Tumor-targeted self-assembly nano preparation based on protein degradation technology and preparation method and application thereof

Abstract

The invention discloses a tumor targeted self-assembly nano preparation based on a protein degradation technology and a preparation method and application thereof, and belongs to the technical field of pharmacy. The structure of the protein degradation agent is optimized on the basis of the tumor-targeted protein degradation agent, so that PROTAC molecules can be self-assembled into nanoparticles in a water solution, and other nano materials are not needed. Therefore, the invention provides a brand-new nano preparation of a protein degradation agent as well as a preparation method and application of the nano preparation, the nano preparation shows good target protein degradation activity, and the technology can realize inhibition on tumors by degrading model protein EGFR, has certain broad-spectrum antitumor activity, can obviously delay tumor growth, and can be applied to EGFR-mediated tumor diseases.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a tumor-targeted self-assembly nano preparation based on protein degradation technology, 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 insurmountable defects: 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. Therefore, how to enhance the tumor targeting of PROTAC molecules and improve their water solubility and cell permeability is a key scientific issue that needs to be urgently addressed 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 optimize the PROTAC molecule itself without introducing foreign substances, especially when self-assembling to form nanoparticles, it will have great application prospects. Summary of the invention

[0005] The purpose of the present invention is to provide a tumor-targeted self-assembling nanoformulation based on protein degradation technology, a preparation method and application thereof, aiming at the deficiencies of the prior art, overcoming the defects of existing drug treatments, optimizing its structure based on the targeted EGFR protein degrader, so that the PROTAC molecule itself can self-assemble into nanoparticles in water. Therefore, not only the water solubility of PROTAC is improved, but also the advantages of nanoparticles can be brought into play, providing a solution to improve the problems faced by PROTAC.

[0006] To achieve the above object, the present invention provides a tumor-targeted self-assembling nanoformulation based on protein degradation technology, wherein the tumor-targeted self-assembling nanoformulation based on protein degradation technology is a compound or a pharmaceutically acceptable salt of any chemical structure of general formula (I), general formula (II), general formula (III) or general formula (IV); the general structural formulas of general formula (I), general formula (II), general formula (III) and general formula (IV) are as follows:

[0007]

[0008] Wherein, X, R and R1 are one of saturated or unsaturated straight chain hydrocarbon groups, oxygen heterochains and phenyl groups having 1 to 14 carbon atoms.

[0009] Preferably, X is also a heterocyclic group or one of the following linking groups, wherein n=0-5,

[0010]

[0011] The heterocyclic group includes piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl.

[0012] Preferably, R is a heterocyclic group or one of the following linking groups, wherein n=0-5,

[0013]

[0014] The heterocyclic group includes piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl.

[0015] Preferably, R1 is also a heterocyclic group or one of the following linking groups, wherein n=0-5,

[0016]

[0017] The heterocyclic group includes piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl.

[0018] Preferably, the pharmaceutically acceptable salt of the compound is an acid addition salt formed by a compound of any chemical formula of general formula (I), general formula (II), general formula (III) or general formula (IV) 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 or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid.

[0019] The present invention also provides a method for preparing the above-mentioned tumor-targeted self-assembly nanoformulation based on protein degradation technology, comprising the following steps:

[0020] a. Compound 1 and compound 2 are dissolved in dimethylacetamide, and N,N-diisopropylethylamine and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate are added and reacted at room temperature for 4-8 hours to obtain compound 3;

[0021] b. Compound 3 was dissolved in a mixed solvent of methanol, water and tetrahydrofuran, and LiOH was added to react for 2-4h to obtain compound 4;

[0022] c. Compound 4 and compound 5 were dissolved in DMF, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine were added and reacted for 4-6h to obtain compound 8.

[0023] d. Compound 8 was dissolved in methanol, 1M 1,4-dioxane hydrochloric acid solution was added and the mixture was reacted at room temperature for 2-4 hours to obtain compound 9;

[0024] e. Compound 9 was dissolved in ethanol or DMSO, added dropwise to ddH2O and stirred at 500 rpm on a magnetic stirrer, and the ethanol in the solution was evaporated or the DMSO was removed by dialysis to obtain a spherical structure of a tumor-targeted self-assembled nanoformulation based on protein degradation technology;

[0025] The compound 1 is: N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-(piperazin-1-yl)propoxy)quinazolin-4-amine;

[0026] The compound 2 is

[0027] The chemical structure of compound 5 is as follows: Compound 5 is (2S, 4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-((S)-1-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide,

[0028] or (2S,4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide,

[0029] or (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, or (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide;

[0030] 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:(1-8).

[0031] Preferably, when compound 5 is (2S, 4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-((S)-1-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, the general formula of compound 9 is general formula (I),

[0032] Or when compound 5 is (2S,4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide, the general formula of compound 9 is general formula (II);

[0033] or when compound 5 is (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, the general formula of compound 9 is general formula (III);

[0034] Or when compound 5 is (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide, the general formula of compound 9 is general formula (IV).

[0035] Preferably, (2S,4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-((S)-1-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide is prepared from (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0036] (2S, 4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide is prepared from (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide.

[0037] The specific preparation steps are as follows: (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide or (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide is dissolved in DMF, N,N-diisopropylethylamine and 2-(7-benzotriazole oxide)-N,N,N' , N'-tetramethyluronium hexafluorophosphate is reacted at room temperature for 4-8 hours, then dissolved in dichloromethane and TFA is added to react at room temperature for 4-6 hours to obtain (2S, 4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-((S)-1-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide or (2S, 4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide.

[0038] The tumor-targeted self-assembly nanoformulation based on protein degradation technology provided by the present invention is applied to the preparation of medicines for tumors related to EGFR expression.

[0039] 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.

[0040] Therefore, the present invention has the following beneficial effects:

[0041] 1. For the first time, the present invention optimizes the PROTAC molecular structure to enable PROTAC molecules to self-assemble into nanoparticles in aqueous solution without adding any excipients, thereby avoiding the toxic side effects of these additives in the human body.

[0042] 2. The compounds of the present invention exhibit good EGFR protein degradation activity, achieve tumor inhibition by degrading EGFR protein, have a certain broad-spectrum anti-tumor activity, can significantly delay tumor growth, and can be applied to EGFR-mediated tumor diseases.

[0043] 3. As the first reported EGFR-based anti-tumor drug, this type of compound has further development and research value.

[0044] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is a preparation flow chart of the general formula (Ⅰ) of the present invention;

[0046] Figure 2 is a preparation flow chart of the general formula (III) of the present invention;

[0047] Figure 3 This is a preparation flow chart of Example 1 of the present invention;

[0048] Figure 4 are the structural expressions of the compounds of Examples 3 to 6 of the present invention, wherein (a) is the structural expression of the compound of Example 3, (b) is the structural expression of the compound of Example 4, (c) is the structural expression of the compound of Example 5, and (d) is the structural expression of the compound of Example 6;

[0049] Figure 5 is the hydrated particle size distribution diagram of the compounds of Examples 3 to 6 of the present invention;

[0050] Figure 6This is a TEM image of Example 5 J-83-2 of the present invention;

[0051] Figure 7 The degradation effects of J-83-2 and J-123-2 on EGFR protein in the application examples of the present invention. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0053] 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.

[0054] The present invention provides a tumor-targeted self-assembling nanoformulation based on protein degradation technology. The tumor-targeted self-assembling nanoformulation based on protein degradation technology is a compound or a pharmaceutically acceptable salt of any chemical structure of general formula (I), general formula (II), general formula (III) or general formula (IV); the general structures of general formula (I), general formula (II), general formula (III) and general formula (IV) are as follows:

[0055]

[0056] Wherein, X, R and R1 are one of saturated or unsaturated straight chain hydrocarbon groups, oxygen heterochains and phenyl groups having 1 to 14 carbon atoms.

[0057] X is also a heterocyclic group or one of the following linking groups, wherein n=0-5,

[0058]

[0059] Heterocyclic groups include piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl.

[0060] R is a heterocyclic group or one of the following linking groups, wherein n=0-5,

[0061]

[0062] Heterocyclic groups include piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl.

[0063] R1 is also a heterocyclic group or one of the following linking groups, wherein n=0-5,

[0064]

[0065] Heterocyclic groups include piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl.

[0066] The pharmaceutically acceptable salt of the compound is an acid addition salt formed by a compound of any chemical formula of general formula (I), general formula (II), general formula (III) or general formula (IV) 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 or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid.

[0067] The present invention also provides a method for preparing the above-mentioned tumor-targeted self-assembly nanoformulation based on protein degradation technology, comprising the following steps:

[0068] a. Compound 1 and compound 2 were dissolved in dimethylacetamide, and N,N-diisopropylethylamine and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate were added to react at room temperature for 4-8 hours to obtain compound 3.

[0069] b. Compound 3 was dissolved in a mixed solvent of methanol, water and tetrahydrofuran, and LiOH was added to react for 2-4 hours to obtain compound 4.

[0070] c. Compound 4 and compound 5 were dissolved in DMF, and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine were added to react for 4-6 hours to condense to obtain compound 8.

[0071] d. Compound 8 was dissolved in methanol and 1M 1,4-dioxane hydrochloric acid solution was added to react at room temperature for 2-4 hours to obtain compound 9.

[0072] e. Dissolve compound 9 in ethanol or DMSO, add it dropwise into ddH2O and stir it on a magnetic stirrer at 500 rpm. Wait until the ethanol in the solution evaporates or the DMSO is removed by dialysis to obtain a spherical structure of a tumor-targeted self-assembled nanoformulation based on protein degradation technology.

[0073] The chemical structural formula of compound 1 is: N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-(piperazin-1-yl)propoxy)quinazolin-4-amine.

[0074] Compound 2 is

[0075] When compound 5 is (2S,4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-((S)-1-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, the general formula of compound 9 is general formula (I),

[0076] Or when compound 5 is (2S,4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide, the general formula of compound 9 is general formula (II);

[0077] or when compound 5 is (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, the general formula of compound 9 is general formula (III);

[0078] Or when compound 5 is (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide, the general formula of compound 9 is general formula (IV).

[0079] 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:(1-8).

[0080] (2S,4R)-1-((S)-3,3-dimethyl-2-ureidobutanoyl)-4-hydroxy-N-((S)-1-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide was prepared from (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0081] (2S,4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide was prepared from (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide.

[0082] The specific preparation steps are: (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide or (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide The mixture was dissolved in DMF, and N,N-diisopropylethylamine and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate were added and reacted at room temperature for 4-8h to obtain tert-butyl (3-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl))carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1- tert-butyl (2-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)amino)-2-oxoethyl)carbamate, then dissolved in dichloromethane, TFA was added and the mixture was heated to room temperature. The reaction is carried out under temperature conditions for 4-6 hours to obtain (2S, 4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-((S)-1-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide or (2S, 4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide.

[0083] The preparation flow chart of general formula (Ⅰ) is as follows Figure 1As shown in the figure, compound 7 is (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, and compound 5 is (2S, 4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-((S)-1-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide. The preparation process of general formula (II) is similar to that of general formula (I). During the preparation, compound 7 is (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide, and compound 5 is (2S, 4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide. The preparation flow chart of general formula (III) is as follows: Figure 2 As shown in the figure, compound 5 is (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide. The preparation process of general formula (IV) is similar to that of general formula (III). During the preparation, compound 5 is (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide.

[0084] The above-mentioned tumor-targeted self-assembly nanoformulation based on protein degradation technology provided by the present invention is applied to the preparation of medicine 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.

[0085] 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.

[0086] Embodiment 1

[0087] This embodiment provides a tumor-targeted self-assembly nanoformulation based on protein degradation technology, tert-butyl ((R)-5-(4-(3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazin-1-yl)-1-((3-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl))ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropyl)amino)-1,5-dioxolan-2-yl)carbamate, the preparation steps are as follows:

[0088] Step a: The compound (N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-(piperazin-1-yl)propoxy)quinazolin-4-amine and (R)-5-(benzyloxy)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoic acid were dissolved in DMF, and DIPEA was added dropwise to the solution. After stirring at room temperature for 4 hours, the solution was purified by silica gel column chromatography with the eluent being dichloromethane and methanol in a ratio of 100:3 to obtain a white solid, benzyl (R)-2-((tert-butoxycarbonyl)amino)-5-(4-(3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy))piperazin-1-yl)-5-oxopentanoic acid propyl ester.

[0089] Step b: The compound obtained in step a was dissolved in a mixed solvent of CH3OH, H2O, and THF, and LiOH was added to the solution. 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, (R)-2-((tert-butoxycarbonyl)amino)-5-(4-(3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazin-1-yl)-5-oxopentanoic acid.

[0090] Step c: Compound (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl))ethyl)pyrrolidine-2-carboxamide and 3-((tert-butoxycarbonyl)amino)propionic acid were dissolved in DMF, and DIPEA was added dropwise to the solution. After stirring at room temperature for 4 hours, the solution was purified by silica gel column chromatography with a ratio of dichloromethane to methanol of 100:2 as the eluent to obtain a white solid, tert-butyl (3-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl))carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropyl)carbamate.

[0091] Step d: The compound obtained in step c was dissolved in dichloromethane, and TFA was added to the solution. After reacting at room temperature for 6 hours, the mixture was purified by silica gel column chromatography with the eluent being dichloromethane and methanol in a ratio of 100:6 to obtain a white solid, (2S,4R)-1-((S)-2-(3-aminopropionamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0092] Step e: The compound obtained in step d and the compound obtained in step b were dissolved in DMF, and DIPEA was added dropwise to the solution. 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 100:3 to obtain a white solid, tert-butyl ((R)-5-(4-(3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazin-1-yl)-1-((3-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl))ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropyl)amino)-1,5-dioxolan-2-yl)carbamate.

[0093] Step f: The compound obtained in step e was dissolved in methanol, and 1,4-dioxane hydrochloride solution was added to the solution. After reacting at room temperature for 4 hours, the mixture was purified by silica gel column chromatography with an eluent of dichloromethane and methanol in a ratio of 100:8 to obtain a white solid, tert-butyl ((R)-5-(4-(3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazin-1-yl)-1-((3-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl))ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropyl)amino)-1,5-dioxolan-2-yl)carbamate.

[0094] Embodiment 2

[0095] This embodiment provides a tumor-targeted self-assembly nanoformulation based on protein degradation technology, tert-butyl ((R)-5-(4-(3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)piperazin-1-yl)-1-((3-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl))ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropyl)amino)-1,5-dioxolan-2-yl)carbamate hydrochloride, which is synthesized by adding the product prepared in Example 1 to hydrochloric acid. The specific preparation steps are as follows: Figure 3 shown.

[0096] Embodiment 3

[0097] This embodiment provides a tumor-targeted self-assembled nanoformulation based on protein degradation technology, wherein compound 2 is (R)-5-(benzyloxy)-4-(tert-butyloxycarbonyl)amino)-5-oxopentanoic acid; compound 7 is (2R, 4S)-1-((R)-2-(3-aminopropionamide)-3,3-dimethylbutyryl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, and J-145-2 is (2S, 4R)-1-((S) -2-(7-((R)-2-amino-5-(4-(3-((4-((3-chloro-4-fluorophenyl)amino))-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-5-oxopentanamido)heptamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride, 1 mg of the above compound was dissolved in 20 μL of ethanol and then slowly added dropwise to 1 mL of ddH2O at a speed of 500 rpm, and the ethanol was completely evaporated to obtain a tumor-targeted self-assembled nanoformulation solution based on protein degradation technology with a concentration of 1 mg / mL, which was numbered J-145-2.

[0098] Embodiment 4

[0099] This embodiment provides a tumor-targeted self-assembled nanoformulation based on protein degradation technology, wherein compound 2 is (R)-5-(benzyloxy)-4-(tert-butyloxycarbonyl)amino)-5-oxopentanoic acid; compound 7 is (2R, 4S)-1-((R)-2-(5-aminopentaamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, and J-83-2 is (2S, 4R)-1-((S) -2-(5-((R)-2-amino-5-(4-(3-((4-((3-chloro-4-fluorophenyl)amino))-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-5-oxopentanamido)pentanamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride, 1 mg of the above compound was dissolved in 20 μL of ethanol and then slowly added dropwise to 1 mL of dd H2O at a speed of 500 rpm, and the ethanol was completely evaporated to obtain a tumor-targeted self-assembled nanoformulation solution based on protein degradation technology with a concentration of 1 mg / mL, numbered J-83-2.

[0100] Embodiment 5

[0101] This embodiment provides a tumor-targeted self-assembled nanoformulation based on protein degradation technology, wherein compound 2 is (R)-5-(benzyloxy)-4-(tert-butyloxycarbonyl)amino)-5-oxopentanoic acid; compound 7 is (2R, 4S)-1-((R)-2-(7-aminoheptylamide)-3,3-dimethylbutyryl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, and J-123-2 is (2S, 4R)-1-((S) -2-(7-((R)-2-amino-5-(4-(3-((4-((3-chloro-4-fluorophenyl)amino))-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-5-oxopentanamido)heptamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride, 1 mg of the above compound was dissolved in 20 μL of ethanol and then slowly added dropwise to 1 mL of ddH2O at a speed of 500 rpm, and the ethanol was completely evaporated to obtain a tumor-targeted self-assembled nanoformulation solution based on protein degradation technology with a concentration of 1 mg / mL, which was numbered J-123-2.

[0102] Embodiment 6

[0103] This embodiment provides a tumor-targeted self-assembled nanoformulation based on protein degradation technology, wherein compound 2 is (R)-5-(benzyloxy)-4-(tert-butyloxycarbonyl)amino)-5-oxopentanoic acid; compound 7 is (2R, 4S)-1-((R)-2-(9-aminononanamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, and J-133-2 is (2S, 4R)-1-((S) -2-(9-((R)-2-amino-5-(4-(3-((4-((3-chloro-4-fluorophenyl)amino))-7-methoxyquinazolin-6-yl)oxy)propyl)piperazine-1-yl)-5-oxopentanamido)nonanamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride, 1 mg of the above compound was dissolved in 20 μL of ethanol and then slowly added dropwise to 1 mL of dd H2O at a speed of 500 rpm, and the ethanol was completely evaporated to obtain a tumor-targeted self-assembled nanoformulation solution based on protein degradation technology with a concentration of 1 mg / mL, which was numbered J-133-2.

[0104] The structures of the tumor-targeted self-assembled nanoformulations based on protein degradation technology provided in Examples 3 to 6 are as follows: Figure 4 As shown, and its 1 HNMR, 13 CNMR and MS data are shown in Table 1 below:

[0105] Table 1 Structure of Embodiments 3 to 6 and their 1 HNMR, 13 CNMR and MS data

[0106]

[0107]

[0108]

[0109] The compounds of Examples 3 to 6 were characterized by nanoparticles, and the hydrated particle size of the nanoparticles was measured by a particle size analyzer. The particle size distribution results of J-145-2, J-83-2, J-123-2 and J-133-2 are as follows: Figure 5 The hydrated particle size (Size), polymer dispersibility index (PDI) and potential (Zeta pontential) of the nanoparticles are shown in Table 2 below:

[0110] Table 2 Nanoparticle characterization results

[0111]

[0112] The actual particle size of J-123-2 was photographed by transmission electron microscopy. Figure 6 shown.

[0113] Application Examples

[0114] The tumor-targeted self-assembled nanoformulations (J-83-2, J-123-2, and J-133-2) based on protein degradation technology of Examples 3 to 5 of the present invention were tested for their ability to inhibit the proliferation of HCC-827 tumor cells. The test method used the conventional MTT method. HCC-827 tumor cells in the logarithmic growth phase were digested with trypsin, and then the cells were diluted and suspended into a single cell suspension with 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 .

[0115] Experimental results: The half inhibitory concentration IC of nanoparticles prepared by the compound of the present invention on tumor cells 50 The values ​​are shown in Table 3. The degradation effects of J-83-2 and J-123-2 on EGFR protein are shown in Figure 7 As shown in Figure 2, the test results showed that this series of compounds exhibited moderate to excellent anti-tumor activity, among which J-123-2 showed the best IC50 for the inhibitory activity against HCC-827. 50 value.

[0116] Table 3 IC50 of target nanoparticles on tumor cells 50 (Unit: nmol / L)

[0117] <![CDATA[NP s ]]> <![CDATA[IC 50 ]]> J-83-2 132 J-123-2 48 J-133-2 101

[0118] Therefore, the present invention provides a tumor-targeted self-assembled nanoformulation based on protein degradation technology, and a preparation method and application thereof, which exhibits good EGFR protein degradation activity, achieves tumor inhibition by degrading EGFR protein, has a certain broad-spectrum anti-tumor activity, can significantly delay tumor growth, and can be applied to EGFR-mediated tumor diseases.

[0119] 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. A tumor-targeted self-assembly nanoformulation based on protein degradation technology, characterized in that: The tumor-targeted self-assembly nanoformulation based on protein degradation technology is a compound or a pharmaceutically acceptable salt of any chemical structure of general formula (I), general formula (II), general formula (III) or general formula (IV); the general structures of general formula (I), general formula (II), general formula (III) and general formula (IV) are as follows: Wherein, X, R and R1 are one of saturated or unsaturated straight chain hydrocarbon groups, oxygen heterochains and phenyl groups having 1 to 14 carbon atoms.

2. The tumor-targeted self-assembly nanoformulation based on protein degradation technology 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 includes piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl.

3. The tumor-targeted self-assembly nanoformulation based on protein degradation technology according to claim 1, characterized in that: R is a heterocyclic group or one of the following linking groups, wherein n=0-5, The heterocyclic group includes piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl.

4. The tumor-targeted self-assembly nanoformulation based on protein degradation technology according to claim 1, characterized in that: R1 is also a heterocyclic group or one of the following linking groups, wherein n=0-5, The heterocyclic group includes piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl.

5. The tumor-targeted self-assembly nanoformulation based on protein degradation technology according to claim 1, characterized in that: The pharmaceutically acceptable salt of the compound is an acid addition salt formed by a compound of any chemical formula of general formula (I), general formula (II), general formula (III) or general formula (IV) 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 or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid.

6. A method for preparing a tumor-targeted self-assembled nanoformulation based on protein degradation technology according to 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 and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate are added and reacted at room temperature for 4-8 hours to obtain compound 3; b. Compound 3 was dissolved in a mixed solvent of methanol, water and tetrahydrofuran, and LiOH was added to react for 2-4h to obtain compound 4; c. Compound 4 and compound 5 were dissolved in DMF, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine were added and reacted for 4-6h to obtain compound 8. d. Compound 8 was dissolved in methanol, 1M 1,4-dioxane hydrochloric acid solution was added and the mixture was reacted at room temperature for 2-4 hours to obtain compound 9; e. Compound 9 was dissolved in ethanol or DMSO, added dropwise to ddH2O and stirred at 500 rpm on a magnetic stirrer, and the ethanol in the solution was evaporated or the DMSO was removed by dialysis to obtain a spherical structure of a tumor-targeted self-assembled nanoformulation based on protein degradation technology; The compound 1 is: N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-(piperazin-1-yl)propoxy)quinazolin-4-amine; The compound 2 is The compound 5 is (2S, 4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-((S)-1-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, or (2S,4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide, or (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, or (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide; 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:(1-8).

7. The method for preparing a tumor-targeted self-assembled nanoformulation based on protein degradation technology according to claim 6, characterized in that: When compound 5 is (2S,4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-((S)-1-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, the general formula of compound 9 is general formula (I), Or when compound 5 is (2S,4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide, the general formula of compound 9 is general formula (II); or when compound 5 is (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide, the general formula of compound 9 is general formula (III); Or when compound 5 is (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide, the general formula of compound 9 is general formula (IV).

8. The method for preparing a tumor-targeted self-assembly nanoformulation based on protein degradation technology according to claim 7, characterized in that: (2S,4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-((S)-1-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide is prepared from (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide. (2S, 4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide is prepared from (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide. The specific preparation steps are as follows: (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide or (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide is dissolved in DMF, N,N-diisopropylethylamine and 2-(7-benzotriazole oxide)-N,N,N' , N'-tetramethyluronium hexafluorophosphate is reacted at room temperature for 4-8 hours, then dissolved in dichloromethane and TFA is added to react at room temperature for 4-6 hours to obtain (2S, 4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-((S)-1-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide or (2S, 4R)-1-((S)-3,3-dimethyl-2-ureidobutyryl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide.

9. An application of the tumor-targeted self-assembly nanoformulation based on protein degradation technology as claimed in claim 1, characterized in that: The tumor-targeted self-assembly nanoformulation based on protein degradation technology is applied to the preparation of medicines for tumors related to EGFR expression.

10. The use of a tumor-targeted self-assembly nanoformulation based on protein degradation technology 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.

Images