CN114573563B - Bifunctional molecular compound for inducing PD-L1 protein degradation and preparation and application thereof - Google Patents

Abstract

The invention relates to a bifunctional molecular compound for inducing PD-L1 protein degradation, and preparation and application thereof, belonging to the technical field of chemical biology. The compound takes PD-L1 as a target protein binding ligand, can target and degrade PD-L1 protein, can be used as a PD-L1 protein targeting degradation agent, and can provide a very potential treatment scheme for PD-L1 mediated infection, tumor, autoimmune diseases and/or other diseases through research. The structural formula (I) and the B and L in each structural formula are described in the specification.

Description

Bifunctional molecular compound for inducing PD-L1 protein degradation and preparation and application thereof

Technical Field

The invention belongs to the technical field of chemical biology, and particularly relates to a bifunctional molecular compound for inducing PD-L1 protein degradation, and preparation and application thereof. The compound can degrade PD-L1 in A375 and B16F 10 cell lines and can increase the effect of T cells in killing tumors.

Background

The apoptosis protein 1 ligand 1 (PD-L1, B7-H1, CD 274) is a member of the B7 family of cell surface ligands that regulate T cell activation and immune responses. PD-L1 is similar in structure to a B7 family member and comprises extracellular IgV and IgC domains and a short cytoplasmic domain. The PD-L1 ligand binds to the PD-L1 transmembrane receptor and inhibits T cell activation. Studies have shown that PD-L1 is expressed in antigen presenting cells, activated T cells, placenta, heart and lung tissues. In addition, PD-L1 is expressed in cells of many tumor types such as melanoma, ovarian cancer, colon cancer, lung cancer, breast cancer, and renal cell carcinoma. Expression of PD-L1 in cancer cells is associated with tumor-infiltrating lymphocytes, and PD-L1 expression can be mediated by release of interferon gamma. Other studies have shown that PD-L1 expression is associated with cancer associated with viral infection.

The concept of protein targeting chimera gradually enters the field of drug development, and the protein targeting chimera utilizes a ubiquitination mechanism to degrade target proteins so as to inhibit the target proteins. Traditional small molecules, antibodies and the like have the effect of treating diseases by inhibiting the function of target proteins through an 'occupation driving' action mode, and the action mode needs high concentration of the inhibitor or the monoclonal antibody to occupy the active site of a target point so as to block the transduction of a downstream signal path. PROTAC, in turn, acts through an "event-driven" mechanism, which does not affect protein function, mediating degradation of the target protein.

At present, the medicines used for tumor immunotherapy mainly comprise monoclonal antibodies, and the curative effect of the PD-L1 small molecule inhibitor is not ideal. The effect of the small molecule inhibitor on the poor action of PD-L1 can be well solved by utilizing PROTAC technology, so that the design of the PD-L1 related small molecule degradation agent can obviously improve the clinical immunotherapy effect.

Disclosure of Invention

The invention aims to complement the prior art and provides a bifunctional molecule compound for inducing PD-L1 protein degradation, and preparation and application thereof, in particular to a non-natural PD-L1 degradation agent synthesized based on biphenyl compounds and application thereof in tumor immunotherapy. By degrading intracellular PD-L1, the direct killing effect of T cells on tumor cells is enhanced. Researches show that PROTAC molecules taking biphenyl compound BMS-37 as PD-L1 ligand have certain degradation effect on PD-L1 no matter CRBN or VHL is selected as E3 ligase ligand. The compound synthesized in the research can be combined with two E3 ligands to degrade PD-L1, and the phenomenon can be reversed by a proteasome inhibitor MG132, which proves that the compound acts through an ubiquitin proteasome pathway. In addition, the research results show that the compounds can obviously increase the killing effect of T cells. Therefore, the PD-L1 protein degradation agent based on the biphenyl compound is designed to provide a new treatment strategy for tumor immunotherapy.

The specific scheme of the invention is as follows:

the invention provides five bifunctional molecule compounds shown in the following formulas I, II, III, IV and V for inducing PD-L1 protein degradation, or pharmaceutically acceptable salts, hydrates or prodrugs thereof;

Wherein B is ubiquitin ligase E3 ligand, more preferably one of CRBN, VHL, MDM, cIAP, UBR7, RNF114, CBLB and KEAP 1.

Wherein, the CRBN is preferably one of the following structural formulas:

Wherein:

W is selected from one of CH ₂、C＝O、SO₂, NH and N-alkyl; alkyl is preferably C1-C4 alkyl;

x is selected from one or two of O, S;

z is selected from one of hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl and halogen;

G. G' each independently represents H, C-C4 alkyl, -OH, C1-C4 alkyl substituted 5-10 membered heterocyclyl containing 1-3 heteroatoms N, O or S;

R ¹ is selected from H, D, halogen, nitro, amino, cyano, hydroxy, C1-C4 alkyl, halogenated C1-C4 alkyl, deuterated C1-C4 alkyl.

The structural formula of the VHL is as follows:

Wherein:

R ² is selected from one of CH ₃ and H.

The structural formula of the MDM2 is as follows:

Wherein:

r ³ is piperazinyl, piperidinyl, a heterocyclic group, or one of the linking groups of the following structure:

in the linking group, n is an integer of 0 to 3.

Wherein:

The heterocyclic group is one of piperazinonyl, pyrrolyl, pyrazolyl, furyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl.

The structure of the cIAP is as follows:

r ⁴ is H or Boc.

The L is preferably one of the following structures:

wherein: m is selected from integers between 1 and 10.

The present invention is preferably a bifunctional compound of formula VI and formula VII below that induces the degradation of PD-L protein, or a pharmaceutically acceptable salt, hydrate or prodrug thereof:

Of the bifunctional compounds of the above formulae VI and VII which induce the degradation of PD-L1 protein, any of the following structures is more preferred:

m is selected from integers between 1 and 10.

More preferred in the present invention are bifunctional compounds of formula VIII below that induce degradation of PD-L1 protein, or pharmaceutically acceptable salts, hydrates or prodrugs thereof:

in the bifunctional compound shown in the formula VIII for inducing the degradation of PD-L1, L is preferably the following structure:

m is selected from integers between 1 and 5.

Preferred bifunctional compounds of the present invention that induce degradation of PD-L1 proteins, represented by the following structural formula, include, but are not limited to:

According to the invention, pharmaceutically acceptable salts of bifunctional compounds that induce the degradation of PD-L1 protein include addition salts formed with: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, and the like. Hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, maleic acid, benzenesulfonic acid, succinic acid and similar known acceptable acids.

In addition, prodrugs of the derivatives of the invention are also encompassed by the invention. They may themselves have a weaker activity or even no activity, but are converted to the corresponding biologically active form after administration under physiological conditions (e.g. by metabolism, solvolysis or otherwise).

The invention also provides a preparation method of the bifunctional molecule compound for inducing PD-L1 protein degradation, which comprises the following steps:

The synthetic route of the PD-L1 protein degradation agent shown in the formula I is as follows:

in this synthetic route, L is M is selected from integers between 1 and 7;

Step 1-1: synthesis of Compound 3

4-Hydroxy-2, 6-dimethoxybenzaldehyde (910 mg,5 mmol), triphenylphosphine (1.44 g,5.5 mmol) and (2-methyl- [1,1' -biphenyl ] -3-yl) methanol (990 mg,5 mmol) were weighed into anhydrous THF, a solution of diisopropyl azodicarboxylate (1.11 g,5.5 mmol) in anhydrous THF was added dropwise to the above reaction system under ice bath conditions, the reaction was continued overnight at room temperature, the reaction mixture was poured into H ₂ O, the mixture was then extracted with DCM, the organic phase was washed with brine, dried over anhydrous sodium sulfate, concentrated in vacuo to give a residue, which was purified by silica gel chromatography, gradient eluted with ethyl acetate/petroleum ether to give compound 3;

Step 1-2: synthesis of Compound 6

4-Pentanoic acid (200 mg,2 mmol) was dissolved with DCM, then N-Boc-ethylenediamine (320 mg,2 mmol), EDCI (400 mg,2.1 mmol), HOBt (270 mg,2 mmol) and DIPEA (0.4 mL) were added to a round bottom flask, the mixture was stirred at room temperature overnight, the reaction system was diluted with DCM, the organic phase was washed twice with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, the mixture was concentrated under reduced pressure, the product was purified by silica gel chromatography and then deprotected with trifluoroacetic acid to give compound 6;

step 1-3: synthesis of Compound 7

Compound 3 (3.4 mmol,1.23 g) and compound 6 (5.1 mmol, 710 mg) were sequentially added to a round bottom flask, reacted at room temperature for 2 hours, then sodium triacetoxyborohydride (4.1 mmol,870 mg) and one drop of acetic acid were added, the reaction system was stirred overnight at room temperature, the organic phase was washed twice with a saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and purified by silica gel chromatography to give compound 7;

step 1-4: synthesis of Compounds of formula I

Sequentially adding the compound 7 (0.035 mmol,17 mg), B-linker, cuSO ₄ (0.044 mmol,7 mg) and VcNa (0.105 mmol,21 mg) into a mixed solution of THF and water (1:1), stirring at room temperature for 2 hours, concentrating to remove tetrahydrofuran, adding water, extracting with dichloromethane for 3 times, drying an organic phase with anhydrous sodium sulfate, concentrating to obtain a crude product, and purifying by silica gel column chromatography to obtain the compound of the formula I;

(II) the synthetic route of the PD-L1 protein degradation agent shown in the formula II and the formula III is as follows:

in this synthetic route, L is M is selected from integers between 1 and 7;

step 2-1: synthesis of compound 9;

5-chloro-2, 4-dihydroxybenzaldehyde (863 mg,5 mmol), triphenylphosphine (1.44 g,5.5 mmol) and (2-methyl- [1,1' -biphenyl ] -3-yl) methanol (990 mg,5 mmol) were weighed into anhydrous THF, a solution of diisopropyl azodicarboxylate (1.11 g,5.5 mmol) in anhydrous THF was added dropwise to the above reaction system under ice bath conditions, the reaction was continued overnight at room temperature, the reaction mixture was poured into H ₂ O, the mixture was then extracted with DCM, the organic phase was washed with brine, dried over anhydrous sodium sulfate, concentrated in vacuo to give a residue, which was purified by silica gel chromatography, gradient elution was performed with ethyl acetate/petroleum ether to give compound 3;

Step 2-2: synthesis of Compound 11

Compound 9 (0.21 mmol,74 mg) was dissolved in DMF, then 3- (bromomethyl) benzonitrile (0.25 mmol,49 mg) was added sequentially, reacted at 80 ℃ for 30min, water was added to the reaction system, extracted with ethyl acetate, the organic phase was washed 3 times with saturated brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure to give crude product, which was purified with tert-butyl methyl ether, filtered to give compound 11;

Step 2-3: synthesis of Compound 13

A mixture of compound 11 (0.13 mmol,63 mg), 2-amino-2-methylpropanoic acid (0.54 mmol,56 mg), acOH (2 d) and NaBH ₃ CN (0.67 mmol,42 mg) in 5mL DMF was reacted at 80℃for 1h, ethyl acetate and water were added to the reaction mixture, the organic extract was concentrated, the crude product was chromatographed on silica gel eluting with methanol and dichloromethane solution to give compound 13;

step 2-4: synthesis of Compound 13y

Compound 13 (0.02 mmol,13 mg) was dissolved in DMF and propargylamine (0.03 mmol, 2. Mu.L), HATU (0.03 mmol,11 mg) and DIPEA (0.04 mmol, 6. Mu.L) were reacted at room temperature for 2 hours to give the product by silica gel chromatography of the petroleum ether ethyl acetate system to give compound 13y;

Step 2-5: synthesis of Compounds of formula II

Sequentially adding the compound 13y (0.035 mmol,17 mg), B-linker, cuSO ₄ (0.044 mmol,7 mg) and VcNa (0.105 mmol,21 mg) into a mixed solution of THF and water (1:1), stirring at room temperature for 2 hours, concentrating to remove tetrahydrofuran, adding water, extracting with dichloromethane for 3 times, drying an organic phase with anhydrous sodium sulfate, concentrating to obtain a crude product, and purifying by silica gel column chromatography to obtain a compound of formula II;

step 2-6: synthesis of Compound of formula III

Compound 13 (0.04 mmol,22 mg), EDCI (0.08 mmol,15 mg), HOBt (0.08 mmol,11 mg), DIPEA (0.16 mmol, 28. Mu.L) and B-linker were dissolved in DCM and stirred at room temperature for 2h, the reaction mixture was extracted with water and DCM, the organic layer was washed 3 times with water, dried over anhydrous sodium sulfate, concentrated by distillation under reduced pressure to give a residue, which was purified by silica gel chromatography to give formula III;

(III) the synthetic route of the PD-L1 protein degradation agent shown in the formula IV is as follows:

in this synthetic route, L is M is selected from integers between 1 and 7;

step 3-1: synthesis of Compound 16

To a solution of 2-methylbiphenyl-3-methanol (1.45 g,7.31 mmol) in dichloromethane at room temperature was added in portions dess-martin reagent (3.26 g,7.68 mmol), the resulting mixture was stirred at room temperature for 30min, then quenched with sodium bicarbonate solution and sodium sulfate solution, the mixture was extracted with dichloromethane, the combined extracts were dried over anhydrous sodium sulfate and concentrated, and the residue was purified by column chromatography to give compound 16;

step 3-2: synthesis of Compound 18y

Methyl 3-amino-4-hydroxybenzoate (49 mg,0.29 mmol), compound 16 (69 mg,0.35 mmol) and zinc triflate (10 mg,0.03 mmol) were dissolved in ethanol (1.5 mL) and refluxed overnight, after which the reaction mixture was cooled to room temperature, then concentrated, the residue was dissolved in dichloromethane, then dichlorodicyanobenzoquinone (100 mg,0.6 mmol) was added, the mixture was stirred at room temperature for 0.5 hours, then diluted with ethyl acetate and washed with sodium bicarbonate solution, sodium sulfate solution, water and brine, the organic layer was dried over anhydrous sodium sulfate and concentrated, and the residue was purified by column chromatography to give compound 18y;

Step 3-3: synthesis of Compound 18

Compound 18y was treated under LiOH, methanol/water conditions, then extracted and concentrated by ethyl acetate to give compound 18;

Step 3-4: synthesis of Compound 19, synthesis of Compound 19 and Compound 13y;

Step 3-5: synthesis of compounds of formula IV;

Sequentially adding the compound 19 (0.035 mmol,13 mg), B-linker, cuSO ₄ (0.044 mmol,7 mg) and VcNa (0.105 mmol,21 mg) into a mixed solution of THF and water (1:1), stirring at room temperature for 2 hours, concentrating to remove tetrahydrofuran, adding water, extracting with dichloromethane for 3 times, drying an organic phase with anhydrous sodium sulfate, concentrating to obtain a crude product, and purifying by silica gel column chromatography to obtain the compound of the formula II;

(IV) the synthetic route of the PD-L1 protein degradation agent shown in the formula V is as follows:

in this synthetic route, L is M is selected from integers between 1 and 7;

Step 4-1: synthesis of Compound 22

After 6-chloropyridazin-3-amine (5 mmol, 488 mg) was dissolved in 10mL of dioxane, naHCO ₃ (7.5 mmol,630 mg) and ethyl 3-bromo-2-oxopropionate (5.5 mmol,1.07 g) were sequentially added, the mixture was reacted at 100℃for 3 hours, the reaction system was filtered, the filtrate was diluted with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to give a crude black colloid, and the product was purified by silica gel column chromatography;

Step 4-2: synthesis of Compound 24

A mixture of compound 22 (10.1 mmol,2.5 g), K ₂CO₃ (30.5 mmol,4.2 g), tert-butyl (R) -pyrrolidin-3-ylcarbamate (10.1 mmol,1.9 g) and 40mL DMF was reacted at 110℃for 12 hours, diluted with water, extracted 3 times with ethyl acetate, the organic phase was washed 3 times with saturated brine, dried over anhydrous sodium sulfate, concentrated to give crude product, and purified by column chromatography to give compound 24;

Step 4-3: synthesis of compound 25, synthesis of compound 25 and compound 18;

step 4-4: synthesis of Compound 27

Boc-D-histidine (2 mmol,511 mg) and K ₂CO₃ (6 mmol, 8239 mg) were added to 20mL DMF, then benzyl bromide (2.2 mmol, 261. Mu.L) was added dropwise and reacted at room temperature for 4h, the residue was purified by silica gel flash chromatography to give compound 27;

step 4-5: synthesis of Compound 28, deprotection of Compound 27 with trifluoroacetic acid, boc, gives Compound 28;

Step 4-6: synthesis of Compound 29

2-Methyl- [1,1' -biphenyl ] -3-methanol (1 g,5 mmol) was dissolved in methylene chloride, triethylamine (5.5 mmol,0.7 mL) and methylene chloride solution of triphosgene (600 mg,2 mmol) were added dropwise at-20 ℃, then the reaction was carried out for 1.5 hours at 0 ℃, 30mL of n-hexane was added to the reaction system, stirred for 10 minutes and then filtered, the filter cake was washed 3 times with 10mL of mixed solution of methylene chloride and n-hexane (1:2), and the filtrate was concentrated to give compound 29, which was directly put into the next reaction without further purification because the product was unstable;

Step 4-7: synthesis of Compound 30

Compound 24 (1.86 mmol,650 mg) was dissolved in a mixed solution of THF and water, naHCO ₃ (5.6 mmol,470 mg) was added, a solution of compound 29 in THF (6.5 mL) was added dropwise at 0 ℃, the mixture was transferred to room temperature and stirring was continued for 2 hours, the reaction was quenched with water and extracted 3 times with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, concentrated after filtration, and the residue was purified by silica gel column chromatography to give compound 30;

Step 4-8: synthesis of compound 31;

Compound 28 (0.04 mmol,15 mg), compound 30 (0.04 mmol,19 mg), EDCI (0.08 mmol,15 mg), HOBt (0.08 mmol,11 mg), DIPEA (0.16 mmol, 28. Mu.L) were dissolved in DCM and stirred at room temperature for 2h, the reaction mixture was extracted with water and DCM, the organic layer was washed 3 times with water, dried over anhydrous sodium sulfate, concentrated by distillation under reduced pressure to give a residue which was purified by silica gel chromatography to give Compound 31;

Step 4-9: synthesis of compound 31 y;

Compound 31 (0.04 mmol,32 mg), propynylamine (0.08 mmol,4 mg), EDCI (0.08 mmol,15 mg), HOBt (0.08 mmol,11 mg), DIPEA (0.16 mmol, 28. Mu.L) were dissolved in DCM and stirred at room temperature for 2h, the reaction mixture was extracted with water and DCM, the organic layer was washed 3 times with water, dried over anhydrous sodium sulfate, concentrated by distillation under reduced pressure to give a residue which was purified by silica gel chromatography to give Compound 31;

Step 4-10: synthesis of a compound of formula V.

Sequentially adding the compound 31y (0.035 mmol,25.8 mg), B-linker, cuSO ₄ (0.044 mmol,7 mg) and VcNa (0.105 mmol,21 mg) into a mixed solution of THF and water (1:1), stirring at room temperature for 2 hours, concentrating to remove tetrahydrofuran, adding water, extracting with dichloromethane for 3 times, drying an organic phase with anhydrous sodium sulfate, concentrating to obtain a crude product, and purifying by silica gel column chromatography to obtain a compound of formula V;

the synthetic route of ubiquitin ligase E3 ligand with L (linker) is as follows:

(1) When the ubiquitin ligase E3 ligand is CRBN, th (thalidomide derivative) is preferred, the method of synthesis is:

Dissolving compound Th (thalidomide derivative) in DMF, adding DIPEA,1.0mol linker, reacting at 90deg.C for 2 hr, adding water, extracting with ethyl acetate, collecting organic layer, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and purifying by silica gel column chromatography to obtain compound Th-L;

(2) When the ubiquitin ligase E3 ligand is MDM2, the synthesis method comprises the following steps:

dissolving 1.0mol of MDM2 in DCM, adding 1.1mol of EDCI, 1.1mol of HOBt and 1.5mol of DIPEA in ice bath, adding 1.0mol of linker, reacting for 5 hours, adding a proper amount of DCM for dilution extraction, collecting an organic layer, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and purifying by silica gel column chromatography to obtain a compound MDM2-L;

(3) When ubiquitin ligase E3 ligand is VHL, the synthesis route is the same as MDM2-L, the difference is that MDM2 is replaced by VHL, and VHL-L is prepared correspondingly;

(4) When the ubiquitin ligase E3 ligand is cIAP, the synthetic route is the same as MDM2-L, except that VHL is replaced with cIAP, and cIAP-L is prepared accordingly.

A pharmaceutical composition comprising a therapeutically effective amount of a bifunctional molecular compound inducing the degradation of PD-L1 protein as an active ingredient.

A pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent, adjuvant, vehicle, or combination thereof.

Wherein, the dosage form of the pharmaceutical composition is any one of injection, tablet and capsule.

The invention also provides application of the bifunctional molecule compound for inducing PD-L1 protein degradation and pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparation of medicines for treating and/or preventing infection, cancer or autoimmune diseases.

The infection is skin infection, gastrointestinal tract infection, genitourinary system infection, systemic infection, or virus infection caused by one or more of influenza, hepatitis C virus, human papilloma virus, cytomegalovirus, epstein-Barr virus, polio virus, aquatics zona virus, coxsackie virus and human immunodeficiency virus;

The cancer is one or more of bone cancer, lung cancer, gastric cancer, colon cancer, membrane adenocarcinoma, breast cancer, prostate cancer, lung cancer, brain cancer, ovarian cancer, cancer of the breast, cervical cancer, cancer of the ball of the lung, renal cancer, cancer of the head and neck, lymphoma, leukemia and skin cancer.

The autoimmune disease is one or more of rheumatoid arthritis, systemic lupus erythematosus, mixed connective tissue disease, systemic scleroderma, dermatomyositis, nodular vasculitis, nephropathy, endocrine related diseases, liver diseases, psoriasis and autoimmune reaction caused by infection.

The invention has the beneficial effects that:

The invention takes PROTAC technology as support and takes PD-L1 inhibitor as raw material to synthesize bifunctional molecule compounds with different Linker lengths for inducing PD-L1 protein degradation.

The invention effectively targets and degrades PD-L1; similar to catalytic reactions, the drug onset dose is low; only provides binding activity, is event driven, is different from the traditional occupation driving, and does not need to directly inhibit the functional activity of target protein; the drug does not need to bind to the target protein for a long period of time and with high strength. The bifunctional molecule compound for inducing the degradation of the PD-L1 protein provides a novel treatment mode for treating tumors and/or other diseases mediated by PD-L1.

Drawings

FIG. 1 shows the degradation of PD-L1 by BMS-37-C1-C5 and BMS37-3C-V2, BMS37-5C-V2, BMS37-7C-V2 of the invention;

FIG. 2 shows the time dependence of BMS-37-C1-C5 and BMS37-3C-V2, BMS37-5C-V2, BMS37-7C-V2 on PD-L1 degradation and MG 132 reversal experiments of the invention;

FIG. 3 shows changes in PD-L1 expression detected by flow cytometry in BMS-37-C1, BMS-37-C3 and BMS37-5C-V2 of the present invention;

FIG. 4 shows the effect of BMS-37-C1 and BMS-37-C3 of the present invention on T cell killing tumor cells;

FIG. 5 shows affinity binding curves of BMS-37-C1, BMS-37-C3 and BMS37-5C-V2 to PD-L1 as measured by MST in capillaries treated according to the inventive examples.

Detailed Description

The invention is further illustrated by means of examples. It should not be construed that the scope of the invention is limited to the following examples.

Example 1 Synthesis of intermediate Th-L2 (m=2)

In this embodiment, L isM is 2;

b is CRBN, the synthetic route is as follows:

58mg of compound Th (thalidomide derivative, commercially available) are placed in a eggplant-shaped bottle, 3mL of DMF is added, 50mg of azido-2 PEG-amine and 47 mu L of DIPEA are sequentially added under stirring to react for 3-4 hours at 90 ℃, 30mL of water and 30mL of ethyl acetate are added to extract, an organic layer is dried with anhydrous sodium sulfate and concentrated to obtain a crude product, and the crude product is purified by silica gel column chromatography, petroleum ether-ethyl acetate is eluted in a gradient from 1:2 to 1:4 to obtain 33.2mg of yellow oily substance, and the yield is 41%.

Th-L2，¹H NMR(400MHz,CDCl₃)δ9.06(br s,1H),7.49(t,J＝7.8Hz,1H),7.09(dd,J＝

7.2,2.0Hz,1H),6.93(d,J＝8.4Hz,1H),6.50(t,J＝5.6Hz,1H),4.96–4.92(m,1H),3.74(t,J＝5.2Hz,2H),3.70–3.67(m,6H),3.48(q,J＝5.6Hz,2H),3.38(t,J＝4.4Hz,2H),2.80–2.73(m,3H),2.13–2.10(m,1H);¹³C NMR(100MHz,CDCl₃)δ171.7,169.4,168.8,167.7,146.9,136.1,132.6,116.9,111.6,110.3,70.75,70.72,70.1,69.6,50.7,48.9,42.4,31.5,22.8.

Example 2 Synthesis of intermediate M-L1 (m=1)

In this embodiment, B is MDM2;

In this embodiment, L is M is 1;

320mg of compound M is put into a eggplant-shaped bottle, 3mL of DCM is added, 65mg of azido-1 PEG-amine, 132 mu L of DIPEA, 74mg of EDCI and 92mg of HOBt are sequentially added under ice bath, the mixture is reacted for 3 to 4 hours, 30mL of water and 30mL of DCM are added for extraction, an organic layer is dried by anhydrous sodium sulfate, a crude product is obtained after concentration, and 237.5mg of white solid is obtained after silica gel column chromatography purification, and the yield is: 63.2%.

M-L1，¹H NMR(600MHz,DMSO-d₆)δ7.96(t,J＝5.7Hz,1H),7.56–7.51(m,1H),7.17–

7.14(m,2H),7.12(d,J＝8.3Hz,2H),7.07–7.02(m,2H),6.98(d,J＝8.0Hz,2H),6.62(d,J＝7.3Hz,2H),5.66(d,J＝9.7Hz,1H),5.58(d,J＝9.7Hz,1H),4.72(hept,J＝6.0Hz,1H),3.83(s,4H),3.76–3.67(m,2H),3.65–3.55(m,3H),3.43(t,J＝5.9Hz,2H),3.38(t,J＝4.9Hz,3H),3.21(q,J＝5.8Hz,3H),3.00(h,J＝7.3Hz,2H),1.27(d,J＝6.0Hz,3H),1.22(d,J＝6.0Hz,3H).HRMS(ESI⁺):m/z calculated for C₃₆H₄₀Cl₂N₈O₆[M+H]⁺,751.2448;found,751.2443.

EXAMPLE 3 Synthesis of intermediate V-2L

B in this embodiment is VHL;

In this embodiment, L is M is 1;

215mg of compound V is put into a eggplant-shaped bottle, 3mL of DCM is added, 57.5mg of 3-azidopropionic acid, 132 mu L of DIPEA, 74mg of EDCI and 92mg of HOBt are sequentially added in ice bath for reaction for 3-4 hours, 30mL of water and 30mL of DCM are added for extraction, an organic layer is dried by anhydrous sodium sulfate, a crude product is obtained after concentration, and 279mg of white solid is obtained after silica gel column chromatography and purification, the yield is: 53%.

V-2L，¹H NMR(600MHz,DMSO-d₆)δ8.98(s,1H),8.57(t,J＝6.0Hz,1H),8.12(d,J＝9.4

Hz,1H),7.47–7.36(m,4H),5.13(d,J＝3.6Hz,1H),4.58(d,J＝9.4Hz,1H),4.47–4.40(m,2H),4.39–4.33(m,1H),4.21(dd,J＝15.8,5.5Hz,1H),3.69(dd,J＝10.5,4.2Hz,1H),3.62(dt,J＝10.8,1.8Hz,1H),3.52(ddd,J＝12.2,7.9,5.6Hz,1H),3.47(dt,J＝12.2,6.1Hz,1H),2.63–2.53(m,1H),2.46–2.45(m,1H),2.45(s,3H),2.07–1.99(m,1H),1.91(ddd,J＝12.9,8.5,4.6Hz,1H),0.95(s,9H).HRMS(ESI⁺):m/z calculated for C₂₅H₃₃N₇O₄S[M+H]⁺,528.2315;found,528.2320.

EXAMPLE 4 Synthesis of Compound 3

The specific operation process is shown in the step 1-1, namely light yellow solid and yield :58％.¹H NMR(400MHz,CDCl₃)δ10.37(s,1H),7.42–7.27(m,8H),6.20(s,2H),5.15(s,2H),3.88(s,6H),2.27(s,3H).

EXAMPLE 5 Synthesis of Compound 6

The specific operation process is shown in step 1-2, yellow oily matter .¹H NMR(400MHz,CDCl₃)δ6.29(s,1H),4.92(s,1H),3.40–3.36(m,2H),3.31–3.28(m,2H),2.55–2.51(m,2H),2.42–2.39(m,2H),2.01(t,J＝2.4Hz,1H),1.44(s,9H).

EXAMPLE 6 Synthesis of Compound 7

The specific operation process is shown in the steps 1-3, and the yield is ：48％.¹H NMR(400MHz,CDCl₃)δ7.48–7.27(m,8H),6.24(s,2H),5.08(s,2H),4.61(s,2H),3.96(s,2H),3.84(s,6H),3.47–3.44(m,2H),2.82(t,J＝4.8Hz,2H),2.51–2.49(m,2H),2.47–2.43(m,2H),2.27(s,3H),1.94(t,J＝2.4Hz,1H).

EXAMPLE 7 Synthesis of Compound 11

The specific operation process is shown in step 2-2, the white solid product is 70mg, the yield ：71％.¹H NMR(600MHz,CDCl₃)δ10.32(s,1H),7.91(s,1H),7.72(td,J＝1.8,0.8Hz,1H),7.68(ddt,J＝7.8,6.2,1.3Hz,2H),7.54(t,J＝7.8Hz,1H),7.46–7.40(m,3H),7.40–7.34(m,1H),7.32–7.27(m,4H),6.63(s,1H),5.20(d,J＝9.6Hz,4H),2.27(s,3H).

EXAMPLE 8 Synthesis of Compound 13

The specific operation process is shown in the step 2-3, white solid 28mg, yield 38％.¹H NMR(600MHz,DMSO-d₆)δ8.03(s,1H),7.89(d,J＝7.9Hz,1H),7.83(d,J＝7.8Hz,1H),7.62(t,J＝7.8Hz,1H),7.56(s,1H),7.50–7.44(m,3H),7.42–7.36(m,1H),7.33–7.26(m,3H),7.21(dd,J＝7.6,1.4Hz,1H),7.12(s,1H),5.29(d,J＝3.5Hz,4H),3.87(s,2H),2.23(s,3H),1.90(s,1H),1.27(s,6H).

EXAMPLE 9 Synthesis of Compound 13y

The specific operation process is shown in step 2-4, white solid ,8mg,70％.¹H NMR(600MHz,DMSO-d₆)δ8.13(s,1H),7.96(s,1H),7.84–7.79(m,2H),7.62(t,J＝7.8Hz,1H),7.49–7.42(m,4H),7.40–7.37(m,1H),7.33–7.30(m,2H),7.27(t,J＝7.6Hz,1H),7.21(dd,J＝7.7,1.5Hz,1H),7.06(s,1H),5.31(s,2H),5.24(s,2H),3.83(dd,J＝6.0,2.6Hz,2H),3.49(s,2H),3.02(t,J＝2.5Hz,1H),2.23(s,3H),1.99(dq,J＝15.9,6.8,6.2Hz,1H),1.25–1.22(m,3H),1.20(s,3H).

EXAMPLE 10 Synthesis of Compound 16

The specific operation process is shown in step 3-1, white solid 430mg, yield ：86％.¹H NMR(400MHz,CDCl₃)δ10.39(s,1H),7.84(dd,J＝7.6,1.6Hz,1H),7.48-7.38(m,5H),7.29-7.27(m,2H),2.55(s,3H).

EXAMPLE 11 Synthesis of Compound 22

The specific operation process is shown in step 4-1, yield ：52％.¹H NMR(600MHz,DMSO-d₆)δ8.89(s,1H),8.30(d,J＝9.6Hz,1H),7.50(d,J＝9.6Hz,1H),4.35(q,J＝7.1Hz,3H),1.33(t,J＝7.1Hz,4H).

EXAMPLE 12 Synthesis of Compound 24

The specific operation process is shown in step 4-2, black colloid 1.9g, yield 44.8％.¹H NMR(600MHz,DMSO)δ8.35(d,J＝0.7Hz,1H),7.83(d,J＝10.0Hz,1H),7.25(d,J＝6.7Hz,1H),6.98(d,J＝9.9Hz,1H),4.27(q,J＝7.1Hz,2H),4.13(d,J＝6.8Hz,1H),3.65(dd,J＝10.8,6.3Hz,1H),3.57(dt,J＝10.2,7.2Hz,1H),3.47(ddd,J＝10.3,7.9,5.7Hz,1H),3.28(dd,J＝10.8,4.6Hz,1H),2.14(dq,J＝13.6,6.9Hz,1H),1.91(dq,J＝12.7,6.2Hz,1H),1.39(s,10H),1.30(t,J＝7.1Hz,3H).

EXAMPLE 13 Synthesis of Compound 27

The specific operation process is shown in the step 4-4, and the yield is ：49％.¹H NMR(600MHz,DMSO-d₆)δ7.33(t,J＝7.7Hz,1H),7.28(dd,J＝8.3,6.4Hz,5H),7.24–7.16(m,3H),6.82(s,3H),5.11(d,J＝1.9Hz,2H),5.03(q,J＝12.5Hz,2H),4.31–4.24(m,1H),2.84–2.80(m,2H),1.34(s,9H).

EXAMPLE 14 Synthesis of Compound 30

The specific operation process is shown in the steps 4-7, the yellow solid is 512mg, and the yield is ：55％.¹H NMR(600MHz,DMSO-d₆)δ8.35(s,1H),7.84(dd,J＝10.0,0.7Hz,1H),7.73(d,J＝6.6Hz,1H),7.44(t,J＝7.5Hz,2H),7.40–7.33(m,2H),7.28–7.26(m,2H),7.24(d,J＝7.6Hz,1H),7.16(d,J＝7.6Hz,1H),6.98(d,J＝9.9Hz,1H),5.12(s,2H),4.28(q,J＝7.1Hz,2H),4.23(q,J＝6.0Hz,1H),3.68(dd,J＝10.9,6.2Hz,1H),3.58(dt,J＝10.3,7.3Hz,1H),3.50(ddd,J＝10.1,7.7,5.2Hz,1H),3.37(dd,J＝11.1,4.4Hz,1H),2.22–2.17(m,1H),2.16(s,3H),2.00–1.91(m,1H),1.30(t,J＝7.1Hz,3H).

EXAMPLE 15 Synthesis of Compound BMS37-Cn (n=1-5, for example n=1)

Compound 7 (0.035 mmol,17 mg), th-L1 (0.035 mmol,13.5 mg), cuSO ₄ (0.044 mmol,7 mg) and VcNa (0.105 mmol,21 mg) were added sequentially to a mixed solution of THF and water (1:1), and stirred at room temperature for 2 hours. Concentrating to remove tetrahydrofuran, adding water, extracting with dichloromethane for 3 times, drying the organic phase with anhydrous sodium sulfate, concentrating to obtain crude product, purifying by silica gel column chromatography to obtain 13mg solid, and obtaining yield ：42.5％.¹H NMR(400MHz,CDCl₃)δ7.72(s,1H),7.62(s,1H),7.52–7.48(m,1H),7.43–7.41(m,3H),7.38–7.28(m,4H),7.12(d,J＝7.2Hz,1H),6.88(d,J＝8.4Hz,1H),6.47(t,J＝5.2Hz,1H),6.24(s,2H),5.10(s,2H),4.97–4.42(m,1H),4.54–4.51(m,2H),4.12(s,2H),3.88–3.83(m,2H),3.82(s,6H),3.67(t,J＝4.8Hz,2H),3.50–3.48(m,2H),3.43(q,J＝5.2Hz,2H),3.03(td,J＝6.8,2.8Hz,3H),2.92(t,J＝4.8Hz,2H),2.86–2.73(m,4H),2.55(t,J＝7.6Hz,2H),2.27(s,3H),2.09–2.07(m,1H).HRMS m/z calcd for C₄₇H₅₃N₈O₉ 873.3936,found873.3930[M+H⁺].

EXAMPLE 16 Synthesis of Compound NP19-nP-Th (n=2-3, n=2 as an example)

Specific procedures and ratios reference example 15.

NP19-2P-Th, yellow solid, yield 48％.¹H NMR(600MHz,DMSO-d₆)δ11.09(s,1H),8.25(s,1H),7.93(s,1H),7.83(s,1H),7.80–7.75(m,2H),7.59(t,J＝7.8Hz,1H),7.54(dd,J＝8.6,7.1Hz,1H),7.46(q,J＝7.9Hz,3H),7.40–7.34(m,2H),7.32–7.29(m,2H),7.26(t,J＝7.6Hz,1H),7.20(dd,J＝7.7,1.5Hz,1H),7.07–6.99(m,3H),6.55(t,J＝5.9Hz,1H),5.26(s,2H),5.21(s,2H),5.07(dd,J＝12.7,5.6Hz,1H),4.48(t,J＝5.2Hz,2H),4.30(d,J＝5.7Hz,2H),3.80(t,J＝5.2Hz,2H),3.56(t,J＝5.5Hz,2H),3.48(s,2H),3.41–3.38(m,2H),3.17(dd,J＝5.3,1.1Hz,1H),2.88(ddd,J＝16.8,13.6,5.4Hz,1H),2.62–2.52(m,2H),2.22(s,3H),2.01(tdd,J＝12.6,8.0,5.1Hz,2H),1.46(s,1H),1.32–1.25(m,2H),1.23(s,3H),1.21(s,3H).HRMS m/z calcd for C₅₅H₅₇ClN₉O₉ 1022.3962,found 1022.4005[M+H⁺].

Example 17 Synthesis of Compound MP-PC-nC (n=4, 6, for example n=4)

Compound 13 (0.04 mmol,22 mg), EDCI (0.08 mmol,15 mg), HOBt (0.08 mmol,11 mg), DIPEA (0.16 mmol, 28. Mu.L) and Th-4L (0.04 mmol,13.76 mg) were dissolved in DCM and stirred at room temperature for 2h. The mixture was diluted with water and extracted with DCM. The combined organic layers were washed with water, dried over anhydrous sodium sulfate, filtered under reduced pressure and concentrated to give a residue. Purified by silica gel chromatography to give yellow solid 15mg, yield :43％.¹H NMR(600MHz,CDCl₃)δ8.17(s,1H),7.68(d,J＝1.7Hz,1H),7.62(ddt,J＝10.8,7.8,1.4Hz,2H),7.50(t,J＝7.7Hz,2H),7.46–7.40(m,4H),7.38–7.35(m,1H),7.31(d,J＝1.5Hz,1H),7.29(d,J＝0.9Hz,2H),7.26(d,J＝1.4Hz,1H),7.25(s,1H),7.04(d,J＝7.1Hz,1H),6.85(d,J＝8.5Hz,1H),6.59(s,1H),6.19(s,1H),5.09(s,4H),4.89(dd,J＝12.5,5.3Hz,1H),3.61(s,2H),3.25(q,J＝6.6Hz,2H),3.19(q,J＝6.7Hz,2H),2.85(ddd,J＝16.9,3.6,2.4Hz,1H),2.79–2.66(m,2H),2.25(s,3H),2.12–2.07(m,1H),1.63–1.60(m,2H),1.55(dt,J＝10.5,4.4Hz,2H),1.35(s,6H).HRMS m/z calcd for C₅₀H₅₀ClN₆O₇ 881.3424,found 881.3432[M+H⁺].

EXAMPLE 18 Synthesis of Compound NP19-2C-V2

Specific operations and ratios reference example 18.

NP19-2C-V2, yellow solid, yield :42％.¹H NMR(600MHz,DMSO-d₆)δ8.98(s,1H),8.38(d,J＝7.8Hz,1H),7.98–7.91(m,2H),7.89(s,1H),7.81(dd,J＝7.8,1.7Hz,2H),7.62(d,J＝7.7Hz,1H),7.49(s,1H),7.48–7.45(m,2H),7.44(d,J＝5.4Hz,1H),7.42(s,1H),7.40–7.35(m,3H),7.32–7.29(m,2H),7.27(s,1H),7.21(d,J＝1.4Hz,1H),7.06(s,1H),5.35–5.32(m,1H),5.31(d,J＝4.9Hz,2H),5.24(s,2H),4.91(s,1H),4.53(d,J＝9.3Hz,1H),4.42(t,J＝8.0Hz,1H),4.27(p,J＝3.3Hz,1H),3.60(d,J＝3.4Hz,2H),3.54–3.49(m,2H),3.27(p,J＝6.5Hz,1H),3.21(p,J＝6.5Hz,2H),2.45(s,3H),2.35–2.25(m,1H),2.23(s,3H),2.05–1.95(m,4H),1.23(s,6H),1.18(d,J＝4.5Hz,3H),0.90(s,9H).HRMS m/z calcd for C₅₉H₆₆ClN₇O₇S 1052.4506,found 1052.4512[M+H⁺]

EXAMPLE 19 Synthesis of Compound NB series (exemplified by NB-C1 Compounds)

Specific procedures and ratios reference example 15.

NB-C1, yellow solid, yield :85％.¹H NMR(400MHz,CDCl₃)δ9.28(s,1H),8.24(d,J＝1.2Hz,1H),8.10(dd,J＝6.8,2.4Hz,1H),7.93(s,1H),7.90(dd,J＝8.8,1.6Hz,1H),7.58(d,J＝8.4Hz,1H),7.47-7.33(m,9H),7.10(d,J＝6.8Hz,1H),6.85(d,J＝8.4Hz,1H),6.47(t,J＝5.2Hz,1H),5.08(dd,J＝12.0,5.2Hz,1H),4.84-4.72(m,2H),4.62-4.58(m,2H),3.90-3.88(m,2H),3.70(t,J＝5.2Hz,2H),3.45-3.42(m,2H),2.88-2.76(m,3H),2.62(s,3H),2.18-2.14(m,1H).HRMS m/z calcd for C₄₁H₃₆N₈O₇Na 775.2599,found 775.2613[M+Na⁺].

EXAMPLE 20 Synthesis of Compound Pd-nC-Th (n=2, 4,6, for example n=2)

Specific operations and ratios reference example 18.

Pd-2C-Th, yellow solid, yield :53％.¹H NMR(600MHz,DMSO-d₆)δ11.09(s,1H),8.24–8.18(m,2H),8.15(s,1H),7.84(d,J＝9.9Hz,1H),7.72(d,J＝6.6Hz,2H),7.58–7.53(m,1H),7.44(t,J＝7.5Hz,2H),7.40–7.33(m,2H),7.29–7.26(m,2H),7.25–7.21(m,2H),7.21–7.18(m,2H),7.16(dd,J＝8.8,2.8Hz,2H),7.15–7.11(m,2H),7.01(dd,J＝7.0,1.3Hz,1H),6.97(d,J＝9.9Hz,1H),6.91(s,1H),6.75(d,J＝4.9Hz,1H),5.11(d,J＝15.4Hz,4H),5.04(ddd,J＝12.8,5.5,1.8Hz,1H),4.67–4.62(m,1H),4.23(q,J＝5.7Hz,1H),3.70(dd,J＝10.8,6.1Hz,1H),3.59(q,J＝7.8,5.4Hz,1H),3.53–3.49(m,1H),3.37(dd,J＝10.8,4.1Hz,2H),3.27(t,J＝6.5Hz,2H),3.25–3.15(m,2H),2.96(s,2H),2.92–2.83(m,1H),2.60–2.54(m,1H),2.20(dt,J＝12.6,6.9Hz,1H),2.16(s,3H),2.04–1.92(m,4H).HRMS m/z calcd for C₅₄H₅₃N₁₂O₈ 997.4104,found 997.4083[M+H⁺].

Example 21 Western Blot detection of changes in related proteins

Alterations in PD-L1 protein expression

(1) Cells grown logarithmically and having reached 90% confluency were collected by digestion, adjusted for cell concentration by addition of fresh medium, and then counted using a cell counting plate. The cells are uniformly inoculated in a large dish, the culture wall is attached in an incubator and starts to be in the logarithmic division phase, and the culture medium containing the medicines is replaced and then placed in the incubator for continuous culture for 48 hours. After waiting for the end of the culture, the cells were digested with pancreatin and collected and labeled. The excess EDTA in the pancreatin was removed by washing twice with PBS for 5min each and the cells were collected in 1.5mL EP tubes. After the cleaning is finished, adding a proper amount of RIPA lysate according to the cell number, adding phosphatase inhibitor in advance into the lysate, adding holoenzyme inhibitor and adding PMSF in time. PMSF is an irreversible inhibitor of serine proteases, which inhibits serine proteases (trypsin and chymotrypsin), and also inhibits cysteine proteases and acetylcholinesterase, thereby preventing degradation of proteins. After adding the lysate, the cells were mixed using a vortex shaker and protein was prevented from degradation by lysing on ice for 20min. After completion of the lysis, centrifugation was performed using a centrifuge at 4℃and a rotational speed of 11500rpm for 10min. Waiting for the end of centrifugation, and sucking supernatant, wherein the supernatant is protein;

(2) Protein content was detected by BCA method in this experiment. Under alkaline conditions, reducing Cu ²⁺ into Cu ⁺,Cu⁺ by the protein to form a purple blue complex with the BCA reagent, measuring the absorption value of the complex at 562nm, and comparing the complex with a standard curve to calculate the concentration of the protein to be detected;

(3) The protein sample with the end of the quantification was added to the corresponding volume of 6X Loading buffer according to its volume. DTT is added to 6X Loading buffer before use, and the DTT can open disulfide bond on cys to form linear polypeptide, so that antibody can recognize and bind to specific site. Mixing the samples with vortex oscillator, and decocting in 100deg.C water bath for 5-10min to thoroughly denature protein. If the amino acid composition of a protein contains more hydrophobic amino acids (tryptophan W, alanine A, valine V, leucine L, isoleucine I, proline P, phenylalanine F and the like), and the protein has strong hydrophobicity, aggregates can be easily formed after the protein is boiled, and the protein rich in the hydrophobic amino acids can be incubated for 1h at 37 ℃ or boiled in a water bath kettle at 60 ℃ for 30min instead of 100 ℃ for 5-10min;

(4) Preparing SDS-PAGE gel, and loading the prepared sample;

(5) The PVDF membrane was put into anhydrous methanol for 30s to activate the PVDF membrane. The membrane is then immersed in the wet transfer solution. The electric rotating clamp is sequentially placed with sponge, filter paper and PVDF film on the white surface, and then sequentially placed with glue, filter paper and sponge to manufacture the sandwich structure. Air bubbles are avoided between the glue and the membrane. The sandwich structure is inserted into the electric rotating groove, and the electric rotating device is placed into the ice box mixed with ice water, so that the failure of film rotating caused by excessive heat generated in the film rotating process is avoided. Transferring the protein for 2h by using 300 mA;

(6) After the protein transfer printing is finished, the sandwich structure is opened, the film is dyed by using ponceau to check the transfer printing effect, and the film is cleaned by using pure water. The membrane was cut according to the protein molecular weight, then the filter paper was air dried to PVDF membrane, and again activated with methanol for a second time. After activation, the methanol on the surface is cleaned by pure water, then put into 5% skimmed milk and sealed for 2 hours by using a shaking table;

(7) The membrane after the sealing was rinsed with TBST solution to clean the surface of the membrane with milk. The 5% bsa liquid was used to prepare the desired antibodies by primary anti-dilution according to the dilution ratios indicated in the antibody instructions. Then placing PVDF film in TBST on a wet box for primary antibody incubation, and placing the wet box in a refrigerator at 4 ℃ for overnight incubation;

(8) The wet cartridges were removed and the membranes were washed three times with TBST for 5min each on a shaker. Different secondary antibodies were selected according to the primary antibody instructions. Placing the cleaned membrane on a wet box, adding secondary antibody liquid and sealing for two hours at room temperature;

(9) The target strip was exposed using a gel imager.

As shown in FIGS. 1 and 2, the experimental results showed that the compounds BMS-37-C3 and BMS-37-C1 were most effective in degrading in the A375 cell line. These two compounds began to inhibit the expression level of PD-L1 protein at 0.3 μm. C2 also has degradation capacity but is less powerful than C3. C4 and C5 had no effect in the a375 cell line. Also in the B16F 10 cell line BMS-37-C3 and C1 still inhibited PD-L1 more than other compounds. BMS-37-VHL series also have inhibitory effect on PD-L1 expression. Further use of MG132 on cells, the inhibition of PD-L1 by BMS-37-C1 and BMS-37-C3 was reversed, further indicating that these compounds degrade proteins via the ubiquitination pathway to reduce the expression of PD-L1.

Example 22 flow cytometry detection of changes in related proteins

(1) Cells grown logarithmically and having reached 90% confluency were collected by digestion, adjusted for cell concentration by addition of fresh medium, and then counted using a cell counting plate. Cells were inoculated evenly in six well plates, adhered in an incubator and started in the logarithmic division phase, and after changing the culture medium containing the drugs, placed in the incubator for further culture for 48 hours.

(2) After waiting for the end of the incubation, the cells were digested with pancreatin and collected into 1.5mL sample tubes, which were labeled. Centrifuging the sample at 4deg.C for 5min, discarding supernatant, adding PBS, washing for one time to remove excessive EDTA in pancreatin and serum contained in the culture medium, dividing the control group into two groups, wherein one group is negative control, and the other group is positive control, centrifuging at 4deg.C for 5min again for 400g, and discarding supernatant.

(3) A PBS solution containing 5% BSA was prepared, and the antibody and PBS were mixed in a volume ratio of 1:60 (murine) and 1:40 (human) under light-shielding conditions, and finally, igG and PD-L1 antibody were added to the sample tube in a volume of 30. Mu.L, respectively.

(4) Incubate at 4deg.C for 40min in the dark, again 4deg.C, 400g, centrifuge for 5min, discard supernatant, and add 200. Mu.L PBS solution into the sample tube for on-machine detection.

As shown in FIG. 3, the experimental results showed that BMS-37-C1 and BMS-37-VHL reduced the expression of cell surface PD-L1 between 0.1 and 1. Mu.M in both B16-F10 and A375 cells, but showed HOCK effect at 3 to 10. Mu.M; BMS-37-C3 can reduce the content of PD-L1 on the cell surface in a concentration-dependent manner in B16-F10 and A375 cells, and has better effect.

Example 23 Effect of Compounds on T cell killing tumor cells

(1) Cells grown logarithmically and having reached 90% confluency were collected by digestion, adjusted for cell concentration by addition of fresh medium, and then counted using a cell counting plate. Cells were evenly seeded in ninety-six well plates and T cells were added after cell attachment, eventually allowing tumor cells and T cells to continue co-cultivation for 48h at a ratio of 1:0,1:1,1:2,1:3,1:4, 1:6.

(2) After the co-cultivation is finished, taking out ninety-six pore plates, slowly sucking the supernatant, adding 100 mu L of PBS into each pore to rinse both sides, adding 4% paraformaldehyde, fixing for 20min at room temperature, rinsing both sides with PBS again, dyeing for 15-20 min with DAPI (DAPI) staining solution at room temperature, and detecting on a machine after PBS rinsing to determine the final co-cultivation proportion.

(3) Cells grown logarithmically and having reached 90% confluency were collected by digestion, adjusted for cell concentration by addition of fresh medium, and then counted using a cell counting plate. Uniformly inoculating cells in a ninety-six pore plate, adding corresponding culture medium containing the drugs, placing the cells into an incubator, after the cells adhere to the wall, replacing the culture medium containing the drugs and the T cells with the above proportion, and placing the cells into the incubator for continuous culture for 48 hours.

(4) Taking out the co-culture plates, slowly collecting the supernatant, adding 100 mu L of PBS into each hole to rinse both sides, adding 4% paraformaldehyde, fixing for 20min at room temperature, rinsing both sides with PBS again, dyeing with DAPI (DAPI) staining solution at room temperature for 15-20 min, rinsing with PBS, and performing machine inspection.

FIG. 4A is a microscopic view of stimulated T cells after primary culture, as shown in FIG. 4; b is discussion of the co-culture proportion of T cells and tumor cells; c is the cytotoxicity of BMS-37, BMS-37-C1, BMS-37-C3 and T cells on A375 cells after co-culture; d is a statistical graph of T cell killing activity of BMS-37, BMS-37-C1, BMS-37-C3 and atizomib; the experimental results show that: when the co-culture target ratio of the tumor cells and the T cells is explored, the T cells can kill about 50% of the tumor cells when the tumor cells and the T cells are co-cultured in a ratio of 1:3, so that the fixed target ratio is 1:3 in the subsequent experiments. When the corresponding medicine is added and co-cultured with T cells, the PD-L1 mono PROTAC BMS-37-C1 and BMS-37-C3 can promote anti-tumor immunity between 0.3 and 1 mu M, and have concentration dependence, and the effect is stronger than that of the bulk drugs BMS-37 and PD-L1 monoclonal antibody Atezolizumab.

Example 24 micro thermal surge experiment

PD-L1 protein (50 nM) was labeled with Monolith NT ^TM Protein Labeling Kit RED (Cat#L001) and samples were diluted in 20mM Tris-HCl (pH 7.5) and 0.5 (v/v)% Tween-20. BMS-37-C1/BMS-37-C3/BMS-37-5C-V2 powder was dissolved in 5% DMSO at a concentration of 200. Mu.M, diluted in a double ratio. After incubation for 10 minutes at room temperature, the samples were loaded into MonolithTM standard treated capillaries and after incubation for 30 minutes on a Monolith nt.115 instrument (NanoTemper Technologies, munchen, germany), thermophoresis was measured at 25 ℃. The laser power was set to 40% using an on time of 30 seconds. The power of the LED was set to 100%. The dissociation constant Kd values were fitted by using NTANALYSIS software (NanoTemper Technologies, munchen, germany).

As shown in FIG. 5, the experimental results showed that the equilibrium dissociation constants Kd of BMS-37-C1/BMS-37-C3/BMS-37-5C-V2 were 28.70.+ -. 4.94. Mu.M, 1.61.+ -. 0.37. Mu.M and 9.10.+ -. 2.11. Mu.M, respectively, indicating that the synthesized PD-L1 PROTACs could bind well to PD-L1.

In general, the invention detects the degradation level of PD-L1 protein through in vitro binding force, intracellular Western Blot and flow cytometry, and finally finds a powerful and effective PD-L1 degradation agent BMS-37-C3, and the compound has the effect on immunotherapy, can enhance the killing capacity of T cells, and provides a new treatment mode for tumor immunotherapy.

Claims (3)

1. A bifunctional molecule compound which induces the degradation of PD-L1 protein and is represented by the following formula BMS37-C1 to BMS37-C3, or a pharmaceutically acceptable salt thereof, characterized by specifically being a compound represented by the following structural formula:

2. A pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of the bifunctional molecular compound of claim 1, which induces degradation of PD-L1 protein, further comprising a pharmaceutically acceptable carrier, diluent, adjuvant, vehicle, or combination thereof.

3. The use of a bifunctional compound of claim 1, or a pharmaceutically acceptable salt thereof, that induces the degradation of PD-L1 protein, or a pharmaceutical composition of claim 2, for the manufacture of a medicament for the treatment and/or prophylaxis of an infection, cancer or autoimmune disease, wherein the infection is one or more of a skin infection, a gastrointestinal infection, a genitourinary infection, a systemic infection, or a viral infection caused by one or more of influenza, hepatitis c virus, human papilloma virus, cytomegalovirus, epstein barr virus, polio virus, coxsackievirus and human immunodeficiency virus;

The cancer is one or more of bone cancer, lung cancer, gastric cancer, colon cancer, membranous adenocarcinoma, breast cancer, prostatic cancer, lung cancer, brain cancer, ovarian cancer, bladder cancer, cervical cancer, testicular cancer, kidney cancer, head and neck cancer, lymph cancer, leukemia and skin cancer;

The autoimmune disease is one or more of rheumatoid arthritis, systemic lupus erythematosus, mixed connective tissue disease, systemic scleroderma, dermatomyositis, nodular vasculitis, nephropathy, endocrine related diseases, liver diseases, psoriasis and autoimmune reaction caused by infection.