the mixture was extracted with ethyl acetate (30 mL. Times.3). The organic phase was washed with brine (30 mL. Times.2), dried over anhydrous Na ₂ SO ₄ Drying and vacuum concentration gave a residue. The crude product was purified by silica gel chromatography (eluting with volume ratio petroleum ether/ethyl acetate=40/1) to give 0.67g of a white solid in 56.1% yield.

Step 4, intermediate 8:3- ({ [5- ({ [3- (2-fluorophenyl) -2-methylphenyl)]Methyl } oxy) -2-formylphenyl]To a solution of intermediate 6 (0.67 g,1.99 mmol) and m-cyanoborobenzyl compound 7 (0.33 g,2.19 mmol) in DMF (15 mL) was added cesium carbonate (0.97 g,2.99 mmol) and sodium iodide (0.30 g,1.99 mmol). Heating to 60 ℃, stirring for 3h, and TLC shows that the reaction is complete. Pouring the mixture into H ₂ O (30 mL) and then the mixture was extracted with ethyl acetate (30 mL. Times.3). The organic phase was washed with brine (30 mL. Times.2), dried over anhydrous Na ₂ SO ₄ Drying and vacuum concentration gave a residue. The crude product was purified by beating (volume ratio petroleum ether/ethyl acetate=10/1 beating) to give 0.67g of white solid with a yield of 52.3%.

Step 5, intermediate 11: (2- ((2- (2, 6-Dioxypiperidin-3-yl) -1, 3-Dioxyisoindolin-4-yl) amino) ethyl) carbamic acid tert-butyl ester

Compound 9 (50 mg,0.18 mmol), mono-Boc-ethylenediamine (compound 10, 32mg,0.20 mmol), N, N-diisopropylethylamine (46.8 mg,0.36 mmol) was dissolved in N-methylpyrrolidone 4mL at room temperature. After the addition, the temperature is raised to 90 ℃ for reaction for 3 hours. After the reaction was completed, the reaction solution was cooled to room temperature, the reaction solution was poured into 10mL of water, the aqueous phase was extracted with ethyl acetate (10 ml×3), the organic phases were combined, washed with water, dried over anhydrous sodium sulfate, suction-filtered, and evaporated to dryness to give 75mg of a yellowish green oil with a yield of 99.9%. The crude product was directly taken to the next step without purification.

Step 6, intermediate 12:4- [ (2-aminoethyl) amino ] -2- (2, 6-dioxopiperidin-3-yl) isoindoline-1, 3-dione

Intermediate 11 (75 mg,0.18 mmol) was dissolved in 1mL of dichloromethane under ice-bath conditions, and 1.0mL of trifluoroacetic acid was slowly added. After the addition, the reaction was carried out at room temperature for 0.5h. After the reaction, the mixture was evaporated to dryness to give 56.88mg of a dark green fluorescent viscous liquid compound in a yield of 99.1%. The crude product was directly taken to the next step without purification.

Step 7,3- { [ (2- { [ (2- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo-isoindol-4-yl ] amino } ethyl) amino ] methyl } -5- ({ [3- (2-fluorophenyl) -2-methylphenyl ] methyl } oxy) phenyl) oxy ] methyl } benzonitrile

Intermediate compound 12 was added directly to a solution of intermediate 8 (81.2 mg,0.18 mmol) in DMF (2 mL) at room temperature. Dripping triethylamine to adjust pH to 5-6, adding NaBH ₃ CN (34.0 mg,0.54 mmol) was reacted at room temperature overnight. After the reaction was completed, the reaction solution was cooled to room temperature, the reaction solution was poured into 6mL of water, the aqueous phase was extracted with ethyl acetate (10 ml×3), the organic phases were combined, washed with water, dried over anhydrous sodium sulfate, suction-filtered, evaporated to dryness, and the residue was purified by silica gel column chromatography (volume ratio dichloromethane: methanol=20:1 to 10:1), to give compound 13a as green fluorescent powder 31.5mg, yield 23.3%; analytical HPLC purity was 97.6%.

¹ H NMR(400MHz,Chloroform-d)δ7.71(s,1H),7.63(d,J＝8.0Hz,1H),7.58(d,J＝8.1Hz,1H),7.46(dq,J＝7.6,3.8,3.3Hz,3H),7.39(d,J＝6.7Hz,1H),7.24(q,m,4H),7.17(t,J＝9.0Hz,1H),7.07(d,J＝7.1Hz,1H),6.87(d,J＝8.2Hz,1H),6.63(d,J＝8.3Hz,1H),6.60–6.48(m,2H),5.08(s,4H),4.92(dd,J＝12.1,5.5Hz,1H),3.90(s,2H),3.54–3.31(m,2H),2.95(q,J＝7.5,5.7Hz,2H),2.89–2.64(m,3H),2.20(s,3H),2.13–2.01(m,1H).

Example 2: preparation of 4- { [ (2- { [ (3-cyanophenyl) methyl ] oxy } -4- ({ [3- (2-fluorophenyl) -2-methylphenyl ] methyl } oxy) phenyl) methyl ] amino } -N- [2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindol-4-yl ] butanamide (18 a) the chemical formula of 18a:

chemical reaction procedure for the preparation of compound 18a:

step 8, intermediate 16: tert-butyl (4- ((2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) amino) -4-oxobutyl) carbamate

In a 25mL flask, lenalidomide (50 mg,0.193 mmol), boc-4-aminobutyric acid (43.1 mg,0.21 mmol), EDCI (147.5 mg,0.772 mmol), DMAP (4.7 mg,0.04 mmol) and DMF (10 mL) were mixed well. Stir at room temperature overnight. After the reaction, the mixture obtained is poured into H ₂ O (30 mL) and then the mixture was extracted with ethyl acetate (30 mL. Times.3). The organic phase was washed with brine (30 mL. Times.2), dried over anhydrous Na ₂ SO ₄ Drying and concentration in vacuo gave 79mg of a white oily solid in 92.9% yield.

Step 6, intermediate 17: 4-amino-N- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl) butanamide

The preparation is described in example 1.

Step 7, end product 18a:4- (2- (3-cyanobenzyloxy) -4-fluoro-2-methyl-1, 1' -biphenyl-3-ylmethoxy) amino) -N- (2, 6-dioxapiperidin-3-yl) -1-oxoisoindoline-4-butyramide

The preparation is described in example 1. The yield was 36.9%; analytical HPLC purity 95.7%.

¹ H NMR(400MHz,Chloroform-d)δ7.74(s,1H),7.68(d,J＝7.9Hz,1H),7.62–7.53(m,3H),7.45(t,J＝7.7Hz,2H),7.42–7.29(m,3H),7.28–7.20(m,4H),7.16(ddd,J＝9.6,8.2,1.1Hz,1H),6.59(dd,J＝8.4,2.3Hz,1H),6.54(d,J＝2.3Hz,1H),5.04(s,5H),4.37(s,2H),3.93(s,2H),2.88(d,J＝6.5Hz,2H),2.78–2.65(m,2H),2.62–2.49(m,2H),2.18(d,J＝1.5Hz,4H),2.11–1.92(m,3H).

Example 3: preparation 25a of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (2- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo-isoindolin-4-yl ] amino } ethyl) amino ] methyl } phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 a) chemical formula:

chemical reaction procedure for the preparation of compound 25 a:

example 3 was prepared according to the general procedure described in example 1, except that an equivalent molar amount of 3-bromo-2-chlorobenzyl alcohol was used in place of 3-bromo-2-methylbenzyl alcohol in Step 1, and an equivalent molar amount of 5- (chloromethyl) nicotinonitrile was used in place of m-cyanobenzyl bromide in Step 4. The yield was 24.7%; analytical HPLC purity 95.5%.

¹ H NMR(400MHz,Chloroform-d)δ8.84(m,2H),8.07(s,1H),7.60(m,2H),7.48–7.30(m,6H),7.27–7.05(m,4H),6.97–6.84(m,1H),6.74–6.57(m,2H),6.57–6.45(m,1H),5.29–5.09(m,4H),4.92(dd,J＝12.0,5.9Hz,1H),3.89(s,1H),3.48(m,3H),3.10(q,J＝7.3Hz,2H),2.97(d,J＝8.6Hz,2H),2.05(d,J＝11.3Hz,2H).

Example 4: preparation of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (3- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoidenedioxyisoindolin-4-yl ] amino } propyl) amino ] methyl } phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 b) chemical formula 25 b:

example 4 was prepared according to the general procedure described in example 3, except that an equivalent molar amount of mono-Boc-propylenediamine was used instead of mono-Boc-ethylenediamine at Step 5, following the procedure described in example 3. The yield was 22.9%; analytical HPLC purity was 99.6%.

¹ H NMR(400MHz,Chloroform-d)δ8.96–8.69(m,2H),8.08(m,1H),7.55(d,J＝6.7Hz,1H),7.47–7.29(m,5H),7.19(dt,J＝28.2,8.2Hz,3H),6.96(d,J＝6.1Hz,1H),6.78(s,1H),6.58(m,2H),6.31(s,1H),5.27–5.05(m,4H),4.95(s,1H),4.04(s,2H),3.90(s,1H),3.27(s,2H),3.06–2.56(m,5H),2.03(m,3H).

Example 5: preparation of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (4- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo-isoindolin-4-yl ] amino } butyl) amino ] methyl } phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 c) the chemical formula of 25 c:

example 5 was prepared according to the general procedure described in example 3, except that an equivalent molar amount of mono-Boc-butanediamine was used instead of mono-Boc-ethylenediamine at Step 5, following the procedure described in example 3. The yield was 32.2%; analytical HPLC purity was 98.3%.

¹ H NMR(400MHz,Chloroform-d)δ8.95–8.74(m,2H),8.28–7.98(m,1H),7.66–7.50(m,1H),7.49–7.29(m,6H),7.27–7.12(m,3H),6.97(s,1H),6.87(d,J＝6.9Hz,1H),6.60(d,J＝14.7Hz,2H),6.23(s,1H),5.19(d,J＝7.3Hz,4H),4.93(s,1H),4.02(m,2H),3.21(m,2H),2.81(m,5H),1.86–1.52(m,4H).

Example 6: preparation of 25d of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (5- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo-isoindolin-4-yl ] amino } pentyl) amino ] methyl } phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 d) has the chemical formula:

example 6 was prepared according to the general procedure described in example 3 except that an equivalent molar amount of mono-Boc-pentamethylenediamine was used instead of mono-Boc-ethylenediamine at Step 5, following the procedure described in example 3. The yield was 38.7%; analytical HPLC purity was 97.4%.

¹ H NMR(400MHz,Chloroform-d)δ8.91(m,2H),8.25(s,1H),7.63–7.30(m,7H),7.21(dt,J＝28.0,8.1Hz,2H),7.06(d,J＝7.1Hz,1H),6.85(d,J＝8.2Hz,1H),6.64(d,J＝10.1Hz,2H),6.22(s,1H),5.15(d,J＝34.4Hz,4H),4.96–4.85(m,1H),3.92(s,2H),3.51(s,2H),3.22(q,J＝6.5Hz,2H),2.92–2.63(m,5H),1.78(m,2H),1.59(t,J＝7.6Hz,2H),1.45–1.35(m,2H).

Example 7: preparation of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (6- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoidenedioxyisoindolin-4-yl ] amino } hexyl) amino ] methyl } phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 e) the chemical formula of 25 e:

Example 7 was prepared according to the general procedure described in example 3, except that an equivalent molar amount of mono-Boc-hexamethylenediamine was used instead of mono-Boc-ethylenediamine at Step 5, following the procedure described in example 3. The yield thereof was found to be 39.9%; analytical HPLC purity 96.8%.

¹ H NMR(400MHz,Chloroform-d)δ8.94(d,J＝2.1Hz,1H),8.86(d,J＝2.0Hz,2H),8.24(t,J＝1.9Hz,1H),7.56(dd,J＝7.4,2.1Hz,2H),7.51–7.30(m,8H),7.28–7.13(m,3H),7.06(d,J＝7.0Hz,1H),6.87(d,J＝8.6Hz,1H),6.64(d,J＝8.3Hz,2H),6.22(t,J＝5.6Hz,1H),5.16(d,J＝22.1Hz,4H),4.90(dd,J＝12.0,5.5Hz,1H),3.99(s,2H),3.23(q,J＝6.5Hz,2H),2.89–2.69(m,6H),2.18–2.06(m,2H),1.82–1.51(m,8H).

Example 8: preparation of 25f of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (7- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl ] amino } heptyl) amino ] methyl } phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 f) has the chemical formula:

example 8 was prepared according to the general procedure described in example 3 except that an equivalent molar amount of mono-Boc-heptanediamine was used instead of mono-Boc-ethylenediamine at Step 5, following the procedure described in example 3. The yield was 41.6%; analytical HPLC purity was 99.2%.

¹ H NMR(400MHz,Chloroform-d)δ8.97(s,1H),8.73(s,1H),8.11(s,1H),7.64–7.54(m,1H),7.53–7.29(m,6H),7.28–7.14(m,4H),7.08(d,J＝7.0Hz,1H),6.90(d,J＝8.4Hz,1H),6.66(s,1H),6.61(s,1H),5.19(d,J＝19.4Hz,4H),4.94–4.82(m,1H),4.11(s,2H),3.28(s,2H),2.87(m,5H),2.27(t,J＝7.6Hz,1H),2.13(d,J＝9.1Hz,1H),2.08–1.98(m,2H),1.64(s,4H),1.37(m,6H).HRMS[M+H] ⁺ calcd.for C ₄₇ H ₄₅ ClFN ₆ O ₆ ；843.3068；found,843.3029.

Example 9: preparation of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (8- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo-isoindolin-4-yl ] amino } octyl) amino ] methyl } phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 g) 25g of the chemical formula:

example 9 was prepared according to the general procedure described in example 3, except that an equivalent molar amount of mono-Boc-octanediamine was used instead of mono-Boc-ethylenediamine at Step 5, following the procedure described in example 3. The yield was 31.0%; analytical HPLC purity 96.9%.

¹ H NMR(400MHz,Chloroform-d)δ8.92(s,1H),8.85(s,1H),8.14(s,1H),7.58(d,J＝7.4Hz,1H),7.49(t,J＝7.8Hz,1H),7.45–7.30(m,5H),7.21(dt,J＝28.5,8.2Hz,2H),7.06(d,J＝7.1Hz,1H),6.90(d,J＝8.6Hz,1H),6.66(d,J＝9.0Hz,2H),6.24(t,J＝5.6Hz,1H),5.20(d,J＝5.6Hz,4H),4.89(dd,J＝11.9,5.3Hz,1H),4.09(s,2H),3.73(s,2H),3.27(q,J＝6.5Hz,2H),2.79–2.69(m,2H),2.07(m,3H),1.64(m,6H),1.45–1.30(m,6H).

Example 10: preparation of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- (1- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo-isoindol-4-yl ] amino } -6-aza-3-oxahept-7-yl) phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 h) chemical formula:

example 10 was prepared according to the general procedure described in example 3 except that an equivalent molar amount of tert-butyl [2- (2-aminoethoxy) ethyl ] carbamate was used in place of mono-Boc-ethylenediamine at Step 5, following the procedure described in example 3. The yield was 35.5%; analytical HPLC purity was 97.4%.

¹ H NMR(400MHz,Chloroform-d)δ8.89(s,1H),8.78(s,1H),8.07(s,1H),7.59(d,J＝7.5Hz,1H),7.53–7.30(m,6H),7.27–7.15(m,2H),7.07(d,J＝7.1Hz,1H),6.88(d,J＝8.6Hz,1H),6.69–6.59(m,2H),6.40(t,J＝5.6Hz,1H),5.29–5.11(m,4H),4.90–4.77(m,1H),4.25–3.99(m,2H),3.72(dt,J＝28.8,4.8Hz,4H),3.42(q,J＝5.2Hz,2H),3.10(q,J＝7.4Hz,3H),2.87–2.59(m,3H),2.13–2.04(m,1H).

Example 11: preparation of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- (1- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo-isoindol-4-yl ] amino } -9-aza-3, 6-dioxadec-10-yl) phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 i) the chemical formula:

example 11 was prepared according to the general procedure described in example 3 except that an equivalent molar amount of tert-butyl 2- (2- (2-aminoethoxy) ethoxy) ethylcarbamate was used in place of the mono-Boc-ethylenediamine at Step 5, following the procedure described in example 3. The yield was 34.2%; analytical HPLC purity was 98.3%.

¹ H NMR(400MHz,Chloroform-d)δ8.90(d,J＝2.1Hz,1H),8.81(d,J＝1.9Hz,1H),8.14(t,J＝2.1Hz,1H),7.64–7.53(m,1H),7.51–7.29(m,7H),7.24(t,J＝7.5Hz,1H),7.18(t,J＝9.1Hz,1H),7.06(d,J＝7.0Hz,1H),6.85(d,J＝8.5Hz,1H),6.58(d,J＝9.1Hz,2H),6.44(t,J＝5.5Hz,1H),5.16(d,J＝7.3Hz,4H),4.91(dd,J＝12.3,5.5Hz,1H),4.16(s,2H),3.78(t,J＝5.0Hz,2H),3.72–3.56(m,6H),3.38(q,J＝5.3Hz,2H),3.13(t,J＝5.0Hz,2H),2.86–2.59(m,3H),2.15–1.93(m,2H). ¹³ C NMR(600MHz,Chloroform-d)δ171.79,169.50,169.29,167.57,160.54,158.77,157.30,152.03,151.83,146.63,138.53,136.14,135.71,134.76,133.08,132.83,132.47,132.05,131.50(d,J＝2.9Hz),131.22,130.05(d,J＝8.2Hz),128.45,126.75,124.03(d,J＝3.5Hz),116.81,116.41,115.73,115.58,113.91,111.73,110.28,110.03,106.62,100.34,70.33,70.04,68.97,67.71,66.75,60.41,48.97,46.65,46.42,42.17,31.36,22.83.HRMS(m/z):[M+H] ⁺ calcd.for C ₄₆ H ₄₃ ClFN ₆ O _8, 861.2809；found,861.2770.

Example 12: preparation of 25j of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- (1- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo-isoindol-4-yl ] amino } -12-aza-3, 6, 9-trioxatridec-13-yl) phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 j) has the chemical formula:

Example 12 was prepared according to the general procedure described in example 3 except that an equivalent molar amount of tert-butyl (2- (2- (2-aminoethoxy) ethoxy) ethyl) carbamate was used instead of mono Boc-ethylenediamine in Step 5, following the procedure described in example 3. The yield was 32.7%; analytical HPLC purity was 97.3%.

¹ H NMR(400MHz,Chloroform-d)δ8.92(s,1H),8.83(s,1H),8.14(s,1H),7.59(dd,J＝13.5,7.3Hz,1H),7.52–7.30(m,7H),7.25(t,J＝7.4Hz,1H),7.19(t,J＝9.1Hz,1H),7.07(d,J＝7.0Hz,1H),6.88(d,J＝8.6Hz,1H),6.67–6.54(m,2H),6.47(t,J＝5.5Hz,1H),5.28–5.14(m,4H),4.91(q,J＝5.7Hz,1H),4.10(s,2H),3.82–3.53(m,11H),3.40(q,J＝5.2Hz,2H),3.06(t,J＝5.0Hz,2H),2.88–2.64(m,3H),2.17–2.00(m,2H). ¹³ C NMR(400MHz,Chloroform-d)δ171.65,169.32,160.39,157.20,151.93,151.90,146.70,138.32,136.08,135.73,134.80,132.83,132.48,132.09,131.53,131.50,131.24,130.10,130.01,128.46,126.77,124.05,124.01,116.81,116.37,115.79,115.57,111.58,110.17,110.09,106.61,100.30,70.72,70.36,70.28,70.20,69.06,67.77,66.91,66.67,48.93,42.14,31.47,22.83.HRMS(m/z):[M+H] ⁺ calcd.for C ₄₈ H ₄₇ ClFN ₆ O ₉ ；905.3072；found,905.3043.

Example 13: preparation of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- (1- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxoisoindol-4-yl ] amino } -15-aza-3, 6,9, 12-tetraoxahexadecan-16-yl) phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 k)

25k of the formula:

example 13 was prepared according to the general procedure described in example 3 except that an equivalent molar amount of tert-butyl (14-amino-3, 6,9, 12-tetraoxatetradecyl) carbamate was used in place of mono-Boc-ethylenediamine in Step 5, following the procedure described in example 3. The yield was 30.2%; analytical HPLC purity was 98.2%.

¹ H NMR(400MHz,Chloroform-d)δ8.90(d,J＝25.5Hz,2H),8.13(s,1H),7.58(d,J＝7.4Hz,1H),7.53–7.30(m,5H),7.27–7.14(m,2H),7.07(d,J＝7.1Hz,1H),6.88(d,J＝8.4Hz,1H),6.74–6.54(m,2H),6.45(d,J＝5.7Hz,1H),5.22(s,4H),4.93(q,J＝5.9Hz,1H),4.15(s,2H),3.85–3.32(m,18H),3.11(d,J＝4.8Hz,2H),2.80–2.67(m,2H),2.10(d,J＝24.8Hz,2H).

Example 14: preparation of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- (1- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo isoindol-4-yl ] amino } -18-aza-3, 6,9,12, 15-pentoxanonadec-19-yl) phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 l)

25l of the formula:

example 14 was prepared according to the general procedure described in example 3, except that an equivalent molar amount of tert-butyl (17-amino-3, 6,9,12, 15-pentaoxaheptadecane) carbamate was used in place of mono-Boc-ethylenediamine in Step 5, following the procedure described in example 3. The yield was 22.0%; analytical HPLC purity 95.6%.

¹ H NMR(400MHz,Chloroform-d)δ8.91(d,J＝2.1Hz,1H),8.87(d,J＝2.0Hz,1H),8.18(t,J＝2.0Hz,1H),7.59(dd,J＝7.6,1.9Hz,1H),7.51–7.30(m,6H),7.28–7.16(m,2H),7.09(d,J＝7.1Hz,1H),6.89(d,J＝8.5Hz,1H),6.64(dd,J＝8.3,2.3Hz,1H),6.60(d,J＝2.3Hz,1H),6.50(t,J＝5.5Hz,1H),5.21(d,J＝21.3Hz,4H),4.90(dd,J＝12.1,5.3Hz,1H),4.06(s,2H),3.71(q,J＝5.1,4.3Hz,4H),3.64(s,6H),3.63–3.57(m,9H),3.43(q,J＝5.3Hz,2H),3.06–2.96(m,2H),2.89–2.68(m,3H),2.16–2.06(m,1H),2.03(s,1H).

Example 15: preparation of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- (1- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo isoindol-4-yl ] amino } -14-aza-4, 7, 10-trioxapentadec-15-yl) phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 m)

25m of the formula:

example 15 was prepared according to the general procedure described in example 3 except that tert-butyl (3- (2- (3-aminopropoxy) ethoxy) propyl) carbamate was used instead of mono-Boc-ethylenediamine at Step 5. The yield was 31.2%; analytical HPLC purity 94.3%.

¹ H NMR(400MHz,Chloroform-d)δ8.92(d,J＝2.1Hz,1H),8.87(d,J＝2.0Hz,1H),8.17(t,J＝2.1Hz,1H),7.57(dd,J＝7.5,1.9Hz,1H),7.50–7.38(m,4H),7.37–7.30(m,3H),7.27–7.22(m,1H),7.22–7.16(m,1H),7.06(d,J＝7.0Hz,1H),6.87(d,J＝8.5Hz,1H),6.67(dd,J＝8.4,2.3Hz,1H),6.60(d,J＝2.3Hz,1H),6.54(t,J＝5.4Hz,1H),5.28–5.17(m,4H),4.91(td,J＝7.9,6.8,3.8Hz,1H),4.13(s,2H),3.65(dd,J＝9.2,4.0Hz,3H),3.58(m,7H),3.53(d,J＝4.3Hz,2H),3.36(q,J＝6.0Hz,2H),3.14(dt,J＝7.7,4.5Hz,2H),2.80–2.69(m,3H),2.16–2.08(m,1H),1.98(t,J＝5.6Hz,2H),1.89(q,J＝6.0Hz,3H).

Example 16: preparation of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [4- (2- { [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo-isoindol-4-yl ] amino } ethyl) piperazin-1-yl ] methyl } phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 n) the chemical structural formula 25 n:

Example 16 was prepared according to the general procedure described in example 3 except that tert-butyl 4- (2-aminoethyl) piperazine-1-carboxylate was used instead of mono Boc-ethylenediamine in Step 5, following the procedure described in example 3. The yield was 9.2%; analytical HPLC purity 96.3%.

¹ H NMR(400MHz,Chloroform-d)δ9.29(s,1H),8.95(d,J＝2.1Hz,1H),8.89(d,J＝2.0Hz,1H),8.15(s,1H),7.60(d,J＝6.0Hz,1H),7.55–7.29(m,6H),7.25(t,J＝7.4Hz,1H),7.18(t,J＝9.1Hz,1H),7.09(d,J＝7.1Hz,1H),6.88(d,J＝8.5Hz,1H),6.69(dd,J＝8.4,2.3Hz,1H),6.62(d,J＝2.8Hz,2H),5.20(d,J＝36.3Hz,4H),4.96(dd,J＝12.0,5.1Hz,1H),3.81(s,2H),3.36(q,J＝5.7Hz,2H),3.05–2.43(m,13H),2.14(td,J＝7.1,3.2Hz,1H).

Example 17: preparation of 5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [4- ({ 4- [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo-isoindol-4-yl ] piperazin-1-yl } methyl) piperidin-1-yl ] methyl } phenyl ] oxy } methyl) pyridine-3-carbonitrile (25 o) the chemical formula:

chemical reaction procedure for preparation of Compound 25o

Intermediate 26: tert-butyl 4- (2, 6-dioxapiperidin-3-yl) -1, 3-dioxoisoindolin-4-yl) piperazine-1-carboxylate was prepared according to the Step 5 method described in example 1, except that tert-butyl 4- (2-aminoethyl) piperazine-1-carboxylate was used instead of mono Boc-ethylenediamine.

Intermediate 27:2- (2, 6-Dioxypiperidin-3-yl) -4- (piperazin-1-yl) isoindoline-1, 3-dione was prepared according to the Step 6 procedure described in example 1, except that intermediate 11 was replaced with intermediate 26.

Intermediate 28:5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [4- (hydroxymethyl) piperidin-1-yl ] methyl } phenyl ] oxy } methyl) pyridine-3-carbonitrile was prepared according to the Step 7 procedure described in example 3, except that 4-piperidinemethanol was used instead of intermediate 12.Step 9: intermediate 29:5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- [ (4-formylpiperidin-1-yl) methyl ] phenyl ] oxy } methyl) pyridine-3-carbonitrile

Intermediate 28 (36 mg,0.06 mmol) was dissolved in dichloromethane (3 mL) and under ice-bath conditions, dess-Martin oxidant (80 mg,0.18 mmol) was slowly added and reacted at room temperature for 2h after the addition, TLC detection was completed and saturated NaHCO was added ₃ The solution (3 mL) and saturated sodium thiosulfate solution (3 mL) were collected, and the dichloromethane layer was concentrated under reduced pressure to give a residue. The crude product was purified by chromatography on silica gel (with volume ratio of dichloromethaneMethanol=20/1 elution) to give 33mg of a white solid in 92.6% yield.

Final product 25o:5- ({ [5- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [4- ({ 4- [2- (2, 6-dioxopiperidin-3-yl) -1, 3-dioxo-isoindol-4-yl ] piperazin-1-yl } methyl) piperidin-1-yl ] methyl } phenyl ] oxy } methyl) pyridine-3-carbonitrile

Prepared according to the Step 7 method described in example 3 except that intermediate 27 was used instead of intermediate 12 and intermediate 29 was used instead of intermediate 24. The yield was 22.2%; analytical HPLC purity was 99.8%.

¹ H NMR(400MHz,Chloroform-d)δ8.93(d,J＝2.2Hz,1H),8.89(d,J＝2.0Hz,1H),8.14(t,J＝2.1Hz,1H),7.66–7.57(m,2H),7.47–7.39(m,3H),7.35(m,4H),7.27–7.16(m,3H),6.69(dd,J＝8.3,2.3Hz,1H),6.62(d,J＝2.4Hz,1H),5.22(d,J＝32.7Hz,4H),4.98(dd,J＝12.2,5.3Hz,1H),3.71(d,J＝17.6Hz,2H),3.36(m,4H),3.14(brs,2H),2.97–2.72(m,4H),2.64(brs,4H),2.30(d,J＝6.9Hz,2H),2.03(q,J＝6.4Hz,2H),1.84(m,5H).

Example 18: preparation of 4- ({ [4- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (5-cyanopyridin-3-yl) methyl ] oxy } phenyl ] methyl } amino) -N- [2- (2, 6-dioxopiperidin-3-yl) -1-oxoindol-4-yl ] butanamide (32 a) the chemical formula of 32 a:

Chemical reaction step

Example 18 was prepared according to the general procedure described in example 2 except that intermediate 24 was used instead of intermediate 8 at Step 7. The yield was 46.7%; analytical HPLC purity was 98.5%.

¹ H NMR(400MHz,Chloroform-d)δ8.84(d,J＝20.0Hz,1H),8.72(d,J＝17.4Hz,1H),8.08(d,J＝10.4Hz,1H),7.72(d,J＝7.9Hz,1H),7.52(dd,J＝7.4,1.9Hz,1H),7.42–7.36(m,2H),7.33(d,J＝7.6Hz,1H),7.31–7.29(m,2H),7.27(d,J＝1.9Hz,1H),7.24–7.20(m,2H),7.13(t,J＝8.4Hz,1H),6.58–6.53(m,2H),5.30(s,1H),5.13–5.06(m,4H),4.34(s,1H),4.02(d,J＝21.0Hz,2H),3.48(s,1H),2.98–2.88(m,2H),2.64(s,1H),2.52(s,1H),2.27(s,2H),2.02–1.91(m,3H),1.75(s,1H).

Example 19: preparation of 4- ({ [4- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (3-cyanophenyl) methyl ] oxy } phenyl ] methyl } amino) -N- [2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindol-4-yl ] butanamide (34 a) the chemical formula of 34 a:

the chemical reaction steps are as follows:

example 19 the procedure for the synthesis of intermediate 33 was prepared according to the general method described for the synthesis of intermediate 24 in example 3, except that m-cyanoborobenzyl was used instead of 5- (chloromethyl) nicotinonitrile at Step 4, and the procedure for the synthesis of final product 34a was prepared according to the general method described in example 2, except that Step 7 was used intermediate 33 instead of intermediate 8. The yield was 41.2%; analytical HPLC purity was 98.4%.

¹ H NMR (400 mhz, chloro form-d) delta 7.76 (d, j=8.0 hz, 1H), 7.68 (s, 1H), 7.57 (d, j=7.8 hz, 1H), 7.51-7.46 (m, 2H), 7.41-7.36 (m, 3H), 7.35-7.30 (m, 2H), 7.28 (d, j=1.6 hz, 1H), 7.25-7.18 (m, 3H), 7.15-7.11 (m, 1H), 6.51 (d, j=9.2 hz, 2H), 5.07-5.02 (m, 4H), 4.98 (dd, j=13.1, 5.5hz, 1H), 4.37 (s, 2H), 4.03 (s, 2H), 2.93 (s, 2H), 2.60 (s, 2H), 2.54 (s, 2H), 2.26 (d, 1H), 6.1.25-7.18 (d, 1 hz, 2H), 5.07-5.2H). 3- {4- [ ({ [4- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl group ]Methyl } oxy) -2- { [ (5-cyanopyridin-3-yl) methyl]Oxy } phenyl]Methyl } amino) methyl group]-1,2, 3-triazacyclopent-1-yl } -N- [2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl } -]Propionamide(53a) Is prepared from

53a of the formula:

the chemical reaction steps are as follows:

the synthesis of intermediate 50 was prepared according to the general procedure described in example 3, 25a, except Step7 used propargylamine instead of intermediate 12.

Step10, intermediate 51: 3-bromo-N- [2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl ] propanamide

Intermediate 14 (160 mg,0.62 mmol) was dissolved in tetrahydrofuran (4 ml), 3-bromopropionyl chloride (529.69 mg,3.09 mmol) was slowly added dropwise at room temperature, and after the addition, the temperature was raised to 66℃for 3 hours, 1ml of methanol was added to the reaction solution, and concentrated in vacuo to give a residue. The crude product was purified by beating (petroleum ether/ethyl acetate=10/1) to give 202.9mg of a white solid in 98.2% yield. Step11, intermediate 52: 3-azido-N- [2- (2, 6-dioxopiperidin-3-yl) -1-oxoisoindolin-4-yl ] propanamide

Intermediate 51 (150 mg,0.45 mmol) was dissolved in DMF (4 mL), sodium azide (60 mg,0.92 mmol) was added, the temperature was raised to 50 ℃, after the reaction was completed overnight, the reaction solution was cooled to room temperature, the reaction solution was poured into 20mL of water, the aqueous phase was extracted with ethyl acetate (10 ml×3), the organic phases were combined, washed with water, dried over anhydrous sodium sulfate, suction filtered, evaporated to dryness, and the crude product was purified by beating (volume ratio petroleum ether/ethyl acetate=10/1) to give 130.2mg of white solid in 85.1% yield.

Step12, end product 53a:3- {4- [ ({ [4- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (5-cyanopyridin-3-yl) methyl ] oxy } phenyl ] methyl } amino) methyl ] -1,2, 3-triazacyclopent-1-yl } -N- [2- (2, 6-dioxopiperidin-3-yl) -1-oxyisoindol-4-yl ] propanamide

Intermediate 52 (28.2 mg,0.08 mmol) intermediate 50 (50 mg,0.08 mmol) was dissolved in 4mL of a mixed solution (tetrahydrofuran: water=1:1), copper sulfate pentahydrate (3.08 mg,0.01 mmol), sodium ascorbate (4.8 mg,0.024 mmol) was added, the temperature was raised to 50 ℃ and the reaction was allowed to stand overnight, after the reaction was completed, the reaction solution was poured into 20mL of water, the aqueous phase was extracted with ethyl acetate (10 ml×3), the organic phases were combined, washed with water, dried over anhydrous sodium sulfate, suction filtered, evaporated to dryness, and the crude product was purified by silica gel chromatography (eluting with dichloromethane/methanol=10/1) to give 34.12mg of a white solid in 49.6% yield. The yield was 24.2%; analytical HPLC purity was 99.0%.

¹ H NMR(400MHz,Chloroform-d)δ9.30(s,1H),8.88(d,J＝19.6Hz,2H),8.17(s,1H),7.96(s,1H),7.74(s,1H),7.58(t,J＝8.5Hz,2H),7.47–7.31(m,4H),7.17(d,J＝8.9Hz,2H),6.71–6.52(m,2H),5.18(m,4H),4.74(s,1H),4.30(q,J＝18.8,18.2Hz,2H),3.90(m,3H),3.40(s,1H),3.12(s,2H),2.76(s,2H),2.61(s,2H),2.08(s,2H).

Example 21: preparation of 6- {4- [ ({ [4- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (5-cyanopyridin-3-yl) methyl ] oxy } phenyl ] methyl } amino) methyl ] -1,2, 3-triazapentan-1-yl } -N- [2- (2, 6-dioxopiperidin-3-yl) -1-oxyidenei-ndolin-4-yl ] hexanamide (53 b)

53b of the formula:

the chemical reaction steps are as follows:

example 21 was prepared according to the general procedure described in example 20 except that Step8 was used instead of Step10 in the synthesis of intermediate 54. Step8 was performed according to the procedure for the preparation of synthetic intermediate 16 in example 2, except that 6-bromohexanoic acid was used instead of Boc-4-aminobutyric acid.

The yield was 15.7%; analytical HPLC purity 95.3%.

¹ H NMR(400MHz,Methanol-d ₄ )δ8.88(dd,J＝6.3,2.0Hz,2H),8.30(t,J＝2.1Hz,1H),7.95(s,1H),7.67–7.57(m,4H),7.48–7.42(m,3H),7.40(d,J＝7.6Hz,1H),7.33(ddd,J＝7.5,3.7,1.9Hz,2H),7.30–7.24(m,3H),7.19(ddd,J＝9.7,8.3,1.1Hz,1H),6.80(d,J＝2.3Hz,1H),6.69(dd,J＝8.3,2.3Hz,1H),5.25(d,J＝16.8Hz,4H),4.44(d,J＝7.4Hz,5H),4.08(s,2H),3.98(s,2H),2.43(t,J＝7.1Hz,3H),2.24–2.12(m,2H),2.07–2.00(m,1H),1.95(p,J＝6.9Hz,3H),1.72(p,J＝7.1Hz,3H).

Example 22: preparation of 2- (4- { [4- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (5-cyanopyridin-3-yl) methyl ] oxy } phenyl ] methyl } piperazin-1-yl) -N- [2- (2, 6-dioxopiperidin-3-yl) -1-oxoidenei-ndolin-4-yl ] acetamide (53 c) the chemical formula of 53 c:

the chemical reaction steps are as follows:

step10, intermediate 56: prepared according to the general procedure described for intermediate 51 of example 20 except that chloroacetyl chloride was used instead of bromopropionyl chloride.

Step13, intermediate 57:4- (2- { [2- (2, 6-Dioxypiperidin-3-yl) -1-oxoindol-4-yl ] amino } -2-oxoethyl) piperazine-1-carboxylic acid 2-methylpropan-2-yl ester

Intermediate 51 (30 mg,0.09 mmol) was dissolved in DMF (2 mL) and mono-Boc piperazine (36 mg,0.18 mmol), DIPEA (58 mg,0.45 mmol), naI (27 mg,0.18 mmol) was added after the reaction was completed overnight, the reaction mixture was cooled to room temperature, the reaction mixture was poured into 20mL of water, the aqueous phase was extracted with ethyl acetate (10 mL. Times.3), the organic phases were combined, washed with water, dried over anhydrous sodium sulfate, suction filtered and evaporated to dryness to give 36.1mg of colorless oily liquid in 99.9% yield.

End product 53c was prepared according to the general procedure described in example 3. The yield was 33.5%; analytical HPLC purity 96.8%.

¹ H NMR(400MHz,Chloroform-d)δ9.25(s,2H),8.99(d,J＝2.1Hz,1H),8.91(d,J＝2.0Hz,1H),8.13(t,J＝2.1Hz,1H),7.86(d,J＝7.9Hz,1H),7.73(d,J＝7.5Hz,1H),7.62(dd,J＝7.6,1.9Hz,1H),7.51(t,J＝7.8Hz,1H),7.44(td,J＝7.7,1.9Hz,1H),7.39(t,J＝7.6Hz,1H),7.36–7.29(m,3H),7.26(td,J＝7.4,1.2Hz,1H),7.19(ddd,J＝9.6,8.2,1.1Hz,1H),6.68(dd,J＝8.3,2.4Hz,1H),6.62(d,J＝2.3Hz,

1H),5.33–5.11(m,5H),4.56–4.37(m,2H),3.58(s,2H),3.22(d,J＝2.3Hz,2H),2.97–2.80(m,2H),2.72(bs,4H),2.60(bs,4H),2.49–2.30(m,2H),2.29–1.95(m,5H).

Example 23: preparation of 2- {4- [2- ({ [4- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (5-cyanopyridin-3-yl) methyl ] oxy } phenyl ] methyl } amino) ethyl ] piperazin-1-yl } -N- [2- (2, 6-dioxopiperidin-3-yl) -1-oxyidenei-ndolin-4-yl ] acetamide (53 d)

53d of the formula:

example 23 was prepared according to the general procedure described in example 22 except that 1- (N-Boc-aminoethyl) piperazine was used instead of mono-Boc piperazine in Step 13. The yield was 22.9%; analytical HPLC purity was 98.8%.

¹ H NMR(400MHz,Chloroform-d)δ9.18(s,1H),8.84(t,J＝2.1Hz,2H),8.65(s,1H),8.06(d,J＝2.2Hz,1H),7.77(dd,J＝44.5,7.9Hz,1H),7.58(dd,J＝7.5,1.8Hz,1H),7.48(t,J＝7.7Hz,1H),7.44–7.27(m,6H),7.26–6.96(m,3H),6.60(dd,J＝8.4,2.4Hz,1H),6.53(d,J＝2.3Hz,1H),5.14(m,5H),4.50–4.32(m,1H),3.65(s,2H),3.13(s,1H),2.94–2.74(m,2H),2.72–2.30(m,8H),2.27–2.15(m,1H),2.14–1.69(m,7H).

Example 24: preparation of (2S, 4R) -1- [ (7R) -1- [4- ({ [ 2-chloro-3- (2-fluorophenyl) phenyl ] methyl } oxy) -2- { [ (5-cyanopyridin-3-yl) methyl ] oxy } phenyl ] -7- (2-methylpropan-2-yl) -5, 8-dioxan-2, 6-diazaoct-8-yl ] -4-hydroxy-N- [ (1S) -1- [4- (4-methyl-1, 3-thiazapentan-5-yl) phenyl ] ethyl ] tetrahydropyran-2-carboxamide (61 a)

61a of the formula:

the chemical reaction steps are as follows:

example 24 was prepared according to the general procedure described in example 2 except that E3 ligand VHL was used in Step8 instead of intermediate 14, intermediate Boc-3-aminopropionic acid instead of Boc-4-aminobutyric acid. The yield was 38.9%; analytical HPLC purity 96.7%.

¹ H NMR(400MHz,Chloroform-d)δ9.01(d,J＝77.1Hz,3H),8.28(s,1H),7.56(d,J＝8.3Hz,1H),7.48–7.29(m,10H),7.26–7.14(m,3H),6.72–6.60(m,2H),5.24(s,4H),5.10(s,1H),4.68(s,1H),4.51(s,1H),4.29-4.16(m,7H),3.65(s,1H),3.20(s,2H),2.74(s,2H),2.58(s,3H),2.38(s,1H),2.20(s,1H),1.49(s 3H),1.03(s,9H).

Compound biological activity investigation:

ability of compounds to degrade PD-L1 proteins

The degradation condition of the compound on PD-L1 in the whole cell lysate is evaluated by using a Western Blot experiment, and the degradation of the compound on PD-L1 is specifically performed as follows:

(1) And (3) paving: tumor cells in the logarithmic growth phase and at a confluence of 80% -90% were collected, resuspended and counted at a cell number of 3X 10 per well ⁵ Inoculating in a 6-well plate;

(2) Compound incubation: when the cells grow to 70%, the original medium is removed. Compounds were added to the wells at the indicated concentrations, control groups were added the same volumes of DMSO and incubated for 24h.

(3) Extraction of protein: whole cell lysates were obtained using NP-40 lysates.

(4) Protein quantification and sample preparation: protein sample concentrations were detected using BCA protein concentration assay kit. A40. Mu.L sample was prepared, and after mixing well, it was heated in a metal bath at 100deg.C for 10min, and then kept at 4deg.C for further use.

(5) Gel electrophoresis: SDS-PAGE gel was prepared and an electrophoresis apparatus was set up, the electrophoresis voltage was 80V, when bromophenol blue was observed to enter the separation gel, the voltage was adjusted to 110V, and when Marker was observed to be sufficiently wide, electrophoresis was stopped.

(6) Transferring: cutting gel, reserving needed holes, cutting proper PVDF film, activating with methanol, and washing with 1X film-transferring buffer for later use. Taking out the gel, and preparing a sandwich structure according to the sponge-filter paper-gel-PVDF membrane-filter paper-sponge. And (5) assembling film transferring equipment, carrying out ice water bath at 80V, and transferring for 90min.

(7) Blocking and incubation of primary antibody: the PVDF membrane was removed with the protein facing down and the blocking solution was added. The shaker was closed at room temperature for 2h. Then primary antibody (1:1000) was added and the mixture was shaken overnight at 4 ℃.

(8) Incubating a secondary antibody: after the incubation, the incubation was washed with 1 XTBST buffer. Subsequently, secondary antibodies were added and incubated for 2h in a shaker at room temperature. Washing with 1 XTBST buffer is still required after incubation.

(9) Exposure: the developer was developed and the results were recorded using a gel imager.

The performance test is carried out on the compound of the invention, the Western Blot experiment is used for determining the change condition of the PD-L1 expression level in the whole cell lysate of HCC-827 in the presence of PROTAC molecules, and the effect is shown in figure 1 (A). In addition, compounds 25i and 25j also induced degradation of PD-L1 protein in tumor cells SK-N-AS, SY5Y, 786-O and MC38, and the experimental effect is shown in FIG. 1 (B). The results of the quantitative graphs of the degradation activity of compounds 25i and 25j on HCC-827 are shown in FIG. 1 (C).

Based on Western Blot immunoblot analysis, as shown in FIG. 1 (A), two representative compounds 25i (20. Mu.M, 60.87%) and 25j (10. Mu.M, 58.05%) were screened, which were effective in inducing PD-L1 degradation of non-small cell lung cancer HCC-827 cells and exhibited dose dependence. In addition, tumor cells with different PD-L1 expression levels (human SK-N-AS, SY5Y and 786-O which moderately express PD-L1 and murine MC38 which highly express PD-L1) are selected, and the degradation conditions of the active compounds 25i and 25j are continuously examined. As shown in fig. 1 (B), the compounds 25i and 25j also have significant degradation activity against different cells, and the degradation activity thereof has a certain broad spectrum.

Degradation of intracellular and membrane proteins by Compounds 25i and 25j

The PD-L1 on the HCC-827 membrane is surface-labeled by using a flow cytometer to obtain the influence of two compounds 25i and 25j on the protein on the membrane, and the specific method is as follows:

(1) And (3) paving: cells were collected by digestion, counted and seeded in 6-well plates with 2.5X10 cells per well ⁵ And each.

(2) Compound incubation: when the cells grow to 70%, the original medium is removed. Compounds were added to the wells at the indicated concentrations, control groups were added the same volumes of DMSO and incubated for 24h.

(3) Collecting a sample: after the incubation time of the compounds was completed, cells were collected by pancreatin digestion and placed in a centrifuge tube labeled in advance, then gently rinsed 2-3 times with PBS, and centrifuged to discard the supernatant.

(4) Dyeing: fluorescent antibody was added, incubated for 20min in the dark, washed twice with 3ml PBS, and finally, cells in each centrifuge tube were lightly resuspended in 200 μl PBS and transferred to flow tubes.

(5) And (5) detecting on the machine.

The results of compounds 25i and 25j inducing PD-L1 degradation on the cell membrane and cytoplasm of HCC-827 are shown in FIG. 2. As shown in fig. 2 (a), the flow surface labeling results showed that the compound treated group showed a significant decrease compared to the control group, i.e., the compound degraded the protein on the membrane. In addition, cytoplasmic and membrane proteins were extracted using ProteinExt Mammalian membrane protein extraction kit, respectively, and Western Blot experiments were performed on the obtained samples. As shown in FIG. 2 (B), compounds 25i and 25j were effective in inducing degradation of PD-L1 proteins on and in the cell membrane of HCC-827. This is consistent with flow results, demonstrating that compounds 25i and 25j can effectively degrade the levels of PD-L1 on and in the membrane.

Compounds 25i and 25j activate an in vitro immune pathway

The degradation of PD-L1 can interrupt the connection between PD-1 and PD-L1, so that T cells are activated, an immune response is generated, and the effect of killing tumor cells is achieved through an immune pathway. To evaluate the ability of compounds 25i and 25j to degrade PD-L1 and restore immune responses, a Jurkat cell and HCC-827 co-culture model was constructed, reflecting the degree of immune activation by the killing effect of Jurkat on HCC-827 cells. The specific method comprises the following steps:

(1) Determination of co-culture concentration:

the co-culture concentration without cytotoxicity was determined by comprehensive cytotoxicity detection on HCC-827 and Jurkat.

(2) Co-cultivation:

a. and (3) paving: the cells were collected by digestion, counted and seeded in 96-well plates with 2X 103 cells per well.

Jurkat activation: the cells were collected and counted, the required amount of cells was separated according to a ratio of tumor cells to T cells of 1:10, inoculated into a petri dish, and activated with PMA for 2 hours.

c. Adding the medicine: after HCC-827 had adhered, the original medium was aspirated, jurkat which had been activated was added, and the test compound, 1% DMSO, was added, and then the well plate was placed in an incubator for further culture for 24 hours.

d. CCK-8 was added and absorbance was measured.

As shown in FIG. 3, the concentration of the co-culture system was determined on the basis of the obtained cytotoxicity of 25i and 25j with HCC-827 and T cells alone, on the one hand, the effect of the compound's own toxicity was excluded, and on the other hand, it was ensured that at higher concentrations, a significant immune activation was exhibited. In the co-culture system, the compound can indirectly promote the anti-tumor immune to kill tumor cells, the survival rate of the co-culture system is 50.65% at the concentration of 25i of 10 mu M, and the survival rate of the co-culture system is 73.22% due to the strong toxicity to Jurkat of 25j, wherein the concentration of the co-culture system is 5 mu M. The results show that 25i and 25j can activate Jurkat, play a role in killing tumor cells and have concentration dependence in an in vitro model.

Compounds 25i and 25j trigger proteasome and lysosome dual pathway synergistic degradation

First, to verify that degradation of PD-L1 is mediated by the PROTAC molecule, the effect of a56 and CRBN ligands on PD-L1 expression was experimentally verified, and the results of the mechanism study of compounds 25i and 25j to target degradation of PD-L1 are shown in fig. 4. As shown in fig. 4 (a), ligand alone treatment did not affect PD-L1 expression, co-treatment group PD-L1 degradation was hindered, indicating that simultaneous binding of both ligands to their receptors was necessary for efficient degradation of PD-L1 protein, i.e. indicating the necessity of ternary complex formation.

To further verify the pathway by which PD-L1 degradation occurs, we used protease inhibitor MG132 and lysosomal inhibitor Bafilomycin pretreatment, as shown in fig. 4 (B) and 4 (C), after which the active compound was significantly hindered from PD-L1 protein degradation. The results indicate that the degradation of PD-L1 by active compounds 25i and 25j is synergistically accomplished by the ubiquitin-proteasome pathway and the lysosomal pathway.

In vivo antitumor Activity of Compounds 25i and 25j

To evaluate the tumor-inhibiting ability of compounds 25i and 25j in mice, first, a C57BL/6 mouse MC38 colon cancer model (n=6) was constructed. Growth after tumor implantation reaches 50mm ³ After that, the cells were randomly grouped and administered by intraperitoneal injection (day 1), followed by 15 days of continuous administration, with cyclophosphamide as a positive control. As shown in FIG. 5, after 15 days of treatment, the average tumor volume growth inhibition (TGI) was 25i 48.72% (10 mg/kg group) and 57.35% (20 mg/kg group), and 25j 34.61% (10 mg/kg group) and 54.92% (20 mg/kg group), respectively.

The results of safety evaluation of the compounds 25i and 25j are shown in fig. 6, and according to the blood sample (fig. 6 (B)), the HE staining of the viscera (fig. 6 (a)) and the comparative analysis of the weight between groups, no significant difference appears, which indicates that the compounds have no significant toxicity to experimental animals and have a certain safety. In vivo immunomodulatory effects of Compounds 25i and 25j

Immunohistochemistry and Western Blotting experiments are adopted to evaluate the influence of the compound on the PD-L1 protein of the tumor tissue; qPCR and flow methods were used to assess chemokine mRNA expression levels and density of infiltrating lymphocytes. Various angles demonstrate the effect of a compound on immune activation in vivo. The specific method comprises the following steps:

flow cytometry:

(1) Preparation of tumor tissue single cell suspension: a portion of fresh tumor tissue was taken, washed with PBS and the tumor was minced with scissors. Centrifugation and removal of supernatant, collagenase IV (2 mg/ml) addition, digestion in a 37℃water bath for 60min and termination of digestion with complete medium. Preparation of a lymphocyte suspension: fresh lymph node tissue was taken, mildly ground, and cells were released.

(2) Sieving: sieving with 200 mesh sieve, centrifuging the filtrate, and washing with PBS 2 times.

(3) Count, reserve 1×10 ⁶ Individual cells.

(4) Dyeing: mu.LFITC-CD 3, 2. Mu.LPE-CD 4, 2. Mu.LAPC-CD 8 were added. After gentle mixing, the mixture was reacted at 4℃for 30min in the dark. (5) washing and loading: 3mL PBS was washed 2 times, centrifuged, the supernatant discarded, and finally transferred to a flow tube using 200 μLPBS and put on-line.

Fluorescent quantitative PCR

(1) Extracting total RNA: part of tumor tissue was taken, added to Trizon, homogenized using a tissue disrupter. Chloroform is added, the mixture is fully and evenly shaken, and centrifuged, and the transparent clear liquid (three layers are all arranged from top to bottom: transparent-milky-pink) on the upper layer is sucked. Adding equal volume of isopropanol, turning upside down, standing, centrifuging to obtain trace RNA precipitate, 75% ethanol (DEPC water configuration), and washing the precipitate. Centrifuging to remove the supernatant, inverting to absorb water, drying to obtain an RNA sample, and adding a proper amount of DEPC water to dissolve RNA precipitate.

(2) Detecting the concentration and purity of RNA: and (3) detecting and evaluating indexes by using an enzyme-labeled instrument: 1.8< OD260/OD280<2.2.

(3) Reverse transcription: a reverse transcription solution system was prepared and added to a 200. Mu. LEP tube, followed by placing in a PCR instrument. Reverse transcription conditions: 42 ℃ (15 min), 95 ℃ (3 min), 4 ℃ (5 min).

(4) qPCR: preparing a corresponding solution system, comprising: 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, 5. Mu.L of SYBR Green, 3. Mu.L of LDEPC water. The cDNA obtained by reverse transcription was diluted 10-fold, and 1. Mu.L was collected therefrom. Sequentially adding the mixture to a qPCR plate, sealing a membrane and centrifuging.

(5) And (5) running the detection, and processing the data by Prism software.

Immunohistochemistry and Westernblotting analysis of tumor tissue showed that treatment with compounds 25i and 25j resulted in significant downregulation of PD-L1 protein, consistent with cell-level results (fig. 7). To further investigate the effect of compounds on the regulation of the immune system in vivo, mRNA expression levels of chemokines CXCR3, CXCL9, CXCL10 were first assessed, which levels were positively correlated with activation of immunity. As shown in fig. 7B, compound-treated mice exhibited significantly increased mRNA levels of CXCR3, CXCL9, and CXCL10 in tumor tissues, and the intensity of change was also positively correlated with tumor suppression rate, compared to the control group. In addition, the analysis of tumor-infiltrating lymphocytes of tumor tissues and lymph nodes by using flow cytometry shows that the compound group is CD8 compared with the control group ⁺ /CD4 ⁺ The significant increase, which indicates that the compounds significantly activated the immune system after action (fig. 8), and at the same time, the expression levels of granorubicin B and perforin, which are key mediators of cd8+ T cell cytotoxicity, were also found to be significantly up-regulated by immunohistochemistry (fig. 7).

Taken together, the results show that in vitro, the active compounds 25i and 25j degrade PD-L1 through proteasome and lysosome dual-pathway complex, cut off the connection between PD-1 and PD-L1, and significantly activate the immune response in Jurkat/HCC-827 co-culture model. In addition, in vitro, the active compounds 25i and 25j can effectively reduce the PD-L1 level of a mouse tumor model, induce the autoimmunity of the mouse and play a role in inhibiting the growth of the tumor.

In summary, compounds 25i and 25j exhibit good anti-tumor activity in vitro and in vivo, providing valuable insight for the successful introduction of the PROTAC technology into the development of immune checkpoint inhibitors, indicating that they can be used as a potential cancer treatment method, bringing new opportunities for tumor immunotherapy.

Claims (5)

1. The proteolytic targeting chimera of the target degradable PD-L1 is PROTAC molecule with the structural general expression as shown in the formula I:

the dibenzyl ether in formula I represents a ligand moiety that binds to a PD-L1 protein;

in formula I, E3 ligand represents an E3 ubiquitin ligase ligand; linker is a Linker, i.e. a Linker, connecting the PD-L1 protein-bound ligand and the E3 ubiquitin ligase ligand;

wherein: r is R ₁ Hydrogen or halogen substituted in the 2, 3 or 4 position of the benzene ring; r is R ₂ Hydrogen, halogen or methyl substituted in the 2, 4, 5 or 6 position of the benzene ring; r is R ₃ Is hydrogen,

Linker includes: one of saturated fatty chain, unsaturated fatty chain, polyethylene glycol chain, nitrogen-containing five-membered/six-membered heterocyclic ring, substituted benzene ring and amino acid condensed short polypeptide chain;

e3 ligand has any one of the structures shown in the following formulas or an isomer form thereof:

2. the proteolytic targeting chimera for targeted degradation of PD-L1 according to claim 1, wherein the Linker has the structure according to the formula:

Wherein Z is ₀ 、Z ₁ 、Z ₂ Each independently selected fromAny one of the groups, m0, m1, m2, m3, m4, m5, m6 are each independently selected from any one of integers from 0 to 15;

or the Linker has one of the structures shown in the following formula:

wherein Cx is selected from a 3-8 membered heterocycle, a 3-8 membered cycloalkyl, a 6-8 membered aryl, or a single bond, said heterocycle containing 1-4 heteroatoms, the heteroatoms being selected from at least one of O, S, N; cy is selected from a 3-8 membered heterocycle, a 3-8 membered cycloalkyl, a 6-8 membered aryl or a single bond, said heterocycle containing 1-4 heteroatoms, the heteroatoms being selected from at least one of O, S, N; z is-CH ₂ -NH or-O; m is an integer between 1 and 6, and n is an integer between 0 and 6.

3. The proteolytic targeting chimera for the targeted degradation of PD-L1 according to claim 1, or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture of several thereof, or a pharmaceutically acceptable salt thereof, characterized in that: representative structural formulas of the proteolytic targeting chimeras are selected from one of the following:

4. the use of a proteolytic targeting chimera for targeted degradation of PD-L1 according to claim 1 for the preparation of antitumor drugs.

5. The use according to claim 4, wherein the proteolytically targeted chimera is used for the preparation of an antitumor drug targeting PD-L1 for the treatment of non-small cell lung cancer or breast cancer.