Design and use of novel cancer PROTAC drug compounds based on unstable amino acids

Abstract

The invention relates to a PROTAC compound for inducing EGFR (epidermal growth factor receptor) and ALK (anaplastic lymphoma kinase) degradation based on unstable amino acid or pharmaceutically acceptable salt thereof and application of the PROTAC compound, and belongs to the field of biological medicine. The molecular structure of the compound comprises a single amino acid (such as Gly, Pro, Thr and the like) and a ligand Brigatinib derivative BA (Brigatinib analog), and the single amino acid (such as Gly, Pro, Thr and the like) and the ligand Brigatinib derivative BA (Brigatinib analog) are connected through a PEG linker. The compound designed and synthesized by the invention is proved to be a specific degradation agent for EGFR (L858R / T790M) and ALK (anaplastic lymphoma kinase) in an in-vitro experiment, and the degradation speed of EGFR and ALK can be regulated and controlled. According to the present invention, the new treatment strategy can be provided for the non-small cell lung cancer (NSCLC) and other cancers, and the drug can be easily transplanted to other target drugs, such that the wide application feasibility is provided, and the new effective method is provided for the human disease treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a novel cancer PROTAC compound based on unstable amino acids or a pharmaceutically acceptable small molecule thereof and applications thereof. Background Art

[0002] In 2001, Raymond Deshaies, Craig Crews and others proposed the concept of PROTAC and successfully designed and synthesized the first batch of peptide PROTACs for the degradation of methionyl aminopeptidase 2 (MetAP-2). In 2019, ARV-110, developed by industry pioneer Professor Crews, officially entered the clinical trial stage as the world's first PROTAC drug, and protein degradation targeting chimera PROTAC (Proteolysis targeting chimeria) has received more and more attention.

[0003] PROTAC consists of three parts: a ligand that binds to the E3 ligase to guide protein degradation; a ligand that binds to the target protein to guide the targeted positioning of small molecules; and a linker responsible for the chimera of the two ligands. After the PROTAC molecule enters the cell, the target protein (Protein of Interest, POI) ligand in its structure can specifically bind to the corresponding target protein, while the other end can recruit the E3 ligase to form a POI-Linker-E3 ligase ternary complex, in which the E3 ligase can mediate the polyubiquitination of -POI, and the ubiquitin-tagged POI is recognized and degraded by the proteasome. PROTAC compounds are a technology based on protein degradation caused by the ubiquitin-proteasome system. The technical principle is as shown in the attached Figure 1 shown.

[0004] Theoretically, PROTAC is an event-driven pharmacological action mode. This process does not require the target protein ligand to occupy the binding site for a long time. Only the brief formation of the ternary complex is required to instantaneously complete the ubiquitination of the target protein, and the PROTAC molecule can be recycled multiple times in the cell. Therefore, compared with traditional small molecule inhibitors and large molecule antibodies, PROTAC has obvious advantages and is expected to target those difficult-to-drug proteins for drug development. It may also have the advantages of small dosage, low toxicity, independence from affinity, high selectivity, and overcoming drug resistance caused by target protein mutations / overexpression.

[0005] With the continuous deepening of research on PROTAC technology, according to the different structures of Linkers, it can be divided into alkyl chains and PEG chains. There are also more rigid alkynyl bipiperidine rings, spiro rings or bridge rings containing nitrogen atoms as Linkers to limit the flexibility and freedom of PROTAC molecules. The commonly used Linker length is generally 4-15 carbon atoms (or heteroatoms). Researchers have found that the length of the Linker structure itself and the structure at the molecular level have a very important influence on the activity of PROTAC drugs. In addition, depending on the target, the length of the Linker has different effects on the degradation activity. However, there is currently no general rule for Linker structure design. In addition, Click chemistry is often used in the Linker of PROTAC molecules to connect molecules at both ends due to its milder reaction conditions and higher efficiency.

[0006] The E3 ligases currently reported in the literature that are used in PROTACs are mainly CRBN, VHL, cIAP, and MDM2. The E3 ligases with better effects and the highest frequency of use are mainly CRBN (60.1%) and VHL (30.1%). Among them, the ligands of CRBN are mainly lenalidomide, pomalidomide and its analogs, and the ligands of VHL are mainly VHL-L. In the process of PROTAC research and development, Dr. Craig Crews of Yale University took the lead in making a key breakthrough. The PROTAC small molecule they designed can recognize the estrogen receptor at one end, and the other end interacts with the ubiquitin ligase VHL, resulting in the ubiquitination and degradation of the estrogen receptor, thereby preventing the growth of breast tumors. At the same time, the Bradner group at Harvard Medical School also designed a PROTAC compound that uses CRBN ubiquitin ligase to degrade the transcription factor BRD4, which plays an important role in cancer. Both studies successfully inhibited tumors in mouse models, overcoming a major obstacle to the use of protein knockdown methods for treatment. Since then, PROTAC has received widespread attention from the biomedical and industrial communities as an emerging technology, especially Arvinas, which recently had two PROTAC compounds show good Phase II clinical results. Unlike the "occupancy-driven" mode of traditional small molecule inhibitors to inhibit protein function, PROTAC adopts an "event-driven" pharmacological mode of action, which means that only a catalytic amount of drugs is needed to degrade the target protein in the cell, providing a new way to discover inhibitors of difficult-to-drug targets.

[0007] Although PROTAC technology has made great progress in recent years, there are still some problems: (1) PROTAC drugs have a large molecular weight and are difficult to penetrate the cell membrane; (2) The length of the linker and the binding position will affect the degradation ability of PROTAC; (3) As one of the components of PROTAC, E3 ubiquitin ligase plays a vital role. Although E3 ligase is a large protein family, more than 600 E3 ligases have been found to play a role in human cells, but currently only VHL, CRBN, IAPs, and MDM2 are E3 ligase ligands that can be truly used for PROTAC design. The lack of available E3 ligases and ligands has limited the development of PROTAC technology. (4) Although VHL, CRBN, IAPs, and MDM2 have been used in PROTAC so far, they all lack the regulation of the degradation rate of the target protein, because too fast or too slow degradation of the protein may be harmful in some cases; (5) In addition, the two most commonly used ubiquitin ligases VHL and CRBN in PROTAC also have some defects, such as their low activity in some cells, which may not be easy to use for tumor treatment, such as blood cancer, brain cancer, lung cancer and kidney cancer. Therefore, how to make PROTAC more generally applicable remains a key and urgent issue.

[0008] Epidermal growth factor receptor (EGFR) belongs to the ErbB family and is a transmembrane receptor tyrosine kinase (RTK) that plays a key role in cell proliferation, differentiation and motility. Homodimerization or heterodimerization of EGFR and other ErbB family members activates the cytoplasmic tyrosine kinase domain, thereby initiating intracellular signal transduction. Overexpression or activating mutations of EGFR are associated with the manifestation of many types of cancers, such as pancreatic cancer, breast cancer, glioblastoma multiforme, head and neck cancer, and non-small cell lung cancer. It has been determined that activating mutations in the EGFR tyrosine kinase domain are oncogenic drivers of NSCLC. EGFR mutations are common driver gene mutations in NSCLC, and different EGFR mutations have different sensitivities to different EGFR-tyrosine kinase inhibitors (EGFR-TKIs). The first-generation EGFR inhibitors mainly include Gefitinib and Erlotinib, which can effectively inhibit EGFR (Del19) and EGFR (L858R) mutants, but the first-generation inhibitors often produce EGFR (T790M) resistance mutations after one year of use, thereby reducing the binding of the inhibitors; the second-generation EGFR inhibitors mainly include Afatinib and Dacomitinib, which can not only inhibit EGFR (Del19) and EGFR (L858R) mutants, but also inhibit EGFG (T790M) mutants, but because they have no selectivity for wild-type EGFR (WT), they often have greater side effects; the third-generation inhibitors mainly include Osimertinib (Olmutinib), which solves the problem of the second-generation inhibitors, that is, it does not affect EGFR (WT) and inhibits EGFR (Del19), EGFR (L858R) and EGFG (T790M) mutants, but with the extension of the medication time, EGFR (C797S) resistance mutations will be generated. Therefore, there is an urgent need to develop new targeted drugs that can overcome the drug resistance of EGFR-mutated non-small cell lung cancer (NSCLC).

[0009] Anaplastic lymphoma kinase (ALK) is a receptor-type tyrosine kinase that consists of an extracellular domain, a single-pass transmembrane domain, and an intracellular kinase domain. The extracellular domain of ALK includes two methyldopa-A5 protein-receptor type protein tyrosine phosphatase (MAM) domains, a low-density lipoprotein (LDLa) domain, and a glycine-rich extracellular domain (G-rich); a single transmembrane (TM) domain located between the extracellular and intracellular parts; and an intracellular tyrosine kinase (PTK) domain. FAM150 is the ligand of ALK, and when the ligand binds to the extracellular domain, the receptor protein-tyrosine kinase is activated by inducing receptor dimerization or oligomerization. The possible mechanism of ligand and dimer-induced ALK activation involves the phosphorylation of one or more membrane-proximal tyrosine residues (Tyr 1078, 1092, 1096, and 1131), followed by sequential phosphorylation until the active form of ALK is formed.

[0010] Fusion and rearrangement of the ALK gene are important causes of many cancers, including non-small cell lung cancer (NSCLC) and anaplastic large cell lymphoma (ALCL). The proportion of ALK fusion mutations in non-small cell lung adenocarcinoma is 3-5%, and the proportion in China is about 5.3%. The incidence is higher in non-small cell lung adenocarcinoma, young patients (less than 60 years old) and non-smokers. ALK fusion is the second largest NSCLC molecular subtype after EGFR mutation, and the corresponding targeted drugs are completely different from EGFR molecular subtypes. ALK can fuse and rearrange with a variety of paired genes, resulting in its constitutive activation. The most common fusion occurs in echinoderm microtubule-associated protein 4-anaplastic lymphoma kinase (EML4-ALK). To date, several ALK-TKIs have been approved for the treatment of ALK-positive NSCLC patients, including the first-generation ALK-TKI crizotinib, the second-generation ALK-TKIs ceritinib, alectinib, brigatinib and ensartinib, and the third-generation ALK-TKI lorlatinib. Unfortunately, patients treated with ALK-TKIs will develop drug resistance and cause disease progression. Therefore, it is of great significance to develop new targeted drugs that overcome drug resistance in ALK-positive non-small cell lung cancer (NSCLC).

[0011] Studies have found that when Gly or Pro is located at the N-terminus of a protein, the protein becomes unstable and can be degraded by the E3 ligase ZYG11B / ZER1 or GID4 through the ubiquitin proteasome system. Summary of the invention

[0012] The purpose of the present invention is to propose a novel and unique PROTAC small molecule, which uses the cell's own degradation pathway to specifically degrade the L858R / T790M double-mutated EGFR receptor tyrosine protein kinase and EML4-ALK fusion protein. The PROTAC small molecule uses single amino acids such as Pro and Gly as ligands of E3 ligases GID4, ZYG11B, etc., and the other end is bound to the target protein EGFR (L858R / T790M) or EML4-ALK through an analog (BA) of the ligand Brigatinib. The two ligands are then connected by PEG linkers of different lengths. Such a chemical molecule connects the E3 ligase to EGFR (L858R / T790M) or EML4-ALK, so that EGFR (L858R / T790M) and EML4-ALK are ubiquitinated and degraded by the proteasome.

[0013] The technical solution of the present invention is achieved in this way:

[0014] The present invention provides a compound of the following formula (M), or its stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates, isotope-labeled derivatives or pharmaceutical compositions:

[0015]

[0016] The compound of formula (M) includes a single amino acid AA and a ligand brigatinib derivative BA (brigatinib analog), which are connected by a PEG linker. Alkyl chains, more rigid alkynyl bipiperidine rings, spiro rings or bridge rings containing nitrogen atoms can also be used as linkers.

[0017] Among them, AA is selected from Gly, Ala, Val, Leu, Ile, Phe, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Asn, Gln, Glu, Thr, Lys, Arg, Orn or His, preferably Gly, Pro, Thr, Lys or Orn.

[0018] Wherein, the structure of the ligand Brigatinib derivative BA (Brigatinib analog) is shown in the following formula:

[0019]

[0020] It is obtained by removing a methyl group and a piperidine from Brigatinib:

[0021]

[0022] The PEG linker includes but is not limited to diethylene glycol (PEG1), triethylene glycol (PEG2), tetraethylene glycol (PEG3), and pentaethylene glycol (PEG4).

[0023] As a further improvement of the present invention, the compound comprises Gly-PEG3-BA: Pro-PEG3-BA: Thr-PEG3-BA: Lys-PEG3-BA: Orn-PEG3-BA: Pro-PEG1-BA: Pro-PEG2-BA: Pro-PEG4-BA:

[0024] The present invention also provides a pharmaceutical composition comprising a compound of formula (M) or its stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates, isotope-labeled derivatives and pharmaceutically acceptable carriers or excipients.

[0025] In the present invention, depending on the desired mode of administration, a pharmaceutically acceptable composition will contain from about 1 to about 99% by weight of a compound of the invention and from 99 to 1% by weight of a suitable carrier or pharmaceutical excipient. Preferably, the composition contains from about 5 to 75% by weight of a compound of the invention, with the remainder being a suitable carrier or pharmaceutical excipient. More preferably, the composition contains from about 10 to 50% by weight of a compound of the invention, with the remainder being a suitable carrier or pharmaceutical excipient.

[0026] The present invention further provides a pharmaceutical composition, which contains a therapeutically effective amount of the compound of the above formula (M) or its stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates, isotope-labeled derivatives and a pharmaceutically acceptable carrier.

[0027] The pharmaceutical composition according to the present invention can be formulated for various conventional or specific routes of administration, such as oral administration, parenteral administration and rectal administration, etc. Oral dosage forms, such as tablets, capsules (including sustained release or timed release prescriptions), pills, powders, granules, elixirs, tinctures, suspensions (including nanosuspensions, microsuspensions, spray-dried dispersions), syrups and emulsions; parenteral administration, such as by subcutaneous, intravenous, intramuscular or intrasternal injection, or infusion technology (such as as a sterile injectable aqueous solution or non-aqueous solution or suspension); nasal administration, such as administration to the nasal mucosa, by inhalation spray; topical administration, such as in the form of creams or ointments; rectal administration, such as in the form of suppositories. The compounds according to the present invention can be administered alone, but will usually be administered together with a pharmaceutical carrier selected according to the selected route of administration and standard pharmaceutical operations.

[0028] "Pharmaceutically acceptable carrier" refers to a medium generally accepted in the art for delivering biologically active agents to animals, especially mammals, and includes, for example, adjuvants, excipients or excipients, such as diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants and dispersants, depending on the mode of administration and the nature of the dosage form.

[0029] Pharmaceutically acceptable carriers are within the purview of those of ordinary skill in the art and can be selected and formulated based on a number of factors. They include, but are not limited to, the type and nature of the active agent being formulated, the subject to whom the composition containing the agent is to be administered, the intended route of administration of the composition and the target therapeutic indication, etc. Pharmaceutically acceptable carriers include both aqueous and non-aqueous media as well as a variety of solid and semisolid dosage forms. In addition to the active agent, such carriers may include many different ingredients and additives, and it is well known to those of ordinary skill in the art to include additional ingredients in the formulation for a variety of reasons (e.g., stabilization of the active agent, adhesives, etc.).

[0030] The dosage regimen for the compounds of the invention can certainly be varied according to known factors, such as the pharmacodynamic characteristics of the specific agent and the mode and route of administration thereof, the species, age, sex, health, medical condition and weight of the recipient, the nature and extent of the symptoms, the type of coexisting treatment, the frequency of treatment, the route of administration, the function of the kidney and liver of the patient, and the desired effect. The therapeutically effective dose of the compound, pharmaceutical composition or combination thereof depends on the subject species, weight, age and individual condition, the treated condition or disease or its severity. A physician, clinician or veterinarian with ordinary skills can easily determine the effective amount of each active ingredient required to prevent, treat or inhibit the progression of a condition or disease.

[0031] The present invention also provides the use of the above-mentioned compound of formula (M) or its stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates, isotope-labeled derivatives or the above-mentioned pharmaceutical compositions in the preparation of drugs for treating cancer.

[0032] Epidermal growth factor receptor (EGFR) is widely distributed on the cell surface of mammalian epithelial cells, fibroblasts, glial cells, etc. The EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation and differentiation. EGFR mutation is also the most common mutation type in NSCLC patients, especially in Asian populations, accounting for 40% to 50%. In addition, ALK was first discovered in a subtype of anaplastic large cell lymphoma (ALCL), so it was named anaplastic lymphoma kinase (ALK). Subsequently, before the discovery of ALK gene rearrangement in non-small cell lung cancer, multiple types of ALK gene rearrangements were found in diffuse large B-cell lymphoma and inflammatory myofibroblastic tumor (IMT), indicating that ALK is a strong oncogenic driver gene.

[0033] Therefore, the present invention also provides a method for treating cancer, which comprises administering to a patient a therapeutically effective amount of the above-mentioned compound or its stereoisomer, tautomer, pharmaceutically acceptable salt, prodrug, hydrate, solvate, isotope-labeled derivative or the above-mentioned pharmaceutical composition. The above-mentioned cancers include lymphoma, non-Hodgkin's lymphoma, ovarian cancer, cervical cancer, prostate cancer, colorectal cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, melanoma, leukemia, gastric cancer, endometrial cancer, lung cancer, hepatocellular carcinoma, gastrointestinal stromal tumor (GIST), acute myeloid leukemia (AML), bile duct cancer, renal cancer, thyroid cancer, anaplastic large cell lymphoma, mesothelioma, multiple myeloma, melanoma.

[0034] The present invention also provides the use of the above-mentioned compound or its stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates, isotope-labeled derivatives or the above-mentioned pharmaceutical compositions in the preparation of drugs for treating EGFR and / or ALK-related cancers. The above-mentioned cancers include lymphoma, non-Hodgkin's lymphoma, ovarian cancer, cervical cancer, prostate cancer, colorectal cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, melanoma, leukemia, gastric cancer, endometrial cancer, lung cancer, hepatocellular carcinoma, gastrointestinal stromal tumor (GIST), acute myeloid leukemia (AML), bile duct cancer, renal cancer, thyroid cancer, anaplastic large cell lymphoma, mesothelioma, multiple myeloma, and melanoma.

[0035] In some embodiments of the present invention, the above-mentioned cancer is lung cancer.

[0036] Description and Definitions:

[0037] Unless otherwise specified, the following terms and phrases used herein have the following meanings. A particular term or phrase should not be considered ambiguous or unclear without special definition, but should be understood according to the ordinary meaning.

[0038] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0039] The term "pharmaceutically acceptable salt" refers to a derivative of the compound of the present invention prepared with a relatively non-toxic acid or base. These salts can be prepared during the synthesis, separation, and purification of the compound, or the purified free form of the compound can be used alone to react with a suitable acid or base. When the compound contains a relatively acidic functional group, it reacts with an alkali metal, alkaline earth metal hydroxide or an organic amine to obtain a base addition salt, including cations based on alkali metals and alkaline earth metals and non-toxic ammonium, quaternary ammonium and amine cations, and also covers salts of amino acids. When the compound contains a relatively basic functional group, it reacts with an organic acid or an inorganic acid to obtain an acid addition salt.

[0040] The compounds provided by the present invention also include prodrug forms, which refer to compounds that are rapidly converted in vivo to the parent compound of the above formula, which can be converted into the compounds of the present invention by chemical or biochemical methods in an in vivo or in vitro environment, such as by hydrolysis in the blood.

[0041] The compounds of the present invention can exist in unsolvated and solvated forms, including solvates, which include hydrates. In general, the pharmaceutical effects of the solvates are equivalent to those of the unsolvated forms, and are also encompassed within the scope of the present invention.

[0042] The compounds of the present invention exist as "tautomers", which refers to a type of functional isomer that has different points of attachment due to the displacement of one or more double bonds, for example, a ketone and its enol form are keto-enol tautomers.

[0043] The compounds of the present invention exist in geometric isomers and stereoisomers, such as cis-trans isomers, enantiomers, diastereomers, racemic mixtures and other mixtures, and all of these mixtures belong to the scope of the present invention.

[0044] The term "enantiomer" refers to stereoisomers that are mirror images of one another.

[0045] The term "diastereomer" refers to stereoisomers that have two or more chiral centers and are not mirror images of each other.

[0046] The term "cis-trans isomers" refers to configurations in which double bonds or single bonds of ring carbon atoms in a molecule cannot rotate freely.

[0047] Stereoisomers of the compounds of the present invention can be prepared by chiral synthesis or chiral reagents or other conventional techniques. For example, one enantiomer of a compound of the present invention can be prepared by asymmetric catalysis technology or chiral auxiliary derivatization technology. Alternatively, a compound of a single stereo configuration can be obtained from a mixture by chiral resolution technology. Alternatively, it can be directly prepared using chiral starting materials. The separation of optically pure compounds in the present invention is usually completed using preparative chromatography, using a chiral chromatographic column to achieve the purpose of separating chiral compounds.

[0048] The present invention also includes isotope-labeled derivatives. Comprise the isotope of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine, for example 2H, 3H, 13C, 11C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F and 36Cl respectively. The compounds of the present invention that contain other isotopic compounds of the present invention of above-mentioned isotope and / or other atoms all belong to scope of the present invention.

[0049] The term "effective preventive or therapeutic amount" refers to a sufficient amount of the compound of the present invention or its stereoisomer, tautomer or pharmaceutically acceptable salt, prodrug, hydrate, solvate, isotope-labeled derivative to treat the disorder at a reasonable effect / risk ratio applicable to any medical treatment and / or prevention. However, it should be recognized that the total daily dosage of the compound of formula (M) of the present invention or its stereoisomer, tautomer or pharmaceutically acceptable salt, prodrug, hydrate, solvate, isotope-labeled derivative and composition must be determined by the attending physician within the scope of reliable medical judgment.

[0050] For any particular patient, the specific therapeutically effective dosage level must be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex and diet; the administration time, route of administration and excretion rate of the specific compound used; the duration of treatment; drugs used in combination or concomitantly with the specific compound used; and similar factors known in the medical field. For example, the practice in the art is to start the dose of the compound from a level lower than that required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is obtained. In general, the dosage of a compound of formula (M) of the present invention or a pharmaceutically acceptable salt thereof for mammals, especially humans, can be between 0.001 and 1000 mg / kg body weight / day, such as between 0.01 and 100 mg / kg body weight / day, such as between 0.01 and 10 mg / kg body weight / day.

[0051] The compounds described herein can be prepared by methods known to those skilled in the art.

[0052] The present invention further protects the use of a compound of the above formula (M) or a pharmaceutically acceptable salt thereof in the preparation of a drug for degrading the protein level of EGFR and / or ALK in vivo.

[0053] The present invention further protects the use of a compound of the above formula (M) or a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs.

[0054] As a further improvement of the present invention, the tumor includes but is not limited to blood cancer, brain cancer, lung cancer and kidney cancer.

[0055] PROTAC (proteolysis targeting chimeras) is a drug development technology that uses the ubiquitin-proteasome system to degrade the target protein (POI). Here, the present invention designs a novel and unique class of PROTAC small molecules that use the cell's own degradation pathway to specifically degrade the L858R / T790M double-mutated EGFR receptor tyrosine protein kinase and EML4-ALK fusion protein, that is, the PROTAC small molecules synthesized by the present invention use single amino acids such as Pro and Gly as ligands of E3 ligases GID4, ZYG11B, etc., and the other end is bound to the target protein EGFR (L858R / T790M) or EML4-ALK fusion protein through an analog (BA) of the ligand Brigatinib, and the two ligands are then connected by PEG linkers of different lengths. Such chemical molecules link the E3 ligase to EGFR (L858R / T790M) or EML4-ALK fusion protein, causing the EGFR (L858R / T790M) or EML4-ALK fusion protein to be ubiquitinated and degraded by the proteasome.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] 1. The advantages of PROTAC of the present invention are that the target protein can be degraded through a very small degradation signal (single amino acid), thereby significantly reducing the size of the PROTAC molecule to increase cell permeability and effectiveness; secondly, by replacing different amino acids (such as Pro, Gly, etc.), these small molecules with only differences in the N-terminus change the degradation rate of the target protein due to binding to different E3 ligases, thereby optimizing the regulation of protein stability.

[0058] 2. The Gly-PEG3-BA, Pro-PEG3-BA, Thr-PEG3-BA, Pro-PEG2-BA and Pro-PEG4-BA designed and synthesized by the present invention are proved to be specific degraders of EGFR (L858R / T790M) and EML4-ALK fusion proteins in in vitro experiments, and can regulate the degradation rate of EGFR (L858R / T790M) and EML4-ALK fusion proteins. It can not only provide a new treatment strategy for non-small cell lung cancer (NSCLC), but also can be easily transplanted to a variety of cancers and other target drugs, and has the feasibility of wide application, bringing new and effective methods to the treatment of human diseases, and therefore has extremely important significance for the treatment of diseases and improving the survival and quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0060] Figure 1 This is a schematic diagram of the PROTAC technology principle;

[0061] Figure 2 It is the schematic diagram of N degron pathway;

[0062] Figure 3 is the structural formula of PROTAC with different amino acids;

[0063] Figure 4 The effects of different PROTACs and BAs on EGFR (WT), EGFR (Del19), and EGFR (L858R / T790M);

[0064] Figure 5 The degradation effects of Pro-PEG(1-4)-BA with different lengths of PEG Linker on EGFR(L858R / T790M) and ALK;

[0065] Figure 6 The effects of PROTACs with different amino acids on the degradation ability and degradation rate of EGFR (L858R / T790M) and ALK;

[0066] Figure 7 The effect of Pro-PEG3-BA on the cell cycle and apoptosis of EGFR (L858R / T790M) double mutant and ALK-positive NSCLC cells. DETAILED DESCRIPTION

[0067] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0068] The preparation methods of PROTAC compounds of the series Gly-PEG3-BA, Pro-PEG3-BA, Thr-PEG3-BA, Lys-PEG3-BA, Orn-PEG3-BA, Pro-PEG1-BA, Pro-PEG2-BA and Pro-PEG4-BA in the present invention are feasible through experiments, and the experimental conditions are reproducible and the yield is stable.

[0069] Synthesis steps

[0070] Synthesis of Brigatinib analog (BA)

[0071]

[0072] In a reaction flask, 2-(dimethoxyphosphino)aniline (500 mg, 2.96 mmol, 1.0 eq), 2,4,5-trichloropyrimidine (542.1 mg, 2.96 mmol, 1.0 eq), potassium carbonate (857.9 mg, 6.2 mmol, 2.1 eq), tetrabutylammonium hydrogen sulfate (100.4 mg, 0.3 mmol, 0.1 eq) were dissolved in 7 ml of DMF and stirred at 65°C overnight. After the reaction was completed by monitoring by thin layer chromatography (TLC), potassium carbonate was removed by filtration, the filtrate was concentrated in vacuo, and purified by column chromatography using DCM:MeOH=50:1 to obtain 1 as a white solid (757 mg, 2.4 mmol, 81%). HRMS (ESI + )m / z:calcd.for C 12 H 12 Cl2N3OP[M+H] + 316.0095, found 316.0168.

[0073] In a 10 mL reaction tube, compound 1 (100 mg, 0.32 mmol, 1.0 eq), tert-butyl 4-(4-amino-3-methoxyphenyl)piperazine-1-carboxylate (117 mg, 0.38 mmol, 1.2 eq) and 2 ml of isopropanol solution were added and stirred until dissolved, followed by addition of TFA (25.9 μL, 0.35 mmol, 1.1 eq), and the reaction tube was stirred at 95 ° C overnight. The progress of the reaction was tracked using analytical HPLC, and the reaction mixture was directly purified by preparative HPLC to give a gray-green powder 2 after freeze drying. The gray-green powder was dissolved in 20% TFA / DCM under an ice bath, and the reaction solution was then allowed to warm to room temperature and stirred for 1 hour. The reaction was monitored using analytical HPLC, and after the reaction was completed, the solvent was evaporated under reduced pressure, and purified by preparative HPLC to give a yellow-green powder 3 (t R =7.246min, 140mg, 0.29mmol, 91%), namely Brigatinibanalog (BA). HRMS (ESI + )m / z:calcd.for C 23 H 28 C1N6O2P[M+H] + 487.1700, found 487.1776.

[0074] Synthesis of X-PEG3-BA

[0075]

[0076] Take the synthesis of compound 7 (Pro-PEG3-BA) as an example

[0077] In a 10 ml reaction tube, compound 3 (59 mg, 0.121 mmol, 1.0 eq), compound 4 (64.5 mg, 0.146 mmol, 1.2 eq), HATU (55.4 mg, 0.146 mmol, 1.2 eq) and 2 ml of anhydrous DMF were added and stirred until dissolved, followed by the addition of DIEA (200.6 μL, 1.214 mmol, 10.0 eq) and reacted at room temperature for 2 hours. The reaction progress was followed by analytical HPLC. The reaction mixture was directly purified by preparative HPLC to obtain white powder 5 (88.9 mg, 0.098 mmol, 80%) after freeze drying. Compound 5 (20 mg, 0.022 mmol, 1.0 eq) was dissolved in a mixed solution of DEA / DCM (4 ml, 1:1), reacted at room temperature for 1 hour, and monitored by HPLC. After the reaction was completed, the solvent was evaporated under reduced pressure to obtain the crude amine 6 with the Fmoc protection group removed without further purification. The crude deprotected amine 6, BOC-L-proline (4.5 mg, 0.021 mmol, 1.2 eq.) and HATU (7.9 mg, 0.021 mmol, 1.2 eq.) were dissolved in 1 ml of anhydrous DMF, and DIEA (28.8 μl, 2.228 mmol, 10.0 eq.) was added after stirring and dissolving. The mixture was reacted at room temperature for 2 hours, and the progress of the reaction was monitored by HPLC. After the reaction was completed, the mixture was directly purified by preparative HPLC to obtain a white powder after freeze drying. The white powder was dissolved in 20% TFA / DCM under an ice bath, and then the reaction solution was warmed to room temperature and stirred for 1 hour. After the reaction was completed, it was purified by HPLC and freeze dried to obtain a white powder 7 (t R =8.376min, 3.3 mg, 0.004 mmol, 35%), namely Pro-PEG3-BA. HRMS (ESI + )m / z:calcd.for C 37 H 52 ClN8O7NaP[M+Na] + 809.3385, found 809.3277.

[0078] Compound 8 (Gly-PEG3-BA), compound 9 (Orn-PEG3-BA) and compound 10 (Lys-PEG3-BA) were synthesized by the same experimental steps, and their relevant data are as follows:

[0079] Gly-PEG3-BA t R =7.914, Yield=52%, HRMS(ESI + ) m / z: calcd. for C 34 H 48 ClN8O7NaP [M+Na] + 769.3072, found 769.2964.

[0080] Orn-PEG3-BA t R =7.472, Yield=57%, HRMS(ESI + ) m / z: calcd. for C 37 H 56 ClN9O7P [M+H] + 804.3651, found 804.3723.

[0081] Lys-PEG3-BA t R =7.501, Yield=62%, HRMS(ESI + ) m / z: calcd. for C 38 H 58 C1N9O7P [M+H] + 818.3807, found 818.3880.

[0082] Synthesis of Pro-PEGX-BA

[0083]

[0084] Taking the synthesis of Pro-PEG1-BA as an example

[0085] In a 10 ml reaction tube, compound 3 (20 mg, 0.041 mmol, 1.0 eq), Fmoc-NH-PEG1-C2-acid (17.5 mg, 0.049 mmol, 1.2 eq), HATU (18.8 mg, 0.049 mmol, 1.2 eq) and 2 ml of anhydrous DMF were added and stirred until dissolved. Subsequently, DIEA (53.2 μL, 0.411 mmol, 10.0 eq) was added and reacted at room temperature for 2 hours. The reaction progress was tracked using analytical HPLC. The reaction mixture was directly purified by preparative HPLC to obtain white powder 12 (26.3 mg, 0.032 mmol, 78%) after freeze drying. Compound 12 (10 mg, 0.012 mmol, 1.0 eq) was dissolved in a mixture of DEA / DCM (2 ml, 1:1) and reacted at room temperature for 1 hour and monitored by HPLC. After the reaction was completed, the solvent was evaporated under reduced pressure to obtain the crude amine 13 without Fmoc protection group without further purification. The crude deprotected amine 13, BOC-L-proline (3.1 mg, 0.014 mmol, 1.2 eq) and HATU (5.54 mg, 0.015 mmol, 1.2 eq) were dissolved in 1 ml of anhydrous DMF, followed by the addition of DIEA (20 μL, 0.121 mmol, 10.0 eq) and reacted at room temperature for 2 hours. The progress of the reaction was followed by analytical HPLC. The reaction mixture was directly purified by preparative HPLC to obtain 14 (9.35 mg, 0.012 mmol, 96%) as a white powder after freeze drying. The white powder was dissolved in 20% TFA / DCM in an ice bath, and then the reaction solution was warmed to room temperature and stirred for 1 hour. After the reaction was completed, it was purified by HPLC and freeze-dried to obtain a white powder 15 (t R =8.371min, 1.58mg, 0.002mmol, 19%), namely Pro-PEG1-BA. HRMS (ESI + )m / z:calcd.for C 33 H 44 C1N8O5P[M+H] + 699.2861, found 699.2934.

[0086] Pro-PEG2-BA, Pro-PEG3-BA and Pro-PEG4-BA were synthesized by the same experimental steps.

[0087] Pro-PEG2-BA R =8.526, Yield=26%, HRMS(ESI+)m / z: calcd.forC 35 H 48ClN8O6NaP[M+Na]+765.3123,found 765.3015.

[0088] Pro-PEG3-BA t R =8.376,Yield=35%,HRMS(ESI+)m / z:calcd.forC 37 H 52 C1N s O7NaP[M+Na] + 809.3385,found 809.3277。

[0089] Pro-PEG4-BA t R =8.801,Yield=32%,HRMS(ESI + )m / z:calcd.forC 39 H 56 C1N8O8NaP[M+Na] + 853.3647,found 853.3540.

[0090] Example 1 N degron pathway

[0091] PROTAC small molecules synthesized using the N degron pathway start with single amino acids Pro, Gly, etc., and are fused to BA through PEG linkers of different lengths. Through this Pro(Gly)-PEG(1-4)-BA small molecule intermediate bridge, E3 ligases such as GID4, ZYG11B / ZER1 are connected to EGFR(L858R / T790M) or EML-ALK, which causes EGFR(L858R / T790M) or EML-ALK to be ubiquitinated and significantly reduce its level, thereby inhibiting NSCLC growth. A major advantage of protein degradation is that, unlike typical enzyme inhibition-based methods, it only requires transient drug-target interactions, rather than direct inhibition of kinase activity. Our improvements to the existing commonly used PROTACs are: (1) a very small degradation signal (such as a single amino acid Pro, Gly) significantly reduces the size of the PROTAC compound to increase cell permeability and effectiveness; (2) by comparing PEG linkers of different lengths, there are different degrees of degradation effects on the target protein, so as to find the shortest linker to achieve the best degradation effect; (3) the E3 ligases GID4 and ZYG11B used are widely expressed and highly active E3 ubiquitin ligases; (4) by replacing different amino acids (such as Pro, Gly, etc.), these small molecules with only N-terminal differences change the degradation rate due to the use of different E3 ligases, thereby optimizing the regulation of protein stability. Our goal is to use the smallest degradation signal - a single amino acid and to design novel PROTAC small molecules by replacing different amino acids, thereby improving cell permeability and achieving better therapeutic effects, as well as better controlling the level of EGFR double mutant protein through unique adjustable degradation capabilities, see Figure 2 .

[0092] Example 2 PROTAC structures targeting EGFR or ALK based on N degradation subpathway

[0093] Brigatinib is a selective ALK inhibitor, and existing studies have found that it can also inhibit IGF-1R, FLT3, and EGFR. [1] Brigatinib with one methyl group and one piperidine removed (i.e., BA) can also bind to EGFR mutants (see Figure 3 A), so we designed PROTAC as follows: BA was used as the ligand of EGFR or ALK, and amino acids Pro, Gly, and Lys were used as the ligands of E3 ligases, respectively, and BA and Pro / Gly / Lys were connected by three PEG linkers (Lysine Lys was used as a degrader of the UBR family, which we have reported before). [2]As a positive control, ornithine (Orn) was used as a negative control because it is not an amino acid in the N degron pathway (see Figure 3 B).

[0094] [1]ZHANG H, XIE R, AI-FURAS H, et al. Design, synthesis, and biological evaluation of novel EGFR PROTACs targeting Del19 / T790M / C797S mutation[J]. ACSMedicinal Chemistry Letters, 2022, 13(2): 278-283.

[0095] [2]ZHANG J, MAC, YU Y, et al.Single amino acid-based PROTACs triggerdegradation of the oncogenic kinase BCR-ABL in chronic myeloid leukemia(CML)[J].Journal of Biological Chemistry, 2023, 299(8)

[0096] Example 3 Degradation of wild-type and different mutant EGFR by Pro(Gly, Orn)-PEG3-BA

[0097] Immunoblotting experiments were performed to determine whether Pro(Gly)-PEG3-BA reduced the level of wild-type EGFR(WT) protein: A549 and H1299 cells expressing endogenous EGFR(WT) genes were plated in 12-well plates and treated with Pro-PEG3-BA, Gly-PEG3-BA, Lys-PEG3-BA (positive control), and Orn-PEG3-BA (negative control). After each PROTAC was treated with 6 different concentrations of 0, 0.01, 0.1, 1, 10, and 20 μM for 72 hours, the cells were lysed with RIPA for 30 minutes, and the BCA protein was quantified. At each concentration, 50 μg of protein samples were used for immunoblotting experiments to detect the protein level of EGFR(WT) at different drug concentrations.

[0098] Immunoblotting experiments were performed to determine whether Pro(Gly)-PEG3-BA reduced the protein level of EGFR(Del19) single mutants: PC9 cells expressing the endogenous EGFR(Del19) gene were plated in 12-well plates and treated with Pro-PEG3-BA, Gly-PEG3-BA, Lys-PEG3-BA, and Orn-PEG3-BA, respectively. After each PROTAC was treated with 6 different concentrations of 0, 0.01, 0.1, 1, 10, and 20 μM for 72 hours, the cells were lysed with RIPA for 30 minutes, and the BCA protein was quantified. At each concentration, 50 μg of protein samples were used for immunoblotting experiments to detect the protein level of EGFR(Del19) at different drug concentrations.

[0099] Immunoblotting experiments were performed to determine whether Pro(Gly)-PEG3-BA reduced the protein level of the EGFR(L858R / T790M) double mutant: H1975 cells expressing the endogenous EGFR(L858R / T790M) gene were plated in 12-well plates and treated with Pro-PEG3-BA, Gly-PEG3-BA, Lys-PEG3-BA, and Orn-PEG3-BA, respectively. After each PROTAC was treated with 6 different concentrations of 0, 0.01, 0.1, 1, 10, and 20 μM for 72 hours, the cells were lysed with RIPA for 30 minutes, and the BCA protein was quantified. At each concentration, 50 μg of protein samples were used for immunoblotting experiments to detect the protein level of EGFR(L858R / T790M) at different drug concentrations.

[0100] Immunoblotting showed that Pro-PEG3-BA, Lys-PEG3-BA, Gly-PEG3-BA, Orn-PEG3-BA and BA had no effect on the protein levels of EGFR(WT) and EGFR(Del19). Pro-PEG3-BA and Lys-PEG3-BA showed that they could reduce the protein level of EGFR(L858R / T790M) at 10 μM, and Gly-PEG3-BA showed that it could reduce the protein level of EGFR(L858R / T790M) at 20 μM, while Orn-PEG3-BA and BA had no effect on the protein level of EGFR(L858R / T790M) (see Figure 4 ).

[0101] Therefore, the PROTAC we designed can specifically degrade the EGFR (L858R / T790M) double mutant.

[0102] Example 4 Determination of the effect of Pro-PEG(1-4)-BA with different lengths of PEG linkers on EGFR(L858R / T790M) and EML-ALK

[0103] We selected the better PROTACs with Pro as E3 ligase ligand and designed linkers of different lengths: Pro-PEG1-BA, Pro-PEG2-BA, Pro-PEG3-BA, Pro-PEG4-BA (see Figure 5 A), and each PROTAC was used to treat H1975 or H3122 cells with 6 different concentrations of 0, 0.01, 0.1, 1, 10, 20 μM for 72 hours. Immunoblotting showed that Pro-PEG1-BA had no effect on the protein level of EGFR (L858R / T790M), while Pro-PEG2-BA, Pro-PEG4-BA and Pro-PEG3-BA could reduce the protein level of EGFR (L858R / T790M) at 20 μM (see Figure 5 B), Pro-PEG1-BA, Pro-PEG2-BA, Pro-PEG3-BA and Pro-PEG4-BA were able to reduce the protein level of EML-ALK at 1 μM (see Figure 5 C), while Pro-PEG3-BA showed the best degradation effect and could reduce the protein level of EGFR (L858R / T790M) at 10 μM (see Figure 5 B), similarly, Pro-PEG3-BA had the best degradation effect on EML-ALK at 1 μM (see Figure 5 C) Our results show that the PROTAC molecule with three PEG3 linkers has the best effect on target protein degradation

[0104] Example 5 Determination of the effects and degradation rates of different PROTACs on EGFR (L858R / T790M) and EML-ALK

[0105] H1975 cells were treated with different concentrations of Pro-PEG3-BA, Gly-PEG3-BA, and Lys-PEG3-BA, and immunoblotting determined that the DC50 of Pro-PEG3-BA for EGFR (L858R / T790M) was 69% at 13.50 μM and 20 μM, the DC50 of Gly-PEG3-BA was 43% at 20 μM, and the DC50 of Lys-PEG3-BA was 52% at 19.66 μM and 20 μM (see Figure 6A and 6C). CCK-8 cell proliferation assay found that the above four compounds can significantly reduce cell activity, among which Pro-PEG3-BA IC50: 8.8μM, Gly-PEG3-BA IC50: 20.74μM, Lys-PEG3-BA IC50: 18.56μM and BA IC50: 0.89μM (see Figure 6 B and 6C).

[0106] Comparison of the degradation rates of EGFR (L858R / T790M) by Pro-PEG3-BA, Gly-PEG3-BA, and Lys-PEG3-BA: We treated H1975 cells with 20 μM of the above three PROTACs for 0, 12, 24, 36, 48, and 72 h, and found that the time for the three PROTACs to degrade EGFR (L858R / T790M) at 20 μM was Pro-PEG3-BA: ~12 h, Gly-PEG3-BA: ~24 h, Lys-PEG3-BA: ~48 h (see Figure 6 D).

[0107] H3122 cells were treated with different concentrations of Pro-PEG3-BA, Gly-PEG3-BA, and Lys-PEG3-BA, and immunoblotting determined that the DC50 of Pro-PEG3-BA on EML-ALK was 96% at 0.42 μM and 10 μM, the DC50 of Gly-PEG3-BA was 98% at 0.5 μM and 10 μM, and the DC50 of Lys-PEG3-BA was 91% at 1.32 μM and 10 μM (see Figure 6 E and 6G). CCK-8 cell proliferation assay found that the above four compounds can significantly reduce cell activity, among which Pro-PEG3-BA IC50: 0.156μM, Gly-PEG3-BA IC50: 0.843μM, Lys-PEG3-BA IC50: 5.298μM and BA IC50: 0.303μM (see Figure 6 F and 6G).

[0108] Comparison of the degradation rates of Pro-PEG3-BA, Gly-PEG3-BA, and Lys-PEG3-BA on EGFR (L858R / T790M): We treated H3122 cells with 5 μM of the above three PROTACs for 0, 0.25, 0.75, 1.5, 3, 6, 12, and 24 h. We found that the time for the three PROTACs to degrade EML-ALK at 5 μM was Pro-PEG3-BA: ~0.75 h, Gly-PEG3-BA: ~0.25 h, and Lys-PEG3-BA: ~3 h (see Figure 6 H)

[0109] Our results show that PROTAC designed by replacing different unstable amino acids Gly, Pro, and Lys can regulate the degradation rate of the target protein.

[0110] Example 6 Determination of the effect of Pro-PEG3-BA on cell cycle and apoptosis of EGFR (L858R / T790M) double mutant and EML-ALK positive NSCLC cells

[0111] Evaluation of the effect of Pro-PEG3-BA on the cell cycle and apoptosis of EGFR (L858R / T790M) double mutants: H1975 cells were treated with different concentrations of 0, 10, and 20 μM Pro-PEG3-BA for 72 h. Flow cytometry analysis showed that Pro-PEG3-BA treatment could effectively block the G1 phase of cells and significantly inhibit the proportion of cells in the S phase and G2 phase ( Figure 7 A-7C). H1975 cells were treated with different concentrations of 0, 10, and 20 μM Pro-PEG3-BA for 72 h. Flow cytometry analysis showed that Pro-PEG3-BA treatment could effectively induce early and late apoptosis of cells (see Figure 7 D and 7E).

[0112] Evaluation of the effect of Pro-PEG3-BA on the cell cycle and apoptosis of ALK-positive NSCLC cells: H3122 cells were treated with different concentrations of 0, 5, and 10 μM Pro-PEG3-BA for 48 h. Flow cytometry analysis showed that Pro-PEG3-BA treatment could effectively block the G1 phase of cells and significantly inhibit the proportion of cells in the S and G2 phases ( Figure 7 F-7H). H3122 cells were treated with different concentrations of 0, 5, and 10 μM Pro-PEG3-BA for 48 h. Flow cytometry analysis showed that Pro-PEG3-BA treatment could effectively induce early and late apoptosis of cells (see Figure 7 I and 7J).

[0113] Our results show that PROTACs based on unstable amino acids can effectively block cell cycle progression and promote apoptosis.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound of formula (M), or a stereoisomer, tautomer, pharmaceutically acceptable salt, prodrug, hydrate, solvate, or isotope-labeled derivative thereof, characterized in that: The molecular structure of the compound is as follows: In the compound of formula (M), AA is a single amino acid, BA is a ligand buguiaridine derivative, and the two are connected by a PEG linker. Alkyl chains, more rigid alkynyl bipiperidine rings, spiro rings or bridge rings containing nitrogen atoms can also be used as linkers.

2. A compound of formula (M) according to claim 1, or a stereoisomer, tautomer, pharmaceutically acceptable salt, prodrug, hydrate, solvate, or isotope-labeled derivative thereof, characterized in that: The single amino acid is selected from Gly, Ala, Val, Leu, Ile, Phe, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Asn, Gln, Glu, Thr, Lys, Arg, Orn or His, preferably Gly, Pro, Thr, Lys or Orn.

3. A compound of formula (M) according to claim 1, or a stereoisomer, tautomer, pharmaceutically acceptable salt, prodrug, hydrate, solvate, or isotope-labeled derivative thereof, characterized in that: The BA structure is shown in Formula I:

4. A compound of formula (M) according to claim 1, or a stereoisomer, tautomer, pharmaceutically acceptable salt, prodrug, hydrate, solvate, or isotope-labeled derivative thereof, characterized in that: The PEG linker is selected from diethylene glycol, triethylene glycol, tetraethylene glycol or pentaethylene glycol.

5. A compound of formula (M) according to claim 1, or a stereoisomer, tautomer, pharmaceutically acceptable salt, prodrug, hydrate, solvate, or isotope-labeled derivative thereof, characterized in that: The compound is selected from any one of the following structures:

6. A pharmaceutical composition comprising a compound of formula (M) as described in any one of claims 1 to 5 or its stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates, isotope-labeled derivatives and a pharmaceutically acceptable carrier or excipient.

7. Use of a compound of formula (M) as described in any one of claims 1 to 5 or its stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates, isotope-labeled derivatives in the preparation of drugs for degrading the protein level of EGFR and / or ALK in vivo.

8. Use of a compound of formula (M) as claimed in any one of claims 1 to 5 or its stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs, hydrates, solvates, isotope-labeled derivatives in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The tumor is selected from lymphoma, non-Hodgkin's lymphoma, ovarian cancer, cervical cancer, prostate cancer, colorectal cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, melanoma, leukemia, gastric cancer, endometrial cancer, lung cancer, hepatocellular carcinoma, gastrointestinal stromal tumor (GIST), acute myeloid leukemia (AML), bile duct cancer, renal cancer, thyroid cancer, anaplastic large cell lymphoma, mesothelioma, multiple myeloma or melanoma.

10. The use according to claim 9, characterized in that: The tumor is selected from lung cancer.

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