WO2026016338A1 - 基于N-degron的新型无连接子的微型PROTAC化合物及其用途 - Google Patents

基于N-degron的新型无连接子的微型PROTAC化合物及其用途

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Publication number
WO2026016338A1
WO2026016338A1 PCT/CN2024/129836 CN2024129836W WO2026016338A1 WO 2026016338 A1 WO2026016338 A1 WO 2026016338A1 CN 2024129836 W CN2024129836 W CN 2024129836W WO 2026016338 A1 WO2026016338 A1 WO 2026016338A1
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Prior art keywords
cancer
pharmaceutically acceptable
compound
pro
abl
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PCT/CN2024/129836
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English (en)
French (fr)
Inventor
饶海
张建超
房丽晶
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Shenzhen Institute of Advanced Technology of CAS
Southern University of Science and Technology
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Shenzhen Institute of Advanced Technology of CAS
Southern University of Science and Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system

Definitions

  • This application belongs to the field of pharmaceutical technology, specifically relating to a novel linkerless micro PROTAC compound based on N-degron and its uses.
  • PROTACs protein degradation-targeting chimeras
  • PROTACs consist of three parts: a ligand that binds to an E3 ligase to guide protein degradation; a ligand that binds to a target protein to guide the targeted localization of the small molecule; and a linker responsible for integrating the two ligands.
  • the target protein (POI) ligand in the PROTAC structure specifically binds to the corresponding target protein, while the other end recruits an E3 ligase to form a POI-linker-E3 ligase ternary complex.
  • the E3 ligase mediates polyubiquitination of the -POI, and the ubiquitin-labeled POI is recognized and degraded by the proteasome.
  • PROTAC compounds utilize a technology based on protein degradation induced by the ubiquitin-proteasome system.
  • PROTAC is an event-driven pharmacological mode of action. This process does not require the target protein ligand to occupy the binding site for a long time. The ubiquitination of the target protein can be completed instantaneously with only the brief formation of the ternary complex. Furthermore, PROTAC molecules 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 proteins that are difficult to drug and overcome drug resistance caused by target protein mutations/overexpression.
  • PROTAC technology still has some problems, such as the large molecular weight of PROTACs, the limited number of E3 ligases available for PROTAC design, and the lack of control over the degradation rate of target proteins. These problems limit the application of PROTACs.
  • this application provides a novel linkerless micro PROTAC compound based on N-degron and its uses.
  • this application relates to the following aspects:
  • X is a single amino acid or its derivative
  • Y is a ligand of the target protein
  • a pharmaceutical composition comprising the compound described in items 1-7 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the cancer is selected from one or more of the following: non-small cell lung cancer, anaplastic large cell lymphoma, chronic myeloid leukemia, acute lymphoblastic leukemia, atypical chronic myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, B-cell leukemia/lymphoma, lymphoblastic lymphoma, Tarton-Brown-Rachman syndrome or acute promyelocytic leukemia, small cell lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, gastric cancer, kidney cancer, Ovarian cancer, multiple myeloma.
  • a method of treating BCR-ABL or EML4-ALK mediated diseases comprising administering to a subject a therapeutically effective amount of the compound described in items 1-7 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition described in item 8.
  • the cancer is selected from one or more of the following: non-small cell lung cancer, anaplastic large cell lymphoma, chronic myeloid leukemia, acute lymphoblastic leukemia, atypical chronic myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, B-cell leukemia/lymphoma, lymphoblastic lymphoma, Tarton-Brown-Rachman syndrome or acute promyelocytic leukemia, small cell lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, gastric cancer, kidney cancer, ovarian cancer, and myeloma.
  • Figure 1 shows the ALK protein level detected by Western blotting after treating H3122 cells with the compounds of this application.
  • A represents the results of treating H3122 cells with different concentrations of Pro-BA for 24 hours;
  • B represents the results of treating H3122 cells with different concentrations of Gly-BA for 24 hours;
  • C represents the results of treating H3122 cells with 250 nM Pro-BA for different times;
  • D represents the results of treating H3122 cells with 250 nM Gly-BA for different times.
  • Figure 2 shows the effects of Pro-BA or Gly-BA on cell proliferation as detected by CCK8 assay.
  • A represents Pro-BA
  • B represents Gly-BA.
  • Figure 3 shows the flow cytometry results of H3122 cells treated with different concentrations of 0, 250 nM, and 500 nM Pro-BA for 48 h.
  • Figure 4 shows the flow cytometry results of H3122 cells treated with different concentrations of 0, 250 nM, and 500 nM Pro-BA for 48 h.
  • Figure 5 shows the results of the Pro-BA tumor growth inhibition assay.
  • A is a tumor photograph; B is the tumor size; C is the tumor growth curve; D is body weight; E and F are the ALK protein levels detected by Western blotting.
  • Figure 6 shows the results of the oral Pro-BA tumor growth inhibition assay.
  • A is a tumor photograph; B is the tumor growth curve; C is the immunohistochemical staining result of the tumor tissue; and D is the body weight.
  • Figure 7 shows the BCR-ABL levels detected by Western blotting after treating K562 cells with the compounds of this application. Protein levels.
  • A represents the results of treating K562 cells with different concentrations of Pro-DA for 24 hours; B represents the results of treating K562 cells with different concentrations of Gly-DA for 24 hours; C represents the results of treating K562 cells with 10 nM Pro-DA for different durations; and D represents the results of treating K562 cells with 10 nM Gly-DA for different durations.
  • Figure 8 shows the effects of Pro-BA or Gly-BA on cell proliferation as detected by CCK8 assay.
  • A represents Pro-BA
  • B represents Gly-BA.
  • a “ligand” is a molecule that forms a complex with a biomolecule to exert a biological function. Ligand complexes can be formed via ionic bonds, covalent bonds, van der Waals interactions, and/or hydrogen bonds.
  • a “target protein ligand” is a molecule that forms a complex with a target protein to exert a biological function.
  • “Pharmaceutically acceptable salt” is a salt formed by the compound of this application with an acid or base, wherein the acid is selected from hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, or ferulic acid, and the base is selected from alkali metal cation bases, alkaline earth metal cation bases, ammonium cation bases, or choline.
  • the acid is selected from hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesul
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs, as well as other components such as pharmaceutically acceptable carriers.
  • the purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.
  • treatment is to prevent or slow undesirable physiological changes or disorders.
  • beneficial or desired clinical outcomes include, but are not limited to, detectable or undetectable: relief of symptoms, reduction of disease severity, stabilization (i.e., no worsening) of the disease state, delay or slowing of disease progression, and improvement of... To improve or alleviate the state of a disease, and to relieve (partially or completely) it.
  • Treatment can also refer to extending survival time compared to the expected survival without treatment.
  • “Pharmaceutically acceptable” means a compound, material, composition, and/or dosage form suitable for contact with human or animal tissues without causing excessive toxicity, irritation, allergic reactions, other problems, or complications, and with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable compounds, materials, compositions, and/or dosage forms refer to those compounds, materials, compositions, and/or dosage forms approved by regulatory agencies (such as the U.S. Food and Drug Administration, the China National Medical Products Administration, and the European Medicines Agency) or listed in recognized pharmacopoeias (such as the United States Pharmacopeia, the Chinese Pharmacopoeia, and the European Pharmacopoeia) for use in animals, particularly humans.
  • “Pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or delivery vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, used to carry or transport the compound of this application from one site, body fluid, tissue, organ (internal or external), or body part to another site, body fluid, tissue, organ, or body part.
  • a pharmaceutically acceptable carrier may be a delivery vehicle, diluent, excipient, or other material that can be used in contact with animal tissues without excessive toxicity or adverse reactions.
  • Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars, starches, cellulose, malt, astragalus gum, gelatin, Ringer's solution, alginate, isotonic saline, buffers, etc.
  • Subject refers to an organism, tissue, or cell. Subjects may include human subjects for medical purposes (e.g., diagnosis and/or treatment of an existing condition or disease, or preventative treatment to prevent the onset of a condition or disease), or animal subjects for medical veterinary purposes or developmental purposes. Subjects also include sample materials from tissue cultures, cell cultures, organ replication, stem cell production, etc. Suitable animal subjects include mammals and birds.
  • mammal as used herein includes, but is not limited to, primates (e.g., humans, monkeys, apes, etc.), cattle (e.g., bulls), sheep (e.g., sheep, goats, etc.), pigs, horses, cats, dogs, rabbits, rodents (e.g., mice, rats, etc.).
  • animal as used herein includes, but is not limited to, chickens, ducks, geese, quails, turkeys, pheasants, etc.
  • the subject is a mammal or mammalian cell.
  • the subject is a human or human cell.
  • Subjects include, but are not limited to, fetuses, newborns, infants, adolescents, and adult subjects. Furthermore, “subject” may include patients who have or are suspected of having a condition or disease. Therefore, the terms “subject” and “patient” are used interchangeably herein. Subjects can also refer to cells in the laboratory or biological processing media in the test.
  • the term "effective amount” refers to an amount of drug or pharmaceutical reagent that will elicit a biological or medical response in a tissue, system, animal, or human, such response being, for example, determined by a researcher or clinician.
  • therapeutic effective amount means any amount that, compared to a corresponding control subject who did not receive such an amount, results in improvement, cure, prevention, or relief of disease, disorder, or side effects, or a decrease in the rate of progression of the disease or disorder.
  • the term also includes, within its scope, amounts that effectively enhance normal physiological function.
  • the therapeutically effective amount of one or more compounds of this application is known to those skilled in the art or can be readily determined by standard methods known in the art.
  • PROTAC technology still has the following problems:
  • PROTAC has too large a molecular weight
  • PROTAC molecules typically consist of three parts: a POI ligand, a linker, and an E3 ligase ligand. Their molecular weight is usually around 1000 Da, violating Lipinski's fifth rule (Ro5). This results in relatively poor permeability, solubility, pharmacokinetics, and oral bioavailability for PROTAC drugs. Furthermore, their synthesis is relatively difficult and costly. The type and length of the linker are unpredictable, requiring experimentation with various possibilities, and sometimes the linker is too large. PROTACs need to enter cells to mobilize intracellular UPS (Unified Cellular Utilization), therefore membrane permeability is crucial for their function. Currently, the permeation mechanism of PROTACs is not fully understood.
  • UPS Unified Cellular Utilization
  • PROTACs have a molecular weight of 1000-2000 Da and cross the cell membrane primarily through passive diffusion and active transport. However, due to their larger molecular weight and larger exposed polar surface area, the cell/tissue permeability of PROTACs is far inferior to that of smaller molecules. Therefore, researchers have developed various strategies to enhance PROTAC permeability. Common methods include limiting its molecular weight to below 1000 Da or splitting the molecule into two smaller precursors that bind in the cell to form the mature PROTAC (CLIPTAC).
  • CLIPTAC mature PROTAC
  • long, flexible linker fragments can be introduced to form intramolecular hydrogen bonds to reduce partial polarity, or cell-permeable peptides (such as poly-D-arginine sequences) can be linked to E3 ligands to increase PROTAC cell permeability.
  • cell-permeable peptides such as poly-D-arginine sequences
  • E3 ligands to increase PROTAC cell permeability.
  • nanoparticles such as liposomes to deliver PROTAC can also significantly enhance cellular uptake.
  • the human genome contains over 600 E3 ligases; however, 90% of the E3 ligases currently used in PROTACs are CRBN and VHL. Both are tumor suppressor proteins, and CRBN and VHL-based PROTACs are prone to developing drug resistance in cancer treatment due to mutations and deletions of the E3 ligases. Furthermore, the tissue-specific distribution of CRBN and VHL limits their applicability.
  • NAMPT nicotinamide phosphoribosyltransferase
  • BCR-ABL The formation of the BCR-ABL gene is due to a rearrangement of chromosomes 9 and 22, namely t(9;22)(q34;q11), resulting in an abnormal chromosome 22 called the Philadelphia (Ph) chromosome.
  • This chromosomal translocation leads to the fusion of the BCR and ABL genes, forming the BCR-ABL gene ( Figure 2), which results in constitutive activation of the ABL tyrosine kinase.
  • Abnormal activation of BCR-ABL is closely related to the development of chronic myeloid leukemia (CML). Therefore, BCR-ABL is an important therapeutic target for CML.
  • BCR-ABL-TKIs BCR-ABL tyrosine kinase inhibitors
  • CML patients treated with BCR-ABL-TKIs develop resistance after about one year of treatment. Therefore, the development of novel drugs to overcome BCR-ABL resistance is of great significance.
  • Anaplastic lymphoma kinase is a receptor-type tyrosine kinase composed of an extracellular domain, a single-channel transmembrane domain, and an intracellular kinase domain.
  • the extracellular domains of ALK include 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 lies between the extracellular and intracellular portions; and an intracellular tyrosine kinase (PTK) domain.
  • FAM150 is the ligand for ALK, and when the ligand binds to the extracellular domain, the receptor-type protein-tyrosine kinase is activated by inducing receptor dimerization or oligomerization.
  • Possible mechanisms by which ligand and dimer induce ALK activation involve phosphorylation of one or more juxtamembrane tyrosine residues (Tyr 1078, 1092, 1096, and 1131), followed by sequential phosphorylation until the active form of ALK is formed.
  • ALK gene fusions and rearrangements are a significant cause of various cancers, including non-small cell lung cancer (NSCLC) and anaplastic large cell lymphoma (ALCL).
  • NSCLC non-small cell lung cancer
  • ACL anaplastic large cell lymphoma
  • the proportion of ALK fusion mutation-positive cases in NSCLC is 3-5%, with approximately 5.3% in China. The incidence is higher in NSCLC patients, younger patients (under 60 years old), and non-smokers.
  • ALK fusions are a subsequent mutation following EGFR mutations.
  • the second largest molecular subtype of NSCLC, its corresponding targeted drugs are completely different from those of the EGFR molecular subtype.
  • ALK can fuse and rearrange with various paired genes, leading to its constitutive activation.
  • ALK-TKIs have been approved for the treatment of ALK-positive NSCLC patients, including the first-generation ALK-TKI crizotinib, second-generation ALK-TKIs such as ceritinib, alectinib, brigatinib, and ensartinib, and the third-generation ALK-TKI loratinib.
  • NSCLC ALK-positive non-small cell lung cancer
  • the N-degron pathway refers to the process by which the stability and half-life of a protein depend on its N-terminal amino acid residues.
  • the classic N-degron pathway was first discovered in Alexander Varshavsky's laboratory in 1986. Studies have shown that this degradation pathway exists in mammals, plants, and bacteria, playing a crucial role in fundamental life activities such as cell cycle and cell division, signal transduction, gene expression, and DNA repair.
  • the unstable amino acid residue located at the N-terminus of a target protein is called an "N-degron.”
  • the N-degron pathway mainly includes the Arg/N-degron, Pro/N-degron, and Gly/N-degron pathways, among which the Arg/N-degron pathway was the first to be discovered and is the most widely studied.
  • an unstable N-terminal residue including Arg, Lys, His, Leu, Phe, Tyr, Trp, and Ile
  • UBR family E3 ligases UBR1/2/4/5 thereby inducing the protein to degrade via the ubiquitin-proteasome.
  • the Pro/N-degron pathway targets proteins with a proline (Pro) residue at the N-terminus.
  • GID4 and GID10 in the multi-subunit RING-type E3 ubiquitin ligase CTLH complex are responsible for recognizing the Pro residue at the N-terminus.
  • the Gly/N-degron pathway discovered in 2019, involves CRL2 ZER1 & ZYG11B , which recognizes the glycine (Gly) residue at the first position at the N-terminus of a protein.
  • this application provides a novel linkerless micro PROTAC compound based on N-degron or a pharmaceutically acceptable salt thereof.
  • micro-PROTAC compound is shown in formula (I):
  • X is a single amino acid or its derivative
  • Y is a ligand of the target protein
  • the monoamino acid can be any of the natural or non-natural amino acids known in the art, such as one of Gly, Ala, Val, Leu, Ile, Phe, Pro, Trp, Ser, Tyr, Cys, Asp, Asn, Gln, Glu, Thr, Lys, Arg, and His.
  • the single amino acid is Gly, Pro, or Arg.
  • the target protein is an EML4-ALK fusion protein or a BCR-ABL fusion protein.
  • Y can be a ligand for various EML4-ALK fusion proteins or BCR-ABL fusion proteins known in the art.
  • Y is a bukatebin derivative.
  • Y is a buffalo derivative as shown in the following formula:
  • Y is a dasatinib derivative.
  • the compound is selected from one of the following:
  • This application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-described compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • composition depends on several criteria, including, for example, route of administration, disease severity, or dosage.
  • the pharmaceutical composition may be formulated for delivery to the subject via appropriate routes, including but not limited to oral, injectable (e.g., intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural, intraperitoneal, etc.), mucosal (e.g., intranasal, intraoral, etc.), sublingual, rectal, percutaneous, intraocular, and pulmonary routes.
  • injectable e.g., intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural, intraperitoneal, etc.
  • mucosal e.g., intranasal, intraoral, etc.
  • sublingual rectal, percutaneous, intraocular, and pulmonary routes.
  • This application also provides the use of the above-mentioned compounds or their pharmaceutically acceptable salts or pharmaceutical compositions in the preparation of medicaments for treating BCR-ABL or EML4-ALK mediated diseases.
  • This application also provides a method for treating BCR-ABL or EML4-ALK mediated diseases, comprising administering to a subject a therapeutically effective amount of the above-mentioned compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof.
  • the disease is cancer.
  • the cancer is selected from one or more of the following: non-small cell lung cancer, anaplastic large cell lymphoma, chronic myeloid leukemia, acute lymphoblastic leukemia, atypical chronic myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, B-cell leukemia/lymphoma, lymphoblastic lymphoma, Tarton-Brown-Rachman syndrome or acute promyelocytic leukemia, small cell lung cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, gastric cancer, kidney cancer, ovarian cancer, hematological diseases, and myeloma.
  • the disease mediated by BCR-ABL is chronic myeloid leukemia.
  • EML4-ALK mediated diseases are non-small cell lung cancer or anaplastic large cell lymphoma.
  • this application provides the use of Pro-DA or Gly-DA in the preparation of a medicament that degrades the BCR-ABL fusion protein.
  • this application provides the use of Pro-DA or Gly-DA in the preparation of a BCR-ABL fusion protein degrader.
  • this application provides the use of Pro-DA or Gly-DA in the preparation of a medicament for treating BCR-ABL-mediated diseases.
  • the BCR-ABL-mediated disease is chronic myeloid leukemia.
  • this application provides a method for treating BCR-ABL-mediated disease, comprising administering a therapeutically effective amount of Pro-DA or Gly-DA to a subject.
  • the BCR-ABL-mediated disease is chronic myeloid leukemia.
  • this application provides the use of Pro-BA or Gly-BA in the preparation of a medicament that degrades the EML4-ALK fusion protein.
  • this application provides the use of Pro-BA or Gly-BA in the preparation of an EML4-ALK fusion protein degrader.
  • this application provides the use of Pro-BA or Gly-BA in the preparation of a medicament for treating EML4-ALK-mediated diseases.
  • the EML4-ALK-mediated disease is non-small cell lung cancer or anaplastic large cell lymphoma.
  • this application provides a method for treating EML4-ALK-mediated diseases, comprising administering a therapeutically effective amount of Pro-BA or Gly-BA to a subject.
  • the EML4-ALK-mediated disease is non-small cell lung cancer or anaplastic large cell lymphoma.
  • UV absorbance was measured at 254 nm.
  • the preparative HPLC gradient started with 1% phase B (B: acetonitrile, A: 0.1% aqueous trifluoroacetic acid) and increased to 100% over 20 minutes.
  • High-resolution mass spectrometry was performed using an ABIQ-star Elite.
  • 2-(dimethoxyphospho)aniline 500 mg, 2.96 mmol, 1.0 equivalent
  • 2,4,5-trichloropyrimidine 542.1 mg, 2.96 mmol, 1.0 equivalent
  • potassium carbonate 857.9 mg, 6.2 mmol, 2.1 equivalent
  • tetrabutylammonium bisulfate 100.4 mg, 0.3 mmol, 0.1 equivalent
  • N-Dehydroxyethyldasatinib (20 mg, 0.045 mmol, 1.0 equivalence), Boc-L-proline (11.7 mg, 0.054 mmol, 1.2 equivalence), and HATU (20.6 mg, 0.054 mmol, 1.2 equivalence) were dissolved in 1 mL of anhydrous DMF. After stirring and dissolving, DIEA (22.4 ⁇ L, 0.135 mmol, 3.0 equivalence) was added, and the reaction was carried out at room temperature for 2 h, with the reaction progress monitored by HPLC. After the reaction was completed, the reaction solution was extracted with ethyl acetate, the organic phase was collected, evaporated to dryness, and pumped for 30 min.
  • N-Dehydroxyethyldasatinib (20 mg, 0.045 mmol, 1.0 equivalence), Boc-L-glycine (9.48 mg, 0.054 mmol, 1.2 equivalence), and HATU (20.6 mg, 0.054 mmol, 1.2 equivalence) were dissolved in 1 mL of anhydrous DMF. After stirring and dissolving, DIEA (22.4 ⁇ L, 0.135 mmol, 3.0 equivalence) was added, and the reaction was carried out at room temperature for 2 h, with the reaction progress monitored by HPLC.
  • N-Dehydroxyethyldasatinib (20 mg, 0.045 mmol, 1.0 equivalence), Boc-Arg(Pbf)-OH (28.5 mg, 0.054 mmol, 1.2 equivalence), and HATU (20.6 mg, 0.054 mmol, 1.2 equivalence) were dissolved in 1 mL of anhydrous DMF. After stirring and dissolving, DIEA (22.4 ⁇ L, 0.135 mmol, 3.0 equivalence) was added, and the reaction was carried out at room temperature for 2 h, with the reaction progress monitored by HPLC.
  • H3122 cells expressing endogenous EML4-ALK were seeded into 12-well plates and treated with different concentrations (final concentrations of 0, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, 250 nM, and 500 nM) of Pro-BA and Gly-BA for 24 hours. Cells were then lysed with RIPA for 30 mins, and BCA protein was quantified. For each concentration, 50 ⁇ g of protein sample was used for Western blotting to detect the protein level of ALK at different drug concentrations ( Figures 1A and 1B).
  • H3122 cells were treated with different concentrations of Pro-BA (final concentrations of 0, 250 nM, and 500 nM, in DMSO solvent) for 48 hours, fixed with 70% ethanol, stained with PI, and analyzed by flow cytometry to determine the cell cycle.
  • Pro-BA treatment increased the proportion of cells in G1 phase and decreased the proportion of cells in S and G2 phases ( Figures 3A and 3B).
  • H3122 cells were treated with different concentrations of Pro-BA (final concentrations of 0, 250 nM, and 500 nM, with DMSO as the solvent) for 48 hours.
  • Cell cycle analysis was performed using Annexin V-FITC staining and flow cytometry. The results showed that Pro-BA treatment increased the number of early and late apoptotic cells ( Figures 4A and 4B).
  • H3122 cells were ectopically transplanted subcutaneously into nude mice.
  • Pro-BA (10 mg/kg)
  • Pro-PEG3-BA prepared according to the method disclosed in CN2023118151005
  • bufotalin 10 mg/kg
  • 90% corn oil + 10% DMSO (as the vehicle group) were administered intraperitoneally, and the tumor size was measured.
  • mice were euthanized after 29 days. Tumors were removed and photographed (Figure 5A), tumor size was recorded ( Figure 5B), and tumor growth curves were plotted (Figure 5C).
  • the results showed that Pro-BA, Pro-PEG3-BA, and bufotalidine all inhibited tumor growth, with Pro-BA showing the best inhibitory effect. Furthermore, these drugs did not significantly affect the mouse body weight (Figure 5D).
  • Western blot analysis of ALK in the tumor tissue revealed that only the degradative agents Pro-BA and Pro-PEG3-BA reduced ALK protein levels ( Figures 5E and 5F).
  • Pro-BA (2 mg/kg dissolved in ddH2O ) was administered to mice via tail vein (IV), and Pro-BA (10 mg/kg dissolved in ddH2O ) was administered orally (PO).
  • Plasma samples were collected at 0.083 h, 0.25 h, 0.5 h, 1 h, 3 h, 6 h, 8 h, 10 h, and 12 h, and Pro-BA blood concentrations were measured.
  • Pharmacokinetic (PK) parameters were calculated using Phoenix WinNonlin 8.1 software. The results are shown in Table 1.
  • H3122 cells were subcutaneously inoculated into four-week-old female nude mice. When the tumors reached an average size of 54 mm3 , Pro-BA (25 mg/kg) was administered orally every two days for a total of eight times. Oral administration of the same dose of ddH2O (vehicle) served as a control. Tumors were harvested and photographed (Figure 6A), and tumor growth curves were plotted (Figure 6B), showing that Pro-BA significantly inhibited tumor growth. Immunohistochemical staining of tumor tissues to detect ALK expression levels showed that Pro-BA significantly reduced ALK protein levels (Figure 6C). Mouse body weight was also measured. Pro-BA was found to have no significant effect on mouse body weight, indicating that Pro-BA has no obvious toxicity (Figure 6D).

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Abstract

本申请提供一种基于N-degron的新型无连接子的微型PROTAC化合物及其用途。本申请提供一种新颖独特的无连接子的微型PROTAC小分子，可以利用细胞自身的降解途径来特异性降解BCR-ABL和EML4-ALK融合蛋白。与普通的PROTAC中利用连接子的长度和类型来调节靶蛋白与特定E3泛素连接酶的空间位置不同，本申请的微型PROTAC通过十九种不同的N-degron氨基酸来分别招募不同的E3泛素连接酶，以便形成空间上有利于实现对靶蛋白进行泛素化标记的最优"E3—AA-miniPROTAC—POI"三元复合物，进而实现靶蛋白的高效降解。

Description

基于N-degron的新型无连接子的微型PROTAC化合物及其用途 技术领域
本申请属于医药技术领域，具体地，涉及一种基于N-degron的新型无连接子的微型PROTAC化合物及其用途。
背景技术
2001年Raymond.Deshaies和Craig.Crews等人提出了蛋白降解靶向嵌合体(Proteolysis targeting chimeria，PROTAC)这个概念，并成功地设计合成了第一批多肽PROTAC用于降解甲硫氨酰氨肽酶2(MetAP-2)。到了2019年，行业先驱Crews教授开发的ARV-110，以全球首个PROTAC药物的身份正式进入临床试验阶段，PROTAC越来越受到关注。
PROTAC由三部分组成：一个与E3连接酶结合的配体，用于引导蛋白降解；一个与靶蛋白结合的配体，用于引导小分子的靶向定位；以及一个负责嵌合两个配体的连接子。PROTAC分子进入细胞后，其结构中的目标蛋白(Protein of Interest，POI)配体可特异性地与相应的靶蛋白结合，而另一端可以募集E3连接酶从而形成POI-连接子-E3连接酶三元复合物，其中E3连接酶可介导-POI的多泛素化，被泛素标记的POI被蛋白酶体识别并降解。PROTAC化合物是基于泛素-蛋白酶体系统引起的蛋白质降解的技术。
理论上PROTAC是事件驱动的药理作用模式，此过程无需靶蛋白配体长时间占据结合位点，只需三元复合物短暂的形成便可瞬时完成目标蛋白的泛素化，并且PROTAC分子在细胞内可多次循环利用，因此，与传统的小分子抑制剂和大分子抗体相比，PROTAC具有明显的优势，有望将那些难以成药的蛋白靶向成药、克服靶蛋白突变/过表达引起的耐药等优点。
但是PROTAC技术仍然存在一些问题，例如PROTAC分子量太大、用于PROTAC设计的E3连接酶有限、PROTAC缺乏对靶蛋白的降解速率的控制等。这些问题都制约了PROTAC的应用。
发明内容
针对现有技术存在的问题，本申请提供一种基于N-degron的新型无连接子的微型PROTAC化合物及其用途。
具体来说，本申请涉及如下方面：
1.一种式(I)所示的化合物或其药学上可接受的盐：
X-Y
式(I)
其中，X为单氨基酸或其衍生物，Y为靶蛋白的配体。
2.根据项1所述的化合物或其药学上可接受的盐，其中所述单氨基酸选自Gly、Ala、Val、Leu、Ile、Phe、Pro、Trp、Ser、Tyr、Cys、Asp、Asn、Gln、Glu、Thr、Lys、Arg、His中的一种。
3.根据项1所述的化合物或其药学上可接受的盐，其中所述单氨基酸为Gly、Pro或Arg。
4.根据项1-3中任一项所述的化合物或其药学上可接受的盐，其中所述靶蛋白为EML4-ALK融合蛋白或BCR-ABL融合蛋白。
5.根据项1-4中任一项所述的化合物或其药学上可接受的盐，其中Y为下式所示的布吉他滨衍生物：
6.根据项1-4中任一项所述的化合物或其药学上可接受的盐，其中Y为下式所示的达沙替尼衍生物：
7.根据项1-6中所述的化合物或其药学上可接受的盐，其中所述化合物选自 以下一种：
8.一种药物组合物，包括项1-7中所述的化合物或其药学上可接受的盐，以及药学上可接受的载体。
9.项1-7中所述的化合物或其药学上可接受的盐或项8所述的药物组合物在制备降解BCR-ABL或EML4-ALK融合蛋白的药物中的用途。
10.项1-7中所述的化合物或其药学上可接受的盐或项8所述的药物组合物在制备BCR-ABL或EML4-ALK融合蛋白降解剂中的用途。
11.项1-7中所述的化合物或其药学上可接受的盐或项8所述的药物组合物在制备治疗BCR-ABL或EML4-ALK介导的疾病的药物中的用途。
12.根据项11所述的用途，其中所述疾病为癌症。
13.根据项12所述的用途，其中所述癌症选自以下一种或两种以上：非小细胞肺癌、间变性大细胞淋巴瘤、慢性髓系白血病、急性淋巴细胞白血病、非典型慢性粒细胞白血病、急性淋巴母细胞白血病、急性髓系白血病、B淋巴细胞白血病/淋巴瘤、淋巴母细胞淋巴瘤、塔顿-布朗-拉赫曼综合征或急性早幼粒细胞白血病、小细胞肺癌、结直肠癌、乳腺癌、前列腺癌、肝癌、胰腺癌、胃癌、肾癌、 卵巢癌、骨髓瘤。
14.一种治疗BCR-ABL或EML4-ALK介导的疾病的方法，包括向受试者施用治疗有效量的项1-7中所述的化合物或其药学上可接受的盐或项8所述的药物组合物。
15.根据项14所述的方法，其中所述疾病为癌症。
16.根据项15所述的方法，其中所述癌症选自以下一种或两种以上：非小细胞肺癌、间变性大细胞淋巴瘤、慢性髓系白血病、急性淋巴细胞白血病、非典型慢性粒细胞白血病、急性淋巴母细胞白血病、急性髓系白血病、B淋巴细胞白血病/淋巴瘤、淋巴母细胞淋巴瘤、塔顿-布朗-拉赫曼综合征或急性早幼粒细胞白血病、小细胞肺癌、结直肠癌、乳腺癌、前列腺癌、肝癌、胰腺癌、胃癌、肾癌、卵巢癌、骨髓瘤。
附图说明
图1显示使用本申请的化合物处理H3122细胞后，WB检测的ALK蛋白水平。其中A为不同浓度Pro-BA处理H3122细胞24h的结果；B为不同浓度Gly-BA处理H3122细胞24h的结果；C为用250nM Pro-BA处理H3122不同时间的结果；D为用250nM Gly-BA处理H3122不同时间的结果。
图2显示CCK8检测的Pro-BA或Gly-BA对细胞增殖的影响。其中A为Pro-BA，B为Gly-BA。
图3显示用不同浓度0、250nM、500nM Pro-BA处理H3122细胞48h的流式细胞分析结果。
图4显示用不同浓度0、250nM、500nM Pro-BA处理H3122细胞48h的流式细胞分析结果。
图5显示Pro-BA抑制肿瘤生长试验结果。其中A为肿瘤照片；B为肿瘤大小；C为制肿瘤生长曲线；D为体重情况；E和F为WB检测的ALK蛋白水平。
图6显示口服Pro-BA抑制肿瘤生长试验结果。其中A为肿瘤照片；B为制肿瘤生长曲线；C为对肿瘤组织进行免疫组化染色的结果；D为体重情况。
图7显示使用本申请的化合物处理K562细胞后，WB检测的BCR-ABL 蛋白水平。其中A为不同浓度Pro-DA处理K562细胞24h的结果；B为不同浓度Gly-DA处理K562细胞24h的结果；C为用10nM Pro-DA处理K562不同时间的结果；D为用10nM Gly-DA处理K562不同时间的结果。
图8显示CCK8检测的Pro-BA或Gly-BA对细胞增殖的影响。其中A为Pro-DA，B为Gly-DA。
具体实施方式
下面结合实施例进一步说明本申请，应当理解，实施例仅用于进一步说明和阐释本申请，并非用于限制本申请。
除非另外定义，本说明书中有关技术的和科学的术语与本领域内的技术人员所通常理解的意思相同。虽然在实验或实际应用中可以应用与此间所述相似或相同的方法和材料，本文还是在下文中对材料和方法做了描述。在相冲突的情况下，以本说明书包括其中定义为准，另外，材料、方法和例子仅供说明，而不具限制性。以下结合具体实施例对本申请作进一步的说明，但不用来限制本申请的范围。
定义
“配体”是指与生物分子形成复合物以发挥生物学功能的分子。配体复合物可以经由离子键、共价键、范德华相互作用和/或氢键形成。“靶蛋白的配体”是指与靶蛋白形成复合物以发挥生物学功能的分子。
“药学上可接受的盐”是由本申请的化合物与酸或碱形成的盐，所述酸选自盐酸、氢溴酸、硫酸、磷酸、甲磺酸、苯磺酸、对甲苯磺酸、萘磺酸、柠檬酸、酒石酸、乳酸、丙酮酸、乙酸、马来酸、琥珀酸、富马酸、水杨酸、苯基乙酸、杏仁酸或阿魏酸，所述碱选自碱金属阳离子碱、碱土金属阳离子碱、铵阳离子碱或胆碱。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上可药用的盐或前体药物，以及其他组分例如药学上可接受的载体的混合物。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
“治疗”的目的是防止或减慢不期望的生理学变化或紊乱。为了本申请的目的，有益的或期望的临床结果包括但不限于可检测的或不可检测的：减轻症状，降低疾病程度小，稳定(即，不恶化)疾病状态，延迟或减缓疾病进展，改 善或缓和疾病状态，以及缓解(部分或是全部)。“治疗”也可以指相比不接受治疗的预期存活延长存活时间。
“药学上可接受的”是指适用于接触人类或动物的组织而不会产生过度的毒性、刺激、过敏反应、其他问题或并发症，具有合理的效益/风险比的化合物、材料、组合物和/或剂型。在一些实施方式中，药学上可接受的化合物、材料、组合物和/或剂型是指由监管机构(如美国食品和药物管理局、中国国家药品监督管理局、欧洲药品管理局)批准的或列在公认药典(如美国药典、中国药典、欧洲药典)中的用于动物，特别是人类的那些化合物、材料、组合物和/或剂型。
“药学上可接受的载体”是指将本申请的化合物从一个位置、体液、组织、器官(内部或外部)或身体部分携带或运输至另一位置、体液、组织、器官或身体部分时所涉及的药学上可接受的材料、组合物或运载体，如液体或固体填料、稀释剂、赋形剂、溶剂或封装材料。药学上可接受的载体可为运载体、稀释剂、赋形剂或可用于接触动物组织而无过度毒性或不良反应的其他材料。示例性的药学上可接受的载体包括但不限于糖、淀粉、纤维素、麦芽、黄芪胶、明胶、林格氏溶液、海藻酸、等渗盐水、缓冲剂等。
“受试者”是指生物体、组织或细胞。受试者可包括用于医疗目的(例如已有病症或疾病的诊断和/或治疗，或预防病症或疾病发作的预防性治疗)的人类受试者，或用于医疗兽医学目的或开发目的的动物受试者。受试者也包括来自组织培养物、细胞培养物、器官复制、干细胞生产等的样品材料。合适的动物受试者包括哺乳动物和鸟类。本文所用的术语“哺乳动物”包括但不限于灵长类(例如人类、猴、猿等)、牛(例如公牛等)、羊(例如绵羊、山羊等)、猪、马、猫、犬、兔、啮齿动物(例如小鼠、大鼠等)等。本文所用的术语“鸟类”包括但不限于鸡、鸭、鹅、鹌鹑、火鸡、野鸡等。在一些实施方式中，受试者为哺乳动物或哺乳动物细胞。在一些实施方式中，受试者为人类或人类细胞。人类受试者包括但不限于胎儿、新生儿、幼儿、青少年和成人受试者。此外，“受试者”可包括患有或疑似患有某种病症或疾病的患者。因此，术语“受试者”和“患者”在本文可互换使用。受试者也可指实验室中的细胞或测试中的生物加工培养基。
本文所用的术语“有效量”是指这样的药物或药学试剂的量：其将引起组织、系统、动物或人的生物学或医学应答，所述应答是例如研究人员或临床医师所 追求的。此外，术语“治疗有效量”意指这样的任何量：与没有接受这种量的相应对受试者比，其导致疾病、障碍或副作用的改善的治疗、治愈、预防或缓解，或者疾病或障碍的进展速度的下降。该术语在其范围内也包括有效地增强正常生理功能的量。一种或多种本申请的化合物的所述治疗有效量是技术人员已知的，或者可以通过本领域已知的标准方法容易地确定。
化合物
如上所述，PROTAC技术仍然存在以下问题：
(1)PROTAC分子量太大
PROTAC分子通常由POI配体、连接子和E3连接酶配体三部分组成，其分子量通常都在1000Da左右，违反了Lipinski的五法则(Ro5)，所以导致PROTAC药物的渗透性、溶解度、药代动力学和口服生物利用度差都相对较差，另外合成难度和成本也相对较高。连接子使用上，其类型及长度都无法预测，需要试验多种可能，有时过大。PROTAC需要进入细胞以调动细胞内的UPS，因此膜通透性是PROTAC发挥作用的关键。目前，PROTAC的渗透机制尚未阐明，大多数PROTAC的分子量为1000-2000Da，主要通过被动扩散和主动转运的方式穿过细胞膜。然而，由于较大的分子量和较大的暴露极性表面积，PROTAC的细胞/组织渗透性远不如小分子。因此，研究人员已开发了多种用于提高PROTAC渗透性的策略，常见的方法是将其分子量限制在1000Da以下或将分子拆分成两个较小的前体并在细胞中结合为成熟的PROTAC(CLIPTAC)。此外，可以引入长的柔性连接片段形成分子内氢键以降低部分极性，或将细胞渗透性肽(如聚-D-精氨酸序列)连接到E3配体上，进而增加PROTAC的细胞渗透性。除了修饰PROTAC本身，应用脂质体等纳米颗粒来递送PROTAC也可以显著增强了PROTAC的细胞摄取。
(2)用于PROTAC设计的E3连接酶有限
人类基因组中有超过600个E3连接酶，然而目前应用到PROTAC中的E3连接酶90％是CRBN和VHL。二者都是抑癌蛋白，且基于CRBN和VHL的PROTAC在癌症治疗中由于E3连接酶的突变和缺失易产生耐药性，另外由于CRBN和VHL存在组织特异性分布，从而限制了其适用性。
(3)PROTAC缺乏对靶蛋白的降解速率的控制
细胞的正常生命活动需要关键的细胞调节因子保持在合适的范围内，因为 在某些情况下，这些蛋白质的过多或过少都可能变的有害，从而引起疾病。例如烟酰胺磷酸核糖转移酶NAMPT，可以让细胞维持充足的NAD+水平，在抗衰老中具有重要作用，另一方面在多种肿瘤中NAMPT高表达，是肿瘤治疗的一个靶点。所以，将NAMPT的蛋白水平控制在合理的范围至关重要。由此可见，对于有些靶蛋白，PROTAC并不一定降解效果越强越好但目前的PROTAC基本上降解速度都是固定的，不容易调节。
BCR-ABL基因的形成是由于9号染色体和22号染色体重排，即t(9；22)(q34；q11)，产生一种异常的22号染色体，称为费城(Ph)染色体，这种染色体的相互易位导致BCR和ABL基因融合，形成BCR-ABL基因(图2)，导致ABL的酪氨酸激酶组成型激活。BCR-ABL的异常激活与慢性髓系白血病(chronic myeloid leukemia，CML)的发生密切相关。因此BCR-ABL是CML的重要治疗靶标，迄今为止，多款BCR-ABL的酪氨酸激酶抑制剂(BCR-ABL-TKI)已获得批准用于治疗CML患者，包括第一代TKI伊马替尼(imatinib)，第二代TKI达沙替尼(dasatinib)、尼洛替尼(nilotinib)和博舒替尼(bosutinib)等，以及第三代ALK-TKI泊那替尼(ponatinib)和泊那替尼(ponatinib)。但遗憾的是，接受BCR-ABL-TKI治疗的CML患者在用药一年左右均会出现耐药。因此，研发克服BCR-ABL耐药的新型药物具有重大意义。
间变性淋巴瘤激酶(anaplastic lymphoma kinase，ALK)是一种受体型酪氨酸激酶，由胞外结构域、单通道跨膜结构域和胞内激酶结构域组成。ALK的胞外结构域包括两个甲基多巴-A5蛋白-受体型蛋白酪氨酸磷酸酶(MAM)结构域、一个低密度脂蛋白(LDLa)结构域和一个富含甘氨酸的胞外结构域(G-rich)；一个单一的跨膜(TM)域位于细胞外和细胞内部分之间；以及细胞内的酪氨酸激酶(PTK)结构域。FAM150是ALK的配体，当配体与胞外结构域结合时，受体蛋白-酪氨酸激酶通过诱导受体二聚或寡聚而被激活。配体和二聚体诱导ALK活化的可能机制涉及近膜酪氨酸残基的一个或多个磷酸化(Tyr 1078、1092、1096和1131)，然后依次进行连续磷酸化，直到形成ALK的活性形式。
ALK基因的融合与重排是导致多种癌症的重要原因，包括非小细胞肺癌(NSCLC)和间变性大细胞淋巴瘤(ALCL)。非小细胞肺腺癌中ALK融合突变阳性的比例为3-5％，中国的比例约5.3％，在非小细胞肺腺癌、年轻患者(小于60岁)以及不吸烟的人群中发生率较高，ALK融合是既EGFR突变之后的 第二大NSCLC分子亚型，相对应的靶向药物与EGFR分子亚型完全不同。ALK可与多种配对基因发生融合重排，从而导致其组成型激活，最常见融合发生在棘皮动物微管相关类蛋白4-间变性淋巴瘤激酶(EML4-ALK)。迄今为止，多款ALK-TKIs已获得批准用于治疗ALK阳性的NSCLC患者，包括第一代ALK-TKI克唑替尼，第二代ALK-TKIs塞瑞替尼、阿来替尼、布格替尼和恩莎替尼等，以及第三代ALK-TKI洛拉替尼。但遗憾的是，接受ALK-TKIs治疗的患者均会出现耐药而导致疾病进展。因此，研发克服ALK阳性非小细胞肺癌(NSCLC)耐药的新型靶向药物具有重大意义。
N-degron pathway是指蛋白的稳定性和半衰期取决于其N末端氨基酸残基。经典的N-degron pathway最早是由Alexander Varshavsky实验室在1986年发现。研究显示该降解途径从哺乳动物、植物到细菌中都存在，在调控细胞周期和细胞分裂、信号转导、基因表达、DNA修复等生命基本活动中发挥重要作用。位于目标蛋白N-末端的不稳定性氨基酸残基称为“N端降解子(N-degron)”。N-degron途径主要包括Arg/N-degron、Pro/N-degron和Gly/N-degron途径等，其中Arg/N-degron途径是最早被发现，也是研究最为广泛的。当蛋白的N端暴露出不稳定N-末端残基(包括Arg、Lys、His、Leu、Phe、Tyr、Trp、Ile)，其能被UBR家族E3连接酶UBR1/2/4/5识别，从而诱导蛋白通过泛素蛋白酶体降解。Pro/N-degron途径于2017年被发现，可以靶向N-末端为脯氨酸(Pro)的蛋白质，多亚基RING型E3泛素连接酶CTLH复合体中的GID4和GID10负责识别蛋白N端的Pro。Gly/N-degron途径于2019年被发现，由CRL2ZER1&ZYG11B能够识别蛋白N端第一个位置的甘氨酸(Gly)残基。
针对现有技术存在的问题，本申请提供一种基于N-degron的新型无连接子的微型PROTAC化合物或其药学上可接受的盐。
所述微型PROTAC化合物如式(I)所示：
X-Y
式(I)
其中，X为单氨基酸或其衍生物，Y为靶蛋白的配体。
单氨基酸可以为本领域已知的各种天然或非天然氨基酸，例如Gly、Ala、Val、Leu、Ile、Phe、Pro、Trp、Ser、Tyr、Cys、Asp、Asn、Gln、Glu、Thr、Lys、Arg、His中的一种。
在一个具体的实施方式中，所述单氨基酸为Gly、Pro或Arg。
在一个具体的实施方式中，所述靶蛋白为EML4-ALK融合蛋白或BCR-ABL融合蛋白。Y可以是本领域已知的各种EML4-ALK融合蛋白或BCR-ABL融合蛋白的配体。
在一个具体的实施方式中，Y为布吉他滨衍生物。
在一个具体的实施方式中，Y为下式所示的布吉他滨衍生物：
在一个具体的实施方式中，Y为达沙替尼衍生物。
在一个具体的实施方式中，Y为下式所示的达沙替尼衍生物：
在一个具体的实施方式中，所述化合物选自以下一种：

本领域技术人员可以理解，本申请的化合物可以以多种不同的形式存在，所有均包括在本申请的范围内。这些形式包括，例如，互变异构体、立体异构体、外消旋混合物、前药、溶剂化形式、不同的晶型或多晶型等。
药物组合物
本申请还提供一种药物组合物，包括上述的化合物或其药学上可接受的盐，以及药学上可接受的载体。
药物组合物的形式取决于多个标准，包括例如施用途径、疾病程度，或施用剂量等。
在一些实施方式中，药物组合物可配制为通过适当的途径递送到对象中，包括但不限于，通过口服途径、注射途径(如静脉注射、肌肉注射、皮下注射、皮内注射、心内注射、鞘内注射、胸膜腔内注射、腹腔内注射等)、粘膜途径(如鼻腔内施用、口腔内施用等)、舌下途径、直肠途径、经皮途径、眼内途径、肺部途径。根据所需的施用途径，药物组合物可配制为片剂、胶囊、丸剂、糖衣丸、粉剂、颗粒、扁囊、锭剂、栓剂、悬浮液、乳剂、糖浆、气雾剂(作为固体或在液体介质中)、喷雾剂、膏剂、糊剂、贴剂、霜剂、洗剂、凝胶、 吸入剂等。
治疗方法、治疗用途
本申请还提供上述化合物或或其药学上可接受的盐或药物组合物在制备降解BCR-ABL或EML4-ALK融合蛋白的药物中的用途。
本申请还提供上述化合物或或其药学上可接受的盐或药物组合物在制备BCR-ABL或EML4-ALK融合蛋白降解剂中的用途。
本申请还提供上述化合物或或其药学上可接受的盐或药物组合物在制备治疗BCR-ABL或EML4-ALK介导的疾病的药物中的用途。
本申请还提供一种治疗BCR-ABL或EML4-ALK介导的疾病的方法，包括向受试者施用治疗有效量的上述化合物或或其药学上可接受的盐或药物组合物。
在本申请中，“BCR-ABL介导的疾病”旨在包括任何与BCR-ABL基因或蛋白相关的疾病。“EML4-ALK介导的疾病”旨在包括任何与EML4-ALK基因或蛋白相关的疾病。
在一个具体的实施方式中，BCR-ABL或EML4-ALK介导的疾病是通过降解BCR-ABL或EML4-ALK可治疗的疾病。
在一个具体的实施方式中，所述疾病为癌症。
在一个具体的实施方式中，所述癌症选自以下一种或两种以上：非小细胞肺癌、间变性大细胞淋巴瘤、慢性髓系白血病、急性淋巴细胞白血病、非典型慢性粒细胞白血病、急性淋巴母细胞白血病、急性髓系白血病、B淋巴细胞白血病/淋巴瘤、淋巴母细胞淋巴瘤、塔顿-布朗-拉赫曼综合征或急性早幼粒细胞白血病、小细胞肺癌、结直肠癌、乳腺癌、前列腺癌、肝癌、胰腺癌、胃癌、肾癌、卵巢癌、血液病、骨髓瘤。
在一个具体的实施方式中，BCR-ABL介导的疾病是慢性髓系白血病。
在一个具体的实施方式中，EML4-ALK介导的疾病是非小细胞肺癌或间变性大细胞淋巴瘤。
在一个具体的实施方式中，本申请提供Pro-DA或Gly-DA在制备降解BCR-ABL融合蛋白的药物中的用途。
在一个具体的实施方式中，本申请提供Pro-DA或Gly-DA在制备BCR-ABL融合蛋白降解剂中的用途。
在一个具体的实施方式中，本申请提供Pro-DA或Gly-DA在制备治疗BCR-ABL介导的疾病的药物中的用途。在一个具体的实施方式中，BCR-ABL介导的疾病是慢性髓系白血病。
在一个具体的实施方式中，本申请提供一种治疗BCR-ABL介导的疾病的方法，包括向受试者施用治疗有效量的Pro-DA或Gly-DA。在一个具体的实施方式中，BCR-ABL介导的疾病是慢性髓系白血病。
在一个具体的实施方式中，本申请提供Pro-BA或Gly-BA在制备降解EML4-ALK融合蛋白的药物中的用途。
在一个具体的实施方式中，本申请提供Pro-BA或Gly-BA在制备EML4-ALK融合蛋白降解剂中的用途。
在一个具体的实施方式中，本申请提供Pro-BA或Gly-BA在制备治疗EML4-ALK介导的疾病的药物中的用途。在一个具体的实施方式中，EML4-ALK介导的疾病是非小细胞肺癌或间变性大细胞淋巴瘤。
在一个具体的实施方式中，本申请提供一种治疗EML4-ALK介导的疾病的方法，包括向受试者施用治疗有效量的Pro-BA或Gly-BA。在一个具体的实施方式中，EML4-ALK介导的疾病是非小细胞肺癌或间变性大细胞淋巴瘤。
实施例
材料：2,4,5-三氯嘧啶、2-(二甲氧磷基)苯胺、和Boc-Arg(Pbf)-OH购买于上海毕得试剂公司；Boc-L-脯氨酸购买于百灵威试剂公司；4-(4-氨基-3-甲氧基苯基)哌嗪-1-羧酸叔丁酯、N-脱羟乙基达沙替尼购买于江苏艾康试剂公司；四丁基硫酸氢铵购买于阿拉丁试剂公司；Boc-甘氨酸、2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)、N,N-二异丙基乙胺(DIEA)、三氟乙酸(TFA)和乙腈购买于安耐吉试剂公司。
表征方法：样品分析是在配备DAD-UV检测器的安捷伦1260高效液相系统上进行的，使用安捷伦Poroshell 120，EC-C18色谱柱(4.6mm×100mm，2.7μm)。分析型HPLC梯度从10％的B相(B：乙腈，A：0.1％三氟乙酸的水溶液)开始，然后在20分钟内增加到100％，流速为0.5mL/min。分析型HPLC测定的化合物的纯度(>95％)用于生物学研究。样品纯化是在ULTIMAT 3000(DIONEX)制备型HPLC仪器上进行，使用光电二极管阵列检测器在220和 254nm处测量紫外吸光度。制备型HPLC梯度从1％的B相(B：乙腈，A：0.1％三氟乙酸的水溶液)开始，然后在20分钟内增加到100％。高分辨率质谱是用ABIQ-star Elite测量的。
布吉他滨衍生物的合成：
布吉他滨衍生物的合成步骤如下：
在反应瓶中，将2-(二甲氧磷基)苯胺(500毫克，2.96毫摩尔，1.0当量)、2,4,5-三氯嘧啶(542.1毫克，2.96毫摩尔，1.0当量)、碳酸钾(857.9毫克，6.2毫摩尔，2.1当量)、四丁基硫酸氢铵(100.4毫克，0.3毫摩尔，0.1当量)溶于7毫升DMF，并在65℃下搅拌过夜。用薄层层析法(TLC)监测反应完成后，过滤除去碳酸钾，真空浓缩滤液，用DCM：MeOH＝50：1柱层析纯化，得白色固体1(757毫克，2.4毫摩尔，产率为81％)。HRMS(ESI+)m/z：calcd.for C12H13Cl2N3OP[M+H]+316.0168，found 316.0161。
在10mL反应管中，加入化合物1(100毫克，0.32毫摩尔，1.0当量)、4-(4-氨基-3-甲氧基苯基)哌嗪-1-羧酸叔丁酯(117毫克，0.38毫摩尔，1.2当量)和2毫升异丙醇溶液，搅拌至溶解，随后加入TFA(25.9微升，0.35毫摩尔，1.1当量)，并将反应管于95℃搅拌过夜。使用分析型HPLC跟踪反应进度，反应混合物直接通过制备型HPLC纯化，冷冻干燥后得到灰绿色粉末化合物2。将灰绿色粉末在冰浴下溶于20％ TFA/DCM中，然后使反应液升至室温并搅拌1小时。使用分析型HPLC监测反应，反应结束后，在减压下蒸发溶剂，并通过制备型HPLC纯化，冷冻干燥后得到黄绿色粉末化合物3(tR＝7.246min,140毫克，0.29毫摩尔，产率为91％)，即布吉他滨衍生物(BA)。HRMS(ESI+)m/z：calcd.for C23H29O2N6ClP[M+H]+487.1773，found 487.1775。
实施例1 Pro-BA(化合物4)的合成
将化合物3(50.5毫克，0.104毫摩尔，1.0当量)、Boc-L-脯氨酸(26.8毫克，0.125毫摩尔，1.2当量)和HATU(47.4毫克，0.125毫摩尔，1.2当量)溶于1毫升无水DMF中，搅拌溶解后加入DIEA(51.5微升，0.312毫摩尔，3.0当量)，室温反应2小时，并通过HPLC监测反应进程。反应结束后，将反应液用乙酸乙酯萃取，收集有机相，旋干，用油泵抽30分钟，然后将残留物在冰浴下溶于20％ TFA/DCM中，随后使反应液升至室温并搅拌1小时。反应结束后，通过HPLC纯化，冷冻干燥后得到白色粉末4(tR＝7.828min，28.9毫克，0.050毫摩尔，产率为60％)，即Pro-BA。HRMS(ESI+)m/z：calcd.for C28H36O3N7ClP[M+H]+584.2300，found 584.2299。
实施例2 Gly-BA(化合物5)的合成
将化合物3(21.09毫克，0.043毫摩尔，1.0当量)、Boc-L-甘氨酸(9.11毫克，0.052毫摩尔，1.2当量)和HATU(19.8毫克，0.052毫摩尔，1.2当量)溶于1毫升无水DMF中，搅拌溶解后加入DIEA(21.5微升，0.130毫摩尔，3.0当量)，室温反应2小时，并通过HPLC监测反应进程。反应结束后，将反应液用乙酸乙酯萃取，收集有机相，旋干，用油泵抽30分钟，然后将残留物在冰浴下溶于20％ TFA/DCM中，随后使反应液升至室温并搅拌1小时。反应结束后，通过HPLC纯化，冷冻干燥后得到白色粉末5(tR＝7.348min，12.1毫克，0.022毫摩尔，产率为65％)，即Gly-BA。HRMS(ESI+)m/z：calcd.for C25H32O3N7ClP[M+H]+544.1987，found 544.1989。
实施例3 Arg-BA(化合物6)的合成
将化合物3(39.43毫克，0.081毫摩尔，1.0当量)、Boc-Arg(Pbf)-OH(51.3毫克，0.097毫摩尔，1.2当量)和HATU(37.0毫克，0.097毫摩尔，1.2当量)溶于1毫升无水DMF中，搅拌溶解后加入DIEA(40.2微升，0.243毫摩尔，3.0当量)，室温反应2小时，并通过HPLC监测反应进程。反应结束后，将反应液用乙酸乙酯萃取，收集有机相，旋干，用油泵抽30分钟，然后将残留物在冰浴下溶于30％ TFA/DCM中，随后使反应液升至室温并搅拌1小时。反应结束后，通过HPLC纯化，冷冻干燥后得到白色粉末6(tR＝7.147min，23.6毫克，0.037毫摩尔，产率为58％)，即Arg-BA。HRMS(ESI+)m/z：calcd.for C29H41O3N10ClP[M+H]+643.2784，found 643.2786。
实施例4 Pro-DA的合成
将N-脱羟乙基达沙替尼(20毫克，0.045毫摩尔，1.0当量)、Boc-L-脯氨酸(11.7毫克，0.054毫摩尔，1.2当量)和HATU(20.6毫克，0.054毫摩尔，1.2当量)溶于1毫升无水DMF中，搅拌溶解后加入DIEA(22.4微升，0.135毫摩尔，3.0当量)，室温反应2小时，并通过HPLC监测反应进程。反应结束后，将反应液用乙酸乙酯萃取，收集有机相，旋干，用油泵抽30分钟，然后将残留物在冰浴下溶于20％ TFA/DCM中，随后使反应液升至室温并搅拌1小时。反应结束后，通过HPLC纯化，冷冻干燥后得到白色粉末化合物Pro-DA(12.6毫克，0.023毫摩尔，产率为65％)。HRMS(ESI+)m/z：calcd.for C25H30O2N8ClS[M+H]+541.1896，found 541.1895。
实施例5 Gly-DA的合成
将N-脱羟乙基达沙替尼(20毫克，0.045毫摩尔，1.0当量)、Boc-L-甘氨酸(9.48毫克，0.054毫摩尔，1.2当量)和HATU(20.6毫克，0.054毫摩尔，1.2当量)溶于1毫升无水DMF中，搅拌溶解后加入DIEA(22.4微升，0.135毫摩尔，3.0当量)，室温反应2小时，并通过HPLC监测反应进程。反应结束后，将反应液用乙酸乙酯萃取，收集有机相，旋干，用油泵抽30分钟，然后将残留物在冰浴下溶于20％ TFA/DCM中，随后使反应液升至室温并搅拌1小时。反应结束后，通过HPLC纯化，冷冻干燥后得到白色粉末化合物Gly-DA(13.3毫克，0.027毫摩尔，产率为73％)。HRMS(ESI+)m/z：calcd.for C22H26O2N8ClS[M+H]+501.1583，found 501.1591。
实施例6 Arg-DA的合成
将N-脱羟乙基达沙替尼(20毫克，0.045毫摩尔，1.0当量)、Boc-Arg(Pbf)-OH(28.5毫克，0.054毫摩尔，1.2当量)和HATU(20.6毫克，0.054毫摩尔，1.2当量)溶于1毫升无水DMF中，搅拌溶解后加入DIEA(22.4微升，0.135毫摩尔，3.0当量)，室温反应2小时，并通过HPLC监测反应进程。反应结束后，将反应液用乙酸乙酯萃取，收集有机相，旋干，用油泵抽30分钟，然后将残留物在冰浴下溶于30％ TFA/DCM中，随后使反应液升至室温并搅拌1小时。反应结束后，通过HPLC纯化，冷冻干燥后得到白色粉末Arg-DA(11.6毫克，0.019毫摩尔，产率为53％)。HRMS(ESI+)m/z：calcd.for C26H35O2N11ClS[M+H]+600.2379，found 600.2382。
试验例
试验例1诱导ALK降解试验
将表达内源性EML4-ALK的H3122细胞铺到12孔板，用不同浓度(终浓度分别为0、5nM、10nM、25nM、50nM、75nM、100nM、250nM、500nM)的Pro-BA和Gly-BA分别处理细胞24小时后，用RIPA裂解细胞30mins，BCA蛋白定量，每个浓度都用50μg蛋白样品进行免疫印迹实验以检测ALK在不同浓度药物下的蛋白水平(图1A和1B)。比较Pro-BA、Gly-BA对ALK的降解速度：用250nM的Pro-BA和Gly-BA分别处理H3122细胞不同时间0、0.5h、1h、3h、6h、12h、18h、24h，发现Pro-BA和Gly-BA在250nM条件下能够降解EML-ALK的T1/2时间分别是Pro-BA：7.84h、Gly-BA：10.25h(图1C和1D)。
试验例2抑制H3122细胞增殖试验
用不同浓度(终浓度分别为0、2.5nM、5nM、10nM、25nM、50nM、100nM、250nM、500nM)的Pro-BA和Gly-BA分别处理H3122细胞48小时后，CCK-8细胞增殖实验发现Pro-BA和Gly-BA均能显著降低H3122细胞活性，其中Pro-BA的IC50为34.05nM，Gly-BA的IC50为68.78nM(图2A和2B)。
试验例3阻滞H3122细胞周期试验
用不同浓度(终浓度分别为0、250nM、500nM，溶剂为DMSO)的Pro-BA处理H3122细胞48小时后，利用70％乙醇固定，然后PI染色，流式细胞术分析细胞周期。发现Pro-BA处理后G1期细胞比例变多，S和G2期细胞比例变少(图3A和3B)。
试验例4诱导H3122细胞凋亡试验
用不同浓度(终浓度分别为0、250nM、500nM，溶剂为DMSO)的Pro-BA处理H3122细胞48小时后，利用Annexin V-FITC染色，流式细胞术分析细胞周期，发现Pro-BA处理后早期凋亡和晚期凋亡细胞增多(图4A和4B)。
试验例5 Pro-BA抑制肿瘤生长试验
将四周龄雌性小鼠20只，随机分成4组，每组5只。将H3122细胞异位移植裸鼠皮下，当肿瘤长到45mm3后，通过腹腔注(biw)Pro-BA(10mg/kg)、Pro-PEG3-BA(制备方法参照CN2023118151005所公开的方法)(10mg/kg)、布吉他滨(10mg/kg)和90％的玉米油+10％DMSO(作为媒介(Vehicle)组)，并测量肿瘤大小。
其中，29天后将小鼠安乐死。取出肿瘤拍照(图5A)，统计肿瘤大小(图5B)，绘制肿瘤生长曲线(图5C)。结果显示Pro-BA、Pro-PEG3-BA和布吉他滨均能抑制肿瘤生长，相比较而言Pro-BA的抑制效果最好。并且这些药物没有对小鼠体重造成明显影响(图5D)。通过Western blot分析肿瘤组织ALK发现只有降解剂Pro-BA和Pro-PEG3-BA二者能降低ALK的蛋白水平(图5E和5F)。
试验例6 Pro-BA的药代动力学研究
分别将Pro-BA(2mg/kg溶于ddH2O中)，通过尾静脉(I.V.)注射到小鼠，将Pro-BA(10mg/kg溶于ddH2O中)，通过口服(P.O.)注射到小鼠。取0.083h，0.25h，0.5h，1h，3h，6h，8h，10h和12h的血浆，并测量Pro-BA血药浓度。通过Phoenix WinNonlin 8.1软件计算PK参数。结果如表1所示。
表1
试验例7口服Pro-BA有效抑制了肿瘤生长
将H3122细胞皮下接种到四周龄雌性裸鼠，待肿瘤长到平均54mm3每两天口服给药Pro-BA(25mg/kg)，总共8次。其中口服相同剂量的ddH2O(vehicle)作为对照。取小鼠肿瘤并拍照(图6A)，并绘制肿瘤生长曲线(图6B)，显示Pro-BA显著抑制了肿瘤生长。对肿瘤组织进行免疫组化染色检测ALK的表达水平，显示Pro-BA明显降低了ALK蛋白水平(图6C)。通过测量小鼠体重， 发现Pro-BA并没有明显影响小鼠体重，表明Pro-BA无明显毒性(图6D)。
试验例8诱导BCR-ABL降解试验
将表达内源性BCR-ABL的K562细胞铺到12孔板，用不同浓度(终浓度分别为0、2.5nM、5nM、7.5nM、10nM、20nM)的Pro-DA和Gly-DA分别处理细胞48小时后，用RIPA裂解细胞30mins，BCA蛋白定量，每个浓度都用50μg蛋白样品进行免疫印迹实验以检测BCR-ABL在不同浓度药物下的蛋白水平(图7A和7B)。比较Pro-DA、Gly-DA对BCR-ABL的降解速度：用10nM的Pro-DA和Gly-DA分别处理K562细胞不同时间0、6h、12h、24h、36、48h，发现Pro-DA和Gly-DA在10nM条件下能够降解BCR-ABL的T1/2时间分别是Pro-DA：17.95h、Gly-DA：27.89h(图7C和7D)。
试验例9抑制K562细胞增殖试验
用不同浓度(终浓度分别为0、0.1nM、0.25nM、0.5nM、0.75nM、1nM、2.5nM、5nM、10nM)的Pro-DA和Gly-DA分别处理K562细胞48小时后，CCK-8细胞增殖实验发现Pro-DA和Gly-DA均能显著降低K562细胞活性，其中Pro-DA的IC50为0.91nM，Gly-DA的IC50为0.604nM(图8A和8B)。

Claims (16)

  1. 一种式(I)所示的化合物或其药学上可接受的盐：
    X-Y
    式(I)
    其中，X为单氨基酸或其衍生物，Y为靶蛋白的配体。
  2. 根据权利要求1所述的化合物或其药学上可接受的盐，其中所述单氨基酸选自Gly、Ala、Val、Leu、Ile、Phe、Pro、Trp、Ser、Tyr、Cys、Asp、Asn、Gln、Glu、Thr、Lys、Arg、His中的一种。
  3. 根据权利要求1所述的化合物或其药学上可接受的盐，其中所述单氨基酸为Gly、Pro或Arg。
  4. 根据权利要求1-3中任一项所述的化合物或其药学上可接受的盐，其中所述靶蛋白为EML4-ALK融合蛋白或BCR-ABL融合蛋白。
  5. 根据权利要求1-4中任一项所述的化合物或其药学上可接受的盐，其中Y为下式所示的布吉他滨衍生物：
  6. 根据权利要求1-4中任一项所述的化合物或其药学上可接受的盐，其中Y为下式所示的达沙替尼衍生物：
  7. 根据权利要求1-6中所述的化合物或其药学上可接受的盐，其中所述化合物选自以下一种：
  8. 一种药物组合物，包括权利要求1-7中所述的化合物或其药学上可接受的盐，以及药学上可接受的载体。
  9. 权利要求1-7中所述的化合物或其药学上可接受的盐或权利要求8所述的药物组合物在制备降解BCR-ABL或EML4-ALK融合蛋白的药物中的用途。
  10. 权利要求1-7中所述的化合物或其药学上可接受的盐或权利要求8所述的药物组合物在制备BCR-ABL或EML4-ALK融合蛋白降解剂中的用途。
  11. 权利要求1-7中所述的化合物或其药学上可接受的盐或权利要求8所述的药物组合物在制备治疗BCR-ABL或EML4-ALK介导的疾病的药物中的用途。
  12. 根据权利要求11所述的用途，其中所述疾病为癌症。
  13. 根据权利要求12所述的用途，其中所述癌症选自以下一种或两种以上：非小细胞肺癌、间变性大细胞淋巴瘤、慢性髓系白血病、急性淋巴细胞白血病、非典型慢性粒细胞白血病、急性淋巴母细胞白血病、急性髓系白血病、B淋巴细胞白血病/淋巴瘤、淋巴母细胞淋巴瘤、塔顿-布朗-拉赫曼综合征或急性早幼粒细胞白血病、小细胞肺癌、结直肠癌、乳腺癌、前列腺癌、肝癌、胰腺癌、胃癌、肾癌、卵巢癌、骨髓瘤。
  14. 一种治疗BCR-ABL或EML4-ALK介导的疾病的方法，包括向受试者施用治疗有效量的权利要求1-7中所述的化合物或其药学上可接受的盐或权利要求8所述的药物组合物。
  15. 根据权利要求14所述的方法，其中所述疾病为癌症。
  16. 根据权利要求15所述的方法，其中所述癌症选自以下一种或两种以上：非小细胞肺癌、间变性大细胞淋巴瘤、慢性髓系白血病、急性淋巴细胞白血病、非典型慢性粒细胞白血病、急性淋巴母细胞白血病、急性髓系白血病、B淋巴细胞白血病/淋巴瘤、淋巴母细胞淋巴瘤、塔顿-布朗-拉赫曼综合征或急性早幼粒细胞白血病、小细胞肺癌、结直肠癌、乳腺癌、前列腺癌、肝癌、胰腺癌、胃癌、肾癌、卵巢癌、骨髓瘤。
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