CN117986320A

CN117986320A - PROTAC compound for targeted degradation of intracellular BCL-3 as well as preparation method and application thereof

Info

Publication number: CN117986320A
Application number: CN202311779897.8A
Authority: CN
Inventors: 赵静; 李宾宾; 李永淑; 毕波; 张常华; 何裕隆
Original assignee: Seventh Affiliated Hospital Of Sun Yat Sen University Shenzhen
Current assignee: Seventh Affiliated Hospital Of Sun Yat Sen University Shenzhen
Priority date: 2023-12-22
Filing date: 2023-12-22
Publication date: 2024-05-07

Abstract

The invention relates to a method for targeted degradation of intracellular BCL-3 protein and application thereof, belonging to the technical field of biological medicine. The structural formula of PROTAC compounds of targeted degradation intracellular BCL-3 protein is shown as a general formula (I): Compared with the existing BCL3 inhibitor, the PROTAC compound for targeted degradation of intracellular BCL-3 can controllably degrade BCL3 protein through a ubiquitin-protein system to cause apoptosis of colorectal cancer cells, and has less influence on normal cells.

Description

PROTAC compound for targeted degradation of intracellular BCL-3 as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of protein degradation targeting chimeras, in particular to a PROTAC compound for targeting degradation of intracellular BCL-3 as well as a preparation method and application thereof.

Background

BCL-3 (B-cell lysate 3) is an atypical member of the inhibitory NF- κB family and is an important regulator of NF- κB activity. It can promote or inhibit the expression of NF- κB target gene according to the cell type and stimulus, and has effect on various cell functions including proliferation and differentiation, apoptosis induction, immune response, etc. In recent years, more and more researches show that BCL-3 has over-expression in various malignant tumors, such as diffuse B cell lymphoma, cervical cancer, hepatocellular carcinoma, colorectal cancer (CRC) and the like, and plays roles in promoting tumorigenesis, development, drug resistance, metastasis and the like by regulating and controlling oncogene expression. In colorectal cancer CRC, BCL-3 can promote cancer cell survival and metastasis by activating PI3K-AKT signaling pathway, and can promote colorectal cancer cell proliferation by stabilizing c-MYC. In addition, BCL-3 can also be used as a co-regulator of beta-catenin/TCF transcription complex to synergistically promote the dryness characteristics of CRC cells. Recent studies have also found that BCL-3 in CRC cells can also be resistant to tumor radiotherapy by enhancing DNA damage repair. In addition, BCL-3 can also modulate intestinal epithelial cell proliferation-mediated (AOM/DSS) -induced colon tumors. Thus, specific targeting of the activity of degrading intracellular BCL-3 in inflammatory bowel disease may be an effective strategy to inhibit tumors associated with intestinal inflammation.

In 2021, a small molecule inhibitor JS6 specific to BCL-3 was first reported by Brancale, which affects NF- κB downstream signaling by inhibiting BCL-3 interaction with p50 protein, thereby inhibiting breast cancer cell metastasis. However, in colorectal cancer, BCL3 also has NF- κb independent pro-neoplastic effects, so that BCL-3 specific small molecule inhibitor JS6 only affects BCL3 binding to p50, with no killing activity on colorectal cancer cells. Besides small molecule inhibitors, BCL3 genes can be sheared by a gene editing technology or the expression of BCL3 can be regulated down by an RNAi technology, but exogenous genes have poor physiological stability, and the genes can be only acted by mediating the expression of the genes in vivo by a gene delivery system, so that the method is complex and has higher cost. Thus, methods and pathways how to regulate intracellular BCL-3 protein levels and further induce colorectal cancer tumor cell death remain to be further investigated.

PROTAC (PROteolysis TARGETING CHIMERA) protein targeted degradation chimera is a technology for inducing targeted protein degradation by using a ubiquitin-protease system naturally existing in cells. PROTAC consists of a target protein ligand, a linker, an E3 ubiquitin ligase ligand 3 moiety. PROTAC molecules specifically recognize and bind to a target through a target protein ligand at one end of the PROTAC molecules, and specifically recognize and bind to an E3 ligand through an E3 ubiquitin ligase ligand at the other end of the PROTAC molecules, so that a target protein-PROTAC-E3 ligase ternary complex is formed. In this complex, the target protein is ubiquitously modified by the E3 ligase, and the ubiquitinated modified target protein is recognized and degraded by the proteasome, thereby inhibiting the function of the target protein.

PROTAC has several potential advantages over traditional small molecule inhibitors for cancer treatment: it is event-driven (rather than space-occupying-directly inhibiting the functional activity of the target protein), so that very low doses can have very good efficacy; PROTAC causes protein degradation, then separates from the compound, and enters the next catalytic cycle, so that the medicine has high action efficiency; each PROTAC can degrade a plurality of protein molecules, has the potential of breaking through a target point of non-patent medicine, and currently, more than ten types of PROTAC medicines enter clinical experiments.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide PROTAC compounds for targeted degradation of intracellular BCL-3, and a preparation method and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

In a first aspect, the invention provides a PROTAC compound for targeted degradation of intracellular BCL-3, wherein the structural formula of the PROTAC compound for targeted degradation of intracellular BCL-3 is shown as a general formula (I):

Wherein n represents a natural number of 1 to 5; r is an E3 ligase ligand; the E3 ligase ligand is

One of the VHL, MDM2, clAP1 or CRBN ligands is shown by the following structural formula:

In the general formula (I), an ether bond of- (CH 2-CH 2-O) n-is connected with an E3 ligase ligand.

The invention selects F para position in benzene ring as Linker position: the site is a Br site on a benzene ring of a JS6 molecular of a precursor BCL-3 inhibitor of PROTAC compounds for targeted degradation of intracellular BCL-3. The JS6 molecule is known as 2- { [ (5-bromo-2-fluorophenyl) carbonyl ] amino } -N- [2- (1, 4-oxaazepin-4-yl) ethyl ] benzamide. Based on the structure-activity relationship between JS6 molecule and BCL-3, the link and PROTAC formed by E3 ligase ligand are connected at the site to degrade intracellular BCL-3 in a better targeting way, and meanwhile, the site is easy to carry out crosslink reaction, so that the site is the optimal link connector connecting site.

As a preferred embodiment of the present invention, R is VHL; when R is VHL, the structural formula of the PROTAC compound targeted to degrade intracellular BCL-3 has a structure shown in a general formula (II):

the ether linkage of the- (CH 2-O) n-is linked to the acetamido group of the VHL ligand. By exchanging the terminal acetyl groups of VHL with a suitable linking group, the ether linkage in Linker can be easily linked to VHL via an amide linkage.

As a preferred embodiment of the present invention, the hydroxyl group on the VHL is modified with tetramethylene sulfoxide.

The hydroxyl groups on VHL were modified with tetramethylene sulfoxide (TMSO). The TMSO protects the hydroxyl of VHL in advance, avoids the competition of the E3 ligase ligand with amino and boric acid reaction in Gul reaction, and ensures the selectivity of the reaction. The molecule may also be deprotected to reduce TMSO to the hydroxy form before the PROTAC compound targeted to degrade intracellular BCL-3 is conjugated to folic acid.

As a preferred embodiment of the present invention, R is- (CH 2-O) n, n represents a natural number of 1 to 5. Further, n is 3 and R is- (CH 2-O) 3.

As a preferred embodiment of the invention, the PROTAC compound for targeted degradation of intracellular BCL-3 has a structural formula shown as formula (III):

As a preferred embodiment of the present invention, the compound of formula (II) is named PJ1799. Compared with the prior BCL-3 inhibitor JS6, the PJ1799 compound can degrade intracellular BCL-3 and further control and kill tumors, in particular colorectal cancer cells. Compared with JS6, the PJ1799 compound has obvious killing effect on human CRC cells and weaker killing effect on human normal cells.

In a second aspect, the invention provides a derivative of PROTAC compounds targeted to degrade intracellular BCL-3, which is a derivative of PROTAC compounds targeted to degrade intracellular BCL-3 coupled to folic acid.

According to the invention, through coupling PROTAC compounds of targeted degradation intracellular BCL-3 with folic acid, folic acid molecules play a role in targeting drugs, so that aggregation of PROTAC compounds of targeted degradation intracellular BCL-3 in tumor cells is increased, toxic and side effects of a system are reduced, and the modified drugs have stronger killing effect on human CRC cells.

In a third aspect, the present invention provides a method for preparing PROTAC compounds for targeted degradation of intracellular BCL-3, comprising the steps of:

adding an E3 ligase ligand, triethylamine and carboxylic acid to perform condensation reaction to synthesize a compound A;

Dissolving JS6 molecules and a compound A in an organic reagent, adding a catalyst into the system, stirring at room temperature for reaction for 18-48 hours, and purifying to obtain PROTAC compounds of targeted degradation intracellular BCL-3;

The JS6 molecule is 2- { [ (5-bromo-2-fluorophenyl) carbonyl ] amino } -N- [2- (1, 4-oxaazepin-4-yl) ethyl ] benzamide.

Further, the catalyst is Cu (OAc) ₂.

As a preferred embodiment of the present invention, the equivalent ratio of compound A, JS molecules to Cu (OAc) ₂ is compound a: JS6 molecule: cu (OAc) ₂ =1:1:4.

As a preferred embodiment of the present invention, JS6 molecule and compound a are dissolved in MeCN to perform a room temperature reaction; the room temperature is 20-25 ℃.

As a preferred embodiment of the present invention, when the PROTAC compound targeted to degrade intracellular BCL-3 has a structural formula as shown in formula (III), the preparation of the compound A comprises the following steps:

S1, dissolving VHL, triethylamine and (E) -14, 14-dimethyl-3, 6, 9-trioxa-12-aza-heptadec-12-enoic acid in MeCN, stirring and heating to 40 ℃, and reacting overnight;

s2, cooling the product of the overnight reaction in the step S1 to room temperature, and adding hydrochloric acid for acidification;

s3, purifying the acidized product of the step S2, and stirring for 2-24 hours at room temperature under the conditions of trimethylchlorosilane and imidazole to finish the preparation of the compound A.

As a preferred embodiment of the present invention, the molar ratio of VHL, triethylamine and (E) -14, 14-dimethyl-3, 6, 9-trioxa-12-aza-heptadec-12-enoic acid is VHL: triethylamine: (E) -14, 14-dimethyl-3, 6, 9-trioxa-12-aza-heptadec-12-enoic acid = 0.1-1): (0.1-2.5): (0.1-1).

Further, the ratio of VHL, triethylamine and (E) -14, 14-dimethyl-3, 6, 9-trioxa-12-aza-heptadec-12-enoic acid is VHL: triethylamine: (E) -14, 14-dimethyl-3, 6, 9-trioxa-12-aza-heptadec-12-enoic acid = 1:2.5:1.

As a preferred embodiment of the present invention, in preparing compound a, the organic solvent is dichloromethane; the concentration of the hydrochloric acid is 1M; the volume ratio of the dichloromethane to the hydrochloric acid is 10:1.

In a fourth aspect, the invention provides an application of PROTAC compounds for targeted degradation of intracellular BCL-3 in preparation of tumor therapeutic drugs.

As a preferred embodiment of the use of the present invention, the tumor therapeutic agent includes, but is not limited to, tumor therapeutic agents for small cell lung cancer, non-small cell lung cancer, colorectal cancer, biliary tract tumor, breast cancer, ovarian cancer, pancreatic cancer, stomach cancer, liver cancer, kidney cancer, prostate cancer, cervical cancer, testicular cancer, endometrial cancer, uterine cancer, bladder cancer, urothelial cancer, neuroendocrine cancer, thyroid cancer, medullary thyroid cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, oral cancer, vaginal cancer, penile cancer, anal cancer, thymus cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, mekel cell cancer, glioblastoma, glioma, sarcoma, soft tissue sarcoma, mesothelioma, myeloma, lymphoma, leukemia and/or myelodysplastic syndrome.

Further, the tumor therapeutic drugs are colorectal cancer, breast cancer, ovarian cancer and gastric cancer therapeutic drugs; more preferably, the tumor therapeutic agent is a therapeutic agent for colorectal cancer.

As a preferred embodiment of the present invention, the preparing a tumor therapeutic agent comprises: loading PROTAC compound of targeted degradation intracellular BCL-3 into a nano-carrier; preferably, the nanocarrier is albumin.

In a fifth aspect, the present invention provides a medicament for treating tumors, where the medicament for treating tumors is formed by loading PROTAC compounds for targeted degradation of intracellular BCL-3 on nano-carriers. Preferably, the nanocarrier is albumin.

Further, the nano-package is an albumin package, and the protein is serum albumin. According to the preparation targeting strategy, albumin is used for wrapping, so that the PROTAC compound for targeting and degrading the intracellular BCL-3 is increased in the tumor cells, and the toxic and side effects of the system are reduced. The serum albumin structure is provided with a plurality of hydrophobic cavities which can be loaded with hydrophobic drugs. Hydrophobic PROTAC molecules are wrapped in albumin by an ultrasonic auxiliary method so as to increase tumor targeting and endocytic efficiency of tumor cells. The modified medicine has stronger killing effect on human CRC cells.

As a preferred embodiment of the present invention, the medicament for treating tumors comprises the PROTAC compound targeted to degrade intracellular BCL-3 and a pharmacologically or physiologically acceptable salt thereof, or comprises a formulation-targeted modified form or a drug-targeted modified form of the PROTAC compound targeted to degrade intracellular BCL-3.

As a preferred embodiment of the invention, the medicament for treating tumors comprises a pharmaceutical composition composed of PROTAC compounds targeted to degrade intracellular BCL-3 and a pharmaceutically acceptable carrier.

As a preferred embodiment of the present invention, the pharmaceutically acceptable carrier comprises a solvent, a solubilizer, a cosolvent, an emulsifier, a flavoring agent, a coloring agent, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a pH regulator, a stabilizer, a surfactant, and/or a preservative.

Compared with the prior art, the invention has the beneficial effects that:

1. The PROTAC compound for targeted degradation of intracellular BCL-3 provided by the invention can thoroughly degrade BCL-3 protein in cells by using ubiquitin-proteasome system compared with the existing BCL-3 inhibitor. . The PJ1799 compound can kill colorectal cancer cells effectively and has less influence on normal cells.

2. The invention provides a preparation method of PROTAC compounds for targeted degradation of intracellular BCL-3, which designs the conformational relation between an inhibitor and BCL-3 protein, and the molecular locus is more suitable for crosslink reaction; meanwhile, the E3 ligand is protected, so that the selectivity of the reaction process is ensured, the reaction condition is mild, and the method is simple and convenient.

3. The invention provides an application of PROTAC compounds for targeted degradation of intracellular BCL-3, which increases PROTAC aggregation in tumor cells and reduces systemic toxic and side effects through two strategies of drug targeting (folic acid molecule modification) and preparation targeting (albumin encapsulation).

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of JS6 molecules synthesized in example 1;

FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of PJ1799 molecule synthesized in example 1;

FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of PJ1799-fo molecule of folic acid coupled PJ1799 synthesized in example 2;

FIG. 4 is a graph of PJ1799-alb nanoparticle data for albumin-coated PJ1799 synthesized in example 2;

FIG. 5 is a graph showing the expression of BCL-3 and K-M survival prognosis information in TCGA RNA-seq data of colorectal cancer patients.

FIG. 6 is a graph showing comparison of BCL-3 protein expression in human knot CRC cell lines and human normal cell lines;

FIG. 7 is a graph showing experimental comparisons of JS6 and PJ1799 in drug CCK 8;

FIG. 8 is a graph showing the killing performance of PJ1799 drug against patient-derived CRC tumor organoids;

FIG. 9 shows BCL-3 protein expression patterns in CRC cells treated with PJ1799 molecules at different concentrations;

FIG. 10 shows BCL-3 protein expression patterns in CRC cells treated with PJ1799 molecules at various times;

FIG. 11 is a graph showing the comparison of BCL-3 protein expression in CRC cells after treatment with PJ179 molecules and proteasome inhibitors and lysosomal inhibitors;

FIG. 12 is a graph showing the cell cycle of the PJ1799 molecule after the CRC cell treatment;

FIG. 13 is a graph showing comparison of drug tumor killing performance after targeting modification of PJ1799 molecule;

FIG. 14 is a graph showing experimental results of different PROTAC molecules in a mouse colorectal carcinoma subcutaneous tumor model;

FIG. 15 is a graph showing experimental results of various PROTAC molecules in a mouse peritoneal disseminated tumor model.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.

EXAMPLE 1 Synthesis and characterization of PROTAC Compound PJ1799, a target degrading intracellular BCL-3

The structure of PROTAC compound PJ1799 of targeted degradation cell BCL-3 is shown as the following formula:

The nuclear magnetic resonance hydrogen spectrum of PROTAC compound PJ1799 compound of the targeted degradation cell BCL-3 is shown in figure 2, and the structural correctness is confirmed through the nuclear magnetic resonance hydrogen spectrum. The data of nuclear magnetic resonance hydrogen spectrum are ：0.94(s,9H),2.22(s,3H),2.23(dd,J＝2.4,8.0 1H),2.38-2.58(m,7H),3.42-3.65(m,22H),3.70-3.75(m,1H),4.10-4.40(m,4H),5.30(s,1H)7.16-7.20(m,2H),7.29-7.50(m,5H),7.78-7.88(m,3H),8.22(brs,1H),8.40(brs,1H)8.56(d,J＝7.2Hz,1H),9.05(s,1H),8.82(brs,1H),11.8(s,1H).

Synthesis of BCL-3 small molecule inhibitor JS6

The synthesis was carried out using the method reported by Soukupova (Mol CANCER THER, 2021). Specifically, anthranilic acid (1.0 g,7.29 mmol) and fluorobenzyloxy chloride (2.54 g,16 mmol) were added to 10ml of pyridine, and after stirring at room temperature for 5 hours, the reaction solution was slowly poured into 50ml of a 10% aqueous sodium carbonate solution to precipitate to obtain intermediate 2- (2-fluorophenyl) -4H-benzo [ d ] [1,3] oxazin-4-one.

The intermediate was dissolved in 10ml of DMF at room temperature, and N, N-diisopropylamine (0.72 mL,4.15 mmol) and 2- (aminoethyl) morpholine (0.60 mL,4.56 mmol) were added and reacted for 18 hours after complete dissolution. After the reaction, the reaction mixture was diluted with 50ml of distilled water, extracted with ethyl acetate, washed, dried and purified to obtain JS6 small molecule. The nuclear magnetic resonance hydrogen spectrum of JS6 is shown in FIG. 1, and the structure is correct by the nuclear magnetic resonance hydrogen spectrum, and the yield is about 73%. The synthesis route of JS6 small molecule is:

2. Design and synthesis of PROTAC compound PJ1799 for targeted degradation of intracellular BCL-3

Based on the structure-activity relationship of J6S in BCL-3, the benzene ring where Br is located in JS6 is easy to perform crosslink reaction, so that Br groups are optimal Linker connecting sites, glycol connecting chains with better solubility are selected as Linker, VHL is used as a ligand of E3 ligase, and PROTAC compound PJ1799 based on BCL-3 and used for targeted degradation of intracellular BCL-3 is designed and synthesized.

Specifically, the synthesis step of the molecule PJ1799 comprises the following steps:

VHL, triethylamine (0.5 mmol,506 mg) and (E) -14, 14-dimethyl-3, 6, 9-trioxa-12-aza-heptadec-12-enoic acid (0.2 mmol,55.0 mg) were dissolved in 50ml of dichloromethane, the reaction was stirred and heated to 40℃overnight, the reaction system was cooled to room temperature, 5ml of hydrochloric acid (1M) was added to acidify the reaction product, and the resultant was concentrated by separation, and purified by chromatography to give compound A. The hydroxyl group on VHL in compound a was modified with tetramethylene sulfoxide (TMSO). The TMSO protects the hydroxyl in advance, so that amino competition and boric acid reaction are avoided in the next reaction with Gul, and the reaction selectivity is ensured. The synthetic route for compound a is as follows:

according to the structure-activity relationship based on J6S in BCL-3, the benzene ring where Br is located in JS6 is easy to carry out crosslink reaction, so that Br groups are optimal Linker connecting sites. The reaction route for Miyaura boronation of the Br site on the JS6 benzene ring obtained in example 1 is as follows:

The compound A and the JS6 small molecule (B) obtained after site selection are dissolved in 20ml of MeCN, 4 equivalents of Cu (OAc) ₂ are added into the mixed solution, the mixture is stirred for hours at room temperature, and the obtained product is purified by a chromatographic column to obtain a PROTAC compound PJ1799 compound for targeted degradation of intracellular BCL-3. The nuclear magnetic resonance hydrogen spectrum of PROTAC compound PJ1799 compound of targeted degradation intracellular BCL-3 is shown in figure 2, and the structural correctness is confirmed by the nuclear magnetic resonance hydrogen spectrum, and the yield is about 67%. The synthetic route of PROTAC compound PJ1799 compound targeted to degrade intracellular BCL-3 is as follows:

example 2 Targeted modification of PROTAC Compound PJ1799 targeting degradation of intracellular BCL-3

And performing folic acid coupling on PROTAC compounds of targeted degradation intracellular BCL-3 to obtain PJ1799-fo, wherein the structure is shown in the following formula. The nuclear magnetic resonance hydrogen spectrum of PJ1799-fo is shown in FIG. 3.

1. Drug targeting modification: coupling of PJ1799 with folic acid TMSO on the VHL ligand of the PJ1799 compound obtained in example 1 was deprotected and reacted with folic acid at 120℃for 2 hours, the reaction system further comprising HCl (1M), dichloromethane and ethanol as organic solvents, the synthetic route being as follows:

After the reaction, PROTAC molecules PJ1799-fo compound modified by folic acid is obtained, the nuclear magnetic resonance hydrogen spectrum is shown in figure 3, and the yield is 87%. The data of nuclear magnetic resonance hydrogen spectrum are 0.94(s,9H),2.20-2.35(m,8H),2.38-2.58(m,7H),3.42-3.65(m,22H),3.70-3.75(m,1H),4.10-4.40(m,6H),4.55(t,J＝6.8Hz),5.30(s,1H),6.28(s,1H),6.6(s,2H),6.85(d,J＝8.0Hz),7.16-7.20(m,2H),7.29-7.50(m,5H),7.57(d,J＝8.0Hz),7.78-7.88(m,3H),8.02(s,1H),8.22(brs,1H),8.40(brs,1H)8.56(d,J＝7.2Hz,1H),9.05(s,1H),8.82(brs,1H),11.8(s,1H).

2. And (3) preparation targeting modification: albumin encapsulation by PJ1799

The PJ1799 compound obtained in the example 1 is dissolved in a small amount of tetrahydrofuran, the solution is added into an albumin solution in a dropwise manner under the assistance of ultrasound, the tetrahydrofuran is removed by rotary evaporation after the solution is stable, and the solution is preserved at 4 ℃ in a dark environment. The particle size and morphology of the nano-drug are regulated and controlled by regulating the reaction conditions such as reactant concentration, solution pH value, ultrasonic power and the like, and the formed nano-particle is PJ1799-alb. Nanoparticles of uniform size and particle size of 150-200nm are preferred for in vivo/in vitro biological evaluation. The structure and particle size of nanoparticle PJ1799-alb are shown in FIG. 4.

EXAMPLE 3 comparison of tumor killing Activity of drugs of JS6 and PJ1799

Taking CRC colorectal cancer as an example, the invention downloads and sorts RNA-seq data of 698 colorectal cancer tumor tissues and 51 paracancerous tissues from a TCGA database, and analysis finds that the expression of BCL-3 of the tumor tissues is obviously higher than that of the paracancerous tissues, and the high expression of BCL-3 protein of a patient is inversely related to the survival rate of the CRC patient, and the expression quantity of BCL-3 protein of CRC cells of most people is obviously higher than that of normal cells through Western Blotting, as shown in fig. 5 and 6.

JS6 is known as an inhibitor of BCL-3, and JS6 and PJ1799 of the application are adopted to carry out CCK8 cell proliferation and toxicity detection experiments on different colorectal cancer tumor cells, and objects comprise human colorectal adenocarcinoma cells HCT15, human colon carcinoma cells HCT116, colorectal adenocarcinoma epithelial cells DLD1, human ileocecum carcinoma cells HCT8, human colon carcinoma cells sw620, a low-differentiation colon carcinoma cell line RKO and primary colon adenocarcinoma cells sw480.

The tumor killing activity of the drugs of JS6 and PJ1799 is shown in figure 7, and the result shows that the BCL-3 inhibitor JS6 has no obvious effect on killing human CRC cells, but the PJ1799 prepared in the embodiment 1 of the invention has obvious effect on killing human CRC cells. Moreover, as the concentration of PJ1799 increases, the activities of human renal cortex proximal tubular epithelial cells HK2, neutrophil GES1 and human peritoneal mesothelial cells HMrCv remain at 70-100%, indicating that PJ1799 compounds kill human normal cells poorly; in the results of IC ₅₀ values for the cancer and normal groups, IC ₅₀ values for PJ1799 were significantly reduced in the cancer group, indicating that PJ1799 is effective in killing colorectal cancer cells while not affecting normal cells.

EXAMPLE 4 study of the mechanism of drug action of PJ1799 on CRC

Experiments were performed by collecting patient-derived CRC tumor cells and using PJ1799, the killing performance of PJ1799 drug on patient-derived CRC tumor organoids is shown in fig. 8, where fig. 8 (a) is a white light picture and fig. 8 (b) is an AM/PI stained picture.

Further, by Western Blotting experiments, we found that PJ1799 has a concentration and time dependence on intracellular BCL-3 protein degradation. Beta-actin is used as reference protein, and experiments are carried out under the conditions of different concentrations and different treatment times respectively. As shown in FIG. 9, western Blotting analysis of BCL-3 protein expression in CRC cells treated with PJ1799 molecules at different concentrations shows that the inhibition effect on BCL-3 is better as the concentration of PJ1799 increases. Western Blotting analysis of BCL-3 protein expression in CRC cells over various time periods with PJ1799 molecule treatment is shown in FIG. 10, and shows that PJ1799 compounds are effective in inhibiting BCL-3 formation after 12-36 hours of treatment.

On the other hand, the route of action of PJ1799 was studied by comparison of PJ1799 with the treatment of human CRC cells with the proteasome inhibitor MG132, the lysosome inhibitor NH ₄ Cl. Analysis by Western Blotting experiments, as shown in fig. 11, showed that PJ1799 was degrading BCL-3 protein by intracellular UPS system, independent of lysosomal degradation pathway.

The difference in cell cycle after treatment of CRC cells with PJ1799 molecule was studied by flow cytometry, and the results are shown in fig. 12, which show that PJ1799 can block the CRC cell cycle in G0/G1 phase, thereby inhibiting tumor cell proliferation.

EXAMPLE 5 tumor killing Properties of Targeted modified drugs of PJ1799

CCK8 cell experiments were performed on PJ1799, PJ1799-fo and PJ1799-alb in example 1 and example 2, with human colorectal adenocarcinoma cells HCT15 and human colon carcinoma cells HCT116, respectively. The results are shown in FIG. 13, wherein FIG. 13 (a) shows the PJ1799 molecule prepared in example 1, FIG. 13 (b) shows the PJ1799-fo of folic acid coupled with the PJ1799 molecule, and FIG. 13 (c) shows the PJ1799-alb drug of albumin coated with the PJ1799 molecule, and the results show that the modified drug can further reduce the cell activity to 0% and has stronger killing effect on human CRC cells by coupling folic acid ligand and albumin coated with the PJ1799 drug. Because folic acid receptor is over-expressed in various types of tumors, PJ1799-fo increases the aggregation of BCL-3PROTAC in tumor cells by utilizing the characteristic, and further improves the killing effect of the drug on human CRC cells. And also seen a significant anti-tumor effect on the mouse colorectal carcinoma subcutaneous tumor model, as shown in fig. 14, the tumor killing ability of PJ1799-fo was greater than PJ1799 fig. 14. On the other hand, the serum albumin structure is provided with a plurality of hydrophobic cavities, and can be loaded with hydrophobic drugs. The PROTAC compound of the hydrophobic targeted degradation intracellular BCL-3 is wrapped in albumin by an ultrasonic auxiliary method so as to increase the tumor targeting and the tumor endocytosis efficiency of the albumin, and the tumor model is spread in the abdominal cavity of a mouse to show stronger tumor killing performance, as shown in figure 15.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A PROTAC compound for targeted degradation of intracellular BCL-3, wherein the structural formula of the PROTAC compound for targeted degradation of intracellular BCL-3 has a structure represented by general formula (i):

Wherein n represents a natural number of 1 to 5; r is an E3 ligase ligand; the E3 ligase ligand is one of VHL, MDM2, clAP1 or CRBN ligand, and the structural formula is shown as follows:

2. the PROTAC compound that targets and degrades intracellular BCL-3 of claim 1, wherein preferably, R is a VHL ligand; when R is a VHL ligand, the structural formula of the PROTAC compound for targeted degradation of intracellular BCL-3 has a structure shown in a general formula (II):

3. The PROTAC compound for targeted degradation of intracellular BCL-3 of claim 2, wherein the PROTAC compound for targeted degradation of intracellular BCL-3 has the structural formula shown in formula (iii):

4. a derivative of a PROTAC compound that targets degradation of intracellular BCL-3, wherein the derivative is a derivative of a PROTAC compound that targets degradation of intracellular BCL-3 coupled to folic acid as claimed in any one of claims 1 to 3.

5. A method of preparing a PROTAC compound targeted to degrade intracellular BCL-3 according to any one of claims 1-3, comprising the steps of:

6. The method of claim 5, wherein when the PROTAC compound targeted to degrade intracellular BCL-3 has the structural formula shown in formula (iii), the preparation of compound a comprises the steps of:

S1, dissolving VHL, triethylamine and (E) -14, 14-dimethyl-3, 6, 9-trioxa-12-aza-heptadec-12-enoic acid in an organic reagent, stirring and heating to 40 ℃, and reacting overnight;

7. The method of claim 6, wherein the ratio of VHL, triethylamine, and (E) -14, 14-dimethyl-3, 6, 9-trioxa-12-aza-heptadec-12-enoic acid is VHL: triethylamine: (E) -14, 14-dimethyl-3, 6, 9-trioxa-12-aza-heptadec-12-enoic acid= (0.1-1): (0.1-2.5):

(0.1-1)。

8. use of a PROTAC compound that targets degradation of intracellular BCL-3 according to any one of claims 1-3 in the manufacture of a medicament for the treatment of a tumor.

9. The use of claim 8, wherein preparing a tumor therapeutic comprises: loading PROTAC compound of targeted degradation intracellular BCL-3 into a nano-carrier; preferably, the nanocarrier is albumin.

10. The medicine for treating tumor is characterized in that the medicine for treating tumor is formed by loading PROTAC compound of targeted degradation intracellular BCL-3 on nano carrier.