CN113321700B

CN113321700B - Bifunctional compound for degrading target protein and application thereof

Info

Publication number: CN113321700B
Application number: CN202110649234.9A
Authority: CN
Inventors: 苏向东; 冯小龙; 白明杰; 王金戌
Original assignee: Taibirui Pharmaceutical Technology Shijiazhuang Co ltd
Current assignee: Taibirui Pharmaceutical Technology Shijiazhuang Co ltd
Priority date: 2020-06-02
Filing date: 2021-06-02
Publication date: 2022-07-01
Anticipated expiration: 2041-06-02
Also published as: CN113321700A

Abstract

The invention relates to the field of biomedicine, and particularly provides a bifunctional compound for degrading a target protein or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a polymorph thereof. The compound structure includes 3 functional moieties: LGP-LK-LGE, wherein LGP is a ligand connected to a target protein, LGE is a ligand connected to E3 ubiquitin ligase, and LK is a bridge chain connecting the above two ligands. The compounds inhibit the growth and replication of tumor cells by degrading and inhibiting specific target proteins, particularly tumor-related kinases, and further play a role in treating tumor-related diseases. The invention utilizes the improved ligand of E3 ubiquitin ligase to improve the inhibition effect on the growth of tumor cells.

Description

Bifunctional compound for degrading target protein and application thereof

Technical Field

The invention relates to the field of biomedicine, and particularly provides a bifunctional compound for degrading a target protein or a pharmaceutically acceptable salt, a stereoisomer, a solvate or a polymorph thereof. The compound structure includes 3 functional moieties: LGP-LK-LGE, wherein LGP is a ligand linked to a target protein, LGE is a ligand linked to E3 ubiquitin ligase, and LK is a bridge linking the above two ligands. The compounds inhibit the growth and replication of tumor cells by degrading and inhibiting specific target proteins, particularly tumor-related kinases, and further play a role in treating tumor-related diseases. The invention utilizes the improved ligand of E3 ubiquitin ligase to improve the inhibition effect on the growth of tumor cells.

Background

Two protein degradation pathways mainly exist in eukaryotic cells, wherein the ubiquitin-proteasome pathway is a high-efficiency and specific protein negative regulation mode, and 80% -90% of ubiquitinated proteins in the cells can be degraded by the ubiquitin-proteasome pathway. This ubiquitinated protein degradation plays an extremely important role in maintaining the levels of various proteins in cells, and involves almost all vital activities such as regulation of cell cycle, proliferation, apoptosis, metastasis, gene expression, and signal transmission. Ubiquitin proteins are highly conserved proteins that are ubiquitous in eukaryotic cells and consist of 76 amino acids. Ubiquitinated proteins can be transported to the 26S proteasome or enter lysosomal (lysosome) for digestion and degradation. The ubiquitination of the protein is carried out under the synergistic action of Ubiquitin activating enzyme (Ubiquitin activating enzyme) E1, Ubiquitin conjugating enzyme (Ubiquitin conjugating enzyme) E2 and E3 Ubiquitin ligase (Ubiquitin ligase). Firstly, ubiquitin is linked to E1 as ubiquitin in an activated state by the formation of a high energy thioester bond between the carboxyl group on its C-terminal glycine and the essential cysteine thiol group on ubiquitin activating enzyme E1; secondly, the activated ubiquitin is transferred from ubiquitin activating enzyme E1 to ubiquitin conjugating enzyme E2; finally, under the action of E3 ubiquitin ligase, the ubiquitin molecule connected with ubiquitin conjugating enzyme E2 is connected to the substrate protein by means of covalent peptide bond connection. The substrate proteins are ubiquitinated and then degraded in the proteasome. The specific recognition capability of E3 ubiquitin ligase to substrate protein determines that ubiquitin-mediated protein degradation has specificity

Protein degradation Targeting chimera protac (proteolytic Targeting chimera) technology utilizes the intracellular ubiquitin-proteasome system to degrade specific proteins. The hybrid small molecule compound utilizes a small molecule ligand which can be combined with a target protein, and is connected with a ligand of E3 ubiquitin ligase through a bridge chain fragment to form a bifunctional compound. By optimizing the size and other physicochemical properties of the bridge chain, ligands at two ends of the PROTAC molecule are simultaneously combined with the target protein and the E3 ubiquitin ligase to form a target protein-PROTACs-E3 ligase ternary complex, so that the target protein is ubiquitinated and degraded by a protease system. The PROTAC technology has the advantages of being capable of being used for degrading protein which is difficult to be prepared into drugs, strong in degradation effect, capable of keeping catalytic degradation effect at low concentration and the like. Meanwhile, because the protein degradation mode of the technology is repeated iteration, the technology has better tolerance compared with the traditional medicine under the conditions of target protein mutation and the like. The technical difficulty of PROTAC is that the conformation and site of linkage, modification of the length and composition of the bridge chain, and concentration of the target protein ligand, E3 ubiquitin ligase ligand all affect the formation of ternary complex and its stability, so the regulation is more challenging.

Although more than 600E 3 ubiquitin ligases are known to humans, there are only a limited number of compounds that are practically used in PROTAC, including VHL species, CRBN species, MDM2 species, cIAP1 species. These E3 ubiquitin ligases confer substrate specificity to achieve target protein ubiquitination. The development of ligands for E3 ubiquitin ligases has proven challenging. Von Hippel-Lindau (VHL) tumor suppressor is an important E3 ubiquitin ligase, which consists of extensin B and C, Cul2 and Rbx 1. The major substrates of VHL are hypoxia inducible factor 1(HIF-1), a transcription factor that upregulates genes such as the pro-angiogenic growth factor VEGF and the red blood cell-inducing cytokine erythropoietin in response to low oxygen levels. Patent CN 108601764 a was studied for ligands of E3 ubiquitin ligase VHL. Based on the crystal structure of VHL and ligand that has been obtained, it was demonstrated that small molecule compounds can mimic the binding pattern of the transcription factor HIF-1 mimicking the major substrate of a transcription factor). By using reasonable design, the small molecule ligand taking Von Hippel Lindau (VHL) as a receptor can be used as a substrate recognition subunit of E3 ubiquitin ligase VCB, and plays a therapeutic role in various important protein targets such as cancer, chronic anemia, ischemia, antivirus and the like. In practical application, the binding property of the ligand of VHL and E3 ubiquitin ligase is relatively weak, so that the target protein is not completely degraded, and even off-target effect is caused. The Journal of Medicinal Chemistry 2014,57,8657 and 8663 literature was also investigated for this purpose and it is believed that the important reason for the weak activity of the early VHL ligand is due to its too strong hydrophilicity. The invention performs a targeted optimization study on VHL ligands.

The tumor is a disease with high morbidity and mortality in the global scope, seriously threatens the human health and becomes one of the important social problems facing all countries in the world. Liver cancer is the fourth most advanced tumor in our country, about 46 ten thousand new patients with liver cancer are newly added each year, and the tumor with the second most mortality rate in our country (the mortality rate is 26/10 ten thousand). Worldwide, there are over 84 million new liver cancer cases each year, and china accounts for about 50% of the world. Liver cancer lacks effective drugs, and its five-year survival rate is only 10%. The prevention and treatment of liver cancer is very severe. The invention designs and prepares the bifunctional compound capable of degrading target protein by combining the optimized VHL ligand and the kinase ligand which influences the growth and the propagation of tumor cells, and the result proves that the bifunctional compound can effectively inhibit the growth of the tumor cells.

Disclosure of Invention

The invention is realized by the following aspects:

first aspect of the invention: provided is a compound and pharmaceutically acceptable salts, solvates, polymorphs, isotopic labels, stereoisomers, metabolites or prodrugs thereof, wherein said compound has the structural composition LGP-LK-LGE; wherein LGP is a ligand that can bind to a target protein, preferably a ligand that can bind to a kinase; LGE is a ligand that can bind E3 ubiquitin ligase, preferably a ligand that can bind Von Hippel-Lindau tumor suppressor (pVHL); LK is a molecular fragment linking LGP to LGE.

Second aspect of the invention: the compound is characterized in that LGE has a structure represented by the following general formula:

wherein R is₁And R₂Each independently selected from H, F, NR₅R₆；

Or R₁R₂Together forming NOR₇A group;

R₃is C1-C8 alkyl, C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl containing 1, 2 or 3 heteroatoms selected from O, N, S; preferably isopropyl, tert-butyl, cyclohexyl or tetrahydropyranyl; more preferably a tert-butyl group;

R₄is H or C1-C6 alkyl;

R₅、R₆one of (A) is H and the other is selected from the group consisting of H, OH, -COR₇Or C1-C6 alkyl;

R₇is H or C1-C6 alkyl.

A third aspect of the present invention: characterized in that the LGE of the above compound preferably has the following structure:

the fourth aspect of the present invention: LK, as described in the above compounds, is linked at one end to LGP by a covalent bond and at the other end to LGE by a covalent bond, and is further characterized in that it is selected from the following structural units:

wherein m is optionally a natural number from 0 to 5; n is a natural number of 0-20; p is a natural number of 0-4; q is a natural number of 0-20; r is selected from 1-3 natural numbers; s is optionally a natural number of 1-5; x is optionally CH₂、O、S、NR₈，R₈Optionally H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy-substituted C1-C6 alkyl;

fifth aspect of the present invention: the compound as described above, characterized in that LGP is a ligand that can bind to a cancer-associated protein target; preferred are ligands that can bind to cancer-associated kinases.

The sixth aspect of the present invention: the compound, wherein the LGP-binding protein target is ABL1-2, ALK, AURKA-C, AXL, BLK, BTK, CDK, CSF1R, DDR1-2, EGFR, EPHA1-8, EPHB1-6, ERK, FGFR1-4, FLT3, FRK, KIT, MET, P38 alpha-delta, PDGFR A-B, RAF, RET, SCFR, TIE1-2, TRKA-C, VEGFR1-3, or a combined multi-kinase target.

Seventh aspect of the present invention: the above compound, characterized in that LGE can bind to Von Hippel-Lindau tumor suppressor (Von Hippel-Lindau tumor suppressor, pVHL) and accomplish ubiquitination labeling of LGP-bound protein targets.

The eighth aspect of the present invention: the LGP in the compound is selected from the following structural units:

wherein R is₉Selected from H, C1-C6 alkyl; r₁₀Selected from H or halogen.

The ninth aspect of the present invention: the compound is characterized in that the degradation of the target protein is promoted by ubiquitination of the LGP binding marker, and the growth and the reproduction of tumor cells are further inhibited.

The tenth aspect of the present invention: pharmaceutical compositions of the above compounds are provided, wherein the above compounds or pharmaceutically acceptable salts, stereoisomers, solvates or polymorphs thereof, are in combination with a carrier, additive or excipient.

Eleventh aspect of the present invention: the compounds and pharmaceutical compositions are characterized by being useful for the prevention, treatment or diagnosis of tumor-related diseases.

The twelfth aspect of the present invention: the compound of the present invention is a compound having the following structure:

drawings

FIG. 1-NH-1¹HNMR mapping.

FIG. 2-NH-1¹³A CNMR map.

FIG. 3 LC-MS spectrum of NH-1.

FIG. 4-NH-2¹HNMR atlas.

FIG. 5-NH-2¹³A CNMR map.

FIG. 6 LC-MS spectrum of NH-2.

FIG. 7 growth inhibition of huh-7 cells by compounds NH-1, NH-2 and lenvatinib.

FIG. 8 degradation of the target protein p38alpha, p38delta by NH-1, NH-2.

Detailed Description

The invention is further illustrated by the following specific examples in connection with the accompanying drawings in the examples of the invention. These examples are for the purpose of more detailed specification only and are not to be construed as limiting the invention in any way. The invention can be implemented in a number of different ways, which are defined and covered by the claims.

The materials used in the tests and the experimental methods are described generically and specifically. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. Hereinafter, the materials used and the methods of operation are well known in the art, unless otherwise specified.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The synthesis of the target compounds NH-1 and NH-2 adopts the methods shown in the reaction flow chart I and the reaction flow chart II. The structure and purity of the product are confirmed by nuclear magnetic resonance, mass spectrum and liquid chromatogram. The tumor cell inhibitory activity of NH-1 and NH-2 was evaluated on the huh-7 cell line and compared to the positive control lenvatinib. The results show that the inhibition effect of the tumor cell growth is better than that of lenvatinib. The degradation capability of NH-1 and NH-2 to the target protein P38alpha, P38delta is evaluated, and the result shows that both can effectively degrade the target protein.

The synthesis of the target compound NH-1 in the examples is based on the following reaction scheme. The key intermediate 7 is obtained by conventional chemical transformation starting from compound 1. The intermediate is subjected to amide condensation reaction with LK bridge chain 7A containing terminal carboxyl, then condensed with LGP part 9A containing terminal carboxyl after removing corresponding protecting group, and finally removed protecting group to obtain the target product (see reaction scheme I).

EXAMPLE 1 Synthesis of Compound 2

Compound 1A (5.1g, 28mmol), Compound 1(5.56g, 56mmol), Potassium acetate (5.5 g)56mmol), palladium (II) acetate (63mg, 0.28mmol) in DMA (50mL) was heated to 120 ℃ overnight. The reaction was poured into water and extracted with EA (100mL × 2). Combine the organic layers with Na₂SO₄Drying, evaporation under reduced pressure and purification on silica gel gave compound 2(2.5g, 45%) as a yellow solid.

EXAMPLE 2 Synthesis of Compound 3

To a solution of compound 2(5.55g, 27.7mmol) and cobalt chloride (9.9g, 41.6mmol) in methanol (280mL) was added sodium borohydride (5.2g, 139mmol) in portions at 0 deg.C, and the reaction was stirred at room temperature overnight. The reaction was poured into water and extracted with DCM (100mL × 2). Combine the organic layers with Na₂SO₄Drying, evaporation under reduced pressure and purification on silica gel gave compound 3 as a black oil (1.5g, 27%).

EXAMPLE 3 Synthesis of Compound 4

To a solution of compound 3(3g, 14.7mmol) and compound 3A (7.2g, 16.2mmol) in DMF (30mL) were added HOBT (2.9g, 22mmol), EDCI (4.2g, 22mmol) and TEA (2.9g, 44 mmol). The mixture was stirred at room temperature overnight. The reaction was poured into water and extracted with EA (50mL x 2). The combined organic layers were dried over Na2SO4, evaporated under reduced pressure and purified by preparative chromatography to give compound 4(2.2g, 22.6%) as a white solid.

EXAMPLE 4 Synthesis of Compound 5

A solution of compound 4(6g,9.4mmol) in HCl/EA (60mL,5M) was stirred at room temperature overnight. The compound was filtered under reduced pressure to give compound 5(4.8g, 94%) as a white solid.

EXAMPLE 5 Synthesis of Compound 6

To a solution of compound 5(2g, 3.7mmol), compound 5A (944mg, 4.1mmol) in DMF (20mL) were added HOBT (750mg, 5.6mmol), EDCI (1.06g,5.6mmol) and TEA (750mg,7.4mmol), respectively. The mixture was stirred at room temperature overnight. The reaction was poured into water and extracted with EA (20mL x 2). Combine the organic layers with Na₂SO₄Drying, evaporation under reduced pressure and purification on silica gel gave compound 6 as a yellow solid (1.5g, 53%).

EXAMPLE 6 Synthesis of Compound 7

A solution of compound 6(5g, 6.65mmol) in HCl/EA (50mL,5M) was stirred at room temperature overnight. The mixture was filtered and the filter cake was dried under reduced pressure to give compound 7 as a white solid (3.8g, 88%).

EXAMPLE 7 Synthesis of Compound 8

To a solution of compound 7(3g, 4.6mmol) and compound 7A (1.3g, 5.07mmol) in DMF (20mL) were added HOBT (933mg,6.91mmol), EDCI (1.32g,6.91mmol) and TEA (930mg,9.2mmol). The mixture was stirred at room temperature overnight. The reaction was poured into water and extracted with EA (30mL x 2). Combine the organic layers with Na₂SO₄Drying, evaporation under reduced pressure and purification on silica gel gave compound 8 as a white solid (1.6g, 34%).

EXAMPLE 8 Synthesis of Compound 9

A solution of compound 8(1g,1.11mmol) in HCl/EA (5mL,5M) was stirred at room temperature overnight. The mixture was filtered and the filter cake was dried under reduced pressure to give compound 9(780mg, 87%) as a white solid.

Example 9 Synthesis of NH-1

To a solution of compound 9(300mg, 0.376mmol) and compound 9A (177mg, 0.414mmol) in DMF (5mL) were added HOBT (76mg,0.565mmol), EDCI (108mg,0.565mmol) and TEA (114mg,1.1mmol), respectively. The mixture was stirred at room temperature overnight. Piperidine (1mL) was added to the reaction mixture at room temperature, and the mixture was stirred at room temperature for 2 hours. Purification by preparative liquid chromatography gave NH-1(70mg, 18%) as a white solid.

Example 10: identification of Compound NH-1

The purity of compound NH-1 was determined by hplc to be > 99% (95% acetonitrile-water (containing 0.05% trifluoroacetic acid; retention time 4.6 min (C18 reverse phase column 5um 4.6mm x 150 mm).

The structure of the compound NH-1 is confirmed by a nuclear magnetic resonance spectrum and a liquid chromatography-mass spectrometry spectrum, and the spectra are shown in the attached figure 1, the attached figure 2 and the attached figure 3 of the specification. Specific values are as follows:

¹H NMR(400MHz,CDCl3):δ0.71(s,2H),0.88(d,2H,J＝5.2Hz),0.92(s,9H),1.65(m,1H),2.42-2.47(m,1H),2.49(s,3H),2.65(s,1H),3.51-3.56(m,1H),3.65-3.83(m,10H),4.03(s,2H),4.07(s,3H),4.23-4.28(dd,1H),4.43-4.46(m,1H)4.50-4.56(dd,1H),4.60(d,1H,J＝9.6Hz),5.71(br,1H),6.44(d,1H,J＝5.6Hz),7.04-7.07(m,1H),7.16(s,1H),7.28-7.36(m,5H),7.44(t,1H,J＝5.2Hz),7.51(s,1H),7.76(s,1H),8.37(s,1H),8.39(s,1H),8.61(d,1H,J＝5.2Hz),8.66(s,1H),9.11(s,1H)。

¹C NMR(400MHz,CDCl3):δ7.49,16.03,22.62,26.35,29.69,35.53,36.49,39.75,43.14,50.98,56.27,56.34,56.48,58.96,70.11,70.15,70.35,71.37,103.06,108.14,115.61,120.57,121.75,121.96,122.91,123.29,127.22,128.15,129.43,130.87,131.59,133.85,138.13,148.31,148.39,150.29,152.11,153.34,155.93,158.37,162.70,164.97,169.61,170.76,171.15。

mass spectrum showed MS 986(M +2H)⁺。

The target compound, NH-2, was prepared based on the procedure shown in scheme two in the examples. Intermediate 12 was obtained by conventional chemical transformation starting from compound 7A. The intermediate is subjected to amide condensation with 7, followed by removal of the corresponding protecting group, condensation with LGP moiety 14A containing a terminal carboxyl group, and final removal of the protecting group to give the desired NH-2 (see scheme II).

EXAMPLE 11 Synthesis of Compound 11

To a solution of compound 7A (4g, 15.2mmol) in DMF (40mL) was added 60% NaH (1.8g,45.6mol), and the reaction was stirred at 0 ℃ for 1 hour. MeI (21.6g,152mmol) was then added dropwise to the mixture at 0 ℃. The reaction was stirred at room temperature overnight. NH for the mixture₄The reaction was quenched with Cl and then extracted with EA (2 × 50 mL). The combined organic layers were washed with brine (2 × 50mL) and Na₂SO₄Dried and concentrated in vacuo to afford crude compound 11 as a yellow liquid (4.3g, 97% yield).

EXAMPLE 12 Synthesis of Compound 12

To a solution of compound 11(2g, 6.8mmol) in THF (20mL) and water (10mL) was added NaOH (554mg,13.6mmol) at room temperature and the mixture was heated to 50 ℃ for reaction overnight. The reaction solution was adjusted to pH 2-3 with 1N hydrochloric acid (20mL) to 1N (20mL) and then extracted with EA (30mL × 2). For organic layersWashed with water and then Na₂SO₄Dried and concentrated in vacuo to afford crude compound 12(1.2g, 63%) as a yellow oil.

Example 13 Synthesis of Compound 13

To a solution of compound 4(500mg, 0.76mmol) and compound 3(237mg, 0.844mmol) in DMF (5mL) were added HOBT (155mg,1.15mmol), EDCI (220mg,1.15mmol) and TEA (232mg,2.3 mmol). The mixture was stirred at room temperature overnight, the reaction was poured into water and extracted with EA (30mL x 2). Combine the organic layers with Na₂SO₄Drying, evaporation under reduced pressure and purification on silica gel gave compound 13 as a white solid (410mg, 58.7%).

EXAMPLE 14 Synthesis of Compound 14

A solution of compound 13(1g,1.11mmol) in HCl/EA (10mL,5M) was stirred at room temperature overnight. The mixture was filtered and the filter cake was dried under reduced pressure to give compound 14 as a white solid (820mg, 92%).

EXAMPLE 15 Synthesis of Compound NH-2

To a solution of compound 14(1g, 1.23mmol) and compound 14A (612mg, 1.35mmol) in DMF (10mL) was added HOBT (250mg,1.85mmol), EDCI (353mg,1.85mmol) and TEA (374mg,3.7 mmol). The mixture was stirred at room temperature overnight. 3mL of piperidine was added at room temperature, and the reaction was stirred at room temperature for 2 hours. The mixture was purified using preparative liquid chromatography to give NH-2 as a white solid (140mg, 9.15%).

Example 16: identification of Compound NH-2

The purity of compound NH-2 was determined by hplc to be > 98% (95% acetonitrile-water (containing 0.05% trifluoroacetic acid; retention time 6.2 min (C18 reverse phase column 5um 4.6mm x 150 mm).

The structure of the compound NH-2 is confirmed by a nuclear magnetic resonance spectrum and a liquid chromatography-mass spectrometry spectrum, and the spectra are shown in the attached figure 4, the attached figure 5 and the attached figure 6 of the specification. The specific values are as follows:

¹H NMR(400MHz,CDCl3):δ0.96(s,9H),1.26(m,1H),1.81-1.85(m,1H),2.42-2.49(m,4H),3.12(d,3H,J＝12.4Hz),3.52-3.64(m,7H),3.75-3.85(m,4H),3.89-4.00(m,2H),4.34-4.39(m,1H),443-4.49(m,1H),4.55(d,1H,J＝8.8Hz),4.69(t,1H,J＝6.8Hz),6.89-6.99(m,4H),7.18-7.41(m,9H),7.57(d,1H,J＝8.8Hz),7.69(d,1H,J＝8.0Hz),8.31-8.41(m,2H),8.48(s,1H),8.68(s,1H)。

¹C NMR(400MHz,CDCl3):δ16.05,26.36,26.57,29.69,34.35,35.19,35.21,36.65,36.78,38.36,43.19,47.86,50.54,50.96,50.99,56.26,56.87,59.17,59.24,68.61,69.05,70.27,70.33,70.42,70.50,70.86,71.12,110.06,110.72,113.25,117.47,117.51,121.22,121.38,121.44,121.49,122.54,124.09,124.61,128.04,128.21,128.52,130.01,131.55,131.58,131.75,136.81,137.83,137.89,138.34,148.36,148.45,150.33,150.38,150.53,152.82,155.77,155.89,166.12,166.23,169.32,169.81,169.84,170.02,170.97,171.06,171.34,171.39。

mass spectrum showed MS 1023(M +1H)⁺。

Example 17 cell growth inhibition assay

Collecting hepatocarcinoma cell (huh-7) with 2 × 10⁴The cells/mL were plated in 96-well plates with 100uL per well. Incubate overnight.

Adherence was confirmed by microscopic examination of the cells every other day. Preparing a compound, respectively weighing a certain amount of NH series compounds or a control drug lenvatinib, firstly preparing a mother solution with higher concentration by using pure DMSO, adopting a gradient dilution method to prepare a liquid medicine with 10% DMSO-containing compound concentration of 10nM/100nM/300nM/1000nM/3000nM, and shaking to fully and uniformly mix the liquid medicine. The control group was prepared with lenvatinib in the same solvent and manner.

After preparation, 10uL of the liquid medicine is added into each hole of a 96-hole plate, and each dose is repeated three times. The cells and the drug solution were repeatedly contacted by gentle shaking, and incubated in an incubator for 72 hours. After 72 hours, the cells were removed, developed with cck-8 kit, 10uL cck-8 reagent was added to each well, shaken gently and incubated in an incubator for two hours. After two hours, the wells were removed and the absorbance of each well was measured using a microplate reader at a wavelength of 450nm according to the formula: the cell growth inhibition rate is [1- (experimental absorbance-medium control absorbance)/(blank control absorbance-medium control absorbance) ] × 100%, and the growth inhibition ability of the compound on the liver cancer cells at the current dose and time can be obtained. And a graph is drawn according to the concentration gradient, so that the result can be visually compared. See figure 7 for results. GI50 values (see table one) were calculated by the program GRAPHPAD PRISM.

The results show that NH-1, NH-2 has better tumor cell growth inhibition than lenvatinib.

Example 18: target protein degradation assay

Collecting hepatocarcinoma cell (huh-7 in this patent) with 4 × 10⁵The cells were seeded at a concentration of 2mL per well in 6-well plates and incubated to wait for the cells to adhere.

And (3) after 24h, preparing compounds, namely weighing a certain amount of NH series compounds of the patent, preparing mother liquor with higher concentration by using pure DMSO, preparing a liquid medicine with a solvent of 10% DMSO-containing compound concentration of 10nM/30nM/100nM/300nM/1000nM or 1nM/3nM/10nM/30nM/100nM by adopting a gradient dilution method, and shaking to fully and uniformly mix the liquid medicine. 200uL of liquid medicine is added into each hole according to the ratio of the liquid medicine to the culture medium of 1: 10.

After 24 hours incubation, the cells were removed, the medium was aspirated with a pipette and washed 3 times with PBS. The liquid in the well plate was blotted dry, 150uL RIPA lysate was added to each well and the cells were lysed by incubation on ice for 30 min.

Collecting cell lysate, centrifuging at 16000rpm for 10 min, taking supernatant, leveling total protein content with BCA kit, and detecting p38alpha and p38delta content of target protein by Western blot method.

The results were analyzed by ImageJ, the grey values of the individual bands were measured, and the proteins were quantified and analyzed by comparison according to the amount of protein-experimental grey value/blank grey value, see figure 8 and table two.

In addition to P38alpha and P38delta proteins, the NH series of compounds of this patent also showed superior degradation effects on ABL1-2, ALK, AURKA-C, AXL, BLK, BTK, CDK, CSF1R, DDR1-2, EGFR, EPHA1-8, EPHB1-6, ERK, FGFR1-4, FLT3, FRK, KIT, MET, P38 beta, P38gamma, PDGFR A-B, RAF, RET, SCFR, TIE1-2, TRKA-C, VEGFR1-3, and the like, according to the method of example 18.

It is to be understood that the specific examples and embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention, and that various further modifications or changes included within the spirit and scope of the present application are intended to be included within the scope of the present disclosure.

Claims

1. A compound and pharmaceutically acceptable salts thereof, wherein said compound has the structural composition LGP-LK-LGE;

wherein the LGP is a ligand capable of binding kinase, and is characterized in that the LGP-bound protein target is P38 alpha-delta and is selected from the following structural units:

wherein R is₉Selected from H, C1-C6 alkyl;

LGE is a ligand that binds Von Hippel-Lindau tumor suppressor (Von Hippel-Lindau tumor suppressor, pVHL) characterized in that the LGE has the following structure:

；

LK is a molecular fragment linking LGP to LGE.

2. The compound of claim 1, wherein LK is linked at one end to LGP by a covalent bond and at the other end to LGE by a covalent bond, and is further characterized by being selected from the group consisting of the following structural units:

wherein r is a natural number of 1-3, and m is a natural number of 1-3.

3. A compound and pharmaceutically acceptable salts thereof, wherein the compound has the structure:

4. a pharmaceutical composition comprising a compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, which may be a solvate or polymorph, an isotopic label, an additive or an excipient.

5. Use of a compound as claimed in any one of claims 1 to 3, and pharmaceutically acceptable salts thereof, or a pharmaceutical composition as claimed in claim 4, in the manufacture of a medicament for the treatment of tumours.