WO2018103739A1 - Antibody-drug conjugate, preparation method, intermediate, pharmaceutical composition and use - Google Patents
Antibody-drug conjugate, preparation method, intermediate, pharmaceutical composition and use Download PDFInfo
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- WO2018103739A1 WO2018103739A1 PCT/CN2017/115281 CN2017115281W WO2018103739A1 WO 2018103739 A1 WO2018103739 A1 WO 2018103739A1 CN 2017115281 W CN2017115281 W CN 2017115281W WO 2018103739 A1 WO2018103739 A1 WO 2018103739A1
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- 238000010367 cloning Methods 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 229940126214 compound 3 Drugs 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 231100000050 cytotoxic potential Toxicity 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
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- 238000011033 desalting Methods 0.000 description 1
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- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 108010087914 epidermal growth factor receptor VIII Proteins 0.000 description 1
- ZANNOFHADGWOLI-UHFFFAOYSA-N ethyl 2-hydroxyacetate Chemical compound CCOC(=O)CO ZANNOFHADGWOLI-UHFFFAOYSA-N 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 102000045108 human EGFR Human genes 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 229940044173 iodine-125 Drugs 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 210000001853 liver microsome Anatomy 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- WKPWGQKGSOKKOO-RSFHAFMBSA-N maytansine Chemical compound CO[C@@H]([C@@]1(O)C[C@](OC(=O)N1)([C@H]([C@@H]1O[C@@]1(C)[C@@H](OC(=O)[C@H](C)N(C)C(C)=O)CC(=O)N1C)C)[H])\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 WKPWGQKGSOKKOO-RSFHAFMBSA-N 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 125000000250 methylamino group Chemical group [H]N(*)C([H])([H])[H] 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 229910002055 micronized silica Inorganic materials 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- LPNBBFKOUUSUDB-UHFFFAOYSA-N p-toluic acid Chemical compound CC1=CC=C(C(O)=O)C=C1 LPNBBFKOUUSUDB-UHFFFAOYSA-N 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229940097886 phosphorus 32 Drugs 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 230000004537 potential cytotoxicity Effects 0.000 description 1
- 229940069328 povidone Drugs 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- YORCIIVHUBAYBQ-UHFFFAOYSA-N propargyl bromide Chemical compound BrCC#C YORCIIVHUBAYBQ-UHFFFAOYSA-N 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 229940082787 spirulina Drugs 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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- 231100000027 toxicology Toxicity 0.000 description 1
- 229940043263 traditional drug Drugs 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000001573 trophoblastic effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
- C07D498/14—Ortho-condensed systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5383—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with heterocyclic ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
-
- 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
- A61K47/68—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 an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
-
- 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
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Definitions
- the present invention relates to an antibody drug conjugate, a process for the preparation thereof, an intermediate, a pharmaceutical composition and use thereof.
- a typical antibody drug conjugate comprises monoclonal antibodies (mAbs) that bind to cancer cell surface specific antigens and bind these specific antibodies to a highly toxic drug via a cleavable linker.
- the mechanism of action of ADC is to recognize and bind to specific antigens by antibodies, trigger a series of reactions, and then enter the cytoplasm through endocytosis, and lysosomal enzymes release strong toxic drugs to kill cancer cells.
- mAbs monoclonal antibodies
- mAbs monoclonal antibodies
- Monoclonal antibodies in ADCs include proteins on the surface of immune system B cells and T cells, such as CD20, CD22, and human epidermal growth factor receptor 2 (Her2) and prostate specific membrane antigen (PSMA).
- proteins on the surface of immune system B cells and T cells such as CD20, CD22, and human epidermal growth factor receptor 2 (Her2) and prostate specific membrane antigen (PSMA).
- anthracyclines are antibiotic compounds that exhibit cytotoxic activity, including doxorubicin, epirubicin, idarubicin, and daunorubicin. Studies have shown that anthracycline antibiotics can kill cells through a variety of different mechanisms, including: 1) embedding drug molecules in the DNA of cells to inhibit DNA-dependent nucleic acid synthesis; 2) by free radical drug production and reaction with cellular macromolecules Causes damage to cells; 3) Interaction of drug molecules with cell membranes. Due to the cytotoxic potential of anthracyclines, they have been used to treat a variety of cancers including leukemia, breast cancer, lung cancer, ovarian cancer and the like.
- PNU-159682 has stronger in vitro and in vivo tumor model cytotoxicity than nemoxil (Beulz-Riche, et. al. Fundamental & Clinical Pharmacology, 2001, 15, 373,; Quintieri, L., Geroni, C., et. al .Clinical. Cancer. Research, 2005, 11, 1608; EP 0889898; WO 2004/082689; WO 2004/082579).
- the use of antibody drug conjugates can target the toxin drugs capable of killing or always tumor cells to the tumor, and the general formula for seeking the maximum efficacy of the drug can significantly improve the selectivity and reduce the side effects of the antitumor drugs (Xie). Et al., Expert. Opin. Biol. Ther., 2006, 6, 281.; Kovtun et al., Cancer. Res., 2006, 66, 3214.; Law et al, Cancer. Res., 2006, 66, 2328. Wu et al, Nature. Biotech. 2005, 23, 1137.; Lamber J. et al. Current. Opin. in Pharmacol. 2005, 5, 543.; Hamann P. et al., Expert. Opin. Ther. Patents. 2005, 15, 1087.; Payne, G., Cancer. Cell. 2003, 3, 207.; Trail et al, Cancer. Immunol. Immunother., 2003, 52, 328.).
- antibody drug conjugates In a typical antibody drug conjugate, the choice of antibody and drug will depend on the particular disease, which has a significant impact on the safety and efficacy of the antibody drug conjugate. Factors that determine the efficacy of antibody drug conjugates include the stability of the linker unit and its cleavage sensitivity, cell surface internalization, transport, and release of cytotoxin.
- T-DM1 which is much less cytotoxic than some anthracycline antibiotics, is prone to prematurely degrade and release toxins before endocytosis of antigen-binding protein (Abu), causing side effects, and the drug/antibody obtained by coupling it with antibodies
- Abu antigen-binding protein
- the ratio is low and the distribution changes are relatively large, and it is difficult to accurately control the efficacy and safety (such as patent application WO2012/061590 A1 or WO201139721).
- the technical problem to be solved by the present invention is to overcome the prior art antibody drug conjugate which lacks cytotoxin release efficiency, high cytotoxicity and good anticancer effect, and provides an antibody drug conjugate and preparation method thereof. , intermediates, pharmaceutical compositions and applications.
- the antibody drug conjugate of the invention has high cytotoxicity, good anticancer effect and good market application prospect.
- the present invention provides an antibody drug conjugate of the formula IB, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, and a pharmaceutically acceptable thereof. Salt or its prodrug,
- the mAb is a monoclonal antibody fragment
- the monoclonal antibody includes, but is not limited to, Herceptin, an anti-TPBG (embryonic trophoblastic glycoprotein) antibody, an anti-CD70 antibody or an anti-EGFRVIII (epidermal growth factor receptor) antibody
- the monoclonal antibody may be a monoclonal antibody conventional in the art as long as it can form an antibody drug conjugate with a fragment other than the mAb in the antibody drug conjugate of the formula IB.
- 0 ⁇ k ⁇ 8 (preferably, 0 ⁇ k ⁇ 6, such as 0.3, 0.73, 3.21, 3.76, 2.65, 3.96, 3.31, 2.98, 1.80, 2.23, 2.57, 1.89, 1.63, 2.63, 3.05, 1.53, 1.68, 3.10, 1.47, 3.20, 1.56, 1, 2, 3, 4, 5 or 6);
- R 1 is hydrogen, hydroxy, C 1-6 alkoxy (eg methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy or tert-butoxy), C 1-6 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl), -NR a R b or -C(O)NR c R d ;
- R a , R b , R c and R d are each independently hydrogen, C 1-6 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl) Or a C 6-10 aryl group (eg phenyl);
- R 2 and R 3 are each independently C 1-6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl), C 1-6 alkoxy a group (eg methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy or tert-butoxy) or a C 1-6 alkylthio (eg methylthio, Ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio or tert-butylthio);
- C 1-6 alkyl e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutylthio or tert-butylthio
- C 1-6 alkyl e.g
- Y is (preferably R 4a and R 4b are each independently hydrogen, C 1-4 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl), C 3 ⁇ 6 cycloalkyl substituted C 1-4 alkyl (the C 1-4 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl;
- the C 3-6 cycloalkyl group such as a cyclopropyl group;
- the C 3-6 cycloalkyl substituted C 1-4 alkyl group is preferably a cyclopropylmethyl group, a C 3 -6 cycloalkyl group (for example) a cyclopropyl), C 2-8 heteroalkyl group (the hetero atom in the C 2-8 heteroalkyl group may be one
- Z is Or a 4-6 membered heteroarylene group
- the hetero atom in the 4-6 membered heteroarylene group is preferably one or more of N, O and S; the number of hetero atoms is preferably 1, 2 or 3; the 4-6 membered heteroarylene group may be a 4 member, 5 or 6 membered heteroarylene; the 5-membered heteroarylene group is preferably R 5a and R 5b are each independently hydrogen or C 1-4 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl);
- R 6 is hydrogen, C 1-4 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl), C 2-8 heteroalkyl (described
- the hetero atom in the C 2-8 heteroalkyl group may be one or more of O, S and N, and the number of hetero atoms may be 1, 2, 3 or 4; preferably, the C 2 is -8heteroalkyl is CH 3 OCH 2 -), or -(OCH 2 CH 2 ) j2 OH; j2 is 1, 2, 3, 4, 5, 6, 7, or 8;
- p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and when Z is When p is not 0;
- n 0 or 1
- R 7a and R 7b are each independently hydrogen, C 1-4 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl), C 3-6 a cycloalkyl-substituted C 1-4 alkyl group (the C 1-4 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl; a C 3-6 cycloalkyl group such as a cyclopropyl group; the C 3-6 cycloalkyl substituted C 1-4 alkyl group is preferably a cyclopropylmethyl group), a C 3 -6 cycloalkyl group (for example, a cyclopropyl group) a C 2-8 heteroalkyl group (the hetero atom in the C 2-8 heteroalkyl group may be one or
- n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; when there are multiple Es (ie, when n is greater than or equal to 2), E may be the same or different ( For example, it can be ),
- u is 0 or 1
- R 8 is hydrogen, C 1-4 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl), C 3-6 cycloalkyl substituted C a 1-4 alkyl group (the C 1-4 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl; the C 3-6 ring
- the alkyl group is, for example, a cyclopropyl group;
- the C 3-6 cycloalkyl-substituted C 1-4 alkyl group is preferably a cyclopropylmethyl group, a C 3-6 cycloalkyl group (e.g., a cyclopropyl group), C 2- a heteroalkyl group (the hetero atom in the C 2-8 heteroalkyl group may be one or more
- q is 0, 1, 2, 3, 4, 5, 6, 7, 8 , 9, or 10; when a plurality of -C(R 8 )- are contained (that is, when q is greater than or equal to 2), -C(R 8 ) - can be the same or different ( For example, it can be );
- Y is an integer from 0 to 24 (for example, 0, 1, 2, 3, 4, 5 or 6);
- s is 0 or 1;
- Q is t 1 , t 3 and t 4 are each independently 0, 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9, or 10.
- k represents a molar ratio of a drug molecule to a mAb (also referred to as DAR, that is, a drug antibody coupling ratio), and is preferably understood to be: an antibody-conjugated drug obtained by coupling a single monoclonal antibody molecule to a drug.
- the average of the molar ratio of the drug molecule to the monoclonal antibody molecule can be generally hydrophobic-interaction Chromatography (HIC), polyacrylamide-SDS gel electrophoresis (SDS-PAGE, electrophoresis), liquid chromatography mass spectrometry (liquid) Chromatograph-mass spectrometer, LC-MS) was measured.
- k can be 0.43, 0.49, 2.56, 5.64, 2.26, 2.17 or 2.83.
- E is
- E e1, e2, e3, e4, e5, and e6 are each independently 0, 1, 2, 3, 4, 5, 6, 7 , or 8;
- R 7a , R 7b , R 7c , R 7d , R 7e And R 7f and R 7h are each independently hydrogen, C 1-4 alkyl, C 3-6 cycloalkyl substituted C 1-4 alkyl, C 3-6 cycloalkyl, C 2-8 heteroalkyl , -(OCH 2 CH 2 ) j3 OH or J3 is 1, 2, 3, 4, 5, 6, 7, or 8;
- r1, r2, r3 and r4 are independently 0, 1 , 2, 3, 4 , 5 , 6, 7, 8 , 9 or 10;
- R 6e is hydrogen, C 1-4 alkyl, C 2-8 Heteroalkyl or -(OCH 2 CH 2 ) j2a OH;
- j2a is 1, 2, 3, 4, 5, 6, 7, or 8;
- R 8e is hydrogen, C 1-4 alkyl, C 3-6 naphthenic a substituted C 1-4 alkyl group, a C 3-6 cycloalkyl group, a C 2-8 heteroalkyl group or a -(OCH 2 CH 2 ) j4a OH;
- j4a is 1, 2, 3, 4, 5, 6, 7 or 8;
- R e is a glycosyl group (eg, glucosyl) Or maltosyl), Fmoc Or -(OCH 2 CH 2 ) en OH, en is 0, 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10,
- the -NR a R b is a methylamino group or a dimethylamino group.
- m is preferably 1.
- Z is preferably p is preferably 1; m is preferably 0; n is preferably 1; E is preferably u is preferably 0 or 1; q is preferably 2; y is preferably 2; s is preferably 0 or 1 (when u is 1, S is preferably 0); G is preferred Q is preferably (and when G is When Q is preferred ).
- Z is preferably
- R 4a , R 4b , R 5a and R 5b have the same meanings as defined above.
- R 4a , R 4b , R 5a , R 5b and R 6 have the same meanings as defined above.
- Drug molecule fragment of the antibody drug conjugate as shown in Formula IB of the present invention may refer to such drug molecules in the field (ie, anthracyclines such as doxorubicin, epirubicin, idarubicin, idarubicin, naimo) Conventional selection of spirulina, PNU-159682, etc., for example Optimal Most preferred
- the antibody drug conjugate as shown in Formula IB is preferably a compound of Formula IB-1:
- R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q, y, G, s and Q are as defined above.
- the antibody drug conjugate as shown in Formula IB is selected from any one of the following compounds, wherein the mAb in the following compounds is Herceptin, an anti-TPBG antibody, an anti-CD70 antibody or an anti-EGFRVIII antibody:
- the antibody drug conjugate of formula IB is the following compound:
- the monoclonal antibody is reacted with a thiol group or an amino group in an amino acid residue to Connected
- a thiol group or an amino group in an amino acid residue to Connected
- Q is Wherein the S atom is derived from a thiol group in the monoclonal antibody
- Q is Wherein NH is derived from the amino group of the monoclonal antibody.
- the present invention also provides a method for preparing the antibody drug conjugate according to Formula IB, which comprises the steps of: IA compound and single in an organic solvent at a pH of 6-8.
- the cloning antibody is subjected to a coupling reaction as shown below to obtain the antibody drug conjugate as shown in Formula IB;
- k, R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q, y, G, s, Q and mAb are as defined above ;
- Q 1 is The definitions of t 1 , t 3 and t 4 are as described above.
- the present invention particularly preferably as follows:
- the organic solvent is preferably one or more of an amide solvent, a sulfoxide solvent, and an ether solvent.
- the amide solvent is preferably N,N dimethylformamide (DMF) and/or dimethylacetamide (DMA); and the sulfoxide solvent is preferably dimethyl sulfoxide (DMSO).
- the ether solvent is preferably tetrahydrofuran.
- the mass to volume ratio of the IA compound to the organic solvent is preferably from 0.1 mg/mL to 100 mg/mL.
- the molar ratio of the IA compound to the monoclonal antibody is preferably from 1 to 10 (e.g., 6), preferably from 1 to 5.
- the pH is preferably 7.5.
- the pH of 6-8 can be achieved by adding a buffer solution to the reaction solution; the buffer solution is generally a low salt buffer, preferably a phosphate buffer solution (for example, potassium phosphate and potassium dihydrogen phosphate). Buffer solution) or borate buffer solution (for example, a buffer solution of boric acid and sodium borate).
- the temperature of the coupling reaction is preferably 4 ° C to 37 ° C, more preferably 10 to 35 ° C.
- the monoclonal antibody is preferably a monoclonal antibody after dialysis.
- the method of dialysis can be a conventional method of dialysis of monoclonal antibodies in the art.
- the coupling reaction is preferably carried out under gas protection; when the coupling reaction is carried out under gas protection, the gas is preferably nitrogen.
- the method comprises the steps of: dialysis of the monoclonal antibody and the IA compound in a buffer solution having a pH of 6-8; The organic solvent is mixed and the coupling reaction is carried out.
- the mixing order of "mixing the dialyzed monoclonal antibody with the IA compound and the organic solvent" is not particularly limited, and generally the IA compound and the organic solvent are added to the The dialysis monoclonal antibody can be used.
- the progress of the coupling reaction can be monitored by a conventional test method (such as TLC, HPLC or NMR) in the art, generally when the IA compound disappears. The end of the reaction.
- a conventional test method such as TLC, HPLC or NMR
- the IA compound can be prepared by a person skilled in the art according to the disclosure of the specific embodiments of the present invention, combined with the conventional experimental techniques in the art.
- the invention also provides an IA compound,
- R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q, y, G, s and Q 1 have the same meanings as defined above.
- the IA compound is preferably a compound represented by IA-1:
- R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q, y, G, s and Q1 are as defined above.
- the IA compound is selected from any of the following compounds:
- the IA compound is the following compound:
- the invention also provides an IC-1, IC-2 or IC-3 compound:
- R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q and y are as defined above, and R 9 is
- Drug molecule fragments in the IC-1, IC-2 and IC-3 compounds The definition is the same as above.
- the IC-1 compound is preferably an IC-1-1 compound:
- the IC-1 compound is more preferably any of the following compounds:
- the IC-1 compound is preferably any of the following compounds:
- the IC-2 compound is preferably an IC-2-1 compound:
- the IC-3 compound is preferably an IC-3-1 compound:
- the IC-3 compound is more preferably any of the following compounds:
- IC-1 compound, the IC-2 compound and the IC-3 compound can be prepared by those skilled in the art according to the disclosure of the specific embodiments of the present invention in combination with conventional experimental techniques in the art.
- the present invention also provides the antibody drug conjugate, the tautomer, the optical isomer, the hydrate, the solvate, the polymorph, the isotope compound, and the pharmaceutically acceptable compound thereof, as shown in Formula IB.
- the present invention also provides the IA compound, its tautomer, optical isomer, hydrate, solvate, polymorph, pharmaceutically acceptable salt thereof or prodrug thereof for preparation for treatment And/or the use of drugs for the prevention of cancer.
- the present invention also provides the IC-1, IC-2 or IC-3 compound, tautomer, optical isomer, hydrate, solvate, polymorph, isotopic compound thereof, and pharmaceutically thereof thereof.
- the cancer of the present invention may be a cancer conventional in the art, including but not limited to breast cancer, lymphoma, lung cancer, liver cancer, colon cancer, head and neck cancer, bladder cancer, kidney cancer, esophageal cancer, gallbladder cancer, ovarian cancer, Pancreatic cancer, stomach cancer, Cervical cancer, thyroid cancer, prostate cancer, skin cancer including squamous cell carcinoma; leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma , non-Hodgkin's lymphoma, hairy cell lymphoma, Burkitt's lymphoma, acute and chronic myeloid leukemia, myelodysplastic syndrome, promyelocytic leukemia, fibrosarcoma, rhabdomyosarcoma, astrocytoma, nerve Head cell tumor, glioma, schwannomas, melanoma
- the tumor cells of the cancer include, but are not limited to, Her2 positive human BT474 breast tumor cells, Her2 low expressed human MCF-7 breast tumor cells, or human breast tumor MCF7-Her2 stable cells transduced with Her2 in MCF-7. Strain.
- the present invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising the antibody drug conjugate of the formula IB, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph.
- the present invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising the IA compound, a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, which is pharmaceutically acceptable a salt or a prodrug thereof, and one or more pharmaceutically acceptable excipients.
- the present invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising the IC-1, IC-2 or IC-3 compound, tautomer, optical isomer, hydrate, solvate, polymorph thereof a compound, an isotope compound, a pharmaceutically acceptable salt thereof or a prodrug thereof, and one or more pharmaceutically acceptable excipients.
- the pharmaceutically acceptable excipient refers to a conventional pharmaceutical excipient in the pharmaceutical field, and is an antibody drug conjugate of the present invention added to solve the moldability, effectiveness, stability and safety of the preparation.
- All other conventional pharmaceutical materials such as diluents (such as sodium carboxymethyl starch), binders (such as povidone, etc.), disintegrants (such as microcrystalline cellulose, etc.), lubricants (such as stearic acid) Magnesium, micronized silica gel, etc.), as well as other adjuvants.
- the above-mentioned excipients may be selected as needed, and the antibody drug conjugate of the present invention is formulated into a pharmaceutical preparation according to a conventional method in the art; the pharmaceutical preparations are various conventional dosage forms in the art, such as tablets, powders, pills, capsules. Agents, granules, oral liquids, dry suspensions or pills.
- isotopic compound refers to an antibody drug conjugate of the present invention, the IA compound, the IC-1, IC-2 or IC-3 compound, its tautomer, optical isomerism.
- the body, hydrate, solvate, polymorph, isotope compound, pharmaceutically acceptable salt thereof or prodrug thereof contains one or more atomic isotopes of natural or non-natural abundance.
- Non-natural abundance of atomic isotopes including, but not limited to, hydrazine ( 2 H or D), hydrazine ( 3 H or T), iodine-125 ( 125 I), phosphorus-32 ( 32 P), carbon-13 ( 13 C) Or carbon-14 ( 14 C).
- the aforementioned isotopic compounds can also be used as therapeutic or diagnostic agents (i.e., in vivo developers), or as research tools. All isotopic variations of the compounds of the invention, whether or not they are radioactive, are included within the scope of the invention.
- halogen means fluorine, chlorine, bromine, iodine or hydrazine.
- cyano means -CN.
- a heteroalkyl group (C 2-8 heteroalkyl group) generally means that one or more CH 2 structures in an alkyl group (including a branched or linear alkyl group) are heteroatoms (for example, O, S or NH). Instead, it is attached to other groups by a carbon atom, such as CH 2 OCH 2 -, CH 3 CH 2 OCH 2 - or CH 2 OCH(CH 3 )- and the like.
- the present invention relates to a group containing two linking sites (such as G or Q, etc.), the manner of which is generally understood to be linked to the corresponding compound or structure in a left-right sequential manner according to the structure of the group, for example when G for In the case of the compound of formula IB, the linkage is as follows:
- the room temperature referred to in the present invention refers to an ambient temperature of 10 ° C to 35 ° C.
- the reagents and starting materials used in the present invention are commercially available.
- the present invention uses a highly cytotoxic anthracycline antibiotic as a toxin, and designs and synthesizes a series of novel antibody drug conjugates which are stable to acid and peptidase cathepsins, preferably substituted with water-soluble glycosyl groups.
- the use of a stable ether bond for attachment significantly improves water solubility and stability. In vitro activity tests indicate that it has higher cytotoxicity and has good market application prospects.
- the antibody drug conjugate of the present invention exhibits higher cancer cell killing activity than an antibody drug conjugate having a traditional drug such as maytansin, Auristatin or the like as a toxin.
- CL-081 is:
- CL-066 is:
- SMCC is:
- the reaction product 2-1 (1.4 g, 8 mmol) was dissolved in 50 mL of THF.
- propargyl bromide 1.4 mL, 16 mmol
- 10 mL of saturated ammonium chloride solution dropwise to quench the reaction
- extract with ethyl acetate 3 times 40 mL ⁇ 3
- LCMS (ESI) m / z 236.7 (M + H) +.
- reaction product of the previous step 9-1 (40 mg, 0.12 mmol) and the compound 5-1 (40 mg, 0.06 mmol) were weighed into an eggplant type bottle, and 2 mL of t-butanol and 2 mL of methanol were added thereto, followed by sodium ascorbate (Sodium Ascorbate) (6 mg). , 0.03 mmol) and copper sulfate (10 mg, 0.06 mmol), and the reaction mixture was stirred at room temperature overnight.
- sodium ascorbate sodium Ascorbate
- copper sulfate (10 mg, 0.06 mmol
- reaction product 10-1 (15 mg, 0.015 mol) in the previous step was dissolved in 1 mL of acetonitrile, cooled to 0 ° C in an ice water bath, 0.5 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours, and acetonitrile and diethylamine were removed under reduced pressure at room temperature. And using diethylamine with diethylamine twice, 15 mg of crude product 11-1 was obtained as a red solid, which was directly used for the next reaction.
- LCMS (ESI) m / z 780.3 (M + H) +.
- reaction product 5-2 (5 mg, 0.007 mmol) from the previous step was dissolved in 2 mL of acetonitrile, cooled to 0 ° C in an ice water bath, and 1 mL of diethylamine was added and stirred for 1 hour. Concentration and removal of the organic solvent gave 4 mg of crude compound 6-2 as a red solid. The crude product was used directly in the next step.
- LCMS (ESI) m / z 754.1 (M + H) +.
- CL-055 is:
- reaction product 3-3 (20mg, 0.018mol) was dissolved in 2mL acetonitrile in the previous step, cooled to 0 ° C in ice water bath, 1 mL of diethylamine was added, stirred for 1.5 hours, acetonitrile and diethylamine were removed under reduced pressure at room temperature, and dichlorochloride was used. Methane was taken twice with diethylamine to give 15 mg of crude product 4-3 as a red solid which was used directly for the next reaction.
- LCMS (ESI) m / z 888.3 (M + H) +.
- reaction product 6-3 (30mg, 0.03mol) in the previous step was dissolved in 2mL acetonitrile, cooled to 0 ° C in ice water bath, 1 mL of diethylamine was added, stirred for 1.5 hours, acetonitrile and diethylamine were removed under reduced pressure at room temperature, and dichloroethylene was used. Methane was taken twice with diethylamine to give 22 mg of crude product 7-3 as a red solid which was used directly for the next reaction.
- LCMS (ESI) m / z 756.3 (M + H) +.
- reaction product 2-9 (12 g, 24.8 mmol) was dissolved in a mixed solvent of 50 mL of isopropanol and 250 mL of chloroform, and 5 g of silica gel was added thereto, and the mixture was cooled to 0 ° C in an ice water bath, and sodium borohydride (1.4 g, 37.2 mmol) was slowly added in portions. After stirring for 2 hours, the reaction solution was poured into 300 mL of ice water, and the organic phase was separated, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and evaporated. Solid, yield 83%.
- LCMS (ESI) m / z 508.0 (M + Na) +.
- reaction product 5-9 (374 mg, 0.5 mmol) and PNP-CO-PNP (bis(p-nitrophenyl) carbonate, 304 mg, 1 mmol) were dissolved in 10 mL of N,N-dimethylformamide and added to DIPEA. (170 ⁇ L, 1 mmol), stirred at room temperature overnight.
- Example 42 The procedure for coupling a linker toxin to an antibody is as follows:
- Antibody proteins eg Herceptin, anti-TPBG antibodies, anti-CD70 antibodies or anti-EGFRVIII antibodies
- dialysis buffer 25 mM Sodium Borate, sodium borate
- 25 mM NaCl 25 mM NaCl
- 1 mM DTPA final pH 7.4.
- the volume of the dialysis buffer is more than 500 times the volume of the antibody protein.
- the concentration was measured using A280 (i.e., A280 (nm) ultraviolet absorption method for measuring protein concentration).
- TCEP tris(2-carboxyethyl)phosphine hydrochloride
- the freshly prepared TCEP (tris(2-carboxyethyl)phosphine hydrochloride) solution is added to the antibody in a set ratio to reduce it by 2 to 5 times the amount of the antibody substance.
- the antibody was switched to a coupling buffer (20 mM succinate 150 mM NaCl, 2 mM EDTA, using a desalting column). About 75mM Tris.).
- the compound to be coupled was dissolved to a concentration of 10 mM using DMSO.
- the final concentration of 10-30% DMSO was slowly added, and immediately mixed; the dissolved compound was added to the antibody solution in a certain ratio.
- the antibody substance After reacting at 25 ° C for 2 hours, dialysis was carried out using a dialysis membrane having a pore size of 10 kDa and exchanged once, and dialyzed overnight at 4 °C.
- the volume of the dialysis buffer needs to be more than 500 times the volume of the antibody-compound.
- the antibody-compound was taken out, filtered through a 0.22 ⁇ m green membrane, and the concentration was measured.
- Her2-positive human BT474 breast tumor cells (abbreviated as BT474), Her2 low-expression human MCF-7 breast tumor cells (abbreviated as MCF-7), and MCF-7-expressing Her2 human breast tumor MCF7-Her2 stable cells Strain (MCF-7-Her2 for short)
- MCF-7 Her2-positive human BT474 breast tumor cells
- MCF-7 Her2 low-expression human MCF-7 breast tumor cells
- MCF-7-Her2 Her2-positive human BT474 breast tumor cells
- MCF-7 Her2 low-expression human MCF-7 breast tumor cells
- MCF-7-expressing Her2 human breast tumor MCF7-Her2 stable cells Strain (MCF-7-Her2 for short)
- BT474, MCF7-Her2 and MCF-7 were digested with 0.25% (vol/vol) trypsin to detach the cells, then suspended in 100 ⁇ L of complete medium, and 2,000 cells were seeded in 96-well plates for culture. Incubate at 37 ° C overnight, then add 100 ⁇ L of antibody drug conjugate containing different concentration gradients and complete medium. Add 50 ⁇ L after 120 hours Fluorescent cell activity assay reagent Luminescent, Promega) performs relative cell proliferation assays. The activity data is shown in Table 1.
- mAb indicates the monoclonal antibody Herceptin
- the antibody conjugate of the present invention has a good inhibitory activity against MCF-Her2 cells, and its inhibitory activity is substantially higher than that of the control drug T-DM1.
- the small molecule toxin PNU-159682 has comparable activity to BT474, negative MCF-7 cells, and positive MCF-7-Her2 cells, and is essentially non-selective.
- the antibody drug conjugate of the present invention greatly reduced the activity of the negative MCF-7 cells, and the activity of MCF-7-Her2 positive cells was substantially equal or slightly decreased, and the selectivity thereof was significantly improved, which was against MCF-
- the selectivity of 7 and MCF-Her2 is almost similar to that of T-DM1, reducing potential cytotoxicity.
- TPBG-positive human MBA-MB-468 breast cancer cells (abbreviated as MBA-MB-468) and TPBG-low expressed human NCI-H1975 non-small cell lung cancer cells (abbreviated as NCI-H1975) were used.
- the growth inhibition of tumor cells by the antibody drug conjugate of the present invention was evaluated.
- MBA-MB-468 and NCI-H1975 were digested with 0.25% (v/v) trypsin, the cells were exfoliated, and then suspended in 100 ⁇ L of complete medium, and 2,000 cells were seeded in 96-well plates for culture. Incubate at 37 ° C overnight, then add 100 ⁇ L of antibody drug conjugate containing different concentration gradients and complete medium. Add 50 ⁇ L after 120 hours Fluorescent cell activity assay reagent Luminescent, Promega) performs relative cell proliferation assays. The activity data is shown in Table 2.
- mAb represents the monoclonal antibody TPBG
- the antibody drug conjugate of the present invention is TPBG-positive human MBA-MB-468 breast cancer cell (abbreviated as MBA-MB-468) compared to the antibody drug conjugate using DM1 and MMAF as a toxin.
- Human H1568 non-small cell lung cancer cells with high expression of TPBG have higher inhibitory activity.
- NCI-H1975 non-small cell lung cancer cells (NCI-H1975) with low expression of TPBG the cell cytotoxic activity of the antibody drug conjugate of the present invention is much higher than the corresponding control with DM1 and MMAF as toxin.
- the growth inhibition of tumor cells by the antibody drug conjugate of the present invention was evaluated using the CD70 recombinant cell line CHOK-hCD70 (prepared in accordance with MCF7-Her2) (abbreviated as CHOK-hCD70) and the negative cell CHOk1 not expressing CD70.
- CD70-positive human renal cancer cell line 786-O (abbreviation 786-O)
- CD70 low-expression human kidney cancer cell Caki-1 abbreviated as Caki-1)
- Caki-2 abbreviated as Caki-2
- CHOK-hCD70, CHOK1, 786-O, Caki-1 and Caki-2 were digested with 0.25% (vol/vol) trypsin to detach the cells, then suspended in 100 ⁇ L of complete medium, and 2,000 cells were seeded in 96-well plates. Cultivate. Incubate at 37 ° C overnight, then add 100 ⁇ L of antibody drug conjugate containing different concentration gradients and complete medium. Add 50 ⁇ L after 120 hours Fluorescent cell activity assay reagent Luminescent, Promega) performs relative cell proliferation assays. The activity data is shown in Table 3.
- mAb represents the monoclonal antibody CD70.
- the antibody drug conjugate of the present invention is equivalent to the CD70-positive recombinant cell line CHOK-hCD70 (referred to as CHOK-hCD70, the preparation method is consistent with MCF7-Her2) compared with the antibody drug conjugate using DM1 as a toxin.
- human kidney cancer cell line 786-O (abbreviation 786-O) has near or higher inhibitory activity.
- Caki-1 Caki-1
- Caki-2 Caki-2
- the antibody drug conjugate of the present invention has a near or higher cell killing power.
- the recombinant cell line CHOK1-EGFR (CHOK1-EGFR) expressing human EGFR protein
- CHOK1-EGFRvIII (referred to as CHOK1-EGFRvIII) expressing human GFRvIII protein
- the negative cell CHOk1 not expressing EGFR were evaluated.
- the antibody drug conjugate of the present invention inhibits the growth of tumor cells.
- CHOK1-EGFR, CHOK1-EGFRvIII, and CHOk1 were digested with 0.25% (v/v) trypsin, and the cells were detached, and then suspended in 100 ⁇ L of complete medium, and 2,000 cells were seeded in a 96-well plate for culture.
- mAb represents the monoclonal antibody EGFRVIII.
- the antibody drug conjugate of the present invention is EGFR-positive recombinant cell line CHOK1-EGFR (referred to as CHOK1-EGFR, and the preparation method is consistent with MCF7-Her2) compared with the antibody drug conjugate using MMAF as a toxin.
- CHOK1-EGFR EGFR-positive recombinant cell line
- CHOK1-EGFRvIII EGFRvIII-positive recombinant cell line CHOK1-EGFRvIII, which is produced in accordance with MCF7-Her2
- CHOK1-EGFRvIII which is produced in accordance with MCF7-Her2
Abstract
Provided are an antibody-drug conjugate, a preparation method, an intermediate, a pharmaceutical composition and the use thereof. The antibody-drug conjugate has high cytotoxicity and good anti-cancer effects, and has a market application prospect.
Description
本申请要求申请日为2016年12月9日的中国专利申请CN201611129379.1的优先权。本申请引用上述中国专利申请的全文。The present application claims priority from Chinese Patent Application No. CN201611129379.1, filed on Dec. 9, 2016. This application cites the entire text of the above-mentioned Chinese patent application.
本发明涉及一种抗体药物偶联物、其制备方法、中间体、药物组合物及应用。The present invention relates to an antibody drug conjugate, a process for the preparation thereof, an intermediate, a pharmaceutical composition and use thereof.
典型的抗体药物偶联物(ADC)包含能够与癌细胞表面特异性抗原相结合的单克隆抗体(mAbs)，并通过一个可裂解的连接单元将这些特异性抗体与强毒性药物进行连接。ADC的作用机理是通过抗体识别并与特异性抗原结合，引发一系列反应，进而通过细胞内吞进入细胞质内，溶酶体酶将强毒性药物释放杀死癌细胞。相比传统化疗对正常组织的无差别损伤，ADC通过靶向给药，可以使药物直接作用于癌细胞，减少了其对正常细胞的损害。A typical antibody drug conjugate (ADC) comprises monoclonal antibodies (mAbs) that bind to cancer cell surface specific antigens and bind these specific antibodies to a highly toxic drug via a cleavable linker. The mechanism of action of ADC is to recognize and bind to specific antigens by antibodies, trigger a series of reactions, and then enter the cytoplasm through endocytosis, and lysosomal enzymes release strong toxic drugs to kill cancer cells. Compared with traditional chemotherapy, there is no differential damage to normal tissues. By targeted drug delivery, ADC can directly act on cancer cells and reduce its damage to normal cells.
ADC中的单克隆抗体包括免疫***B细胞和T细胞表面的一些蛋白，比如CD20、CD22以及人表皮生长因子受体2(Her2)和***特异性膜抗原(PSMA)等。Monoclonal antibodies in ADCs include proteins on the surface of immune system B cells and T cells, such as CD20, CD22, and human epidermal growth factor receptor 2 (Her2) and prostate specific membrane antigen (PSMA).
目前大部分抗体药物偶联物使用Seattle Genetics的auristatins、ImmunoGen的美登素类，Spirogen公司的PBD dimer类，或蒽环类抗生素作为有效毒素。蒽环类抗生素为表现细胞毒素活性的抗生素化合物，包括阿霉素(doxorubicin)、表柔比星(epirubicin)、伊达比星(idarubicin)和伊达比星(daunorubicin)等。研究表明蒽环类抗生素可通过多种不同机制杀死细胞，包括：1)将药物分子嵌入细胞的DNA中，抑制DNA依赖性核酸合成；2)通过自由基药物的产生并与细胞大分子反应引起细胞的损伤；3)药物分子与细胞膜的相互作用。由于蒽环类抗生素的细胞毒性潜力，它们已经用于治疗包括白血病、乳腺癌、肺癌、卵巢癌等多种癌症。Most of the current antibody drug conjugates use the auristatins of Seattle Genetics, the maytansinoid of ImmunoGen, the PBD dimer of Spirogen, or an anthracycline antibiotic as an effective toxin. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity, including doxorubicin, epirubicin, idarubicin, and daunorubicin. Studies have shown that anthracycline antibiotics can kill cells through a variety of different mechanisms, including: 1) embedding drug molecules in the DNA of cells to inhibit DNA-dependent nucleic acid synthesis; 2) by free radical drug production and reaction with cellular macromolecules Causes damage to cells; 3) Interaction of drug molecules with cell membranes. Due to the cytotoxic potential of anthracyclines, they have been used to treat a variety of cancers including leukemia, breast cancer, lung cancer, ovarian cancer and the like.
近几年来，人们合成了一些结构新颖的高细胞毒性的蒽环类抗生素，奈莫柔比星具
有强烈的抗肿瘤活性，目前正在进行肝癌治疗的临床实验，具有较高的开发前景(J.W.Lown,Bioactive Molecules,1988,6,55；C.Sessa,O.Valota,C.Geroni,Cardiovascular Toxicology,2007,7,75)。M.Caruso等也报道了来自肝微体的奈莫柔比星代谢产物PNU-159682等一系列高抗癌活性的三环吗啉取代蒽环类衍生物(WO 98/02446)。PNU-159682在具有比奈莫柔比星更强的体外和体内肿瘤模型细胞毒性(Beulz-Riche,et.al.Fundamental&Clinical Pharmacology,2001,15,373,；Quintieri,L.,Geroni,C.,et.al.Clinical.Cancer.Research.,2005,11,1608；EP 0889898；WO 2004/082689；WO 2004/082579)。In recent years, some novel and highly cytotoxic anthracycline antibiotics have been synthesized, and it has been synthesized.
Has strong anti-tumor activity, is currently undergoing clinical trials for liver cancer treatment, and has high development prospects (JWLown, Bioactive Molecules, 1988, 6, 55; C. Sessa, O. Valota, C. Geroni, Cardiovascular Toxicology, 2007, 7, 75). M. Caruso et al. also reported a series of high anticancer activity tricyclic morpholine substituted anthracycline derivatives such as the nemoxine star metabolite PNU-159682 from liver microsomes (WO 98/02446). PNU-159682 has stronger in vitro and in vivo tumor model cytotoxicity than nemoxil (Beulz-Riche, et. al. Fundamental & Clinical Pharmacology, 2001, 15, 373,; Quintieri, L., Geroni, C., et. al .Clinical. Cancer. Research, 2005, 11, 1608; EP 0889898; WO 2004/082689; WO 2004/082579).
这类高活性蒽环类抗生素表现出的毒性与其给药剂量密切相关，这大大限制了其治疗效果。通过将其与抗体或其他不同的载体进行连接，可以改善它们的治疗效果(Tolcher.,et al.,J.Clin.Oncology.,1999,17,478,；Ajani et.al.,Cancer.Jour.,2000,6,78,；Saleh et al.,J.Clin.Oncology.,2000,18,2282,；Nagy.,et al.,Proc.Natl.Acad.Sci.,2000,97,829,；Dubowchik et al.,Bioorg.&Med.Chem.Lett.；2002,12,1529,；King.,et al.,J.Med.Chem.,2002,45,4336,；Torgov.,et al.,Bioconj.Chem.,2005,16,717,；Jeffrey.,et al.,Bioorganic&Med.Chem.Lett.；2006,16,358,；Kratz.,et al.,Current.Med.Chem.,2006,13,477,；Bristol Mayers,EP0328147；Farmitalia Carlo Erba,WO 9202255；Pharmacia&Upjohn,US 5776458Genentech,WO2009/099741；WO2010/009124；CN 102159248(2011)；US8389697；US 8470984；Shang-Fan Yu et.al.；Clin.Cancer.Res.；2015,21,3298；WO2015/089344；WO2015/095223；WO 2016/040825)。在癌症治疗中，使用抗体药物偶联物将能够杀伤或者一直肿瘤细胞的毒素药物靶向输送到肿瘤，在寻求药物最大功效的通式可以显著提高选择性，降低抗肿瘤药物的毒副作用(Xie et al.,Expert.Opin.Biol.Ther.,2006,6,281.；Kovtun et al.,Cancer.Res.,2006,66,3214.；Law et al,Cancer.Res.,2006,66,2328.；Wu et al,Nature.Biotech.2005,23,1137.；Lamber J.et al.Current.Opin.in Pharmacol.2005,5,543.；Hamann P.et al.,Expert.Opin.Ther.Patents.,2005,15,1087.；Payne,G.,Cancer.Cell.2003,3,207.；Trail et al,Cancer.Immunol.Immunother.,2003,52,328.)。
The toxicity exhibited by such highly active anthracyclines is closely related to the dose administered, which greatly limits the therapeutic effect. Their therapeutic effects can be improved by linking them to antibodies or other different carriers (Tolcher., et al., J. Clin. Oncology., 1999, 17, 478, Ajani et. al., Cancer. Jour., 2000, 6, 78,; Saleh et al., J. Clin. Oncology., 2000, 18, 2282, Nagy., et al., Proc. Natl. Acad. Sci., 2000, 97, 829,; Dubowchik et al ., Bioorg. & Med. Chem. Lett.; 2002, 12, 1529,; King., et al., J. Med. Chem., 2002, 45, 4336,; Torgov., et al., Bioconj. , 2005, 16, 717,; Jeffrey., et al., Bioorganic & Med. Chem. Lett.; 2006, 16, 358,; Kratz., et al., Current. Med. Chem., 2006, 13, 477,; Bristol Mayers, EP 0328147; Carlo Erba, WO 9202255; Pharmacia & Upjohn, US 5776458 Genentech, WO2009/099741; WO2010/009124; CN 102159248 (2011); US8389697; US 8470984; Shang-Fan Yu et.al.; Clin. Cancer. Res.; 2015, 21, 3298; WO2015/089344; WO2015/095223; WO 2016/040825). In the treatment of cancer, the use of antibody drug conjugates can target the toxin drugs capable of killing or always tumor cells to the tumor, and the general formula for seeking the maximum efficacy of the drug can significantly improve the selectivity and reduce the side effects of the antitumor drugs (Xie). Et al., Expert. Opin. Biol. Ther., 2006, 6, 281.; Kovtun et al., Cancer. Res., 2006, 66, 3214.; Law et al, Cancer. Res., 2006, 66, 2328. Wu et al, Nature. Biotech. 2005, 23, 1137.; Lamber J. et al. Current. Opin. in Pharmacol. 2005, 5, 543.; Hamann P. et al., Expert. Opin. Ther. Patents. 2005, 15, 1087.; Payne, G., Cancer. Cell. 2003, 3, 207.; Trail et al, Cancer. Immunol. Immunother., 2003, 52, 328.).
典型的抗体药物偶联物中，抗体和药物的选择取决于特定的疾病，其对抗体药物偶联物的安全性和有效性具有重要影响。决定抗体药物偶联物疗效的因素包括连接单元的稳定性及其断裂敏感性，细胞表面激发内化、转运和细胞毒素的释放。例如T-DM1，其细胞毒性远低于部分蒽环类抗生素，且容易在抗原结合蛋白(Abu)内吞之前过早降解并释放毒素，引起副作用，同时其与抗体偶联得到的药物/抗体比例较低而且分布变化比较大，药效和安全性精确控制的难度较大(如专利申请WO2012/061590 A1或WO201139721)。In a typical antibody drug conjugate, the choice of antibody and drug will depend on the particular disease, which has a significant impact on the safety and efficacy of the antibody drug conjugate. Factors that determine the efficacy of antibody drug conjugates include the stability of the linker unit and its cleavage sensitivity, cell surface internalization, transport, and release of cytotoxin. For example, T-DM1, which is much less cytotoxic than some anthracycline antibiotics, is prone to prematurely degrade and release toxins before endocytosis of antigen-binding protein (Abu), causing side effects, and the drug/antibody obtained by coupling it with antibodies The ratio is low and the distribution changes are relatively large, and it is difficult to accurately control the efficacy and safety (such as patent application WO2012/061590 A1 or WO201139721).
因此，寻找稳定性高，水溶性好的连接体，进而得到细胞毒素的释放效率高、特异性好、细胞毒性高、抗癌效果好的抗体药物偶联物是目前亟需解决的技术问题。Therefore, it is an urgent technical problem to find an antibody drug conjugate with high stability, good water solubility and high cytotoxin release efficiency, specificity, high cytotoxicity and good anticancer effect.
发明内容Summary of the invention
本发明所要解决的技术问题是为了克服现有技术中缺乏细胞毒素的释放效率高、细胞毒性高、抗癌效果好的抗体药物偶联物，而提供了一种抗体药物偶联物、制备方法、中间体、药物组合物及应用。本发明的抗体药物偶联物细胞毒性高，抗癌效果好，具有良好的市场应用前景。The technical problem to be solved by the present invention is to overcome the prior art antibody drug conjugate which lacks cytotoxin release efficiency, high cytotoxicity and good anticancer effect, and provides an antibody drug conjugate and preparation method thereof. , intermediates, pharmaceutical compositions and applications. The antibody drug conjugate of the invention has high cytotoxicity, good anticancer effect and good market application prospect.
本发明提供了一种如式IB所示的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，The present invention provides an antibody drug conjugate of the formula IB, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, and a pharmaceutically acceptable thereof. Salt or its prodrug,
其中，mAb为单克隆抗体片段(所述的单克隆抗体包括但不限于赫赛汀、抗TPBG(胚胎滋养层糖蛋白)抗体、抗CD70抗体或抗EGFRVIII(表皮生长因子受体)抗
体；所述的单克隆抗体可为本领域常规的单克隆抗体，只要能与如式IB所示的抗体药物偶联物中除mAb外的片段形成抗体药物偶联物即可。)Wherein, the mAb is a monoclonal antibody fragment (the monoclonal antibody includes, but is not limited to, Herceptin, an anti-TPBG (embryonic trophoblastic glycoprotein) antibody, an anti-CD70 antibody or an anti-EGFRVIII (epidermal growth factor receptor) antibody
The monoclonal antibody may be a monoclonal antibody conventional in the art as long as it can form an antibody drug conjugate with a fragment other than the mAb in the antibody drug conjugate of the formula IB. )
0<k≤8(优选地，0<k≤6，例如0.3、0.73、3.21、3.76、2.65、3.96、3.31、2.98、1.80、2.23、2.57、1.89、1.63、2.63、3.05、1.53、1.68、3.10、1.47、3.20、1.56、1、2、3、4、5或6)；0 < k ≤ 8 (preferably, 0 < k ≤ 6, such as 0.3, 0.73, 3.21, 3.76, 2.65, 3.96, 3.31, 2.98, 1.80, 2.23, 2.57, 1.89, 1.63, 2.63, 3.05, 1.53, 1.68, 3.10, 1.47, 3.20, 1.56, 1, 2, 3, 4, 5 or 6);
R1为氢、羟基、C1-6烷氧基(例如甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基或叔丁氧基)、C1-6烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基)、-NRaRb或-C(O)NRcRd；Ra、Rb、Rc和Rd各自独立地为氢、C1-6烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基)或C6-10芳基(例如苯基)；R 1 is hydrogen, hydroxy, C 1-6 alkoxy (eg methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy or tert-butoxy), C 1-6 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl), -NR a R b or -C(O)NR c R d ; R a , R b , R c and R d are each independently hydrogen, C 1-6 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl) Or a C 6-10 aryl group (eg phenyl);
R2和R3各自独立地为C1-6烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基)、C1-6烷氧基(例如甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基或叔丁氧基)或C1-6烷硫基(例如甲硫基、乙硫基、正丙硫基、异丙硫基、正丁硫基、异丁硫基或叔丁硫基)；R 2 and R 3 are each independently C 1-6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl), C 1-6 alkoxy a group (eg methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy or tert-butoxy) or a C 1-6 alkylthio (eg methylthio, Ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio or tert-butylthio);
X为
X is
Y为
(优选为
)；R4a和R4b各自独立地为氢、C1～4烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基)、C3～6环烷基取代的C1～4烷基(所述的C1～4烷基例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；所述的C3～6环烷基例如环丙基；所述的C3～6环烷基取代的C1～4烷基优选环丙基甲基)、C3～6环烷基(例如环丙基)、C2-8杂烷基(所述的C2-8杂烷基中的杂原子可为O、S和N中的一种或多种，杂原子的个数可为1、2、3或4；优选地，所述的C2-8杂烷基为CH3OCH2-)、或-(OCH2CH2)j1OH；j1为1、2、3、4、5、6、7或8；Y is (preferably R 4a and R 4b are each independently hydrogen, C 1-4 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl), C 3 ～6 cycloalkyl substituted C 1-4 alkyl (the C 1-4 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl; The C 3-6 cycloalkyl group such as a cyclopropyl group; the C 3-6 cycloalkyl substituted C 1-4 alkyl group is preferably a cyclopropylmethyl group, a C 3 -6 cycloalkyl group (for example) a cyclopropyl), C 2-8 heteroalkyl group (the hetero atom in the C 2-8 heteroalkyl group may be one or more of O, S and N, and the number of hetero atoms may be 1 2, 3 or 4; preferably, the C 2-8 heteroalkyl group is CH 3 OCH 2 -), or -(OCH 2 CH 2 ) j1 OH; j1 is 1, 2, 3, 4, 5 , 6, 7 or 8;
Z为
或4-6元亚杂芳基(所述的4-6元亚杂芳基中的杂原子优选N、O和S中的一种或多种；杂原子个数优选1个、2个或3个；所述的4-6元亚杂芳基可为4元、5或元6元亚杂芳基；所述的5元亚杂芳基优选
)；R5a和R5b各自独立地为氢或C1～4烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或
叔丁基)；Z is Or a 4-6 membered heteroarylene group (the hetero atom in the 4-6 membered heteroarylene group is preferably one or more of N, O and S; the number of hetero atoms is preferably 1, 2 or 3; the 4-6 membered heteroarylene group may be a 4 member, 5 or 6 membered heteroarylene; the 5-membered heteroarylene group is preferably R 5a and R 5b are each independently hydrogen or C 1-4 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl);
或者，
为
(优选地，当所述的X为
时，
为
)；or, for (preferably, when said X is Time, for );
R6为氢、C1～4烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基)、C2-8杂烷基(所述的C2-8杂烷基中的杂原子可为O、S和N中的一种或多种，杂原子的个数可为1、2、3或4；优选地，所述的C2-8杂烷基为CH3OCH2-)、或-(OCH2CH2)j2OH；j2为1、2、3、4、5、6、7或8；R 6 is hydrogen, C 1-4 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl), C 2-8 heteroalkyl (described The hetero atom in the C 2-8 heteroalkyl group may be one or more of O, S and N, and the number of hetero atoms may be 1, 2, 3 or 4; preferably, the C 2 is -8heteroalkyl is CH 3 OCH 2 -), or -(OCH 2 CH 2 ) j2 OH; j2 is 1, 2, 3, 4, 5, 6, 7, or 8;
p为0、1、2、3、4、5、6、7、8、9或10；且当Z为
时，p不为0；p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and when Z is When p is not 0;
m为0或1；m is 0 or 1;
E为
R7a和R7b各自独立地为氢、C1～4烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基)、C3～6环烷基取代的C1～4烷基(所述的C1～4烷基例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；所述的C3～6环烷基例如环丙基；所述的C3～6环烷基取代的C1～4烷基优选环丙基甲基)、C3～6环烷基(例如环丙基)、C2-8杂烷基(所述的C2-8杂烷基中的杂原子可为O、S和N中的一种或多种，杂原子个数可为1、2、3、4、5或6，所述的C2-8杂烷基例如CH3OCH2-)、或-(OCH2CH2)j3OH；j3为1、2、3、4、5、6、7或8；E is R 7a and R 7b are each independently hydrogen, C 1-4 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl), C 3-6 a cycloalkyl-substituted C 1-4 alkyl group (the C 1-4 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl; a C 3-6 cycloalkyl group such as a cyclopropyl group; the C 3-6 cycloalkyl substituted C 1-4 alkyl group is preferably a cyclopropylmethyl group), a C 3 -6 cycloalkyl group (for example, a cyclopropyl group) a C 2-8 heteroalkyl group (the hetero atom in the C 2-8 heteroalkyl group may be one or more of O, S and N, and the number of heteroatoms may be 1, 2 3, 4, 5 or 6, said C 2-8 heteroalkyl group such as CH 3 OCH 2 -), or -(OCH 2 CH 2 ) j3 OH; j3 is 1, 2, 3, 4, 5, 6 , 7 or 8;
n为0、1、2、3、4、5、6、7、8、9或10；当有多个E时(即当n大于等于2时)，E可以相同或不同(
例如可为
)，n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; when there are multiple Es (ie, when n is greater than or equal to 2), E may be the same or different ( For example, it can be ),
u为0或1；u is 0 or 1;
R8为氢、C1～4烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基)、C3～6环烷基取代的C1～4烷基(所述的C1～4烷基例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；所述的C3～6环烷基例如环丙基；所述的C3～6环烷基取代的C1～4烷
基优选环丙基甲基)、C3～6环烷基(例如环丙基)、C2-8杂烷基(所述的C2-8杂烷基中的杂原子可为O、S和N中的一种或多种，杂原子个数可为1、2、3、4、5或6，所述的C2-
8杂烷基例如CH3OCH2-)、或-(OCH2CH2)j4OH；j4为1、2、3、4、5、6、7或8；R 8 is hydrogen, C 1-4 alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl), C 3-6 cycloalkyl substituted C a 1-4 alkyl group (the C 1-4 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl; the C 3-6 ring The alkyl group is, for example, a cyclopropyl group; the C 3-6 cycloalkyl-substituted C 1-4 alkyl group is preferably a cyclopropylmethyl group, a C 3-6 cycloalkyl group (e.g., a cyclopropyl group), C 2- a heteroalkyl group (the hetero atom in the C 2-8 heteroalkyl group may be one or more of O, S and N, and the number of hetero atoms may be 1, 2, 3, 4, 5 or 6, a C 2 -8 heteroalkyl group such as CH 3 OCH 2 -), or -(OCH 2 CH 2 ) j4 OH; j4 is 1, 2, 3, 4, 5, 6, 7, or 8;
q为0、1、2、3、4、5、6、7、8、9或10；当含有多个-C(R8)-时(即当q大于等于2时)，-C(R8)-可以相同或不同(
例如可为
)；q is 0, 1, 2, 3, 4, 5, 6, 7, 8 , 9, or 10; when a plurality of -C(R 8 )- are contained (that is, when q is greater than or equal to 2), -C(R 8 ) - can be the same or different ( For example, it can be );
y为0～24的整数(例如0、1、2、3、4、5或6)；Y is an integer from 0 to 24 (for example, 0, 1, 2, 3, 4, 5 or 6);
G为
G is
s为0或1；s is 0 or 1;
Q为
t1、t3和t4各自独立地为0、1、2、3、4、5、6、7、8、9或10。Q is t 1 , t 3 and t 4 are each independently 0, 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9, or 10.
本发明中，k表示药物分子与mAb的摩尔比(又称DAR，即药物抗体偶联比率)，优选地理解为是：单个单克隆抗体分子与药物偶联后得到的抗体偶联药物中的药物分子与单克隆抗体分子的摩尔比的平均值，一般可以采用疏水层析(Hydrophobic-Interaction Chromatography，HIC)，聚丙烯酰胺-SDS凝胶电泳(SDS-PAGE，electrophoresis)，液相质谱(liquid chromatograph-mass spectrometer，LC-MS)等方式测定得到。In the present invention, k represents a molar ratio of a drug molecule to a mAb (also referred to as DAR, that is, a drug antibody coupling ratio), and is preferably understood to be: an antibody-conjugated drug obtained by coupling a single monoclonal antibody molecule to a drug. The average of the molar ratio of the drug molecule to the monoclonal antibody molecule can be generally hydrophobic-interaction Chromatography (HIC), polyacrylamide-SDS gel electrophoresis (SDS-PAGE, electrophoresis), liquid chromatography mass spectrometry (liquid) Chromatograph-mass spectrometer, LC-MS) was measured.
在本发明一优选实施方案中，k可为0.43、0.49、2.56、5.64、2.26、2.17或2.83。In a preferred embodiment of the invention, k can be 0.43, 0.49, 2.56, 5.64, 2.26, 2.17 or 2.83.
在本发明一优选实施方案中，E为
In a preferred embodiment of the invention, E is
E中，e1、e2、e3、e4、e5和e6各自独立地为0、1、2、3、4、5、6、7或8；R7a、R7b、R7c、R7d、R7e、R7f和R7h各自独立地为氢、C1～4烷基、C3～6环烷基取代的C1～4烷基、C3～6环烷基、C2-8杂烷基、-(OCH2CH2)j3OH或
j3为1、2、3、4、5、6、7或8；In E, e1, e2, e3, e4, e5, and e6 are each independently 0, 1, 2, 3, 4, 5, 6, 7 , or 8; R 7a , R 7b , R 7c , R 7d , R 7e And R 7f and R 7h are each independently hydrogen, C 1-4 alkyl, C 3-6 cycloalkyl substituted C 1-4 alkyl, C 3-6 cycloalkyl, C 2-8 heteroalkyl , -(OCH 2 CH 2 ) j3 OH or J3 is 1, 2, 3, 4, 5, 6, 7, or 8;
所述的如式IB所示的抗体药物偶联物中：
优选
Said antibody drug conjugate as shown in formula IB: Optimal
优选地，R1中，所述的-NRaRb为甲胺基或二甲胺基。Preferably, in R 1 , the -NR a R b is a methylamino group or a dimethylamino group.
优选地，当Z为
时，p不为0。Preferably, when Z is When p is not 0.
优选地，当所述的Z为
时，p优选为0，m优选为1。Preferably, when said Z is When p is preferably 0, m is preferably 1.
优选地，当Y为
时(优选地为当X为
Y为
时)，Z优选为
p优选为1；m优选为0；n优选为1；E优选为
u优选为0或1；q优选为2；y优选为2；s优选为0或1(当u为1时，S优选为0)；G优选
Q优选为
(且当G为
时，Q优选为
)。Preferably, when Y is Time (preferably when X is Y is Time), Z is preferably p is preferably 1; m is preferably 0; n is preferably 1; E is preferably u is preferably 0 or 1; q is preferably 2; y is preferably 2; s is preferably 0 or 1 (when u is 1, S is preferably 0); G is preferred Q is preferably (and when G is When Q is preferred ).
优选地，当Y为
时(优选为当X为
Y为
时)，Z优选为
Preferably, when Y is Time (preferably when X is Y is Time), Z is preferably
优选地，当Y为
Z
时，p不为0。Preferably, when Y is Z When p is not 0.
优选地，
为以下任一结构：Preferably, For any of the following structures:
其中，R4a、R4b、R5a和R5b的定义同上所述。Wherein R 4a , R 4b , R 5a and R 5b have the same meanings as defined above.
更优选地，所述的
为以下任一结构：More preferably, said For any of the following structures:
优选地，所述的
为以下任一结构：Preferably said For any of the following structures:
其中，R4a、R4b、R5a、R5b和R6的定义同上所述。Wherein R 4a , R 4b , R 5a , R 5b and R 6 have the same meanings as defined above.
更优选地，所述的
为以下任一结构：More preferably, said For any of the following structures:
最优选地，所述的
为以下任一结构：
Most preferably, said For any of the following structures:
在本发明一优选实施方案中，所述的
为以下结构：In a preferred embodiment of the invention, the For the following structure:
本发明所述的如式IB所示的抗体药物偶联物中的药物分子片段
中涉及的取代基种类和手性中心的构型可参照本领域该类药物分子(即蒽环类抗生素，如阿霉素、表柔比星、伊达比星、伊达比星、奈莫柔比星、PNU-159682等)的常规进行选择，例如
优选
最优选
Drug molecule fragment of the antibody drug conjugate as shown in Formula IB of the present invention The types of substituents and chiral centers involved in the reference may refer to such drug molecules in the field (ie, anthracyclines such as doxorubicin, epirubicin, idarubicin, idarubicin, naimo) Conventional selection of spirulina, PNU-159682, etc., for example Optimal Most preferred
如式IB所示的抗体药物偶联物优选为式IB-1所示的化合物：The antibody drug conjugate as shown in Formula IB is preferably a compound of Formula IB-1:
其中，R1、R2、R3、X、Y、Z、R6、p、m、E、n、u、R8、q、y、G、s和Q的定义同上所述。Wherein R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q, y, G, s and Q are as defined above.
优选地，所述的如式IB所示的抗体药物偶联物选自以下任一化合物，下述化合物中的mAb为赫赛汀、抗TPBG抗体、抗CD70抗体或抗EGFRVIII抗体：Preferably, the antibody drug conjugate as shown in Formula IB is selected from any one of the following compounds, wherein the mAb in the following compounds is Herceptin, an anti-TPBG antibody, an anti-CD70 antibody or an anti-EGFRVIII antibody:
在本发明一优选实施方案中，所述的如式IB所示的抗体药物偶联物为下列化合物：In a preferred embodiment of the invention, the antibody drug conjugate of formula IB is the following compound:
所述的如式IB所示的抗体药物偶联物中，所述的单克隆抗体通过氨基酸残基中的巯基或氨基进行反应从而与所述的
相连生成了
这一结构，当Q为
时，其中的S原子来自所述单克隆抗体中的巯基；当Q为
时，其中的NH来自所述单克隆抗体的氨基。In the antibody drug conjugate of the formula IB, the monoclonal antibody is reacted with a thiol group or an amino group in an amino acid residue to Connected This structure, when Q is Wherein the S atom is derived from a thiol group in the monoclonal antibody; when Q is Wherein NH is derived from the amino group of the monoclonal antibody.
本发明还提供了一种所述的如式IB所示的抗体药物偶联物的制备方法，其包括以下步骤：有机溶剂中，在pH值为6-8的条件下，将IA化合物与单克隆抗体进行如下所示的偶联反应，得到所述的如式IB所示的抗体药物偶联物，即可；The present invention also provides a method for preparing the antibody drug conjugate according to Formula IB, which comprises the steps of: IA compound and single in an organic solvent at a pH of 6-8. The cloning antibody is subjected to a coupling reaction as shown below to obtain the antibody drug conjugate as shown in Formula IB;
其中，k、R1、R2、R3、X、Y、Z、R6、p、m、E、n、u、R8、q、y、G、s、Q和mAb定义同上所述；
Wherein k, R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q, y, G, s, Q and mAb are as defined above ;
Q1为
t1、t3和t4的定义同上所述。Q 1 is The definitions of t 1 , t 3 and t 4 are as described above.
所述的如式IB所示的抗体药物偶联物的制备方法，可参照本领域中该类偶联反应的常规方法和条件，具体可参照文献Gail D.Lewis Phillips,et al.,Cancer.Res.,2008,68,9280和Teemu T.Junttila,et al.,Breast.Cancer.Res.Treat.,2011,128,34，本发明特别优选如下方法：For the preparation method of the antibody drug conjugate as shown in Formula IB, refer to the conventional methods and conditions of the coupling reaction in the art, and specifically refer to the literature Gail D. Lewis Phillips, et al., Cancer. Res., 2008, 68, 9280 and Teemu T. Junttila, et al., Breast. Cancer. Res. Treat., 2011, 128, 34, the present invention particularly preferably as follows:
所述的有机溶剂优选酰胺类溶剂、亚砜类溶剂和醚类溶剂中的一种或多种。所述的酰胺类溶剂优选N,N二甲基甲酰胺(DMF)和/或二甲基乙酰胺(DMA)；所述的亚砜类溶剂优选二甲亚砜(DMSO)。所述的醚类溶剂优选四氢呋喃。The organic solvent is preferably one or more of an amide solvent, a sulfoxide solvent, and an ether solvent. The amide solvent is preferably N,N dimethylformamide (DMF) and/or dimethylacetamide (DMA); and the sulfoxide solvent is preferably dimethyl sulfoxide (DMSO). The ether solvent is preferably tetrahydrofuran.
所述的IA化合物与所述的有机溶剂的质量体积比优选0.1mg/mL-100mg/mL。The mass to volume ratio of the IA compound to the organic solvent is preferably from 0.1 mg/mL to 100 mg/mL.
所述的IA化合物与所述的单克隆抗体的摩尔比值优选1-10(例如6)，优选1-5。The molar ratio of the IA compound to the monoclonal antibody is preferably from 1 to 10 (e.g., 6), preferably from 1 to 5.
所述的pH值优选7.5。所述的pH值为6-8的条件可通过在反应溶液中添加缓冲溶液实现；所述的缓冲溶液一般为低盐缓冲液，优选磷酸盐缓冲溶液(例如磷酸钾与磷酸二氢钾形成的缓冲溶液)或硼酸缓冲溶液(例如硼酸和硼酸钠形成的缓冲溶液)。The pH is preferably 7.5. The pH of 6-8 can be achieved by adding a buffer solution to the reaction solution; the buffer solution is generally a low salt buffer, preferably a phosphate buffer solution (for example, potassium phosphate and potassium dihydrogen phosphate). Buffer solution) or borate buffer solution (for example, a buffer solution of boric acid and sodium borate).
所述的偶联反应的温度优选4℃～37℃，更优选10～35℃。The temperature of the coupling reaction is preferably 4 ° C to 37 ° C, more preferably 10 to 35 ° C.
所述的单克隆抗体优选透析后的单克隆抗体。所述的透析的方法可为本领域单克隆抗体透析常规的方法。The monoclonal antibody is preferably a monoclonal antibody after dialysis. The method of dialysis can be a conventional method of dialysis of monoclonal antibodies in the art.
所述的偶联反应优选在气体保护下进行；当所述的偶联反应在气体保护下进行时，所述的气体优选氮气。The coupling reaction is preferably carried out under gas protection; when the coupling reaction is carried out under gas protection, the gas is preferably nitrogen.
所述的如式IB所示的抗体药物偶联物的制备方法中，优选包括以下步骤：在pH值为6-8的缓冲溶液中，将透析后的单克隆抗体与所述的IA化合物和所述的有机溶剂混合，进行所述的偶联反应，即可。所述的“将透析后的单克隆抗体与所述的IA化合物和所述的有机溶剂混合”的混合顺序没有特殊的限定，一般为将所述的IA化合物和所述的有机溶剂加入到所述的透析后的单克隆抗体中即可。In the method for preparing an antibody drug conjugate as shown in Formula IB, preferably, the method comprises the steps of: dialysis of the monoclonal antibody and the IA compound in a buffer solution having a pH of 6-8; The organic solvent is mixed and the coupling reaction is carried out. The mixing order of "mixing the dialyzed monoclonal antibody with the IA compound and the organic solvent" is not particularly limited, and generally the IA compound and the organic solvent are added to the The dialysis monoclonal antibody can be used.
所述的式IB抗体药物偶联物的制备方法中，所述的偶联反应的进程可以采用本领域中的常规测试方法(如TLC、HPLC或NMR)进行监控，一般以IA化合物消失时为反应的终点。
In the preparation method of the antibody conjugate of the formula IB antibody, the progress of the coupling reaction can be monitored by a conventional test method (such as TLC, HPLC or NMR) in the art, generally when the IA compound disappears. The end of the reaction.
所述的IA化合物，本领域技术人员根据本发明具体实施方式公开的内容，结合本领域常规实验技术手段均可以制备得到。The IA compound can be prepared by a person skilled in the art according to the disclosure of the specific embodiments of the present invention, combined with the conventional experimental techniques in the art.
本发明还提供了一种IA化合物，The invention also provides an IA compound,
其中，R1、R2、R3、X、Y、Z、R6、p、m、E、n、u、R8、q、y、G、s和Q1的定义同上所述。Wherein R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q, y, G, s and Q 1 have the same meanings as defined above.
所述的IA化合物中的药物分子片段
的定义同上所述。a drug molecule fragment in the IA compound The definition is the same as above.
所述的IA化合物优选为IA-1所示的化合物：The IA compound is preferably a compound represented by IA-1:
其中，R1、R2、R3、X、Y、Z、R6、p、m、E、n、u、R8、q、y、G、s和Q1的定义同上所述。
Wherein R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q, y, G, s and Q1 are as defined above.
优选地，所述的IA化合物选自以下任一化合物：Preferably, the IA compound is selected from any of the following compounds:
在本发明一优选实施方案中，所述的IA化合物为下列化合物：
In a preferred embodiment of the invention, the IA compound is the following compound:
本发明还提供了一种IC-1、IC-2或IC-3化合物：
The invention also provides an IC-1, IC-2 or IC-3 compound:
其中，R1、R2、R3、X、Y、Z、R6、p、m、E、n、u、R8、q和y的定义同上所述，R9为
Wherein R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q and y are as defined above, and R 9 is
所述的IC-1、IC-2和IC-3化合物中的药物分子片段
的定义同上
所述。Drug molecule fragments in the IC-1, IC-2 and IC-3 compounds The definition is the same as above.
所述的IC-1化合物优选IC-1-1化合物：The IC-1 compound is preferably an IC-1-1 compound:
所述的IC-1化合物更优选以下任一化合物：The IC-1 compound is more preferably any of the following compounds:
在本发明一优选实施方案中，所述的IC-1化合物优选以下任一化合物：In a preferred embodiment of the invention, the IC-1 compound is preferably any of the following compounds:
所述的IC-2化合物优选为IC-2-1化合物：The IC-2 compound is preferably an IC-2-1 compound:
更优选以下化合物：More preferred are the following compounds:
所述的IC-3化合物优选为IC-3-1化合物：
The IC-3 compound is preferably an IC-3-1 compound:
所述的IC-3化合物更优选为以下任一化合物：The IC-3 compound is more preferably any of the following compounds:
所述的IC-1化合物、IC-2化合物和IC-3化合物，本领域技术人员根据本发明具体实施方式公开的内容，结合本领域常规实验技术手段均可以制备得到。The IC-1 compound, the IC-2 compound and the IC-3 compound can be prepared by those skilled in the art according to the disclosure of the specific embodiments of the present invention in combination with conventional experimental techniques in the art.
本发明还提供了所述的如式IB所示的抗体药物偶联物、其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药在制备用于治疗和/或预防癌症的药物中的应用。The present invention also provides the antibody drug conjugate, the tautomer, the optical isomer, the hydrate, the solvate, the polymorph, the isotope compound, and the pharmaceutically acceptable compound thereof, as shown in Formula IB. The use of a salt or a prodrug thereof for the preparation of a medicament for the treatment and/or prevention of cancer.
本发明还提供了所述的IA化合物、其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、其药学上可接受的盐或其前药在制备用于治疗和/或预防癌症的药物中的应用。The present invention also provides the IA compound, its tautomer, optical isomer, hydrate, solvate, polymorph, pharmaceutically acceptable salt thereof or prodrug thereof for preparation for treatment And/or the use of drugs for the prevention of cancer.
本发明还提供了所述的IC-1、IC-2或IC-3化合物、其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药在制备用于治疗和/或预防癌症的药物中的应用。The present invention also provides the IC-1, IC-2 or IC-3 compound, tautomer, optical isomer, hydrate, solvate, polymorph, isotopic compound thereof, and pharmaceutically thereof thereof. Use of an acceptable salt or prodrug thereof for the manufacture of a medicament for the treatment and/or prevention of cancer.
本发明所述的癌症可以为本领域中常规的癌症，包括但不限于乳腺癌、淋巴癌、肺癌、肝癌、结肠癌、头颈癌、膀胱癌、肾癌、食道癌、胆囊癌、卵巢癌、胰腺癌、胃癌、
***、甲状腺癌、***癌、皮肤癌包括鳞状细胞癌；白细胞过多症、急性淋巴细胞性白血病、急性成淋巴细胞性白血病、B细胞淋巴瘤、T细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、毛细胞淋巴瘤、伯基特淋巴瘤、急性和慢性髓细胞性白血病、骨髓增生异常综合征、前髓细胞白血病、纤维肉瘤、横纹肌肉瘤、星形细胞瘤、神经目细胞瘤、胶质瘤、神经鞘瘤、黑色素瘤、***瘤、畸胎癌、骨肉瘤、着色性干皮病、角质黄色瘤、甲状腺滤泡癌和卡波西肉瘤。所述的癌症的肿瘤细胞包括但不限于Her2阳性的人BT474乳腺肿瘤细胞、Her2低表达的人MCF-7乳腺肿瘤细胞、或在MCF-7外转Her2的人乳腺肿瘤MCF7-Her2稳转细胞株。The cancer of the present invention may be a cancer conventional in the art, including but not limited to breast cancer, lymphoma, lung cancer, liver cancer, colon cancer, head and neck cancer, bladder cancer, kidney cancer, esophageal cancer, gallbladder cancer, ovarian cancer, Pancreatic cancer, stomach cancer,
Cervical cancer, thyroid cancer, prostate cancer, skin cancer including squamous cell carcinoma; leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma , non-Hodgkin's lymphoma, hairy cell lymphoma, Burkitt's lymphoma, acute and chronic myeloid leukemia, myelodysplastic syndrome, promyelocytic leukemia, fibrosarcoma, rhabdomyosarcoma, astrocytoma, nerve Head cell tumor, glioma, schwannomas, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratinous xanthomas, thyroid follicular carcinoma and Kaposi's sarcoma. The tumor cells of the cancer include, but are not limited to, Her2 positive human BT474 breast tumor cells, Her2 low expressed human MCF-7 breast tumor cells, or human breast tumor MCF7-Her2 stable cells transduced with Her2 in MCF-7. Strain.
本发明还提供了一种药物组合物，其包含所述的如式IB所示的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，以及一种或多种药学上可接受的辅料。The present invention also provides a pharmaceutical composition comprising the antibody drug conjugate of the formula IB, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph. An isotope compound, a pharmaceutically acceptable salt thereof or a prodrug thereof, and one or more pharmaceutically acceptable excipients.
本发明还提供了一种药物组合物，其包含所述的IA化合物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，以及一种或多种药学上可接受的辅料。The present invention also provides a pharmaceutical composition comprising the IA compound, a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, which is pharmaceutically acceptable a salt or a prodrug thereof, and one or more pharmaceutically acceptable excipients.
本发明还提供了一种药物组合物，其包含所述的IC-1、IC-2或IC-3化合物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，以及一种或多种药学上可接受的辅料。The present invention also provides a pharmaceutical composition comprising the IC-1, IC-2 or IC-3 compound, tautomer, optical isomer, hydrate, solvate, polymorph thereof a compound, an isotope compound, a pharmaceutically acceptable salt thereof or a prodrug thereof, and one or more pharmaceutically acceptable excipients.
本发明中，所述的药学上可接受的辅料是指药学领域常规的药用辅料，是为解决制剂的成型性、有效性、稳定性、安全性加入的除本发明的抗体药物偶联物以外的一切常规药用物料，如稀释剂(如羧甲淀粉钠等)、粘合剂(如聚维酮等)、崩解剂(如微晶纤维素等)、润滑剂(如硬脂酸镁、微粉硅胶等)、以及其它辅助剂。根据需要，可选择上述辅料，按本领域常规方法，将本发明的抗体药物偶联物制成药物制剂；所述的药物制剂为本领域各种常规剂型，如片剂、散剂、丸剂、胶囊剂、颗粒剂、口服液、干混悬剂或滴丸剂等。In the present invention, the pharmaceutically acceptable excipient refers to a conventional pharmaceutical excipient in the pharmaceutical field, and is an antibody drug conjugate of the present invention added to solve the moldability, effectiveness, stability and safety of the preparation. All other conventional pharmaceutical materials, such as diluents (such as sodium carboxymethyl starch), binders (such as povidone, etc.), disintegrants (such as microcrystalline cellulose, etc.), lubricants (such as stearic acid) Magnesium, micronized silica gel, etc.), as well as other adjuvants. The above-mentioned excipients may be selected as needed, and the antibody drug conjugate of the present invention is formulated into a pharmaceutical preparation according to a conventional method in the art; the pharmaceutical preparations are various conventional dosage forms in the art, such as tablets, powders, pills, capsules. Agents, granules, oral liquids, dry suspensions or pills.
本文所用术语“同位素化合物”是指本发明的抗体药物偶联物、所述的IA化合物、所述的IC-1、IC-2或IC-3化合物、其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药中含有一个或多个天然或非天然丰度的原子同位素。非天然丰度的原子同位素包括但不限于氘(2H或D)、氚(3H或T)、碘-125(125I)、磷-32(32P)、碳-13(13C)或碳-14(14C)。前述同位素化合物还可用作治疗或诊断剂(即，体内显影剂)，或研究工具。本发明的化合物的所有同位素变体，无论是否具有放射性，都包括在本发明的范围内。
The term "isotopic compound" as used herein refers to an antibody drug conjugate of the present invention, the IA compound, the IC-1, IC-2 or IC-3 compound, its tautomer, optical isomerism. The body, hydrate, solvate, polymorph, isotope compound, pharmaceutically acceptable salt thereof or prodrug thereof contains one or more atomic isotopes of natural or non-natural abundance. Non-natural abundance of atomic isotopes including, but not limited to, hydrazine ( 2 H or D), hydrazine ( 3 H or T), iodine-125 ( 125 I), phosphorus-32 ( 32 P), carbon-13 ( 13 C) Or carbon-14 ( 14 C). The aforementioned isotopic compounds can also be used as therapeutic or diagnostic agents (i.e., in vivo developers), or as research tools. All isotopic variations of the compounds of the invention, whether or not they are radioactive, are included within the scope of the invention.
本发明中，所述的术语“卤素”表示氟、氯、溴、碘或砹。In the present invention, the term "halogen" means fluorine, chlorine, bromine, iodine or hydrazine.
本发明中，所述的术语“氰基”表示-CN。In the present invention, the term "cyano" means -CN.
本发明中，杂烷基(C2-8杂烷基)一般指烷基(包括支链或直链烷基)中的一个或多个CH2结构被杂原子(例如O、S或NH)替代，且以碳原子与其他基团进行连接，例如CH2OCH2-、CH3CH2OCH2-或CH2OCH(CH3)-等等。In the present invention, a heteroalkyl group (C 2-8 heteroalkyl group) generally means that one or more CH 2 structures in an alkyl group (including a branched or linear alkyl group) are heteroatoms (for example, O, S or NH). Instead, it is attached to other groups by a carbon atom, such as CH 2 OCH 2 -, CH 3 CH 2 OCH 2 - or CH 2 OCH(CH 3 )- and the like.
本发明涉及含有两个连接位点的基团(如G或Q等)，其连接方式一般理解为按照该基团的所示结构的左右顺序方式连接到相应的化合物或结构中，例如当G为
时，在式IB化合物中其连接方式如下所示：The present invention relates to a group containing two linking sites (such as G or Q, etc.), the manner of which is generally understood to be linked to the corresponding compound or structure in a left-right sequential manner according to the structure of the group, for example when G for In the case of the compound of formula IB, the linkage is as follows:
又如，当Q为
时，在式IB化合物中其连接方式一般如下所示：
Another example is when Q is In the case of the compound of formula IB, the manner of attachment is generally as follows:
本发明所述的室温指环境温度，为10℃～35℃。The room temperature referred to in the present invention refers to an ambient temperature of 10 ° C to 35 ° C.
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。The above preferred conditions can be arbitrarily combined without departing from the ordinary knowledge in the art, that is, preferred embodiments of the present invention.
本发明所用试剂和原料均市售可得。The reagents and starting materials used in the present invention are commercially available.
本发明的积极进步效果在于：The positive effects of the present invention are:
本发明使用高细胞毒性的蒽环类化合物抗生素为毒素，设计合成了一系列结构新颖、对酸和肽酶组织蛋白酶稳定的抗体药物偶联物，优选地使用水溶性好的糖基进行取代并使用稳定性好的醚键进行连接，明显提高了水溶性和稳定性，体外活性测试表明其具有更高的细胞毒性，有良好的市场应用前景。The present invention uses a highly cytotoxic anthracycline antibiotic as a toxin, and designs and synthesizes a series of novel antibody drug conjugates which are stable to acid and peptidase cathepsins, preferably substituted with water-soluble glycosyl groups. The use of a stable ether bond for attachment significantly improves water solubility and stability. In vitro activity tests indicate that it has higher cytotoxicity and has good market application prospects.
相对于以美登醇，Auristatin等传统药物为毒素的抗体药物偶联物，本发明的抗体药物偶联物表现出更高癌细胞杀伤活性。The antibody drug conjugate of the present invention exhibits higher cancer cell killing activity than an antibody drug conjugate having a traditional drug such as maytansin, Auristatin or the like as a toxin.
IA-1和IA-2的合成路线：
Synthetic route of IA-1 and IA-2:
C-L-081为：
CL-081 is:
C-L-004为：
CL-004 is:
实施例1 IA-1的合成Example 1 Synthesis of IA-1
(1)化合物2的合成(1) Synthesis of Compound 2
取原料1(224mg,0.35mmol)溶于40mL甲醇和30mL水中，加入高碘酸钠(90mg,0.42mmol)溶于5mL水后的溶液。反应液室温搅拌1.5小时，LCMS检测原料已经完全转化为产物，减压移除甲醇，剩余物冷冻干燥得粗产品，加入100mL二氯甲烷溶解产物，过滤，滤液浓缩得粗产品210mg，直接用于下一步反应。LCMS(ESI)m/z＝628.7(M+H)+。Raw material 1 (224 mg, 0.35 mmol) was dissolved in 40 mL of methanol and 30 mL of water, and a solution of sodium periodate (90 mg, 0.42 mmol) dissolved in 5 mL of water was added. The reaction solution was stirred at room temperature for 1.5 hours. The material was completely converted to product by LCMS. Methanol was removed under reduced pressure. The residue was lyophilized to give a crude product. The product was dissolved in 100 mL of dichloromethane, filtered, and the filtrate was concentrated to give a crude product 210 mg. The next step is to react. LCMS (ESI) m / z = 628.7 (M + H) +.
(2)化合物3的合成(2) Synthesis of Compound 3
上一步制得的化合物2(188mg,0.3mol)溶于20mL干燥二氯甲烷中，加入HATU(228mg,0.6mmol)和DIPEA(0.25mL，1.5mmol)，再加入胺2a(156mg,0.45mmol)，反应液室温搅拌2小时。减压移除溶剂，剩余物硅胶柱层析(二氯甲烷/丙酮＝5:1)纯化得
产品120mg，为红色固体，产率44％，LCMS(ESI)m/z＝920.7(M+H)+。The compound 2 (188 mg, 0.3 mol) obtained in the previous step was dissolved in 20 mL of dry methylene chloride. EtOAc ( 228 g, 0.6 mmol) and DIPEA (0.25 mL, 1.5 mmol), and then the amine 2a (156mg, 0.45mmol) The reaction solution was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure. EtOAc mjjjjjjjjj ) + .
(3)化合物4的合成(3) Synthesis of Compound 4
将化合物3(28mg,0.03mmol)溶于2mL乙腈中，冰水浴冷却至0℃，加入二乙胺1mL升至室温搅拌1.5小时，常温减压移除乙腈和二乙胺，并用二氯甲烷带二乙胺两遍，得到30mg粗产品化合物4(红色固体)，直接用于下一步反应。LCMS(ESI)m/z＝698.7(M+H)+。The compound 3 (28 mg, 0.03 mmol) was dissolved in 2 mL of acetonitrile, cooled to 0 ° C in an ice water bath, 1 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours, and acetonitrile and diethylamine were removed under reduced pressure at room temperature and taken with dichloromethane. Two times of diethylamine, 30 mg of crude product compound 4 (red solid) was obtained, which was directly used for the next reaction. LCMS (ESI) m / z = 698.7 (M + H) +.
(4)化合物IA-1的合成(4) Synthesis of compound IA-1
将上一步制得的反应粗产物4(21mg,0.03mmol)溶于2mL干燥二氯甲烷中，加入C-L-081(28mg,0.06mol)和三乙胺(6μL,0.045mmol)。反应液室温搅拌2小时，减压移除溶剂，剩余物制备TLC(二氯甲烷/乙酸乙酯/甲醇＝50:50:1)纯化得13mg产品IA-1，红色固体，产率41％。LCMS(ESI)m/z＝1072.3(M+H)+。The crude reaction product 4 (21 mg, 0.03 mmol) obtained in the previous step was dissolved in 2 mL of dry methylene chloride, and EtOAc (EtOAc, EtOAc (EtOAc) The reaction was stirred at room temperature for 2 hr. EtOAc was evaporated. LCMS (ESI) m / z = 1072.3 (M + H) +.
1HNMR(400MHz,d-CDCl3)δppm 1.34-1.25(m,6H),1.38(d,J＝6.4Hz,3H),1.55-1.60(m,2H),1.73-1.79(m,1H),1.91-2.00(m,1H),2.18(t,J＝7.2Hz,2H),2.35-2.42(m,1H),2.62(t,J＝7.2Hz,2H),2.69-2.82(m,2H),2.94-3.08(m,4H),3.37-3.62(m,20H),4.72-4.94(m,4H),4.05(d,J＝5.6Hz,2H),4.09(s,3H),4.47(s,1H),4.71(s,1H),5.32-5.37(m,1H),5.54(t,J＝6.0Hz,1H),6.68(s,2H),7.40(d,J＝8.4Hz,1H),8.81(t,J＝8.4Hz,1H),8.04(d,J＝8.0Hz,1H),13.36(s,1H),13.95(s,1H)。 1 H NMR (400 MHz, d-CDCl 3 ) δ ppm 1.34-1.25 (m, 6H), 1.38 (d, J = 6.4 Hz, 3H), 1.55-1.60 (m, 2H), 1.73-1.79 (m, 1H), 1.91-2.00 (m, 1H), 2.18 (t, J = 7.2 Hz, 2H), 2.35-2.42 (m, 1H), 2.62 (t, J = 7.2 Hz, 2H), 2.69-2.82 (m, 2H) , 2.94-3.08 (m, 4H), 3.37-3.62 (m, 20H), 4.72-4.94 (m, 4H), 4.05 (d, J = 5.6 Hz, 2H), 4.09 (s, 3H), 4.47 (s , 1H), 4.71 (s, 1H), 5.32-5.37 (m, 1H), 5.54 (t, J = 6.0 Hz, 1H), 6.68 (s, 2H), 7.40 (d, J = 8.4 Hz, 1H) , 8.81 (t, J = 8.4 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 13.36 (s, 1H), 13.95 (s, 1H).
实施例2 IA-2的合成Example 2 Synthesis of IA-2
上一步反应粗产物4(21mg,0.03mmol)溶于2mL N,N-二甲基甲酰胺中，加入C-L-004(44mg,0.06mol)和三乙胺(6μL,0.045mmol)。反应液室温搅拌2小时，减压移除溶剂，剩余物制备TLC(二氯甲烷/丙酮/甲醇＝50:50:1)纯化得6mg产品IA-2，为红色固体，产率15％。LCMS(ESI)m/z＝1296.7(M+H)+。The crude product 4 (21 mg, 0.03 mmol) was dissolved in 2 mL of N,N-dimethylformamide, and then EtOAc (EtOAc, EtOAc (EtOAc) The reaction mixture was stirred at room temperature for 2 hr. EtOAc was evaporated. LCMS (ESI) m / z = 1296.7 (M + H) +.
化合物IA-3，037和039的合成路线：Synthetic route of compound IA-3, 037 and 039:
其中，C-L-066为：
Among them, CL-066 is:
SMCC为：
SMCC is:
McOSu：
McOSu:
实施例3 IA-3的合成Example 3 Synthesis of IA-3
(1)化合物2-1的合成(1) Synthesis of Compound 2-1
N-甲基乙醇胺(2g,26.7mmol)溶于50mL二氯甲烷中，加入Boc2O(5.9g,27.2mmol)。反应液室温搅拌2小时，浓缩，剩余物硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得产品4.5g，为无色油状物，产率96％。N-methylethanolamine (2 g, 26.7 mmol) was dissolved in 50 mL dichloromethane and Boc 2 O (5.9 g, 27.2 mmol). The reaction mixture was stirred at room temperature for 2 hr. EtOAc (EtOAc)
(2)化合物3-1的合成(2) Synthesis of Compound 3-1
上一步反应产物2-1(1.4g,8mmol)溶于50mL四氢呋喃中，冰水浴冷却至0℃，分批缓慢加入氢化钠(384mg,60％悬浮于矿物油，9.6mmol)，0℃搅拌20分钟，加入炔丙基溴(1.4mL,16mmol)，逐步升至室温搅拌2小时，冰水浴冷却至0℃，逐滴缓慢加入10mL饱和氯化铵溶液淬灭反应，乙酸乙酯萃取3次(40mL×3)，合并有机相，无水硫酸钠干燥，浓缩，剩余物硅胶柱层析(石油醚/乙酸乙酯＝10:1至5:1)纯化得1.5g产品3-1，为浅黄色油状物，产率79％。LCMS(ESI)m/z＝236.7(M+H)+。
The reaction product 2-1 (1.4 g, 8 mmol) was dissolved in 50 mL of THF. In a minute, add propargyl bromide (1.4 mL, 16 mmol), gradually warm to room temperature and stir for 2 hours, cool to 0 ° C in an ice water bath, slowly add 10 mL of saturated ammonium chloride solution dropwise to quench the reaction, and extract with ethyl acetate 3 times ( 40 mL × 3), the organic phase was combined, dried over anhydrous sodium sulfate, and concentrated, and the residue was purified by silica gel column chromatography ( petroleum ether / ethyl acetate = 10:1 to 5:1) to obtain 1.5 g of product 3-1. Yellow oil, yield 79%. LCMS (ESI) m / z = 236.7 (M + H) +.
(3)化合物4-1的合成(3) Synthesis of Compound 4-1
上一步反应产物3-1(640mg,3mmol)溶于5mL二氯甲烷中，冰水浴冷却至0℃，加入盐酸气的1,4-二氧六环溶液(10mL,4N,40mmol)，反应液室温搅拌2小时，减压浓缩，得700mg粗产品4-1，为浅黄色油状物，直接用于下一步反应。LCMS(ESI)m/z＝114.7(M+H)+。The reaction product of the previous step 3-1 (640 mg, 3 mmol) was dissolved in 5 mL of dichloromethane, cooled to 0 ° C in an ice water bath, and a solution of 1,4-dioxane (10 mL, 4 N, 40 mmol) of hydrochloric acid was added. The mixture was stirred at room temperature for 2 hr. LCMS (ESI) m / z = 114.7 (M + H) +.
(4)化合物5-1的合成(4) Synthesis of Compound 5-1
上一步反应产物4-1(1.13g,10mmol)溶于50mL二氯甲烷中，加入三乙胺(2.8mL,20mmol)，冰水浴冷却至0℃，分批加入Fmoc-Cl(3.1g,12mmol)。反应液升温至室温搅拌2小时，减压浓缩，往剩余物中加入50mL乙酸乙酯，饱和食盐水洗3次(30mL×3)，无水硫酸钠干燥，浓缩，粗产品经硅胶柱层析纯化(石油醚/乙酸乙酯＝5:1to 2:1)得1.5g产品5-1，为浅黄色油状物，产率50％。LCMS(ESI)m/z＝336.1(M+H)+。The reaction product of the previous step 4-1 (1.13 g, 10 mmol) was dissolved in 50 mL of dichloromethane, and then triethylamine (2.8 mL, 20 mmol) was added, and the mixture was cooled to 0 ° C in ice water, and Fmoc-Cl (3.1 g, 12 mmol) was added in portions. ). The reaction mixture was warmed to room temperature and stirred for 2 hr. EtOAc was evaporated, evaporated, evaporated, evaporated. (Petroleum Ether / Ethyl Acetate = 5:1 to 2:1) afforded 1.5 g of product 5-1 as a pale yellow oil, yield 50%. LCMS (ESI) m / z = 336.1 (M + H) +.
(5)化合物7-1的合成(5) Synthesis of Compound 7-1
化合物6-1(2g,3.5mmol)溶解在120mL的甲醇和1,4-二氧六环(1:2)混合溶剂中，加入原甲酸酯(2mL,18.3mmol)，混合物常温搅拌30分钟。然后逐滴加入液溴的氯仿溶液(w/v＝1:9,6.8mL,4.3mmol)，反应液在30℃下搅拌40分钟，待反应完全后直接倒入2L的无水***中，过滤，得到的粗产物用丙酮和***(1:1,25mL)重结晶，得到1.8g的化合物7-1，其为红色固体，收率78％。LCMS(ESI)m/z＝606.4(M+H)+。Compound 6-1 (2 g, 3.5 mmol) was dissolved in 120 mL of a mixed solvent of methanol and 1,4-dioxane (1:2), and then the toluate (2 mL, 18.3 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. . Then, a solution of liquid bromine in chloroform (w/v = 1:9, 6.8 mL, 4.3 mmol) was added dropwise, and the reaction solution was stirred at 30 ° C for 40 minutes. After the reaction was completed, it was poured into 2 L of anhydrous diethyl ether and filtered. The obtained crude product was recrystallized from acetone and diethyl ether (1:1, 25 mL) to afford 1.8 g of Compound 7-1 as a red solid. LCMS (ESI) m / z = 606.4 (M + H) +.
(6)化合物8-1的合成(6) Synthesis of Compound 8-1
化合物7-1(1.5g,3mmol)溶解在50mL的四氢呋喃中，加入叠氮化钠(390mg,6mmol)，上述混合物常温搅拌过夜。浓缩，得到的粗产物用100mL甲醇溶解后加入硅胶(20g,100-200目)，旋干后经过快速分离柱层析纯化(二氯甲烷/甲醇＝10:1)，得到750mg产物8-1，其为红色固体，产率53％。LCMS(ESI)m/z＝569.2(M+H)+。Compound 7-1 (1.5 g, 3 mmol) was dissolved in 50 mL of tetrahydrofuran, sodium azide (390 mg, 6 mmol) was added, and the mixture was stirred at room temperature overnight. Concentration, the obtained crude product was dissolved in 100 mL of methanol, and then added to silica gel (20 g, 100-200 mesh), which was purified by flash column chromatography (dichloromethane/methanol = 10:1) It was a red solid with a yield of 53%. LCMS (ESI) m / z = 569.2 (M + H) +.
(7)化合物9-1的合成(7) Synthesis of Compound 9-1
参考专利(GB296495、WO1998/002446、WO2012/073217,WO2014/177441，如WO1998/002446第7页实施例1)合成PNU-159682的方法，制备得到化合物9-1，其为红色固体，产率23％。LCMS(ESI)m/z＝667.2(M+H)+。Reference to the patent (GB296495, WO1998/002446, WO2012/073217, WO2014/177441, such as Example 1 of WO1998/002446, page 7) for the synthesis of PNU-159682, which gave compound 9-1 as a red solid, yield 23 %. LCMS (ESI) m / z = 667.2 (M + H) +.
(8)化合物10-1的合成(8) Synthesis of Compound 10-1
称取上一步反应产物9-1(40mg,0.12mmol)和化合物5-1(40mg,0.06mmol)于茄型瓶中，加入2mL叔丁醇和2mL甲醇，依次加入抗坏血酸钠(Sodium Ascorbate)(6mg,0.03mmol)和硫酸铜(10mg,0.06mmol)，反应液室温搅拌过夜。加入5mL水，乙酸乙酯萃取3次(20mL×3)，无水硫酸钠干燥，浓缩，粗产品经硅胶柱层析(二氯甲烷
/乙酸乙酯＝1:4)纯化得34mg产品10-1，为红色固体，产率57％。LCMS(ESI)m/z＝1002.7(M+H)+。The reaction product of the previous step 9-1 (40 mg, 0.12 mmol) and the compound 5-1 (40 mg, 0.06 mmol) were weighed into an eggplant type bottle, and 2 mL of t-butanol and 2 mL of methanol were added thereto, followed by sodium ascorbate (Sodium Ascorbate) (6 mg). , 0.03 mmol) and copper sulfate (10 mg, 0.06 mmol), and the reaction mixture was stirred at room temperature overnight. Add 5 mL of water, extract with ethyl acetate 3 times (20 mL × 3), dry over anhydrous sodium sulfate, and then concentrate, and the crude product is purified by silica gel column chromatography (dichloromethane / ethyl acetate = 1:4) 1, a red solid with a yield of 57%. LCMS (ESI) m / z = 1002.7 (M + H) +.
(9)化合物11-1的合成(9) Synthesis of Compound 11-1
上一步反应产物10-1(15mg,0.015mol)溶于1mL乙腈中，冰水浴冷却至0℃，加入二乙胺0.5mL，升至室温搅拌1.5小时，常温减压移除乙腈和二乙胺，并用二氯甲烷带二乙胺两遍，得到15mg粗产品11-1，为红色固体，直接用于下一步反应。LCMS(ESI)m/z＝780.3(M+H)+。The reaction product 10-1 (15 mg, 0.015 mol) in the previous step was dissolved in 1 mL of acetonitrile, cooled to 0 ° C in an ice water bath, 0.5 mL of diethylamine was added, and the mixture was stirred at room temperature for 1.5 hours, and acetonitrile and diethylamine were removed under reduced pressure at room temperature. And using diethylamine with diethylamine twice, 15 mg of crude product 11-1 was obtained as a red solid, which was directly used for the next reaction. LCMS (ESI) m / z = 780.3 (M + H) +.
(10)化合物IA-3的合成(10) Synthesis of Compound IA-3
化合物11-1(23mg,0.03mmol)溶解在10mL的二氯甲烷中，氮气条件下，加入化合物C-L-066(23mg,0.06mmol)，然后加入9μL的三乙胺，反应混合物在室温氮气条件下搅拌2小时。浓缩除掉有机溶剂，粗品通过PREP-TLC(二氯甲烷/乙酸乙酯/甲醇＝35:10:5)分离纯化，得到7mg的化合物IA-3，收率22％，其为红色固体。LCMS(ESI)m/z＝1071.1(M+Na)+。Compound 11-1 (23 mg, 0.03 mmol) was dissolved in 10 mL of dichloromethane, and then, under nitrogen, compound CL-066 (23 mg, 0.06 mmol) was added, then 9 μL of triethylamine was added, and the reaction mixture was stirred at room temperature under nitrogen. Stir for 2 hours. The organic solvent was removed by concentration, and the crude material was purified and purified eluting with EtOAc (EtOAc/EtOAc/EtOAc/EtOAc LCMS (ESI) m / z = 1071.1 (M + Na) +.
实施例4 化合物IA-4的合成Example 4 Synthesis of Compound IA-4
(1)化合物12-1的合成(1) Synthesis of Compound 12-1
化合物11-1(30mg,0.038mmol)溶解在10mL的二氯甲烷中，氮气条件下，加入化合物11a(26mg,0.076mmol)，然后加入10μL的三乙胺，反应混合物在氮气条件下室温搅拌2小时。浓缩除掉有机溶剂，粗品通过PREP-TLC(DCM/EA/MeOH＝35:10:5)分离纯化，得到15mg的化合物12-1，收率39％，其为红色固体。LCMS(ESI)m/z＝1002.3(M+Na)+。Compound 11-1 (30 mg, 0.038 mmol) was dissolved in 10 mL of dichloromethane, and then, under nitrogen, compound 11a (26 mg, 0.076 mmol) was added, then 10 μL of triethylamine was added, and the reaction mixture was stirred at room temperature under nitrogen. hour. The organic solvent was removed by EtOAc (EtOAc) (EtOAc:EtOAc: LCMS (ESI) m / z = 1002.3 (M + Na) +.
(2)化合物13-1的合成(2) Synthesis of Compound 13-1
上一步反应产物12-1(15mg,0.015mmol)溶解在乙酸乙酯和甲醇(1:1)的混合溶剂(2mL)中，在氮气体系下，加入pH＝7.5的磷酸钾缓冲溶液(PBS buffer)1mL和DTT(二硫苏糖醇12mg,0.075mmol)，反应混合物室温搅拌16小时后，加入pH＝6.0的磷酸钾缓冲溶液5mL淬灭反应。乙酸乙酯萃取(10mL×3)，有机相无水硫酸钠干燥，过滤、浓缩，粗品直接用于下一步。LCMS(ESI)m/z＝956.3(M+H)+。The reaction product of the previous step 12-1 (15 mg, 0.015 mmol) was dissolved in a mixed solvent of ethyl acetate and methanol (1:1) (2 mL), and a potassium phosphate buffer solution (PBS buffer) of pH=7.5 was added under a nitrogen atmosphere. 1 mL and DTT (dithiothreitol 12 mg, 0.075 mmol), the reaction mixture was stirred at room temperature for 16 hours, and then quenched by adding 5 mL of a potassium phosphate buffer solution of pH = 6.0. It was extracted with ethyl acetate (10 mL × 3), dried over anhydrous sodium sulfate, filtered and evaporated. LCMS (ESI) m / z = 956.3 (M + H) +.
(3)化合物IA-4的合成(3) Synthesis of Compound IA-4
化合物13-1(12mg,0.012mmol)溶于2mL四氢呋喃中，加入pH＝6的磷酸钾缓冲溶液0.05mL和SMCC(21mg,0.063mmol)，氮气保护下室温搅拌过夜。浓缩，直接由Prep-TLC(展开剂为甲醇/二氯甲烷＝1:10)纯化得4.6mg产品IA-4，为红色固体，产率28％。LCMS(ESI)m/z＝1290.5(M+H)+。
Compound 13-1 (12 mg, 0.012 mmol) was dissolved in 2 mL of tetrahydrofuran, and then added to a solution of potassium phosphate buffer of pH=6, 0.05 mL, and SMCC (21 mg, 0.063 mmol). Concentration, directly purified by Prep-TLC (developing solvent: methanol / methylene chloride = 1 : 10) afforded 4.6 g of product IA-4 as a red solid, yield 28%. LCMS (ESI) m / z = 1290.5 (M + H) +.
实施例5 IA-5的合成Example 5 Synthesis of IA-5
化合物11-1(23mg,0.03mmol)溶解在10mL的二氯甲烷中，氮气条件下，加入化合物McOSu(19mg,0.06mmol)，然后加入9μL的三乙胺，反应混合物在室温氮气条件下搅拌2小时。浓缩除掉有机溶剂，粗品通过PREP-TLC(二氯甲烷/乙酸乙酯/甲醇＝35:10:5)分离纯化，得到7.6mg的化合物IA-5，收率26％，其为红色固体。LCMS(ESI)m/z＝995.7(M+Na)+。1HNMR(400MHz,CDCl3)δppm 1.44(d,J＝5.6Hz,3H),1.56-1.78(m,8H),2.10-2.14(m,1H),2.28-2.36(m,2H),2.60(d,J＝11.2Hz,1H),2.73-2.84(m,2H),2.89&3.04(s,3H),3.32-3.42(m,2H),3.46(s,3H),3.48-3.52(m,2H),3.57-3.61(m,3H),3.64-3.69(m,2H),3.92(t,J＝7.6Hz,,1H),4.05-4.07(m,1H),4.09(s,3H),4.11(d,J＝6.0Hz,1H),4.47(d,J＝4.0Hz,1H),4.68(d,J＝9.2Hz,2H),4.71-4.72(m,1H),5.16(s,1H),5.37(s,1H),5.52(t,J＝4.0Hz,1H),5.72(t,J＝14.0Hz,1H),5.87&5.83(d,J＝4.2Hz,1H),6.67(s,2H),7.58(d,J＝9.2Hz,1H),7.73(t,J＝6.0Hz,1H),7.97(d,J＝6.0Hz,1H),13.21(s,1H),13.84(s,1H)。Compound 11-1 (23 mg, 0.03 mmol) was dissolved in 10 mL of dichloromethane, and then, under nitrogen, was added to the compound of EtOAc (19 mg, 0.06 mmol), then 9 μL of triethylamine was added, and the reaction mixture was stirred at room temperature under nitrogen. hour. The organic solvent was removed by concentration, and the crude material was purified and purified eluting with EtOAc (EtOAc/EtOAc/EtOAc/EtOAc LCMS (ESI) m / z = 995.7 (M + Na) +. 1 H NMR (400 MHz, CDCl 3 ) δ ppm 1.44 (d, J = 5.6 Hz, 3H), 1.56-1.78 (m, 8H), 2.10-2.14 (m, 1H), 2.28-2.36 (m, 2H), 2.60 ( d, J = 11.2 Hz, 1H), 2.73 - 2.84 (m, 2H), 2.89 & 3.04 (s, 3H), 3.32-3.42 (m, 2H), 3.46 (s, 3H), 3.48-3.52 (m , 2H), 3.57-3.61 (m, 3H), 3.64-3.69 (m, 2H), 3.92 (t, J = 7.6 Hz, 1H), 4.05-4.07 (m, 1H), 4.09 (s, 3H) , 4.11 (d, J = 6.0 Hz, 1H), 4.47 (d, J = 4.0 Hz, 1H), 4.68 (d, J = 9.2 Hz, 2H), 4.71-4.72 (m, 1H), 5.16 (s, 1H), 5.37 (s, 1H), 5.52 (t, J = 4.0 Hz, 1H), 5.72 (t, J = 14.0 Hz, 1H), 5.87 & 5.83 (d, J = 4.2 Hz, 1H), 6.67 (s, 2H), 7.58 (d, J = 9.2 Hz, 1H), 7.73 (t, J = 6.0 Hz, 1H), 7.97 (d, J = 6.0 Hz, 1H), 13.21 (s, 1H), 13.84 (s, 1H).
化合物IA-6，IA-7，IA-8的合成路线：Synthetic route of compound IA-6, IA-7, IA-8:
实施例6 IA-6的合成Example 6 Synthesis of IA-6
化合物1(100mg,0.16mmol)溶解在10mL的二氯甲烷中，依次加入甲醇(10mL)，三乙酰氧基硼氢化钠(50mg,0.23mmol)，上述混合物在氮气室温条件下搅拌1.5小时，
然后加入另外50mg的三乙酰氧基硼氢化钠，氮气条件下室温搅拌1小时。减压除去溶剂后，残余物通过快速分离柱色谱分离纯化(二氯甲烷/甲醇＝10:1)，得到70mg的化合物IA-6，收率70％，其为红色固体。LCMS(ESI)m/z＝644.2(M+H)+。Compound 1 (100 mg, 0.16 mmol) was dissolved in 10 mL of dichloromethane, and then methanol (10 mL), sodium triacetoxyborohydride (50 mg, 0.23 mmol), and the mixture was stirred at room temperature under nitrogen for 1.5 hours, then An additional 50 mg of sodium triacetoxyborohydride was added and the mixture was stirred at room temperature for 1 hour under nitrogen. After the solvent was removed under reduced pressure, the residue was purified (jjjjjjjjjjj LCMS (ESI) m / z = 644.2 (M + H) +.
1HNMR(400MHz,CDCl3)δppm 1.42(t,J＝5.2Hz,3H),1.63-1.79(m,6H),1.92-1.96(m,2H),2.60-2.88(m,6H),3.21-3.69(m,8H),3.89-3.93(m,3H),4.01-4.08(m,5H),4.48(s,1H),4.70-4.72(m,2H),5.30(s,1H),5.49(q,J1＝4.8Hz,J2＝4.4Hz,1H),7.38(d,J＝8.0Hz,1H),7.77(t,J＝8.8Hz,1H),8.02(d,J＝7.6Hz,1H),13.32(d,J＝8.0Hz,1H),13.88(d,J＝4.4Hz,1H)。 1 H NMR (400 MHz, CDCl 3 ) δ ppm 1.42 (t, J = 5.2 Hz, 3H), 1.63-1.79 (m, 6H), 1.92-1.96 (m, 2H), 2.60-2.88 (m, 6H), 3.21. 3.69 (m, 8H), 3.89-3.93 (m, 3H), 4.01-4.08 (m, 5H), 4.48 (s, 1H), 4.70-4.72 (m, 2H), 5.30 (s, 1H), 5.49 ( q, J 1 = 4.8 Hz, J 2 = 4.4 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.77 (t, J = 8.8 Hz, 1H), 8.02 (d, J = 7.6 Hz, 1H), 13.32 (d, J = 8.0 Hz, 1H), 13.88 (d, J = 4.4 Hz, 1H).
实施例7 IA-7的合成Example 7 Synthesis of IA-7
(1)化合物2-2的合成(1) Synthesis of Compound 2-2
021(50mg,0.078mmol)溶解在20mL的二氯甲烷中，加入戴斯-马丁氧化剂(Dess-Matin,50mg,0.117mmol)，上述混合物在氮气室温条件下搅拌2小时，上述混合物用10％的Na2S2O3溶液(10mL)和饱和碳酸氢钠溶液(10mL)淬灭，用二氯甲烷(50mL×5)萃取，有机相无水硫酸钠干燥，过滤和浓缩，得到40mg的粗品化合物2-2，其为红色固体，粗品直接用于下一步。LCMS(ESI)m/z＝642.1(M+H)+。021 (50 mg, 0.078 mmol) was dissolved in 20 mL of dichloromethane, and then added with Dess-Matin oxidant (Dess-Matin, 50 mg, 0.117 mmol). The mixture was stirred at room temperature for 2 hours under nitrogen, and the mixture was 10%. na 2 S 2 O 3 solution (10mL) and saturated sodium bicarbonate solution (10 mL) quenched and extracted with methylene chloride (50mL × 5), the organic phase dried over anhydrous sodium sulfate, filtered and concentrated to give 40mg of the crude compound 2-2, which is a red solid, the crude product is used directly in the next step. LCMS (ESI) m / z = 642.1 (M + H) +.
(2)化合物IA-7的合成(2) Synthesis of Compound IA-7
化合物2-2(40mg,0.062mmol)溶解在10mL的二氯甲烷中，加入化合物
(25mg，0.075mmol)，上述混合物在氮气室温条件下搅拌2小时，减压除去溶剂后，残余物通过快速分离柱色谱分离纯化(二氯甲烷/乙酸乙酯/甲醇＝35:10:5)，得到15mg的化合物IA-7，收率35％，其为红色固体。LCMS(ESI)m/z＝698.1(M+H)+。Compound 2-2 (40 mg, 0.062 mmol) was dissolved in 10 mL of dichloromethane and compound was added (25 mg, 0.075 mmol), the mixture was stirred at room temperature for 2 hr under N2HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH 15 mg of compound IA-7 was obtained in a yield of 35% as a red solid. LCMS (ESI) m / z = 698.1 (M + H) +.
实施例8 IA-8的合成Example 8 Synthesis of IA-8
(1)化合物3-2的合成(1) Synthesis of Compound 3-2
在0℃氩气保护条件下，化合物035(12mg,0.017mmoL)用5mL的0.1N的氢氧化钠溶液处理，反应混合物在同一条件下搅拌2小时，然后加入5mL水，调节pH＝2，二氯甲烷萃取(20mL×5),有机相用无水硫酸钠干燥，过滤，浓缩，得到5mg的粗品，粗品直接用于下一步。LCMS(ESI)m/z＝684.2(M+H)+。Compound 035 (12 mg, 0.017 mmol) was treated with 5 mL of 0.1 N sodium hydroxide solution under argon atmosphere at 0 ° C. The reaction mixture was stirred under the same conditions for 2 hours, then 5 mL of water was added to adjust pH = 2, The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give 5 mg of crude material. LCMS (ESI) m / z = 684.2 (M + H) +.
(2)化合物5-2的合成(2) Synthesis of Compound 5-2
上一步反应产物3-2(10mg,0.015mmol)溶解在5mL二氯甲烷中，氮气条件下，依次加入化合物2a(15mg,0.043mmol)，HATU(25mg,0.066mmol),然后加入DIPEA(18μL,0.1mmol)，反应混合物在氮气条件下室温搅拌2小时。浓缩除掉溶剂，粗品通过快速分离柱(二氯甲烷/乙酸乙酯/甲醇＝30:10:5)分离纯化，得到5mg的化合物5-2，收率36％，
其为红色固体。LCMS(ESI)m/z＝976.2(M+H)+。The reaction product of the previous step 3-2 (10 mg, 0.015 mmol) was dissolved in 5 mL of dichloromethane, and then, under nitrogen, compound 2a (15 mg, 0.043 mmol), HATU (25 mg, 0.066 mmol), then DIPEA (18 μL, 0.1 mmol), the reaction mixture was stirred at room temperature under nitrogen for 2 h. The solvent was removed by concentration, and the crude material was purified and purified, mjjjjjjjjjjj LCMS (ESI) m / z = 976.2 (M + H) +.
(3)化合物6-2的合成(3) Synthesis of Compound 6-2
上一步反应产物5-2(5mg,0.007mmol)溶解在2mL的乙腈中，冰水浴冷却至0℃，加入1mL的二乙胺，搅拌1小时。浓缩移除有机溶剂，得到4mg的粗品化合物6-2，其为红色固体。粗品直接用于下一步。LCMS(ESI)m/z＝754.1(M+H)+。The reaction product 5-2 (5 mg, 0.007 mmol) from the previous step was dissolved in 2 mL of acetonitrile, cooled to 0 ° C in an ice water bath, and 1 mL of diethylamine was added and stirred for 1 hour. Concentration and removal of the organic solvent gave 4 mg of crude compound 6-2 as a red solid. The crude product was used directly in the next step. LCMS (ESI) m / z = 754.1 (M + H) +.
(4)化合物IA-8的合成(4) Synthesis of compound IA-8
化合物6-2(4mg,0.003mmol)溶解在2mL的二氯甲烷中，氮气条件下，加入化合物McOSu(4mg,0.013mmol)，然后加入2μL三乙胺，反应混合物在氮气条件下室温搅拌2小时。浓缩，粗品通过PREP-TLC(二氯甲烷/乙酸乙酯/甲醇＝10:5:1)分离纯化，得到1.2mg的化合物IA-8，收率24％，其为红色固体。LCMS(ESI)m/z＝947.3(M+H)+。Compound 6-2 (4 mg, 0.003 mmol) was dissolved in 2 mL of dichloromethane, and then, under nitrogen, the compound of EtOAc (4 mg, 0.013 mmol) was added, then 2 μL of triethylamine was added, and the reaction mixture was stirred at room temperature under nitrogen for 2 hours. . Concentration, the crude was purified by EtOAc (EtOAc/EtOAc/EtOAc) LCMS (ESI) m / z = 947.3 (M + H) +.
化合物IA-9,IA-10,IA-11,IA-12的合成路线：Synthetic route of compound IA-9, IA-10, IA-11, IA-12:
其中，C-L-055为：
Among them, CL-055 is:
实施例9 IA-9的合成Example 9 Synthesis of IA-9
化合物8-1(60mg,0.09mmol)溶解在5mL的THF中，依次加入化合物1a-3(61mg,0.18mmol)，三苯基膦(47mg)和饱和碳酸氢钠(0.1mL)，上述反应混合物在常温搅拌过夜。浓缩，得到的粗产物通过快速分离柱层析纯化(二氯甲烷/丙酮＝5:1)，得到30mg化
合物IA-9，为红色固体，产率39％。LCMS(ESI)m/z＝863.2(M+H)+。1HNMR(400MHz,CDCl3)δppm 1.40-1.48(m,6H),1.61(d,J＝7.2Hz,1H),1.82-1.89(m,1H),2.07-2.21(m,2H),2.43(s,3H),2.86(s,1H),2.91-2.95(m,2H),3.04(d,J＝19.2Hz,1H),3.17(d,J＝8.4Hz,1H),3.25-3.32(m,1H),3.45(s,3H),3.54-3.63(m,2H),3.69-3.82(m,8H),3.99(d,J＝6.4Hz,1H),4.09(s,3H),4.21(d,J＝6.8Hz,1H),4.51-4.63(m,1H),4.67(s,1H),4.81(s,1H),5.34(s,1H),5.57(t,J＝4.8Hz,1H),7.39(d,J＝8.0Hz,1H),7.60(t,J＝5.6Hz,1H),7.78(t,J＝7.6Hz,1H),8.03(d,J＝7.6Hz,1H),13.26(s,1H),13.90(s,1H)。Compound 8-1 (60 mg, 0.09 mmol) was dissolved in 5 mL of THF, and then compound 1a-3 (61 mg, 0.18 mmol), triphenylphosphine (47 mg) and saturated sodium hydrogen carbonate (0.1 mL) Stir at room temperature overnight. Concentration, the obtained crude product was purified by flash column chromatography (dichloromethane/acetone = 5:1) to yield 30 mg of compound IA-9 as a red solid. LCMS (ESI) m / z = 863.2 (M + H) +. 1 H NMR (400 MHz, CDCl 3 ) δ ppm 1.40-1.48 (m, 6H), 1.61 (d, J = 7.2 Hz, 1H), 1.82-1.89 (m, 1H), 2.07-2.21 (m, 2H), 2.43 ( s, 3H), 2.86 (s, 1H), 2.91-2.95 (m, 2H), 3.04 (d, J = 19.2 Hz, 1H), 3.17 (d, J = 8.4 Hz, 1H), 3.25-3.32 (m , 1H), 3.45 (s, 3H), 3.54-3.63 (m, 2H), 3.69-3.82 (m, 8H), 3.99 (d, J = 6.4 Hz, 1H), 4.09 (s, 3H), 4.21. d, J = 6.8 Hz, 1H), 4.51-4.63 (m, 1H), 4.67 (s, 1H), 4.81 (s, 1H), 5.34 (s, 1H), 5.57 (t, J = 4.8 Hz, 1H) ), 7.39 (d, J = 8.0 Hz, 1H), 7.60 (t, J = 5.6 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H), 13.26 (s, 1H), 13.90 (s, 1H).
实施例10 IA-10的合成Example 10 Synthesis of IA-10
(1)化合物2-3的合成(1) Synthesis of Compound 2-3
上一步反应产物IA-9(30mg,0.035mmol)溶解在乙酸乙酯和甲醇(1:1)的混合溶剂(3mL)中，在氮气体系下，加入PH＝7.5的磷酸钾缓冲溶液1.5mL和DTT(27mg,0.175mmol)，反应混合物室温搅拌16小时后，加入PH＝6.0的磷酸钾缓冲溶液5ml淬灭反应。乙酸乙酯萃取(10mL×3)，有机相无水硫酸钠干燥，过滤、浓缩，粗品直接用于下一步。LCMS(ESI)m/z＝817.2(M+H)+。The reaction product of the previous step, IA-9 (30 mg, 0.035 mmol) was dissolved in a mixed solvent of ethyl acetate and methanol (1:1) (3 mL), and 1.5 mL of a potassium phosphate buffer solution of pH=7.5 was added under a nitrogen atmosphere. After DTT (27 mg, 0.175 mmol), the reaction mixture was stirred at room temperature for 16 hr, and then quenched by adding 5 ml of potassium phosphate buffer solution of pH = 6.0. It was extracted with ethyl acetate (10 mL × 3), dried over anhydrous sodium sulfate, filtered and evaporated. LCMS (ESI) m / z = 817.2 (M + H) +.
(2)化合物IA-10的合成(2) Synthesis of Compound IA-10
化合物2-3(25mg,0.03mmol)溶于3mL N,N-二甲基甲酰胺中，加入PH＝6的磷酸钾缓冲溶液0.1mL和SMCC(50mg,0.15mmol)，氮气保护下室温搅拌过夜。反应液过滤，直接由制备HPLC纯化得12mg产品IA-10，为红色固体，产率30％。Compound 2-3 (25 mg, 0.03 mmol) was dissolved in 3 mL of N,N-dimethylformamide, and 0.1 mL of a potassium phosphate buffer solution of pH=6 and SMCC (50 mg, 0.15 mmol) were added and stirred overnight at room temperature under nitrogen atmosphere. . The reaction mixture was filtered and purified by preparative HPLC to afford 12 g of IA-10 as a red solid.
LCMS(ESI)m/z＝1150.3(M+H)+。1HNMR(400MHz,CDCl3)δppm 1.06(q,J＝11.6Hz,2H),1.25(s,3H),1.43(s,3H),1.52(q,J＝10.8Hz,2H),1.71-1.80(m,4H),2.00-2.24(m,4H),2.46-2.59(m,2H),2.81(s,4H),2.90-3.06(m,2H),3.12-3.28(m,4H),3.38(d,J＝6.8Hz,3H),3.45(s,3H),3.60-4.03(m,12H),4.09(s,3H),4.22(d,J＝6.8Hz,1H),4.45-4.60(m,2H),4.67(s,1H),4.81(s,1H),5.33(s,1H),5.56(t,J＝5.6Hz,1H),7.39(d,J＝8.8Hz,1H),7.53(d,J＝5.6Hz,1H),7.78(t,J＝7.6Hz,1H),8.03(d,J＝7.6Hz,1H),13.28(s,1H),13.89(s,1H)。LCMS (ESI) m / z = 1150.3 (M + H) +. 1 H NMR (400 MHz, CDCl 3 ) δ ppm 1.06 (q, J = 11.6 Hz, 2H), 1.25 (s, 3H), 1.43 (s, 3H), 1.52 (q, J = 10.8 Hz, 2H), 1.71-1.80 (m, 4H), 2.00-2.24 (m, 4H), 2.46-2.59 (m, 2H), 2.81 (s, 4H), 2.90-3.06 (m, 2H), 3.12-3.28 (m, 4H), 3.38 (d, J = 6.8 Hz, 3H), 3.45 (s, 3H), 3.60-4.03 (m, 12H), 4.09 (s, 3H), 4.22 (d, J = 6.8 Hz, 1H), 4.45 - 4.60 ( m, 2H), 4.67 (s, 1H), 4.81 (s, 1H), 5.33 (s, 1H), 5.56 (t, J = 5.6 Hz, 1H), 7.39 (d, J = 8.8 Hz, 1H), 7.53 (d, J = 5.6 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H), 13.28 (s, 1H), 13.89 (s, 1H).
实施例11 IA-11的合成Example 11 Synthesis of IA-11
(1)化合物3-3的合成(1) Synthesis of Compound 3-3
化合物8-1(60mg,0.09mmol)溶解在5mL四氢呋喃中，依次加入化合物1b-3(105mg,0.18mmol)，三苯基膦(47mg,0.18mmol)和饱和碳酸氢钠(0.1mL)，上述反应混合物在室温搅拌过夜。浓缩，得到的粗产物通过快速分离柱层析纯化(二氯甲烷/丙酮＝5:1)，得到20mg化合物3-3，为红色固体，产率20％。LCMS(ESI)m/z 1110.2(M+H)+。Compound 8-1 (60 mg, 0.09 mmol) was dissolved in 5 mL of tetrahydrofuran, and then compound 1b-3 (105 mg, 0.18 mmol), triphenylphosphine (47 mg, 0.18 mmol) and saturated sodium hydrogen carbonate (0.1 mL), The reaction mixture was stirred at room temperature overnight. Concentration, the obtained crude product was purified by flash column chromatography (dichloromethane/acetone = 5:1) to afford 20 mg of compound 3-3 as a red solid. LCMS (ESI) m / z 1110.2 (M + H) +.
(2)化合物4-3的合成
(2) Synthesis of compound 4-3
上一步反应产物3-3(20mg,0.018mol)溶于2mL乙腈中，冰水浴冷却至0℃，加入二乙胺1mL，搅拌1.5小时，常温减压移除乙腈和二乙胺，并用二氯甲烷带二乙胺两遍，得到15mg粗产品4-3，为红色固体，直接用于下一步反应。LCMS(ESI)m/z＝888.3(M+H)+。The reaction product 3-3 (20mg, 0.018mol) was dissolved in 2mL acetonitrile in the previous step, cooled to 0 ° C in ice water bath, 1 mL of diethylamine was added, stirred for 1.5 hours, acetonitrile and diethylamine were removed under reduced pressure at room temperature, and dichlorochloride was used. Methane was taken twice with diethylamine to give 15 mg of crude product 4-3 as a red solid which was used directly for the next reaction. LCMS (ESI) m / z = 888.3 (M + H) +.
(3)化合物IA-11的合成(3) Synthesis of compound IA-11
化合物4-3(15mg,0.017mmol)溶解在2mL的二氯甲烷中，氮气条件下，加入化合物C-L-055(10mg,0.034mmol)，反应混合物在氮气条件下室温搅拌2小时。浓缩，粗品通过PREP-TLC(二氯甲烷/乙酸乙酯/甲醇＝35:10:5)分离纯化，得到1.0mg的化合物IA-11，收率5.7％，其为红色固体。LCMS(ESI)m/z＝1039.4(M+Na)+。Compound 4-3 (15 mg, 0.017 mmol) was dissolved in dichloromethane (2 mL). Concentration, the crude was purified by EtOAc (EtOAc/EtOAc/EtOAc) LCMS (ESI) m / z = 1039.4 (M + Na) +.
实施例12 IA-12的合成Example 12 Synthesis of IA-12
(1)化合物6-3的合成(1) Synthesis of Compound 6-3
化合物8-1(60mg,0.09mmol)溶解在5mL四氢呋喃中，依次加入化合物1c-3(81mg,0.18mmol)，三苯基膦(47mg,0.18mmol)和饱和碳酸氢钠(0.1mL)，上述混合物在室温搅拌过夜。浓缩，得到的粗产物通过快速分离柱层析纯化(二氯甲烷/丙酮＝5:1)，得到30mg化合物6-3，为红色固体，产率34％。LCMS(ESI)m/z 978.2(M+H)+。Compound 8-1 (60 mg, 0.09 mmol) was dissolved in 5 mL of tetrahydrofuran, and then compound 1c-3 (81 mg, 0.18 mmol), triphenylphosphine (47 mg, 0.18 mmol) and saturated sodium hydrogen carbonate (0.1 mL), The mixture was stirred at room temperature overnight. Concentration, the obtained crude product was purified by flash column chromatography (dichloromethane/acetone = 5:1) to afford 30 mg of Compound 6-3 as a red solid. LCMS (ESI) m / z 978.2 (M + H) +.
(2)化合物7-3的合成(2) Synthesis of compound 7-3
上一步反应产物6-3(30mg,0.03mol)溶于2mL乙腈中，冰水浴冷却至0℃，加入二乙胺1mL，搅拌1.5小时，常温减压移除乙腈和二乙胺，并用二氯甲烷带二乙胺两遍，得到22mg粗产品7-3，为红色固体，直接用于下一步反应。LCMS(ESI)m/z＝756.3(M+H)+。The reaction product 6-3 (30mg, 0.03mol) in the previous step was dissolved in 2mL acetonitrile, cooled to 0 ° C in ice water bath, 1 mL of diethylamine was added, stirred for 1.5 hours, acetonitrile and diethylamine were removed under reduced pressure at room temperature, and dichloroethylene was used. Methane was taken twice with diethylamine to give 22 mg of crude product 7-3 as a red solid which was used directly for the next reaction. LCMS (ESI) m / z = 756.3 (M + H) +.
(3)化合物IA-12的合成(3) Synthesis of compound IA-12
化合物7-3(22mg,0.029mmol)溶解在2mL的二氯甲烷中，氮气条件下，加入化合物C-L-066(15mg,0.058mmol)，反应混合物在氮气条件下室温搅拌2小时。浓缩除掉有机溶剂，粗品通过PREP-TLC(二氯甲烷/乙酸乙酯/甲醇＝35:10:5)分离纯化，得到5.0mg的化合物IA-12，收率16.7％，其为红色固体。LCMS(ESI)m/z＝1025.4(M+Na)+。1HNMR(400MHz,CDCl3)δppm 0.81(d,J＝6.0Hz,3H),1.35(d,J＝5.2Hz,3H),1.95-2.11(m,2H),2.15(d,J＝11.2Hz,1H),2.44(d,J＝11.2Hz,1H),2.50(t,J＝4.4Hz,1H),2.66(s,3H),2.68-2.76(m,2H),2.97(d,J＝14.8Hz,1H),3.19(d,J＝14.8Hz,1H),3.30-3.35(m,1H),3.36(d,J＝4.0Hz,2H),3.39(s,3H),3.41-3.61(m,8H),3.66(t,J＝4.4Hz,2H),3.70(t,J＝4.0Hz,2H),3.82-4.07(m,3H),3.94(s,2H),4.03(s,3H),4.39-4.54(m,2H),4.62-4.71(m,2H),5.28(t,J＝4.0Hz,2H),5.44(t,J＝4.0Hz,1H),6.64(s,2H),6.99(t,J＝4.8Hz,1H),7.26(t,J＝4.8Hz,1H),7.33(d,J＝6.8
Hz,1H),7.72(t,J＝6.4Hz,1H),7.97(d,J＝6.4Hz,1H),13.22(s,1H),13.85(s,1H)。Compound 7-3 (22 mg, 0.029 mmol) was dissolved in dichloromethane (2 mL). The organic solvent was removed by concentration. EtOAc (EtOAc m. LCMS (ESI) m / z = 1025.4 (M + Na) +. 1 H NMR (400 MHz, CDCl 3 ) δ ppm 0.81 (d, J = 6.0 Hz, 3H), 1.35 (d, J = 5.2 Hz, 3H), 1.95-2.11 (m, 2H), 2.15 (d, J = 11.2 Hz) , 1H), 2.44 (d, J = 11.2 Hz, 1H), 2.50 (t, J = 4.4 Hz, 1H), 2.66 (s, 3H), 2.68-2.76 (m, 2H), 2.97 (d, J = 14.8 Hz, 1H), 3.19 (d, J = 14.8 Hz, 1H), 3.30-3.35 (m, 1H), 3.36 (d, J = 4.0 Hz, 2H), 3.39 (s, 3H), 3.41-3.61 ( m, 8H), 3.66 (t, J = 4.4 Hz, 2H), 3.70 (t, J = 4.0 Hz, 2H), 3.82-4.07 (m, 3H), 3.94 (s, 2H), 4.03 (s, 3H) ), 4.39-4.54 (m, 2H), 4.62-4.71 (m, 2H), 5.28 (t, J = 4.0 Hz, 2H), 5.44 (t, J = 4.0 Hz, 1H), 6.64 (s, 2H) , 6.99 (t, J = 4.8 Hz, 1H), 7.26 (t, J = 4.8 Hz, 1H), 7.33 (d, J = 6.8 Hz, 1H), 7.72 (t, J = 6.4 Hz, 1H), 7.97 (d, J = 6.4 Hz, 1H), 13.22 (s, 1H), 13.85 (s, 1H).
化合物IA-13，IA-14，IA-15，IA-16的合成路线：Synthetic route of compound IA-13, IA-14, IA-15, IA-16:
实施例13 IA-13的合成Example 13 Synthesis of IA-13
化合物1(64mg,0.1mmol)溶解在10mL二氯甲烷中，加入三乙胺(70μL,0.5mmol)和对甲苯磺酰氯(57mg,0.3mmol)。反应液室温搅拌过夜。减压移除溶剂，剩余物硅胶柱层析纯化(二氯甲烷/甲醇＝20:1)得22mg目标化合物2-4，为红色固体，产率47％，LCMS(ESI)m/z＝796.2(M+H)+
Compound 1 (64 mg, 0.1 mmol) was dissolved in 10 mL dichloromethane, triethylamine (EtOAc, < The reaction was stirred at room temperature overnight. The solvent was removed under reduced pressure. EtOAcjjjjjjjjjjj (M+H) +
化合物2-4(80mg,0.1mmol)和乙醇酸乙酯(28μL,0.3nnol)溶解在10mL的N,N-二
甲基甲酰胺中，冰水浴冷却至0℃，氮气条件下，加入NaH(12mg,0.3mmoL)，反应混合物在室温搅拌2小时，饱和NH4Cl淬灭反应，用乙酸乙酯(30mL×3)萃取，合并有机相，水洗(20mL×3)，无水硫酸钠干燥，过滤，减压浓缩除掉溶剂，残余物通过快速分离柱(二氯甲烷/丙酮＝5:1)分离纯化，得到18mg的化合物IA-13，为红色固体，产率26％。LCMS(ESI)m/z＝700.2(M+H)+。1HNMR(400MHz,CDCl3)δppm 1.33(t,J＝8.8Hz,3H),1.39(d,J＝6.0Hz,3H),1.70-1.77(m,1H),1.98-2.04(m,2H),2.08&2.12(dd,J1＝14.8Hz,J2＝3.6Hz,1H),2.22(t,J＝7.6Hz,1H),2.50(d,J＝14.8Hz,1H),2.71-2.83(m,2H),3.04(d,J＝18.8Hz,1H),3.21-3.28(m,1H),3.36-3.40(m,1H),3.46(s,3H),3.56-3.60(m,1H),3.89-3.94(m,1H),3.99-4.04(m,1H),4.07-4.09(m,1H),4.09(s,3H),4.22-4.26(m,2H),4.47(s,1H),4.71(s,1H),4.76-4.97(m,3H),5.34(t,J＝4.8Hz,1H),5.48(t,J＝5.2Hz,1H),7.40(d,J＝8.4Hz,1H),7.78(t,J＝8.0Hz,1H),8.03(d,J＝7.6Hz,1H),13.27(s,1H),13.89(s,1H)。Compound 2-4 (80 mg, 0.1 mmol) and ethyl glycolate (28 μL, 0.3 nnol) were dissolved in 10 mL of N,N-dimethylformamide, cooled to 0 ° C in an ice water bath, and NaH was added under nitrogen. 12mg, 0.3mmoL), the reaction mixture was stirred at room temperature for 2 hours, quenched with saturated NH 4 Cl, extracted with ethyl acetate (30mL × 3), combined organic phases were washed with water (20mL × 3), dried over anhydrous sodium sulfate, Filtration, concentrating under reduced pressure and solvent was evaporated. mjjjjjjj LCMS (ESI) m / z = 700.2 (M + H) +. 1 H NMR (400 MHz, CDCl 3 ) δ ppm 1.33 (t, J = 8.8 Hz, 3H), 1.39 (d, J = 6.0 Hz, 3H), 1.70-1.77 (m, 1H), 1.98-2.04 (m, 2H) , 2.08 & 2.12 (dd, J 1 = 14.8 Hz, J 2 = 3.6 Hz, 1H), 2.22 (t, J = 7.6 Hz, 1H), 2.50 (d, J = 14.8 Hz, 1H), 2.71-2.83 (m, 2H), 3.04 (d, J = 18.8 Hz, 1H), 3.21-3.28 (m, 1H), 3.36-3.40 (m, 1H), 3.46 (s, 3H), 3.56-3.60 (m, 1H) ), 3.89-3.94 (m, 1H), 3.99-4.04 (m, 1H), 4.07-4.09 (m, 1H), 4.09 (s, 3H), 4.22-4.26 (m, 2H), 4.47 (s, 1H) ), 4.71 (s, 1H), 4.76-4.97 (m, 3H), 5.34 (t, J = 4.8 Hz, 1H), 5.48 (t, J = 5.2 Hz, 1H), 7.40 (d, J = 8.4 Hz) , 1H), 7.78 (t, J = 8.0 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H), 13.27 (s, 1H), 13.89 (s, 1H).
实施例14 IA-14的合成Example 14 Synthesis of IA-14
(1)化合物3-4的合成(1) Synthesis of Compound 3-4
化合物IA-13(95mg,0.13mmol)溶解在12mL的四氢呋喃中，依次加入乙醇(12mL)，水(5mL)，碳酸钾(218mg)，反应混合物在室温条件下搅拌1小时，加入10mL水，浓缩除掉四氢呋喃和乙醇，水相用2N稀盐酸调pH＝5，用二氯甲烷(30mL×5)萃取，有机相无水硫酸钠干燥，过滤，浓缩，得到95mg的粗品化合物3-4，其为红色固体，粗品直接用于下一步。LCMS(ESI)m/z＝700.2(M+H)+。Compound IA-13 (95 mg, 0.13 mmol) was dissolved in 12 mL of tetrahydrofuran, and then ethanol (12mL), water (5mL), potassium carbonate (218mg), and the reaction mixture was stirred at room temperature for 1 hour, 10 mL of water was added and concentrated. The tetrahydrofuran and ethanol were removed, the aqueous phase was adjusted to pH=5 with 2N diluted hydrochloric acid, and extracted with dichloromethane (30 mL×5), dried over anhydrous sodium sulfate, filtered and concentrated to give 95 mg of crude compound 3-4. It was a red solid and the crude was used directly in the next step. LCMS (ESI) m / z = 700.2 (M + H) +.
(2)化合物4-4的合成(2) Synthesis of Compound 4-4
化合物2-4(65mg,0.093mmol)溶解在50mL的二氯甲烷中，氮气条件下，加入化合物2a(48mg,0.14mmol)，HATU(71mg,0.19mmol)，然后加入DIPEA(77μL,0.47mmol)，反应混合物在室温氮气条件下搅拌12小时。浓缩除掉溶剂，粗品通过快速分离柱(二氯甲烷/乙酸乙酯/甲醇＝10:2:1)分离纯化，得到56mg的化合物4-4，收率61％，其为红色固体。LCMS(ESI)m/z＝992.3(M+H)+。Compound 2-4 (65 mg, 0.093 mmol) was dissolved in 50 mL of methylene chloride. Compound 2a (48 mg, 0.14 mmol), HATU (71 mg, 0.19 mmol), then DIPEA (77 μL, 0.47 mmol) The reaction mixture was stirred at room temperature under nitrogen for 12 hours. The solvent was removed by concentration, and the crude material wasjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj LCMS (ESI) m / z = 992.3 (M + H) +.
(3)化合物5-4的合成(3) Synthesis of compound 5-4
化合物3-4(56mg,0.057mmol)溶解在5mL的乙腈中，在冰浴条件下，加入2.5mL的二乙胺，并搅拌1小时。浓缩除掉有机溶剂，得到50mg的粗品化合物5-4，其为红色固体，粗品直接用到下一步。LCMS(ESI)m/z＝770.3(M+H)+。Compound 3-4 (56 mg, 0.057 mmol) was dissolved in 5 mL of acetonitrile, and 2.5 mL of diethylamine was added and stirred for 1 hour under ice bath. Concentration and removal of the organic solvent gave 50 mg of crude compound 5-4 as a red solid. LCMS (ESI) m / z = 770.3 (M + H) +.
(4)化合物IA-14的合成(4) Synthesis of compound IA-14
化合物5-4(20mg,0.026mmol)溶解在10mL的二氯甲烷中，氮气条件下，加入化合物McOSu(20mg,0.065mmol)，然后加入10μL的DIPEA，反应混合物在室温氮气条件
下搅拌3小时。浓缩除掉溶剂，粗品通过快速分离柱(二氯甲烷/乙酸乙酯/甲醇＝10:2:1)分离纯化，得到15mg的化合物IA-14，收率60％，其为红色固体。LCMS(ESI)m/z＝963.3(M+H)+。1HNMR(400MHz,CDCl3)δppm 1.25-1.52(m,10H),1.64-2.01(m,2H),2.10-2.13(m,1H),2.20-2.36(m,3H),2.51(d,J＝14.4Hz,1H),2.75-2.82(m,2H),2.90-3.07(m,6H),3.15-3.17(m,1H),3.38-3.59(m,10H),3.61-3.80(m,1H),3.91(t,J＝9.2Hz,1H),4.01-4.12(m,4H),4.21-4.39(m,2H),4.46(s,1H),4.70(s,1H),4.82-4.91(m,2H),5.32-5.36(m,2H),5.47-5.51(m,1H),6.68(s,2H),7.34-7.36(m,1H),7.69-7.72(m,1H),8.02(s,1H),13.23(brs,1H),13.90(s,1H)。Compound 5-4 (20 mg, 0.026 mmol) was dissolved in 10 mL of methylene chloride. Compound EtOAc (20 mg, EtOAc) The solvent was removed by concentration, and the crude material was purified and purified, mjjjjlililililililililililililili LCMS (ESI) m / z = 963.3 (M + H) +. 1 H NMR (400 MHz, CDCl 3 ) δ ppm 1.25-1.52 (m, 10H), 1.64-2.01 (m, 2H), 2.10-2.13 (m, 1H), 2.20-2.36 (m, 3H), 2.51 (d, J) =14.4 Hz, 1H), 2.75-2.82 (m, 2H), 2.90-3.07 (m, 6H), 3.15-3.17 (m, 1H), 3.38-3.59 (m, 10H), 3.61-3.80 (m, 1H) ), 3.91 (t, J = 9.2 Hz, 1H), 4.01-4.12 (m, 4H), 4.21-4.39 (m, 2H), 4.46 (s, 1H), 4.70 (s, 1H), 4.82-4.91 ( m, 2H), 5.32-5.36 (m, 2H), 5.47-5.51 (m, 1H), 6.68 (s, 2H), 7.34-7.36 (m, 1H), 7.69-7.72 (m, 1H), 8.02 ( s, 1H), 13.23 (brs, 1H), 13.90 (s, 1H).
实施例15 IA-15的合成Example 15 Synthesis of IA-15
化合物5-4(25mg,0.033mmol)溶解在5mL的N,N-二甲基甲酰胺中，氮气条件下，加入化合物C-L-004(20mg,0.026mmol)，然后加入5.4μL的三乙胺，反应混合物在室温氮气条件下搅拌2小时。用10mL的饱和食盐水淬灭反应，乙酸乙酯(20mL×3)萃取，有机相用饱和食盐水(20mL×5)洗涤，有机相用无水硫酸钠干燥，过滤，浓缩。粗品通过快速分离柱(二氯甲烷/乙酸乙酯/甲醇＝30:15:5,然后二氯甲烷/甲醇＝10:1)分离纯化，得到12mg的化合物IA-15，收率27％，其为红色固体。LCMS(ESI)m/z＝1368.6(M+H)+。Compound 5-4 (25 mg, 0.033 mmol) was dissolved in 5 mL of N,N-dimethylformamide, and compound CL-004 (20 mg, 0.026 mmol) was added under nitrogen, and then 5.4 μL of triethylamine was added. The reaction mixture was stirred at room temperature under nitrogen for 2 hours. The reaction was quenched with EtOAc (EtOAc m. The crude product was isolated and purified by a flash column (dichloromethane/ethyl acetate/methanol=30:15:5, then dichloromethane/methanol = 10:1) to afford 12 mg of compound IA-15 (yield 27%) It is a red solid. LCMS (ESI) m / z = 1368.6 (M + H) +.
实施例15IA-16的合成Example 15 Synthesis of IA-16
化合物5-4(25mg,0.033mmol)溶解在10mL的二氯甲烷中，氮气条件下，加入化合物C-L-066(25mg,0.065mmol)，然后加入9μL的TEA，反应混合物在室温氮气条件下搅拌2小时。浓缩除掉有机溶剂，粗品通过PREP-TLC(二氯甲烷/乙酸乙酯/甲醇＝35:10:5)分离纯化，得到13mg的化合物IA-16，收率38％，其为红色固体。LCMS(ESI)m/z＝1039.2(M+H)+。1HNMR(400MHz,CDCl3)δppm 1.39(d,J＝6.4Hz,3H),1.97-2.11(m,4H),2.22(t,J＝7.6Hz,1H),2.49(d,J＝14Hz,1H),2.72-2.99(m,5H),3.00-3.08(m,7H),3.24(d,J＝19.2Hz,1H),3.38-3.45(m,1H),3.49(s,3H),3.54-3.73(m,13H),3.88-3.93(m,1H),4.03-4.06(m,1H),4.09(s,3H),4.18(s,1H),4.21-4.28(m,3H),4.46(s,1H),4.70(s,1H),4.08-4.91(m,3H),5.34(t,J＝5.2Hz,1H),5.48(t,J＝5.2Hz,1H),6.70(s,2H),7.39(d,J＝8.8Hz,1H),7.78(t,J＝8.0Hz,1H),8.03(d,J＝7.2Hz,1H),13.29(s,1H),13.91(s,1H)。Compound 5-4 (25 mg, 0.033 mmol) was dissolved in 10 mL of dichloromethane, and then, under nitrogen, compound CL-066 (25 mg, 0.065 mmol) was added, then 9 μL of TEA was added, and the reaction mixture was stirred at room temperature under nitrogen. hour. The organic solvent was removed by concentration, and the crude material was purified and purified, mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj LCMS (ESI) m / z = 1039.2 (M + H) +. 1 H NMR (400 MHz, CDCl 3 ) δ ppm 1.39 (d, J = 6.4 Hz, 3H), 1.97-2.11 (m, 4H), 2.22 (t, J = 7.6 Hz, 1H), 2.49 (d, J = 14 Hz, 1H), 2.72-2.99 (m, 5H), 3.00-3.08 (m, 7H), 3.24 (d, J = 19.2 Hz, 1H), 3.38-3.45 (m, 1H), 3.49 (s, 3H), 3.54 -3.73 (m, 13H), 3.88-3.93 (m, 1H), 4.03-4.06 (m, 1H), 4.09 (s, 3H), 4.18 (s, 1H), 4.21-4.28 (m, 3H), 4.46 (s, 1H), 4.70 (s, 1H), 4.08-4.91 (m, 3H), 5.34 (t, J = 5.2 Hz, 1H), 5.48 (t, J = 5.2 Hz, 1H), 6.70 (s, 2H), 7.39 (d, J = 8.8 Hz, 1H), 7.78 (t, J = 8.0 Hz, 1H), 8.03 (d, J = 7.2 Hz, 1H), 13.29 (s, 1H), 13.91 (s, 1H).
化合物IA-17,IA-18合成路线：
Compound IA-17, IA-18 synthetic route:
实施例16 IA-17的合成Example 16 Synthesis of IA-17
化合物5-4(20mg,0.026mmol)溶解在5mL的二氯甲烷中，依次加入化合物1a-5(10mg，0.039mmoL),HATU(20mg,0.052mmoL),DIPEA(22μL,0.13mmoL),上述混合物在室温氮气条件下搅拌3小时。浓缩除掉有机溶剂，残余物通过快速分离柱(二氯甲烷/乙酸乙酯/甲醇＝1:1:0.1)分离纯化，得到18mg的化合物IA-17，收率69％，其为红色固体。LCMS(ESI)m/z＝992.3(M+H)+。Compound 5-4 (20 mg, 0.026 mmol) was dissolved in 5 mL of dichloromethane, and then compound 1a-5 (10 mg, 0.039 mmol), HATU (20 mg, 0.052mmoL), DIPEA (22 μL, 0.13mmoL), Stir at room temperature under nitrogen for 3 hours. The organic solvent was removed by EtOAc (EtOAc m.) LCMS (ESI) m / z = 992.3 (M + H) +.
实施例16IA-18的合成Example 16 Synthesis of IA-18
(1)化合物2-5的合成(1) Synthesis of Compound 2-5
化合物IA-17(18mg,0.018mmol)溶解在5mL的甲醇中，依次加入5mL的乙酸乙酯，5mL的PH＝7.5的磷酸钾缓冲溶液，和38mg的DTT，反应混合物在室温条件下搅拌1小时。加入10mL的PH＝6.0磷酸钾缓冲液淬灭反应，室温条件下搅拌10分钟，用乙酸乙酯(50mL×2)萃取,有机相通过无水硫酸钠干燥，过滤，浓缩，得到粗品。粗品通过快速分离柱(二氯甲烷/甲醇＝10:1)分离纯化,得到10mg的化合物2-5，收率59％，其为红色固体。LCMS(ESI)m/z＝946.3(M+H)+。Compound IA-17 (18 mg, 0.018 mmol) was dissolved in 5 mL of methanol, followed by 5 mL of ethyl acetate, 5 mL of a potassium phosphate buffer solution of pH = 7.5, and 38 mg of DTT, and the reaction mixture was stirred at room temperature for 1 hour. . The reaction was quenched by the addition of 10 mL of EtOAc EtOAc (EtOAc)EtOAc. The crude product was isolated and purified using a flash column (dichloromethane/methanol = 10:1) to afford 10 mg of compound 2-5 as a red solid. LCMS (ESI) m / z = 946.3 (M + H) +.
(2)化合物IA-18的合成(2) Synthesis of compound IA-18
化合物2-5(8mg,0.008mmol)溶解在5mL的N,N-二甲基甲酰胺中，氮气条件下，加入20mg的化合物SMCC，然后加入10μL的TEA，反应混合物在室温氮气条件下搅拌12小时。用10mL的饱和食盐水淬灭反应，乙酸乙酯(20mL×3)萃取，有机相用饱和食盐水(20mL×5)洗涤，无水硫酸钠干燥，过滤，浓缩。粗品通过快速分离柱(二氯甲烷/乙腈＝3:1,然后二氯甲烷/甲醇＝10:1)分离纯化,得到2.5mg的化合物IA-18，收率23％，其为红色固体。LCMS(ESI)m/z＝1280.3(M+H)+。Compound 2-5 (8 mg, 0.008 mmol) was dissolved in 5 mL of N,N-dimethylformamide. Under nitrogen, 20 mg of compound SMCC was added, then 10 μL of TEA was added, and the reaction mixture was stirred at room temperature under nitrogen. hour. The reaction was quenched with EtOAc (EtOAc)EtOAc. The crude product was isolated and purified by flash chromatography eluting eluting eluting eluting eluting eluting LCMS (ESI) m / z = 1280.3 (M + H) +.
化合物IA-19，IA-20，IA-21的合成路线：
Synthetic route of compound IA-19, IA-20, IA-21:
实施例17 IA-19的合成Example 17 Synthesis of IA-19
(1)化合物2-6的合成(1) Synthesis of compound 2-6
化合物3-4(30mg,0.043mmol)溶解在10mL的二氯甲烷中，氮气条件下，加入化合
物1a-6(26mg,0.064mmol)，HATU(33mg,0.086mmol),然后加入DIPEA(35μL,0.22mmol)，反应混合物在氮气条件下室温搅拌12小时。减压移除溶剂，粗品通过快速分离柱(二氯甲烷/乙酸乙酯/甲醇＝35:10:5)分离纯化，得到30mg的化合物2-6，收率67％，其为红色固体。LCMS(ESI)m/z＝1052.3(M+H)+。Compound 3-4 (30 mg, 0.043 mmol) was dissolved in 10 mL of dichloromethane. Compound 1a-6 (26 mg, 0.064 mmol), HATU (33 mg, 0.086 mmol), then DIPEA (35 μL, 0.22) was added under nitrogen. (mmol), the reaction mixture was stirred at room temperature under nitrogen for 12 h. The solvent was removed under reduced pressure and the crude material was purified mjjjjlilililililililililililililililili LCMS (ESI) m / z = 1052.3 (M + H) +.
(2)化合物3-6的合成(2) Synthesis of compound 3-6
化合物2-6(30mg,0.028mmol)溶解在6mL的乙腈中，冰水浴冷却至0℃，加入3mL的二乙胺，并搅拌1小时。浓缩除掉有机溶剂，得到23mg的粗品化合物3-6，其为红色固体，粗品直接用到下一步。LCMS(ESI)m/z＝852.2(M+H)+。Compound 2-6 (30 mg, 0.028 mmol) was dissolved in 6 mL of acetonitrile, cooled to 0 ° C in ice water, and 3 mL of diethylamine was added and stirred for 1 hour. Concentration and removal of the organic solvent gave 23 mg of crude compound 3-6 as a red solid. LCMS (ESI) m / z = 852.2 (M + H) +.
(3)化合物IA-19的合成(3) Synthesis of compound IA-19
化合物3-6(23mg,0.028mmol)溶解在10mL的二氯甲烷中，氮气条件下，加入化合物C-L-066(22mg,0.056mmol)，然后加入7.7μL的三乙胺，反应混合物在氮气条件下室温搅拌2小时。浓缩除掉有机溶剂，粗品通过PREP-TLC(二氯甲烷/乙酸乙酯/甲醇＝100:35:10)分离纯化，得到3.5mg的化合物IA-19，收率12％，其为红色固体。LCMS(ESI)m/z＝1099.3(M+H)+。Compound 3-6 (23 mg, 0.028 mmol) was dissolved in 10 mL of dichloromethane, and then, under nitrogen, compound CL-066 (22 mg, 0.056 mmol) was added, then 7.7 μL of triethylamine was added, and the reaction mixture was under nitrogen. Stir at room temperature for 2 hours. The organic solvent was removed by concentration, and the crude material was purified and purified, mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj LCMS (ESI) m / z = 1099.3 (M + H) +.
实施例18 IA-20的合成Example 18 Synthesis of IA-20
(1)化合物4-6的合成(1) Synthesis of compound 4-6
化合物3-4(30mg,0.043mmol)溶解在10mL的二氯甲烷中，氮气条件下，加入化合物1b-6(21mg,0.064mmol)，HATU(33mg,0.086mmol),然后加入DIPEA(35μL,0.22mmol)，反应混合物在室温氮气条件下搅拌12小时。减压移除溶剂，粗品通过快速分离柱(二氯甲烷/乙酸乙酯/甲醇＝40:4:10)分离纯化，得到27mg的化合物4-6，收率66％，其为红色固体。LCMS(ESI)m/z＝964.2(M+H)+。Compound 3-4 (30 mg, 0.043 mmol) was dissolved in 10 mL of dichloromethane. Compound 1b-6 (21 mg, 0.064 mmol), HATU (33 mg, 0.086 mmol), then DIPEA (35 μL, 0.22) was added under nitrogen. (mmol), the reaction mixture was stirred at room temperature under nitrogen for 12 hours. The solvent was removed under reduced pressure and the crude was purified mjjjjlililililililililililililililili LCMS (ESI) m / z = 964.2 (M + H) +.
(2)化合物5-6的合成(2) Synthesis of compound 5-6
化合物4-6(27mg,0.028mmol)溶解在6mL的乙腈中，在冰水浴条件下，加入3mL的二乙胺，并搅拌1小时。浓缩除掉有机溶剂，得到20mg的粗品化合物5-6，其为红色固体，粗品直接用到下一步。LCMS(ESI)m/z＝742.2(M+H)+。Compound 4-6 (27 mg, 0.028 mmol) was dissolved in 6 mL of acetonitrile, and 3 mL of diethylamine was added under ice-water bath and stirred for 1 hour. Concentration and removal of the organic solvent gave 20 mg of crude compound 5-6 as a red solid. LCMS (ESI) m / z = 742.2 (M + H) +.
(3)化合物IA-20的合成(3) Synthesis of Compound IA-20
化合物5-6(20mg,0.027mmol)溶解在10mL的二氯甲烷中，氮气条件下，加入化合物C-L-066(21mg,0.054mmol)，然后加入7.5μL的三乙胺，反应混合物在室温氮气条件下搅拌2小时。浓缩除掉有机溶剂，粗品通过PREP-TLC(二氯甲烷/乙酸乙酯/甲醇＝10:3:1)分离纯化，得到7mg的化合物IA-20，收率26％，其为红色固体。LCMS(ESI)m/z＝1011.2(M+H)+。1HNMR(400MHz,d-CDCl3)δppm 1.39(d,J＝5.6Hz,3H),2.00-2.02(m,2H),2.08-
2.12(m,1H),2.48-2.51(m,1H),2.73-2.80(m,3H),3.02(d,J＝15.2Hz,1H),3.25(d,J＝14Hz,1H),3.37-3.49(m,5H),3.57-3.65(m,12H),3.73(t,J＝4.8Hz,2H),3.89-4.09(m,11H),4.47(s,1H),4.70(s,1H),4.80(s,2H),4.89(s,1H),5.30-5.34(m,1H),5.49(t,J＝4.4Hz,1H),6.71(s,2H),7.40(d,J＝6.4Hz,1H),7.49(brs,2H),7.79(t,J＝6.8Hz,1H),8.04(d,J＝5.2Hz,1H),13.29(s,1H),13.90(s,1H)。Compound 5-6 (20 mg, 0.027 mmol) was dissolved in 10 mL of dichloromethane, and then, under nitrogen, compound CL-066 (21 mg, 0.054 mmol) was added, then 7.5 μL of triethylamine was added, and the reaction mixture was stirred at room temperature under nitrogen. Stir under 2 hours. The organic solvent was removed by EtOAc (EtOAc m.) LCMS (ESI) m / z = 1011.2 (M + H) +. 1 H NMR (400 MHz, d-CDCl 3 ) δ ppm 1.39 (d, J = 5.6 Hz, 3H), 2.00-2.02 (m, 2H), 2.08- 2.12 (m, 1H), 2.48-2.51 (m, 1H), 2.73-2.80 (m, 3H), 3.02 (d, J = 15.2 Hz, 1H), 3.25 (d, J = 14 Hz, 1H), 3.37-3.49 (m, 5H), 3.57-3.65 (m, 12H), 3.73 (t, J = 4.8 Hz, 2H), 3.89-4.09 (m, 11H), 4.47 (s, 1H), 4.70 (s, 1H), 4.80 (s, 2H), 4.89 (s, 1H), 5.30 -5.34 (m, 1H), 5.49 (t, J = 4.4 Hz, 1H), 6.71 (s, 2H), 7.40 (d, J = 6.4 Hz, 1H), 7.49 (brs, 2H), 7.79 (t, J = 6.8 Hz, 1H), 8.04 (d, J = 5.2 Hz, 1H), 13.29 (s, 1H), 13.90 (s, 1H).
实施例19 IA-21的合成Example 19 Synthesis of IA-21
(1)化合物6-6的合成(1) Synthesis of compound 6-6
化合物3-4(80mg,0.114mmol)溶解在50mL的二氯甲烷中，氮气条件下，加入化合物1c-6(56mg,0.17mmol)，HATU(87mg,0.228mmol),然后加入DIPEA(94μL,0.57mmol)，反应混合物在室温氮气条件下搅拌12小时。浓缩除掉溶剂，粗品通过快速分离柱(二氯甲烷/乙酸乙酯/甲醇＝10:2:1)分离纯化，得到60mg的化合物6-6，收率52％，其为红色固体。LCMS(ESI)m/z＝1008.3(M+H)+。Compound 3-4 (80 mg, 0.114 mmol) was dissolved in 50 mL of methylene chloride. Compound 1c-6 (56 mg, 0.17 mmol), HATU (87 mg, 0.228 mmol), then DIPEA (94 μL, 0.57) was added under nitrogen. (mmol), the reaction mixture was stirred at room temperature under nitrogen for 12 hours. The solvent was removed by concentration, and the crude material was purified mjjjjlilililililililililililililililili LCMS (ESI) m / z = 1008.3 (M + H) +.
(2)化合物7-6的合成(2) Synthesis of compound 7-6
化合物6-6(60mg,0.060mmol)溶解在5mL的乙腈中，在冰水浴条件下，加入2.5mL的二乙胺，并搅拌1小时。浓缩除掉有机溶剂，得到50mg的粗品化合物7-6，其为红色固体，粗品直接用到下一步。LCMS(ESI)m/z＝786.8(M+H)+。Compound 6-6 (60 mg, 0.060 mmol) was dissolved in 5 mL of acetonitrile, and 2.5 mL of diethylamine was added under ice-water bath and stirred for 1 hour. Concentration of the organic solvent afforded 50 mg of crude compound 7-6 as a red solid. LCMS (ESI) m / z = 786.8 (M + H) +.
(3)化合物IA-21的合成(3) Synthesis of Compound IA-21
化合物7-6(50mg,0.064mmol)溶解在10mL的二氯甲烷中，氮气条件下，加入化合物C-L-066(37mg,0.096mmol)，反应混合物在氮气条件下室温搅拌3小时。减压移除溶剂，粗品通过PREP-TLC(二氯甲烷/乙酸乙酯/甲醇＝35:10:5)分离纯化，得到25mg的化合物IA-21，收率48％，其为红色固体。LCMS(ESI)m/z＝1055.4(M+H)+。1HNMR(400MHz,d-CDCl3)δppm 1.39(d,J＝5.6Hz,3H),1.97-2.05(m,2H),2.08&2.10(m,1H),2.22(t,J＝6.0Hz,1H),2.48-2.51(m,1H),2.72-2.77(m,1H),2.81&2.83(2×t,J＝2.0Hz,1H),3.00&3.04(2×s,1H),3.22&3.26(2×s,1H),3.39(q,J＝4.8Hz,1H),3.48-3.54(m,5H),3.57-3.68(m,14H),3.73(t,J＝4.8Hz,2H),3.89-4.03(m,2H),4.00(s,3H),4.10-4.12(m,6H),4.47(s,1H),4.70(s,1H),4.82(s,2H),4.88(s,1H),5.32(br,1H),5.34(t,J＝4.0Hz,1H),5.47(t,J＝4.8Hz,1H),6.71(s,2H),7.35(t,J＝4.0Hz,1H),7.40(d,J＝6.4Hz,1H),7.79(t,J＝6.4Hz,1H),8.04(d,J＝5.6Hz,1H),13.28(s,1H),13.90(s,1H)。Compound 7-6 (50 mg, 0.064 mmol) was dissolved in dichloromethane (10 mL). The solvent was removed under reduced pressure and the crude purified title mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj LCMS (ESI) m / z = 1055.4 (M + H) +. 1 H NMR (400 MHz, d-CDCl 3 ) δ ppm 1.39 (d, J = 5.6 Hz, 3H), 1.97-2.05 (m, 2H), 2.08 & 2.10 (m, 1H), 2.22 (t, J = 6.0 Hz) , 1H), 2.48-2.51 (m, 1H), 2.72-2.77 (m, 1H), 2.81 & 2.83 (2×t, J=2.0 Hz, 1H), 3.00&3.04 (2×s, 1H) , 3.22 & 3.26 (2 × s, 1H), 3.39 (q, J = 4.8 Hz, 1H), 3.48-3.54 (m, 5H), 3.57-3.68 (m, 14H), 3.73 (t, J = 4.8 Hz, 2H), 3.89-4.03 (m, 2H), 4.00 (s, 3H), 4.10-4.12 (m, 6H), 4.47 (s, 1H), 4.70 (s, 1H), 4.82 (s, 2H) , 4.88 (s, 1H), 5.32 (br, 1H), 5.34 (t, J = 4.0 Hz, 1H), 5.47 (t, J = 4.8 Hz, 1H), 6.71 (s, 2H), 7.35 (t, J=4.0 Hz, 1H), 7.40 (d, J=6.4 Hz, 1H), 7.79 (t, J=6.4 Hz, 1H), 8.04 (d, J=5.6 Hz, 1H), 13.28 (s, 1H) , 13.90 (s, 1H).
化合物IA-22的合成路线：
Synthetic route of compound IA-22:
实施例20 IA-22的合成Example 20 Synthesis of IA-22
(1)化合物2-7的合成(1) Synthesis of Compound 2-7
化合物3-4(60mg,0.086mmol)溶解在30mL二氯甲烷中，氮气条件下，加入化合物1a-7(21mg,0.13mmol)，HATU(65mg,0.172mmol),然后加入DIPEA(71μL,0.43mmol)，反应混合物在氮气条件下室温搅拌12小时。浓缩除掉溶剂，粗品通过快速分离柱(二氯甲烷/乙酸乙酯/甲醇＝10:2:1)分离纯化，得到36mg的化合物2-7，收率51％，其为红色固体。LCMS(ESI)m/z＝819.2(M+H)+。Compound 3-4 (60 mg, 0.086 mmol) was dissolved in 30 mL of dichloromethane. Compound 1a-7 (21 mg, 0.13 mmol), HATU (65 mg, 0.172 mmol) was added under nitrogen, then DIPEA (71 μL, 0.43 mmol) was added. The reaction mixture was stirred at room temperature for 12 hours under nitrogen. The solvent was concentrated and concentrated, and then purified and purified, mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj LCMS (ESI) m / z = 819.2 (M + H) +.
(2)化合物3-7的合成(2) Synthesis of compound 3-7
化合物2-7(36mg,0.044mmol)溶解在2mL的甲醇中，依次加入2mL的乙酸乙酯，2mL的pH＝7.5的磷酸钾缓冲溶液，和14mg的DTT，反应混合物在室温条件下搅拌1小时。加入5mL的PH＝6.0磷酸钾缓冲液淬灭反应，室温条件下搅拌10分钟，用乙酸乙酯(20mL×2)萃取,有机相通过无水硫酸钠干燥，过滤，浓缩，得到30mg的粗品化合物3-7，其为红色固体，粗品直接用到下一步。LCMS(ESI)m/z＝773.2(M+H)+。Compound 2-7 (36 mg, 0.044 mmol) was dissolved in 2 mL of methanol, followed by 2 mL of ethyl acetate, 2 mL of potassium phosphate buffer solution of pH = 7.5, and 14 mg of DTT, and the reaction mixture was stirred at room temperature for 1 hour. . The reaction was quenched by the addition of 5 mL of aq. 3-7, which is a red solid, and the crude product is used directly in the next step. LCMS (ESI) m / z = 773.2 (M + H) +.
(3)化合物IA-22的合成(3) Synthesis of compound IA-22
化合物3-7(30mg,0.039mmol)溶解在5mL的四氢呋喃中，氮气条件下，加入26mg的化合物SMCC，然后加入2mL的pH＝6.0的磷酸钾缓冲溶液，反应混合物在氮气条件下室温搅拌12小时。反应液直接冷冻干燥。粗品通过PREP-TLC(二氯甲烷/乙酸乙酯/甲醇＝35:10:5)分离纯化，得到11.9mg的化合物IA-22，收率28％，其为红色固体。LCMS(ESI)m/z＝1093.3(M+H)+。1HNMR(400MHz,d-CDCl3)δppm 0.81(t,J＝5.6Hz,3H),1.01(d,J＝9.2Hz,2H),1.07(d,J＝4.8Hz,2H),1.33(d,J＝5.2Hz,3H),1.63-1.74(m,2H),1.92-1.97(m,
2H),1.99-2.04(m,1H),2.09(d,J＝11.2Hz,2H),2.15(t,J＝6.4Hz,1H),2.41-2.53(m,2H),2.66-2.83(m,3H),2.92-2.98(m,1H),3.07-3.19(m,3H),3.33(d,J＝5.6Hz,3H),3.39(s,3H),3.50-3.70(m,3H),3.81-3.87(m,2H),3.93(q,J＝5.6Hz,1H),3.99-4.07(m,2H),4.03(s,3H),4.40(d,J＝1.6Hz,1H),4.63(d,J＝1.6Hz,1H),4.76(d,J＝6.0Hz,2H),4.82(s,1H),5.25(br,1H),5.27(t,J＝4.0Hz,1H),5.41(t,J＝4.4Hz,1H),7.33(d,J＝6.8Hz,1H),7.40(q,J＝4.8Hz,1H),7.72(t,J＝6.4Hz,1H),7.98(d,J＝5.6Hz,1H),13.23(s,1H),13.83(s,1H)。Compound 3-7 (30 mg, 0.039 mmol) was dissolved in 5 mL of tetrahydrofuran. Under nitrogen, 26 mg of compound SMCC was added, then 2 mL of a potassium phosphate buffer solution of pH=6.0 was added, and the reaction mixture was stirred at room temperature for 12 hours under nitrogen. . The reaction solution was directly freeze-dried. The crude product was purified by EtOAc (EtOAc/EtOAc/EtOAc/EtOAc) LCMS (ESI) m / z = 1093.3 (M + H) +. 1 H NMR (400 MHz, d-CDCl 3 ) δ ppm 0.81 (t, J = 5.6 Hz, 3H), 1.01 (d, J = 9.2 Hz, 2H), 1.07 (d, J = 4.8 Hz, 2H), 1.33 (d) , J = 5.2 Hz, 3H), 1.63-1.74 (m, 2H), 1.92-1.97 (m, 2H), 1.99-2.04 (m, 1H), 2.09 (d, J = 11.2 Hz, 2H), 2.15 ( t, J=6.4 Hz, 1H), 2.41-2.53 (m, 2H), 2.66-2.83 (m, 3H), 2.92-2.98 (m, 1H), 3.07-3.19 (m, 3H), 3.33 (d, J=5.6 Hz, 3H), 3.39 (s, 3H), 3.50-3.70 (m, 3H), 3.81-3.87 (m, 2H), 3.93 (q, J = 5.6 Hz, 1H), 3.99-4.07 (m) , 2H), 4.03 (s, 3H), 4.40 (d, J = 1.6 Hz, 1H), 4.63 (d, J = 1.6 Hz, 1H), 4.76 (d, J = 6.0 Hz, 2H), 4.82 (s , 1H), 5.25 (br, 1H), 5.27 (t, J = 4.0 Hz, 1H), 5.41 (t, J = 4.4 Hz, 1H), 7.33 (d, J = 6.8 Hz, 1H), 7.40 (q) , J = 4.8 Hz, 1H), 7.72 (t, J = 6.4 Hz, 1H), 7.98 (d, J = 5.6 Hz, 1H), 13.23 (s, 1H), 13.83 (s, 1H).
化合物IA-23，IA-24的合成路线：Synthetic route of compound IA-23, IA-24:
实施例21 IA-23的合成Example 21 Synthesis of IA-23
(1)化合物2-8的合成(1) Synthesis of Compound 2-8
化合物1-8(600mg,3.75mmol)溶解在20mL的饱和碳酸氢钠溶液中，冰水浴冷却，然后加入化合物1a-8(793mg,4.69mmoL)，反应混合物在0℃条件下搅拌30分钟，然后室温下搅拌1小时。反应混合物用二氯甲烷萃取(50mL×3)，合并有机相，无水硫酸钠干燥，过滤、浓缩。粗品通过快速分离柱分离纯化(石油醚/乙酸乙酯＝1:1)，得到525mg的化合物2-8，其为白色固体，收率58％。LCMS(ESI)m/z＝185.1(M+H)+。1HNMR(400MHz,d-CDCl3)δppm 1.40(s,9H),3.31-3.35(m,2H),3.66(t,J＝4.4Hz,2H),4.65-4.77(m,1H),6.70(s,2H)。Compound 1-8 (600 mg, 3.75 mmol) was dissolved in 20 mL of saturated sodium bicarbonate solution, cooled in an ice water bath, then compound 1a-8 (793 mg, 4.69 mmol) was added, and the reaction mixture was stirred at 0 ° C for 30 minutes, then Stir at room temperature for 1 hour. The reaction mixture was extracted with EtOAc (EtOAc m. The crude product was purified by EtOAc (EtOAc/EtOAc) LCMS (ESI) m / z = 185.1 (M + H) +. 1 H NMR (400 MHz, d-CDCl 3 ) δ ppm 1.40 (s, 9H), 3.31-3.35 (m, 2H), 3.66 (t, J = 4.4 Hz, 2H), 4.65-4.77 (m, 1H), 6.70 ( s, 2H).
(2)化合物3-8的合成(2) Synthesis of compound 3-8
化合物2-8(500mg,2.08mmol)溶解在10mL的二氯甲烷中，冰水浴冷却至0℃，然后加入10mL的氯化氢的1,4-二氧六环溶液，反应混合物0℃下搅拌1小时，常温减压浓缩移除溶剂，粗品悬浮在10mL的二氯甲烷中，过滤，滤饼用10mL的二氯甲烷洗涤，收集固体，真空干燥，得到240mg的化合物3-8。LCMS(ESI)m/z＝141.1(M+H)+。1HNMR(400MHz,d-DMSO)δppm 2.93-2.97(m,2H),3.67(t,J＝5.2Hz,2H),7.06(s,2H),8.22(brs,3H)。Compound 2-8 (500 mg, 2.08 mmol) was dissolved in 10 mL of dichloromethane, cooled to 0 ° C in an ice water bath, then 10 mL of hydrogen chloride in 1,4-dioxane solution was added, and the reaction mixture was stirred at 0 ° C for 1 hour. The solvent was concentrated under reduced pressure at room temperature, and the crude material was evaporated, evaporated, evaporated, evaporated, LCMS (ESI) m / z = 141.1 (M + H) +. 1 H NMR (400 MHz, d-DMSO) δ ppm 2.93-2.97 (m, 2H), 3.67 (t,J = 5.2 Hz, 2H), 7.06 (s, 2H), 8.22 (brs, 3H).
(3)化合物IA-23的合成(3) Synthesis of compound IA-23
化合物3-4(20mg,0.029mmol)溶解在5mL的二氯甲烷中，氮气条件下，加入化合物3-8(8mg,0.043mmol)，HATU(22mg,0.057mmol),然后加入DIPEA(23μL,0.14mmol)，反应混合物在氮气条件下室温搅拌2小时。减压浓缩溶剂，粗品通过PREP-TLC(二氯甲烷/甲醇＝10:1)分离纯化，得到10mg的化合物IA-23，收率43％，其为红色固体。LCMS(ESI)m/z＝822.2(M+H)+。1HNMR(400MHz,d-CDCl3)δppm 1.39(d,J＝5.2Hz,3H),1.70-1.76(m,1H),1.99-2.04(m,1H),2.07-2.11(m,1H),2.51(d,J＝12Hz,1H),2.71-2.82(m,1H),3.02(d,J＝15.2Hz,1H),3.28(d,J＝15.6Hz,1H),3.36-3.40(m,1H),3.46(s,3H),3.50-3.59(m,4H),3.71-3.73(m,2H),3.89-3.93(m,1H),3.99-4.04(m,4H),4.09(s,3H),4.46(d,
J＝1.2Hz,1H),4.70(d,J＝1.6Hz,1H),4.81(d,J＝9.2Hz,2H),4.86(s,1H),5.31-5.32(m,1H),5.48(t,J＝4.4Hz,1H),6.73(s,2H),7.40(d,J＝6.8Hz,1H),7.48(t,J＝4.8Hz,1H),7.79(t,J＝6.8Hz,1H),8.03(d,J＝6.4Hz,1H),13.30(s,1H),13.90(s,1H)。Compound 3-4 (20 mg, 0.029 mmol) was dissolved in 5 mL of dichloromethane. Compound 3-8 (8 mg, 0.043 mmol), HATU (22 mg, 0.057 mmol), then DIPEA (23 μL, 0.14) was added under nitrogen. (mmol), the reaction mixture was stirred at room temperature under nitrogen for 2 h. The solvent was concentrated under reduced pressure and the crude purified mjjjjjjjjjjjjj LCMS (ESI) m / z = 822.2 (M + H) +. 1 H NMR (400 MHz, d-CDCl 3 ) δ ppm 1.39 (d, J = 5.2 Hz, 3H), 1.70-1.76 (m, 1H), 1.99-2.04 (m, 1H), 2.07-2.11 (m, 1H), 2.51 (d, J = 12 Hz, 1H), 2.71-2.82 (m, 1H), 3.02 (d, J = 15.2 Hz, 1H), 3.28 (d, J = 15.6 Hz, 1H), 3.36-3.40 (m, 1H), 3.46 (s, 3H), 3.50-3.59 (m, 4H), 3.71-3.73 (m, 2H), 3.89-3.93 (m, 1H), 3.99-4.04 (m, 4H), 4.09 (s, 3H), 4.46 (d, J = 1.2 Hz, 1H), 4.70 (d, J = 1.6 Hz, 1H), 4.81 (d, J = 9.2 Hz, 2H), 4.86 (s, 1H), 5.31-5.32 ( m,1H), 5.48 (t, J = 4.4 Hz, 1H), 6.73 (s, 2H), 7.40 (d, J = 6.8 Hz, 1H), 7.48 (t, J = 4.8 Hz, 1H), 7.79 ( t, J = 6.8 Hz, 1H), 8.03 (d, J = 6.4 Hz, 1H), 13.30 (s, 1H), 13.90 (s, 1H).
实施例22 IA-24的合成Example 22 Synthesis of IA-24
(1)化合物5-8的合成(1) Synthesis of compound 5-8
化合物4-8(296mg,2mmol)溶解在5mL二氯甲烷中，冰水浴冷却到0℃，Boc2O(196mg,0.9mmoL)溶解在5mL的二氯甲烷，然后慢慢滴加入上述溶液中，混合物升到室温并搅拌过夜。加入20mL水淬灭反应，用二氯甲烷萃取(50mL×3),合并有机相，无水硫酸钠干燥，过滤、浓缩。粗品通过快速分离柱分离纯化(二氯甲烷/甲醇/氨水＝10:1:0.1)，得到143mg化合物5-8，收率29％。LCMS(ESI)m/z＝249.1(M+H)+。Compound 4-8 (296 mg, 2 mmol) was dissolved in 5 mL of dichloromethane, cooled to 0 ° C in an ice water bath, and Boc 2 O (196 mg, 0.9 mmoL) was dissolved in 5 mL of dichloromethane, and then slowly added dropwise to the above solution. The mixture was warmed to room temperature and stirred overnight. The reaction was quenched with EtOAc (EtOAc)EtOAc. The crude product was purified by flash column (dichloromethane/methanol/methanol = 10:1:0.1) to afford 143 mg of compound 5-8. LCMS (ESI) m / z = 249.1 (M + H) +.
(2)化合物6-8的合成(2) Synthesis of compound 6-8
化合物5-8(248mg,1mmol)溶解在10mL的饱和碳酸氢钠溶液中，冰水浴冷却，然后加入化合物1b-8(229mg,1.25mmoL)，混合物在0℃条件下搅拌30分钟，然后室温下搅拌1小时。反应混合物用二氯甲烷萃取(50mL×3)，合并有机相，无水硫酸钠干燥，然后过滤、浓缩。粗品通过快速分离柱分离纯化(石油醚/乙酸乙酯＝1:1),得到152mg的化合物6-8，收率46％。LCMS(ESI)m/z＝351.1(M+H)+。Compound 5-8 (248 mg, 1 mmol) was dissolved in 10 mL of saturated sodium bicarbonate solution, cooled in an ice-water bath, then compound 1b-8 (229mg, 1.25mmoL) was added, and the mixture was stirred at 0 ° C for 30 minutes, then at room temperature Stir for 1 hour. The reaction mixture was extracted with dichloromethane (50 mL×3). The crude product was separated and purified (yield: petroleum ether / ethyl acetate = 1:1) to afford 152 mg of compound 6-8. LCMS (ESI) m / z = 351.1 (M + H) +.
(3)化合物7-8的合成(3) Synthesis of compound 7-8
化合物6-8(150mg,0.46mmol)溶解在2mL的二氯甲烷中，冰水浴冷却，然后加入6mL的氯化氢的1,4-二氧六环溶液，混合物0℃下搅拌2小时，浓缩除掉溶剂，粗品真空干燥1小时，得到120mg的化合物7-8(粗品)。LCMS(ESI)m/z＝229.1(M+H)+。Compound 6-8 (150 mg, 0.46 mmol) was dissolved in 2 mL of dichloromethane, cooled in ice water, then 6 mL of hydrogen chloride in 1,4-dioxane solution, and the mixture was stirred at 0 ° C for 2 hours, concentrated and removed. The solvent and the crude product were dried under vacuum for 1 hour to give 120 mg of Compound 7-8 (cr.). LCMS (ESI) m / z = 229.1 (M + H) +.
(4)化合物IA-24的合成(4) Synthesis of compound IA-24
化合物3-4(35mg,0.050mmol)溶解在10mL的二氯甲烷中，氮气条件下，加入化合物7-8(20mg,0.075mmol)，HATU(38mg,0.100mmol),然后加入DIPEA(42μL,0.25mmol)，反应混合物在氮气条件下室温搅拌2小时。浓缩除掉溶剂，粗品通过PREP-TLC(二氯甲烷/乙酸乙酯/甲醇＝10:3.5:1)分离纯化，得到20mg的化合物IA-24，收率43％，其为红色固体。LCMS(ESI)m/z＝910.1(M+H)+。1HNMR(400MHz,d-CDCl3)δppm 1.39(d,J＝5.2Hz,3H),1.70-1.74(m,1H),1.99-2.11(m,3H),2.49(d,J＝11.6Hz,1H),2.74-2.76(m,1H),2.81-2.83(m,1H),3.02(d,J＝15.2Hz,1H),3.24(d,J＝15.2Hz,1H),3.38-3.40(m,1H),3.46(s,3H),3.49-3.66(m,10H),3.73(t,J＝4.4Hz,2H),3.89-3.94(m,1H),4.00-4.02(m,1H),4.06-4.09(m,6H),4.47(d,J＝1.2Hz,1H),4.70(d,J＝1.2Hz,1H),4.82(d,J＝2Hz,2H),4.86(s,1H),5.32-5.34(m,1H),5.48(t,J＝4.4Hz,1H),6.71(s,2H),7.31-7.33(m,1H),7.40(d,J＝6.8
Hz,1H),7.79(t,J＝6.4Hz,1H),8.04(d,J＝6.0Hz,1H),13.28(s,1H),13.89(s,1H)。Compound 3-4 (35 mg, 0.050 mmol) was dissolved in 10 mL of dichloromethane, and then, under nitrogen, compound 7-8 (20 mg, 0.075 mmol), HATU (38 mg, 0.100 mmol), then DIPEA (42 μL, 0.25) (mmol), the reaction mixture was stirred at room temperature under nitrogen for 2 h. The solvent was concentrated, and the crude was purified and purified eluted eluted eluted eluted elut elut elut elut elut elut elut LCMS (ESI) m / z = 910.1 (M + H) +. 1 H NMR (400 MHz, d-CDCl 3 ) δ ppm 1.39 (d, J = 5.2 Hz, 3H), 1.70-1.74 (m, 1H), 1.99-2.11 (m, 3H), 2.49 (d, J = 11.6 Hz, 1H), 2.74-2.76 (m, 1H), 2.81-2.83 (m, 1H), 3.02 (d, J = 15.2 Hz, 1H), 3.24 (d, J = 15.2 Hz, 1H), 3.38-3.40 (m) , 1H), 3.46 (s, 3H), 3.49-3.66 (m, 10H), 3.73 (t, J = 4.4 Hz, 2H), 3.89-3.94 (m, 1H), 4.00-4.02 (m, 1H), 4.06-4.09(m,6H), 4.47(d,J=1.2Hz,1H), 4.70(d,J=1.2Hz,1H),4.82(d,J=2Hz,2H),4.86(s,1H) , 5.32-5.34 (m, 1H), 5.48 (t, J = 4.4 Hz, 1H), 6.71 (s, 2H), 7.31-7.33 (m, 1H), 7.40 (d, J = 6.8 Hz, 1H), 7.79 (t, J = 6.4 Hz, 1H), 8.04 (d, J = 6.0 Hz, 1H), 13.28 (s, 1H), 13.89 (s, 1H).
化合物IA-25、IA-26的合成路线：Synthetic route of compound IA-25, IA-26:
实施例23 IA-25的合成
Example 23 Synthesis of IA-25
(1)化合物2-9的合成(1) Synthesis of compound 2-9
原料1-9(10g,25.25mmol)溶于250mL乙腈中，依次加入4-羟基-3-硝基苯甲醛(7.1g,42.58mmol)和氧化银(26.1g,113.63mmol)，氮气保护下避光搅拌4小时，硅藻土过滤，滤液减压移除乙腈，剩余物溶于400mL乙酸乙酯中，饱和碳酸氢钠洗涤3次(200mL×3)，水洗(200mL)，饱和食盐水洗(200mL)，无水硫酸钠干燥，浓缩，得12g粗产品2-9，为黄色固体，直接用于下一步反应。LCMS(ESI)m/z＝506.0(M+Na)+。Starting materials 1-9 (10 g, 25.25 mmol) were dissolved in 250 mL of acetonitrile, followed by 4-hydroxy-3-nitrobenzaldehyde (7.1 g, 42.58 mmol) and silver oxide (26.1 g, 113.63 mmol). The mixture was stirred for 4 hours, filtered over celite, and the filtrate was evaporated to remove acetonitrile. The residue was dissolved in ethyl acetate (400 mL), washed twice with saturated sodium hydrogen carbonate (200 mL×3), washed with water (200 mL), and brine (200 mL) Drying with anhydrous sodium sulfate and concentrating to give 12 g of crude product 2-9 as a yellow solid. LCMS (ESI) m / z = 506.0 (M + Na) +.
(2)化合物3-9的合成(2) Synthesis of compound 3-9
上一步反应产物2-9(12g,24.8mmol)溶于50mL异丙醇和250mL氯仿的混合溶剂中，加入5g硅胶，冰水浴冷却至0℃，分批缓慢加入硼氢化钠(1.4g,37.2mmol)，搅拌2小时，反应液倒入300mL冰水中，分出有机相，饱和食盐水洗(100mL)，无水硫酸钠干燥，浓缩，剩余物***洗涤，过滤，得10g产品3-9，为白色固体，收率83％。LCMS(ESI)m/z＝508.0(M+Na)+。The reaction product 2-9 (12 g, 24.8 mmol) was dissolved in a mixed solvent of 50 mL of isopropanol and 250 mL of chloroform, and 5 g of silica gel was added thereto, and the mixture was cooled to 0 ° C in an ice water bath, and sodium borohydride (1.4 g, 37.2 mmol) was slowly added in portions. After stirring for 2 hours, the reaction solution was poured into 300 mL of ice water, and the organic phase was separated, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and evaporated. Solid, yield 83%. LCMS (ESI) m / z = 508.0 (M + Na) +.
(3)化合物4-9的合成(3) Synthesis of compound 4-9
上一步反应产物3-9(2g,4.1mmol)溶于300mL乙酸乙酯中，加入0.3g二氧化铂，置换氢气3次，氢气氛下室温搅拌过夜。过滤，滤液浓缩，剩余物硅胶柱层析(石油醚/乙酸乙酯＝1:3)纯化得1.7g产品4-9，为白色固体，收率91％。LCMS(ESI)m/z＝456.1(M+H)+。The reaction product 3-9 (2 g, 4.1 mmol) in the previous step was dissolved in 300 mL of ethyl acetate, and 0.3 g of platinum dioxide was added thereto, and the hydrogen gas was replaced three times, and stirred at room temperature overnight under a hydrogen atmosphere. Filtration, the filtrate was concentrated, and the residue was purified mjjjjjjjjj LCMS (ESI) m / z = 456.1 (M + H) +.
(4)化合物5-9的合成(4) Synthesis of compound 5-9
上一步反应产物4-9(1.7g,3.74mmol)和Fmoc保护的β-氨基丙酸(1.74g,5.6mmol)溶于30mL二氯甲烷和15mL甲醇的混合溶剂中，加入EEDQ(1.85g,7.48mmol)，室温搅拌过夜。减压移除溶剂，剩余物硅胶柱层析(石油醚/乙酸乙酯＝1:3)纯化得1.8g产品5-9，为白色固体，收率65％。LCMS(ESI)m/z＝749.0(M+H)+。The reaction product 4-9 (1.7 g, 3.74 mmol) and the Fmoc-protected β-aminopropionic acid (1.74 g, 5.6 mmol) were dissolved in a mixture of 30 mL of dichloromethane and 15 mL of methanol, and EEDQ (1.85 g, 7.48 mmol), stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was purifiedjjjjjjjjjjj LCMS (ESI) m / z = 749.0 (M + H) +.
(5)化合物6-9的合成(5) Synthesis of compound 6-9
上一步反应产物5-9(374mg,0.5mmol)和PNP-CO-PNP(二(对硝基苯)碳酸酯，304mg,1mmol)溶于10mL N,N-二甲基甲酰胺中，加入DIPEA(170μL,1mmol)，室温搅拌过夜。加入30mL水，乙酸乙酯萃取3次(30mL×3)，合并有机相，水洗(30mL×3)，饱和食盐水洗(30mL×3)，无水硫酸钠干燥，浓缩，剩余物硅胶柱层析(石油醚/乙酸乙酯＝1:1)纯化得450mg产品6-9，为浅黄色油状物，收率98％。LCMS(ESI)m/z＝914.7(M+H)+。The reaction product 5-9 (374 mg, 0.5 mmol) and PNP-CO-PNP (bis(p-nitrophenyl) carbonate, 304 mg, 1 mmol) were dissolved in 10 mL of N,N-dimethylformamide and added to DIPEA. (170 μL, 1 mmol), stirred at room temperature overnight. After adding 30 mL of water, ethyl acetate was extracted three times (30 mL×3), and the organic phase was combined, washed with water (30 mL×3), brine (30 mL×3), dried over anhydrous sodium sulfate (Petroleum ether / ethyl acetate = 1:1) was purified to give 450 mg of product 6-9 as pale yellow oil, yield 98%. LCMS (ESI) m / z = 914.7 (M + H) +.
(6)化合物8-9的合成(6) Synthesis of compound 8-9
化合物5-4(40mg,0.052mmol)溶解在5mL的N,N-二甲基甲酰胺中，依次加入化合物6-9(50mg,0.052mmoL)，三乙胺(15μL,0.104mmoL)，混合物在氮气条件下，室温
搅拌2小时，然后用10mL饱和食盐水淬灭反应。乙酸乙酯萃取(50mL×3)，合并有机相，并用饱和食盐水洗涤(50mL×3)，无水硫酸钠干燥，过滤、浓缩，得到粗品。粗品通过快速分离柱色谱(二氯甲烷/甲醇＝20:1)分离纯化，得到31mg化合物8-9，收率39％。LCMS(ESI)m/z＝773.4(1/2M+H)+。Compound 5-4 (40 mg, 0.052 mmol) was dissolved in 5 mL of N,N-dimethylformamide, and then compound 6-9 (50 mg, 0.052 mmol), triethylamine (15 μL, 0.104 mmoL), The mixture was stirred at room temperature for 2 hours under nitrogen, and then quenched with 10 mL of brine. The organic layer was combined with EtOAc (EtOAc m. The crude product was separated and purified by flash column chromatography (dichloromethane / methanol = 20:1) to afford 31 mg of compound 8-9. LCMS (ESI) m / z = 773.4 (1 / 2M + H) +.
(7)化合物9-9的合成(7) Synthesis of compound 9-9
化合物8-9(30mg,0.019mmol)溶解在5mL的甲醇中，冰水浴冷却到0℃，然后加入10mL的氢氧化钠溶液(0.1N)，混合物在0℃搅拌2小时，然后用1N的盐酸调节pH值到7。将上述溶液冷冻干燥，粗品悬浮在二氯甲烷和甲醇的混合溶剂(10:1)中，过滤、滤液浓缩，得到粗品化合物9-9。粗品直接用于下一步。LCMS(ESI)m/z＝1426.3(M+H)+。Compound 8-9 (30 mg, 0.019 mmol) was dissolved in 5 mL of methanol, cooled to 0 ° C in an ice water bath, then 10 mL sodium hydroxide solution (0.1 N) was added, and the mixture was stirred at 0 ° C for 2 hours, then with 1 N hydrochloric acid. Adjust the pH to 7. The above solution was freeze-dried, and the crude product was suspended in a mixed solvent of dichloromethane and methanol (10:1), filtered, and concentrated to give crude compound 9-9. The crude product was used directly in the next step. LCMS (ESI) m / z = 1426.3 (M + H) +.
(8)化合物10-9的合成(8) Synthesis of compound 10-9
化合物9-9(30mg,0.021mmol)溶解在2mL的N,N-二甲基甲酰胺中，冰水浴冷却到0℃，然后加入1mL的二乙胺，混合物0℃下搅拌1小时，冷冻干燥除掉溶剂，得到粗品化合物10-9。粗品直接用于下一步。LCMS(ESI)m/z＝1183.4(M+H)+。Compound 9-9 (30 mg, 0.021 mmol) was dissolved in 2 mL of N,N-dimethylformamide, cooled to 0 ° C in an ice water bath, then 1 mL of diethylamine was added, and the mixture was stirred at 0 ° C for 1 hour, freeze-dried The solvent was removed to give the crude compound 10-9. The crude product was used directly in the next step. LCMS (ESI) m / z = 1183.4 (M + H) +.
(9)化合物IA-25的合成(9) Synthesis of compound IA-25
化合物10-9(20mg,0.017mmol)溶解在2mL的N,N-二甲基甲酰胺中，氮气条件下，加入McOSu(11mg,0.034mmol)，然后加入4.7μL的TEA，反应混合物在氮气条件下室温搅拌1小时。冷冻干燥除掉DMF，粗品通过PREP-TLC(二氯甲烷/甲醇＝8:1)分离纯化，得到2.6mg的化合物IA-25，收率11％，其为红色固体。LCMS(ESI)m/z＝1375.3(M+H)+。Compound 10-9 (20 mg, 0.017 mmol) was dissolved in 2 mL of N,N-dimethylformamide. Under a nitrogen atmosphere, EtOAc (11 mg, 0.034 mmol) was added, then 4.7 μL of TEA was added, and the reaction mixture was under nitrogen. Stir at room temperature for 1 hour. The DMF was removed by lyophilization, and the crude material was purified by EtOAc (EtOAc/MeOH: EtOAc) LCMS (ESI) m / z = 1375.3 (M + H) +.
(10)化合物IA-26的合成(10) Synthesis of Compound IA-26
化合物10-9(20mg,0.017mmol)溶解在2mL的N,N-二甲基甲酰胺中，氮气条件下，加入C-L-066(13mg,0.034mmol)，然后加入4.7μL的TEA，反应混合物在氮气条件下室温搅拌1小时。冷冻干燥除掉DMF，粗品通过PREP-TLC(二氯甲烷/甲醇＝8:1)分离纯化，得到3.5mg的化合物IA-26，收率14％，其为红色固体。LCMS(ESI)m/z＝1451.3(M+H)+。Compound 10-9 (20 mg, 0.017 mmol) was dissolved in 2 mL of N,N-dimethylformamide. Under nitrogen, CL-066 (13 mg, 0.034 mmol) was added, then 4.7 μL of TEA was added and the reaction mixture was Stir at room temperature for 1 hour under nitrogen. The DMF was removed by lyophilization, and the crude material was purified and purified eluting with EtOAc (EtOAc/MeOH) LCMS (ESI) m / z = 1451.3 (M + H) +.
化合物IA-27的合成路线：
Synthetic route of compound IA-27:
实施例34 化合物IA-27的合成Example 34 Synthesis of Compound IA-27
化合物1(46mg,0.072mmoL)溶解在5mL的溴丙炔中，依次加入硫酸钙(460mg)，氧化银(35mg,0.14mmoL),上述混合物在40℃氮气条件下搅拌3天。反应液通过硅藻土过滤，滤饼用5mL的甲醇洗涤，然后滤液浓缩，得到粗品。粗品通过快速分离柱色谱分离纯化(二氯甲烷/甲醇＝10:1～4:1),得到28mg化合物IA-27，产率57％,其为红色固体。LCMS(ESI)m/z 680.0(M)+。Compound 1 (46 mg, 0.072 mmol) was dissolved in 5 mL of bromopropyne, and calcium sulfate (460 mg), silver oxide (35 mg, 0.14 mmol) was added in that order, and the mixture was stirred at 40 ° C for 3 days under nitrogen. The reaction solution was filtered through celite, and then filtered, and then filtered. The crude product was purified by flash column chromatography (dichloromethane/methanol = 10:1 to 4:1) to afford 28 mg of Compound IA-27 as a red solid. LCMS (ESI) m / z 680.0 (M) +.
化合物IA-28的合成路线Synthetic route of compound IA-28
实施例35:化合物IA-28的合成Example 35: Synthesis of Compound IA-28
化合物2-11的合成
Synthesis of Compound 2-11
化合物1-11(2.82g，10mmoL)溶解在200mL二氯甲烷中，加入DMAP(244mg，2mmoL)，三乙胺(13.9mL，100mmoL)，反应混合物冰水浴冷却到0℃，分批缓慢加入对甲苯磺酰氯(7.6g，40mmoL)，加完后，反应液室温搅拌过夜。加入100mL的饱和碳酸氢钠，淬灭反应，然后用二氯甲烷(100mL×2)萃取，合并有机相，用饱和碳酸氢钠(100mL×2)洗涤，合并有机相，无水硫酸钠干燥，过滤、浓缩，得到粗品。粗品通过快速分离柱色谱分离纯化(石油醚/乙酸乙酯＝1:2)，得到5g的化合物2-11，为棕色油状物，收率85％。LCMS(ESI)m/z＝591.2(M+H)+.Compound 1-11 (2.82g, 10mmoL) was dissolved in 200mL of dichloromethane, DMAP (244mg, 2mmoL), triethylamine (13.9mL, 100mmoL) was added, the reaction mixture was cooled to 0 ° C in ice water, slowly added in batches Toluenesulfonyl chloride (7.6 g, 40 mmol) was added, and the mixture was stirred at room temperature overnight. After adding 100 mL of saturated sodium hydrogencarbonate, the reaction was quenched, and then extracted with dichloromethane (100 mL×2). Filtration and concentration gave a crude product. The crude product was purified by flash column chromatography (EtOAc/EtOAc:EtOAc:EtOAc) LCMS (ESI) m / z = 591.2 (M + H) +.
化合物3-11的合成Synthesis of Compound 3-11
化合物2-11(2g，3.39mmoL)溶解在20mL DMF中，加入叠氮化钠(1.3g，20.34mmoL)，升温至50℃搅拌过夜。反应混合物冷却至室温，加入50mL水，淬灭反应，乙酸乙酯(80mL×2)萃取，合并有机相，用饱和食盐水(100mL×5)洗涤，有机相用无水硫酸钠干燥、过滤、浓缩，得到1.12g粗品化合物3-11，粗品未经纯化，直接用于下一步反应。LCMS(ESI)m/z＝355.2(M+23)+.Compound 2-11 (2 g, 3.39 mmol) was dissolved in 20 mL of DMF, sodium azide (1.3 g, 20.34 mmol) was added, and the mixture was warmed to 50 ° C and stirred overnight. The reaction mixture was cooled to room temperature, and then added with 50 mL of water, and the mixture was evaporated. The mixture was evaporated to ethyl acetate (EtOAc (EtOAc) Concentration gave 1.12 g of crude compound 3-11. LCMS (ESI) m / z = 355.2 (M + 23) +.
化合物4-11的合成Synthesis of Compound 4-11
化合物3-11(1.12g,3.37mmoL)溶解在10mL的稀盐酸(5％)中，然后将PPh3(883mg,3.37mmoL)溶解在7mL THF中慢慢加入。加完后，反应混合物室温搅拌过夜。加入20mL水，用二氯甲烷(50mL×3)萃取，去除有机相，水相用2N的氢氧化钠水溶液调节pH到12。然后用二氯甲烷(50mL×3)萃取，合并有机相，无水硫酸钠干燥、过滤、浓缩，得到580mg的粗品化合物4-11，为浅黄色油状物，粗品未经纯化，直接用于下一步反应。LCMS(ESI)m/z＝307.0(M+H)+.Compound 3-11 (1.12 g, 3.37 mmoL) was dissolved in 10 mL of dilute hydrochloric acid (5%), and then PPh 3 (883 mg, 3.37 mmoL) was dissolved in 7 mL of THF and slowly added. After the addition was completed, the reaction mixture was stirred at room temperature overnight. 20 mL of water was added, extracted with dichloromethane (50 mL x 3), the organic phase was removed, and the aqueous phase was adjusted to pH 12 with 2N aqueous sodium hydroxide. Then, it is extracted with dichloromethane (50 mL×3), and the organic layer is evaporated. One step reaction. LCMS (ESI) m / z = 307.0 (M + H) +.
化合物5-11的合成Synthesis of Compound 5-11
化合物4-11(580mg,1.89mmoL)溶解在16mL的碳酸钠溶液(2M)中，然后将BOC2O(536mg,2.48mmoL)溶解在16mL的1,4-二氧六环中缓慢滴加，加完后，反应
液室温搅拌过夜。向反应液中加入20mL水，乙酸乙酯(100mL×2)萃取，合并有机相，用水(100mL×2)洗涤，有机相用无水硫酸钠干燥、过滤、浓缩，得到750mg粗品化合物5-11，为浅黄色油状物。粗品未经纯化，直接用于下一步反应。LCMS(ESI)m/z＝407.3(M+H)+.Compound 4-11 (580 mg, 1.89 mmol) was dissolved in 16 mL of sodium carbonate solution (2M), then BOC 2 O (536 mg, 2.48 mmoL) was dissolved in 16 mL of 1,4-dioxane and slowly added dropwise. After the addition was completed, the reaction solution was stirred at room temperature overnight. To the reaction mixture, 20 mL of water and ethyl acetate (100 mL × 2) was evaporated, and the organic phase was combined and washed with water (100 mL × 2). , a pale yellow oil. The crude product was used in the next reaction without purification. LCMS (ESI) m / z = 407.3 (M + H) +.
化合物6-11的合成Synthesis of Compound 6-11
化合物5-11(750mg，1.85mmoL)溶解在60mL乙醇中，加入10％的Pd/C 100mg，混合物置换氢气三次，并在氢气条件下室温搅拌过夜。反应混合物硅藻土过滤，乙醇(50mL)洗涤，滤液浓缩，得到620mg粗品化合物6-11，粗品未经纯化，直接用于下一步反应。LCMS(ESI)m/z＝381.0(M+H)+.Compound 5-11 (750 mg, 1.85 mmoL) was dissolved in 60 mL of ethanol, 10% Pd/C 100 mg was added, the mixture was replaced with hydrogen three times, and stirred at room temperature under hydrogen overnight. The reaction mixture was filtered over Celite, EtOAc (EtOAc)EtOAc. LCMS (ESI) m / z = 381.0 (M + H) +.
化合物7-11的合成Synthesis of Compound 7-11
化合物6-11(620mg，1.63mmoL)溶解在20mL的饱和碳酸氢钠溶液中，冰水浴冷却至0℃，加入化合物ethyl 2,5-dioxo-2H-pyrrole-1(5H)-carboxylate(340mg,2.04mmoL)。反应液在0℃条件下搅拌30分钟，然后升到室温，搅拌1小时，加入20mL水淬灭反应，二氯甲烷(80mL×2)萃取，合并有机相，无水硫酸钠干燥、过滤、浓缩，得到粗品，粗品通过快速分离柱层析分离纯化(PE/EA＝1:1)，得到240mg的化合物7-11为浅黄色油状物，收率32％。LCMS(ESI)m/z＝483.3(M+H)+.Compound 6-11 (620 mg, 1.63 mmoL) was dissolved in 20 mL of saturated sodium bicarbonate solution, cooled to 0 ° C in an ice water bath, and the compound ethyl 2,5-dioxo-2H-pyrrole-1(5H)-carboxylate (340 mg, 2.04mmoL). The reaction mixture was stirred at 0 ° C for 30 minutes, then warmed to room temperature and stirred for 1 hour. The reaction was quenched with water (20 mL). The crude product was obtained, and the crude product was purified by flash column chromatography (PE/EA = 1:1) to afford 240 mg of compound 7-11 as a pale yellow oil. LCMS (ESI) m / z = 483.3 (M + H) +.
化合物8-11的合成Synthesis of Compound 8-11
化合物7-11(60mg，0.13mmoL)溶解在2mL二氯甲烷中，加入1mL的TFA，混合物室温搅拌2小时，减压移除溶剂，得到粗产物化合物8-11。粗产物未经纯化，直接用于下一步反应。LCMS(ESI)m/z＝361.3(M+H)+.Compound 7-11 (60 mg, 0.13 mmol) was dissolved in 2 mL of dichloromethane, 1 mL of TFA was added, and the mixture was stirred at room temperature for 2 hours, and the solvent was removed under reduced pressure to give crude compound 8-11. The crude product was used in the next step without purification. LCMS (ESI) m / z = 361.3 (M + H) +.
IA-28的合成
Synthesis of IA-28
化合物3-4(50mg，0.072mmoL)溶解在20mL的二氯甲烷中，依次加入HATU(55mg，0.14mmoL)，DIPEA(59μL，0.36mmoL)，在氮气条件下室温搅拌20分钟，然后加入化合物8-11(39mg，0.11mmoL)，反应液在氮气条件下室温搅拌1.5小时，浓缩，粗品通过PREP-TLC(DCM/EA/MeOH＝100:35:10)分离纯化，得到40mg的化合物IA-28，为红色固体，收率54％。LCMS(ESI)m/z＝1041.6(M+H)+.Compound 3-4 (50 mg, 0.072 mmoL) was dissolved in 20 mL of dichloromethane, and then HATU (55 mg, 0.14 mmoL), DIPEA (59 μL, 0.36 mmoL) was added, and stirred under nitrogen for 20 minutes at room temperature, then compound 8 was added. -11 (39 mg, 0.11 mmol), the reaction mixture was stirred at room temperature under nitrogen for 1.5 hr, and concentrated, and the crude was purified by EtOAc (EtOAc/EtOAc/EtOAc/EtOAc , as a red solid, yield 54%. LCMS (ESI) m / z = 1041.6 (M + H) +.
化合物IA-29的合成路线Synthetic route of compound IA-29
实施例36 化合物IA-29的合成Example 36 Synthesis of Compound IA-29
化合物3的合成
Synthesis of Compound 3
化合物3-4(150mg，0.215mmol)溶解在30mL的二氯甲烷中，氮气条件下，加入化合物2-12(132mg，0.325mmol)，HATU(164mg,0.432mmol),然后加入DIPEA(178μL,1.07mmol)，反应混合物在氮气条件下室温搅拌12小时。减压移除溶剂，粗品通过快速分离硅胶柱层析(DCM/EA/MeOH＝35:10:5)分离纯化，得到158mg的化合物3-12，为红色固体，收率70％。LCMS(ESI)m/z＝1052.2(M+H)+.Compound 3-4 (150 mg, 0.215 mmol) was dissolved in 30 mL of dichloromethane. Compound 2-12 (132 mg, 0.325 mmol), HATU (164 mg, 0.432 mmol), then DIPEA (178 μL, 1.07) was added under nitrogen. (mmol), the reaction mixture was stirred at room temperature under nitrogen for 12 h. The solvent was removed under reduced pressure. EtOAc m. m. LCMS (ESI) m / z = 1052.2 (M + H) +.
化合物4-12的合成Synthesis of Compound 4-12
化合物3-12(38mg，0.036mmol)溶解在6mL的乙腈中，冰水浴冷却至0℃，加入3mL的二乙胺，搅拌1小时。减压浓缩，得到30mg的化合物4-12粗品，为红色固体，粗品直接用到下一步反应。LCMS(ESI)m/z＝830.0(M+H)+.Compound 3-12 (38 mg, 0.036 mmol) was dissolved in 6 mL of acetonitrile, cooled to 0 ° C in an ice water bath, and 3 mL of diethylamine was added and stirred for 1 hour. Concentration under reduced pressure gave 30 mg of crude compound 4-12 as a red solid. LCMS (ESI) m / z = 830.0 (M + H) +.
化合物IA-29的合成Synthesis of Compound IA-29
化合物4-12(25mg，0.030mmol)溶解在10mL DCM中，氮气条件下，加入化合物5-12(59mg，0.076mmol)，然后再加入8.4μL的TEA，反应混合物在室温氮气条件下搅拌2小时。浓缩除掉有机溶剂，粗品通过PREP-TLC(DCM/EA/MeOH＝100:25:12)分离纯化，得到4.9mg的化合物IA-29，为红色固体，收率11％。LCMS(ESI)m/z＝746.7(M/2
+H)+.Compound 4-12 (25 mg, 0.030 mmol) was dissolved in 10 mL of DCM, and compound 5-12 (59 mg, 0.076 mmol) was added under nitrogen, then 8.4 μL of TEA was added, and the reaction mixture was stirred at room temperature under nitrogen for 2 hours. . The organic solvent was removed by concentration. EtOAc was purified eluted eluted elute LCMS (ESI) m/z = 746.7 (M/2 + H) + .
化合物IA-30的合成路线Synthetic route of compound IA-30
实施例37:化合物IA-30的合成Example 37: Synthesis of Compound IA-30
化合物2-13的合成Synthesis of Compound 2-13
化合物1(190mg，0.30mmoL)溶解在10mL DCM中，依次加入化合物1-13(330mg，0.089mmoL)，DMAP(181mg，1.48mmoL)，4A MS(200mg)，反应混合物在氮
气条件下，室温搅拌1周。反应混合物过滤、浓缩，得到粗产品，粗产品通过快速分离柱层析分离纯化(二氯甲烷/丙酮/甲醇＝5:1:0.1)，得到110mg的化合物2-13，为红色固体，收率38％。LCMS(ESI)m/z＝978.0(M+H)+.Compound 1 (190 mg, 0.30 mmol) was dissolved in 10 mL DCM, and compound 1-13 (330 mg, 0.089 mmol), DMAP (181 mg, 1.48 mmol), 4A MS (200 mg) was added sequentially, and the reaction mixture was stirred under nitrogen at room temperature. 1 week. The reaction mixture is filtered and concentrated to give a crude material. The crude product is purified by flash column chromatography (dichloromethane/acetone/methanol = 5:1:0.1) to afford 110 mg of compound 2-13 as a red solid. 38%. LCMS (ESI) m / z = 978.0 (M + H) +.
化合物3-13的合成Synthesis of Compound 3-13
化合物2-13(50mg，0.051mmol)溶解在6mL乙腈中，在冰水浴条件下，加入3mL的二乙胺，并搅拌1小时。减压浓缩，得到39mg的粗品化合物3-13，为红色固体，粗产品直接用到下一步反应。LCMS(ESI)m/z＝756.2(M+H)+.Compound 2-13 (50 mg, 0.051 mmol) was dissolved in 6 mL of acetonitrile, and 3 mL of diethylamine was added under ice water bath and stirred for 1 hour. Concentration under reduced pressure gave 39 mg of crude compound 3-13 as a red solid. LCMS (ESI) m / z = 756.2 (M + H) +.
化合物5-13的合成Synthesis of Compound 5-13
化合物3-13(40mg，0.052mmol)溶解在5mL的DMF中，依次加入化合物6-9(50mg，0.052mmoL)，TEA(15μL，0.106mmoL)，反应混合物在氮气条件下，室温搅拌2小时。然后用10mL饱和食盐水淬灭反应，乙酸乙酯萃取(50mL×3)，合并有机相，饱和食盐水洗涤(50mL×3)，无水硫酸钠干燥，过滤、浓缩，得到粗品。粗品通过快速分离柱色谱分离纯化(DCM/MeOH＝20:1)，得到40mg化合物5-13，收率49％。LCMS(ESI)m/z＝1530.2(M+H)+.Compound 3-13 (40 mg, 0.052 mmol) was dissolved in 5 mL of DMF, and then compound 6-9 (50 mg, 0.052 mmol), TEA (15 μL, 0.106 mmol) was added, and the reaction mixture was stirred at room temperature for 2 hours under nitrogen. The reaction was then quenched with EtOAc (EtOAc)EtOAc. The crude product was purified by flash column chromatography (DCM / MeOH = 20:1) to afford 40 mg of compound 5-13. LCMS (ESI) m / z = 1530.2 (M + H) +.
化合物6-13的合成
Synthesis of Compound 6-13
化合物5-13(40mg，0.026mmol)溶解在5mL甲醇中，冰水浴冷却到0℃，然后加入15mL的氢氧化钠溶液(0.1N)，反应混合物于0℃搅拌2小时。然后用1N的盐酸调节pH值到7。将上述溶液冷冻干燥，粗品悬浮在二氯甲烷和甲醇的混合溶剂(10:1)中，过滤，滤液浓缩，得到粗品化合物6-13，粗品直接用于下一步反应。LCMS(ESI)m/z＝1390.1(M+H)+.Compound 5-13 (40 mg, 0.026 mmol) was dissolved in 5 mL of methanol, cooled to 0 ° C in ice water, then 15mL sodium hydroxide solution (0.1 N), and the mixture was stirred at 0 ° C for 2 hours. The pH was then adjusted to 7 with 1 N hydrochloric acid. The above solution was freeze-dried, and the crude product was suspended in a mixed solvent of dichloromethane and methanol (10:1), filtered, and the filtrate was concentrated to give crude compound 6-13. LCMS (ESI) m / z = 1390.1 (M + H) +.
化合物7-13的合成Synthesis of Compound 7-13
化合物6-13(35mg，0.025mmol)溶解在4mL DMF中，冰水浴冷却到0℃，然后加入2mL二乙胺，反应混合物0℃下搅拌1小时，冷冻干燥移除溶剂，得到粗品化合物7-13，粗品直接用于下一步反应。LCMS(ESI)m/z＝1168.2(M+H)+.Compound 6-13 (35 mg, 0.025 mmol) was dissolved in 4 mL of DMF, cooled to 0 ° C in an ice water bath, then 2 mL of diethylamine was added, and the mixture was stirred at 0 ° C for 1 hour, and the solvent was removed by lyophilization to give crude compound 7- 13, the crude product is directly used in the next reaction. LCMS (ESI) m / z = 1168.2 (M + H) +.
化合物IA-30的合成Synthesis of Compound IA-30
化合物7-13(30mg，0.026mmol)溶解在5mL DMF中，氮气条件下，加入化合物8-13(20mg，0.065mmol)，然后加入7.2μL的TEA，反应混合物在氮气条件下室温搅拌1小时。冷冻干燥移除DMF，粗品通过PREP-TLC(DCM/MeOH＝4:1)分离纯化，得到3.1mg的化合物IA-30，为红色固体，收率8.9％。LCMS(ESI)m/z＝1361.1(M+H)+.Compound 7-13 (30 mg, 0.026 mmol) was dissolved in 5 mL of DMF. Compounds 8-13 (20 mg, 0.065 mmol) were added under nitrogen, then 7.2 μL of TEA was added and the reaction mixture was stirred at room temperature under nitrogen for 1 hour. The DMF was removed by lyophilization. The crude material was purified eluting with EtOAc EtOAc (EtOAc) LCMS (ESI) m / z = 1361.1 (M + H) +.
化合物IA-31的合成路线
Synthetic route of compound IA-31
实施例38:化合物IA-31的合成Example 38: Synthesis of Compound IA-31
化合物3-14的合成Synthesis of Compound 3-14
化合物3-4(50mg，0.072mmoL)溶解在10mL的THF，加入10mL的DCM，HATU(41mg，0.11mmoL)，DIPEA(35μL，0.22mmoL)，化合物2-14(38mg，0.29mmoL)，上述混合物在氮气条件下，室温搅拌过夜。浓缩得到粗品。粗品通过快速硅胶柱层析分离纯化(DCM/MeOH＝10:1)，得到41mg的化合物3-14，为红色固体，收率71％。LCMS(ESI)m/z＝814.0(M+H)+.Compound 3-4 (50 mg, 0.072 mmoL) was dissolved in 10 mL of THF, 10 mL of DCM, HATU (41 mg, 0.11 mmoL), DIPEA (35 μL, 0.22 mmol), compound 2-14 (38 mg, 0.29 mmoL), mixture Stir at room temperature overnight under nitrogen. Concentrate to give a crude product. The crude product was purified by flash column chromatography eluting elut elut elut elut elut elut LCMS (ESI) m / z = 814.0 (M + H) +.
化合物IA-31的合成Synthesis of Compound IA-31
化合物3-14(20mg，0.025mmol)溶解在10mL的DCM中，氮气条件下，加入化合物C-L-066(19mg，0.049mmol)，然后加入6.8μL的TEA，反应混合物在氮气条件下室
温搅拌2小时。浓缩，粗品通过PREP-TLC(DCM/EA/MeOH＝100:35:10)分离纯化，得到10mg的化合物IA-31，其为红色固体，收率37％。LCMS(ESI)m/z＝1083.1(M+H)+.Compound 3-14 (20 mg, 0.025 mmol) was dissolved in 10 mL of DCM, mp EtOAc (EtOAc (EtOAc) . Concentration, the crude was purified by EtOAc (EtOAc/EtOAc/EtOAc) LCMS (ESI) m / z = 1083.1 (M + H) +.
化合物IA-32的合成路线Synthesis route of compound IA-32
实施例39:化合物IA-32的合成Example 39: Synthesis of Compound IA-32
化合物3-4(20mg，0.029mmoL)溶解在5mL的DCM中，依次加入HATU(22mg，0.057mmoL)，DIPEA(30μL，0.17mmoL)，化合物2-15(18mg，0.081mmoL)，上述混合物在氮气条件下，室温搅拌过夜。浓缩，得到粗品。粗品通过PREP-HPLC，得到5mg的化合物IA-32，为红色固体，收率19％。LCMS(ESI)m/z＝905.0(M+H)+.Compound 3-4 (20 mg, 0.029 mmol) was dissolved in 5 mL of DCM, followed by HATU (22 mg, 0.057 mmoL), DIPEA (30 μL, 0.17 mmoL), compound 2-15 (18 mg, 0.081 mmoL), the above mixture in nitrogen Stir at room temperature overnight. Concentrate to give a crude product. The crude product was purified by PREP-HPLC to give 5 mg of Compound IA-32 as a red solid. LCMS (ESI) m / z = 905.0 (M + H) +.
化合物IA-33的合成路线Synthetic route of compound IA-33
实施例40:化合物IA-33的合成Example 40: Synthesis of Compound IA-33
化合物3-16的合成
Synthesis of Compound 3-16
化合物3-4(100mg，0.14mmol)溶解在20mL DCM中，氮气条件下，加入化合物2-16(65mg，0.17mmol)，HATU(109mg,0.29mmol),然后加入DIPEA(118μL,0.72mmol)，反应混合物在氮气条件下室温搅拌2小时。浓缩，粗品通过快速分离硅胶柱层析(DCM/MeOH＝20:1)分离纯化，得到130mg的化合物3-16，为红色固体，收率89％。LCMS(ESI)m/z＝1022.0(M+H)+.Compound 3-4 (100 mg, 0.14 mmol) was dissolved in 20 mL of DCM, EtOAc (EtOAc, EtOAc (EtOAc) The reaction mixture was stirred at room temperature for 2 hours under nitrogen. The residue was concentrated and purified by flash chromatography eluting elut elut elut elut elut elut LCMS (ESI) m / z = 1022.0 (M + H) +.
化合物4-16的合成Synthesis of Compound 4-16
化合物3-16(120mg,0.12mmol)溶解在5mL的THF中，依次加入甲醇(5mL),水(2.5mL)，碳酸钾(82mg,0.59mmoL)，反应混合物在室温条件下搅拌1小时，加入20mL水，减压移除THF和甲醇，水相用2N稀盐酸调pH＝7，上述混合物冷冻干燥过夜，得到粗品化合物4-16，粗品直接用于下一步反应。LCMS(ESI)m/z＝1008.2(M+H)+.Compound 3-16 (120 mg, 0.12 mmol) was dissolved in 5 mL of THF, and then methanol (5 mL), water (2.5 mL), potassium carbonate (82 mg, 0.59 mmol), and the mixture was stirred at room temperature for 1 hour. 20 mL of water, THF and methanol were removed under reduced pressure, the aqueous phase was adjusted to pH = 7 with 2N diluted hydrochloric acid, and the mixture was lyophilized overnight to afford crude compound 4-16. LCMS (ESI) m / z = 1008.2 (M + H) +.
化合物IA-33的合成Synthesis of Compound IA-33
化合物4-16(50mg，0.050mmoL)溶解在5mL的DMF中，依次加入HATU(38
mg，0.099mmoL)，DIPEA(41μL，0.25mmoL)，化合物5-16(33mg，0.15mmoL)，上述混合物在氮气条件下，室温搅拌2小时。冷冻干燥得到粗品。粗品通过PREP-TLC分离纯化(DCM/MeOH＝10:1)，得到15mg的化合物IA-33，为红色固体，收率25％。LCMS(ESI)m/z＝1213.2(M+H)+.Compound 4-16 (50 mg, 0.050 mmoL) was dissolved in 5 mL of DMF, followed by HATU (38 mg, 0.099 mmoL), DIPEA (41 μL, 0.25 mmoL), compound 5-16 (33 mg, 0.15 mmoL). The mixture was stirred at room temperature for 2 hours under nitrogen. Freeze to give a crude product. The crude product was purified by EtOAc (EtOAc: EtOAc: EtOAc) LCMS (ESI) m / z = 1213.2 (M + H) +.
化合物IA-34的合成路线Synthetic route of compound IA-34
实施例41:化合物IA-34的合成Example 41: Synthesis of Compound IA-34
化合物3-17的合成
Synthesis of Compound 3-17
化合物1-17(6g，37.5mmoL)溶解在50mL甲醇中，加入化合物2-17(2.7mL，30mmoL)，上述混合物在室温条件下搅拌过夜。浓缩除掉有机溶剂，得到粗品，粗品通过快速硅胶柱层析分离纯化(DCM/MeOH＝10:1)，得到6.1g化合物3-17，为无色油状物，收率83％。LCMS(ESI)m/z＝247.1(M+H)+.Compound 1-17 (6 g, 37.5 mmol) was dissolved in 50 mL of methanol, and compound 2-17 (2.7 mL, 30 mmol) was added, and the mixture was stirred overnight at room temperature. The organic solvent was concentrated to give a crude material. EtOAcjjjjjjj LCMS (ESI) m / z = 247.1 (M + H) +.
化合物5-17的合成Synthesis of Compound 5-17
化合物4-17(2.53g，8.13mmol)溶解在100mL DCM中，氮气条件下，加入化合物3-17(2g，8.13mmol)，HATU(6.18g,16.26mmol),然后加入DIPEA(6.7mL,40.65mmol)，反应混合物在氮气条件下室温搅拌12小时。浓缩，粗品通过快速分离柱(PE/EA＝1:1)分离纯化，得到4.3g的化合物5-17，为浅黄色油状物，收率98％。LCMS(ESI)m/z＝540.0(M+H)+.Compound 4-17 (2.53 g, 8.13 mmol) was dissolved in 100 mL DCM, and then compound 3-17 (2 g, 8.13 mmol), HATU (6.18 g, 16.26 mmol) was added under nitrogen, then DIPEA (6.7 mL, 40.65) was added. (mmol), the reaction mixture was stirred at room temperature under nitrogen for 12 h. After concentration, the crude product was separated and purified using a flash column (PE/EA = 1:1) to afford 4.3 g of compound 5-17 as a pale yellow oil, yield 98%. LCMS (ESI) m / z = 540.0 (M + H) +.
化合物6-17的合成Synthesis of Compound 6-17
化合物5-17(4.38g，8.13mmoL)溶解在30mL THF和30mL甲醇中，冰水浴冷却至0℃，将氢氧化钠(1.62g，40.65mmoL)溶于30mL水中缓慢加入，上述混合物室温搅拌过夜。减压移除THF和甲醇，水相用2N的盐酸调节pH值到7，上述混合物(含化合物6-17)没有进一步处理，直接用于下一步反应。LCMS(ESI)m/z＝304.1(M+H)+.Compound 5-17 (4.38g, 8.13mmoL) was dissolved in 30mL of THF and 30mL of methanol, cooled to 0 ° C in an ice water bath, sodium hydroxide (1.62g, 40.65mmoL) was slowly dissolved in 30mL of water, and the mixture was stirred at room temperature overnight. . The THF and methanol were removed under reduced pressure, the aqueous phase was adjusted to pH 7 using 2N hydrochloric acid, and the mixture (comprising compound 6-17) was used in the next step without further work. LCMS (ESI) m / z = 304.1 (M + H) +.
化合物7-17的合成Synthesis of Compound 7-17
上一步含有化合物6-17(2.46g，8.13mmoL)的溶液用50mL的水稀释，然后依次
加入THF(50mL)、碳酸氢钠(1.36g，16.26mmoL)、Fmoc-Cl(2.52g，9.76mmoL)，上述混合物室温搅拌2小时，减压除掉THF，水相用2N盐酸调节pH到7，用乙酸乙酯(100mL×3)萃取，合并有机相，无水硫酸钠干燥、过滤、浓缩，得到粗品，粗品通过快速分离柱层析分离纯化(DCM/MeOH＝10:1)，得到2.47g的化合物7-17，为白色固体，收率58％。LCMS(ESI)m/z＝548.0(M+H)+.The solution containing the compound 6-17 (2.46 g, 8.13 mmol) in the previous step was diluted with 50 mL of water, then THF (50 mL), sodium hydrogencarbonate (1.36 g, 16.26 mmoL), Fmoc-Cl (2.52 g, 9.76 mmoL). The mixture was stirred at room temperature for 2 hours, THF was evaporated under reduced pressure. EtOAc (EtOAc m. The crude product was obtained. EtOAc mjjjjjjjj LCMS (ESI) m / z = 548.0 (M + H) +.
化合物9-17的合成Synthesis of Compound 9-17
化合物7-17(470mg，0.90mmol)溶解在100mL DCM中，氮气条件下，加入化合物8-17(299mg，1.34mmol)，HATU(680mg,1.79mmol),然后加入DIPEA(738μL,4.48mmol)，反应混合物在氮气条件下室温搅拌12小时。浓缩，粗品通过快速分离柱层析(DCM/MeOH＝5:1)分离纯化，得到280mg的化合物9-17，为浅黄色固体，收率43％。LCMS(ESI)m/z＝731.1(M+H)+.Compound 7-17 (470 mg, 0.90 mmol) was dissolved in 100 mL DCM, EtOAc (EtOAc, EtOAc, EtOAc (EtOAc) The reaction mixture was stirred at room temperature for 12 hours under nitrogen. Concentration, the crude was purified by flash column chromatography eluting elut elut elut elut LCMS (ESI) m / z = 731.1 (M + H) +.
化合物10-17的合成Synthesis of Compound 10-17
化合物9-17(150mg，0.21mmoL)溶解在5mL DCM中，加入氯化氢的乙酸乙酯溶液(3M，20mL)，上述混合物室温搅拌1小时，减压浓缩，得到粗品化合物10-17，粗品未经进一步纯化，直接用于下一步反应。LCMS(ESI)m/z＝631.1(M+H)+.Compound 9-17 (150 mg, 0.21 mmol) was dissolved in 5 mL of EtOAc. EtOAc (EtOAc m. Further purification was used directly in the next reaction. LCMS (ESI) m / z = 631.1 (M + H) +.
化合物12-17的合成Synthesis of Compound 12-17
化合物3-4(130mg，0.19mmol)溶解在5mL DMF中，氮气条件下，加入化合物10-
17(120mg，0.19mmol)，HATU(145mg,0.38mmol)和DIPEA(157μL,0.95mmol)，反应混合物在氮气条件下室温搅拌2小时。冷冻干燥，粗品通过PREP-TLC(DCM/MeOH＝8:1)分离纯化，得到82mg的化合物12-17，收率33％，其为红色固体。LCMS(ESI)m/z＝1311.9(M+H)+.3-4 (130 mg, 0.19 mmol Stir at room temperature for 2 hours under nitrogen. It was lyophilized, and the crude material was purified and purified, mjjjjjjjjjjjjj LCMS (ESI) m / z = 1311.9 (M + H) +.
化合物13-17的合成Synthesis of Compound 13-17
化合物12-17(82mg，0.062mmol)溶解在5mL DCM中，在冰水浴条件下，加入5mL的二乙胺，并搅拌1小时。浓缩，得到68mg的粗品化合物13-17，为红色固体，粗品未经进一步纯化，直接用到下一步反应。LCMS(ESI)m/z＝1089.9(M+H)+.Compound 12-17 (82 mg, 0.062 mmol) was dissolved in 5 mL of DCM, and 5 mL of diethylamine was added under ice-water bath and stirred for 1 hour. Concentration gave 68 mg of crude compound 13-17 as a red solid. LCMS (ESI) m / z = 1089.9 (M + H) +.
化合物IA-34的合成Synthesis of Compound IA-34
化合物13-17(60mg，0.055mmol)溶解在6mL DCM中，氮气条件下，加入2mL DMF，化合物14-17(35mg，0.11mmol)，然后加入15.3μL的TEA，反应混合物在氮气条件下室温搅拌3小时。冷冻干燥，粗品通过PREP-TLC(DCM/MeOH＝8:1)分离纯化，得到10.1mg的化合物IA-34，为红色固体，收率14％。LCMS(ESI)m/z＝1305.4(M+Na)+.Compound 13-17 (60 mg, 0.055 mmol) was dissolved in 6 mL of DCM, and 2 mL of DMF, compound 14-17 (35 mg, 0.11 mmol) was added under nitrogen, then 15.3 μL of TEA was added, and the reaction mixture was stirred at room temperature under nitrogen. 3 hours. It was lyophilized, and the crude product was purified and purified, mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj LCMS (ESI) m / z = 1305.4 (M + Na) +.
实施例42带连接体毒素与抗体偶联步骤如下：Example 42 The procedure for coupling a linker toxin to an antibody is as follows:
将抗体蛋白(例如赫赛汀，抗TPBG抗体，抗CD70抗体或抗EGFRVIII抗体)使用透析缓冲液(25mM Sodium Borate，硼酸钠)，25mM NaCl,and 1mM DTPA,final pH 7.4.)于4℃透析过夜。透析缓冲液的体积是抗体蛋白体积的500倍以上。第二天将抗体取出后，使用0.22μm的滤膜过滤后，使用A280(即A280(nm)紫外光吸收法测定蛋白浓度)测定浓度。使用新鲜配制的TCEP(tris(2-carboxyethyl)phosphine hydrochloride)溶液，以设定的比例加入到抗体中还原，以抗体物质的量的2至5倍为宜。在25℃还原反应2小时后，使用脱盐柱将抗体转换入耦联缓冲液(20mM succinate150mM NaCl,2mM EDTA,
about 75mM Tris.)中。以此同时，使用DMSO将需要耦联的化合物溶解成10mM的浓度。在换盐后的抗体溶液中，缓慢加入终浓度10-30％的DMSO，立刻混匀；再将已溶解的化合物以一定比例加入抗体溶液中。以抗体物质的量5至15倍间加入为宜。于25℃反应2小时后，使用孔径为10kDa的透析膜进行透析并换液一次，4℃透析过夜。透析缓冲液的体积需是抗体-化合物的体积的500倍以上。第三天将抗体-化合物取出后，使用0.22μm的绿膜过滤并测定浓度后。分别进行：疏水层析Hydrophobic-Interaction Chromatography(HIC)，聚丙烯酰胺-SDS凝胶电泳(SDS-PAGE，electrophoresis)，液相质谱liquid chromatograph-mass spectrometer(LC-MS)确定耦联的化合物-抗体比例(即DAR)和效率；分子排阻层析Size-Exclusion Chromatography(SEC)，确定抗体-化合物的分子纯度。具体化合物结构见表1，DAR表示药物与单克隆抗体的摩尔比(即k)。Antibody proteins (eg Herceptin, anti-TPBG antibodies, anti-CD70 antibodies or anti-EGFRVIII antibodies) were dialyzed against dialysis buffer (25 mM Sodium Borate, sodium borate), 25 mM NaCl, and 1 mM DTPA, final pH 7.4.) at 4 °C. overnight. The volume of the dialysis buffer is more than 500 times the volume of the antibody protein. After the antibody was taken out the next day, it was filtered using a 0.22 μm filter, and the concentration was measured using A280 (i.e., A280 (nm) ultraviolet absorption method for measuring protein concentration). The freshly prepared TCEP (tris(2-carboxyethyl)phosphine hydrochloride) solution is added to the antibody in a set ratio to reduce it by 2 to 5 times the amount of the antibody substance. After 2 hours of reduction at 25 ° C, the antibody was switched to a coupling buffer (20 mM succinate 150 mM NaCl, 2 mM EDTA, using a desalting column).
About 75mM Tris.). At the same time, the compound to be coupled was dissolved to a concentration of 10 mM using DMSO. In the antibody solution after salt exchange, the final concentration of 10-30% DMSO was slowly added, and immediately mixed; the dissolved compound was added to the antibody solution in a certain ratio. It is preferred to add 5 to 15 times the amount of the antibody substance. After reacting at 25 ° C for 2 hours, dialysis was carried out using a dialysis membrane having a pore size of 10 kDa and exchanged once, and dialyzed overnight at 4 °C. The volume of the dialysis buffer needs to be more than 500 times the volume of the antibody-compound. On the third day, the antibody-compound was taken out, filtered through a 0.22 μm green membrane, and the concentration was measured. Separate compound-antibody by hydrophobic chromatography, Hydrophobic-Interaction Chromatography (HIC), polyacrylamide-SDS gel electrophoresis (SDS-PAGE, electrophoresis), liquid chromatography-mass spectrometer (LC-MS) Proportion (ie DAR) and efficiency; Size-Exclusion Chromatography (SEC), determine the molecular purity of the antibody-compound. The specific compound structure is shown in Table 1, and DAR represents the molar ratio of the drug to the monoclonal antibody (i.e., k).
效果实施例1生物活性测试Effect Example 1 Biological Activity Test
使用Her2阳性的人BT474乳腺肿瘤细胞(简称BT474)，Her2低表达的人MCF-7乳腺肿瘤细胞(简称MCF-7)，以及在MCF-7外转Her2的人乳腺肿瘤MCF7-Her2稳转细胞株(简称MCF-7-Her2)(参照文献：Teemu T.Junttila,et al.,Breast.Cancer.Res.Treat.,2011,128,347和Jeffrey J.Wallin et al.,Clin.Cancer.Res.,2012,18,3901公开的制备重组细胞方法进行制备，本发明在此引用该文献全文)。评价了得到的抗体药物偶联物对肿瘤细胞的生长抑制。BT474，MCF7-Her2和MCF-7用0.25％(体积/体积)的胰蛋白酶消化，使细胞剥离，然后悬浮于100μL完全培养基，2,000个细胞接种于96孔板进行培养。37℃过夜贴壁生长，然后加入100μL含有不同浓度梯度的抗体药物偶联物以及完全培养基。120小时后加入50μL
萤光细胞活性检测试剂(
Luminescent，Promega)进行相对细胞增殖分析。活性数据见表1。Her2-positive human BT474 breast tumor cells (abbreviated as BT474), Her2 low-expression human MCF-7 breast tumor cells (abbreviated as MCF-7), and MCF-7-expressing Her2 human breast tumor MCF7-Her2 stable cells Strain (MCF-7-Her2 for short) (Reference: Teemu T. Junttila, et al., Breast. Cancer. Res. Treat., 2011, 128, 347 and Jeffrey J. Wallin et al., Clin. Cancer. Res., The preparation of a recombinant cell method disclosed in 2012, 18, 3901 is carried out, the entire disclosure of which is hereby incorporated by reference. The growth inhibition of tumor cells by the obtained antibody drug conjugate was evaluated. BT474, MCF7-Her2 and MCF-7 were digested with 0.25% (vol/vol) trypsin to detach the cells, then suspended in 100 μL of complete medium, and 2,000 cells were seeded in 96-well plates for culture. Incubate at 37 ° C overnight, then add 100 μL of antibody drug conjugate containing different concentration gradients and complete medium. Add 50μL after 120 hours Fluorescent cell activity assay reagent Luminescent, Promega) performs relative cell proliferation assays. The activity data is shown in Table 1.
表1Table 1
*表1中，mAb表示单克隆抗体赫赛汀*In Table 1, mAb indicates the monoclonal antibody Herceptin
由表1可知，本发明的抗体偶联物对MCF-Her2细胞具有很好的抑制活性，其抑制活性基本高于对照药T-DM1。小分子毒素PNU-159682对BT474、阴性MCF-7细胞、阳性MCF-7-Her2细胞活性相当，基本没有选择性。相对于PNU-159682，本发明的抗体药物偶联物对阴性MCF-7细胞活性大大降低，对MCF-7-Her2阳性细胞活性基本相当或略有下降，其选择性显著提高，其对MCF-7和MCF-Her2的选择性基本与T-DM1相近，减小了潜在的细胞毒性。As can be seen from Table 1, the antibody conjugate of the present invention has a good inhibitory activity against MCF-Her2 cells, and its inhibitory activity is substantially higher than that of the control drug T-DM1. The small molecule toxin PNU-159682 has comparable activity to BT474, negative MCF-7 cells, and positive MCF-7-Her2 cells, and is essentially non-selective. Compared with PNU-159682, the antibody drug conjugate of the present invention greatly reduced the activity of the negative MCF-7 cells, and the activity of MCF-7-Her2 positive cells was substantially equal or slightly decreased, and the selectivity thereof was significantly improved, which was against MCF- The selectivity of 7 and MCF-Her2 is almost similar to that of T-DM1, reducing potential cytotoxicity.
效果实施例2生物活性测试Effect Example 2 Biological Activity Test
使用TPBG阳性的人MBA-MB-468乳腺癌细胞(简称MBA-MB-468)，TPBG低表达的人NCI-H1975非小细胞肺癌细胞(简称NCI-H1975)。评价本发明的抗体药物偶联物对肿瘤细胞的生长抑制。MBA-MB-468和NCI-H1975用0.25％(体积/体积)的胰蛋白酶消化，使细胞剥离，然后悬浮于100μL完全培养基，2,000个细胞接种于96孔板进行培养。37℃过夜贴壁生长，然后加入100μL含有不同浓度梯度的抗体药物偶联物以及完全培养基。120小时后加入50μL
萤光细胞活性检测试剂(
Luminescent，Promega)进行相对细胞增殖分析。活性数据见表2。TPBG-positive human MBA-MB-468 breast cancer cells (abbreviated as MBA-MB-468) and TPBG-low expressed human NCI-H1975 non-small cell lung cancer cells (abbreviated as NCI-H1975) were used. The growth inhibition of tumor cells by the antibody drug conjugate of the present invention was evaluated. MBA-MB-468 and NCI-H1975 were digested with 0.25% (v/v) trypsin, the cells were exfoliated, and then suspended in 100 μL of complete medium, and 2,000 cells were seeded in 96-well plates for culture. Incubate at 37 ° C overnight, then add 100 μL of antibody drug conjugate containing different concentration gradients and complete medium. Add 50μL after 120 hours Fluorescent cell activity assay reagent Luminescent, Promega) performs relative cell proliferation assays. The activity data is shown in Table 2.
表2Table 2
*表2中，mAb表示单克隆抗体TPBG*In Table 2, mAb represents the monoclonal antibody TPBG
由表2可知，与使用DM1，MMAF为毒素的抗体药物偶联物相比，本发明的抗体药物偶联物对TPBG阳性的人MBA-MB-468乳腺癌细胞(简称MBA-MB-468)和TPBG高表达的人H1568非小细胞肺癌细胞具有更高的抑制活性。特别是在对TPBG低表达的人NCI-H1975非小细胞肺癌细胞(简称NCI-H1975)本发明的抗体药物偶联物细胞杀伤活性远远高于相应的以DM1和MMAF为毒素的对照物。As can be seen from Table 2, the antibody drug conjugate of the present invention is TPBG-positive human MBA-MB-468 breast cancer cell (abbreviated as MBA-MB-468) compared to the antibody drug conjugate using DM1 and MMAF as a toxin. Human H1568 non-small cell lung cancer cells with high expression of TPBG have higher inhibitory activity. Especially in human NCI-H1975 non-small cell lung cancer cells (NCI-H1975) with low expression of TPBG, the cell cytotoxic activity of the antibody drug conjugate of the present invention is much higher than the corresponding control with DM1 and MMAF as toxin.
效果实施例3生物活性测试Effect Example 3 Biological Activity Test
使用CD70重组细胞系CHOK-hCD70(制备方法与MCF7-Her2一致)(简称CHOK-hCD70)和不表达CD70的阴性细胞CHOk1，评价本发明的抗体药物偶联物对肿瘤细胞的生长抑制。同时也使用CD70阳性的人肾癌细胞786-O(简称786-O)，CD70低表达的人肾癌细胞Caki-1(简称Caki-1)和Caki-2(简称Caki-2)，评价本发明的抗体药物偶联物对肿瘤细胞的生长抑制。CHOK-hCD70、CHOK1、786-O、Caki-1和Caki-2用0.25％(体积/体积)的胰蛋白酶消化，使细胞剥离，然后悬浮于100μL完全培养基，2,000个细胞接种于96孔板进行培养。37℃过夜贴壁生长，然后加入100μL含有不同浓度梯度的抗体药物偶联物以及完全培养基。120小时后加入50μL
萤光细胞活性检测试剂(
Luminescent，Promega)进行相对细胞增殖分析。活性数据见表3。
The growth inhibition of tumor cells by the antibody drug conjugate of the present invention was evaluated using the CD70 recombinant cell line CHOK-hCD70 (prepared in accordance with MCF7-Her2) (abbreviated as CHOK-hCD70) and the negative cell CHOk1 not expressing CD70. At the same time, CD70-positive human renal cancer cell line 786-O (abbreviation 786-O), CD70 low-expression human kidney cancer cell Caki-1 (abbreviated as Caki-1) and Caki-2 (abbreviated as Caki-2) were also used. The antibody drug conjugate of the invention inhibits the growth of tumor cells. CHOK-hCD70, CHOK1, 786-O, Caki-1 and Caki-2 were digested with 0.25% (vol/vol) trypsin to detach the cells, then suspended in 100 μL of complete medium, and 2,000 cells were seeded in 96-well plates. Cultivate. Incubate at 37 ° C overnight, then add 100 μL of antibody drug conjugate containing different concentration gradients and complete medium. Add 50μL after 120 hours Fluorescent cell activity assay reagent Luminescent, Promega) performs relative cell proliferation assays. The activity data is shown in Table 3.
表3table 3
*表3中，mAb表示单克隆抗体CD70.*In Table 3, mAb represents the monoclonal antibody CD70.
由表3可知，与使用DM1为毒素的抗体药物偶联物相比，本发明的抗体药物偶联物对CD70阳性的重组细胞系CHOK-hCD70(简称CHOK-hCD70，制备方法与MCF7-Her2一致)和人肾癌细胞786-O(简称786-O)具有接近或者更高的抑制活性。对CD70低表达的人肾癌细胞Caki-1(简称Caki-1)和Caki-2(简称Caki-2)，本发明的抗体药物偶联物具有接近甚至更高的细胞杀伤力。As can be seen from Table 3, the antibody drug conjugate of the present invention is equivalent to the CD70-positive recombinant cell line CHOK-hCD70 (referred to as CHOK-hCD70, the preparation method is consistent with MCF7-Her2) compared with the antibody drug conjugate using DM1 as a toxin. And human kidney cancer cell line 786-O (abbreviation 786-O) has near or higher inhibitory activity. For human kidney cancer cells Caki-1 (Caki-1) and Caki-2 (Caki-2), which are low expressed by CD70, the antibody drug conjugate of the present invention has a near or higher cell killing power.
效果实施例4生物活性测试Effect Example 4 Biological Activity Test
使用表达人EGFR蛋白的重组细胞系CHOK1-EGFR(简称CHOK1-EGFR)、E表达人GFRvIII蛋白的重组细胞系CHOK1-EGFRvIII(简称CHOK1-EGFRvIII)为阳性细胞和不表达EGFR的阴性细胞CHOk1，评价本发明的抗体药物偶联物对肿瘤细胞的生长抑制。CHOK1-EGFR、CHOK1-EGFRvIII、CHOk1用0.25％(体积/体积)的胰蛋白酶消化，使细胞剥离，然后悬浮于100μL完全培养基，2,000个细胞接种于96孔板进行培养。37℃过夜贴壁生长，然后加入100μL含有不同浓度梯度的抗体药物偶联物以及完全培养基。120小时后加入50μL
萤光细胞活性检测试剂(
Luminescent，
Promega)进行相对细胞增殖分析。活性数据见表4。The recombinant cell line CHOK1-EGFR (CHOK1-EGFR) expressing human EGFR protein, the recombinant cell line CHOK1-EGFRvIII (referred to as CHOK1-EGFRvIII) expressing human GFRvIII protein, and the negative cell CHOk1 not expressing EGFR were evaluated. The antibody drug conjugate of the present invention inhibits the growth of tumor cells. CHOK1-EGFR, CHOK1-EGFRvIII, and CHOk1 were digested with 0.25% (v/v) trypsin, and the cells were detached, and then suspended in 100 μL of complete medium, and 2,000 cells were seeded in a 96-well plate for culture. Incubate at 37 ° C overnight, then add 100 μL of antibody drug conjugate containing different concentration gradients and complete medium. Add 50μL after 120 hours Fluorescent cell activity assay reagent Luminescent, Promega) performs relative cell proliferation assays. The activity data is shown in Table 4.
表4Table 4
*表4中，mAb表示单克隆抗体EGFRVIII.*In Table 4, mAb represents the monoclonal antibody EGFRVIII.
由表4可知，与使用MMAF为毒素的抗体药物偶联物相比，本发明的抗体药物偶联物对EGFR阳性的重组细胞系CHOK1-EGFR(简称CHOK1-EGFR，制备方法与MCF7-Her2一致)和EGFRvIII阳性的重组细胞系CHOK1-EGFRvIII(简称CHOK1-EGFRvIII，制备方法与MCF7-Her2一致)具有接近或者更高的细胞杀伤活性。As can be seen from Table 4, the antibody drug conjugate of the present invention is EGFR-positive recombinant cell line CHOK1-EGFR (referred to as CHOK1-EGFR, and the preparation method is consistent with MCF7-Her2) compared with the antibody drug conjugate using MMAF as a toxin. The EGFRvIII-positive recombinant cell line CHOK1-EGFRvIII (referred to as CHOK1-EGFRvIII, which is produced in accordance with MCF7-Her2) has near or higher cell killing activity.
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。
While the invention has been described with respect to the preferred embodiments of the embodiments of the embodiments of the invention modify. Accordingly, the scope of the invention is defined by the appended claims.
Claims (31)
- 一种如式IB所示的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，An antibody drug conjugate as shown in Formula IB, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof medicine,
其中，mAb为单克隆抗体片段；Wherein, the mAb is a monoclonal antibody fragment;0<k≤8；0<k≤8;R1为氢、羟基、C1-6烷氧基、C1-6烷基、-NRaRb或-C(O)NRcRd；Ra、Rb、Rc和Rd各自独立地为氢、C1-6烷基或C6-10芳基；R 1 is hydrogen, hydroxy, C 1-6 alkoxy, C 1-6 alkyl, -NR a R b or -C(O)NR c R d ; each of R a , R b , R c and R d Independently hydrogen, C 1-6 alkyl or C 6-10 aryl;R2和R3各自独立地为C1-6烷基、C1-6烷氧基或C1-6烷硫基；R 2 and R 3 are each independently a C 1-6 alkyl group, a C 1-6 alkoxy group or a C 1-6 alkylthio group;X为X is
Y为R4a和R4b各自独立地为氢、C1～4烷基、C3～6环烷基取代的C1～4烷基、C3～6环烷基、C2-8杂烷基、或-(OCH2CH2)j1OH；j1为1、2、3、4、5、6、7或8；Y is
R 4a and R 4b are each independently hydrogen, C 1-4 alkyl, C 3-6 cycloalkyl substituted C 1-4 alkyl, C 3-6 cycloalkyl, C 2-8 heteroalkyl, Or -(OCH 2 CH 2 ) j1 OH; j1 is 1, 2, 3, 4, 5, 6, 7, or 8;Z为或4-6元亚杂芳基；R5a和R5b各自独立地为氢或C1～4烷基；Z is
Or a 4-6 membered heteroarylene; each of R 5a and R 5b is independently hydrogen or C 1-4 alkyl;或者，为
or,
forR6为氢、C1～4烷基、C2-8杂烷基、或-(OCH2CH2)j2OH；j2为1、2、3、4、5、6、7或8；R 6 is hydrogen, C 1-4 alkyl, C 2-8 heteroalkyl, or -(OCH 2 CH 2 ) j2 OH; j2 is 1, 2, 3, 4, 5, 6, 7, or 8;p为0、1、2、3、4、5、6、7、8、9或10；且当Z为时，p不为0；p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and when Z is
When p is not 0;m为0或1；m is 0 or 1;E为
E is
E中，e1、e2、e3、e4、e5和e6各自独立地为0、1、2、3、4、5、6、7或8；R7a、R7b、R7c、R7d、R7e、R7f和R7h各自独立地为氢、C1～4烷基、C3～6环烷基取代的C1～4烷基、C3～6环烷基、C2-8杂烷基、-(OCH2CH2)j3OH或j3为1、2、3、4、5、6、7或8；n为0、1、2、3、4、5、6、7、8、9或10；In E, e1, e2, e3, e4, e5, and e6 are each independently 0, 1, 2, 3, 4, 5, 6, 7 , or 8; R 7a , R 7b , R 7c , R 7d , R 7e And R 7f and R 7h are each independently hydrogen, C 1-4 alkyl, C 3-6 cycloalkyl substituted C 1-4 alkyl, C 3-6 cycloalkyl, C 2-8 heteroalkyl , -(OCH 2 CH 2 ) j3 OH or
J3 is 1, 2, 3, 4, 5, 6, 7, or 8; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;u为0或1；u is 0 or 1;R8为氢、C1～4烷基、C3～6环烷基取代的C1～4烷基、C3～6环烷基、C2-8杂烷基、或-(OCH2CH2)j4OH；j4为1、2、3、4、5、6、7或8；R 8 is hydrogen, C 1-4 alkyl, C 3-6 cycloalkyl substituted C 1-4 alkyl, C 3-6 cycloalkyl, C 2-8 heteroalkyl, or -(OCH 2 CH 2 ) j4 OH; j4 is 1, 2, 3, 4, 5, 6, 7, or 8;q为0、1、2、3、4、5、6、7、8、9或10；当含有多个-C(R8)-时，-C(R8)-相同或不同；q is 0, 1, 2, 3, 4, 5, 6, 7, 8 , 9, or 10; when a plurality of -C(R 8 )- are contained, -C(R 8 )- is the same or different;y为0～24的整数；Y is an integer from 0 to 24;G为G is
s为0或1； s is 0 or 1;Q为t1、t3和t4各自独立地为0、1、2、3、4、5、6、7、8、9或10；Q is
t 1 , t 3 and t 4 are each independently 0, 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9, or 10;中，r1、r2、r3和r4独立地为0、1、2、3、4、5、6、7、8、9或10；R6e为氢、C1～4烷基、C2-8杂烷基或-(OCH2CH2)j2aOH；j2a为1、2、3、4、5、6、7或8；R8e为氢、C1～4烷基、C3～6环烷基取代的C1～4烷基、C3～6环烷基、C2-8杂烷基或-(OCH2CH2)j4aOH；j4a为1、2、3、4、5、6、7或8；Re为糖基、Fmoc或-(OCH2CH2)enOH，en为0、1、2、3、4、5、6、7、8、9、10、11或12。
Wherein r1, r2, r3 and r4 are independently 0, 1 , 2, 3, 4 , 5 , 6, 7, 8 , 9 or 10; R 6e is hydrogen, C 1-4 alkyl, C 2-8 Heteroalkyl or -(OCH 2 CH 2 ) j2a OH; j2a is 1, 2, 3, 4, 5, 6, 7, or 8; R 8e is hydrogen, C 1-4 alkyl, C 3-6 naphthenic a substituted C 1-4 alkyl group, a C 3-6 cycloalkyl group, a C 2-8 heteroalkyl group or a -(OCH 2 CH 2 ) j4a OH; j4a is 1, 2, 3, 4, 5, 6, 7 or 8; R e is a glycosyl group, Fmoc or -(OCH 2 CH 2 ) en OH, and en is 0, 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. - 如权利要求1所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，其特征在于，The antibody drug conjugate according to claim 1, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, a pharmaceutically acceptable salt thereof or a prodrug thereof , which is characterized by所述的单克隆抗体为赫赛汀、抗TPBG抗体、抗CD70抗体或抗EGFRVIII抗体；The monoclonal antibody is Herceptin, an anti-TPBG antibody, an anti-CD70 antibody or an anti-EGFRVIII antibody;和/或，0<k≤6；And / or, 0 < k ≤ 6;和/或，所述的为
And / or, said
for和/或，当所述的R1、R2或R3为C1-6烷氧基时，所述的C1-6烷氧基为甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基或叔丁氧基；And/or, when R 1 , R 2 or R 3 is a C 1-6 alkoxy group, the C 1-6 alkoxy group is a methoxy group, an ethoxy group, a n-propoxy group, Isopropoxy, n-butoxy, isobutoxy or tert-butoxy;和/或，当所述的R1、R2或R3为C1-6烷基时，所述的C1-6烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；And/or, when said R 1 , R 2 or R 3 is a C 1-6 alkyl group, said C 1-6 alkyl group is methyl, ethyl, n-propyl, isopropyl, or Butyl, isobutyl or tert-butyl;和/或，当R2或R3为C1-6烷硫基时，所述的C1-6烷硫基为甲硫基、乙硫基、正丙硫基、异丙硫基、正丁硫基、异丁硫基或叔丁硫基；And/or, when R 2 or R 3 is a C 1-6 alkylthio group, the C 1-6 alkylthio group is methylthio, ethylthio, n-propylthio, isopropylthio, or Butylthio, isobutylthio or tert-butylthio;和/或，当Ra、Rb、Rc或Rd为C1-6烷基时，所述的C1-6烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；And/or, when R a , R b , R c or R d is a C 1-6 alkyl group, the C 1-6 alkyl group is a methyl group, an ethyl group, a n-propyl group, an isopropyl group, or a positive Butyl, isobutyl or tert-butyl;和/或，当Ra、Rb、Rc或Rd为C6-10芳基时，所述的C6-10芳基为苯基；And/or, when R a , R b , R c or R d is a C 6-10 aryl group, the C 6-10 aryl group is a phenyl group;和/或，当R4a、R4b、R5a、R5b、R6、R6e、R7a、R7b、R7c、R7d、R7e、R7f、R7h、R8或R8e为C1～4烷基时，所述的C1～4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或 叔丁基；And/or when R 4a , R 4b , R 5a , R 5b , R 6 , R 6e , R 7a , R 7b , R 7c , R 7d , R 7e , R 7f , R 7h , R 8 or R 8e are When C 1-4 alkyl, the C 1-4 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl;和/或，当R4a、R4b、R7a、R7b、R7c、R7d、R7e、R7f、R7h、R8或R8e为C3～6环烷基取代的C1～4烷基时，所述的C1～4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；And / or, when R 4a, R 4b, R 7a , R 7b, R 7c, R 7d, R 7e, R 7f, R 7h, R 8 or R 8e is C 3 ~ 6 cycloalkyl substituted C 1 ~ In the case of a 4- alkyl group, the C 1-4 alkyl group is a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group or a t-butyl group;和/或，当R4a、R4b、R7a、R7b、R7c、R7d、R7e、R7f、R7h、R8或R8e为C3～6环烷基取代的C1～4烷基时，所述的C3～6环烷基为环丙基；And / or, when R 4a, R 4b, R 7a , R 7b, R 7c, R 7d, R 7e, R 7f, R 7h, R 8 or R 8e is C 3 ~ 6 cycloalkyl substituted C 1 ~ When the alkyl group is 4 , the C 3-6 cycloalkyl group is a cyclopropyl group;和/或，当R4a、R4b、R7a、R7b、R7c、R7d、R7e、R7f、R7h、R8或R8e为C3～6环烷基时，所述的C3～6环烷基为环丙基；And/or, when R 4a , R 4b , R 7a , R 7b , R 7c , R 7d , R 7e , R 7f , R 7h , R 8 or R 8e is a C 3-6 cycloalkyl group, C 3-6 cycloalkyl is cyclopropyl;和/或，当R4a、R4b、R6、R6e、R7a、R7b、R7c、R7d、R7e、R7f、R7h、R8或R8e为C2-8杂烷基时，所述的C2-8杂烷基中的杂原子为O、S和N中的一种或多种，杂原子的个数为1、2、3或4；And/or when R 4a , R 4b , R 6 , R 6e , R 7a , R 7b , R 7c , R 7d , R 7e , R 7f , R 7h , R 8 or R 8e are C 2-8 heterocycloalkanes The hetero atom in the C 2-8 heteroalkyl group is one or more of O, S and N, and the number of hetero atoms is 1, 2, 3 or 4;和/或，当Z为4-6元亚杂芳基时，所述的4-6元亚杂芳基中的杂原子为N、O和S中的一种或多种；杂原子个数为1个、2个或3个，所述的4-6元亚杂芳基为4元、5或元6元亚杂芳基；And/or, when Z is a 4-6 membered heteroarylene group, the hetero atom in the 4-6 membered heteroarylene group is one or more of N, O and S; the number of heteroatoms 1 , 2 or 3, the 4-6 membered heteroarylene is a 4 member, 5 or 6 membered heteroarylene;和/或，y为0、1、2、3、4、5或6；And / or, y is 0, 1, 2, 3, 4, 5 or 6;和/或，当Z为时，p不为0；And / or when Z is
When p is not 0;和/或，当所述的Z为时，p为0，m为1。And/or when the Z is
When p is 0, m is 1. - 如权利要求1或2所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，其特征在于，The antibody drug conjugate according to claim 1 or 2, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, a pharmaceutically acceptable salt thereof or Prodrug, characterized in that和/或，所述的为
优选
And / or, said
forOptimal和/或，当R4a、R4b、R7a、R7b、R7c、R7d、R7e、R7f、R7h、R8或R8e为C3～6环烷基取代的C1～4烷基时，所述的C3～6环烷基取代的C1～4烷基为环丙甲基；And / or, when R 4a, R 4b, R 7a , R 7b, R 7c, R 7d, R 7e, R 7f, R 7h, R 8 or R 8e is C 3 ~ 6 cycloalkyl substituted C 1 ~ In the case of a 4- alkyl group, the C 3-6 cycloalkyl-substituted C 1-4 alkyl group is a cyclopropylmethyl group;和/或，当R4a、R4b、R6、R6e、R7a、R7b、R7c、R7d、R7e、R7f、R7h、R8或R8e为C2-8杂烷基时，所述的C2-8杂烷基为CH3OCH2-；And/or when R 4a , R 4b , R 6 , R 6e , R 7a , R 7b , R 7c , R 7d , R 7e , R 7f , R 7h , R 8 or R 8e are C 2-8 heterocycloalkanes The C 2-8 heteroalkyl group is CH 3 OCH 2 -;和/或，当Z为5元亚杂芳基时，所述的5元亚杂芳基为And/or, when Z is a 5-membered heteroarylene, the 5-membered heteroarylene is
和/或，为
and / or,
for和/或，为
and / or,
for - 如权利要求1-3中至少一项所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，其特征在于，An antibody drug conjugate according to at least one of claims 1 to 3, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, which is pharmaceutically acceptable Salt or a prodrug thereof, characterized in that当Y为所述的时，Z为所述的
When Y is as described
When Z is the stated和/或，当Y为所述的时，p为1；And/or when Y is as stated
When p is 1;和/或，当Y为所述的时，m为0；And/or when Y is as stated
When m is 0;和/或，当Y为所述的时，n为1；And/or when Y is as stated
When n is 1;和/或，当Y为所述的时，E为所述的
And/or when Y is as stated
When E is the stated和/或，当Y为所述的时，u为0或1；And/or when Y is as stated
When u is 0 or 1;和/或，当Y为所述的时，q为2；And/or when Y is as stated
When q is 2;和/或，当Y为所述的时，y为2； And/or when Y is as stated
When y is 2;和/或，当Y为所述的时，s为0或1；And/or when Y is as stated
When s is 0 or 1;和/或，当Y为所述的时，G为
And/or when Y is as stated
When G is和/或，当Y为所述的时，Q为
And/or when Y is as stated
When Q is和/或，当Y为所述的Z
时，p不为0。And/or when Y is as stated
ZWhen p is not 0. - 如权利要求1-4中至少一项所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，其特征在于，所述的IB化合物中，为以下任一结构：The antibody drug conjugate according to at least one of claims 1 to 4, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, which is pharmaceutically acceptable Salt or a prodrug thereof, characterized in that, in the IB compound,
For any of the following structures:
其中，R4a、R4b、R5a和R5b的定义如权利要求1～4任一项所述。Wherein R 4a , R 4b , R 5a and R 5b are as defined in any one of claims 1 to 4. - 如权利要求5所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，其特征在于，所述的为以下任一结构：The antibody drug conjugate according to claim 5, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, a pharmaceutically acceptable salt thereof or a prodrug thereof Characterized by
For any of the following structures:
- 如权利要求1-5中至少一项所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，其特征在于，所述的为以下任一结构： The antibody drug conjugate according to at least one of claims 1 to 5, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, which is pharmaceutically acceptable Salt or a prodrug thereof, characterized in that
For any of the following structures:
其中，R4a、R4b、R5a、R5b和R6的定义如权利要求1～5中至少一项所述。Wherein R 4a , R 4b , R 5a , R 5b and R 6 are as defined in any one of claims 1 to 5. - 如权利要求7所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、其药学上可接受的盐或其前药，其特征在于，所述的为以下任一结构：The antibody drug conjugate according to claim 7, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, a pharmaceutically acceptable salt thereof or a prodrug thereof, characterized in that Lie in, said
For any of the following structures:
- 如权利要求1-8中至少一项所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，其特征在于，所述的为以下任一结构：The antibody drug conjugate according to at least one of claims 1 to 8, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, which is pharmaceutically acceptable Salt or a prodrug thereof, characterized in that
For any of the following structures:
- 如权利要求1-9中至少一项所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，其特征在于，所述的抗体药物偶联物为以下任一化合物，其中，所述的mAb为赫赛汀、抗TPBG抗体、抗CD70抗体或抗EGFRVIII抗体；The antibody drug conjugate according to at least one of claims 1 to 9, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, which is pharmaceutically acceptable a salt or a prodrug thereof, wherein the antibody drug conjugate is any one of the following compounds, wherein the mAb is Herceptin, an anti-TPBG antibody, an anti-CD70 antibody or an anti-EGFRVIII antibody;
- 一种如权利要求1～10中至少一项所述的抗体药物偶联物的制备方法，其特征在于，其包括以下步骤：有机溶剂中，在pH值为6-8的条件下，将IA化合物与单克隆抗体进行如下所示的偶联反应，得到所述的抗体药物偶联物，即可； A method for producing an antibody drug conjugate according to at least one of claims 1 to 10, which comprises the step of: IA in an organic solvent at a pH of 6-8 The compound and the monoclonal antibody are subjected to a coupling reaction as shown below to obtain the antibody drug conjugate;
其中，k、R1、R2、R3、X、Y、Z、R6、p、m、E、n、u、R8、q、y、G、s、Q和mAb的定义如权利要求1～10中至少一项所述；Wherein k, R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q, y, G, s, Q and mAb are as defined Claiming at least one of 1 to 10;Q1为t1、t3和t4的定义如权利要求1所述。Q 1 is
The definitions of t 1 , t 3 and t 4 are as set forth in claim 1. - 一种IA化合物， An IA compound,
其中，R1、R2、R3、X、Y、Z、R6、p、m、E、n、u、R8、q、y、G和s的定义如权利要求1～10中至少一项所述；Q1的定义如权利要求11所述。Wherein R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q, y, G and s are as defined in claims 1 to 10 One of the descriptions; Q 1 is as defined in claim 11. - 如权利要求12所述的IA化合物，其特征在于，其选自以下任一化合物：The IA compound according to claim 12, which is selected from any one of the following compounds:
- 一种IC-1、IC-2或IC-3化合物，An IC-1, IC-2 or IC-3 compound,
其中，R1、R2、R3、X、Y、Z、R6、p、m、E、n、u、R8、q和y的定义如权利要求1～10中至少一项所述，R9为Wherein R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q and y are as defined in at least one of claims 1 to 10 , R 9 is
- 如权利要求14所述的IC-1、IC-2或IC-3化合物，其特征在于，The IC-1, IC-2 or IC-3 compound according to claim 14, wherein所述的IC-1化合物为以下任一结构：The IC-1 compound is of any of the following structures:
和/或，所述的IC-2化合物为以下结构：And/or, the IC-2 compound has the following structure:
和/或，所述的IC-3化合物为以下任一结构：And/or, the IC-3 compound is of any of the following structures:
- 一种如权利要求1～10中至少一项所述的抗体药物偶联物、如权利要求12或13所述的IA化合物、或如权利要求14或15所述的IC-1、IC-2或IC-3化合物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药在制备用于治疗和/或预防癌症的药物中的应用；所述的癌症优选乳腺癌、淋巴癌、肺癌、肝癌、结肠癌、头颈癌、膀胱癌、肾癌、食道癌、胆囊癌、卵巢癌、胰腺癌、胃癌、宫颈 癌、甲状腺癌、***癌、皮肤癌包括鳞状细胞癌；白细胞过多症、急性淋巴细胞性白血病、急性成淋巴细胞性白血病、B细胞淋巴瘤、T细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、毛细胞淋巴瘤、伯基特淋巴瘤、急性和慢性髓细胞性白血病、骨髓增生异常综合征、前髓细胞白血病、纤维肉瘤、横纹肌肉瘤、星形细胞瘤、神经目细胞瘤、胶质瘤、神经鞘瘤、黑色素瘤、***瘤、畸胎癌、骨肉瘤、着色性干皮病、角质黄色瘤、甲状腺滤泡癌和卡波西肉瘤；所述的癌症的肿瘤细胞优选Her2阳性的人BT474乳腺肿瘤细胞、Her2低表达的人MCF-7乳腺肿瘤细胞、或在MCF-7外转Her2的人乳腺肿瘤MCF7-Her2稳转细胞株。The antibody drug conjugate according to at least one of claims 1 to 10, the IA compound according to claim 12 or 13, or the IC-1 or IC-2 according to claim 14 or 15. Or an IC-3 compound, a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, an isotope compound, a pharmaceutically acceptable salt thereof or a prodrug thereof, for use in the treatment and/or Or a drug for preventing cancer; the cancer is preferably breast cancer, lymphoma, lung cancer, liver cancer, colon cancer, head and neck cancer, bladder cancer, kidney cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, Cervical Cancer, thyroid cancer, prostate cancer, skin cancer including squamous cell carcinoma; leukopenia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, hairy cell lymphoma, Burkitt's lymphoma, acute and chronic myeloid leukemia, myelodysplastic syndrome, promyelocytic leukemia, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuron Cell tumor, glioma, schwannomas, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratinous xanthoma, thyroid follicular carcinoma and Kaposi's sarcoma; said cancer The tumor cells are preferably Her2-positive human BT474 breast tumor cells, Her2 low-expressing human MCF-7 breast tumor cells, or human breast tumor MCF7-Her2 stable cell lines transfected with Her2 in MCF-7.
- 一种药物组合物，其特征在于，包含如权利要求1～10中至少一项所述的抗体药物偶联物、如权利要求12或13所述的IA化合物、或如权利要求14或15所述的IC-1、IC-2或IC-3化合物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、同位素化合物、其药学上可接受的盐或其前药，以及一种或多种药学上可接受的辅料。A pharmaceutical composition comprising the antibody drug conjugate according to at least one of claims 1 to 10, the IA compound according to claim 12 or 13, or the method according to claim 14 or 15. Compounds of the formula IC-1, IC-2 or IC-3, tautomers, optical isomers, hydrates, solvates, polymorphs, isotopic compounds thereof, pharmaceutically acceptable salts thereof or Prodrug, and one or more pharmaceutically acceptable excipients.
- 一种如式IB所示的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、其药学上可接受的盐或其前药，An antibody drug conjugate of the formula IB, a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, a pharmaceutically acceptable salt thereof or a prodrug thereof,
其中，mAb为单克隆抗体片段；Wherein, the mAb is a monoclonal antibody fragment;0<k≤8；0<k≤8;R1为氢、羟基、C1-6烷氧基、C1-6烷基、-NRaRb或-C(O)NRcRd；Ra、Rb、Rc和Rd各自独立地为氢、C1-6烷基或C6-10芳基；R 1 is hydrogen, hydroxy, C 1-6 alkoxy, C 1-6 alkyl, -NR a R b or -C(O)NR c R d ; each of R a , R b , R c and R d Independently hydrogen, C 1-6 alkyl or C 6-10 aryl;R2和R3各自独立地为C1-6烷基、C1-6烷氧基或C1-6烷硫基； R 2 and R 3 are each independently a C 1-6 alkyl group, a C 1-6 alkoxy group or a C 1-6 alkylthio group;X为X is
Y为R4a和R4b各自独立地为氢、C1～4烷基、C3～6环烷基取代的C1～4烷基、C3～6环烷基、C2-8杂烷基、或-(OCH2CH2)j1OH；j1为1、2、3、4、5、6、7或8；Y is
R 4a and R 4b are each independently hydrogen, C 1-4 alkyl, C 3-6 cycloalkyl substituted C 1-4 alkyl, C 3-6 cycloalkyl, C 2-8 heteroalkyl, Or -(OCH 2 CH 2 ) j1 OH; j1 is 1, 2, 3, 4, 5, 6, 7, or 8;Z为或4-6元亚杂芳基；R5a和R5b各自独立地为氢或C1～4烷基；Z is
Or a 4-6 membered heteroarylene; each of R 5a and R 5b is independently hydrogen or C 1-4 alkyl;或者，为
or,
forR6为氢、C1～4烷基、C2-8杂烷基、或-(OCH2CH2)j2OH；j2为1、2、3、4、5、6、7或8；R 6 is hydrogen, C 1-4 alkyl, C 2-8 heteroalkyl, or -(OCH 2 CH 2 ) j2 OH; j2 is 1, 2, 3, 4, 5, 6, 7, or 8;p为0、1、2、3、4、5、6、7、8、9或10；且当Z为时，p不为0；p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and when Z is
When p is not 0;m为0或1；m is 0 or 1;E为R7a和R7b各自独立地为氢、C1～4烷基、C3～6环烷基取代的C1～4烷基、C3～6环烷基、C2-8杂烷基、或-(OCH2CH2)j3OH；j3为1、2、3、4、5、6、7或8；E is
R 7a and R 7b are each independently hydrogen, C 1-4 alkyl, C 3-6 cycloalkyl substituted C 1-4 alkyl, C 3-6 cycloalkyl, C 2-8 heteroalkyl, Or -(OCH 2 CH 2 ) j3 OH; j3 is 1, 2, 3, 4, 5, 6, 7, or 8;n为0、1、2、3、4、5、6、7、8、9或10；当有多个E时，E相同或不同；n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; when there are multiple E, E is the same or different;u为0或1；u is 0 or 1;R8为氢、C1～4烷基、C3～6环烷基取代的C1～4烷基、C3～6环烷基、C2-8杂烷基、或-(OCH2CH2)j4OH；j4为1、2、3、4、5、6、7或8；R 8 is hydrogen, C 1-4 alkyl, C 3-6 cycloalkyl substituted C 1-4 alkyl, C 3-6 cycloalkyl, C 2-8 heteroalkyl, or -(OCH 2 CH 2 ) j4 OH; j4 is 1, 2, 3, 4, 5, 6, 7, or 8;q为0、1、2、3、4、5、6、7、8、9或10；当含有多个-C(R8)-时，-C(R8)-相同或不同；q is 0, 1, 2, 3, 4, 5, 6, 7, 8 , 9, or 10; when a plurality of -C(R 8 )- are contained, -C(R 8 )- is the same or different;y为0～24的整数； Y is an integer from 0 to 24;G为G is
s为0或1；s is 0 or 1;Q为
t1、t2、t3和t4各自独立地为0、1、2、3、4、5、6、7、8、9或10。Q is
t 1 , t 2 , t 3 and t 4 are each independently 0, 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9, or 10. - 如权利要求18所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、其药学上可接受的盐或其前药，其特征在于，The antibody drug conjugate according to claim 18, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, a pharmaceutically acceptable salt thereof or a prodrug thereof, characterized in that Yes,所述的单克隆抗体为赫赛汀、抗TPBG抗体、抗CD70抗体或抗EGFRVIII抗体；The monoclonal antibody is Herceptin, an anti-TPBG antibody, an anti-CD70 antibody or an anti-EGFRVIII antibody;和/或，0<k≤6；And / or, 0 < k ≤ 6;和/或，所述的为
And / or, said
for和/或，当所述的R1、R2或R3为C1-6烷氧基时，所述的C1-6烷氧基为甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基或叔丁氧基；And/or, when R 1 , R 2 or R 3 is a C 1-6 alkoxy group, the C 1-6 alkoxy group is a methoxy group, an ethoxy group, a n-propoxy group, Isopropoxy, n-butoxy, isobutoxy or tert-butoxy;和/或，当所述的R1、R2或R3为C1-6烷基时，所述的C1-6烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；And/or, when said R 1 , R 2 or R 3 is a C 1-6 alkyl group, said C 1-6 alkyl group is methyl, ethyl, n-propyl, isopropyl, or Butyl, isobutyl or tert-butyl;和/或，当R2或R3为C1-6烷硫基时，所述的C1-6烷硫基为甲硫基、乙硫基、正丙硫基、异丙硫基、正丁硫基、异丁硫基或叔丁硫基； And/or, when R 2 or R 3 is a C 1-6 alkylthio group, the C 1-6 alkylthio group is methylthio, ethylthio, n-propylthio, isopropylthio, or Butylthio, isobutylthio or tert-butylthio;和/或，当Ra、Rb、Rc或Rd为C1-6烷基时，所述的C1-6烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；And/or, when R a , R b , R c or R d is a C 1-6 alkyl group, the C 1-6 alkyl group is a methyl group, an ethyl group, a n-propyl group, an isopropyl group, or a positive Butyl, isobutyl or tert-butyl;和/或，当Ra、Rb、Rc或Rd为C6-10芳基时，所述的C6-10芳基为苯基；And/or, when R a , R b , R c or R d is a C 6-10 aryl group, the C 6-10 aryl group is a phenyl group;和/或，当R4a、R4b、R5a、R5b、R6、R7a、R7b或R8为C1～4烷基时，所述的C1～4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；And/or, when R 4a , R 4b , R 5a , R 5b , R 6 , R 7a , R 7b or R 8 is a C 1-4 alkyl group, the C 1-4 alkyl group is a methyl group, Ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl;和/或，当R4a、R4b、R7a、R7b或R8为C3～6环烷基取代的C1～4烷基时，所述的C1～4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；And/or, when R 4a , R 4b , R 7a , R 7b or R 8 is a C 3-6 cycloalkyl-substituted C 1-4 alkyl group, the C 1-4 alkyl group is a methyl group, Ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl;和/或，当R4a、R4b、R7a、R7b或R8为C3～6环烷基取代的C1～4烷基时，所述的C3～6环烷基为环丙基；And/or, when R 4a , R 4b , R 7a , R 7b or R 8 is a C 3-6 cycloalkyl substituted C 1-4 alkyl group, the C 3 -6 cycloalkyl group is cyclopropyl base;和/或，当R4a、R4b、R7a、R7b或R8为C3～6环烷基时，所述的C3～6环烷基为环丙基；And/or, when R 4a , R 4b , R 7a , R 7b or R 8 is a C 3-6 cycloalkyl group, the C 3-6 cycloalkyl group is a cyclopropyl group;和/或，当R4a、R4b、R6、R7a、R7b或R8为C2-8杂烷基时，所述的C2-8杂烷基中的杂原子为O、S或N中的一种或多种，杂原子的个数为1、2、3或4；And / or, when R 4a, R 4b, R 6 , R 7a, R 7b or R 8 is a C 2-8 heteroalkyl, a heteroatom of the heteroalkyl C 2-8 alkyl group is O, S Or one or more of N, the number of heteroatoms is 1, 2, 3 or 4;和/或，当Z为4-6元亚杂芳基时，所述的4-6元亚杂芳基中的杂原子为N、O和S中的一种或多种；杂原子个数为1个、2个或3个，所述的4-6元亚杂芳基为4元、5或元6元亚杂芳基；And/or, when Z is a 4-6 membered heteroarylene group, the hetero atom in the 4-6 membered heteroarylene group is one or more of N, O and S; the number of heteroatoms 1 , 2 or 3, the 4-6 membered heteroarylene is a 4 member, 5 or 6 membered heteroarylene;和/或，y为0、1、2、3、4、5或6；And / or, y is 0, 1, 2, 3, 4, 5 or 6;和/或，当Z为时，p不为0；And / or when Z is
When p is not 0;和/或，当所述的Z为时，p为0，m为1。And/or when the Z is
When p is 0, m is 1. - 如权利要求18或19所述的抗体偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、其药学上可接受的盐或其前药，其特征在于，The antibody conjugate according to claim 18 or 19, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, a pharmaceutically acceptable salt thereof or a prodrug thereof, Characterized by和/或，所述的为
And / or, said
for和/或，当R4a、R4b、R7a、R7b或R8为C3～6环烷基取代的C1～4烷基时，所述的C3～6环烷基取代的C1～4烷基为环丙甲基；And/or, when R 4a , R 4b , R 7a , R 7b or R 8 is a C 3-6 cycloalkyl substituted C 1-4 alkyl group, the C 3 -6 cycloalkyl substituted C 1 to 4 alkyl is cyclopropylmethyl;和/或，当R4a、R4b、R6、R7a、R7b或R8为C2-8杂烷基时，所述的C2-8杂烷基为CH3OCH2-； And/or, when R 4a , R 4b , R 6 , R 7a , R 7b or R 8 is a C 2-8 heteroalkyl group, the C 2-8 heteroalkyl group is CH 3 OCH 2 -;和/或，当Z为5元亚杂芳基时，所述的5元亚杂芳基为And/or, when Z is a 5-membered heteroarylene, the 5-membered heteroarylene is
和/或，为
and / or,
for和/或，为
and / or,
for - 如权利要求18-20中至少一项所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、其药学上可接受的盐或其前药，其特征在于，The antibody drug conjugate according to at least one of claims 18 to 20, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, a pharmaceutically acceptable salt thereof or a prodrug thereof, characterized in that当Y为所述的时，Z为所述的
When Y is as described
When Z is the stated和/或，当Y为所述的时，p为1；And/or when Y is as stated
When p is 1;和/或，当Y为所述的时，m为0；And/or when Y is as stated
When m is 0;和/或，当Y为所述的时，n为1；And/or when Y is as stated
When n is 1;和/或，当Y为所述的时，E为所述的
And/or when Y is as stated
When E is the stated和/或，当Y为所述的时，u为0或1；And/or when Y is as stated
When u is 0 or 1;和/或，当Y为所述的时，q为2；And/or when Y is as stated
When q is 2;和/或，当Y为所述的时，y为2；And/or when Y is as stated
When y is 2;和/或，当Y为所述的时，s为0或1； And/or when Y is as stated
When s is 0 or 1;和/或，当Y为所述的时，G优选
And/or when Y is as stated
When G is preferred和/或，当Y为所述的时，Q优选为
And/or when Y is as stated
When Q is preferred和/或，当Y为所述的Z
时，p不为0。And/or when Y is as stated
ZWhen p is not 0. - 如权利要求18-21中至少一项所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、其药学上可接受的盐或其前药，其特征在于，所述的IB化合物中，为以下任一结构：The antibody drug conjugate according to at least one of claims 18 to 21, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, a pharmaceutically acceptable salt thereof or a prodrug thereof, characterized in that, in the IB compound,
For any of the following structures:
其中，R4a、R4b、R5a和R5b的定义如权利要求1～4任一项所述。Wherein R 4a , R 4b , R 5a and R 5b are as defined in any one of claims 1 to 4. - 如权利要求22所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、其药学上可接受的盐或其前药，其特征在于，所述的为以下任一结构：The antibody drug conjugate according to claim 22, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, a pharmaceutically acceptable salt thereof or a prodrug thereof, characterized in that Lie in, said
For any of the following structures:
- 如权利要求18-21中至少一项所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、其药学上可接受的盐或其前药，其特征在于，所述的IB化合物中，所述的为以下任一结构：The antibody drug conjugate according to at least one of claims 18 to 21, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, a pharmaceutically acceptable salt thereof or a prodrug thereof, characterized in that said IB compound, said
For any of the following structures:
其中，R4a、R4b、R5a、R5b和R6的定义如权利要求1～4任一项所述。Wherein R 4a , R 4b , R 5a , R 5b and R 6 are as defined in any one of claims 1 to 4. - 如权利要求24所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、其药学上可接受的盐或其前药，其特征在于，所述的为以下任一结构：The antibody drug conjugate according to claim 24, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, a pharmaceutically acceptable salt thereof or a prodrug thereof, which is characterized Lie in, said
For any of the following structures:
- 如权利要求18-25中至少一项所述的抗体药物偶联物，其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、其药学上可接受的盐或其前药，其特征在于，所述的为以下任一结构：The antibody drug conjugate according to at least one of claims 18 to 25, which is a tautomer, an optical isomer, a hydrate, a solvate, a polymorph, a pharmaceutically acceptable salt thereof or a prodrug thereof, characterized in that
For any of the following structures:
- 一种如权利要求18～26中至少一项所述的抗体药物偶联物的制备方法，其特征在于，包括以下步骤：有机溶剂中，pH为6-8条件下，将IA化合物与所述的单克隆抗体进行如下所示的偶联反应，得到所述的式IB抗体药物偶联物，即可； A method for producing an antibody drug conjugate according to at least one of claims 18 to 26, comprising the steps of: IA compound and said organic solvent in a pH of 6-8 The monoclonal antibody is subjected to a coupling reaction as shown below to obtain the antibody conjugate of the antibody of the formula IB;
其中，k、R1、R2、R3、X、Y、Z、R6、p、m、E、n、u、R8、q、y、G、s、Q和mAb的定义如权利要求18～26中至少一项所述；Wherein k, R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q, y, G, s, Q and mAb are as defined Requiring at least one of 18 to 26;Q1为
t1、t2、t3和t4的定义如权利要求18所述。Q 1 is
The definitions of t 1 , t 2 , t 3 and t 4 are as set forth in claim 18. - 一种式IA化合物， a compound of formula IA,
其中，R1、R2、R3、X、Y、Z、R6、p、m、E、n、u、R8、q、y、G和s的定义如权利要求18～26任一项所述；Q1的定义如权利要求27所述。Wherein R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q, y, G and s are as defined in any one of claims 18 to 26 Said; the definition of Q 1 is as defined in claim 27. - 一种如式IC-1、IC-2或IC-3所示的化合物，a compound as shown by the formula IC-1, IC-2 or IC-3,
其中，R1、R2、R3、X、Y、Z、R6、p、m、E、n、u、R8、q和y的定义如权利要求18～26任一项所述，R9为Wherein R 1 , R 2 , R 3 , X, Y, Z, R 6 , p, m, E, n, u, R 8 , q and y are as defined in any one of claims 18 to 26, R 9 is
- 一种如权利要求18～26中至少一项所述的式IB抗体药物偶联物或如权利要求28所述的式IA化合物、其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、其药学上可接受的盐或其前药在制备用于治疗和/或预防癌症的药物中的应用；所述的癌症优选乳腺癌、淋巴癌、肺癌、肝癌、结肠癌、头颈癌、膀胱癌、肾癌、食道癌、胆囊癌、卵巢癌、胰腺癌、胃癌、***、甲状腺癌、***癌、皮肤癌包括鳞状细胞癌；白细胞过多症、急性淋巴细胞性白血病、急性成淋巴细胞性白血病、B细胞淋巴瘤、T细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、毛细胞淋巴瘤、伯基特淋巴瘤、急性和慢性髓细胞性白血病、骨髓增生异常综合征、前髓细胞白血病、纤维肉瘤、横纹肌肉瘤、星形细胞瘤、神经目细胞瘤、胶质瘤、神经鞘瘤、黑色素瘤、***瘤、畸胎癌、骨肉瘤、着色性干皮病、角质黄色瘤、甲状腺滤泡癌和卡波西肉瘤；所述的癌症的肿瘤细胞优选Her2阳性的人BT474乳腺肿瘤细胞、Her2低表达的人MCF-7乳腺肿瘤细胞、或在MCF-7外转Her2的人乳腺肿瘤MCF7-Her2稳转细胞株。An antibody conjugate of the formula IB according to at least one of claims 18 to 26 or a compound of the formula IA according to claim 28, a tautomer, an optical isomer, a hydrate thereof, a solvent Use of a compound, a polymorph, a pharmaceutically acceptable salt thereof or a prodrug thereof for the preparation of a medicament for the treatment and/or prevention of cancer; said cancer preferably breast cancer, lymphoma, lung cancer, liver cancer, colon Cancer, head and neck cancer, bladder cancer, kidney cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, skin cancer including squamous cell carcinoma; leukopenia, acute lymphocytes Leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, Burkitt's lymphoma, acute and chronic myeloid Leukemia, myelodysplastic syndrome, promyelocytic leukemia, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, glioma, schwannomas, melanoma, seminoma, teratocarcinoma, Osteosarcoma, xeroderma pigmentosum, keratinous yellow tumor, thyroid follicular carcinoma and Kaposi's sarcoma; the cancer cells of the cancer are preferably Her2 positive human BT474 breast tumor cells, Her2 low expression human MCF-7 breast tumors The cells, or human breast tumor MCF7-Her2 stable cell line transfected with Her2 in MCF-7.
- 一种药物组合物，其特征在于，包含如权利要求18～26中至少一项所述的式IB抗体药物偶联物、或如权利要求28所述的式IA化合物、其互变异构体、光学异构体、水合物、溶剂化物、多晶型物、其药学上可接受的盐或其前药，以及一种或多种药学上可接受的辅料。 A pharmaceutical composition comprising the antibody conjugate of the formula IB according to at least one of claims 18 to 26, or the compound of the formula IA according to claim 28, a tautomer thereof An optical isomer, hydrate, solvate, polymorph, a pharmaceutically acceptable salt thereof or a prodrug thereof, and one or more pharmaceutically acceptable excipients.
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| CN115043895A (en) * | 2022-07-15 | 2022-09-13 | 戊言医药科技（上海）有限公司 | PNU-159682 and preparation method of intermediate thereof |
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