N-degron-based novel linker-free mini-protac compound and use thereof

Abstract

The present application provides an N-degron-based novel linker-free mini-PROTAC compound, and a use thereof. The present application provides a novel and unique linker-free mini-PROTAC small molecule, which can utilize a cell's own degradation pathway to specifically degrade BCR-ABL and EML4-ALK fusion proteins. Unlike conventional PROTACs, which regulate the spatial position between a target protein and a specific E3 ubiquitin ligase by means of the length and type of a linker, the mini-PROTAC of the present application recruits different E3 ubiquitin ligases by means of nineteen different N-degron amino acids, respectively, so as to form an optimal "E3—AA-miniPROTAC—POI" ternary complex that is spatially favorable for the ubiquitination labeling of a target protein, thereby achieving efficient degradation of the target protein.

Description

Novel N-degron-based micro-protac compounds without linker and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and in particular relates to a novel N-degron-based micro-protac compound without linker and uses thereof. BACKGROUND

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

[0003] PROTAC is composed of three parts: a ligand that binds to an E3 ligase for guiding protein degradation, a ligand that binds to a target protein for guiding the targeted positioning of small molecules, and a linker responsible for the chimeric of the two ligands. After the PROTAC molecule enters the cell, the Protein of Interest (POI) ligand in its structure can specifically bind to the corresponding target protein, and the other end can recruit E3 ligase to form a POI-linker-E3 ligase ternary complex, in which the E3 ligase can mediate the polyubiquitination of the POI, and the ubiquitinated POI is recognized and degraded by the proteasome. PROTAC compound is a technology based on the ubiquitin-proteasome system to cause protein degradation.

[0004] PROTAC is theoretically 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. Only the transient formation of a ternary complex can 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 the druggable proteins, overcome the drug resistance caused by target protein mutations / overexpression, and the like.

[0005] However, there are still some problems in PROTAC technology, such as the large molecular weight of PROTAC, the limited E3 ligases used for PROTAC design, the lack of PROTAC in the control of the degradation rate of target proteins, and the like. These problems restrict the application of PROTAC.

[0006] SUMMARY

[0007] To solve the problems in the prior art, the application provides a novel N-degron-based micro-PROTAC compound without a linker and a use thereof.

[0008] Specifically, the application relates to the following aspects:

[0009] 1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof:

[0010] X-Y

[0011] Formula (I)

[0012] wherein X is a single amino acid or a derivative thereof, and Y is a ligand of a target protein.

[0013] 2. The compound or the pharmaceutically acceptable salt thereof according to item 1, wherein the single amino acid is selected from one of Gly, Ala, Val, Leu, Ile, Phe, Pro, Trp, Ser, Tyr, Cys, Asp, Asn, Gln, Glu, Thr, Lys, Arg and His.

[0014] 3. The compound or the pharmaceutically acceptable salt thereof according to item 1, wherein the single amino acid is Gly, Pro or Arg.

[0015] 4. The compound or the pharmaceutically acceptable salt thereof according to any one of items 1-3, wherein the target protein is an EML4-ALK fusion protein or a BCR-ABL fusion protein.

[0016] 5. The compound or the pharmaceutically acceptable salt thereof according to any one of items 1-4, wherein Y is a birutinib derivative represented by the following formula:

[0017] 6. The compound or the pharmaceutically acceptable salt thereof according to any one of items 1-4, wherein Y is a dasatinib derivative represented by the following formula:

[0018] 7. The compound or the pharmaceutically acceptable salt thereof according to any one of items 1-6, wherein the compound is selected from one of the following:

[0019] 8. A pharmaceutical composition comprising the compound or the pharmaceutically acceptable salt thereof according to any one of items 1-7, and a pharmaceutically acceptable carrier.

[0020] 9. Use of the compound or the pharmaceutically acceptable salt thereof according to any one of items 1-7 or the pharmaceutical composition according to item 8 in the preparation of a medicament for degrading a BCR-ABL or EML4-ALK fusion protein.

[0021] 10. Use of a compound as described in items 1-7 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition described in item 8 in the manufacture of a BCR-ABL or EML4-ALK fusion protein degrader.

[0022] 11. Use of a compound as described in items 1-7 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition described in item 8 in the manufacture of a medicament for the treatment of a BCR-ABL or EML4-ALK mediated disease.

[0023] 12. Use according to item 11, wherein the disease is cancer.

[0024] 13. Use according to item 12, wherein 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-lymphoblastic leukemia / lymphoma, lymphoblastic lymphoma, Taton-Brown-Rahman 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, myeloma.

[0025] 14. A method of treating a BCR-ABL or EML4-ALK mediated disease comprising administering to a subject a therapeutically effective amount of a compound as described in items 1-7 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition described in item 8.

[0026] 15. The method according to item 14, wherein the disease is cancer.

[0027] 16. The method according to item 15, wherein 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-lymphoblastic leukemia / lymphoma, lymphoblastic lymphoma, Taton-Brown-Rahman 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, myeloma. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 shows the ALK protein level detected by WB after treating H3122 cells with the compounds of the present application. Wherein A is the result of treating H3122 cells with different concentrations of Pro-BA for 24h; B is the result of treating H3122 cells with different concentrations of Gly-BA for 24h; C is the result of treating H3122 with 250nM Pro-BA for different time; D is the result of treating H3122 with 250nM Gly-BA for different time.

[0029] Figure 2 shows the effect of Pro-BA or Gly-BA on cell proliferation detected by CCK8. Wherein A is Pro-BA, B is Gly-BA.

[0030] Figure 3 shows the flow cytometry analysis result of treating H3122 cells with different concentrations of 0, 250nM, 500nM Pro-BA for 48h.

[0031] Figure 4 shows the flow cytometry analysis result of treating H3122 cells with different concentrations of 0, 250nM, 500nM Pro-BA for 48h.

[0032] Figure 5 shows the result of Pro-BA inhibiting tumor growth. Wherein A is the tumor photo; B is the tumor size; C is the tumor growth curve; D is the body weight; E and F are the ALK protein level detected by WB.

[0033] Figure 6 shows the result of oral Pro-BA inhibiting tumor growth. Wherein A is the tumor photo; B is the tumor growth curve; C is the result of immunohistochemical staining of tumor tissue; D is the body weight.

[0034] Figure 7 shows the BCR-ABL protein level detected by WB after treating K562 cells with the compounds of the present application. Wherein A is the result of treating K562 cells with different concentrations of Pro-DA for 24h; B is the result of treating K562 cells with different concentrations of Gly-DA for 24h; C is the result of treating K562 with 10nM Pro-DA for different time; D is the result of treating K562 with 10nM Gly-DA for different time.

[0035] Figure 8 shows the effect of Pro-BA or Gly-BA on cell proliferation detected by CCK8. Wherein A is Pro-DA, B is Gly-DA. DETAILED DESCRIPTION

[0036] The present application is further illustrated by the following examples, which should not be construed as limiting the present application.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, methods and materials are described herein as being particular to the present application. In case of conflict, the present specification, including that set forth in the priority document, controls. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the application. However, the application can be practiced without some or all of these specific details. In other instances, well known methods have not been described in detail, in order not to unnecessarily obscure the application.

[0038] Definitions

[0039] "Ligand" refers to a molecule that forms a complex with a biomolecule to exert a biological function. The ligand complex can be formed via ionic bonds, covalent bonds, van der Waals interactions, and / or hydrogen bonds. "Ligand of a target protein" refers to a molecule that forms a complex with a target protein to exert a biological function.

[0040] "Pharmaceutically acceptable salt" is a salt formed by a compound of the present application with an acid 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, or with a base selected from an alkali cation base, an alkaline earth cation base, an ammonium cation base, or choline.

[0041] "Pharmaceutical composition" means a mixture that contains one or more compounds described herein or a physiologically acceptable salt or prodrug thereof, and other ingredients, such as a pharmaceutically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism and to facilitate absorption of the active ingredient into the organism to thereby deliver the active ingredient bioavailable to the organism.

[0042] "Treatment" is intended to mean preventing or slowing an undesired physiological change or disorder. For the purposes of this application, beneficial or desired results include, but are not limited to, detectable or non-detectable: alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether permanent or temporary, irrespective of the course of disease progression. "Treatment" can also mean prolonging survival relative to expected survival if not receiving treatment.

[0043] “Pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that is suitable for use in contact with the tissues of humans or animals without excessive toxicity, irritation, allergic response, other problem, or complication commensurate with a reasonable benefit / risk ratio. In some embodiments, a pharmaceutically acceptable compound, material, composition, and / or dosage form is one that is approved by a regulatory agency of the Federal or a state government of the United States, e.g., the U.S. Food and Drug Administration, the China National Medical Products Administration, the European Medicines Agency, or listed in the U.S. Pharmacopeia, the China Pharmacopeia, the European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0044] “Pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulation material, involved in carrying or transporting the subject compound from one location, body fluid, tissue, organ (internal or external) or part of the body to another location, body fluid, tissue, organ or part of the body. A pharmaceutically acceptable carrier can be a carrier, diluent, excipient, or other material that can be used in conjunction with an animal tissue without excessive toxicity or adverse effects. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars, starches, celluloses, malt, gum tragacanth, gelatin, Ringer’s solution, alginic acid, isotonic salt solutions, buffers, and the like.

[0045] “Subject” means an organism, tissue, or cell. A subject can include a human subject for medical purposes, e.g., diagnosis and / or treatment of an existing condition or disease, or prophylactic treatment to prevent onset of a condition or disease, or an animal subject for medical veterinary purposes or development purposes. A subject also includes sample material from tissue cultures, cell cultures, organ replication, stem cell production, and the like. Suitable animal subjects include mammals and birds. The term “mammal” as used herein includes, but is not limited to, primates (e.g., humans, monkeys, apes, and the like), bovines (e.g., oxen and the like), ovines (e.g., sheep, goats, and the like), porcines, equines, felines, canines, lagomorphs (e.g., rabbits and the like), rodents (e.g., mice, rats, and the like), and the like. The term “bird” as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasants, and the like. In some embodiments, a subject is a mammal or a mammalian cell. In some embodiments, a subject is a human or a human cell. Human subjects include, but are not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient having or suspected of having a certain condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. A subject can also refer to a cell in a laboratory or a bioprocessing medium under test.

[0046] The term "effective amount" as used herein refers to that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means any amount that, when administered to a subject with or without additional therapy, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effects, or a decrease in the rate of advancement of a disease or disorder, as compared to the relevant subject who does not receive such amount. The term also includes within its scope amounts effective to enhance normally physiological function. The therapeutically effective amount of the compound(s) of the present application is known to the skilled artisan or can be readily determined by standard methods known in the art.

[0047] Compound

[0048] As described above, the PROTAC technology still has the following problems:

[0049] (1) PROTAC molecular weight is too large

[0050] PROTAC molecules are usually composed of POI ligand, linker and E3 ligase ligand three parts, and the molecular weight is usually around 1000 Da, which violates Lipinski's five rules (Ro5), so it leads to relatively poor penetration, solubility, pharmacokinetics and oral bioavailability of PROTAC drugs, in addition, the synthesis difficulty and cost are also relatively high. As for the linker, its type and length cannot be predicted, and it needs to be tested for various possibilities, sometimes too large. PROTAC needs to enter the cell to mobilize the UPS in the cell, so membrane permeability is the key to the function of PROTAC. At present, the penetration mechanism of PROTAC has not been elucidated, and the molecular weight of most PROTACs is 1000-2000 Da, which mainly passes through the cell membrane by passive diffusion and active transport. However, due to the larger molecular weight and larger exposed polar surface area, the cell / tissue permeability of PROTAC is far inferior to that of small molecules. Therefore, researchers have developed various strategies to improve the permeability of PROTAC, and the common method is to limit its molecular weight to below 1000 Da or split the molecule into two smaller precursors and combine into a mature PROTAC (CLIPTAC) in the cell. In addition, 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 connected to the E3 ligand, thereby increasing the cell permeability of PROTAC. In addition to modifying PROTAC itself, the application of liposomes and other nanoparticles to deliver PROTAC can also significantly enhance the cellular uptake of PROTAC.

[0051] (2) Limited E3 ligase for PROTAC design

[0052] There are more than 600 E3 ligases in the human genome, but 90% of the E3 ligases currently applied to PROTAC are CRBN and VHL. Both are tumor suppressor proteins, and PROTACs based on CRBN and VHL are prone to drug resistance due to mutations and deletions of E3 ligases in cancer treatment, in addition, due to the tissue-specific distribution of CRBN and VHL, their applicability is limited.

[0053] (3) PROTAC lacks control over the degradation rate of target proteins

[0054] Normal life activities of cells require key cell regulators to be kept within a suitable range, because in some cases, both too much or too little of these proteins can become harmful, thus causing diseases. For example, nicotinamide phosphoribosyltransferase NAMPT can maintain sufficient NAD+ levels in cells, which plays an important role in anti-aging, on the other hand, NAMPT is highly expressed in a variety of tumors, and is a target for tumor treatment. Therefore, it is crucial to control the protein level of NAMPT within a reasonable range. Therefore, for some target proteins, PROTAC does not necessarily have a stronger degradation effect, but the degradation rate of the current PROTAC is basically fixed and not easy to adjust.

[0055] The formation of BCR-ABL gene is due to the rearrangement of chromosome 9 and chromosome 22, i.e. t(9;22)(q34;q11), resulting in an abnormal chromosome 22, called Philadelphia (Ph) chromosome. The reciprocal translocation of this chromosome leads to the fusion of BCR and ABL genes, forming the BCR-ABL gene (Figure 2), resulting in the constitutive activation of ABL tyrosine kinase. Abnormal activation of BCR-ABL is closely related to the occurrence of chronic myeloid leukemia (CML). Therefore, BCR-ABL is an important therapeutic target for CML. So far, several BCR-ABL tyrosine kinase inhibitors (BCR-ABL-TKI) have been approved for the treatment of CML patients, including the first-generation TKI imatinib, the second-generation TKI dasatinib, nilotinib and bosutinib, and the third-generation ALK-TKI ponatinib and ponatinib. Unfortunately, CML patients receiving BCR-ABL-TKI treatment will develop drug resistance after about a year of treatment. Therefore, it is of great significance to develop new drugs to overcome BCR-ABL drug resistance.

[0056] Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase consisting of an extracellular domain, a single-pass transmembrane domain, and an intracellular kinase domain. The extracellular domain of ALK includes two Methyl Dopamine-A5 Protein-Receptor Tyrosine Phosphatase (MAM) domains, a Low Density Lipoprotein (LDLa) domain, and a Glycine-rich extracellular domain (G-rich); a single transmembrane (TM) domain is located between the extracellular and intracellular portions; and an intracellular tyrosine kinase (PTK) domain. FAM150 is a ligand of ALK, 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 dimerization inducing ALK activation involves the phosphorylation of one or more of the juxtamembrane tyrosine residues (Tyr 1078, 1092, 1096, and 1131), followed by sequential phosphorylation in turn until the active form of ALK is formed.

[0057] Fusion and rearrangement of ALK gene is an important cause of various cancers, including non-small cell lung cancer (NSCLC) and anaplastic large cell lymphoma (ALCL). The proportion of ALK fusion mutation positive in non-small cell lung adenocarcinoma is 3-5%, and the proportion in China is about 5.3%, which occurs more frequently in non-small cell lung adenocarcinoma, young patients (less than 60 years old) and non-smoking population. ALK fusion is the second largest NSCLC molecular subtype after EGFR mutation, and the corresponding targeted drugs are completely different from EGFR molecular subtype. ALK can be fused and rearranged with various partner genes, thereby leading to its constitutive activation, the most common fusion occurs in Echinoderm Microtubule-Associated Protein 4-Anaplastic Lymphoma Kinase (EML4-ALK). So far, 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 receiving ALK-TKIs treatment will all develop resistance and lead to disease progression. Therefore, it is of great significance to develop new targeted drugs to overcome resistance of ALK-positive non-small cell lung cancer (NSCLC).

[0058] The N-degron pathway refers to the stability and half-life of a protein depending on its N-terminal amino acid residues. The classical N-degron pathway was first discovered by the Alexander Varshavsky laboratory in 1986. Studies have shown that this degradation pathway exists from mammals, plants to bacteria, and plays an important role in regulating cell cycle and cell division, signal transduction, gene expression, DNA repair and other basic life activities. The instability of amino acid residues located at the N-terminus of the target protein is called "N-degron". The N-degron pathway mainly includes Arg / N-degron, Pro / N-degron and Gly / N-degron pathways, etc. Among them, the Arg / N-degron pathway is the earliest discovered and the most widely studied. When the N-terminus of a protein exposes unstable N-terminal residues (including Arg, Lys, His, Leu, Phe, Tyr, Trp, Ile), it can be recognized by UBR family E3 ligase UBR1 / 2 / 4 / 5, thereby inducing protein degradation through ubiquitin proteasome. The Pro / N-degron pathway was discovered in 2017 and can target proteins with proline (Pro) at the N-terminus. GID4 and GID10 in the multi-subunit RING-type E3 ubiquitin ligase CTLH complex are responsible for recognizing the Pro at the N-terminus of the protein. The Gly / N-degron pathway was discovered in 2019 and can recognize the glycine (Gly) residue at the first position of the N-terminus of the protein by CRL2ZER1&ZYG11B.

[0059] In view of the problems existing in the prior art, the present application provides a novel micro-PROTAC compound based on N-degron or a pharmaceutically acceptable salt thereof without a linker.

[0060] The micro-PROTAC compound is shown as formula (I):

[0061] X-Y

[0062] Formula (I)

[0063] wherein X is a single amino acid or a derivative thereof, and Y is a ligand of a target protein.

[0064] The single amino acid can be various natural or unnatural 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.

[0065] In a specific embodiment, the single amino acid is Gly, Pro or Arg.

[0066] In a specific embodiment, the target protein is EML4-ALK fusion protein or BCR-ABL fusion protein. Y can be a ligand of various EML4-ALK fusion proteins or BCR-ABL fusion proteins known in the art.

[0067] In a specific embodiment, Y is a pegvisomant derivative.

[0068] In a specific embodiment, Y is a pegvisomant derivative represented by the following formula:

[0069] In a specific embodiment, Y is a dasatinib derivative.

[0070] In a specific embodiment, Y is a dasatinib derivative represented by the following formula:

[0071] In a specific embodiment, the compound is selected from one of the following:

[0072] It is understood by those skilled in the art that the compounds of the present application can exist in a variety of different forms, all of which are included within the scope of the present application. These forms include, for example, tautomers, stereoisomers, racemic mixtures, prodrugs, solvated forms, different crystalline or polymorphic forms, etc.

[0073] Pharmaceutical composition

[0074] The present application also provides a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0075] The form of the pharmaceutical composition depends on a number of criteria, including, for example, the route of administration, the extent of the disease, or the dosage to be administered, etc.

[0076] In some embodiments, the pharmaceutical composition can be formulated to be delivered into a subject by an appropriate route, including, but not limited to, by oral route, injection route (such as intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intracardiac injection, intrathecal injection, intrapleural injection, intraperitoneal injection, etc.), mucosal route (such as intranasal administration, intraoral administration, etc.), sublingual route, rectal route, transdermal route, intraocular route, pulmonary route. Depending on the desired route of administration, the pharmaceutical composition can be formulated as tablets, capsules, pills, dragees, powders, granules, sachets, lozenges, suppositories, suspensions, emulsions, syrups, aerosols (as solids or in liquid media), sprays, ointments, pastes, patches, creams, lotions, gels, inhalants, etc.

[0077] Therapeutic methods, therapeutic uses

[0078] The present application also provides use of the above-mentioned compound or or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in the preparation of a medicament for degrading BCR-ABL or EML4-ALK fusion protein.

[0079] The present application also provides use of the above-mentioned compound or or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in the preparation of a BCR-ABL or EML4-ALK fusion protein degrader.

[0080] The present application also provides use of the above-mentioned compound or or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in the preparation of a medicament for treating a BCR-ABL or EML4-ALK mediated disease.

[0081] The present application also provides a method for treating a BCR-ABL or EML4-ALK mediated disease, comprising administering to a subject a therapeutically effective amount of the above-mentioned compound or or a pharmaceutically acceptable salt thereof or a pharmaceutical composition.

[0082] In the present application, "BCR-ABL mediated disease" is intended to include any disease associated with BCR-ABL gene or protein. "EML4-ALK mediated disease" is intended to include any disease associated with EML4-ALK gene or protein.

[0083] In a specific embodiment, the BCR-ABL or EML4-ALK mediated disease is a disease treatable by degrading BCR-ABL or EML4-ALK.

[0084] In a specific embodiment, the disease is cancer.

[0085] In a specific embodiment, 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-lymphoblastic leukemia / lymphoma, lymphoblastic lymphoma, Taton-Brown-Rahman 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 disease, myeloma.

[0086] In a specific embodiment, the BCR-ABL mediated disease is chronic myeloid leukemia.

[0087] In a specific embodiment, the EML4-ALK mediated disease is non-small cell lung cancer or anaplastic large cell lymphoma.

[0088] In one embodiment, the application provides use of Pro-DA or Gly-DA in the manufacture of a medicament for degrading a BCR-ABL fusion protein.

[0089] In one embodiment, the application provides use of Pro-DA or Gly-DA in the manufacture of a BCR-ABL fusion protein degrader.

[0090] In one embodiment, the application provides use of Pro-DA or Gly-DA in the manufacture of a medicament for treating a BCR-ABL mediated disease. In one embodiment, the BCR-ABL mediated disease is chronic myeloid leukemia.

[0091] In one embodiment, the application provides a method of treating a BCR-ABL mediated disease, comprising administering to a subject a therapeutically effective amount of Pro-DA or Gly-DA. In one embodiment, the BCR-ABL mediated disease is chronic myeloid leukemia.

[0092] In one embodiment, the application provides use of Pro-BA or Gly-BA in the manufacture of a medicament for degrading an EML4-ALK fusion protein.

[0093] In one embodiment, the application provides use of Pro-BA or Gly-BA in the manufacture of an EML4-ALK fusion protein degrader.

[0094] In one embodiment, the application provides use of Pro-BA or Gly-BA in the manufacture of a medicament for treating an EML4-ALK mediated disease. In one embodiment, the EML4-ALK mediated disease is non-small cell lung cancer or anaplastic large cell lymphoma.

[0095] In one embodiment, the application provides a method of treating an EML4-ALK mediated disease, comprising administering to a subject a therapeutically effective amount of Pro-BA or Gly-BA. In one embodiment, the EML4-ALK mediated disease is non-small cell lung cancer or anaplastic large cell lymphoma.

[0096] Examples

[0097] Materials: 2,4,5-trichloropyrimidine, 2-(dimethoxyphosphoryl)aniline, and Boc-Arg(Pbf)-OH were purchased from Shanghai Biotech Reagent Co., Ltd; Boc-L-proline was purchased from Bide Pharmatec Co., Ltd; 4-(4-amino-3-methoxyphenyl)piperazine-1-carboxylic acid tert-butyl ester, N-dehydroxyethyl dasatinib were purchased from Jiangsu Enochem Reagent Co., Ltd; Tetrabutylammonium hydrogen sulfate was purchased from Aralab Reagent Co., Ltd; Boc-glycine, 2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA) and acetonitrile were purchased from Anjieji Reagent Co., Ltd.

[0098] Characterization methods: Sample analysis was performed on an Agilent 1260 high performance liquid system equipped with a DAD-UV detector, using an Agilent Poroshell 120, EC-C18 column (4.6 mm x 100 mm, 2.7 μm). The analytical HPLC gradient started at 10% B (B: acetonitrile, A: 0.1% trifluoroacetic acid in water) and increased to 100% over 20 minutes at a flow rate of 0.5 mL / min. The purity of the compounds (>95%) determined by analytical HPLC was used for biological studies. Sample purification was performed on an ULTIMAT 3000 (DIONEX) preparative HPLC instrument using a photo diode array detector to measure UV absorbance at 220 and 254 nm. The preparative HPLC gradient started at 1% B (B: acetonitrile, A: 0.1% trifluoroacetic acid in water) and increased to 100% over 20 minutes. High resolution mass spectrometry was measured with an AB IQ-star Elite.

[0099] Synthesis of busulfan derivatives:

[0100] The synthesis of busulfan derivatives is as follows:

[0101] In a reaction bottle, 2-(dimethoxyphosphoryl)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 monitoring the completion of the reaction by thin layer chromatography (TLC), the potassium carbonate was removed by filtration, the filtrate was concentrated under vacuum, and purified by column chromatography with DCM:MeOH = 50:1 to obtain white solid 1 (757 mg, 2.4 mmol, yield 81%). HRMS (ESI + m / z: calcd. for C 12 H13 Cl2N3OP[M+H] + 316.0168, found 316.0161.

[0102] In a 10 mL reaction tube, compound 1 (100 mg, 0.32 mmol, 1.0 equiv), 4-(4- amino-3-methoxyphenyl)piperazine-1-carboxylic acid tert-butyl ester (117 mg, 0.38 mmol, 1.2 equiv) and 2 mL of isopropanol were added, stirred to dissolution, followed by the addition of TFA (25.9 μL, 0.35 mmol, 1.1 equiv) and the reaction tube was stirred at 95 °C overnight. The reaction progress was followed using analytical HPLC and the reaction mixture was directly purified by preparative HPLC to give compound 2 as a greenish powder after lyophilization. The greenish powder was dissolved in 20% TFA / DCM in an ice bath, then the reaction was allowed to warm to room temperature and stirred for 1 h. The reaction was monitored using analytical HPLC and after completion of the reaction, the solvent was evaporated under reduced pressure and purified by preparative HPLC to give compound 3 as a yellowish green powder after lyophilization (t R = 7.246 min, 140 mg, 0.29 mmol, 91% yield) which is a bortezomib derivative (BA). HRMS (ESI + )m / z: calcd. for C 23 H 29 O2N6ClP[M+H] + 487.1773, found 487.1775.

[0103] Example 1 Synthesis of Pro-BA (compound 4)

[0104] Compound 3 (50.5 mg, 0.104 mmol, 1.0 equiv), Boc-L-proline (26.8 mg, 0.125 mmol, 1.2 equiv) and HATU (47.4 mg, 0.125 mmol, 1.2 equiv) were dissolved in 1 mL of dry DMF, after stirring to dissolution, DIEA (51.5 μL, 0.312 mmol, 3.0 equiv) was added and the reaction was allowed to proceed at room temperature for 2 h and the reaction progress was monitored by HPLC. After completion of the reaction, the reaction was extracted with ethyl acetate, the organic phase was collected, dried on oil pump for 30 min, then the residue was dissolved in 20% TFA / DCM in an ice bath, then the reaction was allowed to warm to room temperature and stirred for 1 h. After completion of the reaction, it was purified by HPLC to give 4 as a white powder after lyophilization (t R = 7.828 min, 28.9 mg, 0.050 mmol, 60% yield) which is Pro-BA. HRMS (ESI + )m / z: calcd. for C28 H 36 O3N7ClP[M+H] + 584.2300,found 584.2299.

[0105] Example 2 Synthesis of Gly-BA (Compound 5)

[0106] Compound 3 (21.09 mg, 0.043 mmol, 1.0 equiv), Boc-L-glycine (9.11 mg, 0.052 mmol, 1.2 equiv) and HATU (19.8 mg, 0.052 mmol, 1.2 equiv) were dissolved in 1 mL of dry DMF, after stirring to dissolve, DIEA (21.5 μL, 0.130 mmol, 3.0 equiv) was added, the reaction was allowed to proceed at room temperature for 2 hours and the progress of the reaction was monitored by HPLC. After the reaction was completed, the reaction was extracted with ethyl acetate, the organic phase was collected, rotary evaporated, pumped with oil pump for 30 minutes, then the residue was dissolved in 20% TFA / DCM in ice bath, then the reaction was allowed to warm to room temperature and stirred for 1 hour. After the reaction was completed, it was purified by HPLC, white powder 5 (t R = 7.348 min, 12.1 mg, 0.022 mmol, 65% yield) was obtained, which is Gly-BA. HRMS (ESI + )m / z: calcd. for C 25 H 32 O3N7ClP[M+H] + 544.1987, found 544.1989.

[0107] Example 3 Synthesis of Arg-BA (Compound 6)

[0108] Compound 3 (39.43 mg, 0.081 mmol, 1.0 equiv), Boc-Arg(Pbf)-OH (51.3 mg, 0.097 mmol, 1.2 equiv) and HATU (37.0 mg, 0.097 mmol, 1.2 equiv) were dissolved in 1 mL of dry DMF, after stirring to dissolve, DIEA (40.2 μL, 0.243 mmol, 3.0 equiv) was added, the reaction was allowed to proceed at room temperature for 2 hours and the progress of the reaction was monitored by HPLC. After the reaction was completed, the reaction was extracted with ethyl acetate, the organic phase was collected, rotary evaporated, pumped with oil pump for 30 minutes, then the residue was dissolved in 30% TFA / DCM in ice bath, then the reaction was allowed to warm to room temperature and stirred for 1 hour. After the reaction was completed, it was purified by HPLC, white powder 6 (t R= 7.147 min, 23.6 mg, 0.037 mmol, 58% yield), i.e. Arg-BA. HRMS (ESI + m / z: calcd for C 29 H 41 O3N 10 ClP[M+H] + 643.2784, found 643.2786.

[0109] Example 4 Synthesis of Pro-DA

[0110] N-desethyl dasatinib (20 mg, 0.045 mmol, 1.0 eq), Boc-L-proline (11.7 mg, 0.054 mmol, 1.2 eq) and HATU (20.6 mg, 0.054 mmol, 1.2 eq) were dissolved in 1 mL of dry DMF, after stirring to dissolve, DIEA (22.4 μL, 0.135 mmol, 3.0 eq) was added, the reaction was allowed to proceed at room temperature for 2 hours, and the reaction progress was monitored by HPLC. After the reaction was completed, the reaction solution was extracted with ethyl acetate, the organic phase was collected, rotary evaporated, pumped for 30 minutes, then the residue was dissolved in 20% TFA / DCM in ice bath, then the reaction solution was allowed to warm to room temperature and stirred for 1 hour. After the reaction was completed, purified by HPLC, white powder compound Pro-DA (12.6 mg, 0.023 mmol, 65% yield) was obtained after freeze-drying. HRMS (ESI + m / z: calcd for C 25 H 30 O2N8ClS[M+H] + 541.1896, found 541.1895.

[0111] Example 5 Synthesis of Gly-DA

[0112] 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 dissolution, DIEA (22.4 μL, 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. The residue was then dissolved in 20% TFA / DCM in an ice bath, and the reaction solution was then brought to room temperature and stirred for 1 h. After the reaction was completed, the solution was purified by HPLC and lyophilized to give a white powder compound Gly-DA (13.3 mg, 0.027 mmol, yield 73%). HRMS (ESI) + m / z: calcd.for C 22 H 26 O2N8ClS[M+H] + 501.1583, found 501.1591.

[0113] Example 6 Synthesis of Arg-DA

[0114] 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 dissolution, DIEA (22.4 μL, 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. The residue was then dissolved in 30% TFA / DCM in an ice bath, and the reaction solution was then brought to room temperature and stirred for 1 h. After the reaction was completed, the solution was purified by HPLC and lyophilized to obtain a white powder Arg-DA (11.6 mg, 0.019 mmol, yield 53%). HRMS (ESI) + m / z: calcd.for C 26 H 35 O2N 11 ClS[M+H] + 600.2379, found 600.2382.

[0115] Test case

[0116] Experimental Example 1: Induced ALK Degradation Test

[0117] H3122 cells expressing endogenous EML4-ALK were plated into 12-well plates, and treated with different concentrations (final concentrations were 0, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, 250 nM, 500 nM, respectively) of Pro-BA and Gly-BA for 24 hours, then the cells were lysed with RIPA for 30 mins, and the protein was quantified by BCA, 50 μg of protein sample was used for immunoblotting to detect the protein level of ALK under different concentrations of drugs (Figures 1A and 1B). The degradation rate of Pro-BA and Gly-BA on ALK was compared: H3122 cells were treated with 250 nM of Pro-BA and Gly-BA for different time periods of 0, 0.5 h, 1 h, 3 h, 6 h, 12 h, 18 h, and 24 h, and it was found that Pro-BA and Gly-BA could degrade EML-ALK at 250 nM, and the time was Pro-BA: 7.84 h, Gly-BA: 10.25 h (Figures 1C and 1D). 1 / 2 The time was Pro-BA: 7.84 h, Gly-BA: 10.25 h (Figures 1C and 1D).

[0118] Test Example 2: Inhibition of H3122 cell proliferation test

[0119] After H3122 cells were treated with different concentrations (final concentrations were 0, 2.5 nM, 5 nM, 10 nM, 25 nM, 50 nM, 100 nM, 250 nM, 500 nM, respectively) of Pro-BA and Gly-BA for 48 hours, CCK-8 cell proliferation test found that Pro-BA and Gly-BA could significantly reduce the activity of H3122 cells, and the IC 50 of Pro-BA was 34.05 nM, and the IC 50 of Gly-BA was 68.78 nM (Figures 2A and 2B).

[0120] Test Example 3: H3122 cell cycle arrest test

[0121] After H3122 cells were treated with different concentrations (final concentrations were 0, 250 nM, 500 nM, and the solvent was DMSO) of Pro-BA for 48 hours, the cells were fixed with 70% ethanol and then stained with PI, and the cell cycle was analyzed by flow cytometry. It was found that the proportion of G1 phase cells increased after Pro-BA treatment, and the proportion of S and G2 phase cells decreased (Figures 3A and 3B).

[0122] Test Example 4: H3122 cell apoptosis induction test

[0123] After H3122 cells were treated with different concentrations (0, 250 nM, 500 nM, final concentration, DMSO as solvent) of Pro-BA for 48 hours, Annexin V-FITC staining was used, and flow cytometry was used to analyze cell cycle. It was found that the number of early and late apoptotic cells increased after Pro-BA treatment (Figures 4A and 4B).

[0124] Test Example 5: Pro-BA inhibits tumor growth test

[0125] Twenty four-week-old female mice were randomly divided into four groups, five in each group. H3122 cells were ectopically transplanted into nude mice subcutaneously, and when the tumor grew to 45 mm 3 Afterwards, Pro-BA (10 mg / kg), Pro-PEG3-BA (the preparation method is disclosed in CN2023118151005) (10 mg / kg), pegsitubicin (10 mg / kg) and 90% corn oil + 10% DMSO (as the Vehicle group) were injected intraperitoneally (biw), and the tumor size was measured.

[0126] After 29 days, the mice were euthanized. The tumors were removed and photographed (Figure 5A), the tumor size was counted (Figure 5B), and the tumor growth curve was drawn (Figure 5C). The results showed that Pro-BA, Pro-PEG3-BA and pegsitubicin could all inhibit tumor growth, and Pro-BA had the best inhibitory effect. Moreover, these drugs did not have a significant impact on the body weight of the mice (Figure 5D). Western blot analysis of tumor tissue ALK showed that only the degradation agents Pro-BA and Pro-PEG3-BA could reduce the protein level of ALK (Figures 5E and 5F).

[0127] Test Example 6: Pharmacokinetic study of Pro-BA

[0128] Pro-BA (2 mg / kg dissolved in ddH2O) was injected into mice through the tail vein (I.V.), and Pro-BA (10 mg / kg dissolved in ddH2O) was injected into mice through oral administration (P.O.). The plasma was taken at 0.083 h, 0.25 h, 0.5 h, 1 h, 3 h, 6 h, 8 h, 10 h and 12 h, and the Pro-BA blood concentration was measured. The PK parameters were calculated by Phoenix WinNonlin 8.1 software. The results are shown in Table 1.

[0129] Table 1

[0130] Test Example 7: Oral Pro-BA effectively inhibits tumor growth

[0131] H3122 cells were subcutaneously inoculated into four-week-old female nude mice, and the tumors were allowed to grow to an average of 54 mm 3 Pro-BA (25 mg / kg) was orally administered every two days for a total of 8 times. The same dose of ddH2O (vehicle) was orally administered as a control. The tumors of the mice were taken and photographed (Figure 6A), and the tumor growth curve was plotted (Figure 6B), showing that Pro-BA significantly inhibited tumor growth. Immunohistochemical staining of tumor tissue to detect the expression level of ALK showed that Pro-BA significantly reduced the ALK protein level (Figure 6C). By measuring the body weight of the mice, it was found that Pro-BA did not significantly affect the body weight of the mice, indicating that Pro-BA had no obvious toxicity (Figure 6D).

[0132] Example 8: BCR-ABL degradation test

[0133] K562 cells expressing endogenous BCR-ABL were plated in a 12-well plate and treated with different concentrations (final concentrations of 0, 2.5 nM, 5 nM, 7.5 nM, 10 nM, 20 nM) of Pro-DA and Gly-DA, respectively, for 48 hours. The cells were lysed with RIPA for 30 mins, and the protein was quantified by BCA. 50 μg of protein sample was used for immunoblotting to detect the protein level of BCR-ABL under different concentrations of drugs (Figures 7A and 7B). Comparison of the degradation rates of Pro-DA and Gly-DA on BCR-ABL: K562 cells were treated with 10 nM of Pro-DA and Gly-DA for different times of 0, 6 h, 12 h, 24 h, 36 h, and 48 h, respectively. It was found that Pro-DA and Gly-DA could degrade BCR-ABL at 10 nM for T 1 / 2 The time was Pro-DA: 17.95 h, Gly-DA: 27.89 h, respectively (Figures 7C and 7D).

[0134] Example 9: K562 cell proliferation inhibition test

[0135] After K562 cells were treated with different concentrations (final concentrations of 0, 0.1 nM, 0.25 nM, 0.5 nM, 0.75 nM, 1 nM, 2.5 nM, 5 nM, 10 nM) of Pro-DA and Gly-DA, respectively, for 48 hours, CCK-8 cell proliferation test found that Pro-DA and Gly-DA could significantly reduce the activity of K562 cells, with an IC 50 of 0.91 nM for Pro-DA and an IC50 of 0.604 nM for Gly-DA (Figures 8A and 8B).

Claims

1. A compound of Formula (I) or a pharmaceutically acceptable salt thereof: X-Y Formula (I) wherein X is a single amino acid or a derivative thereof, and Y is a ligand of a target protein.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the single amino acid is selected from one of Gly, Ala, Val, Leu, lie, Phe, Pro, Trp, Ser, Tyr, Cys, Asp, Asn, Gin, Glu, Thr, Lys, Arg, His.

3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the single amino acid is Gly, Pro or Arg.

4. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein the target protein is an EML4-ALK fusion protein or a BCR-ABL fusion protein.

5. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-4, wherein Y is a buju iticin derivative of the following formula:

6. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-4, wherein Y is a dasatinib derivative of the formula:

7. The compound according to claims 1-6, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from one of the following:

8. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, and a pharmaceutically acceptable carrier.

9. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, or a pharmaceutical composition according to claim 8, in the manufacture of a medicament for degrading a BCR-ABL or EML4-ALK fusion protein.

10. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, or a pharmaceutical composition according to claim 8, in the manufacture of a BCR-ABL or EML4-ALK fusion protein degrader.

11. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, or a pharmaceutical composition according to claim 8, in the manufacture of a medicament for treating a BCR-ABL or EML4-ALK mediated disease.

12. The use according to claim 11, wherein the disease is cancer.

13. The use according to claim 12, wherein the cancer is selected from one or more of 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, Taton-Brown-Rahman 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, myeloma.

14. A method of treating a BCR-ABL or EML4-ALK mediated disease, comprising administering to a subject a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, or a pharmaceutical composition according to claim 8.

15. The method according to claim 14, wherein the disease is cancer.

16. The method of claim 15, wherein 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, Taton-Brown-Rahman 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, myeloma.

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