WO2023088236A1 - Bicyclic peptide ligand of mt1-mmp and conjugate thereof - Google Patents

Bicyclic peptide ligand of mt1-mmp and conjugate thereof Download PDF

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WO2023088236A1
WO2023088236A1 PCT/CN2022/131909 CN2022131909W WO2023088236A1 WO 2023088236 A1 WO2023088236 A1 WO 2023088236A1 CN 2022131909 W CN2022131909 W CN 2022131909W WO 2023088236 A1 WO2023088236 A1 WO 2023088236A1
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ala
hcys
asp
glu
cys
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PCT/CN2022/131909
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French (fr)
Chinese (zh)
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李瑶
陈雷
黄海涛
王浩东
唐平明
余彦
张晨
严庞科
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海思科医药集团股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to a compound containing a polypeptide structure with high affinity for membrane-type matrix metalloproteinase 1 (MT1-MMP) and a polypeptide drug conjugate conjugated to one or more toxin molecules, and the compound and drug conjugate
  • MT1-MMP membrane-type matrix metalloproteinase 1
  • the related use of the compound including the use of preparing a pharmaceutical composition and the use of a medicine for preventing and/or treating a disease overexpressing MT1-MMP.
  • Matrix metalloproteinases are a family of zinc-dependent endoproteases that can degrade most components in the extracellular matrix (extracellular matrix, ECM).
  • ECM extracellular matrix
  • a total of 25 members of the matrix metalloproteinase family have been discovered so far, and 24 species exist in mammals, which are related to various physiological and pathological conditions.
  • Sato et al. identified the first membrane-type matrix metalloproteinase, now called MT1-MMP, and found that it is an agonist of the cell surface MMP-2 zymogen, and it is also a membrane-type matrix metalloproteinase that has been studied more. Protease.
  • Membrane-type matrix metalloproteinase-1 (MT1-MMP) is an important member of the matrix metalloproteinase family, and its encoded protein has a relative molecular mass of 66kDa, which plays an important role in the degradation of the extracellular matrix and is highly Participating in the process of cell invasion to tissue and the formation of epithelial cell polarity, it is considered to be the enzyme most closely related to tumor invasion in the MMP family.
  • MT1-MMP is mainly expressed in fibroblasts, smooth muscle cells and endothelial cells, but it is highly expressed in tumor cells and adjacent stromal cells of most human malignant tumors such as gastric cancer, colon cancer and liver cancer.
  • MT1-MMP is closely related to the poor prognosis of individuals with neuroblastoma, small cell lung cancer, and bladder cancer.
  • the low expression of MT1-MMP is considered to be a good survival indicator in patients with advanced colorectal cancer.
  • MMPs overexpressed proteases
  • MMPs can be used as marker molecular signals for diagnosing tumor cell invasion and migration.
  • MMPs as targets to treat diseases has become a new research hotspot.
  • the research on the function of MT1-MMP at the enzymatic molecular level is relatively thorough, and there is evidence that it can promote the occurrence and development of diseases such as tumors.
  • MT1-MMP is located on the cell surface and is highly expressed in a variety of tumor cells, making MT1-MMP an ideal target for tumor therapy.
  • different types of artificially synthesized MMP inhibitors are used to treat tumors, but the clinical effect is not satisfactory.
  • inhibitors have broad-spectrum specificity, not only inhibiting target enzymes, but also inhibiting some Beneficial enzymes. Therefore, it is of great significance to screen ligands that can specifically bind to MT1-MMP and use them as targeted drug delivery carriers.
  • the present invention firstly provides a compound specific to MT1-MMP, which comprises a polypeptide structure, and also provides a polypeptide drug conjugate in which the compound is conjugated to one or more toxin molecules, and also provides a pharmaceutical composition, and in their Use in medicines for the prevention and/or treatment of diseases overexpressing MT1-MMP.
  • the compounds, drug conjugates and drug compositions of the present invention have high affinity to targets, good selectivity and high stability.
  • the present invention relates to a compound comprising a polypeptide structure and an aromatic molecular scaffold, the polypeptide structure comprising at least three residues independently selected from Cys and Hcys, and at least one of the three residues is an Hcys residue, the The three residues are separated by two amino acid sequences, and the Cys or Hcys residues of the polypeptide structure are connected with the molecular scaffold through a thioether bond, thereby forming two polypeptide rings on the molecular scaffold.
  • the compound comprises a polypeptide structure and an aromatic molecular scaffold, the polypeptide structure comprising at least three residues independently selected from Cys and Hcys, and one of the three residues is a Hcys residue , the three residues are separated by two amino acid sequences, and the Cys or Hcys residues of the polypeptide structure are connected to the molecular scaffold through a thioether bond, thereby forming two polypeptide rings on the molecular scaffold.
  • the compound comprises a polypeptide structure and an aromatic molecular scaffold, the polypeptide structure comprising at least three residues independently selected from Cys and Hcys, and two of the three residues are Hcys residues The three residues are separated by two amino acid sequences, and the Cys or Hcys residues of the polypeptide structure are connected to the molecular scaffold through a thioether bond, thereby forming two polypeptide rings on the molecular scaffold.
  • the aromatic molecular scaffold is selected from TBMB.
  • the compound comprises a polypeptide structure and an aromatic molecular scaffold, the polypeptide structure comprising at least three residues independently selected from Cys and Hcys, and one of the three residues is a Hcys residue , the three residues are separated by two amino acid sequences, and the Cys or Hcys residues of the polypeptide structure are connected to the molecular scaffold through a thioether bond, thereby forming two polypeptide rings on the molecular scaffold, so
  • the molecular scaffold described above is selected from TBMB.
  • the compound comprises a polypeptide structure and an aromatic molecular scaffold, the polypeptide structure comprising at least three residues independently selected from Cys and Hcys, and two of the three residues are Hcys residues base, the three residues are separated by two amino acid sequences, and the Cys or Hcys residues of the polypeptide structure are connected to the molecular scaffold through a thioether bond, thereby forming two polypeptide rings on the molecular scaffold,
  • the molecular scaffold is selected from TBMB.
  • polypeptide structure of the compound comprises an amino acid sequence selected from:
  • a 1 and A 2 represent amino acid residues among Xa 1 , Xa 2 , and Xa 3 , and each of A 1 and A 2 independently comprises 5 or 6 amino acid residues; in some specific embodiments, A 1 , A The amino acid residue that A2 comprises is optionally selected from Hyp, D-Ala, Glu, Leu, 1-Nal, Val, Asn, Pro, Leu, His, D-Asp, Asp, tBu-Gly, Trp, hArg , Ser, Thr, Tyr, Val, Ile and Gly, etc.;
  • Xa 1 , Xa 2 , and Xa 3 are independently Cys or Hcys residues, and at least one of them is a Hcys residue, and the aromatic molecular scaffold forms thioether bonds with Xa 1 , Xa 2 , and Xa 3 of the polypeptide, respectively Thus two polypeptide loops are formed on the molecular scaffold.
  • polypeptide structure comprises the amino acid sequence shown below:
  • polypeptide structure comprises the amino acid sequence shown in any one of SEQ ID NO:1 ⁇ SEQ ID NO:7:
  • Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys (SEQ ID NO: 1) ;
  • polypeptide structure comprises the amino acid sequence shown in any one of SEQ ID NO:8 ⁇ SEQ ID NO:14:
  • the compound is a bicyclic peptide having one of the following structures, optionally containing additional amino acid sequences at the N-terminus and/or C-terminus of the following structures:
  • the -SH group on the Xa 1 , Xa 2 or Xa 3 residue is covalently bonded to the molecular scaffold to form a thioether bond. Therefore, when Xa 1 , Xa 2 or Xa 3 is Cys, correspondingly, n 1 , n 2 or n 3 is 1;
  • n 1 , n 2 or n 3 is 2.
  • the bicyclic peptide of formula I-1 or I wherein:
  • Xa 1 , Xa 2 , Xa 3 are Hcys, n 1 , n 2 , n 3 are 2; or
  • Xa 1 and Xa 3 are Hcys, Xa 2 is Cys, n 1 and n 3 are 2, and n 2 is 1; or
  • Xa 1 and Xa 2 are Cys, Xa 3 is Hcys, n 1 and n 2 are 1, and n 3 is 2.
  • the compound is selected from one of the following structures:
  • the compound containing two polypeptide ring structures of the present invention has protein stability and is stable to plasma protease, epithelial protease, gastric and intestinal protease, lung surface protease, intracellular protease, etc.; has specific targeting to MT1-MMP, and is Peptide ligand specific to MT1-MMP; has long plasma half-life, good pharmacokinetic and pharmacodynamic properties.
  • the present invention also provides the use of the compound as a ligand of MT1-MMP in screening and/or preparing drugs.
  • the present invention also provides a drug conjugate or a pharmaceutically acceptable salt thereof, said conjugate comprising a compound as described above conjugated to one or more effectors and/or functional groups via a linker.
  • said effector is a cytotoxic agent.
  • the cytotoxic agent is selected from one or more of the following group: alkylating agents, antimetabolites, plant alkaloids, terpenoids, podophyllotoxins, and Derivatives, taxanes and their derivatives, topoisomerase inhibitors, antitumor antibiotics.
  • the cytotoxic agent is selected from one or more of the following group: cisplatin, carboplatin, oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, isocycline Phosphoramide, azathioprine, mercaptopurine, pyrimidine analogues, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, paclitaxel, camptothecin, irinotecan, toposide Tecan, Amyridine, Etoposide, Etoposide Phosphate, Teniposide, Actinomycin D, Doxorubicin, Epirubicin, Epothilone and its derivatives, Bleomycin and its derivatives, dactinomycin and its derivatives, plicamycin and its derivatives, mitomycin C, cyspamicin, maytansine and its derivatives, auristatin and its derivative
  • the cytotoxic agent is at least one selected from maytansinoids, monomethyl auristatin, and camptothecin derivatives.
  • the cytotoxic agent is selected from the group consisting of maytansine DM1 (DM1), maytansine DM4 (DM4), monomethyl auristatin E (MMAE), 7-ethyl-10-hydroxyl At least one of the resins (SN38).
  • the linker is a peptide linker, a disulfide linker, or a pH-dependent linker; these linkers are cleavable under certain conditions to release the effector molecule.
  • the disulfide linker is selected from DMDS, MDS, DSDM, NDMDS or the structure of formula II:
  • R 1 , R 2 , R 3 and R 4 are independently selected from H, methyl, ethyl, propyl and isopropyl;
  • p and q are independently 1, 2, 3, 4 or 5;
  • the peptide linker is selected from: -Cit-Val-, -Phe-Lys- and -Val-Lys-;
  • the pH-dependent linker is selected from cis-aconitic anhydride.
  • the above-mentioned linker is mainly used to connect the toxic agent and the peptide ligand, and to release the toxic substance by cleavage under specific conditions.
  • the linker can be appropriately modified, such as in the Peptide ligands or effectors connect some groups to increase the chain length, and increase group modification around the cleavage bond to control the hindrance of the cleavage bond.
  • the linker of the present invention includes linker derivatives modified based on the above.
  • the linker can theoretically be connected to the N-terminal, C-terminal and/or molecular scaffold of the peptide ligand.
  • a functional group linked to it can be modified on the C-terminal or molecular scaffold.
  • the linker is -PABC-Cit-Val-glutaryl- or -PABC-cyclobutyl-Ala-Cit- ⁇ Ala-, where PABC stands for p-aminobenzylcarbamate .
  • the linker is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl linker
  • k is selected from any integer of 1-20.
  • the linker is In some specific embodiments, the linker is In some specific embodiments, the linker is In some specific embodiments, the linker is In respective embodiments, k is selected from any integer of 1-20; in some specific embodiments, k is selected from any integer of 1-10; in some specific embodiments, k is selected from 1, 2, 3, 4, or 5; in some embodiments, k is selected from 2.
  • the drug conjugate has the structure of Formula III, Formula IV, Formula V, or Formula VI:
  • R 1 , R 2 , R 3 and R 4 are independently selected from H, methyl, ethyl, propyl and isopropyl;
  • p and q are independently 1, 2, 3, 4 or 5;
  • k is independently any integer of 1-10;
  • Xa 1 , Xa 2 , and Xa 3 are independently Cys or Hcys residues, and at least one of them is a Hcys residue;
  • n 1 , n 2 or n 3 is 1;
  • n 1 , n 2 or n 3 is 2.
  • the drug conjugate or a pharmaceutically acceptable salt thereof wherein:
  • R 1 , R 2 , R 3 and R 4 are independently selected from H or methyl;
  • p and q are independently 1 or 2;
  • k is independently any integer from 1 to 5;
  • Xa 1 , Xa 2 , Xa 3 are Hcys, n 1 , n 2 , n 3 are 2; or
  • Xa 1 and Xa 3 are Hcys, Xa 2 is Cys, n 1 and n 3 are 2, and n 2 is 1; or
  • Xa 1 and Xa 2 are Cys, Xa 3 is Hcys, n 1 and n 2 are 1, and n 3 is 2.
  • a drug conjugate or a pharmaceutically acceptable salt thereof is selected from one of the following structures:
  • the present invention also relates to a pharmaceutical composition, which contains the aforementioned compound of the present invention (i.e. peptide ligand, or peptide compound) and/or the aforementioned drug conjugate of the present invention or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier and/or excipient.
  • a pharmaceutical composition which contains the aforementioned compound of the present invention (i.e. peptide ligand, or peptide compound) and/or the aforementioned drug conjugate of the present invention or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier and/or excipient.
  • the present invention also relates to a use of the aforementioned compound of the present invention, or the aforementioned drug conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a composition thereof in the preparation of preventing and/or treating overexpression of MT1 - Use of MMPs in medicine for diseases.
  • the disease overexpressing MT1-MMP is selected from at least one of the following diseases: neuroblastoma, small cell lung cancer, bladder cancer, gastric cancer, colon cancer, liver cancer.
  • the present invention also relates to a pharmaceutical composition or pharmaceutical preparation, said pharmaceutical composition or pharmaceutical preparation comprising 1-1500 mg of the peptide compound described in the present invention or the conjugate described in the present invention or its A pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier and/or excipient.
  • the present invention also relates to a method for treating diseases in mammals or humans, the method comprising administering to a subject a therapeutically effective amount of the aforementioned peptide compound of the present invention or the aforementioned conjugate of the present invention or its For pharmaceutically acceptable salts, the therapeutically effective dose is preferably 1-1500 mg, and the disease is preferably at least one of neuroblastoma, small cell lung cancer, bladder cancer, gastric cancer, colon cancer, and liver cancer.
  • the present invention also provides a composition or pharmaceutical preparation, which contains the aforementioned peptide compound, conjugate or pharmaceutically acceptable salt thereof of the present invention, and a pharmaceutically acceptable carrier and/or adjuvant.
  • the pharmaceutical composition may be in unit dosage form (a unit dosage is also referred to as a "dosage strength").
  • composition or pharmaceutical preparation of the present invention contains 1-1500 mg of the aforementioned peptide compound, conjugate or pharmaceutically acceptable salt thereof of the present invention, and pharmaceutically acceptable carriers and/or excipients .
  • the present invention also provides the use of the aforementioned peptide compounds, conjugates or pharmaceutically acceptable salts thereof in the preparation of drugs for the prevention and treatment of diseases or disorders in which MT1-MMP is overexpressed in diseased tissues of tumor individuals .
  • the disease or disease overexpressing MT1-MMP is preferably at least one of neuroblastoma, small cell lung cancer, bladder cancer, gastric cancer, colon cancer, and liver cancer.
  • said individual is a mammal or a human.
  • the present invention also provides a method for treating diseases in mammals or humans, the method comprising administering to a subject a therapeutically effective amount of the aforementioned peptide compounds, conjugates or pharmaceutically acceptable ones shown in the present invention salt, the disease is preferably at least one of neuroblastoma, small cell lung cancer, bladder cancer, gastric cancer, colon cancer, and liver cancer, and preferably the therapeutically effective dose is 1-1500 mg.
  • Effective amount or “therapeutically effective amount” in the present application refers to the administration of a sufficient amount of the compound disclosed in the present application, which will alleviate to some extent one or more symptoms of the disease or disorder being treated. In some embodiments, the result is reduction and/or alleviation of signs, symptoms or causes of disease, or any other desired alteration of a biological system.
  • an "effective amount” for therapeutic use is the amount of a composition comprising a peptide compound disclosed herein, a conjugate, or a pharmaceutically acceptable salt thereof, required to provide a clinically significant reduction in disease symptoms.
  • therapeutically effective amounts include, but are not limited to, 1-1500 mg, 1-1400 mg, 1-1300 mg, 1-1200 mg, 1-1000 mg, 1-900 mg, 1-800 mg, 1-700 mg, 1-600 mg, 1-500 mg, 1 -400mg, 1-300mg, 1-250mg, 1-200mg, 1-150mg, 1-125mg, 1-100mg, 1-80mg, 1-60mg, 1-50mg, 1-40mg, 1-25mg, 1-20mg , 5-1500mg, 5-1000mg, 5-900mg, 5-800mg, 5-700mg, 5-600mg, 5-500mg, 5-400mg, 5-300mg, 5-250mg, 5-200mg, 5-150mg, 5 -125mg, 5-100mg, 5-90mg, 5-70mg, 5-80mg, 5-60mg, 5-50mg, 5-40mg, 5-30mg, 5-25mg, 5-20mg, 10-1500mg, 10-1000mg ,
  • the pharmaceutical composition or preparation of the present invention contains the aforementioned therapeutically effective amount of the peptide compound, conjugate or pharmaceutically acceptable salt thereof of the present invention.
  • the present invention relates to a pharmaceutical composition or pharmaceutical preparation, which comprises a therapeutically effective amount of the peptide compound, conjugate or pharmaceutically acceptable salt thereof and a carrier and/or excipient.
  • the pharmaceutical composition may be in the form of a unit preparation (the amount of the main drug in the unit preparation is also referred to as "preparation specification").
  • the pharmaceutical composition includes, but is not limited to, 1 mg, 1.25 mg, 2.5 mg, 5 mg, 10 mg, 12.5 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg ,70mg,75mg,80mg,85mg,90mg,95mg,100mg,110mg,120mg,125mg,130mg,140mg,150mg,160mg,170mg,180mg,190mg,200mg,210mg,220mg,230mg,240mg,250mg,275mg,300mg . , 1100mg , 1200mg, 1300mg, 1400mg, 1500mg of the peptide compound, conjugate or pharmaceutically acceptable salt thereof of the present invention.
  • the present invention provides a method for treating a disease in a mammal or a human, the method comprising administering to a subject a therapeutically effective amount of a peptide compound of the present invention, a conjugate, or a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier and/or excipient, the therapeutically effective dose is preferably 1-1500 mg, and the disease is preferably neuroblastoma, small cell lung cancer, bladder cancer, gastric cancer, colon cancer, liver cancer at least one.
  • the present invention provides a method for treating a disease in a mammal or a human.
  • the method comprises administering the peptide compound, the conjugate or a pharmaceutically acceptable salt thereof of the present invention, and a pharmaceutically Acceptable carriers and/or excipients are given to the subject at a daily dose of 1-1500 mg/day, and the daily dose can be a single dose or divided doses.
  • the daily dose includes but is not limited to 10 -1500mg/day, 20-1500mg/day, 25-1500mg/day, 50-1500mg/day, 75-1500mg/day, 100-1500mg/day, 200-1500mg/day, 10-1000mg/day, 20-1000mg /day, 25-1000mg/day, 50-1000mg/day, 75-1000mg/day, 100-1000mg/day, 200-1000mg/day, 25-800mg/day, 50-800mg/day, 100-800mg/day , 200-800mg/day, 25-400mg/day, 50-400mg/day, 100-400mg/day, 200-400mg/day, in some embodiments, the daily dosage includes but not limited to 1mg/day, 5mg/day , 10mg/day, 20mg/day, 25mg/day, 50mg/day, 75mg/day, 100mg/day, 10mg/day
  • the present invention also provides a kit, which may include a composition in single dose or multi-dose form, the kit comprising a compound of the present invention, a conjugate or a pharmaceutically acceptable salt thereof, a compound of the present invention, a conjugate, or a pharmaceutically acceptable salt thereof.
  • the amount of the conjugate or its stereoisomer or pharmaceutically acceptable salt is the same as that in the above pharmaceutical composition.
  • Preparation specification refers to the weight of the main drug contained in each tube, tablet or other unit preparation.
  • the peptides of the invention can be prepared synthetically by standard techniques and then reacted with molecular scaffolds in vitro. When doing this, standard chemistry can be used. This enables rapid large-scale preparation of soluble materials for further downstream experiments or validation. Such methods can be achieved using conventional chemistry as disclosed in, for example, Timmerman et al. (2005, ChemBioChem).
  • the drug conjugates of the present invention are synthesized according to the following route:
  • a peptide ligand refers to a compound containing an amino acid sequence (peptide structure) formed by covalently binding a peptide to a molecular scaffold.
  • it can be used as the ligand of membrane-type matrix metalloproteinase-1 (MT1-MMP).
  • the peptides forming such compounds contain two or more reactive groups (i.e. cysteine residues or homocysteine residues) capable of forming covalent bonds (e.g. thioether bonds) with the scaffold , and the opposing sequence between the reactive groups, called the loop sequence, is called a loop sequence because when the peptide binds to the scaffold, it forms a loop.
  • the peptide comprises at least three cysteine residues or homocysteine residues (Cys residues or Hcys residues), which form two loops on the scaffold.
  • Molecular scaffolds include aromatic molecular scaffolds.
  • An aromatic molecular scaffold refers to any molecular scaffold as defined herein comprising an aromatic carbocyclic or aromatic heterocyclic ring system. Examples of suitable aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chemie, International Edition 53(6) 1602-1606.
  • Molecular scaffolds can be small molecules, such as small organic molecules.
  • Polypeptide refers to a compound formed by three or more amino acid molecules linked together by peptide bonds.
  • the unit of amino acid in a polypeptide is called an amino acid residue.
  • Effectors and/or functional groups are pharmacologically or functionally specific molecules or fragments that can be attached (via a linker) to, for example, the N- and/or C-termini of a polypeptide, amino acids within a polypeptide, or the molecular backbone.
  • Suitable effectors and/or functional groups include antibodies and parts or fragments thereof, cytotoxic molecules or fragments, enzyme inhibitor molecules or fragments, metal chelators, and the like.
  • the effector and/or functional group is a drug.
  • cytotoxic agents including alkylating agents, antimetabolites, plant alkaloids, terpenoids, podophyllotoxins and their derivatives, taxanes and their derivatives, topoisomerases Inhibitors, antitumor antibiotics, etc.
  • cisplatin carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide, azathioprine, mercaptopurine, pyrimidine analogs, vincristine, vinca Alkaline, vinorelbine, vindesine, etoposide, teniposide, paclitaxel, camptothecin, irinotecan, topotecan, amyridine, etoposide, etoposide phosphate, teniposide , actinomycin D, doxorubicin, epirubicin, epothilone and its derivatives, bleomycin and its derivatives, dactinomycin and its derivatives, plicamycin and its derivatives substances, mitomycin C, cynistatin, maytansine and its derivatives, auristatin and its derivatives.
  • Derivatives refer to products derived from the substitution of hydrogen atoms or atomic groups in a compound by other atoms or atomic groups.
  • Trp tryptophan
  • Maytansinoids such as DM1 and DM4, are cytotoxic agents, which are thiol-containing derivatives of maytansine.
  • DM1 has the following structure:
  • DM4 has the following structure:
  • MMAE Monomethyl auristatin E
  • monomethyl auristatin E is a synthetic antineoplastic agent and has the following structure:
  • the carbon, hydrogen, oxygen, sulfur, nitrogen or halogens involved in the groups and compounds of the present invention include their isotopes, and the carbon, hydrogen, oxygen, sulfur, Nitrogen or halogen is optionally further replaced by one or more of their corresponding isotopes, wherein isotopes of carbon include 12 C, 13 C and 14 C, isotopes of hydrogen include protium (H), deuterium (deuterium, also known as deuterium ), tritium (T, also known as tritium), the isotopes of oxygen include 16 O, 17 O and 18 O, the isotopes of sulfur include 32 S, 33 S, 34 S and 36 S, and the isotopes of nitrogen include 14 N and 15 N, the isotope of fluorine is 19 F, the isotope of chlorine includes 35 Cl and 37 Cl, and the isotope of bromine includes 79 Br and 81 Br.
  • isotopes of carbon include 12 C, 13 C and 14 C
  • “Pharmaceutically acceptable salt” means that the compound of the present invention maintains the biological effectiveness and characteristics of the free acid or free base, and the free acid is mixed with a non-toxic inorganic base or organic base, and the free base is mixed with a non-toxic inorganic base or organic base.
  • Non-toxic salts obtained by reacting inorganic or organic acids.
  • “Pharmaceutical composition” means a mixture of one or more compounds described herein, or stereoisomers, solvates, pharmaceutically acceptable salts or co-crystals thereof, with other constituents, wherein the other constituents comprise physiological/pharmaceutical acceptable carriers and/or excipients.
  • Carrier refers to: does not cause significant irritation to the organism and does not eliminate the biological activity and characteristics of the administered compound, and can change the way the drug enters the body and its distribution in the body, controls the release rate of the drug and releases the drug.
  • Non-limiting examples of systems for delivery to targeted organs include microcapsules and microspheres, nanoparticles, liposomes, and the like.
  • Excipient means: not itself a therapeutic agent, used as a diluent, adjuvant, binder and/or vehicle, added to a pharmaceutical composition to improve its handling or storage properties or to allow or facilitate The compound or pharmaceutical composition is presented in unit dosage form for administration.
  • pharmaceutical excipients can serve various functions and can be described as wetting agents, buffers, suspending agents, lubricants, emulsifiers, disintegrating agents, absorbing agents, preservatives , surfactants, coloring agents, flavoring agents and sweeteners.
  • Examples of pharmaceutically acceptable excipients include, but are not limited to: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as carboxymethyl Sodium cellulose, ethyl cellulose, cellulose acetate, hydroxypropyl methylcellulose, hydroxypropyl cellulose, microcrystalline cellulose, and croscarmellose (such as croscarmellose sodium) (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, red Flower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as oil (13) a
  • HBTU Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
  • CTC resin 75g, 1.0mmol/g
  • Fmoc-L-citrulline 30.0g, 75.4mmol, 1.0eq
  • dichloromethane 600mL
  • N,N-diisopropylethyl Amine 58.4g, 453mmol, 6.0eq
  • Suction filtration the resin was washed twice with DMF
  • the prepared 20% piperidine/DMF solution was added to the resin mixture, the system reacted for 2 hours
  • suction filtration the resin was washed twice with DMF.
  • Step ten
  • the crude peptide P22 was dissolved in 50% MeCN/H 2 O (500 mL), and TBMB (purchased from WuXi AppTec, 270 mg, 0.60 mmol) was slowly added under stirring at room temperature for more than 30 minutes. Ammonium bicarbonate was added to adjust the pH value to 8, and the reaction solution was stirred at room temperature for 12 hours. LC-MS showed that the reaction was complete. Purification by preparative HPLC (mobile phase, A: 0.075% TFA in H 2 O, B: CH 3 CN) gave a white solid HSK-P22 (42.1 mg, purity 97.3%).
  • HSK-P46, HSK-P47, HSK-P48, HSK-P49, HSK-P50, HSK-P51 were synthesized respectively.
  • HSK-P22 The amino acid sequence is ( ⁇ -Ala)-Sar10-Ala-SEQ ID NO: 1(Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys) (i.e. SEQ ID NO: 8), the molecular scaffold is selected from TBMB.
  • HSK-P46 The amino acid sequence is ( ⁇ -Ala)-Sar10-Ala-SEQ ID NO: 4(Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-cys) (i.e. SEQ ID NO: 11), the molecular scaffold is selected from TBMB.
  • HSK-P47 The amino acid sequence is ( ⁇ -Ala)-Sar10-Ala-SEQ ID NO: 3(Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys) (i.e. SEQ ID NO: 10), the molecular scaffold is selected from TBMB.
  • HSK-P48 The amino acid sequence is ( ⁇ -Ala)-Sar10-Ala-SEQ ID NO: 2(Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys) (i.e. SEQ ID NO:9), the molecular scaffold is selected from TBMB.
  • HSK-P49 The amino acid sequence is ( ⁇ -Ala)-Sar10-Ala-SEQ ID NO: 7(Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys) (i.e. SEQ ID NO: 14), the molecular scaffold is selected from TBMB.
  • HSK-P50 The amino acid sequence is ( ⁇ -Ala)-Sar10-Ala-SEQ ID NO: 6(Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys) (i.e. SEQ ID NO: 13), the molecular scaffold is selected from TBMB.
  • HSK-P51 The amino acid sequence is ( ⁇ -Ala)-Sar10-Ala-SEQ ID NO: 5(Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys) (i.e. SEQ ID NO: 12), the molecular scaffold is selected from TBMB.
  • Step ten
  • HT-1080 cell culture conditions EMEM+10% FBS+1% double antibody, cultured at 37°C, 5% CO 2 incubator.
  • HT-1080 cells in the exponential growth phase were collected and plated on 96-well culture plates, 90 ⁇ L per well, with a plating density of 500 cells/well, and cultured overnight at 37°C in a 5% CO 2 incubator.
  • 10 ⁇ L of different concentrations of compounds were added to each well so that the final concentration of DMSO in each well was 0.1%, and cultured at 37° C. in a 5% CO 2 incubator for 3 days.
  • Inhibition% (1-(RLU compound/RLU control) ⁇ 100% Formula (1)
  • the compound of the present invention has higher inhibitory activity.
  • Human multiple myeloma MOLP8 cells purchased from DSMZ were placed in RPMI-1640 complete medium (supplemented with 20% fetal bovine serum) and cultured at 37°C and 5% CO 2 . Cells in the exponential growth phase were collected, and the cell suspension was adjusted to 8000 cells/135 ⁇ L with medium. Add 135 ⁇ L of cell suspension to each well in a 96-well cell culture plate and incubate overnight. On the second day, different concentrations of compounds were added and cultured in an incubator for 5 days.
  • Test animals male SD rats, about 220 g, 6-8 weeks old, 3 rats/compound. purchased from Chengdu Dashuo Experimental Animal Co., Ltd.
  • NC cannot be calculated
  • the reference compound is BT17BDC-18 of WO2016067035A1, the structural formula is
  • Test animals male balb/c mice, 20-25g, 9/compound. purchased from Chengdu Dashuo Experimental Animal Co., Ltd.
  • Test animals male cynomolgus monkeys, 3-5kg, 3-6 years old, 3 animals/compound. purchased from Suzhou Xishan Biotechnology Co., Ltd.
  • Test method On the test day, 3 monkeys were randomly divided into groups according to body weight. One day before the administration, fasting without water for 14-18 hours, and giving food 4 hours after the administration.
  • 1.0 mL of blood was collected from the veins of the extremities, and placed in EDTAK2 centrifuge tubes. Centrifuge at 5000 rpm for 10 min at 4°C to collect plasma. The time points of blood collection in the vein group were: 0, 2, 5, 15, 30min, 1, 2, 4, 6, 8, 24h. Before analysis and detection, all samples were stored at -80°C, and the samples were quantitatively analyzed by LC-MS/MS.
  • the compounds of the present invention have good pharmacokinetic characteristics in monkeys.
  • the compound of the present invention has good pharmacokinetic characteristics in dogs.

Abstract

Provided are a peptide having high affinity to membrane-type matrix metalloproteinase 1 (MT1-MMP), a drug conjugate, stereoisomer, pharmaceutically acceptable salt, solvate, co-crystal or deuterated product thereof, or a pharmaceutical composition containing same, and a use of the peptide ligand and drug conjugates in the prevention and treatment of diseases or disorders in which MT1-MMP is overexpressed in diseased tissue of individuals suffering from tumors.

Description

MT1-MMP的双环肽配体及其缀合物Bicyclic peptide ligands of MT1-MMP and their conjugates 技术领域technical field
本发明涉及一种具有膜型基质金属蛋白酶1(MT1-MMP)高亲和力的包含多肽结构的化合物及其缀合至一个或多个毒素分子的多肽药物缀合物，及所述化合物和药物缀合物的相关用途，包括制备药物组合物的用途及在预防和/或治疗过度表达MT1-MMP的疾病的药物中的用途。The present invention relates to a compound containing a polypeptide structure with high affinity for membrane-type matrix metalloproteinase 1 (MT1-MMP) and a polypeptide drug conjugate conjugated to one or more toxin molecules, and the compound and drug conjugate The related use of the compound, including the use of preparing a pharmaceutical composition and the use of a medicine for preventing and/or treating a disease overexpressing MT1-MMP.
背景技术Background technique
基质金属蛋白酶(matrix metalloproteinase，MMP)是一类锌离子依赖的内切蛋白酶家族，它们可降解细胞外基质(extracelluar matrix，ECM)中的绝大多数成分。目前发现的基质金属蛋白酶家族成员共有25个，哺乳动物体内存在24种，与多种生理及病理状况相关。1994年，Sato等鉴别出第一个膜型基质金属蛋白酶，现在称为MT1-MMP，并发现它是细胞表面MMP-2酶原的激动剂，也是目前研究较多的一种膜型基质金属蛋白酶。Matrix metalloproteinases (matrix metalloproteinases, MMPs) are a family of zinc-dependent endoproteases that can degrade most components in the extracellular matrix (extracellular matrix, ECM). A total of 25 members of the matrix metalloproteinase family have been discovered so far, and 24 species exist in mammals, which are related to various physiological and pathological conditions. In 1994, Sato et al. identified the first membrane-type matrix metalloproteinase, now called MT1-MMP, and found that it is an agonist of the cell surface MMP-2 zymogen, and it is also a membrane-type matrix metalloproteinase that has been studied more. Protease.
膜型基质金属蛋白酶-1(MT1-MMP)是基质金属蛋白酶家族的一个重要成员，其编码的蛋白质相对分子质量为66kDa，其在细胞外基质的降解过程中扮演了一个重要的角色，并且高度参与到细胞向组织侵袭、上皮细胞极性的形成的过程中，被认为是MMP家族中与肿瘤侵袭关系最密切的酶。MT1-MMP主要在成纤维细胞、平滑肌细胞和内皮细胞表达，但在胃癌，结肠癌，肝癌等大多数人类恶性肿瘤的肿瘤细胞和邻近基质细胞中均有高表达。临床研究发现，MT1-MMP的表达与神经母细胞瘤，小细胞肺癌，膀胱癌等个体的不良预后密切相关。而MT1-MMP的低表达在晚期结直肠癌患者中则被认为是良好的生存指标。Membrane-type matrix metalloproteinase-1 (MT1-MMP) is an important member of the matrix metalloproteinase family, and its encoded protein has a relative molecular mass of 66kDa, which plays an important role in the degradation of the extracellular matrix and is highly Participating in the process of cell invasion to tissue and the formation of epithelial cell polarity, it is considered to be the enzyme most closely related to tumor invasion in the MMP family. MT1-MMP is mainly expressed in fibroblasts, smooth muscle cells and endothelial cells, but it is highly expressed in tumor cells and adjacent stromal cells of most human malignant tumors such as gastric cancer, colon cancer and liver cancer. Clinical studies have found that the expression of MT1-MMP is closely related to the poor prognosis of individuals with neuroblastoma, small cell lung cancer, and bladder cancer. The low expression of MT1-MMP is considered to be a good survival indicator in patients with advanced colorectal cancer.
近年来随着研究的深入科学家们逐渐认识到，过度表达的蛋白酶类(包括MMPs在内)，可以作为诊断肿瘤细胞侵润和迁移的标志性分子信号。以MMPs为靶点治疗疾病已成为新的研究热点。MT1-MMP在酶学分子水平上功能的研究较为透彻，同时有证据表明其可促进肿瘤等疾病的发生、发展。MT1-MMP定位于细胞表面，且在多种肿瘤细胞中表达量较高，使得MT1-MMP成为了一种肿瘤治疗的理想靶点。目前应用人工合成的不同类型的的MMP抑制剂***，临床使用效果并不理想，其中的一个重要原因是，因为这些抑制剂具有广谱的特异性，不仅抑制了靶酶，也抑制了一些有益酶。因此筛选能够与MT1-MMP特异性结合的配体，并作为靶向给药载体具有重要意义。In recent years, with the deepening of research, scientists have gradually realized that overexpressed proteases (including MMPs) can be used as marker molecular signals for diagnosing tumor cell invasion and migration. Using MMPs as targets to treat diseases has become a new research hotspot. The research on the function of MT1-MMP at the enzymatic molecular level is relatively thorough, and there is evidence that it can promote the occurrence and development of diseases such as tumors. MT1-MMP is located on the cell surface and is highly expressed in a variety of tumor cells, making MT1-MMP an ideal target for tumor therapy. At present, different types of artificially synthesized MMP inhibitors are used to treat tumors, but the clinical effect is not satisfactory. One of the important reasons is that these inhibitors have broad-spectrum specificity, not only inhibiting target enzymes, but also inhibiting some Beneficial enzymes. Therefore, it is of great significance to screen ligands that can specifically bind to MT1-MMP and use them as targeted drug delivery carriers.
发明内容Contents of the invention
本发明首先提供了特异于MT1-MMP的化合物，其包含多肽结构，还提供了所述化合物缀合至一个或多个毒素分子的多肽药物缀合物，还提供了药物组合物，及在它们在预防和/或治疗过度表达MT1-MMP的疾病的药物中的用途。本发明所述化合物、药物缀合物、药物组合物，对靶标具有高亲和力，而且选择性好、稳定性高。The present invention firstly provides a compound specific to MT1-MMP, which comprises a polypeptide structure, and also provides a polypeptide drug conjugate in which the compound is conjugated to one or more toxin molecules, and also provides a pharmaceutical composition, and in their Use in medicines for the prevention and/or treatment of diseases overexpressing MT1-MMP. The compounds, drug conjugates and drug compositions of the present invention have high affinity to targets, good selectivity and high stability.
本发明涉及一种化合物，其包含多肽结构和芳香族分子支架，所述多肽结构包含至少三个独立选自Cys和Hcys的残基，且三个残基中至少一个是Hcys残基，所述三个残基被两段氨基酸序列隔开，所述多肽结构的Cys或Hcys残基经硫醚键与所述分子支架连接，从而在所述分子支架上形成两个多肽环。The present invention relates to a compound comprising a polypeptide structure and an aromatic molecular scaffold, the polypeptide structure comprising at least three residues independently selected from Cys and Hcys, and at least one of the three residues is an Hcys residue, the The three residues are separated by two amino acid sequences, and the Cys or Hcys residues of the polypeptide structure are connected with the molecular scaffold through a thioether bond, thereby forming two polypeptide rings on the molecular scaffold.
在一些具体实施方案中，所述化合物，其包含多肽结构和芳香族分子支架，所述多肽结构包含至少三个独立选自Cys和Hcys的残基，且三个残基中一个是Hcys残基，所述三个残基被两段氨基酸序列隔开，所述多肽结构的Cys或Hcys残基经硫醚键与所述分子支架连接，从而在所述分子支架上形成两个多肽环。In some specific embodiments, the compound comprises a polypeptide structure and an aromatic molecular scaffold, the polypeptide structure comprising at least three residues independently selected from Cys and Hcys, and one of the three residues is a Hcys residue , the three residues are separated by two amino acid sequences, and the Cys or Hcys residues of the polypeptide structure are connected to the molecular scaffold through a thioether bond, thereby forming two polypeptide rings on the molecular scaffold.
在一些具体实施方案中，所述化合物，其包含多肽结构和芳香族分子支架，所述多肽结构包含至少三个独立选自Cys和Hcys的残基，且三个残基中两个是Hcys残基，所述三个残基被两段氨基酸序列隔开，所述多肽结构的Cys或Hcys残基经硫醚键与所述分子支架连接，从而在所述分子支架上形成两个多肽环。In some specific embodiments, the compound comprises a polypeptide structure and an aromatic molecular scaffold, the polypeptide structure comprising at least three residues independently selected from Cys and Hcys, and two of the three residues are Hcys residues The three residues are separated by two amino acid sequences, and the Cys or Hcys residues of the polypeptide structure are connected to the molecular scaffold through a thioether bond, thereby forming two polypeptide rings on the molecular scaffold.
在一些具体实施方案中，所述的芳香族分子支架选自TBMB。In some embodiments, the aromatic molecular scaffold is selected from TBMB.
在一些具体实施方案中，所述化合物，其包含多肽结构和芳香族分子支架，所述多肽结构包含至少三个独立选自Cys和Hcys的残基，且三个残基中一个是Hcys残基，所述三个残基被两段氨基酸序列隔开，所述多肽结构的Cys或Hcys残基经硫醚键与所述分子支架连接，从而在所述分子支架上形成两个多肽环，所述的分子支架选自TBMB。In some specific embodiments, the compound comprises a polypeptide structure and an aromatic molecular scaffold, the polypeptide structure comprising at least three residues independently selected from Cys and Hcys, and one of the three residues is a Hcys residue , the three residues are separated by two amino acid sequences, and the Cys or Hcys residues of the polypeptide structure are connected to the molecular scaffold through a thioether bond, thereby forming two polypeptide rings on the molecular scaffold, so The molecular scaffold described above is selected from TBMB.
在一些具体实施方案中，所述化合物，其包含多肽结构和芳香族分子支架，所述多肽结构包含至少三个独立选自Cys和Hcys的残基，且三个残基中两个是Hcys残基，所述三个残基被两段氨基酸序列隔开，所述多肽结构的Cys或Hcys残基经硫醚键与所述分子支架连接，从而在所述分子支架上形成两个多肽环，所述的分子支架选自TBMB。In some specific embodiments, the compound comprises a polypeptide structure and an aromatic molecular scaffold, the polypeptide structure comprising at least three residues independently selected from Cys and Hcys, and two of the three residues are Hcys residues base, the three residues are separated by two amino acid sequences, and the Cys or Hcys residues of the polypeptide structure are connected to the molecular scaffold through a thioether bond, thereby forming two polypeptide rings on the molecular scaffold, The molecular scaffold is selected from TBMB.
在一些具体实施方案中，所述化合物的多肽结构包含选自以下的氨基酸序列：In some specific embodiments, the polypeptide structure of the compound comprises an amino acid sequence selected from:
Xa 1-A 1-Xa 2-A 2-Xa 3， Xa 1 -A 1 -Xa 2 -A 2 -Xa 3 ,
其中，A 1、A 2表示Xa 1、Xa 2、Xa 3之间的氨基酸残基，并且A 1和A 2各自独立地包含5或6个氨基酸残基；在一些具体实施方案中，A 1、A 2包含的氨基酸残基任选地选自Hyp、 D-Ala、Glu、Leu、1-Nal、Val、Asn、Pro、Leu、His、D-Asp、Asp、tBu-Gly、Trp、hArg、Ser、Thr、Tyr、Val、Ile和Gly等； Wherein, A 1 and A 2 represent amino acid residues among Xa 1 , Xa 2 , and Xa 3 , and each of A 1 and A 2 independently comprises 5 or 6 amino acid residues; in some specific embodiments, A 1 , A The amino acid residue that A2 comprises is optionally selected from Hyp, D-Ala, Glu, Leu, 1-Nal, Val, Asn, Pro, Leu, His, D-Asp, Asp, tBu-Gly, Trp, hArg , Ser, Thr, Tyr, Val, Ile and Gly, etc.;
Xa 1、Xa 2、Xa 3独立地为Cys或Hcys残基，且至少有一个是Hcys残基，所述芳香族分子支架与所述多肽的Xa 1、Xa 2、Xa 3分别形成硫醚键从而在所述分子支架上形成两个多肽环。 Xa 1 , Xa 2 , and Xa 3 are independently Cys or Hcys residues, and at least one of them is a Hcys residue, and the aromatic molecular scaffold forms thioether bonds with Xa 1 , Xa 2 , and Xa 3 of the polypeptide, respectively Thus two polypeptide loops are formed on the molecular scaffold.
在一些具体实施方案中，所述多肽结构包含以下所示的氨基酸序列：In some specific embodiments, the polypeptide structure comprises the amino acid sequence shown below:
Xa 1-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Xa 2-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Xa 3。 Xa 1 -(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Xa 2 -Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Xa 3 .
在一些具体实施方案中，其中所述多肽结构包含SEQ ID NO:1～SEQ ID NO:7任一所示的氨基酸序列：In some specific embodiments, wherein said polypeptide structure comprises the amino acid sequence shown in any one of SEQ ID NO:1～SEQ ID NO:7:
Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:1)；Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys (SEQ ID NO: 1) ;
Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:2)；Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys (SEQ ID NO:2) ;
Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:3)；Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys (SEQ ID NO:3) ;
Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys(SEQ ID NO:4)；Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys (SEQ ID NO:4) ;
Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:5)；Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys (SEQ ID NO:5) ;
Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys(SEQ ID NO:6)；Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys (SEQ ID NO: 6) ;
Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys(SEQ ID NO:7)。Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys (SEQ ID NO: 7) .
在一些具体实施方案中，所述多肽结构包含SEQ ID NO:8～SEQ ID NO:14任一所示的氨基酸序列：In some specific embodiments, the polypeptide structure comprises the amino acid sequence shown in any one of SEQ ID NO:8～SEQ ID NO:14:
(β-Ala)-Sar 10-Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:8)； (β-Ala)-Sar 10 -Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Hcys (SEQ ID NO: 8);
(β-Ala)-Sar 10-Ala-Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:9)； (β-Ala)-Sar 10 -Ala-Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Hcys (SEQ ID NO:9);
(β-Ala)-Sar 10-Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:10)； (β-Ala)-Sar 10 -Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Hcys (SEQ ID NO: 10);
(β-Ala)-Sar 10-Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys(SEQ ID NO:11)； (β-Ala)-Sar 10 -Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Cys (SEQ ID NO: 11);
(β-Ala)-Sar 10-Ala-Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:12)； (β-Ala)-Sar 10 -Ala-Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Hcys (SEQ ID NO: 12);
(β-Ala)-Sar 10-Ala-Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys(SEQ ID NO:13)； (β-Ala)-Sar 10 -Ala-Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Cys (SEQ ID NO: 13);
(β-Ala)-Sar 10-Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys(SEQ ID NO:14)。 (β-Ala)-Sar 10 -Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Cys (SEQ ID NO: 14).
在一些具体实施方案中，所述化合物是具有以下结构之一的双环肽，在以下结构的N末端和/或C末端可选地含有其他氨基酸序列：In some embodiments, the compound is a bicyclic peptide having one of the following structures, optionally containing additional amino acid sequences at the N-terminus and/or C-terminus of the following structures:
Figure PCTCN2022131909-appb-000001
Figure PCTCN2022131909-appb-000001
其中，Xa 1、Xa 2或Xa 3残基上的-SH基团与分子支架共价结合形成硫醚键，因此，当Xa 1、Xa 2或Xa 3为Cys时，对应地，n 1、n 2或n 3为1； Wherein, the -SH group on the Xa 1 , Xa 2 or Xa 3 residue is covalently bonded to the molecular scaffold to form a thioether bond. Therefore, when Xa 1 , Xa 2 or Xa 3 is Cys, correspondingly, n 1 , n 2 or n 3 is 1;
当Xa 1、Xa 2或Xa 3为Hcys，对应地，n 1、n 2或n 3为2。 When Xa 1 , Xa 2 or Xa 3 is Hcys, correspondingly, n 1 , n 2 or n 3 is 2.
在一些具体实施方案中，式I-1或I的双环肽，其中：In some specific embodiments, the bicyclic peptide of formula I-1 or I, wherein:
Xa 1、Xa 2、Xa 3为Hcys，n 1、n 2、n 3为2；或 Xa 1 , Xa 2 , Xa 3 are Hcys, n 1 , n 2 , n 3 are 2; or
Xa 1、Xa 3为Hcys，Xa 2为Cys，n 1、n 3为2，n 2为1；或 Xa 1 and Xa 3 are Hcys, Xa 2 is Cys, n 1 and n 3 are 2, and n 2 is 1; or
Xa 1、Xa 2为Cys，Xa 3为Hcys，n 1、n 2为1，n 3为2。 Xa 1 and Xa 2 are Cys, Xa 3 is Hcys, n 1 and n 2 are 1, and n 3 is 2.
在一些具体实施方案中，所述化合物选自以下结构之一：In some embodiments, the compound is selected from one of the following structures:
Figure PCTCN2022131909-appb-000002
Figure PCTCN2022131909-appb-000002
本发明的包含两个多肽环结构的化合物，具有蛋白稳定性，对血浆蛋白酶、上皮蛋白酶、胃和肠蛋白酶、肺表面蛋白酶、细胞内蛋白酶等稳定；对MT1-MMP具有特异靶向性，是特异于MT1-MMP的肽配体；具有较长的血浆半衰期，具有好的药代动力学和药效动力学特性。The compound containing two polypeptide ring structures of the present invention has protein stability and is stable to plasma protease, epithelial protease, gastric and intestinal protease, lung surface protease, intracellular protease, etc.; has specific targeting to MT1-MMP, and is Peptide ligand specific to MT1-MMP; has long plasma half-life, good pharmacokinetic and pharmacodynamic properties.
本发明还提供了所述的化合物作为MT1-MMP的配体在筛选和/或制备药物中的应用。The present invention also provides the use of the compound as a ligand of MT1-MMP in screening and/or preparing drugs.
本发明还提供了一种药物缀合物或其药学上可接受的盐，所述缀合物包含通过连接子缀合至一个或多个效应物和/或官能团的如前所述的化合物。The present invention also provides a drug conjugate or a pharmaceutically acceptable salt thereof, said conjugate comprising a compound as described above conjugated to one or more effectors and/or functional groups via a linker.
在一些具体实施方案中，其中所述效应物为细胞毒性剂。In some embodiments, wherein said effector is a cytotoxic agent.
在一些具体实施方案中，所述细胞毒性剂选自如下组中的一种或多种：烷化剂类、抗代谢物类、植物生物碱类、萜类化合物类、鬼臼毒素类及其衍生物、紫杉烷类及其衍生物、拓扑异构酶抑制剂、抗肿瘤抗生素类。In some specific embodiments, the cytotoxic agent is selected from one or more of the following group: alkylating agents, antimetabolites, plant alkaloids, terpenoids, podophyllotoxins, and Derivatives, taxanes and their derivatives, topoisomerase inhibitors, antitumor antibiotics.
在一些具体实施方案中，所述细胞毒性剂选自如下组中的一种或多种：顺铂、卡铂、奥沙利铂、氮芥、环磷酰胺、苯丁酸氮芥、异环磷酰胺、硫唑嘌呤、巯嘌呤、嘧啶类似物、长 春新碱、长春花碱、长春瑞滨、长春地辛、依托泊苷、替尼泊苷、紫杉醇、喜树碱、伊立替康、拓扑替康、安丫啶、依托泊苷、磷酸依托泊苷、替尼泊苷、放线菌素D、多柔比星、表柔比星、埃博霉素及其衍生物、博来霉素及其衍生物、更生霉素及其衍生物、普卡霉素及其衍生物、丝裂霉素C、刺胞霉素、美登素及其衍生物、奥瑞他汀及其衍生物。In some specific embodiments, the cytotoxic agent is selected from one or more of the following group: cisplatin, carboplatin, oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, isocycline Phosphoramide, azathioprine, mercaptopurine, pyrimidine analogues, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, paclitaxel, camptothecin, irinotecan, toposide Tecan, Amyridine, Etoposide, Etoposide Phosphate, Teniposide, Actinomycin D, Doxorubicin, Epirubicin, Epothilone and its derivatives, Bleomycin and its derivatives, dactinomycin and its derivatives, plicamycin and its derivatives, mitomycin C, cyspamicin, maytansine and its derivatives, auristatin and its derivatives.
在一些具体实施方案中，所述细胞毒性剂选自美登木素生物碱、单甲基奥瑞他汀、喜树碱衍生物中的至少一种。In some specific embodiments, the cytotoxic agent is at least one selected from maytansinoids, monomethyl auristatin, and camptothecin derivatives.
在一些具体实施方案中，所述细胞毒性剂选自美登素DM1(DM1)、美登素DM4(DM4)、一甲基奥瑞他汀E(MMAE)、7-乙基-10-羟基喜树碱(SN38)中的至少一种。In some embodiments, the cytotoxic agent is selected from the group consisting of maytansine DM1 (DM1), maytansine DM4 (DM4), monomethyl auristatin E (MMAE), 7-ethyl-10-hydroxyl At least one of the resins (SN38).
在一些具体实施方案中，所述连接子为肽类连接子、二硫化物连接子或pH依赖型连接子；这些连接子在一定条件下可裂解以释放出效应物分子。In some embodiments, the linker is a peptide linker, a disulfide linker, or a pH-dependent linker; these linkers are cleavable under certain conditions to release the effector molecule.
在一些具体实施方案中，所述二硫化物连接子选自DMDS、MDS、DSDM、NDMDS或式II结构：In some embodiments, the disulfide linker is selected from DMDS, MDS, DSDM, NDMDS or the structure of formula II:
Figure PCTCN2022131909-appb-000003
Figure PCTCN2022131909-appb-000003
其中，R 1、R 2、R 3和R 4独立地选自H、甲基、乙基、丙基和异丙基； Wherein, R 1 , R 2 , R 3 and R 4 are independently selected from H, methyl, ethyl, propyl and isopropyl;
p和q独立地为1、2、3、4或5；p and q are independently 1, 2, 3, 4 or 5;
所述肽类连接子选自：-Cit-Val-、-Phe-Lys-和-Val-Lys-；The peptide linker is selected from: -Cit-Val-, -Phe-Lys- and -Val-Lys-;
所述pH依赖型连接子选自顺乌头酸酐。The pH-dependent linker is selected from cis-aconitic anhydride.
上述连接子主要起连接毒性剂和肽配体，以及在特定条件下裂解释放毒性物的作用，为控制裂解的速率和伴随的效应物分子的释放，连接子可做适当修饰，如在其与肽配体或效应物连接处连接一些基团增加链长度，以及在裂解键周围增加基团修饰控制裂解键的阻碍，本发明的连接子包括基于上述加以修饰的连接子衍生物。The above-mentioned linker is mainly used to connect the toxic agent and the peptide ligand, and to release the toxic substance by cleavage under specific conditions. In order to control the rate of cleavage and the release of the accompanying effector molecule, the linker can be appropriately modified, such as in the Peptide ligands or effectors connect some groups to increase the chain length, and increase group modification around the cleavage bond to control the hindrance of the cleavage bond. The linker of the present invention includes linker derivatives modified based on the above.
本发明中，连接子理论上可以与肽配体的N末端、C末端和/或分子支架相连。当与C末端或分子支架相连时，可在C末端或分子支架上修饰出一个与其链接的官能团。In the present invention, the linker can theoretically be connected to the N-terminal, C-terminal and/or molecular scaffold of the peptide ligand. When linked to the C-terminal or molecular scaffold, a functional group linked to it can be modified on the C-terminal or molecular scaffold.
在一些具体实施方案中，所述连接子为-PABC-Cit-Val-戊二酰-或-PABC-环丁基-Ala-Cit-βAla-，其中PABC代表p-氨基苄基氨基甲酸酯。In some specific embodiments, the linker is -PABC-Cit-Val-glutaryl- or -PABC-cyclobutyl-Ala-Cit-βAla-, where PABC stands for p-aminobenzylcarbamate .
在一些具体实施方案中，所述连接子为In some specific embodiments, the linker is
Figure PCTCN2022131909-appb-000004
Figure PCTCN2022131909-appb-000004
其中k选自1-20的任一整数。Wherein k is selected from any integer of 1-20.
在一些具体实施方案中，所述连接子为
Figure PCTCN2022131909-appb-000005
在一些具体实施方案中，所述连接子为
Figure PCTCN2022131909-appb-000006
在一些具体实施方案中，所述连接子为
Figure PCTCN2022131909-appb-000007
在各自的实施方案中，k选自1-20的任一整数；在一些具体实施方案中，k选自1-10的任一整数；在一些具体实施方案中，k选自1、2、3、4或5；在一些具体实施方案中，k选自2。 In some specific embodiments, the linker is
Figure PCTCN2022131909-appb-000005
In some specific embodiments, the linker is
Figure PCTCN2022131909-appb-000006
In some specific embodiments, the linker is
Figure PCTCN2022131909-appb-000007
In respective embodiments, k is selected from any integer of 1-20; in some specific embodiments, k is selected from any integer of 1-10; in some specific embodiments, k is selected from 1, 2, 3, 4, or 5; in some embodiments, k is selected from 2.
在一些具体实施方案中，所述药物缀合物具有式III、式IV、式V或式VI的结构：In some embodiments, the drug conjugate has the structure of Formula III, Formula IV, Formula V, or Formula VI:
Figure PCTCN2022131909-appb-000008
Figure PCTCN2022131909-appb-000008
其中，R 1、R 2、R 3和R 4独立地选自H、甲基、乙基、丙基和异丙基； Wherein, R 1 , R 2 , R 3 and R 4 are independently selected from H, methyl, ethyl, propyl and isopropyl;
p和q独立地为1、2、3、4或5；p and q are independently 1, 2, 3, 4 or 5;
k独立地为1-10的任一整数；k is independently any integer of 1-10;
Xa 1、Xa 2、Xa 3独立地为Cys或Hcys残基，且至少有一个是Hcys残基； Xa 1 , Xa 2 , and Xa 3 are independently Cys or Hcys residues, and at least one of them is a Hcys residue;
当Xa 1、Xa 2或Xa 3为Cys时，对应地，n 1、n 2或n 3为1； When Xa 1 , Xa 2 or Xa 3 is Cys, correspondingly, n 1 , n 2 or n 3 is 1;
当Xa 1、Xa 2或Xa 3为Hcys，对应地，n 1、n 2或n 3为2。 When Xa 1 , Xa 2 or Xa 3 is Hcys, correspondingly, n 1 , n 2 or n 3 is 2.
在一些具体实施方案中，所述药物缀合物或其药学上可接受的盐，其中：In some specific embodiments, the drug conjugate or a pharmaceutically acceptable salt thereof, wherein:
R 1、R 2、R 3和R 4独立地选自H或甲基； R 1 , R 2 , R 3 and R 4 are independently selected from H or methyl;
p和q独立地为1或2；p and q are independently 1 or 2;
k独立地为1-5的任一整数；且k is independently any integer from 1 to 5; and
Xa 1、Xa 2、Xa 3为Hcys，n 1、n 2、n 3为2；或 Xa 1 , Xa 2 , Xa 3 are Hcys, n 1 , n 2 , n 3 are 2; or
Xa 1、Xa 3为Hcys，Xa 2为Cys，n 1、n 3为2，n 2为1；或 Xa 1 and Xa 3 are Hcys, Xa 2 is Cys, n 1 and n 3 are 2, and n 2 is 1; or
Xa 1、Xa 2为Cys，Xa 3为Hcys，n 1、n 2为1，n 3为2。 Xa 1 and Xa 2 are Cys, Xa 3 is Hcys, n 1 and n 2 are 1, and n 3 is 2.
作为本发明的更具体的技术方案，一种药物缀合物或其药学上可接受的盐，所述药物缀合物选自以下结构之一：As a more specific technical solution of the present invention, a drug conjugate or a pharmaceutically acceptable salt thereof, the drug conjugate is selected from one of the following structures:
Figure PCTCN2022131909-appb-000009
Figure PCTCN2022131909-appb-000009
Figure PCTCN2022131909-appb-000010
Figure PCTCN2022131909-appb-000010
Figure PCTCN2022131909-appb-000011
Figure PCTCN2022131909-appb-000011
本发明还涉及一种药物组合物，其含前述本发明所述的化合物(即肽配体，或称肽化合物)和/或前述本发明所述的药物缀合物或其药学上可接受的盐，以及药学上可接受的载体和/或赋形剂。The present invention also relates to a pharmaceutical composition, which contains the aforementioned compound of the present invention (i.e. peptide ligand, or peptide compound) and/or the aforementioned drug conjugate of the present invention or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier and/or excipient.
本发明还涉及一种用途，前述本发明所述的化合物，或者前述本发明所述的药物缀合物或其药学上可接受的盐，或者其组合物在制备预防和/或治疗过度表达MT1-MMP的疾病的药物中的用途。The present invention also relates to a use of the aforementioned compound of the present invention, or the aforementioned drug conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a composition thereof in the preparation of preventing and/or treating overexpression of MT1 - Use of MMPs in medicine for diseases.
所述过度表达MT1-MMP的疾病选自以下疾病中的至少一种：神经母细胞瘤、小细胞肺癌、膀胱癌、胃癌、结肠癌、肝癌。The disease overexpressing MT1-MMP is selected from at least one of the following diseases: neuroblastoma, small cell lung cancer, bladder cancer, gastric cancer, colon cancer, liver cancer.
本发明还涉及一种药物组合物或药物制剂，所述的药物组合物或药物制剂包含选1-1500mg的权利要求前述本发明所述的肽化合物或前述本发明所述的缀合物或其药学上可接受的盐，以及药学上可接受的载体和/或赋形剂。The present invention also relates to a pharmaceutical composition or pharmaceutical preparation, said pharmaceutical composition or pharmaceutical preparation comprising 1-1500 mg of the peptide compound described in the present invention or the conjugate described in the present invention or its A pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier and/or excipient.
本发明还涉及一种用于治疗哺乳动物或人的疾病的方法，所述方法包括给予受试者治疗有效量的前述本发明所述的肽化合物或前述本发明所述的缀合物或其药学上可接受的盐，治疗有效量优选为1-1500mg，所述的疾病优选为神经母细胞瘤、小细胞肺癌、膀胱癌、胃癌、结肠癌、肝癌中的至少一种。The present invention also relates to a method for treating diseases in mammals or humans, the method comprising administering to a subject a therapeutically effective amount of the aforementioned peptide compound of the present invention or the aforementioned conjugate of the present invention or its For pharmaceutically acceptable salts, the therapeutically effective dose is preferably 1-1500 mg, and the disease is preferably at least one of neuroblastoma, small cell lung cancer, bladder cancer, gastric cancer, colon cancer, and liver cancer.
本发明还提供一种组合物或药物制剂，其中含有前述本发明所述的肽化合物、缀合物或其药学上可接受的盐，以及药学上可接受的载体和/或辅料。该药物组合物可以为单位制剂形式(单位制剂也被称为“制剂规格”)。The present invention also provides a composition or pharmaceutical preparation, which contains the aforementioned peptide compound, conjugate or pharmaceutically acceptable salt thereof of the present invention, and a pharmaceutically acceptable carrier and/or adjuvant. The pharmaceutical composition may be in unit dosage form (a unit dosage is also referred to as a "dosage strength").
进一步地，本发明的组合物或药物制剂，其中含有1-1500mg的前述本发明所述的肽化合物、缀合物或其药学上可接受的盐，以及药学上可接受的载体和/或辅料。Furthermore, the composition or pharmaceutical preparation of the present invention contains 1-1500 mg of the aforementioned peptide compound, conjugate or pharmaceutically acceptable salt thereof of the present invention, and pharmaceutically acceptable carriers and/or excipients .
本发明还提供了前述本发明所述的肽化合物、缀合物或其药学上可接受的盐在制备预防和***个体患病组织中过度表达MT1-MMP的疾病或病症的药物中的用途。进一步地，所述过度表达MT1-MMP的疾病或病症优选为神经母细胞瘤、小细胞肺癌、膀胱癌、胃癌、结肠癌、肝癌中的至少一种。优选地，所述个体为哺乳动物或人。The present invention also provides the use of the aforementioned peptide compounds, conjugates or pharmaceutically acceptable salts thereof in the preparation of drugs for the prevention and treatment of diseases or disorders in which MT1-MMP is overexpressed in diseased tissues of tumor individuals . Further, the disease or disease overexpressing MT1-MMP is preferably at least one of neuroblastoma, small cell lung cancer, bladder cancer, gastric cancer, colon cancer, and liver cancer. Preferably, said individual is a mammal or a human.
本发明还提供了一种用于治疗哺乳动物或人的疾病的方法，所述方法包括给予受试者治疗有效量的前述本发明所示的肽化合物、缀合物或其药学上可接受的盐，所述疾病优选为神经母细胞瘤、小细胞肺癌、膀胱癌、胃癌、结肠癌、肝癌中的至少一种，优选所述治疗有效量为1-1500mg。The present invention also provides a method for treating diseases in mammals or humans, the method comprising administering to a subject a therapeutically effective amount of the aforementioned peptide compounds, conjugates or pharmaceutically acceptable ones shown in the present invention salt, the disease is preferably at least one of neuroblastoma, small cell lung cancer, bladder cancer, gastric cancer, colon cancer, and liver cancer, and preferably the therapeutically effective dose is 1-1500 mg.
本申请中所述“有效量”或“治疗有效量”是指给予足够量的本申请公开的化合物，其将在某种程度上缓解所治疗的疾病或病症的一种或多种症状。在一些实施方案中，结果是减少和/或缓和疾病的体征、症状或原因，或生物***的任何其它希望改变。例如，针对治疗用途的“有效量”是提供临床上显著的疾病症状降低所需的包含本申请公开的肽化合物、缀合物或其药学上可接受的盐的组合物的量。治疗有效量的实例包括但不限于1-1500mg、1-1400mg、1-1300mg、1-1200mg、1-1000mg、1-900mg、1-800mg、1-700mg、1-600mg、1-500mg、1-400mg、1-300mg、1-250mg、1-200mg、1-150mg、1-125mg、1-100mg、1-80mg、1-60mg、1-50mg、1-40mg、1-25mg、1-20mg、5-1500mg、5-1000mg、5-900mg、5- 800mg、5-700mg、5-600mg、5-500mg、5-400mg、5-300mg、5-250mg、5-200mg、5-150mg、5-125mg、5-100mg、5-90mg、5-70mg、5-80mg、5-60mg、5-50mg、5-40mg、5-30mg、5-25mg、5-20mg、10-1500mg、10-1000mg、10-900mg、10-800mg、10-700mg、10-600mg、10-500mg、10-450mg、10-400mg、10-300mg、10-250mg、10-200mg、10-150mg、10-125mg、10-100mg、10-90mg、10-80mg、10-70mg、10-60mg、10-50mg、10-40mg、10-30mg、10-20mg；20-1500mg、20-1000mg、20-900mg、20-800mg、20-700mg、20-600mg、20-500mg、20-400mg、20-350mg、20-300mg、20-250mg、20-200mg、20-150mg、20-125mg、20-100mg、20-90mg、20-80mg、20-70mg、20-60mg、20-50mg、20-40mg、20-30mg；50-1500mg、50-1000mg、50-900mg、50-800mg、50-700mg、50-600mg、50-500mg、50-400mg、50-300mg、50-250mg、50-200mg、50-150mg、50-125mg、50-100mg；100-1500mg、100-1000mg、100-900mg、100-800mg、100-700mg、100-600mg、100-500mg、100-400mg、100-300mg、100-250mg、100-200mg。"Effective amount" or "therapeutically effective amount" in the present application refers to the administration of a sufficient amount of the compound disclosed in the present application, which will alleviate to some extent one or more symptoms of the disease or disorder being treated. In some embodiments, the result is reduction and/or alleviation of signs, symptoms or causes of disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a peptide compound disclosed herein, a conjugate, or a pharmaceutically acceptable salt thereof, required to provide a clinically significant reduction in disease symptoms. Examples of therapeutically effective amounts include, but are not limited to, 1-1500 mg, 1-1400 mg, 1-1300 mg, 1-1200 mg, 1-1000 mg, 1-900 mg, 1-800 mg, 1-700 mg, 1-600 mg, 1-500 mg, 1 -400mg, 1-300mg, 1-250mg, 1-200mg, 1-150mg, 1-125mg, 1-100mg, 1-80mg, 1-60mg, 1-50mg, 1-40mg, 1-25mg, 1-20mg , 5-1500mg, 5-1000mg, 5-900mg, 5-800mg, 5-700mg, 5-600mg, 5-500mg, 5-400mg, 5-300mg, 5-250mg, 5-200mg, 5-150mg, 5 -125mg, 5-100mg, 5-90mg, 5-70mg, 5-80mg, 5-60mg, 5-50mg, 5-40mg, 5-30mg, 5-25mg, 5-20mg, 10-1500mg, 10-1000mg ,10-900mg, 10-800mg, 10-700mg, 10-600mg, 10-500mg, 10-450mg, 10-400mg, 10-300mg, 10-250mg, 10-200mg, 10-150mg, 10-125mg, 10 -100mg, 10-90mg, 10-80mg, 10-70mg, 10-60mg, 10-50mg, 10-40mg, 10-30mg, 10-20mg; 20-1500mg, 20-1000mg, 20-900mg, 20-800mg , 20-700mg, 20-600mg, 20-500mg, 20-400mg, 20-350mg, 20-300mg, 20-250mg, 20-200mg, 20-150mg, 20-125mg, 20-100mg, 20-90mg, 20 -80mg, 20-70mg, 20-60mg, 20-50mg, 20-40mg, 20-30mg; 50-1500mg, 50-1000mg, 50-900mg, 50-800mg, 50-700mg, 50-600mg, 50-500mg , 50-400mg, 50-300mg, 50-250mg, 50-200mg, 50-150mg, 50-125mg, 50-100mg; 100-1500mg, 100-1000mg, 100-900mg, 100-800mg, 100-700mg, 100 - 600mg, 100-500mg, 100-400mg, 100-300mg, 100-250mg, 100-200mg.
在一些实施方案中，本发明的药物组合物或制剂含有上述治疗有效量的本发明所述肽化合物、缀合物或其药学上可接受的盐。In some embodiments, the pharmaceutical composition or preparation of the present invention contains the aforementioned therapeutically effective amount of the peptide compound, conjugate or pharmaceutically acceptable salt thereof of the present invention.
本发明涉及一种药物组合物或药物制剂，所述的药物组合物或药物制剂包含治疗有效量的本发明所述的肽化合物、缀合物或其药学上可接受的盐以及载体和/或赋形剂。该药物组合物可以为单位制剂形式(单位制剂中主药的量也被称为“制剂规格”)。在一些实施方案中，该药物组合物包括但不限于1mg、1.25mg、2.5mg、5mg、10mg、12.5mg、15mg、20mg、25mg、30mg、35mg、40mg、45mg、50mg、55mg、60mg、65mg、70mg、75mg、80mg、85mg、90mg、95mg、100mg、110mg、120mg、125mg、130mg、140mg、150mg、160mg、170mg、180mg、190mg、200mg、210mg、220mg、230mg、240mg、250mg、275mg、300mg、325mg、350mg、375mg、400mg、425mg、450mg、475mg、500mg、525mg、550mg、575mg、600mg、625mg、650mg、675mg、700mg、725mg、750mg、775mg、800mg、850mg、900mg、950mg、1000mg、1100mg、1200mg、1300mg、1400mg、1500mg的本发明的肽化合物、缀合物或其药学上可接受的盐。The present invention relates to a pharmaceutical composition or pharmaceutical preparation, which comprises a therapeutically effective amount of the peptide compound, conjugate or pharmaceutically acceptable salt thereof and a carrier and/or excipient. The pharmaceutical composition may be in the form of a unit preparation (the amount of the main drug in the unit preparation is also referred to as "preparation specification"). In some embodiments, the pharmaceutical composition includes, but is not limited to, 1 mg, 1.25 mg, 2.5 mg, 5 mg, 10 mg, 12.5 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg ,70mg,75mg,80mg,85mg,90mg,95mg,100mg,110mg,120mg,125mg,130mg,140mg,150mg,160mg,170mg,180mg,190mg,200mg,210mg,220mg,230mg,240mg,250mg,275mg,300mg . , 1100mg , 1200mg, 1300mg, 1400mg, 1500mg of the peptide compound, conjugate or pharmaceutically acceptable salt thereof of the present invention.
在一些实施方案中，本发明提供一种用于治疗哺乳动物或人的疾病的方法，所述方法包括给予受试者治疗有效量的本发明肽化合物、缀合物或其药学上可接受的盐，以及药学上可接受的载体和/或赋形剂，治疗有效量优选1-1500mg，所述的疾病优选为神经母细胞瘤、小细胞肺癌、膀胱癌、胃癌、结肠癌、肝癌中的至少一种。In some embodiments, the present invention provides a method for treating a disease in a mammal or a human, the method comprising administering to a subject a therapeutically effective amount of a peptide compound of the present invention, a conjugate, or a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier and/or excipient, the therapeutically effective dose is preferably 1-1500 mg, and the disease is preferably neuroblastoma, small cell lung cancer, bladder cancer, gastric cancer, colon cancer, liver cancer at least one.
在一些实施方案中，本发明提供一种用于治疗哺乳动物或人的疾病的方法所述方法包括，将药物本发明的肽化合物、缀合物或其药学上可接受的盐，以及药学上可接受的载体和/或赋形剂，以1-1500mg/天的日剂量给予受试者，所述日剂量可以为单剂量或分剂量，在一些实施方案中，日剂量包括但不限于10-1500mg/天、20-1500mg/天、25-1500mg/天、50-1500mg/天、75-1500mg/天、100-1500mg/天、200-1500mg/天、10-1000mg/天、20-1000mg/天、25-1000mg/天、50-1000mg/天、75-1000mg/天、100-1000mg/天、200-1000mg/天、25-800mg/天、50-800mg/天、100-800mg/天、200-800mg/天、25-400mg/天、50-400mg/天、100-400mg/天、200-400mg/天，在一些实施方案中，日剂量包括但不限于1mg/天、5mg/天、10mg/天、20mg/天、25mg/天、50mg/天、75mg/天、100mg/天、125mg/天、150mg/天、200mg/天、300mg/天、400mg/天、600mg/天、800mg/天、1000mg/天、1200mg/天、1400mg/天、1500mg/天。In some embodiments, the present invention provides a method for treating a disease in a mammal or a human. The method comprises administering the peptide compound, the conjugate or a pharmaceutically acceptable salt thereof of the present invention, and a pharmaceutically Acceptable carriers and/or excipients are given to the subject at a daily dose of 1-1500 mg/day, and the daily dose can be a single dose or divided doses. In some embodiments, the daily dose includes but is not limited to 10 -1500mg/day, 20-1500mg/day, 25-1500mg/day, 50-1500mg/day, 75-1500mg/day, 100-1500mg/day, 200-1500mg/day, 10-1000mg/day, 20-1000mg /day, 25-1000mg/day, 50-1000mg/day, 75-1000mg/day, 100-1000mg/day, 200-1000mg/day, 25-800mg/day, 50-800mg/day, 100-800mg/day , 200-800mg/day, 25-400mg/day, 50-400mg/day, 100-400mg/day, 200-400mg/day, in some embodiments, the daily dosage includes but not limited to 1mg/day, 5mg/day , 10mg/day, 20mg/day, 25mg/day, 50mg/day, 75mg/day, 100mg/day, 125mg/day, 150mg/day, 200mg/day, 300mg/day, 400mg/day, 600mg/day, 800mg /day, 1000mg/day, 1200mg/day, 1400mg/day, 1500mg/day.
本发明还提供一种试剂盒，该试剂盒可以包括单剂量或多剂量形式的组合物，该试剂盒包含本发明的化合物、缀合物或其药学上可接受的盐，本发明的化合物、缀合物或者其立体异构体或药学上可接受的盐量与上述药物组合物中其量相同。The present invention also provides a kit, which may include a composition in single dose or multi-dose form, the kit comprising a compound of the present invention, a conjugate or a pharmaceutically acceptable salt thereof, a compound of the present invention, a conjugate, or a pharmaceutically acceptable salt thereof. The amount of the conjugate or its stereoisomer or pharmaceutically acceptable salt is the same as that in the above pharmaceutical composition.
本发明中所述化合物、缀合物或者其立体异构体或药学上可接受的盐的量在每种情况下以游离碱的形式换算。The amounts of the compounds, conjugates or stereoisomers or pharmaceutically acceptable salts thereof described in the present invention are in each case calculated in the form of the free base.
“制剂规格”是指每一支、片或其他每一个单位制剂中含有主药的重量。"Preparation specification" refers to the weight of the main drug contained in each tube, tablet or other unit preparation.
合成synthesis
本领域技术人员可以结合已知的有机合成技术制备本发明的化合物，其起始原料为市售化学品和(或)化学文献中所述的化合物。“市售化学品”是从正规商业来源获得的，供应商包括：泰坦科技、安耐吉化学、上海德默、成都科龙化工、韶远化学科技、南京药石、药明康德和百灵威科技等公司。Those skilled in the art can prepare the compounds of the present invention by combining known organic synthesis techniques, starting from commercially available chemicals and/or compounds described in the chemical literature. "Commercially available chemicals" are obtained from formal commercial sources, suppliers include: Titan Technology, Anaiji Chemical, Shanghai Demo, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, Nanjing Yaoshi, WuXi AppTec and Bailingwei Technology, etc. company.
本发明的肽可以通过标准技术合成制备，然后在体外与分子支架反应。当进行此操作时，可以使用标准化学方法。这使得能够快速大规模制备可溶性材料用于进一步的下游实验或验证。这样的方法可以使用例如Timmerman等人(2005，ChemBioChem)中公开的常规化学方法来实现。The peptides of the invention can be prepared synthetically by standard techniques and then reacted with molecular scaffolds in vitro. When doing this, standard chemistry can be used. This enables rapid large-scale preparation of soluble materials for further downstream experiments or validation. Such methods can be achieved using conventional chemistry as disclosed in, for example, Timmerman et al. (2005, ChemBioChem).
一些实施方式中，本发明的药物缀合物按以下路线合成：In some embodiments, the drug conjugates of the present invention are synthesized according to the following route:
路线1：Route 1:
Figure PCTCN2022131909-appb-000012
Figure PCTCN2022131909-appb-000012
路线2：Route 2:
Figure PCTCN2022131909-appb-000013
Figure PCTCN2022131909-appb-000013
更具体的合成步骤参见实施例。For more specific synthesis steps, refer to the examples.
术语the term
肽配体是指肽共价结合至分子支架所形成的含有氨基酸序列(肽结构)的化合物。本发明中可作为膜型基质金属蛋白酶-1(MT1-MMP)的配体。通常，形成此类化合物的肽包含能够与支架形成共价键(如硫醚键)的两个或更多个反应性基团(即半胱氨酸残基或高半胱氨酸残基)、以及在所述反应性基团之间相对的序列(称为环序列)，之所以称为环序列，是因为当肽与支架结合时，其形成环。在本申请的情况下中，肽包含至少三个半胱氨酸残基或高半胱氨酸残基(Cys残基或Hcys残基)，其在支架上形成两个环。A peptide ligand refers to a compound containing an amino acid sequence (peptide structure) formed by covalently binding a peptide to a molecular scaffold. In the present invention, it can be used as the ligand of membrane-type matrix metalloproteinase-1 (MT1-MMP). Typically, the peptides forming such compounds contain two or more reactive groups (i.e. cysteine residues or homocysteine residues) capable of forming covalent bonds (e.g. thioether bonds) with the scaffold , and the opposing sequence between the reactive groups, called the loop sequence, is called a loop sequence because when the peptide binds to the scaffold, it forms a loop. In the case of the present application, the peptide comprises at least three cysteine residues or homocysteine residues (Cys residues or Hcys residues), which form two loops on the scaffold.
分子支架包括芳香族分子支架。芳香族分子支架是指本文定义的任何包含芳香族碳环或芳香族杂环环体系的分子支架。适当的芳香族分子支架的示例描述于Heinis等人(2014)Angewandte Chemie,International Edition 53(6)1602-1606中。分子支架可以是小分子，例如有机小分子。Molecular scaffolds include aromatic molecular scaffolds. An aromatic molecular scaffold refers to any molecular scaffold as defined herein comprising an aromatic carbocyclic or aromatic heterocyclic ring system. Examples of suitable aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chemie, International Edition 53(6) 1602-1606. Molecular scaffolds can be small molecules, such as small organic molecules.
多肽是指三个及以上氨基酸分子以肽键连接在一起而形成的化合物。多肽中的氨基酸单位称为氨基酸残基。Polypeptide refers to a compound formed by three or more amino acid molecules linked together by peptide bonds. The unit of amino acid in a polypeptide is called an amino acid residue.
效应物和/或官能团是可以(通过连接子)连接于例如多肽的N和/或C末端、多肽内的氨基酸、或分子骨架的、具有药理作用或特定功能的分子或片段。合适的效应物和/或官能团包括抗体及其部分或片段、细胞毒分子或片段、酶抑制剂分子或片段、金属螯合剂等。在一些情形中，效应物和/或官能团是药物。特别地是细胞毒性剂，包括烷化剂类、抗代谢物类、植物生物碱类、萜类化合物类、鬼臼毒素类及其衍生物、紫杉烷类及其衍生物、拓扑异构酶抑制剂、抗肿瘤抗生素类等。更具体地是顺铂、卡铂、奥沙利铂、氮芥、环磷酰胺、苯丁酸氮芥、异环磷酰胺、硫唑嘌呤、巯嘌呤、嘧啶类似物、长春新碱、长春花碱、长春瑞滨、长春地辛、依托泊苷、替尼泊苷、紫杉醇、喜树碱、伊立替康、拓扑替康、安丫啶、依托泊苷、磷酸依托泊苷、替尼泊苷、放线菌素D、多柔比星、表柔比星、埃博霉素及其衍生 物、博来霉素及其衍生物、更生霉素及其衍生物、普卡霉素及其衍生物、丝裂霉素C、刺胞霉素、美登素及其衍生物、奥瑞他汀及其衍生物。Effectors and/or functional groups are pharmacologically or functionally specific molecules or fragments that can be attached (via a linker) to, for example, the N- and/or C-termini of a polypeptide, amino acids within a polypeptide, or the molecular backbone. Suitable effectors and/or functional groups include antibodies and parts or fragments thereof, cytotoxic molecules or fragments, enzyme inhibitor molecules or fragments, metal chelators, and the like. In some cases, the effector and/or functional group is a drug. Especially cytotoxic agents, including alkylating agents, antimetabolites, plant alkaloids, terpenoids, podophyllotoxins and their derivatives, taxanes and their derivatives, topoisomerases Inhibitors, antitumor antibiotics, etc. More specifically cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide, azathioprine, mercaptopurine, pyrimidine analogs, vincristine, vinca Alkaline, vinorelbine, vindesine, etoposide, teniposide, paclitaxel, camptothecin, irinotecan, topotecan, amyridine, etoposide, etoposide phosphate, teniposide , actinomycin D, doxorubicin, epirubicin, epothilone and its derivatives, bleomycin and its derivatives, dactinomycin and its derivatives, plicamycin and its derivatives substances, mitomycin C, cynistatin, maytansine and its derivatives, auristatin and its derivatives.
衍生物是指一种化合物中的氢原子或原子团被其他原子或原子团取代而衍生的产物。Derivatives refer to products derived from the substitution of hydrogen atoms or atomic groups in a compound by other atoms or atomic groups.
除非特别说明，所有氨基酸均以L-构型使用。Unless otherwise stated, all amino acids are used in the L-configuration.
Glu：谷氨酸Glu: glutamic acid
1-Nal：1-萘基丙氨酸1-Nal: 1-Naphthylalanine
tBu-Gly：叔丁基甘氨酸tBu-Gly: tert-butylglycine
β-Ala：β-丙氨酸β-Ala: β-alanine
Sar：肌氨酸Sar: sarcosine
Cys：半胱氨酸Cys: cysteine
Hcys：高半胱氨酸Hcys: homocysteine
Hyp：羟脯氨酸Hyp: Hydroxyproline
Asn：天冬酰胺Asn: Asparagine
Pro：脯氨酸Pro: Proline
Leu：亮氨酸Leu: Leucine
His：组氨酸His: Histidine
D-Asp：D-天冬氨酸D-Asp: D-Aspartic Acid
Asp：天冬氨酸Asp: aspartic acid
Trp：色氨酸Trp: tryptophan
hArg：高精氨酸hArg: Homoarginine
Ser：丝氨酸Ser: serine
Thr：苏氨酸Thr: threonine
Phe：苯丙氨酸Phe: Phenylalanine
Tyr：酪氨酸Tyr: Tyrosine
Val：缬氨酸Val: Valine
Ile：异亮氨酸Ile: Isoleucine
Lys：赖氨酸Lys: Lysine
Gly：甘氨酸Gly: Glycine
Cit：瓜氨酸Cit: Citrulline
美登木素生物碱类，如DM1、DM4，是一种细胞毒性剂，其是美登素的含巯基衍生物，DM1具有以下结构：Maytansinoids, such as DM1 and DM4, are cytotoxic agents, which are thiol-containing derivatives of maytansine. DM1 has the following structure:
Figure PCTCN2022131909-appb-000014
Figure PCTCN2022131909-appb-000014
DM4具有以下结构：DM4 has the following structure:
Figure PCTCN2022131909-appb-000015
Figure PCTCN2022131909-appb-000015
单甲基奥瑞他汀E(MMAE，又称为一甲基奥瑞他汀E)是合成的抗肿瘤剂，并具有以下结构：Monomethyl auristatin E (MMAE, also known as monomethyl auristatin E) is a synthetic antineoplastic agent and has the following structure:
Figure PCTCN2022131909-appb-000016
Figure PCTCN2022131909-appb-000016
SN38：7-乙基-10-羟基喜树碱SN38: 7-Ethyl-10-hydroxycamptothecin
DMDS：
Figure PCTCN2022131909-appb-000017
DMDS:
Figure PCTCN2022131909-appb-000017
MDS：
Figure PCTCN2022131909-appb-000018
MDS:
Figure PCTCN2022131909-appb-000018
DSDM：
Figure PCTCN2022131909-appb-000019
DSDM:
Figure PCTCN2022131909-appb-000019
SPDB：
Figure PCTCN2022131909-appb-000020
SPDB:
Figure PCTCN2022131909-appb-000020
NDMDS：
Figure PCTCN2022131909-appb-000021
NDMDS:
Figure PCTCN2022131909-appb-000021
TBMB：
Figure PCTCN2022131909-appb-000022
TBMB:
Figure PCTCN2022131909-appb-000022
本发明所述基团和化合物中所涉及的碳、氢、氧、硫、氮或卤素均包括它们的同位素，及本发明所述基团和化合物中所涉及的碳、氢、氧、硫、氮或卤素任选进一步被一个或多个它们对应的同位素所替代，其中碳的同位素包括 12C、 13C和 14C，氢的同位素包括氕(H)、氘(氘，又称为重氢)、氚(T，又称为超重氢)，氧的同位素包括 16O、 17O和 18O，硫的同位素包括 32S、 33S、 34S和 36S，氮的同位素包括 14N和 15N，氟的同位素 19F，氯的同位素包括 35Cl和 37Cl，溴的同位素包括 79Br和 81Br。 The carbon, hydrogen, oxygen, sulfur, nitrogen or halogens involved in the groups and compounds of the present invention include their isotopes, and the carbon, hydrogen, oxygen, sulfur, Nitrogen or halogen is optionally further replaced by one or more of their corresponding isotopes, wherein isotopes of carbon include 12 C, 13 C and 14 C, isotopes of hydrogen include protium (H), deuterium (deuterium, also known as deuterium ), tritium (T, also known as tritium), the isotopes of oxygen include 16 O, 17 O and 18 O, the isotopes of sulfur include 32 S, 33 S, 34 S and 36 S, and the isotopes of nitrogen include 14 N and 15 N, the isotope of fluorine is 19 F, the isotope of chlorine includes 35 Cl and 37 Cl, and the isotope of bromine includes 79 Br and 81 Br.
“药学上可接受的盐”是指本发明化合物保持游离酸或者游离碱的生物有效性和特性，且所述的游离酸通过与无毒的无机碱或者有机碱，所述的游离碱通过与无毒的无机酸或者有机酸反应获得的盐。"Pharmaceutically acceptable salt" means that the compound of the present invention maintains the biological effectiveness and characteristics of the free acid or free base, and the free acid is mixed with a non-toxic inorganic base or organic base, and the free base is mixed with a non-toxic inorganic base or organic base. Non-toxic salts obtained by reacting inorganic or organic acids.
“药物组合物”表示一种或多种本文所述化合物或其立体异构体、溶剂化物、药学上可接受的盐或共晶，与其他组成成分的混合物，其中其他组分包含生理学/药学上可接受的载体和/赋形剂。"Pharmaceutical composition" means a mixture of one or more compounds described herein, or stereoisomers, solvates, pharmaceutically acceptable salts or co-crystals thereof, with other constituents, wherein the other constituents comprise physiological/pharmaceutical acceptable carriers and/or excipients.
“载体”指的是：不会对生物体产生明显刺激且不会消除所给予化合物的生物活性和特性，并能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系，非限制性的实例包括微囊与微球、纳米粒、脂质体等。"Carrier" refers to: does not cause significant irritation to the organism and does not eliminate the biological activity and characteristics of the administered compound, and can change the way the drug enters the body and its distribution in the body, controls the release rate of the drug and releases the drug. Non-limiting examples of systems for delivery to targeted organs include microcapsules and microspheres, nanoparticles, liposomes, and the like.
“赋形剂”指的是：其本身并非治疗剂，用作稀释剂、辅料、粘合剂和/或媒介物，用于添加至药物组合物中以改善其处置或储存性质或允许或促进化合物或药物组合物形成用于给药的单位剂型。如本领域技术人员所已知的，药用赋形剂可提供各种功能且可描述为润湿剂、缓冲剂、助悬剂、润滑剂、乳化剂、崩解剂、吸收剂、防腐剂、表面活性剂、着色剂、矫味剂及甜味剂。药用赋形剂的实例包括但不限于：(1)糖，例如乳糖、葡萄糖及蔗糖；(2)淀粉，例如玉米淀粉及马铃薯淀粉；(3)纤维素及其衍生物，例如羧甲基纤维素钠、乙基纤维素、乙酸纤维素、羟丙基甲基纤维素、羟丙基纤维素、微晶纤维素及交联羧甲基纤维素(例如交联羧甲基纤维素钠)；(4)黄蓍胶粉；(5)麦芽；(6)明胶；(7)滑石；(8)赋形剂，例如可可脂及栓剂蜡；(9)油，例如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油及大豆 油；(10)二醇，例如丙二醇；(11)多元醇，例如甘油、山梨醇、甘露醇及聚乙二醇；(12)酯，例如油酸乙酯及月桂酸乙酯；(13)琼脂；(14)缓冲剂，例如氢氧化镁及氢氧化铝；(15)海藻酸；(16)无热原水；(17)等渗盐水；(18)林格溶液(Ringer’ssolution)；(19)乙醇；(20)pH缓冲溶液；(21)聚酯、聚碳酸酯和/或聚酐；及(22)其他用于药物制剂中的无毒相容物质。"Excipient" means: not itself a therapeutic agent, used as a diluent, adjuvant, binder and/or vehicle, added to a pharmaceutical composition to improve its handling or storage properties or to allow or facilitate The compound or pharmaceutical composition is presented in unit dosage form for administration. As known to those skilled in the art, pharmaceutical excipients can serve various functions and can be described as wetting agents, buffers, suspending agents, lubricants, emulsifiers, disintegrating agents, absorbing agents, preservatives , surfactants, coloring agents, flavoring agents and sweeteners. Examples of pharmaceutically acceptable excipients include, but are not limited to: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as carboxymethyl Sodium cellulose, ethyl cellulose, cellulose acetate, hydroxypropyl methylcellulose, hydroxypropyl cellulose, microcrystalline cellulose, and croscarmellose (such as croscarmellose sodium) (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, red Flower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as oil (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; ( 18) Ringer's solution (Ringer's solution); (19) ethanol; (20) pH buffer solution; (21) polyester, polycarbonate and/or polyanhydride; Toxic Compatible Substances.
具体实施方式Detailed ways
以下将通过实施例对本发明的内容进行详细描述。实施例中未注明具体条件的，按照常规条件的实验方法进行。所举实施例是为了更好地对本发明的内容进行说明，但并不能理解为本发明的内容仅限于所举实例。本领域常规技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。The content of the present invention will be described in detail below through examples. If specific conditions are not indicated in the embodiments, the experimental method of conventional conditions shall be carried out. The examples given are for better description of the content of the present invention, but it cannot be understood that the content of the present invention is limited to the examples given. Non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above content of the invention still belong to the protection scope of the present invention.
缩写abbreviation
Fmoc：9-芴基甲氧基羰基Fmoc: 9-fluorenylmethoxycarbonyl
Boc：叔丁氧羰基Boc: tert-butoxycarbonyl
HBTU：苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐HBTU: Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate
EEDQ：2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉EEDQ: 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline
HOBT：1-羟基苯并***HOBT: 1-Hydroxybenzotriazole
EDCI：1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDCI: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
化合物的制备Compound preparation
实施例1Example 1
Figure PCTCN2022131909-appb-000023
Figure PCTCN2022131909-appb-000023
第一至第四步：Steps 1 to 4:
Figure PCTCN2022131909-appb-000024
Figure PCTCN2022131909-appb-000024
将CTC树脂(75g，1.0mmol/g)和Fmoc-L-瓜氨酸(30.0g，75.4mmol，1.0eq)加入到二氯甲烷(600mL)中，再加入N,N-二异丙基乙胺(58.4g，453mmol，6.0eq)，反应3小时。抽滤，树脂用DMF洗两次，向树脂混合物中加入配好的20％哌啶/DMF的溶液，体系反应2小时，抽滤，树脂用DMF洗两次。向树脂混合物中依次加入DMF(600mL)，Boc-L-缬氨酸(48.0g，0.22mmol，3.0eq)和HBTU(85.0g，0.21mmol 2.85eq)和N,N-二异丙基乙胺(58.4g，453mmol，6.0eq)，体系反应3小时，抽滤，树脂用DMF洗三次，甲醇洗三次，二氯甲烷洗两次。向树脂混合物中加入配好的20％六氟异丙醇/二氯甲烷溶液，体系反应30分钟，抽滤，再重复一次前面操作。合并母液，浓缩至干，得到25.0g中间体4(四步收率＝88％)。CTC resin (75g, 1.0mmol/g) and Fmoc-L-citrulline (30.0g, 75.4mmol, 1.0eq) were added to dichloromethane (600mL), followed by N,N-diisopropylethyl Amine (58.4g, 453mmol, 6.0eq), reacted for 3 hours. Suction filtration, the resin was washed twice with DMF, the prepared 20% piperidine/DMF solution was added to the resin mixture, the system reacted for 2 hours, suction filtration, the resin was washed twice with DMF. Add DMF (600 mL), Boc-L-valine (48.0 g, 0.22 mmol, 3.0 eq) and HBTU (85.0 g, 0.21 mmol 2.85 eq) and N,N-diisopropylethylamine in sequence to the resin mixture (58.4g, 453mmol, 6.0eq), the system was reacted for 3 hours, filtered with suction, the resin was washed three times with DMF, three times with methanol, and twice with dichloromethane. Add the prepared 20% hexafluoroisopropanol/dichloromethane solution to the resin mixture, react the system for 30 minutes, filter with suction, and repeat the previous operation again. The mother liquors were combined and concentrated to dryness to obtain 25.0 g of intermediate 4 (yield over four steps = 88%).
LCMS m/z＝374.9[M+1] + LCMS m/z=374.9[M+1] +
第五步：the fifth step:
Figure PCTCN2022131909-appb-000025
Figure PCTCN2022131909-appb-000025
将中间体4(25g，66.7mmol)加入到二氯甲烷(250mL)和甲醇(250mL)中，再加入对氨基苯甲醇(9.84g，80.0mmol)和EEDQ(39.5g，80.0mmol)，室温搅拌16h，浓缩至干，柱层析纯化(DCM:MeOH＝10:1)，得到10g中间体5(收率＝31％)。Intermediate 4 (25g, 66.7mmol) was added to dichloromethane (250mL) and methanol (250mL), then p-aminobenzyl alcohol (9.84g, 80.0mmol) and EEDQ (39.5g, 80.0mmol) were added, and stirred at room temperature After 16 hours, it was concentrated to dryness and purified by column chromatography (DCM:MeOH=10:1) to obtain 10 g of intermediate 5 (yield=31%).
LCMS m/z＝480.1[M+1] + LCMS m/z=480.1[M+1] +
第六步：Step six:
Figure PCTCN2022131909-appb-000026
Figure PCTCN2022131909-appb-000026
将中间体5(10.0g，20.8mmol)加入到干燥的二氯甲烷(120mL)和干燥的四氢呋喃(60mL)中，氮气保护，再加入对硝基苯基氯甲酸酯(6.2g，31.2mmol)和吡啶(3.3g，41.6mmo l)，室温搅拌5h，过滤，母液浓缩至干，柱层析纯化(DCM:MeOH＝10:1)得到2.3g中间体6(收率＝16.6％)。Intermediate 5 (10.0g, 20.8mmol) was added to dry dichloromethane (120mL) and dry tetrahydrofuran (60mL), under nitrogen protection, then p-nitrophenyl chloroformate (6.2g, 31.2mmol ) and pyridine (3.3g, 41.6mmol), stirred at room temperature for 5h, filtered, the mother liquor was concentrated to dryness, and purified by column chromatography (DCM:MeOH=10:1) to obtain 2.3g of intermediate 6 (yield=16.6%).
LCMS m/z＝664.3[M+1] + LCMS m/z=664.3[M+1] +
第七步：Step seven:
Figure PCTCN2022131909-appb-000027
Figure PCTCN2022131909-appb-000027
将中间体6(2.1g,3.1mmol)和N,N-二异丙基乙胺(3.6g,31mmol)加入到DMF(40mL)中，氮气保护，室温搅拌10分钟，降温至0℃，再加入MMAE(购自成都华捷明生物科技 有限公司，2.0g，3.1mmol)和HOBT(0.38g，3.1mmol)，保温搅拌30分钟，移至室温搅拌18h，直接C18反相柱纯化(0.1％TFA)(H 2O:ACN＝50:50)，得到2.7g中间体7(收率＝70％)。 Intermediate 6 (2.1g, 3.1mmol) and N,N-diisopropylethylamine (3.6g, 31mmol) were added to DMF (40mL), under nitrogen protection, stirred at room temperature for 10 minutes, cooled to 0°C, and then Add MMAE (purchased from Chengdu Huajieming Biotechnology Co., Ltd., 2.0g, 3.1mmol) and HOBT (0.38g, 3.1mmol), keep stirring for 30 minutes, move to room temperature and stir for 18h, and directly purify on a C18 reverse-phase column (0.1% TFA) (H 2 O:ACN=50:50), yielding 2.7 g of intermediate 7 (yield=70%).
LCMS m/z＝1223.4[M+1] + LCMS m/z=1223.4[M+1] +
第八步：Step Eight:
Figure PCTCN2022131909-appb-000028
Figure PCTCN2022131909-appb-000028
向中间体7(2.0g，1.6mmol)中加入混合均匀的TFA/DCM＝1:10的混合液(34mL)，室温搅拌2h，25℃浓缩干溶剂，加入THF(110mL)和碳酸钾(2.26g，16mmol)，室温搅拌3h。浓缩至干，直接C 18反相柱纯化(0.1％TFA)(H 2O:ACN＝60:40)，得到1.4g中间体8(收率＝76.5％)。 To Intermediate 7 (2.0g, 1.6mmol) was added a well-mixed TFA/DCM=1:10 mixture (34mL), stirred at room temperature for 2h, concentrated the dry solvent at 25°C, added THF (110mL) and potassium carbonate (2.26 g, 16mmol), stirred at room temperature for 3h. Concentrate to dryness, and directly purify by C 18 reverse phase column (0.1% TFA) (H 2 O:ACN=60:40) to obtain 1.4 g of intermediate 8 (yield=76.5%).
LCMS m/z＝1123.4[M+1] + LCMS m/z=1123.4[M+1] +
第九步：Step Nine:
Figure PCTCN2022131909-appb-000029
Figure PCTCN2022131909-appb-000029
将中间体8(1.4g，1.25mmol)，戊二酸酐(0.29g，2.5mmol)和N,N-二异丙基乙胺(0.33g，2.5mmol)加入到DMA(14mL)中，室温搅拌18h，C 18反相制备柱纯化(0.1％TFA)(H 2O:ACN＝50:50)，得到1.3g中间体9(收率＝84.4％)。 Intermediate 8 (1.4g, 1.25mmol), glutaric anhydride (0.29g, 2.5mmol) and N,N-diisopropylethylamine (0.33g, 2.5mmol) were added to DMA (14mL), stirred at room temperature 18h, C 18 reverse phase preparative column purification (0.1% TFA) (H 2 O:ACN=50:50), to obtain 1.3g of intermediate 9 (yield=84.4%).
LCMS m/z＝1237.4[M+1] + LCMS m/z=1237.4[M+1] +
第十步：Step ten:
Figure PCTCN2022131909-appb-000030
Figure PCTCN2022131909-appb-000030
将中间体9(1.3g，1.05mmol)，加入到DMA(58mL)和二氯甲烷(20mL)中，氮气保护，降温至0℃，加入N-羟基丁二酰亚胺(0.37g，3.15mmol)和EDCI(0.61g，3.15mmo l)，室温搅拌18h，C 18反相制备柱纯化(0.1％TFA)(H 2O:ACN＝50:50)，得到1.0g中间体10(收率＝71.4％)。 Intermediate 9 (1.3g, 1.05mmol) was added to DMA (58mL) and dichloromethane (20mL), under nitrogen protection, the temperature was lowered to 0°C, and N-hydroxysuccinimide (0.37g, 3.15mmol) was added ) and EDCI (0.61g, 3.15mmol), stirring at room temperature for 18h, C 18 reverse phase preparative column purification (0.1%TFA) (H 2 O:ACN=50:50), to obtain 1.0g intermediate 10 (yield= 71.4%).
LCMS m/z＝1334.5[M+1] + LCMS m/z=1334.5[M+1] +
第十一步：Eleventh step:
Figure PCTCN2022131909-appb-000031
Figure PCTCN2022131909-appb-000031
将中间体10(10mg，7.5umol)和HSK-P22(20mg，6.2umol)加入到DMA(1mL)中，再加入N,N-二异丙基乙胺(2.4mg，18.6umol)，室温搅拌18h。用液相制备柱分离提纯得到10mg化合物1。Add intermediate 10 (10mg, 7.5umol) and HSK-P22 (20mg, 6.2umol) to DMA (1mL), then add N,N-diisopropylethylamine (2.4mg, 18.6umol), and stir at room temperature 18h. 10 mg of compound 1 was obtained by separation and purification with a liquid phase preparative column.
LCMS m/z＝981.0[M/4+1] +,1307.2[M/3+1] +. LCMS m/z＝981.0[M/4+1] + ,1307.2[M/3+1] + .
双环肽HSK-P22的合成Synthesis of Bicyclic Peptide HSK-P22
Figure PCTCN2022131909-appb-000032
Figure PCTCN2022131909-appb-000032
P22合成方法：P22 synthesis method:
Figure PCTCN2022131909-appb-000033
Figure PCTCN2022131909-appb-000033
多肽的合成采用标准的Fmoc化学方法：Peptides were synthesized using standard Fmoc chemistry:
1.向反应器中加入MBHA树脂(0.5mmol，1.85g，sub:0.27mmol/g)和DMF溶剂，震摇2小时。1. Add MBHA resin (0.5mmol, 1.85g, sub: 0.27mmol/g) and DMF solvent into the reactor and shake for 2 hours.
2.抽干并以DMF淋洗三次。2. Drain and rinse three times with DMF.
3.加入20％的哌啶/DMF，混合30分钟。3. Add 20% piperidine/DMF and mix for 30 minutes.
4.抽干并以DMF淋洗五次。4. Drain and rinse with DMF five times.
5.加入Fmoc保护的氨基酸溶液，混合30秒后加入偶联试剂，氮气鼓泡1小时。5. Add the Fmoc-protected amino acid solution, mix for 30 seconds, add the coupling reagent, and bubble nitrogen for 1 hour.
6.下一个氨基酸偶联重复步骤2-5.6. Repeat steps 2-5 for the next amino acid coupling.
## 原料raw material 偶联试剂Coupling reagent
11 Fmoc-HCys(Trt)-OH(2.0eq.)Fmoc-HCys(Trt)-OH(2.0eq.) HATU(1.90eq.)and DIEA(4.0eq.)HATU(1.90eq.) and DIEA(4.0eq.)
22 Fmoc-tBuGly-OH(2.0eq.)Fmoc-tBuGly-OH (2.0eq.) HATU(1.90eq.)and DIEA(4.0eq.)HATU(1.90eq.) and DIEA(4.0eq.)
33 Fmoc-Asp(OtBu)-OH(3.0eq.)Fmoc-Asp(OtBu)-OH(3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
44 Fmoc-Tyr(tBu)-OH(3.0eq.)Fmoc-Tyr(tBu)-OH(3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
55 Fmoc-Phe-OH(3.0eq.)Fmoc-Phe-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
66 Fmoc-Asp(OtBu)-OH(3.0eq.)Fmoc-Asp(OtBu)-OH(3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
77 Fmoc-Glu(OtBu)-OH(3.0eq.)Fmoc-Glu(OtBu)-OH(3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
88 Fmoc-HCys(Trt)-OH(2.0eq.)Fmoc-HCys(Trt)-OH(2.0eq.) HATU(1.90eq.)and DIEA(4.0eq.)HATU(1.90eq.) and DIEA(4.0eq.)
99 Fmoc-D-Ala-OH(3.0eq.)Fmoc-D-Ala-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
1010 Fmoc-1Nal-OH(3.0eq.)Fmoc-1Nal-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
1111 Fmoc-Glu(OtBu)-OH(3.0eq.)Fmoc-Glu(OtBu)-OH(3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
1212 Fmoc-Asn(Trt)-OH(3.0eq.)Fmoc-Asn(Trt)-OH(3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
1313 Fmoc-D-Ala-OH(3.0eq.)Fmoc-D-Ala-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
1414 Fmoc-HCys(Trt)-OH(2.0eq.)Fmoc-HCys(Trt)-OH(2.0eq.) HATU(1.90eq.)and DIEA(4.0eq.)HATU(1.90eq.) and DIEA(4.0eq.)
1515 Fmoc-Ala-OH(3.0eq.)Fmoc-Ala-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
1616 Fmoc-Sar-OH(3.0eq.)Fmoc-Sar-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
1717 Fmoc-Sar-OH(3.0eq.)Fmoc-Sar-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
1818 Fmoc-Sar-OH(3.0eq.)Fmoc-Sar-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
1919 Fmoc-Sar-OH(3.0eq.)Fmoc-Sar-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
2020 Fmoc-Sar-OH(3.0eq.)Fmoc-Sar-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
21twenty one Fmoc-Sar-OH(3.0eq.)Fmoc-Sar-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
22twenty two Fmoc-Sar-OH(3.0eq.)Fmoc-Sar-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
23twenty three Fmoc-Sar-OH(3.0eq.)Fmoc-Sar-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
24twenty four Fmoc-Sar-OH(3.0eq.)Fmoc-Sar-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
2525 Fmoc-Sar-OH(3.0eq.)Fmoc-Sar-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
2626 Fmoc-β-Ala-OH(3.0eq.)Fmoc-β-Ala-OH (3.0eq.) HATU(2.85eq.)and DIEA(6.0eq.)HATU(2.85eq.) and DIEA(6.0eq.)
20％哌啶DMF溶液，30分钟用于Fmoc保护剂的脱除。使用茚三酮监测偶联反应，树脂以DMF洗涤5次。20% piperidine DMF solution, 30 minutes for the removal of Fmoc protective agent. The coupling reaction was monitored using ninhydrin and the resin was washed 5 times with DMF.
肽的断裂和纯化：Fragmentation and purification of peptides:
1.将多肽侧链保护的多肽加入反应瓶中，加入断裂缓冲液(90％TFA/2.5％TIS/2.5％H 2O/5.0％DTT)，室温搅拌2小时。 1. Add the polypeptide protected by the side chain of the polypeptide into the reaction bottle, add the cleavage buffer (90% TFA/2.5% TIS/2.5% H 2 O/5.0% DTT), and stir at room temperature for 2 hours.
2.加入冰的异丙醚，多肽析出，离心(3min at 3000rpm)。2. Add iced isopropyl ether, the peptide is precipitated, and centrifuged (3min at 3000rpm).
3.用异丙醚洗涤两次。3. Wash twice with isopropyl ether.
4.真空干燥得到白色固体粗品肽化合物P22(1.20g，粗品)。4. Vacuum-dried to obtain white solid crude peptide compound P22 (1.20 g, crude product).
Figure PCTCN2022131909-appb-000034
Figure PCTCN2022131909-appb-000034
HSK-P22的合成方法：The synthetic method of HSK-P22:
将粗品肽P22溶于50％MeCN/H 2O(500mL)，室温搅拌下缓慢加入TBMB(购自药明康德，270mg,0.60mmol)，加料时间30分钟以上，加完，室温搅拌30分钟，加入碳酸氢铵调节PH值至8，反应液室温搅拌12小时，LC-MS显示反应完毕，制备HPLC纯化(流动相，A:0.075％TFA in H 2O,B:CH 3CN)得到白色固体HSK-P22(42.1mg，纯度97.3％)。 The crude peptide P22 was dissolved in 50% MeCN/H 2 O (500 mL), and TBMB (purchased from WuXi AppTec, 270 mg, 0.60 mmol) was slowly added under stirring at room temperature for more than 30 minutes. Ammonium bicarbonate was added to adjust the pH value to 8, and the reaction solution was stirred at room temperature for 12 hours. LC-MS showed that the reaction was complete. Purification by preparative HPLC (mobile phase, A: 0.075% TFA in H 2 O, B: CH 3 CN) gave a white solid HSK-P22 (42.1 mg, purity 97.3%).
按照上述方法，分别合成了HSK-P46，HSK-P47，HSK-P48，HSK-P49，HSK-P50，HSK-P51。According to the above method, HSK-P46, HSK-P47, HSK-P48, HSK-P49, HSK-P50, HSK-P51 were synthesized respectively.
HSK-P22:氨基酸序列为(β-Ala)-Sar10-Ala-SEQ ID NO:1(Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys)(即SEQ ID NO:8)，分子支架选自TBMB。HSK-P22: The amino acid sequence is (β-Ala)-Sar10-Ala-SEQ ID NO: 1(Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys) (i.e. SEQ ID NO: 8), the molecular scaffold is selected from TBMB.
HSK-P46:氨基酸序列为(β-Ala)-Sar10-Ala-SEQ ID NO:4(Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-cys)(即SEQ ID NO:11)，分子支架选自TBMB。HSK-P46: The amino acid sequence is (β-Ala)-Sar10-Ala-SEQ ID NO: 4(Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-cys) (i.e. SEQ ID NO: 11), the molecular scaffold is selected from TBMB.
HSK-P47:氨基酸序列为(β-Ala)-Sar10-Ala-SEQ ID NO:3(Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys)(即SEQ ID NO:10)，分子支架选自TBMB。HSK-P47: The amino acid sequence is (β-Ala)-Sar10-Ala-SEQ ID NO: 3(Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys) (i.e. SEQ ID NO: 10), the molecular scaffold is selected from TBMB.
HSK-P48:氨基酸序列为(β-Ala)-Sar10-Ala-SEQ ID NO:2(Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys)(即SEQ ID NO:9)，分子支架选自TBMB。HSK-P48: The amino acid sequence is (β-Ala)-Sar10-Ala-SEQ ID NO: 2(Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys) (i.e. SEQ ID NO:9), the molecular scaffold is selected from TBMB.
HSK-P49:氨基酸序列为(β-Ala)-Sar10-Ala-SEQ ID NO:7(Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys)(即SEQ ID NO:14)，分子支架选自TBMB。HSK-P49: The amino acid sequence is (β-Ala)-Sar10-Ala-SEQ ID NO: 7(Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys) (i.e. SEQ ID NO: 14), the molecular scaffold is selected from TBMB.
HSK-P50:氨基酸序列为(β-Ala)-Sar10-Ala-SEQ ID NO:6(Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys)(即SEQ ID NO:13)，分子支架选自TBMB。HSK-P50: The amino acid sequence is (β-Ala)-Sar10-Ala-SEQ ID NO: 6(Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys) (i.e. SEQ ID NO: 13), the molecular scaffold is selected from TBMB.
HSK-P51:氨基酸序列为(β-Ala)-Sar10-Ala-SEQ ID NO:5(Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys)(即SEQ ID NO:12)，分子支架选自TBMB。HSK-P51: The amino acid sequence is (β-Ala)-Sar10-Ala-SEQ ID NO: 5(Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu -Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys) (i.e. SEQ ID NO: 12), the molecular scaffold is selected from TBMB.
实施例2Example 2
Figure PCTCN2022131909-appb-000035
Figure PCTCN2022131909-appb-000035
Figure PCTCN2022131909-appb-000036
Figure PCTCN2022131909-appb-000036
第一步：first step:
Figure PCTCN2022131909-appb-000037
Figure PCTCN2022131909-appb-000037
将已知化合物2a(6g，50mmol)和2,2'-二硫二吡啶(25.0g，125mmol)加入到甲醇(60mL)中，室温反应3小时。直接C 18反相柱纯化(0.1％TFA)(H 2O:ACN＝50:50)，得到10g化合物2b(收率＝87％)。 Add known compound 2a (6 g, 50 mmol) and 2,2'-dithiobipyridine (25.0 g, 125 mmol) into methanol (60 mL), and react at room temperature for 3 hours. Direct C 18 reverse phase column purification (0.1% TFA) (H 2 O:ACN=50:50) gave 10 g of compound 2b (yield=87%).
LCMS m/z＝230.1[M+1] + LCMS m/z＝230.1[M+1] +
第二步：Step two:
Figure PCTCN2022131909-appb-000038
Figure PCTCN2022131909-appb-000038
将化合物2b(10.0g，43.6mmol)加入到N，N-二甲基乙酰胺(75mL)和二氯甲烷(25mL)中，再加入N-羟基丁二酰亚胺(6.0g，52.4mmol)和EDCI(25.0g，130.8mmol)，室温搅拌1小时，20℃浓缩除去二氯甲烷，直接C 18反相柱纯化(0.1％TFA)(H 2O:ACN＝40:60)，得到10g化合物2c(收率＝70％)。 Compound 2b (10.0 g, 43.6 mmol) was added to N, N-dimethylacetamide (75 mL) and dichloromethane (25 mL), and N-hydroxysuccinimide (6.0 g, 52.4 mmol) was added and EDCI (25.0g, 130.8mmol), stirred at room temperature for 1 hour, concentrated at 20°C to remove dichloromethane, and directly purified by C 18 reverse phase column (0.1% TFA) (H 2 O:ACN=40:60) to obtain 10g of compound 2c (Yield = 70%).
LCMS m/z＝327.0[M+1] + LCMS m/z=327.0[M+1] +
第三步：third step:
Figure PCTCN2022131909-appb-000039
Figure PCTCN2022131909-appb-000039
将化合物2c(45mg，0.138mmol)加入到N，N-二甲基乙酰胺(3mL)和二氯甲烷(1mL)中，氮气保护，再加入DM1(70mg，0.094mmol)，室温搅拌18小时，20℃浓缩除去二氯甲烷，直接C 18反相柱纯化(0.1％TFA)(H 2O:ACN＝40:60)，得到50mg化合物2d(收率＝55％)。 Compound 2c (45mg, 0.138mmol) was added to N,N-dimethylacetamide (3mL) and dichloromethane (1mL), under nitrogen protection, then DM1 (70mg, 0.094mmol) was added, and stirred at room temperature for 18 hours, Concentrate at 20°C to remove dichloromethane, and directly purify by C 18 reverse phase column (0.1% TFA) (H 2 O:ACN=40:60) to obtain 50mg of compound 2d (yield=55%).
LCMS m/z＝935.3[M+1-H 2O] + LCMS m/z=935.3[M+1-H 2 O] +
第四步：the fourth step:
Figure PCTCN2022131909-appb-000040
Figure PCTCN2022131909-appb-000040
将化合物2d(10mg，10umol)和HSK-P22(20mg，7.4umol)加入到DMA(1mL)中，再加入N,N-二异丙基乙胺(2.4mg，18.6umol)，室温搅拌18h。用液相制备柱分离提纯(液相制备条件：C 18反相制备柱，流动相为含0.1％三氟乙酸的去离子水(A)，含0.1％三氟乙酸的乙腈(B)，梯度洗脱，B含量＝5％～70％，洗脱时间15min，流速12mL/min，柱温：30℃，保留时间：5.64min)得到15mg化合物2(收率＝57％)。 Compound 2d (10mg, 10umol) and HSK-P22 (20mg, 7.4umol) were added to DMA (1mL), then N,N-diisopropylethylamine (2.4mg, 18.6umol) was added, and stirred at room temperature for 18h. Separation and purification with liquid phase preparative column (liquid phase preparation conditions: C 18 reverse phase preparative column, mobile phase is deionized water (A) containing 0.1% trifluoroacetic acid, acetonitrile (B) containing 0.1% trifluoroacetic acid, gradient Elution, B content=5%～70%, elution time 15min, flow rate 12mL/min, column temperature: 30°C, retention time: 5.64min) to obtain 15mg of compound 2 (yield=57%).
LCMS m/z＝1174.8[(M-H 2O)/3+1] + LCMS m/z=1174.8[(MH 2 O)/3+1] +
实施例3Example 3
Figure PCTCN2022131909-appb-000041
Figure PCTCN2022131909-appb-000041
Figure PCTCN2022131909-appb-000042
Figure PCTCN2022131909-appb-000042
第一步：first step:
Figure PCTCN2022131909-appb-000043
Figure PCTCN2022131909-appb-000043
将化合物2c(376mg，1.15mmol)加入到N，N-二甲基乙酰胺(8mL)和饱和碳酸氢钠水溶液(0.9mL)中，氮气保护，再加入DM4(180mg，0.23mmol)，室温搅拌18小时，20℃浓缩除去二氯甲烷，直接C 18反相柱纯化(0.1％TFA)(H 2O:ACN＝40:60)，得到20mg化合物3a(收率＝8.7％)。 Compound 2c (376mg, 1.15mmol) was added to N,N-dimethylacetamide (8mL) and saturated aqueous sodium bicarbonate (0.9mL) under nitrogen protection, then DM4 (180mg, 0.23mmol) was added and stirred at room temperature After 18 hours, dichloromethane was removed by concentration at 20°C, and direct C 18 reverse phase column purification (0.1% TFA) (H 2 O:ACN=40:60) gave 20 mg of compound 3a (yield=8.7%).
第二步：Step two:
Figure PCTCN2022131909-appb-000044
Figure PCTCN2022131909-appb-000044
将化合物3a(14mg，14umol)和HSK-P22(23mg，8.5umol)加入到DMA(1mL)中，再加入N,N-二异丙基乙胺(2.4mg，18.6umol)，室温搅拌18h。用液相制备柱分离提纯(液相制备条件：C 18反相制备柱，流动相为含0.1％三氟乙酸的去离子水(A)，含0.1％三氟乙酸的乙腈(B)，梯度洗脱，B含量＝5％～70％，洗脱时间15min，流速12mL/min，柱温：30℃，保留时间：5.53min)得到15mg化合物3(收率＝50％)。 Compound 3a (14mg, 14umol) and HSK-P22 (23mg, 8.5umol) were added to DMA (1mL), then N,N-diisopropylethylamine (2.4mg, 18.6umol) was added, and stirred at room temperature for 18h. Separation and purification with liquid phase preparative column (liquid phase preparation conditions: C 18 reverse phase preparative column, mobile phase is deionized water (A) containing 0.1% trifluoroacetic acid, acetonitrile (B) containing 0.1% trifluoroacetic acid, gradient Elution, B content=5%～70%, elution time 15min, flow rate 12mL/min, column temperature: 30°C, retention time: 5.53min) to obtain 15mg of compound 3 (yield=50%).
LCMS m/z＝1188.3[(M-H 2O)/3+1] +. LCMS m/z=1188.3[(MH 2 O)/3+1] + .
实施例4Example 4
Figure PCTCN2022131909-appb-000045
Figure PCTCN2022131909-appb-000045
第一步：first step:
Figure PCTCN2022131909-appb-000046
Figure PCTCN2022131909-appb-000046
将已知化合物4a(70g，0.7mol)加入到40％的氢溴酸水溶液(400mL)中，加热至100℃，再加入硫脲(280g，3.68mol)，回流反应24小时。用40℃温水(200mL)稀释反应液，二氯甲烷萃取三次(300mL×3)，再用***(300mL)萃取一次，水相用10N NaOH溶液调PH至12，继续回流反应24小时，冷却至室温，用6N HCl调PH至1，二氯甲烷萃取(300mL×2)，饱和食盐水洗(300mL)，无水硫酸钠干燥，过滤，浓缩得到12g化合物4b(收率＝12.8％)。Add known compound 4a (70g, 0.7mol) into 40% hydrobromic acid aqueous solution (400mL), heat to 100°C, add thiourea (280g, 3.68mol), and reflux for 24 hours. Dilute the reaction solution with 40°C warm water (200mL), extract three times with dichloromethane (300mL×3), then extract once with diethyl ether (300mL), adjust the pH of the aqueous phase to 12 with 10N NaOH solution, continue to reflux for 24 hours, and cool to At room temperature, the pH was adjusted to 1 with 6N HCl, extracted with dichloromethane (300mL×2), washed with saturated brine (300mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 12g of compound 4b (yield = 12.8%).
LCMS m/z＝133.1[M-1] - LCMS m/z=133.1[M-1] -
第二步：Step two:
Figure PCTCN2022131909-appb-000047
Figure PCTCN2022131909-appb-000047
将化合物4b(12.0g，89.5mmol)和2,2'-二硫二吡啶(49.2g，223.7mmol)加入到甲醇(130mL)中，室温反应3小时。柱层析纯化(DCM:MeOH＝10:1)得到18g化合物4c(收率＝83％)。Compound 4b (12.0 g, 89.5 mmol) and 2,2'-dithiobipyridine (49.2 g, 223.7 mmol) were added into methanol (130 mL), and reacted at room temperature for 3 hours. Purification by column chromatography (DCM:MeOH=10:1) gave 18 g of compound 4c (yield=83%).
LCMS m/z＝244.1[M+1] + LCMS m/z=244.1[M+1] +
第三步：third step:
Figure PCTCN2022131909-appb-000048
Figure PCTCN2022131909-appb-000048
将化合物4c(5.3g，21.8mmol)加入到N，N-二甲基乙酰胺(40mL)和二氯甲烷(14mL)中，再加入N-羟基丁二酰亚胺(3.0g，26.2mmol)和EDCI(12.5g，65.4mmol)，室温搅拌1小时，20℃浓缩除去二氯甲烷，加水(80mL)和乙酸乙酯(80mL×2)萃取，乙酸乙酯相用无水硫酸钠干燥，浓缩后柱层析纯化(DCM:MeOH＝10:1)得到7.1g化合物4d(收率＝96％)。Compound 4c (5.3g, 21.8mmol) was added to N,N-dimethylacetamide (40mL) and dichloromethane (14mL), and N-hydroxysuccinimide (3.0g, 26.2mmol) was added and EDCI (12.5g, 65.4mmol), stirred at room temperature for 1 hour, concentrated at 20°C to remove dichloromethane, added water (80mL) and ethyl acetate (80mL×2) for extraction, the ethyl acetate phase was dried over anhydrous sodium sulfate, concentrated Purification by column chromatography (DCM:MeOH=10:1) gave 7.1 g of compound 4d (yield=96%).
LCMS m/z＝341.0[M+1] + LCMS m/z=341.0[M+1] +
第四步：the fourth step:
Figure PCTCN2022131909-appb-000049
Figure PCTCN2022131909-appb-000049
将化合物4d(126mg，0.37mmol)加入到N，N-二甲基乙酰胺(3mL)和二氯甲烷(1mL)中，氮气保护，再加入DM1(182mg，0.25mmol)，室温搅拌18小时，20℃浓缩除去二氯甲烷，C18反相柱纯化(0.1％TFA)(H 2O:ACN＝40:60)，得到180mg化合物4e(收率＝50％)。 Compound 4d (126mg, 0.37mmol) was added to N,N-dimethylacetamide (3mL) and dichloromethane (1mL), under nitrogen protection, then DM1 (182mg, 0.25mmol) was added, and stirred at room temperature for 18 hours, Concentrate at 20°C to remove dichloromethane, and purify by C18 reverse phase column (0.1% TFA) (H 2 O:ACN=40:60) to obtain 180mg of compound 4e (yield=50%).
LCMS m/z＝949.3[M+1-H 2O] + LCMS m/z=949.3[M+1-H 2 O] +
第五步：the fifth step:
Figure PCTCN2022131909-appb-000050
Figure PCTCN2022131909-appb-000050
将化合物4e(13mg，13.3mmol)和HSK-P22(30mg，11.1umol)加入到DMA(1mL)中，再加入N,N-二异丙基乙胺(4.3mg，33.3umol)，室温搅拌18h。用液相制备柱分离提纯(液相制备条件：C18反相制备柱，流动相为含0.1％三氟乙酸的去离子水(A)，含0.1％三氟乙酸的乙腈(B)，梯度洗脱，B含量＝5％～70％，洗脱时间15min，流速12mL/min，柱温：30℃，保留时间：5.34min)得到20mg化合物4(收率＝51％)。Add compound 4e (13mg, 13.3mmol) and HSK-P22 (30mg, 11.1umol) into DMA (1mL), then add N,N-diisopropylethylamine (4.3mg, 33.3umol), and stir at room temperature for 18h . Separation and purification with liquid phase preparative column (liquid phase preparation conditions: C18 reverse phase preparative column, mobile phase is deionized water (A) containing 0.1% trifluoroacetic acid, acetonitrile (B) containing 0.1% trifluoroacetic acid, gradient washing B content = 5% to 70%, elution time 15min, flow rate 12mL/min, column temperature: 30°C, retention time: 5.34min) to obtain 20 mg of compound 4 (yield = 51%).
LCMS m/z＝1179.4[(M-H 2O)/3+1] + LCMS m/z=1179.4[(MH 2 O)/3+1] +
实施例5Example 5
Figure PCTCN2022131909-appb-000051
Figure PCTCN2022131909-appb-000051
合成方法参照实施例4：Synthetic method with reference to embodiment 4:
将化合物4e(9mg，8.9umol)和HSK-P46(20mg，7.4umol)加入到DMA(1mL)中，再加入N,N-二异丙基乙胺(2.8mg，22.2umol)，室温搅拌18h。用液相制备柱分离提纯得到10mg化合物5。Add compound 4e (9mg, 8.9umol) and HSK-P46 (20mg, 7.4umol) to DMA (1mL), then add N,N-diisopropylethylamine (2.8mg, 22.2umol), and stir at room temperature for 18h . 10 mg of compound 5 was obtained by separation and purification with a liquid phase preparative column.
LCMS m/z＝1174.6[(M-H 2O)/3+1]. LCMS m/z=1174.6[( MH2O )/3+1].
实施例6Example 6
Figure PCTCN2022131909-appb-000052
Figure PCTCN2022131909-appb-000052
合成方法参照实施例4：Synthetic method with reference to embodiment 4:
将化合物4e(9mg，8.9umol)和HSK-P47(20mg，7.4umol)加入到DMA(1mL)中，再加入N,N-二异丙基乙胺(2.8mg，22.2umol)，室温搅拌18h。用液相制备柱分离提纯(液相制备条件：C18反相制备柱，流动相为含0.1％三氟乙酸的去离子水(A)，含0.1％三氟乙酸的乙腈(B)，梯度洗脱，B含量＝5％～70％，洗脱时间15min，流速12mL/min，柱温：30℃，保留时间：5.58min)得到11mg化合物6。Add compound 4e (9mg, 8.9umol) and HSK-P47 (20mg, 7.4umol) to DMA (1mL), then add N,N-diisopropylethylamine (2.8mg, 22.2umol), and stir at room temperature for 18h . Separation and purification with liquid phase preparative column (liquid phase preparation conditions: C18 reverse phase preparative column, mobile phase is deionized water (A) containing 0.1% trifluoroacetic acid, acetonitrile (B) containing 0.1% trifluoroacetic acid, gradient washing B content=5%～70%, elution time 15min, flow rate 12mL/min, column temperature: 30°C, retention time: 5.58min) to obtain 11 mg of compound 6.
LCMS m/z＝1174.2[(M-H 2O)/3+1] +. LCMS m/z=1174.2[(MH 2 O)/3+1] + .
实施例7Example 7
Figure PCTCN2022131909-appb-000053
Figure PCTCN2022131909-appb-000053
合成方法参照实施例4：Synthetic method with reference to embodiment 4:
将化合物4e(9mg，8.9umol)和HSK-P48(20mg，7.4umol)加入到DMA(1mL)中，再加入N,N-二异丙基乙胺(2.8mg，22.2umol)，室温搅拌18h。用液相制备柱分离提纯得到10mg化合物7。Add compound 4e (9mg, 8.9umol) and HSK-P48 (20mg, 7.4umol) to DMA (1mL), then add N,N-diisopropylethylamine (2.8mg, 22.2umol), and stir at room temperature for 18h . 10 mg of compound 7 was obtained by separation and purification with a liquid phase preparative column.
LCMS m/z＝1174.3[(M-H 2O)/3+1] +. LCMS m/z=1174.3[(MH 2 O)/3+1] + .
实施例8Example 8
Figure PCTCN2022131909-appb-000054
Figure PCTCN2022131909-appb-000054
合成方法参照实施例4：Synthetic method with reference to embodiment 4:
将化合物4e(9mg，8.9umol)和HSK-P49(20mg，7.4umol)加入到DMA(1mL)中，再加入N,N-二异丙基乙胺(2.8mg，22.2umol)，室温搅拌18h。用液相制备柱分离提纯得到12mg化合物8。Add compound 4e (9mg, 8.9umol) and HSK-P49 (20mg, 7.4umol) to DMA (1mL), then add N,N-diisopropylethylamine (2.8mg, 22.2umol), and stir at room temperature for 18h . 12 mg of compound 8 was obtained by separation and purification with a liquid phase preparative column.
LCMS m/z＝1169.8[(M-H 2O)/3+1] +. LCMS m/z=1169.8[(MH 2 O)/3+1] + .
实施例9Example 9
Figure PCTCN2022131909-appb-000055
Figure PCTCN2022131909-appb-000055
合成方法参照实施例4：Synthetic method with reference to embodiment 4:
将化合物4e(9mg，8.9umol)和HSK-P50(20mg，7.4umol)加入到DMA(1mL)中，再加入N,N-二异丙基乙胺(2.8mg,22.2umol)，室温搅拌18h。用液相制备柱分离提纯得到11mg化合物9。Compound 4e (9mg, 8.9umol) and HSK-P50 (20mg, 7.4umol) were added to DMA (1mL), then N,N-diisopropylethylamine (2.8mg, 22.2umol) was added, and stirred at room temperature for 18h . 11 mg of compound 9 was obtained by separation and purification with a liquid phase preparative column.
LCMS m/z＝1170.1[(M-H 2O)/3+1] +. LCMS m/z=1170.1[(MH 2 O)/3+1] + .
实施例10Example 10
Figure PCTCN2022131909-appb-000056
Figure PCTCN2022131909-appb-000056
合成方法参照实施例4：Synthetic method with reference to embodiment 4:
将化合物4e(9mg，8.9umol)和HSK-P51(20mg，7.4umol)加入到DMA(1mL)中，再加入N,N-二异丙基乙胺(2.8mg，22.2umol)，室温搅拌18h。用液相制备柱分离提纯(液相制备条件：C18反相制备柱，流动相为含0.1％三氟乙酸的去离子水(A)，含0.1％三氟乙酸的乙腈(B)，梯度洗脱，B含量＝5％～70％，洗脱时间15min，流速12mL/min，柱温：30℃，保留时间：5.48min)得到10mg化合物10。Add compound 4e (9mg, 8.9umol) and HSK-P51 (20mg, 7.4umol) to DMA (1mL), then add N,N-diisopropylethylamine (2.8mg, 22.2umol), and stir at room temperature for 18h . Separation and purification with liquid phase preparative column (liquid phase preparation conditions: C18 reverse phase preparative column, mobile phase is deionized water (A) containing 0.1% trifluoroacetic acid, acetonitrile (B) containing 0.1% trifluoroacetic acid, gradient washing elution, B content = 5% to 70%, elution time 15min, flow rate 12mL/min, column temperature: 30°C, retention time: 5.48min) to obtain 10 mg of compound 10.
LCMS m/z＝1169.9[(M-H 2O)/3+1] +. LCMS m/z=1169.9[(MH 2 O)/3+1] + .
实施例11Example 11
Figure PCTCN2022131909-appb-000057
Figure PCTCN2022131909-appb-000057
Figure PCTCN2022131909-appb-000058
Figure PCTCN2022131909-appb-000058
第一步：first step:
Figure PCTCN2022131909-appb-000059
Figure PCTCN2022131909-appb-000059
将化合物4e(1.2g，1.2mmol)和N-芴甲氧羰基-1,2-二氨基乙烷盐酸盐(475mg，1.44mmol)加入到DMA(10mL)中，再加入N,N-二异丙基乙胺(480mg，3.6mmol)，室温搅拌18h。C18反相制备柱纯化(0.1％TFA)(H 2O:ACN＝50:50)，得到1g化合物11a(收率＝71.4％)。 Compound 4e (1.2g, 1.2mmol) and N-fluorenylmethoxycarbonyl-1,2-diaminoethane hydrochloride (475mg, 1.44mmol) were added to DMA (10mL), and then N,N-diaminoethane Isopropylethylamine (480mg, 3.6mmol), stirred at room temperature for 18h. C18 reverse-phase preparative column purification (0.1% TFA) (H 2 O:ACN=50:50) gave 1 g of compound 11a (yield=71.4%).
LCMS m/z＝1116.4[(M-H 2O)+1] +. LCMS m/z=1116.4[(MH 2 O)+1] + .
第二步：Step two:
Figure PCTCN2022131909-appb-000060
Figure PCTCN2022131909-appb-000060
将化合物11a(1.0g，0.88mmol)加入到20％的哌啶/DMF溶液中(10mL)，室温搅拌5分钟。C18反相制备柱纯化(0.1％TFA)(H 2O:ACN＝55:45)，得到500mg化合物11b(收率＝60.0％)。 Compound 11a (1.0 g, 0.88 mmol) was added into 20% piperidine/DMF solution (10 mL), and stirred at room temperature for 5 minutes. C18 reverse-phase preparative column purification (0.1% TFA) (H 2 O:ACN=55:45) gave 500 mg of compound 11b (yield=60.0%).
LCMS m/z＝912.3[M+1] +. LCMS m/z=912.3[M+1] + .
第三步：third step:
Figure PCTCN2022131909-appb-000061
Figure PCTCN2022131909-appb-000061
将17f(394mg，0.48mmol)加入到N,N-二甲基甲酰胺(4mL)中，再加入N,N-二异丙基乙胺(186mg，1.44mmol)，滴加Val-Cit-PABC-MMAE三氟乙酸盐(537mg，0.43mmol)的N,N-二甲基甲酰胺溶液(2mL)，室温搅拌30分钟，再加入N,N-二异丙基乙胺(186mg，1.44mmol)，滴加11b(440mg，0.48mmol)的N,N-二甲基甲酰胺溶液(2mL)，室温搅拌1h，C18反相制备柱纯化(0.1％TFA)(H 2O:ACN＝35:65)，得到700mg 11c(收率＝68.3％)。 Add 17f (394mg, 0.48mmol) to N,N-dimethylformamide (4mL), then add N,N-diisopropylethylamine (186mg, 1.44mmol), add Val-Cit-PABC dropwise -MMAE trifluoroacetate (537mg, 0.43mmol) in N,N-dimethylformamide solution (2mL), stirred at room temperature for 30 minutes, then added N,N-diisopropylethylamine (186mg, 1.44mmol ), add 11b (440mg, 0.48mmol) in N,N-dimethylformamide solution (2mL) dropwise, stir at room temperature for 1h, C18 reverse phase preparative column purification (0.1%TFA) (H 2 O:ACN=35: 65), yielding 700 mg of 11c (yield = 68.3%).
LCMS m/z＝830.2[M/3+1] +,1234.8[(M-H 2O)/2+1] +. LCMS m/z＝830.2[M/3+1] + ,1234.8[(MH 2 O)/2+1] + .
第四步：the fourth step:
Figure PCTCN2022131909-appb-000062
Figure PCTCN2022131909-appb-000062
将化合物11c(0.7g，0.28mmol)加入到20％的哌啶/DMF溶液中(10mL)，室温搅拌5分钟。C18反相制备柱纯化(0.1％TFA)(H 2O:ACN＝55:45)，得到600mg化合物11d(收率＝94.1％)。 Compound 11c (0.7 g, 0.28 mmol) was added into 20% piperidine/DMF solution (10 mL), and stirred at room temperature for 5 minutes. C18 reverse-phase preparative column purification (0.1% TFA) (H 2 O:ACN=55:45) gave 600 mg of compound 11d (yield=94.1%).
LCMS m/z＝755.5[M/3+1] +,1132.2[M/2+1] +. LCMS m/z＝755.5[M/3+1] + ,1132.2[M/2+1] + .
第五步：the fifth step:
Figure PCTCN2022131909-appb-000063
Figure PCTCN2022131909-appb-000063
将11d(600mg，0.265mmol)，戊二酸酐(60.4mg，0.53mmol)和N,N-二异丙基乙胺(102mg，0.795mmol)加入到DMA(6mL)中，室温搅拌18h，C18反相制备柱纯化(0.1％TFA)(H 2O:ACN＝50:50)，得到600mg 11e(收率＝95.2％)。 11d (600mg, 0.265mmol), glutaric anhydride (60.4mg, 0.53mmol) and N,N-diisopropylethylamine (102mg, 0.795mmol) were added to DMA (6mL), stirred at room temperature for 18h, C18 reaction Phase preparative column purification (0.1% TFA) (H 2 O:ACN=50:50) afforded 600 mg of 11e (yield=95.2%).
LCMS m/z＝787.3[(M-H2O)/3+1] +，1181.2[(M-H2O)/2+1] + LCMS m/z=787.3[(M-H2O)/3+1] + , 1181.2[(M-H2O)/2+1] +
第六步：Step six:
Figure PCTCN2022131909-appb-000064
Figure PCTCN2022131909-appb-000064
将11e(440mg，0.185mmol)加入到DMA(4.5mL)和二氯甲烷(1.5mL)中，氮气保护，降温至0℃，加入N-羟基丁二酰亚胺(128mg，1.11mmol)和EDCI(215mg，1.11mmol)，室温搅拌18h，20℃浓缩除去二氯甲烷，C18反相制备柱纯化(0.1％TFA)(H 2O:ACN＝55:45)，得到440mg 11f(收率＝96.0％)。 11e (440mg, 0.185mmol) was added to DMA (4.5mL) and dichloromethane (1.5mL), under nitrogen protection, cooled to 0°C, N-hydroxysuccinimide (128mg, 1.11mmol) and EDCI were added (215mg, 1.11mmol), stirred at room temperature for 18h, concentrated at 20°C to remove dichloromethane, purified by C18 reverse-phase preparative column (0.1%TFA) (H 2 O:ACN=55:45), and obtained 440mg of 11f (yield=96.0 %).
LCMS m/z＝819.8[(M-H2O)/3+1] +，1229.3[(M-H2O)/2+1] + LCMS m/z=819.8[(M-H2O)/3+1] + , 1229.3[(M-H2O)/2+1] +
第七步：Step seven:
Figure PCTCN2022131909-appb-000065
Figure PCTCN2022131909-appb-000065
将11f(40mg，16.1umol)和HSK-P51(50mg，18.7umol)加入到DMA(1mL)中，再加入N,N-二异丙基乙胺(6mg，48umol)，室温搅拌18h。用液相制备柱分离提纯(液相制备条件：C18反相制备柱，流动相为含0.1％三氟乙酸的去离子水(A)，含0.1％三氟乙酸的乙腈(B)，梯度洗脱，B含量＝5％～70％，洗脱时间15min，流速12mL/min，柱温：30℃，保留时间：5.87min)得到40mg化合物11(收率＝49.2％)。11f (40mg, 16.1umol) and HSK-P51 (50mg, 18.7umol) were added to DMA (1mL), then N,N-diisopropylethylamine (6mg, 48umol) was added, and stirred at room temperature for 18h. Separation and purification with liquid phase preparative column (liquid phase preparation conditions: C18 reverse phase preparative column, mobile phase is deionized water (A) containing 0.1% trifluoroacetic acid, acetonitrile (B) containing 0.1% trifluoroacetic acid, gradient washing B content = 5% to 70%, elution time 15min, flow rate 12mL/min, column temperature: 30°C, retention time: 5.87min) to obtain 40mg of compound 11 (yield = 49.2%).
LCMS m/z＝1003.7[(M-H2O)/5+1] +,1254.4[(M-H2O)/4+1] +. LCMS m/z＝1003.7[(M-H2O)/5+1] + ,1254.4[(M-H2O)/4+1] + .
17f的制备：Preparation of 17f:
Figure PCTCN2022131909-appb-000066
Figure PCTCN2022131909-appb-000066
第一步：first step:
Figure PCTCN2022131909-appb-000067
Figure PCTCN2022131909-appb-000067
依次将已知化合物2-(2-(2-氨基乙氧基)乙氧基)乙基氨基甲酸叔丁酯(25.0g，0.1mol)，溴乙酸乙酯(43.2g，0.22mol)，碳酸钠(40.5g，0.25mol)加入到乙腈(1.2L)中，升温至50℃搅拌18h，冷却至室温，过滤，母液浓缩至干，得到42g粗品17b。(收率＝99％)。The known compound 2-(2-(2-aminoethoxy) ethoxy) tert-butyl ethyl carbamate (25.0g, 0.1mol), ethyl bromoacetate (43.2g, 0.22mol), carbonic acid Sodium (40.5g, 0.25mol) was added to acetonitrile (1.2L), heated to 50°C and stirred for 18h, cooled to room temperature, filtered, and the mother liquor was concentrated to dryness to obtain 42g of crude product 17b. (Yield = 99%).
LCMS m/z＝421.3[M+1] + LCMS m/z=421.3[M+1] +
第二、三步：The second and third steps:
Figure PCTCN2022131909-appb-000068
Figure PCTCN2022131909-appb-000068
将化合物17b(42.0g，0.1mol)加入到甲醇(500mL)和水(500mL)中，再加入氢氧化钠(40.3g，1.0mol)，室温搅拌4h，用6N的盐酸调pH至1～2，搅拌0.5h。反应液直接用于下一步反应。Compound 17b (42.0g, 0.1mol) was added to methanol (500mL) and water (500mL), then sodium hydroxide (40.3g, 1.0mol) was added, stirred at room temperature for 4h, and the pH was adjusted to 1-2 with 6N hydrochloric acid. , stirred for 0.5h. The reaction solution was directly used in the next reaction.
LCMS m/z＝265.2[M+1] + LCMS m/z=265.2[M+1] +
第四步：the fourth step:
Figure PCTCN2022131909-appb-000069
Figure PCTCN2022131909-appb-000069
向17d的反应液中依次加入9-芴甲基-N-琥珀酰亚胺基碳酸酯(34.0g，0.1mol)，碳酸氢钠(42.0g，0.5mol)，室温搅拌4h，C18反相柱纯化(0.1％TFA)(H 2O:ACN＝60:40)，得到22g 17e(两步收率＝45.2％)。 Add 9-fluorenylmethyl-N-succinimidyl carbonate (34.0 g, 0.1 mol) and sodium bicarbonate (42.0 g, 0.5 mol) successively to the reaction solution of 17d, stir at room temperature for 4 h, and use a C18 reverse-phase column Purification (0.1% TFA) (H 2 O:ACN=60:40) afforded 22 g of 17e (yield over two steps=45.2%).
LCMS m/z＝487.2[M+1] + LCMS m/z=487.2[M+1] +
第五步：the fifth step:
Figure PCTCN2022131909-appb-000070
Figure PCTCN2022131909-appb-000070
依次将17e(5.0g，0.01mol)，五氟苯酚(3.86g，0.02mol)和N,N-二异丙基碳二亚胺(2.65g，0.02mol)，室温搅拌2h，柱层析纯化(PE:EA＝2:1)得到4.0g 17f(收率＝47.6％)。17e (5.0g, 0.01mol), pentafluorophenol (3.86g, 0.02mol) and N,N-diisopropylcarbodiimide (2.65g, 0.02mol) were sequentially stirred at room temperature for 2h and purified by column chromatography (PE:EA=2:1) yielded 4.0 g of 17f (yield=47.6%).
LCMS m/z＝819.1[M+1] + LCMS m/z＝819.1[M+1] +
实施例12Example 12
Figure PCTCN2022131909-appb-000071
Figure PCTCN2022131909-appb-000071
Figure PCTCN2022131909-appb-000072
Figure PCTCN2022131909-appb-000072
第一步：first step:
Figure PCTCN2022131909-appb-000073
Figure PCTCN2022131909-appb-000073
将12A(20g，188.47mmol)加入无水四氢呋喃(200ml)中，在0℃下缓慢入氢化铝锂(8.58g，226.16mmol)，保持0℃反应2h。TLC监测反应完全。保持在0℃下加入水(8.6ml)，再加入氢氧化钠(1.3g，8.6ml水)。继续加入水(25ml)，搅拌15min。加入无水硫酸钠搅拌干燥多余的水，过滤，滤液减压浓缩掉大部分四氢呋喃后直接用于下一部反应。Add 12A (20g, 188.47mmol) into anhydrous tetrahydrofuran (200ml), slowly add lithium aluminum hydride (8.58g, 226.16mmol) at 0°C, and keep at 0°C for 2h. TLC monitored the completion of the reaction. Water (8.6ml) was added maintaining at 0°C followed by sodium hydroxide (1.3g, 8.6ml water). Continue to add water (25ml) and stir for 15min. Add anhydrous sodium sulfate, stir and dry excess water, filter, and concentrate the filtrate under reduced pressure to remove most of THF, and then directly use it in the next reaction.
第二步：Step two:
Figure PCTCN2022131909-appb-000074
Figure PCTCN2022131909-appb-000074
将上一步粗品12B，2,2'-二硫二吡啶(37.68g，171.03mmol)加入甲醇(200mL)中，室温反应16h。直接将反应液减压浓缩后硅胶柱色谱分离纯化(洗脱剂：DCM/EA＝3/1)得到12C(16g,41.82％)。The crude product 12B from the previous step, 2,2'-dithiobipyridine (37.68 g, 171.03 mmol) was added into methanol (200 mL), and reacted at room temperature for 16 h. The reaction solution was directly concentrated under reduced pressure and purified by silica gel column chromatography (eluent: DCM/EA=3/1) to obtain 12C (16 g, 41.82%).
LCMS m/z＝202.1[M+1] + LCMS m/z=202.1[M+1] +
第三步：third step:
Figure PCTCN2022131909-appb-000075
Figure PCTCN2022131909-appb-000075
将12C(8g，39.74mmol)，吡啶(9.43g，119.22mmol)加入四氢呋喃(70mL)中，再缓慢加入对硝基苯基氯甲酸酯(10.41g，51.66mmol)，室温反应16h。直接将反应液减压浓缩后硅胶柱色谱分离纯化(洗脱剂：EA/PE＝0-25％)得到12D(8g，54.94％)。12C (8g, 39.74mmol), pyridine (9.43g, 119.22mmol) were added into tetrahydrofuran (70mL), and p-nitrophenyl chloroformate (10.41g, 51.66mmol) was added slowly, and reacted at room temperature for 16h. The reaction solution was directly concentrated under reduced pressure and purified by silica gel column chromatography (eluent: EA/PE=0-25%) to obtain 12D (8 g, 54.94%).
LCMS m/z＝367.1[M+1] + LCMS m/z=367.1[M+1] +
第四步：the fourth step:
Figure PCTCN2022131909-appb-000076
Figure PCTCN2022131909-appb-000076
将依喜替康(1.8g，4.15mmol)，12D(1.67g，4.57mmol)，1-羟基苯并***(0.67g，4.96mmol)，吡啶(1.64g，20.75mmol)加入DMF(20mL)中，室温反应16h。LC-MS监测反应。加入水(100ml)，搅拌后过滤，固体用水洗涤后再用(EA/PE＝1:5)(20ml)洗涤，干燥，得到12E(2.2g，79.99％)。Exitecan (1.8g, 4.15mmol), 12D (1.67g, 4.57mmol), 1-hydroxybenzotriazole (0.67g, 4.96mmol), pyridine (1.64g, 20.75mmol) were added to DMF (20mL) In the reaction at room temperature for 16h. LC-MS monitored the reaction. Water (100ml) was added, stirred and filtered, the solid was washed with water and then (EA/PE=1:5) (20ml), dried to give 12E (2.2g, 79.99%).
LCMS m/z＝663.2[M+1] + LCMS m/z=663.2[M+1] +
第五步：the fifth step:
Figure PCTCN2022131909-appb-000077
Figure PCTCN2022131909-appb-000077
将12E(2g，3.02mmol)加入甲醇(20ml)中，再加入4-巯基丁酸(2.18g，18.15mmol)，室温反应16h。TLC监测反应。直接将反应液减压浓缩后硅胶柱色谱分离纯化(洗脱剂：MeOH/DCM＝0-10％)，得到12F(1.8g，88.7％)。12E (2g, 3.02mmol) was added to methanol (20ml), and then 4-mercaptobutyric acid (2.18g, 18.15mmol) was added, and reacted at room temperature for 16h. The reaction was monitored by TLC. The reaction solution was directly concentrated under reduced pressure and purified by silica gel column chromatography (eluent: MeOH/DCM=0-10%) to obtain 12F (1.8 g, 88.7%).
LCMS m/z＝672.2[M+1] + LCMS m/z=672.2[M+1] +
第六步：Step six:
Figure PCTCN2022131909-appb-000078
Figure PCTCN2022131909-appb-000078
将12F(1.8g，2.68mmol)，N-叔丁氧羰基-1,4-丁二胺(0.76g，4.05mmol)，2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(1.53g，4.02mmol)，N,N-二异丙基乙胺(1.73g，13.37mmol)加入DMF(20ml)中，室温反应4h。LC-MS监测反应完全。加入水(100ml)，搅拌后过滤，固体用水洗涤，干燥，得到粗品12G(2.3g)。12F (1.8g, 2.68mmol), N-tert-butoxycarbonyl-1,4-butanediamine (0.76g, 4.05mmol), 2-(7-azobenzotriazole)-N,N, N',N'-tetramethyluronium hexafluorophosphate (1.53g, 4.02mmol), N,N-diisopropylethylamine (1.73g, 13.37mmol) were added to DMF (20ml), and reacted at room temperature for 4h. LC-MS monitored the completion of the reaction. Water (100ml) was added, stirred and filtered, the solid was washed with water and dried to give crude product 12G (2.3g).
LCMS m/z＝842.3[M+1] + LCMS m/z＝842.3[M+1] +
第七步：Step seven:
Figure PCTCN2022131909-appb-000079
Figure PCTCN2022131909-appb-000079
将12G(2.3g，2.73mmol)加入二氯甲烷(20mL)中，再加入TFA(6ml)，室温反应2h。LC-MS监测反应完全。直接减压浓缩后C18反相柱纯化，流动相A，B组成：流动相A：乙腈；流动相B：水(含0.1％TFA)，(A/B＝50％/50％)分离提纯得到12H的三氟乙酸盐(1.8g，77％)。12G (2.3g, 2.73mmol) was added into dichloromethane (20mL), and TFA (6ml) was added, and reacted at room temperature for 2h. LC-MS monitored the completion of the reaction. C18 reverse-phase column purification after direct decompression concentration, mobile phase A, B composition: mobile phase A: acetonitrile; mobile phase B: water (containing 0.1% TFA), (A/B=50%/50%) separation and purification to obtain 12H trifluoroacetate salt (1.8 g, 77%).
第八步：Step Eight:
Figure PCTCN2022131909-appb-000080
Figure PCTCN2022131909-appb-000080
将17f(800mg，0.98mmol)加入到N,N-二甲基甲酰胺(10mL)中，再加入N,N-二异丙基乙胺(380mg，2.94mmol)，滴加Val-Cit-PABC-MMAE(1.09g，0.88mmol)的N,N-二甲基甲酰胺溶液(4mL)，室温搅拌30分钟，再加入N,N-二异丙基乙胺(380mg，2.94mmol)，滴加12H(1.01g，1.18mmol)的N,N-二甲基甲酰胺溶液(4mL)，室温搅拌2h，C18反相制备柱纯化，流动相A,B组成：流动相A：乙腈；流动相B：水(含0.1％TFA)，(A/B＝35％/65％)分离提纯得到12I(1.8g，79％)。Add 17f (800mg, 0.98mmol) to N,N-dimethylformamide (10mL), then add N,N-diisopropylethylamine (380mg, 2.94mmol), add Val-Cit-PABC dropwise -MMAE (1.09g, 0.88mmol) in N,N-dimethylformamide solution (4mL), stirred at room temperature for 30 minutes, then added N,N-diisopropylethylamine (380mg, 2.94mmol), dropwise 12H (1.01g, 1.18mmol) in N,N-dimethylformamide solution (4mL), stirred at room temperature for 2h, C18 reverse-phase preparative column purification, mobile phase A, B composition: mobile phase A: acetonitrile; mobile phase B : Water (containing 0.1% TFA), (A/B=35%/65%) was isolated and purified to obtain 12I (1.8g, 79%).
LCMS m/z＝1158.80[M/2+1] +. LCMS m/z=1158.80[M/2+1] + .
第九步：Step Nine:
Figure PCTCN2022131909-appb-000081
Figure PCTCN2022131909-appb-000081
将12I(1g，0.43mmol)加入到10％哌啶的N,N-二甲基甲酰胺(10mL)溶液中，室温搅拌20分钟，C18反相制备柱纯化,流动相A,B组成：流动相A：乙腈；流动相B：水(含0.1％TFA)，(A/B＝50％/50％)分离提纯得到12J(500mg，55.5％)。Add 12I (1g, 0.43mmol) to 10% piperidine in N,N-dimethylformamide (10mL) solution, stir at room temperature for 20 minutes, C18 reverse phase preparative column purification, mobile phase A, B composition: mobile phase Phase A: acetonitrile; mobile phase B: water (containing 0.1% TFA), (A/B=50%/50%) was separated and purified to obtain 12J (500 mg, 55.5%).
LCMS m/z＝1047.5[M/2+1] +. LCMS m/z=1047.5[M/2+1] + .
第十步：Step ten:
Figure PCTCN2022131909-appb-000082
Figure PCTCN2022131909-appb-000082
将12J(500mg，0.24mmol)和戊二酸酐(55mg，0.48mmol)加入到N,N-二甲基乙酰胺(5mL)溶液中，再加入N,N-二异丙基乙胺(93mg，0.72mmol)，室温搅拌2小时，C18反相制备柱纯化,流动相A,B组成：流动相A：乙腈；流动相B：水(含0.1％TFA)，(A/B＝50％/50％)分离提纯得到12K(430mg，81％)。12J (500mg, 0.24mmol) and glutaric anhydride (55mg, 0.48mmol) were added to N,N-dimethylacetamide (5mL) solution, and N,N-diisopropylethylamine (93mg, 0.72mmol), stirring at room temperature for 2 hours, C18 reverse phase preparative column purification, mobile phase A, B composition: mobile phase A: acetonitrile; mobile phase B: water (containing 0.1% TFA), (A/B=50%/50 %) separation and purification to obtain 12K (430mg, 81%).
LCMS m/z＝1104.4[M/2+1] +. LCMS m/z=1104.4[M/2+1] + .
第十一步：Eleventh step:
Figure PCTCN2022131909-appb-000083
Figure PCTCN2022131909-appb-000083
将12K(430mg，0.19mmol)加入到DMA(5mL)中，氮气保护，降温至0℃，加入N-羟基丁二酰亚胺(220mg，1.91mmol)和EDCI(365mg，1.91mmol)，室温搅拌24h，C18反相制备柱纯化(0.1％TFA)(H 2O:ACN＝55:45)，得到12L(350mg，收率＝79.9％)。 12K (430mg, 0.19mmol) was added to DMA (5mL), under nitrogen protection, cooled to 0°C, N-hydroxysuccinimide (220mg, 1.91mmol) and EDCI (365mg, 1.91mmol) were added, stirred at room temperature After 24h, C18 reverse phase preparative column purification (0.1% TFA) (H 2 O:ACN=55:45) gave 12L (350mg, yield=79.9%).
LCMS m/z＝1153.1[M/2+1] + LCMS m/z=1153.1[M/2+1] +
第十二步：Step twelve:
Figure PCTCN2022131909-appb-000084
Figure PCTCN2022131909-appb-000084
将12L(45mg，19umol)和HSK-P51(40mg，15umol)加入到DMA(2mL)中，再加入N,N-二异丙基乙胺(6mg，45umol)，室温搅拌18h。用液相制备柱分离提纯(液相制备条件：C18反相制备柱，流动相为含0.1％三氟乙酸的去离子水(A)，含0.1％三氟乙酸的乙腈(B)，梯度洗脱，B含量＝5％～70％，洗脱时间15min，流速12mL/min，柱温：30℃，保留时间：5.76min)得到15mg化合物12(收率＝20％)。Add 12L (45mg, 19umol) and HSK-P51 (40mg, 15umol) into DMA (2mL), then add N,N-diisopropylethylamine (6mg, 45umol), and stir at room temperature for 18h. Separation and purification with liquid phase preparative column (liquid phase preparation conditions: C18 reverse phase preparative column, mobile phase is deionized water (A) containing 0.1% trifluoroacetic acid, acetonitrile (B) containing 0.1% trifluoroacetic acid, gradient washing B content = 5% to 70%, elution time 15min, flow rate 12mL/min, column temperature: 30°C, retention time: 5.76min) to obtain 15 mg of compound 12 (yield = 20%).
LCMS m/z＝1217.7[M/4+1] +. LCMS m/z=1217.7[M/4+1] + .
生物测试例biological test case
1.HT-1080细胞增殖抑制实验1. HT-1080 cell proliferation inhibition experiment
HT-1080细胞培养条件：EMEM+10％FBS+1％双抗，培养于37℃，5％CO 2孵箱中。第一天收集指数生长期的HT-1080细胞铺板96孔培养板，每孔90μL，铺板密度为500个/孔，于37℃，5％CO 2孵箱中培养过夜。第二天每孔加入10μL不同浓度化合物，使每孔DMSO终浓度均为0.1％，于37℃，5％CO 2孵箱中培养3天。培养结束后，每孔加入50μL检测液(Cell Viability Assay，Promega，G7573)，混匀2分钟，室温孵育10分钟，酶标仪检测化学发光读数。结果按照式(1)处理，计算出化合物各个浓度的增殖抑制率，并使用origin9.2软件，计算抑制率为50％时化合物的浓度IC 50值。其中RLU compound为药物处理组的读数，RLU control为溶剂对照组的平均值。 HT-1080 cell culture conditions: EMEM+10% FBS+1% double antibody, cultured at 37°C, 5% CO 2 incubator. On the first day, HT-1080 cells in the exponential growth phase were collected and plated on 96-well culture plates, 90 μL per well, with a plating density of 500 cells/well, and cultured overnight at 37°C in a 5% CO 2 incubator. On the second day, 10 μL of different concentrations of compounds were added to each well so that the final concentration of DMSO in each well was 0.1%, and cultured at 37° C. in a 5% CO 2 incubator for 3 days. After the incubation, 50 μL of detection solution (Cell Viability Assay, Promega, G7573) was added to each well, mixed for 2 minutes, incubated at room temperature for 10 minutes, and detected by a microplate reader for chemiluminescence readings. The results were processed according to formula (1), and the proliferation inhibition rate of each concentration of the compound was calculated, and the IC 50 value of the concentration of the compound was calculated when the inhibition rate was 50% using origin9.2 software. Among them, RLU compound is the reading of the drug treatment group, and RLU control is the average value of the solvent control group.
Inhibition％＝(1-(RLU compound/RLU control)×100％     式(1)Inhibition%＝(1-(RLU compound/RLU control)×100% Formula (1)
表1Table 1
化合物compound IC 50(nM) IC 50 (nM) Max inh.％10μMMax inh.%10μM
化合物2Compound 2 99 99.199.1
化合物3Compound 3 11 99.199.1
化合物4Compound 4 2929 97.497.4
化合物6Compound 6 21twenty one 97.797.7
化合物7Compound 7 3333 97.097.0
化合物8Compound 8 2525 97.497.4
化合物9Compound 9 2929 98.298.2
化合物10Compound 10 1414 97.897.8
化合物11Compound 11 7.27.2 --
化合物12Compound 12 8.18.1 --
结论：本发明的化合物具有较高的抑制活性。Conclusion: the compound of the present invention has higher inhibitory activity.
2.MOLP8细胞增殖抑制实验2. MOLP8 cell proliferation inhibition experiment
购自DSMZ的人多发性骨髓瘤MOLP8细胞置于RPMI-1640完全培养基(添加20％胎牛血清)中，在37℃、5％CO 2条件下培养。收集处于指数生长期的细胞，用培养基将细胞悬液调整到8000个/135μL。每孔加135μL细胞悬液于96孔细胞培养板，孵育过夜。第二天，加入不同浓度的化合物，置于孵箱中培养孵育5天。培养结束后，按照CellTiter-Glo试剂盒(Promega，Cat#G7573)操作说明，每孔加入75μL预先融化并平衡至室温的CTG溶液，用微孔板震荡器混匀2分钟，于室温放置10分钟后用Envision 2104读板仪(PerkinElmer)测定萤光信号值。按公式[(1–(RLU compound–RLU blank)/(RLU control–RLU blank))×100％]计算细胞增殖抑制率。使用GraphPad Prism软件通过四参数非线性拟合获得IC 50值。 Human multiple myeloma MOLP8 cells purchased from DSMZ were placed in RPMI-1640 complete medium (supplemented with 20% fetal bovine serum) and cultured at 37°C and 5% CO 2 . Cells in the exponential growth phase were collected, and the cell suspension was adjusted to 8000 cells/135 μL with medium. Add 135 μL of cell suspension to each well in a 96-well cell culture plate and incubate overnight. On the second day, different concentrations of compounds were added and cultured in an incubator for 5 days. After the incubation, according to the operating instructions of the CellTiter-Glo kit (Promega, Cat#G7573), add 75 μL of CTG solution that has been melted and equilibrated to room temperature in advance, mix well with a microplate shaker for 2 minutes, and place at room temperature for 10 minutes Afterwards, the fluorescence signal value was measured with Envision 2104 plate reader (PerkinElmer). The cell proliferation inhibition rate was calculated according to the formula [(1–(RLU compound –RLU blank )/(RLU control –RLU blank ))×100%]. IC50 values were obtained by four-parameter nonlinear fitting using GraphPad Prism software.
结论：本发明的化合物在体外对阴性细胞MOLP8活性远弱于对阳性细胞PC3活性，表明其具有良好的细胞选择性。Conclusion: the activity of the compound of the present invention on negative cell MOLP8 is far weaker than that on positive cell PC3 in vitro, indicating that it has good cell selectivity.
3.大鼠药代动力学测试3. Rat pharmacokinetic test
3.1试验动物：雄性SD大鼠，220g左右，6～8周龄，3只/化合物。购于成都达硕实验动物有限公司。3.1 Test animals: male SD rats, about 220 g, 6-8 weeks old, 3 rats/compound. purchased from Chengdu Dashuo Experimental Animal Co., Ltd.
3.2试验设计：试验当天，6只SD大鼠按体重随机分组。给药前1天禁食不禁水12～14h，给药后4h给食。3.2 Experimental design: On the day of the experiment, 6 SD rats were randomly divided into groups according to body weight. One day before the administration, fasting without water for 12-14 hours, and giving food 4 hours after the administration.
表2.给药信息Table 2. Dosing Information
Figure PCTCN2022131909-appb-000085
Figure PCTCN2022131909-appb-000085
注：50mM Acetate,10％sucrose PH 5Note: 50mM Acetate, 10% sucrose PH 5
于给药前及给药后异氟烷麻醉经眼眶取血0.15ml，置于EDTAK2离心管中，5000rpm，4℃离心10min，收集血浆。静脉组和灌胃组采血时间点均为：0,5,15,30min,1,2,4,6,8,24h。分析检测前，所有样品存于-80℃，用LC-MS/MS对样品进行定量分析。Before and after administration, 0.15 ml of blood was collected through the orbit under isoflurane anesthesia, placed in an EDTAK2 centrifuge tube, centrifuged at 5000 rpm, 4°C for 10 min, and plasma was collected. The time points of blood collection in the intravenous group and intragastric administration group were both: 0, 5, 15, 30min, 1, 2, 4, 6, 8, 24h. Before analysis and detection, all samples were stored at -80°C, and the samples were quantitatively analyzed by LC-MS/MS.
表3.测试化合物在大鼠血浆中的药代动力学参数Table 3. Pharmacokinetic parameters of test compounds in rat plasma
Figure PCTCN2022131909-appb-000086
Figure PCTCN2022131909-appb-000086
-：不适用。NC：无法计算-:not applicable. NC: cannot be calculated
对照化合物为WO2016067035A1的BT17BDC-18，结构式为The reference compound is BT17BDC-18 of WO2016067035A1, the structural formula is
Figure PCTCN2022131909-appb-000087
Figure PCTCN2022131909-appb-000087
结论：化合物4具有良好的大鼠体内药代动力特征。Conclusion: Compound 4 has good pharmacokinetic characteristics in rats.
4.小鼠药代动力学测试4. Pharmacokinetic test in mice
4.1试验动物：雄性balb/c小鼠，20～25g，9只/化合物。购于成都达硕实验动物有限公司。4.1 Test animals: male balb/c mice, 20-25g, 9/compound. purchased from Chengdu Dashuo Experimental Animal Co., Ltd.
4.2试验设计：试验当天，9只balb/c小鼠按体重随机分组。给药前1天禁食不禁水12～14h，给药后4h给食。4.2 Experimental design: On the day of the experiment, 9 balb/c mice were randomly divided into groups according to body weight. One day before the administration, fasting without water for 12-14 hours, and giving food 4 hours after the administration.
表4.给药信息Table 4. Dosing Information
Figure PCTCN2022131909-appb-000088
Figure PCTCN2022131909-appb-000088
注：50mM Acetate,10％sucrose PH 5Note: 50mM Acetate, 10% sucrose PH 5
于给药前及给药后异氟烷麻醉经眼眶取血0.06mL，置于EDTAK2离心管中，5000rpm，4℃离心10min，收集血浆。静脉组和灌胃组采血时间点均为:0,5,15,30min,1,2,4,6,8,24h。分析检测前，所有样品存于-80℃，用LC-MS/MS对样品进行定量分析。Before and after administration, 0.06 mL of blood was collected through the orbit under isoflurane anesthesia, placed in an EDTAK2 centrifuge tube, centrifuged at 5000 rpm, 4°C for 10 min, and plasma was collected. The time points of blood collection in the intravenous group and intragastric group were: 0, 5, 15, 30min, 1, 2, 4, 6, 8, 24h. Before analysis and detection, all samples were stored at -80°C, and the samples were quantitatively analyzed by LC-MS/MS.
表5.测试化合物在小鼠血浆中的药代动力学参数Table 5. Pharmacokinetic parameters of test compounds in mouse plasma
Figure PCTCN2022131909-appb-000089
Figure PCTCN2022131909-appb-000089
结论：化合物4具有良好的小鼠体内药代动力特征。Conclusion: Compound 4 has good pharmacokinetic characteristics in mice.
5.猴药代动力学测试5. Monkey pharmacokinetic test
5.1试验动物：雄性食蟹猴，3～5kg，3～6年龄，3只/化合物。购于苏州西山生物技术有限公司。5.1 Test animals: male cynomolgus monkeys, 3-5kg, 3-6 years old, 3 animals/compound. purchased from Suzhou Xishan Biotechnology Co., Ltd.
5.2试验方法：试验当天，3只猴按体重随机分组。给药前1天禁食不禁水14～18h，给药后4h给食。5.2 Test method: On the test day, 3 monkeys were randomly divided into groups according to body weight. One day before the administration, fasting without water for 14-18 hours, and giving food 4 hours after the administration.
于给药前及给药后通过四肢静脉取血1.0mL，置于EDTAK2离心管中。5000rpm，4℃离心10min，收集血浆。静脉组采血时间点均为：0,2,5,15,30min,1,2,4,6,8,24h。分析检测前，所有样品存于-80℃，用LC-MS/MS对样品进行定量分析。Before and after administration, 1.0 mL of blood was collected from the veins of the extremities, and placed in EDTAK2 centrifuge tubes. Centrifuge at 5000 rpm for 10 min at 4°C to collect plasma. The time points of blood collection in the vein group were: 0, 2, 5, 15, 30min, 1, 2, 4, 6, 8, 24h. Before analysis and detection, all samples were stored at -80°C, and the samples were quantitatively analyzed by LC-MS/MS.
结论：本发明化合物具有良好的猴体内药代动力特征。Conclusion: The compounds of the present invention have good pharmacokinetic characteristics in monkeys.
6.比格犬药代动力学测试6. Beagle pharmacokinetic test
6.1试验动物：雄性比格犬，8～11kg左右，3只/化合物，购于北京玛斯生物技术有限公司。6.1 Experimental animals: male Beagle dogs, about 8-11kg, 3 dogs/compound, purchased from Beijing Masi Biotechnology Co., Ltd.
6.2试验方法：试验当天，3只比格犬按体重随机分组。给药前1天禁食不禁水12～14h，给药后4h给食。6.2 Test method: On the day of the test, 3 Beagle dogs were randomly divided into groups according to body weight. One day before the administration, fasting without water for 12-14 hours, and giving food 4 hours after the administration.
于给药前及给药后通过颈静脉或四肢静脉取血1ml，置于EDTAK2离心管中。5000rpm，4℃离心10min，收集血浆。静脉组和灌胃组采血时间点均为:0,5,15,30min,1,2,4,6,8,10,12,24h。分析检测前，所有样品存于-80℃，用LC-MS/MS对样品进行定量分析。Before and after administration, 1ml of blood was collected from jugular vein or limb vein, and placed in EDTAK2 centrifuge tube. Centrifuge at 5000 rpm for 10 min at 4°C to collect plasma. The time points of blood collection in the intravenous group and intragastric group were: 0, 5, 15, 30min, 1, 2, 4, 6, 8, 10, 12, 24h. Before analysis and detection, all samples were stored at -80°C, and the samples were quantitatively analyzed by LC-MS/MS.
结论：本发明化合物具有良好的犬体内药代动力特征。Conclusion: The compound of the present invention has good pharmacokinetic characteristics in dogs.
Figure PCTCN2022131909-appb-000090
Figure PCTCN2022131909-appb-000090
Figure PCTCN2022131909-appb-000091
Figure PCTCN2022131909-appb-000091
Figure PCTCN2022131909-appb-000092
Figure PCTCN2022131909-appb-000092
Figure PCTCN2022131909-appb-000093
Figure PCTCN2022131909-appb-000093
Figure PCTCN2022131909-appb-000094
Figure PCTCN2022131909-appb-000094
Figure PCTCN2022131909-appb-000095
Figure PCTCN2022131909-appb-000095
Figure PCTCN2022131909-appb-000096
Figure PCTCN2022131909-appb-000096
Figure PCTCN2022131909-appb-000097
Figure PCTCN2022131909-appb-000097
Figure PCTCN2022131909-appb-000098
Figure PCTCN2022131909-appb-000098
Figure PCTCN2022131909-appb-000099
Figure PCTCN2022131909-appb-000099
Figure PCTCN2022131909-appb-000100
Figure PCTCN2022131909-appb-000100
Figure PCTCN2022131909-appb-000101
Figure PCTCN2022131909-appb-000101
Figure PCTCN2022131909-appb-000102
Figure PCTCN2022131909-appb-000102
Figure PCTCN2022131909-appb-000103
Figure PCTCN2022131909-appb-000103
Figure PCTCN2022131909-appb-000104
Figure PCTCN2022131909-appb-000104
Figure PCTCN2022131909-appb-000105
Figure PCTCN2022131909-appb-000105
Figure PCTCN2022131909-appb-000106
Figure PCTCN2022131909-appb-000106
Figure PCTCN2022131909-appb-000107
Figure PCTCN2022131909-appb-000107
Figure PCTCN2022131909-appb-000108
Figure PCTCN2022131909-appb-000108
Figure PCTCN2022131909-appb-000109
Figure PCTCN2022131909-appb-000109
Figure PCTCN2022131909-appb-000110
Figure PCTCN2022131909-appb-000110
Figure PCTCN2022131909-appb-000111
Figure PCTCN2022131909-appb-000111
Figure PCTCN2022131909-appb-000112
Figure PCTCN2022131909-appb-000112

Claims (21)

  1. 一种化合物，其包含多肽结构和芳香族分子支架，所述多肽结构包含三个独立选自Cys和Hcys的残基，且三个残基中至少一个是Hcys残基，所述三个残基被两段氨基酸序列隔开，所述多肽结构的Cys或Hcys残基经硫醚键与所述分子支架连接，从而在所述分子支架上形成两个多肽环。A compound comprising a polypeptide structure and an aromatic molecular scaffold, the polypeptide structure comprising three residues independently selected from Cys and Hcys, and at least one of the three residues is a Hcys residue, the three residues Separated by two amino acid sequences, the Cys or Hcys residues of the polypeptide structure are connected to the molecular scaffold through a thioether bond, thereby forming two polypeptide rings on the molecular scaffold.
  2. 根据权利要求1所述的化合物，其中所述多肽结构包含以下的氨基酸序列：The compound according to claim 1, wherein said polypeptide structure comprises the following amino acid sequence:
    Xa 1-A 1-Xa 2-A 2-Xa 3 Xa 1 -A 1 -Xa 2 -A 2 -Xa 3
    其中，A 1、A 2表示Xa 1、Xa 2、Xa 3之间的氨基酸残基序列，并且A 1和A 2各自独立地包含5或6个氨基酸残基； Wherein, A 1 and A 2 represent the amino acid residue sequence between Xa 1 , Xa 2 , and Xa 3 , and A 1 and A 2 each independently contain 5 or 6 amino acid residues;
    Xa 1、Xa 2、Xa 3独立地为Cys或Hcys残基，且至少有一个是Hcys残基，所述芳香族分子支架与所述多肽的Xa 1、Xa 2、Xa 3分别形成硫醚键从而在所述分子支架上形成两个多肽环。 Xa 1 , Xa 2 , and Xa 3 are independently Cys or Hcys residues, and at least one of them is a Hcys residue, and the aromatic molecular scaffold forms thioether bonds with Xa 1 , Xa 2 , and Xa 3 of the polypeptide, respectively Thus two polypeptide loops are formed on the molecular scaffold.
  3. 根据权利要求2所述的化合物，其中所述多肽结构包含以下所示的氨基酸序列：The compound according to claim 2, wherein the polypeptide structure comprises the amino acid sequence shown below:
    Xa 1-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Xa 2-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Xa 3； Xa 1 -(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Xa 2 -Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Xa 3 ;
    具体的，其中所述多肽结构包含SEQ ID NO:1～SEQ ID NO:7任一所示的氨基酸序列：Specifically, wherein the polypeptide structure comprises the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7:
    Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:1)；Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys (SEQ ID NO: 1) ;
    Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:2)；Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys (SEQ ID NO:2) ;
    Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:3)；Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys (SEQ ID NO:3) ;
    Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys(SEQ ID NO:4)；Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys (SEQ ID NO:4) ;
    Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:5)；Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys (SEQ ID NO:5) ;
    Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys(SEQ ID NO:6)；Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys (SEQ ID NO: 6) ;
    Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys(SEQ ID NO:7)。Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys (SEQ ID NO: 7) .
  4. 根据权利要求3所述的化合物，其中所述多肽结构包含SEQ ID NO:8～SEQ ID NO:14任一所示的氨基酸序列：The compound according to claim 3, wherein the polypeptide structure comprises the amino acid sequence shown in any one of SEQ ID NO:8～SEQ ID NO:14:
    (β-Ala)-Sar 10-Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:8)； (β-Ala)-Sar 10 -Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Hcys (SEQ ID NO: 8);
    (β-Ala)-Sar 10-Ala-Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:9)； (β-Ala)-Sar 10 -Ala-Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Hcys (SEQ ID NO:9);
    (β-Ala)-Sar 10-Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:10)； (β-Ala)-Sar 10 -Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Hcys (SEQ ID NO: 10);
    (β-Ala)-Sar 10-Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys(SEQ ID NO:11)； (β-Ala)-Sar 10 -Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Cys (SEQ ID NO: 11);
    (β-Ala)-Sar 10-Ala-Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Hcys(SEQ ID NO:12)； (β-Ala)-Sar 10 -Ala-Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Hcys (SEQ ID NO: 12);
    (β-Ala)-Sar 10-Ala-Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys(SEQ ID NO:13)； (β-Ala)-Sar 10 -Ala-Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Hcys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Cys (SEQ ID NO: 13);
    (β-Ala)-Sar 10-Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu-Gly)-Cys(SEQ ID NO:14)。 (β-Ala)-Sar 10 -Ala-Hcys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBu- Gly)-Cys (SEQ ID NO: 14).
  5. 根据权利要求3所述的化合物，其是具有以下结构之一的双环肽：The compound according to claim 3, which is a bicyclic peptide having one of the following structures:
    Figure PCTCN2022131909-appb-100001
    Figure PCTCN2022131909-appb-100001
    其中，当Xa 1、Xa 2或Xa 3为Cys时，对应地，n 1、n 2或n 3为1； Wherein, when Xa 1 , Xa 2 or Xa 3 is Cys, correspondingly, n 1 , n 2 or n 3 is 1;
    当Xa 1、Xa 2或Xa 3为Hcys，对应地，n 1、n 2或n 3为2。 When Xa 1 , Xa 2 or Xa 3 is Hcys, correspondingly, n 1 , n 2 or n 3 is 2.
  6. 根据权利要求5所述的化合物，其中：The compound according to claim 5, wherein:
    Xa 1、Xa 2、Xa 3为Hcys，n 1、n 2、n 3为2；或 Xa 1 , Xa 2 , Xa 3 are Hcys, n 1 , n 2 , n 3 are 2; or
    Xa 1、Xa 3为Hcys，Xa 2为Cys，n 1、n 3为2，n 2为1；或 Xa 1 and Xa 3 are Hcys, Xa 2 is Cys, n 1 and n 3 are 2, and n 2 is 1; or
    Xa 1、Xa 2为Cys，Xa 3为Hcys，n 1、n 2为1，n 3为2。 Xa 1 and Xa 2 are Cys, Xa 3 is Hcys, n 1 and n 2 are 1, and n 3 is 2.
  7. 根据权利要求5所述的化合物，其选自以下结构之一：The compound according to claim 5, which is selected from one of the following structures:
    Figure PCTCN2022131909-appb-100002
    Figure PCTCN2022131909-appb-100002
  8. 权利要求1至7中任一项所述的化合物作为MT1-MMP的配体在筛选和/或制备药物中的应用。The application of the compound described in any one of claims 1 to 7 as a ligand of MT1-MMP in screening and/or preparing drugs.
  9. 一种药物缀合物或其药学上可接受的盐，所述缀合物包含通过连接子缀合至一个或多个效应物和/或官能团的权利要求1-7中任意一项所述的化合物。A drug conjugate or a pharmaceutically acceptable salt thereof, the conjugate comprising any one of claims 1-7 conjugated to one or more effectors and/or functional groups via a linker compound.
  10. 根据权利要求9所述的药物缀合物或其药学上可接受的盐，其中所述效应物为细胞毒性剂，所述细胞毒性剂选自美登木素生物碱、单甲基奥瑞他汀、喜树碱衍生物中的至少一种；或 者，所述细胞毒性剂选自美登素DM1、美登素DM4、一甲基奥瑞他汀E、7-乙基-10-羟基喜树碱中的至少一种。The drug conjugate according to claim 9 or a pharmaceutically acceptable salt thereof, wherein the effector is a cytotoxic agent selected from maytansinoids, monomethyl auristatin , at least one of camptothecin derivatives; or, the cytotoxic agent is selected from maytansine DM1, maytansine DM4, monomethyl auristatin E, 7-ethyl-10-hydroxycamptothecin at least one of the
  11. 根据权利要求9所述的药物缀合物或其药学上可接受的盐，其中所述连接子为肽类连接子、二硫化物连接子或pH依赖型连接子。The drug conjugate according to claim 9 or a pharmaceutically acceptable salt thereof, wherein the linker is a peptide linker, a disulfide linker or a pH-dependent linker.
  12. 根据权利要求11所述的药物缀合物或其药学上可接受的盐，所述二硫化物连接子选自DMDS、MDS、DSDM、NDMDS或式II结构：The drug conjugate according to claim 11 or a pharmaceutically acceptable salt thereof, wherein the disulfide linker is selected from DMDS, MDS, DSDM, NDMDS or the structure of formula II:
    Figure PCTCN2022131909-appb-100003
    Figure PCTCN2022131909-appb-100003
    其中，R 1、R 2、R 3和R 4独立地选自H、甲基、乙基、丙基和异丙基； Wherein, R 1 , R 2 , R 3 and R 4 are independently selected from H, methyl, ethyl, propyl and isopropyl;
    p和q独立地为1、2、3、4或5；p and q are independently 1, 2, 3, 4 or 5;
    所述肽类连接子选自：-Cit-Val-、-Phe-Lys-和-Val-Lys-；The peptide linker is selected from: -Cit-Val-, -Phe-Lys- and -Val-Lys-;
    所述pH依赖型连接子选自顺乌头酸酐。The pH-dependent linker is selected from cis-aconitic anhydride.
  13. 根据权利要求11所述的药物缀合物或其药学上可接受的盐，其中所述连接子为-PABC-Cit-Val-戊二酰-或-PABC-环丁基-Ala-Cit-βAla-，其中PABC代表p-氨基苄基氨基甲酸酯。The drug conjugate according to claim 11 or a pharmaceutically acceptable salt thereof, wherein the linker is -PABC-Cit-Val-glutaryl- or -PABC-cyclobutyl-Ala-Cit-βAla -, where PABC stands for p-aminobenzylcarbamate.
  14. 根据权利要求11所述的药物缀合物或其药学上可接受的盐，其中所述连接子为The drug conjugate according to claim 11 or a pharmaceutically acceptable salt thereof, wherein the linker is
    Figure PCTCN2022131909-appb-100004
    Figure PCTCN2022131909-appb-100004
    其中k选自1-20的任一整数。Wherein k is selected from any integer of 1-20.
  15. 根据权利要求9所述的药物缀合物或其药学上可接受的盐，其中所述药物缀合物具有式III、式IV、式V或式VI的结构：The drug conjugate or a pharmaceutically acceptable salt thereof according to claim 9, wherein the drug conjugate has the structure of formula III, formula IV, formula V or formula VI:
    Figure PCTCN2022131909-appb-100005
    Figure PCTCN2022131909-appb-100005
    其中，R 1、R 2、R 3和R 4独立地选自H、甲基、乙基、丙基和异丙基； Wherein, R 1 , R 2 , R 3 and R 4 are independently selected from H, methyl, ethyl, propyl and isopropyl;
    p和q独立地为1、2、3、4或5；p and q are independently 1, 2, 3, 4 or 5;
    k独立地为1-10的任一整数；k is independently any integer of 1-10;
    Xa 1、Xa 2、Xa 3独立地为Cys或Hcys残基，且至少有一个是Hcys残基； Xa 1 , Xa 2 , and Xa 3 are independently Cys or Hcys residues, and at least one of them is a Hcys residue;
    当Xa 1、Xa 2或Xa 3为Cys时，对应地，n 1、n 2或n 3为1； When Xa 1 , Xa 2 or Xa 3 is Cys, correspondingly, n 1 , n 2 or n 3 is 1;
    当Xa 1、Xa 2或Xa 3为Hcys，对应地，n 1、n 2或n 3为2。 When Xa 1 , Xa 2 or Xa 3 is Hcys, correspondingly, n 1 , n 2 or n 3 is 2.
  16. 根据权利要求15所述的药物缀合物或其药学上可接受的盐，其中：The drug conjugate or a pharmaceutically acceptable salt thereof according to claim 15, wherein:
    R 1、R 2、R 3和R 4独立地选自H或甲基； R 1 , R 2 , R 3 and R 4 are independently selected from H or methyl;
    p和q独立地为1或2；p and q are independently 1 or 2;
    k独立地为1-5的任一整数；且k is independently any integer from 1 to 5; and
    Xa 1、Xa 2、Xa 3为Hcys，n 1、n 2、n 3为2；或 Xa 1 , Xa 2 , Xa 3 are Hcys, n 1 , n 2 , n 3 are 2; or
    Xa 1、Xa 3为Hcys，Xa 2为Cys，n 1、n 3为2，n 2为1；或 Xa 1 and Xa 3 are Hcys, Xa 2 is Cys, n 1 and n 3 are 2, and n 2 is 1; or
    Xa 1、Xa 2为Cys，Xa 3为Hcys，n 1、n 2为1，n 3为2。 Xa 1 and Xa 2 are Cys, Xa 3 is Hcys, n 1 and n 2 are 1, and n 3 is 2.
  17. 一种药物缀合物或其药学上可接受的盐，所述药物缀合物选自以下结构之一：A drug conjugate or a pharmaceutically acceptable salt thereof, wherein the drug conjugate is selected from one of the following structures:
    Figure PCTCN2022131909-appb-100006
    Figure PCTCN2022131909-appb-100006
    Figure PCTCN2022131909-appb-100007
    Figure PCTCN2022131909-appb-100007
    Figure PCTCN2022131909-appb-100008
    Figure PCTCN2022131909-appb-100008
  18. 一种药物组合物，其包含权利要求1至7中任一项所述的化合物和/或权利要求9-17任意一项所述的药物缀合物或其药学上可接受的盐，以及药学上可接受的载体和/或赋形剂。A pharmaceutical composition comprising the compound according to any one of claims 1 to 7 and/or the drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 9-17, and a pharmaceutical acceptable carriers and/or excipients.
  19. 权利要求1至7中任一项所述的化合物，权利要求9-17任意一项所述的药物缀合物或其药学上可接受的盐，或者权利要求18所述的组合物，在制备预防和/或治疗过度表达MT1-MMP的疾病的药物中的用途，优选过度表达MT1-MMP的疾病选自以下疾病中的至少一种：神经母细胞瘤、小细胞肺癌、膀胱癌、胃癌、结肠癌、肝癌。The compound according to any one of claims 1 to 7, the drug conjugate according to any one of claims 9-17 or a pharmaceutically acceptable salt thereof, or the composition according to claim 18, during preparation Use in medicines for preventing and/or treating diseases overexpressing MT1-MMP, preferably the diseases overexpressing MT1-MMP are selected from at least one of the following diseases: neuroblastoma, small cell lung cancer, bladder cancer, gastric cancer, Colon cancer, liver cancer.
  20. 一种药物组合物或药物制剂，所述的药物组合物或药物制剂包含选1-1500mg的权利要求1至7中任一项所述的化合物或权利要求9至17中任一项所述的药物缀合物或其药学上可接受的盐，以及药学上可接受的载体和/或赋形剂。A pharmaceutical composition or pharmaceutical preparation, said pharmaceutical composition or pharmaceutical preparation comprising 1-1500 mg of the compound described in any one of claims 1 to 7 or the compound described in any one of claims 9 to 17 A drug conjugate or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or excipient.
  21. 一种用于治疗哺乳动物或人的疾病的方法，所述方法包括给予受试者治疗有效量的权利要求1至7中任一项所述的化合物或权利要求9至17中任一项所述的药物缀合物或其药学上可接受的盐，治疗有效量优选为1-1500mg，所述的疾病优选为神经母细胞瘤、小细胞肺癌、膀胱癌、胃癌、结肠癌、肝癌中的至少一种。A method for treating a disease in a mammal or a human, the method comprising administering to a subject a therapeutically effective amount of the compound of any one of claims 1 to 7 or any one of claims 9 to 17 The above-mentioned drug conjugate or a pharmaceutically acceptable salt thereof, the therapeutically effective dose is preferably 1-1500 mg, and the disease is preferably neuroblastoma, small cell lung cancer, bladder cancer, gastric cancer, colon cancer, liver cancer at least one.
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