GSH-responsive anti-tumor nano preparation coupled with protein degradation and chemotherapeutic drugs as well as preparation method and application of GSH-responsive anti-tumor nano preparation

Abstract

The invention discloses a GSH-responsive anti-tumor nano preparation coupled with protein degradation and chemotherapeutic drugs as well as a preparation method and application of the GSH-responsive anti-tumor nano preparation, and belongs to the technical field of pharmacy. On the basis of a targeted EGFR protein degradation agent, a GSH responsive disulfide bond is coupled with a chemotherapeutic drug DOX to obtain PDC-S, so that PROTAC molecules can be self-assembled into nanoparticles in a water solution. Therefore, not only is the water solubility of the PROTAC improved, but also the advantages of the nanoparticles can be played, and a solution is provided for solving the problems faced by the PROTAC.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to an anti-tumor nano preparation of GSH-responsive protein degradation coupled with a chemotherapeutic drug, and a preparation method and application thereof. Background Art

[0002] PROTAC uses the cell's own ubiquitin proteasome system to enable the target protein to be recognized by the E3 ubiquitin ligase and tagged with ubiquitination, and then the target protein is recognized and degraded by the proteasome. Compared with traditional small molecule inhibitors, PROTAC has significant advantages: 1) Low-dose catalytic degradation function. Unlike the mode of action of traditional small molecule inhibitors, PROTAC only needs a lower compound concentration to achieve good degradation efficiency; 2) High selectivity: PROTAC has higher selectivity for homologous target proteins than traditional small molecule inhibitors; 3) Overcoming drug resistance: The reason for drug resistance of some small molecule inhibitors is often that the target protein mutates, which weakens or even loses the inhibitory effect of small molecule inhibitors on the target protein. PROTAC directly degrades the target protein, which can avoid drug resistance caused by point mutations to a certain extent; 4) Difficult-to-drug targets achieve drugability: proteins that cannot be directly acted on by traditional small molecule inhibitors such as transcription factors, regulatory proteins and other non-enzyme proteins become regulated proteins, expanding the drug target space. At the same time, PROTAC also has some defects that are difficult to overcome: 1) So far, the E3 ligase used by PROTAC is widely expressed in tumor cells and normal tissues, and it is difficult to avoid the occurrence of toxic side effects. Moreover, since PROTAC directly degrades the target protein, its toxic side effects may be greater than those of inhibitors; 2) PROTAC is a heterobifunctional molecule, which is a composite of the target protein ligand and the E3 ligand. It will inevitably produce high molecular weight compounds, making it difficult to maintain a balance between acceptable permeability and solubility in its physical and chemical properties, which seriously affects the drugability of PROTAC; 3) The ligand end of the PROTAC target protein is usually composed of small molecule inhibitors, which may face the drug resistance problem faced by small molecule inhibitors. Therefore, how to enhance the tumor targeting of PROTAC molecules, improve their water solubility and cell permeability, and overcome potential drug resistance are key scientific issues that need to be urgently solved in drug research in this field.

[0003] The PROTAC molecules that have been reported to target and degrade EGFR proteins also face the above problems. At present, it has been reported that several solutions that can overcome the problems of PROTAC include several antibody-drug conjugates (ADCs)-mediated PROTACs that have been developed to degrade proteins by targeting cell membrane receptors (such as HER2 and Erα). However, antibody-based PROTACs are usually composed of a large number of amino acids, and have disadvantages such as high molecular weight, poor cellular uptake, complex pharmacokinetics, and unstable systemic administration, which limits their clinical application. It has been previously reported that the aptamer AS1411 was synthesized by conjugating the von Hippel-Lindau (VHL) ligand part through a cleavable linker-PROTAC. Aptamer-PROTAC exhibits excellent specific protein degradation ability in tumor cells. However, aptamers are easily hydrolyzed by nucleases, and the relatively high molecular weight, stability, and high preparation cost issues greatly hinder in vivo applications.

[0004] Therefore, if it is possible to solve the problems faced by PROTAC itself while also solving the potential drug resistance problem, it will have great application prospects. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-tumor nanoformulation coupled with GSH-responsive protein degradation and chemotherapeutic drugs, and its preparation method and application, in view of the deficiencies of the prior art, overcome the defects of existing drug treatments, and obtain PDC-S by coupling with chemotherapeutic drugs DOX through GSH-responsive disulfide bonds based on targeted EGFR protein degraders, so that the PROTAC molecules themselves can self-assemble into nanoparticles in water. Therefore, the present invention not only improves the water solubility of PROTAC, but also can give play to the advantages of nanoparticles, and provides a solution to improve the problems faced by PROTAC.

[0006] To achieve the above object, the present invention provides an anti-tumor nanoformulation coupled with GSH-responsive protein degradation and chemotherapeutic drugs, wherein the anti-tumor nanoformulation comprises a compound of general formula (I) or general formula (II) or a pharmaceutically acceptable salt of a compound of general formula (I) or general formula (II);

[0007]

[0008] Wherein, in the general formula (I) or the general formula (II), A is S, C, O, n1=1-8, n=0-5;

[0009] X, R, R1, Y and Z are all one of saturated or unsaturated straight chain hydrocarbon groups, oxygen heterochains, phenyl groups and heterocyclic groups having 1 to 14 carbon atoms;

[0010] The heterocyclic group is one of piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl.

[0011] Preferably, X is also one of the following linking groups, wherein n=0-5,

[0012]

[0013] Preferably, R and R1 are also one of the following linking groups, wherein n=0-5,

[0014]

[0015] Preferably, Y is also one of the following linking groups, wherein n=0-5,

[0016]

[0017] Z is also one of the following linking groups, wherein n=0-5,

[0018]

[0019] Preferably, the pharmaceutically acceptable salt is an acid addition salt formed by the compound of general formula (I) or general formula (II) and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0020] The present invention also provides a method for preparing the above-mentioned anti-tumor nano-preparation of GSH-responsive protein degradation coupled with chemotherapeutic drugs, comprising the following steps:

[0021] a. Compound 1 and compound 2 are dissolved in dimethylacetamide, and N,N-diisopropylethylamine is added to react at room temperature for 4-8 hours to obtain compound 3;

[0022] b. Compound 3 and compound 4 were dissolved in dimethylacetamide, and 4-dimethylaminopyridine was added to react for 4-8h to obtain compound 5;

[0023] c. Compound 5 and p-nitrophenyl chloroformate were dissolved in DCM, and N,N-diisopropylethylamine was added to react at room temperature for 8-12h to obtain compound 6;

[0024] d. Compound 6 and compound 7 were dissolved in N,N, dimethylformamide, and N,N-diisopropylethylamine was added to react at room temperature for 1-2h to obtain compound 8;

[0025] e. Compound 8 was dissolved in an appropriate amount of ethanol or DMSO and then added dropwise to ddH2O and stirred at 500 rpm on a magnetic stirrer. After the ethanol in the solution was evaporated or the DMSO was removed by dialysis, a spherical structure of GSH-responsive protein degradation and chemotherapeutic drug-coupled anti-tumor nanoformulation was obtained;

[0026] The general formula of the compound 1 is:

[0027] The compound 2 is N,N'-carbonyldiimidazole;

[0028] The general formula of compound 4 is

[0029] The compound 7 is doxorubicin;

[0030] The above steps ad were purified by silica gel column chromatography, the eluents were dichloromethane and methanol, and the volume of dichloromethane to methanol was 100:(3-8).

[0031] Preferably, when the general formula of compound 1 is When , the general formula of compound 8 is general formula (I).

[0032] Preferably, when the general formula of compound 1 is When , the general formula of compound 8 is general formula (II).

[0033] The anti-tumor nanometer preparation provided by the present invention, which is a combination of GSH-responsive protein degradation and chemotherapeutic drugs, is applied to the preparation of drugs for tumors related to EGFR expression.

[0034] Preferably, tumors associated with EGFR expression include lung cancer, liver cancer, kidney cancer, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, osteosarcoma, and tumors of the central or peripheral nervous system.

[0035] Therefore, the present invention has the following beneficial effects:

[0036] 1. The compounds of the present invention exhibit good EGFR protein degradation activity, achieve tumor inhibition by degrading EGFR protein and releasing the chemotherapeutic drug DOX in tumor cells in a GSH-responsive manner, have a certain broad-spectrum anti-tumor activity, can significantly delay tumor growth, and can be applied to EGFR-mediated tumor diseases.

[0037] 2. For the first time, the present invention optimizes the PROTAC molecular structure and couples it with the chemotherapy drug DOX, so that the PROTAC molecule has the ability to self-assemble into nanoparticles in aqueous solution without adding any excipients or a small amount of DSPE-PEG with good biocompatibility.2000 , avoiding the toxic side effects of a large number of additives in the human body.

[0038] 3. As the first reported EGFR-based anti-tumor drug, this type of compound has further development and research value.

[0039] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The present invention is a preparation flow chart of a GSH-responsive protein degradation and chemotherapeutic drug-coupled anti-tumor nanoformulation of general formula (I);

[0041] Figure 2 The preparation flow chart of the compound PDC-C in Example 1 and the compound PDC-S in Example 2 of the present invention;

[0042] Figure 3 The reaction scheme of step e in Example 2 and Example 3 of the present invention;

[0043] Figure 4 This is a SEM image of the compound PDC-S nanoparticles in Example 2 of the present invention;

[0044] Figure 5 This is a graph of the polymer dispersibility index of the target nanoparticles in the solution of the compound PDC-S nanoparticles in Example 2 of the present invention;

[0045] Figure 6 This is a potential diagram of target nanoparticles in the solution of PDC-S nanoparticles of the compound in Example 2 of the present invention;

[0046] Figure 7 This is a SEM image of the compound PDC-S NPs nanoparticles in Example 3 of the present invention;

[0047] Figure 8 This is a graph of the polymer dispersibility index of the target nanoparticles in the PDC-S NPs nanoparticle solution of the compound in Example 3 of the present invention;

[0048] Fig. 9 The figure is a diagram showing the encapsulation efficiency and drug loading ratio of the compound PDC-S NPs nanoparticle solution in Example 3 of the present invention;

[0049] Fig.10 The appearance diagrams of the compounds in Example 2 and Example 3 of the present invention, wherein (a) is the appearance diagram of the PDC-S nanoparticle solution, and (b) is the appearance diagram of the PDC-S NPs nanoparticle solution;

[0050] Fig.11This is a graph showing the degradation efficiency of PRO on EGFR protein in a concentration- and time-dependent manner in the test case;

[0051] Fig.12 This is a graph showing the degradation efficiency of EGFR protein by PDC-S in a test case in a concentration- and time-dependent manner;

[0052] Fig.13 This is a graph showing the concentration- and time-dependent degradation efficiency of PDS-C on EGFR protein in the test example. DETAILED DESCRIPTION

[0053] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0054] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0055] The present invention provides an anti-tumor nanoformulation of GSH-responsive protein degradation coupled with a chemotherapeutic drug, wherein the EGFR protein degradation nanoformulation is a compound of the general formula (I) or the general formula (II) or a pharmaceutically acceptable salt of the compound of the general formula (I) or the general formula (II);

[0056]

[0057] Wherein, in the general formula (I) or the general formula (II), A is S, C, O, n1=1-8, n=0-5; X, R, R1, Y and Z are all one of saturated or unsaturated straight chain hydrocarbon groups, oxa-chains, phenyl groups and heterocyclic groups having 1 to 14 carbon atoms; the heterocyclic group is one of piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl.

[0058] X is also one of the following linking groups, wherein n=0-5,

[0059]

[0060] R and R1 are also one of the following linking groups, wherein n=0-5,

[0061]

[0062] Preferably, Y is also one of the following linking groups, wherein n=0-5,

[0063]

[0064] Z is also one of the following linking groups, wherein n=0-5,

[0065]

[0066] Pharmaceutically acceptable salts include acid addition salts formed by compounds of general formula (I) or general formula (II) and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0067] The present invention also provides a method for preparing the above-mentioned anti-tumor nano-preparation of GSH-responsive protein degradation coupled with chemotherapeutic drugs, comprising the following steps:

[0068] a. Compound 1 and compound 2 are dissolved in dimethylacetamide, and N,N-diisopropylethylamine is added to react at room temperature for 4-8 hours to obtain compound 3.

[0069] b. Compound 3 and compound 4 were dissolved in dimethylacetamide, 4-dimethylaminopyridine was added and reacted for 4-8 hours to obtain compound 5.

[0070] c. Compound 5 and p-nitrophenyl chloroformate were dissolved in DCM, and N,N-diisopropylethylamine was added to react at room temperature for 8-12 h to obtain compound 6.

[0071] d. Compound 6 and compound 7 were dissolved in N,N, dimethylformamide, and N,N-diisopropylethylamine was added to react at room temperature for 1-2h to obtain compound 8.

[0072] e. Compound 8 was dissolved in an appropriate amount of ethanol or DMSO and then added dropwise to ddH2O and stirred at 500 rpm on a magnetic stirrer. After the ethanol in the solution evaporated or the DMSO was removed by dialysis, a spherical structured nanoformulation targeting EGFR protein degradation was obtained.

[0073] The general formula of compound 1 is:

[0074]

[0075] Compound 2 is N,N'-carbonyldiimidazole.

[0076] The general formula of compound 4 is

[0077] Compound 7 is doxorubicin.

[0078] The above steps ad were purified by silica gel column chromatography, the eluents were dichloromethane and methanol, and the volume ratio of dichloromethane to methanol was 100:(3-8).

[0079] When the general formula of compound 1 is When the general formula of compound 8 is general formula (I), the preparation process is as follows Figure 1 shown.

[0080] When the general formula of compound 1 is When the general formula of compound 8 is general formula (II), the preparation process route is similar to that of general formula (I).

[0081] The anti-tumor nano-preparation provided by the present invention, which is coupled with GSH-responsive protein degradation and chemotherapeutic drugs, is applied to the preparation of drugs for tumors related to EGFR expression. Tumors related to EGFR expression include lung cancer, liver cancer, kidney cancer, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, osteosarcoma, and tumors of the central or peripheral nervous system.

[0082] In order to further illustrate the present invention, the scheme of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0083] Embodiment 1

[0084] This embodiment provides an anti-tumor nanoformulation of GSH-responsive protein degradation coupled with chemotherapeutic drugs, 2-((2-(((((3R,)5R)-1-((R)-2-(11-(4-(3-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazin-1-yl)-11-oxobutanamide)-3,3-dimethylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl The invention discloses a method for preparing a nanometer preparation method of ((2S, 3S, 4S, 6R)-3-hydroxy-2-methyl-6-(((1S, 3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxy-1,2,3,4,6,11-hexahydrotetraoctyl-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamate, and the EGFR protein degradation nanometer preparation is code-named as compound PDC-S.

[0085] a. Compound 1 (2R, 4R)-1-((R)-2-(11-(4-(3-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-11-oxobutanamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide and N,N'-carbonyldiimidazole were dissolved in DCM, DIPEA was added dropwise to the solution, and the mixture was stirred at room temperature for 4 hours. Purification by silica gel column chromatography with an eluent of dichloromethane and methanol in a ratio of 100:3 afforded compound 3 as a white solid, (3R,5R)-1-((R)-2-(11-(4-(3-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-11-oxoundecamide)-3,3-dimethylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl 1H-imidazole-1-carboxylic acid.

[0086] b. Compound 3 and compound 4, compound 4 being (2,2'-disulfanediylbis(ethane-1-ol)) were dissolved in DCM solvent, DMAP was added to the solution, and after stirring at room temperature for 4 hours, the solution was purified by silica gel column chromatography with an eluent of dichloromethane and methanol in a ratio of 100:5 to obtain a white solid compound 5, (3R,5R)-1-((R)-2-(11-(4-(3-(3-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazin-1-yl)-11-oxoundecylamine)-3,3-dimethylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl(2-((2-hydroxyethyl)disulfanyl)ethyl)carbonate.

[0087] c. Compound 5 was dissolved in DCM, and p-nitrophenyl chloroformate was added to the solution. After stirring at room temperature for 8 hours, the solution was purified by silica gel column chromatography with an eluent of dichloromethane and methanol in a ratio of 100:5 to obtain a white solid compound 6, (3R,5R)-1-((R)-2-(11-(4-(3-(3-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazin-1-yl)-11-oxobutanamide)-3,3-dimethylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl(2-((2-(4-nitrophenoxy)carbonyl)oxy)ethyl)disulfonyl)ethyl)carbonate.

[0088] d. Compound 6 was dissolved in dichloromethane, DIPEA and DOX were added to the solution, and the mixture was reacted at room temperature for 1 hour, and then purified by silica gel column chromatography with dichloromethane and methanol in a ratio of 100:6 as the eluent to obtain a red solid compound PDC-C, 6-(((((3R,5R)-1-((R)-2-(11-(4-(3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-11-oxoundecanamide)-3,3-dimethyl (((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)amino)pyrrolidin-3-yl)oxy)carbonyl)oxy)hexyl ((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamate.

[0089] e. Dissolve 1 mg of compound PDC-C in 20 μL DMSO and then slowly add it dropwise into 1 mL of dd H2O at a rotation speed of 500 rpm. Remove DMSO by dialysis to obtain a PDC-C nanoparticle solution with a concentration of 1 mg / mL.

[0090] Embodiment 2

[0091] This embodiment provides an anti-tumor nanoformulation of GSH-responsive protein degradation coupled with chemotherapeutic drugs, 6-(((((3R,5R)-1-((R)-2-(11-(4-(3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-11-oxoundemine)-3,3-dimethylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)aminomethyl The invention discloses a method for preparing a EGFR protein degradation nanoformulation comprising: a (2S, 3S, 4S, 6R)-3-hydroxy-2-methyl-6-(((1S, 3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxy-1,2,3,4,6,11-hexahydrotetraen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamate. The EGFR protein degradation nanoformulation is code-named compound PDC-C.

[0092] a. Compound 1 (2R, 4R)-1-((R)-2-(11-(4-(3-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-11-oxobutanamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide and N,N'-carbonyldiimidazole were dissolved in DCM, DIPEA was added dropwise to the solution, and the mixture was stirred at room temperature for 4 hours. Purification by column chromatography with a ratio of 100:3 of dichloromethane and methanol as the eluent gave compound 3 as a white solid, (3R,5R)-1-((R)-2-(11-(4-(3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazin-1-yl)-11-oxoundecanoyl)-3,3-dimethylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl 1H-imidazole-1-carboxylate.

[0093] b. Compound 3 and compound 4 (hexane-1,6-diol) were dissolved in DCM solvent, DMAP was added to the solution, and after stirring at room temperature for 4 hours, the solution was purified by silica gel column chromatography with an eluent of dichloromethane and methanol in a ratio of 100:5 to obtain a white solid compound 5, (3R,5R)-1-((R)-2-(11-(4-(3-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-11-oxobutanamide)-3,3-dimethylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl(6-hydroxyhexyl) carbonate.

[0094] c. Compound 5 was dissolved in DCM, and p-nitrophenyl chloroformate was added to the solution. After stirring at room temperature for 8 hours, the solution was purified by silica gel column chromatography with an eluent of dichloromethane and methanol in a ratio of 100:5 to obtain a white solid compound 6, (3R,5R)-1-((R)-2-(11-(4-(3-(4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-11-oxobutanamide)-3,3-dimethylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-ylhexane-1,6-diyl(4-nitrophenyl)bis(carbonate).

[0095] d. Compound 6 was dissolved in dichloromethane, and DIPEA and DOX were added to the solution. After reacting at room temperature for 1 hour, the solution was purified by silica gel column chromatography with the eluent being dichloromethane and methanol in a ratio of 100:6 to obtain a red solid compound PDC-C.

[0096] e. Dissolve 1 mg of compound PDC-S in 100 μL of ethanol and then slowly add it dropwise into 1 mL of dd H2O at a speed of 500 rpm. Wait until the ethanol evaporates completely to obtain a PDC-S nanoparticle solution with a concentration of 1 mg / mL.

[0097] The preparation flow chart of the compound PDC-C in Example 1 and the compound PDC-S in Example 2 is as follows: Figure 2 shown.

[0098] Embodiment 3

[0099] This example provides a method for preparing an anti-tumor nanoformulation coupled with GSH-responsive protein degradation and chemotherapeutic drugs, which is the same as the preparation method of Example 2, and the only difference from Example 2 is that in step e: 1 mg of compound PDC-S is mixed with DSPE-PEG 2000 (1:0.15W / W) were mixed or dissolved in 100 μL of ethanol and then slowly added to 1 mL of dd H2O at a rotation speed of 500 rpm. After the ethanol was completely evaporated, a PDC-S NPs nanoparticle solution with a concentration of 1 mg / mL was obtained.

[0100] The preparation method of step e in Example 1 and Example 3 is as follows Figure 3 As shown, compound PDC-S alone or in combination with DSPE-PEG 2000 After mixing, the mixture is dissolved in ethanol or DMSO and slowly added dropwise to the aqueous solution to form nanoparticles.

[0101] The SEM images of the compounds in Example 1 and Example 3: the compound PDC-S nanoparticles and the compound PDC-S NPs nanoparticles are shown as follows: Figure 4 as well as Figure 7 As shown in the results, PDC-S alone or in combination with DSPE-PEG 2000 After mixing, nanoparticles can be formed well.

[0102] The polymer dispersibility index diagrams of compound PDC-S nanoparticles and compound PDC-S NPs nanoparticles are shown as follows: Figure 5 as well as Figure 8 As shown, the results indicate that the two have good dispersion and are suitable for further research.

[0103] Among them, the standard curve for determining the peak area and concentration of the compound PDC-S solution by external standard method is as follows: Figure 6The encapsulation efficiency and drug loading ratio of the compound PDC-S NPs nanoparticle solution are shown in Fig. 9 As shown in Figure 2, the encapsulation efficiency of PDC-S NPs is high with respect to drug loading.

[0104] The appearance of PDC-S nanoparticle solution and PDC-S NPs nanoparticle solution is shown in the figure below. Fig.10 As shown, the red transparent liquid can produce a strong Tyndall effect.

[0105] Embodiment 4

[0106] This embodiment provides an anti-tumor nanoformulation coupled with GSH-responsive protein degradation and chemotherapeutic drugs, which is formed by reacting the compound PDC-S prepared in Example 2 with hydrochloric acid to form 2-((2-(((((3S,5R)-1-((R)-2-(11-(4-(3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-11-oxoundecanoyl)-3,3-dimethylbutanoyl)-5-(((S)-1-(4- ((4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-3-yl)oxy)carbonyl)oxy)ethyl)disulfanyl)ethyl ((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamate hydrochloride; the chemical structure is as follows:

[0107]

[0108] That 1 HNMR, 13 CNMR and MS data are 1H-NMR(DMSO-d6,600MHz)δ:0.92(s,9H),1.11(d,J=6.85Hz,3H),1.14-1.28(m,10H),1.35(d,J=7.31Hz,3H),1.39-1.43(m,4H),1.98-2.02(m,4H),2.04-2.09(m,3H),2.17(t,J=7.81Hz,2H),2.21-2.27(m,3H),2.32(s,2H),2.37(s,2H),2.43(s,3H),2.88(t,J=6.12Hz,2H),2.92-2.98(m,4H),3.42(s,6H),3.50(s,1H),3.71(d,J=9.77Hz,2H),3.93(d,J=12.24Hz,6H),4.07-4.17(m,6H),4.27-4.41(m,4H),4.55(d,J=5.86Hz,2H),4.68(d,J=5.73Hz,1H),4.82-4.91(m,3H),5.09(s,1H),5.19(s,1H),5.42(s,1H),6.77(d,J=7.91Hz,1H),7.15(s,1H),7.34(t,J=8.31Hz,2H),7.38-7.43(m,3H),7.59(t,J=4.92Hz,1H),7.75-7.81(m,3H),7.85(d,J=4.92Hz,2H),8.07-8.09(m,1H),8.39(d,J=7.61Hz,1H),8.45(s,1H),8.96(s,1H),9.48(s,1H),13.24(s,1H),14.02(s,1H). 13C NMR(DMSO-d6,150MHz)δ:16.4,17.5,22.9,25.3,25.9,26.8,29.1,29.2,29.24,29.3,29.34,30.3,32.7,35.0,35.2,36.5,37.3,47.6,48.3 ,53.1,53.7,53.8,54.8,55.4,56.3,57.0,57.3,58.4,62.1,64.1,65 .8,67.1,67.5,68.4,70.3,70.4,75.4,77.5,100.7,102.9,107.7,109 .2,111.2,116.9,119.1,119.3,119.4,120.1,120.5,122.7,123.8,1 26.8,129.3,130.2,131.6,134.5,135.1,136.0,136.6,137.3,145.0, 147.4,148.2,148.8,152.0,152.7,153.0,154.1,154.9,155.5,156.4 ,156.6,161.2,170.2,170.3,171.0,172.9,186.9,187.0,214.2.HRMS m / z Calcd.for C 89 H 108 CIF 10 O 22 S3[M+H] + 1819.6547,Found 1819.6519.

[0109] Embodiment 5

[0110] This embodiment provides an anti-tumor nanoformulation of GSH-responsive protein degradation coupled with chemotherapeutic drugs, which is 2-((2-(((((3R,5R)-1-((R)-2-(11-(4-(3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-11-oxoundecanoyl)-3,3-dimethylbutyryl)-5-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)-1-((4-(3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-11-oxoundecanoyl)-3,3-dimethylbutyryl)-1-((4-(3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-11-oxoundecanoyl ... (((1S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamate hydrochloride; the chemical structure is as follows:

[0111]

[0112] That 1 HNMR, 13 CNMR and MS data are 1 H-NMR (DMSO-d6, 600MHz) δ: 0.98 (s, 9H), 1.18 (d, J = 6.07Hz, 3H), 1.23-1.28 (m, 10H) ,1.37(d,J=7.00Hz,3H),1.44-1.54(m,4H),1.88-1.97(m,2H),2.05(s,2H),2.10-2. 23(m,4H),2.26-2.31(m,4H),2.39(s,2H),2.44(s,2H),2.47(s,1H),2.51(s,2H),2 .95-3.05(m,6H),3.48(d,J=17.27Hz,6H),3.57(s,2H),3.75(s,1H),3.92-4.02(m,7 H),4.14-4.22(m,5H),4.34(s,2H),4.45-4.50(m,2H),4.63(d,J=5.60Hz,2H),4.76 (d,J=6.07Hz,1H),4.92-4.97(m,3H),5.23(s,1H),5.26(s,1H),5.49(s,1H),6.84(d ,J=7.94Hz,1H),7.23(s,1H),7.42-7.56(m,5H),7.67(s,1H),7.83-7.98(m,5H),8. 15-8.23(m,2H),8.53(s,1H),9.03(s,1H),9.56(s,1H),13.30(s,1H),14.07(s,1H). 13C NMR(DMSO-d6,150MHz)δ:16.5,17.5,22.9,25.3,25.8,26.7,29.0,29.2,29.22,29.27,29.3,30.3,32.7,34.8,35.2,36.6,37.3,41.4,45.4 ,47.6,48.1,53.1,53.7,55.4,55.4,56.3,57.0,58.5,62.6,64.2,65. 8,67.1,67.6,68.4,70.3,70.33,75.4,76.8,100.8,102.9,107.7,109 .2,111.2,117.0,119.2,119.3,119.4,120.1,120.4,122.7,123.8,1 27.0,129.2,130.1,131.6,134.5,135.1,135.9,136.6,137.3,144.6, 147.4,148.2,148.8,151.9,152.8,153.0,154.1,154.9,155.5,156.4 ,156.6,161.2,169.9,170.4,170.9,173.3,186.9,187.0,214.3.HRMS m / z Calcd.for C 89 H 108 CIF 10 O 22 S3[M+Na] + 1841.6367,Found 1841.6366.

[0113] Embodiment 6

[0114] This embodiment provides an anti-tumor nanoformulation coupled with GSH-responsive protein degradation and chemotherapeutic drugs, wherein the compound PDC-C prepared in Example 2 is reacted with hydrochloric acid to form 6-(((((3R,5R)-1-((R)-2-(11-(4-(3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-11-oxoundecanamide)-3,3-dimethylbutanoyl)-5-(((S)-1-( 4-(4-methylthiazol-5-yl)phenyl)ethyl)amino)pyrrolidin-3-yl)oxy)carbonyl)oxy)hexyl((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetraphenyl-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamate hydrochloride; the chemical structure is as follows:

[0115]

[0116] That 1 HNMR, 13 CNMR and MS data are 1 H-NMR(DMSO-d6,600MHz)δ:0.92(s,9H),1.10(d,J=6.32Hz,3H),1.19-1.26(m,14H),1.35(d,J=7.11H z,3H),1.44(s,4H),1.54(s,2H),1.81(t,J=13.44Hz,2H),1.95-2.02(m,4H),2.04-2.09(m,3H),2.17 (t,J=6.80Hz,2H),2.23(t,J=7.98Hz,3H),2.32(s,2H),2.37(s,2H),2.43(s,3H),2.93(s,2H),3.41( s,6H),3.50(s,1H),3.71(d,J=9.16Hz,2H),3.83(t,J=6.21Hz,2H),3.92(t,J=13.60Hz,6H),4.01(d, J=10.64Hz,3H),4.13(t,J=7.10Hz,3H),4.34-4.41(m,2H),4.55(d,J=5.91Hz,2H),4.67(d,J=5.62Hz ,1H),4.82-4.91(m,3H),5.08(s,1H),5.19(s,1H),5.42(s,1H),6.59(d,J=8.28Hz,1H),7.14(s,1H), 7.36(t,J=8.28Hz,2H),7.41(d,J=8.57Hz,3H),7.59(s,1H),7.74-7.81(m,3H),7.84(s,2H),8.05-8. 09(m,1H),8.38(d,J=7.69Hz,1H),8.45(s,1H),8.96(s,1H),9.46(s,1H),13.24(s,1H),14.02(s,1H). 13C NMR(DMSO-d6,150MHz)δ:16.4,17.5,22.9,25.3,25.34,25.5,25.8,26.8,28.4,29.0,29.1,29.2,29.23,29.3,29.34,30.3,32.7,35.1,35.2,4 7.4,48.3,53.1,53.7,53.8,54.8,55.4,56.3,57.0,57.3,58.5,64.0,6 4.1,67.1,67.5,68.1,68.5,70.3,70.4,75.4,77.1,100.8,102.9,107. 7,109.2,111.2,116.9,119.1,119.3,119.4,120.1,120.4,122.6,123. 8,126.8,129.3,130.2,131.6,134.5,135.1,136.0,136.6,137.3,145. 0,147.4,148.2,148.8,152.0,152.7,153.0,154.1,154.9,155.9,156. 4,156.6,161.2,170.2,170.3,171.0,172.8,186.9,187.0,214.2.HRMS m / z Calcd.for C 91 H 112 CIF 10 O 22 S[M+H] + 1805.7238,Found 1805.7221.

[0117] Test Case

[0118] The compounds of the present invention were tested for their ability to inhibit proliferation of HCC-827, NCI-H1975, A549 non-small cell lung cancer cells and HEK293 normal cells using the conventional MTT method. Tumor cells in the logarithmic growth phase were digested with trypsin, and then diluted and suspended into a single cell suspension using culture medium (RPMI 1640 + 10% FBS). The cell density was adjusted to 5×10 4 / mL, add 100μL to each well and inoculate in a 96-well plate, place in a 37℃, 5% CO2 incubator for 24 hours, then add different concentrations of nanoparticles, each concentration is parallel to 6 replicates, and set up experimental and control groups. After incubation for 72 hours, add 30μL of MTT solution to each well, then incubate at 37℃ in the dark for 4 hours, pour out the MTT and add 150μL DMSO to each well. After placing in the incubator for 10 minutes, use the Biotek-Synergy microplate reader to measure the 490nm OD value. Calculate the half inhibitory concentration IC 50 .

[0119] The half inhibitory concentration IC of nanoparticles prepared by the compound of the present invention and related compounds on tumor cells 50 The values ​​are shown in Table 1: The tested compounds are all from Example 1 or Example 2.

[0120] Table 1 IC50 of target nanoparticles on tumor cells 50 (Unit: nmol / L)

[0121]

[0122] The test results showed that the above compounds all exhibited moderate to excellent anti-tumor activity, among which compound PDC-S showed the best broad-spectrum anti-tumor IC 50 value.

[0123] The concentration-dependent degradation of EGFR protein by the nanoparticles prepared from the above compounds was investigated, e.g. Fig.11 , Fig.12 As shown in the figure, PRO and PDC-S can strongly degrade EGFR protein. Fig.13 It was shown that PDC-C had a poor degradation effect on EGFR protein.

[0124] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An anti-tumor nanoformulation of GSH-responsive protein degradation coupled with chemotherapeutic drugs, characterized in that: The anti-tumor nanoparticle preparation is a compound of general formula (I) or general formula (II) or a pharmaceutically acceptable salt of a compound of general formula (I) or general formula (II); Wherein, in the general formula (I) or the general formula (II), A is S, C, O, n1=1-8, n=0-5; X, R, R1, Y and Z are each one of a saturated or unsaturated straight chain hydrocarbon group, an oxygen hetero chain and a phenyl group having 1 to 14 carbon atoms.

2. The anti-tumor nanoformulation of GSH-responsive protein degradation coupled with chemotherapeutic drugs according to claim 1, characterized in that: X is also a heterocyclic group or one of the following linking groups, wherein n=0-5, The heterocyclic group is piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl.

3. The anti-tumor nanoformulation of GSH-responsive protein degradation coupled with chemotherapeutic drugs according to claim 1, characterized in that: R and R1 are also heterocyclic groups or one of the following linking groups, wherein n=0-5, The heterocyclic group is piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl.

4. The anti-tumor nanoformulation of GSH-responsive protein degradation coupled with chemotherapeutic drugs according to claim 1, characterized in that: Y is also a heterocyclic group or one of the following linking groups, wherein n=0-5, Z is also a heterocyclic group or one of the following linking groups, wherein n=0-5, The heterocyclic group is piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl.

5. The anti-tumor nanoformulation of GSH-responsive protein degradation coupled with chemotherapeutic drugs according to claim 1, characterized in that: The pharmaceutically acceptable salt is an acid addition salt formed by the compound of general formula (I) or general formula (II) and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

6. A method for preparing an anti-tumor nanoformulation of GSH-responsive protein degradation coupled with a chemotherapeutic drug as claimed in any one of claims 1 to 5, characterized in that: The following steps are involved: a. Compound 1 and compound 2 are dissolved in dimethylacetamide, and N,N-diisopropylethylamine is added to react at room temperature for 4-8 hours to obtain compound 3; b. Compound 3 and compound 4 were dissolved in dimethylacetamide, and 4-dimethylaminopyridine was added to react for 4-8h to obtain compound 5; c. Compound 5 and p-nitrophenyl chloroformate were dissolved in DCM, and N,N-diisopropylethylamine was added to react at room temperature for 8-12h to obtain compound 6; d. Compound 6 and compound 7 were dissolved in N,N, dimethylformamide, and N,N-diisopropylethylamine was added to react at room temperature for 1-2h to obtain compound 8; e. Compound 8 was dissolved in ethanol or DMSO and then added dropwise to ddH2O and stirred at 500 rpm on a magnetic stirrer. The ethanol in the solution was evaporated or the DMSO was removed by dialysis to obtain a spherical structure of a nanoparticle preparation targeting EGFR protein degradation; The chemical structure formula of the compound 1 is: The compound 2 is N,N ’ -Carbonyldiimidazole; The chemical structure of compound 4 is The compound 7 is doxorubicin; The above steps ad were purified by silica gel column chromatography, the eluents were dichloromethane and methanol, and the volume ratio of dichloromethane to methanol was 100:(3-8).

7. The method for preparing an anti-tumor nanoformulation of GSH-responsive protein degradation coupled with chemotherapeutic drugs according to claim 6, characterized in that: When the general formula of compound 1 is When , the general chemical structure formula of compound 8 is general formula (I).

8. The anti-tumor nanoformulation of GSH-responsive protein degradation coupled with chemotherapeutic drugs according to claim 6, characterized in that: When the general formula of compound 1 is When , the general formula of compound 8 is general formula (II).

9. An application of the anti-tumor nanoformulation of GSH-responsive protein degradation coupled with chemotherapeutic drugs as claimed in claim 1, characterized in that: The anti-tumor nano preparation is applied to the preparation of medicine for tumors related to EGFR expression.

10. The use of the anti-tumor nanoformulation of GSH-responsive protein degradation coupled with chemotherapeutic drugs according to claim 9, characterized in that: Tumors associated with EGFR expression include lung cancer, liver cancer, kidney cancer, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, osteosarcoma, and tumors of the central or peripheral nervous system.

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