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WO2022078260A1 - 一种喜树碱类衍生物及其配体-药物偶联物 - Google Patents

一种喜树碱类衍生物及其配体-药物偶联物 Download PDF

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Publication number
WO2022078260A1
WO2022078260A1 PCT/CN2021/122823 CN2021122823W WO2022078260A1 WO 2022078260 A1 WO2022078260 A1 WO 2022078260A1 CN 2021122823 W CN2021122823 W CN 2021122823W WO 2022078260 A1 WO2022078260 A1 WO 2022078260A1
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Prior art keywords
antibody
group
alkyl
aryl
cycloalkylalkyl
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PCT/CN2021/122823
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English (en)
French (fr)
Inventor
朱义
万维李
朱贵莉
于天姿
卓识
杨秀娟
Original Assignee
四川百利药业有限责任公司
成都百利多特生物药业有限责任公司
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Application filed by 四川百利药业有限责任公司, 成都百利多特生物药业有限责任公司 filed Critical 四川百利药业有限责任公司
Priority to IL302054A priority Critical patent/IL302054A/en
Priority to EP21879305.7A priority patent/EP4227310A4/en
Priority to AU2021359457A priority patent/AU2021359457A1/en
Priority to JP2023546378A priority patent/JP2023545581A/ja
Publication of WO2022078260A1 publication Critical patent/WO2022078260A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6863—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from stomach or intestines cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • the present invention relates to a camptothecin derivative and its ligand-drug conjugate.
  • ADCs Ligand-drug conjugates
  • ADCs as new targeted drugs, generally consist of three parts: antibodies or antibody-like ligands, small molecule drugs, and linkers that couple the ligands and drugs.
  • Antibody-drug conjugates utilize the specific recognition of antigens by antibodies to transport drug molecules to the vicinity of target cells and effectively release drug molecules to achieve therapeutic purposes.
  • FDA U.S. Food and Drug Administration
  • Adecteis TM developed by Seattle Genetics for the treatment of Hodgkin lymphoma and relapsed anaplastic large cell lymphoma (ALCL), and its clinical application has been proven. safety and efficacy of these drugs.
  • Camptothecins as small molecule compounds with anti-tumor properties, are known to exhibit anti-tumor effects by inhibiting DNA topoisomerase I, including irinotecan, ixitecan, SN38, etc.
  • Many camptothecin drugs have been widely used in clinic, the main indications are bone cancer, prostate cancer, breast cancer, pancreatic cancer and so on.
  • ixitecan does not require activation by the use of enzymes.
  • ixitecan has stronger topoisomerase I inhibitory activity, and can target a variety of cancer cells in vitro. Has stronger cytocidal activity. In particular, the effect is also shown on cancer cells that show resistance to SN-38 and the like by the expression of P-glycoprotein. Ixinotecan has not been successfully marketed as a single chemotherapeutic drug, presumably associated with its higher cell viability, resulting in a narrow therapeutic window.
  • ADC antibody-drug conjugates
  • ADC drugs in order to ensure safety, the DAR value of the drug will be reduced, resulting in a reduction in the therapeutic effect.
  • the solubility of the drug is one of the important factors affecting the DAR value, and it is also the main factor affecting the bystander effect of ADC drugs.
  • camptothecins have been widely used in clinical practice, their poor water solubility is still an urgent problem to be solved.
  • improving the water solubility of the drug can improve the physical properties of the drug and change its metabolism in the body, thereby improving the efficacy of the drug, enhancing the therapeutic effect, reducing the dosage of the drug and reducing the cost.
  • Poor water solubility can lead to increased aggregation of ADCs, making them easier to be endocytosed by normal cells, resulting in toxic side effects or easier to be metabolized and shortened half-life.
  • the amino chiral isomer compound of ixitecan has significantly better water solubility than ixitecan, and the derivatives synthesized according to it also have better water solubility, and can be synthesized To prepare better anti-tumor camptothecin ADC drugs, so that they have higher safety and efficacy, and better meet clinical needs.
  • the present invention provides a camptothecin derivative represented by general formula D or its tautomer, meso, racemate, enantiomer, diastereomer or its in the form of a mixture, or a pharmaceutically acceptable salt or solvate thereof;
  • R is selected from hydrogen atom, deuterium atom, halogen, alkyl, substituted alkyl, deuterated alkyl, cycloalkylalkyl, alkoxyalkyl, aryl, substituted aryl or heteroaryl;
  • R 1 is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a substituted alkyl group, a deuterated alkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a carboxyl group, a heterocyclic group, an aryl group, a substituted aryl group or a heterocyclic group Aryl;
  • R 2 is selected from hydrogen atom, deuterium atom, halogen, alkyl group, substituted alkyl group, deuterated alkyl group, cycloalkylalkyl group, alkoxyalkyl group, carboxyl group, heterocyclic group, aryl group, substituted aryl group or heterocyclic group Aryl;
  • X is selected from -C(O) -CRaRb- ( CR3R4 ) m - O-, -C(O) -CRaRb- ( CR3R4 ) m - NH- or -C( O)-CR a R b -(CR 3 R 4 ) m -S-;
  • Ra is selected from hydrogen atom, deuterium atom, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl;
  • R b is selected from hydrogen atom, deuterium atom, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl;
  • R a , R b and the carbon atoms to which they are attached constitute C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclyl;
  • R 3 and R 4 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, and a hydroxyalkyl group , cycloalkyl or heterocyclyl;
  • R 3 , R 4 and the carbon atoms to which they are attached constitute C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclyl;
  • n is selected from an integer of 0-4.
  • R 1 is selected from C 1-3 alkyl.
  • R 2 is selected from C 1-3 alkyl groups or fluorine atoms.
  • camptothecin derivative has the structure shown in the following formula D 1 :
  • R is selected from hydrogen atom, deuterium atom, halogen, alkyl, substituted alkyl, deuterated alkyl, cycloalkylalkyl, alkoxyalkyl, aryl, substituted aryl or heteroaryl;
  • X is selected from -C(O) -CRaRb- ( CR3R4 ) m - O-, -C(O) -CRaRb- ( CR3R4 ) m - NH- or -C( O)-CR a R b -(CR 3 R 4 ) m -S-;
  • Ra is selected from hydrogen atom, deuterium atom, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl;
  • R b is selected from hydrogen atom, deuterium atom, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl;
  • R a , R b and the carbon atoms to which they are attached constitute C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclyl;
  • R 3 and R 4 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, and a hydroxyalkyl group , cycloalkyl or heterocyclyl;
  • R 3 , R 4 and the carbon atoms to which they are attached constitute C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclyl;
  • n is selected from an integer of 0-4.
  • the R is selected from C 1-3 alkyl.
  • the X is non-limitingly selected from the following structures:
  • the left wavy line is connected with the camptothecin derivative part, and the right wavy line is connected with the connecting unit.
  • the compound is selected from the structures shown below without limitation:
  • R is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a substituted alkyl group, a deuterated alkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, an aryl group, a substituted aryl group or a heteroaryl group.
  • the compound is non-limitingly selected from the structures shown below:
  • the present invention also provides a linker-drug conjugate comprising a camptothecin derivative or a pharmaceutically acceptable salt or solvate thereof, or a tautomer, meso, racem isomers, enantiomers, diastereomers or mixtures thereof:
  • R is selected from hydrogen atom, deuterium atom, halogen, alkyl, substituted alkyl, deuterated alkyl, cycloalkylalkyl, alkoxyalkyl, aryl, substituted aryl or heteroaryl;
  • R 1 is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a substituted alkyl group, a deuterated alkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a carboxyl group, a heterocyclic group, an aryl group, a substituted aryl group or a heterocyclic group Aryl;
  • R 2 is selected from hydrogen atom, deuterium atom, halogen, alkyl group, substituted alkyl group, deuterated alkyl group, cycloalkylalkyl group, alkoxyalkyl group, carboxyl group, heterocyclic group, aryl group, substituted aryl group or heterocyclic group Aryl;
  • X is selected from -C(O) -CRaRb- ( CR3R4 ) m - O-, -C(O) -CRaRb- ( CR3R4 ) m - NH- or -C( O)-CR a R b -(CR 3 R 4 ) m -S-;
  • Ra is selected from hydrogen atom, deuterium atom, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl;
  • R b is selected from hydrogen atom, deuterium atom, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl;
  • R a , R b and the carbon atoms to which they are attached constitute C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclyl;
  • R 3 and R 4 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, and a hydroxyalkyl group , cycloalkyl or heterocyclyl;
  • R 3 , R 4 and the carbon atoms to which they are attached constitute C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclyl;
  • n is selected from an integer from 0 to 4.
  • L is -L 1 -L 2 -L 3 -L 4 -.
  • -L- is -L 1 -L 2 -L 3 -L 4 -, wherein the L 1 end is connected to the ligand Ab, and the L 4 end is connected to X.
  • L 1 is non-limitingly selected from:
  • L 2 is selected from: -NC(R 5 R 6 )C(O), -NR 7 (CH 2 ) o C(O)-, -NR 7 (CH 2 CH 2 O) o CH 2 C( O)-, -S(CH 2 ) p C(O)- or a chemical bond, wherein o is selected from an integer of 0-20; p is selected from an integer of 0-20;
  • R 5 and R 6 are the same or different, and are independently selected from hydrogen atoms, deuterium atoms, alkyl groups, substituted alkyl groups, deuterated alkyl groups, heteroalkyl groups, carboxyl groups, amino groups, and substituted amino groups;
  • R is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a substituted alkyl group, a deuterated alkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, an aryl group, a substituted aryl group or a heteroaryl group;
  • L 1 and L 2 share N atoms.
  • L is selected from peptide residues consisting of amino acids, wherein the optional amino acids are further selected from deuterium atoms, halogen, hydroxyl, cyano, amino, nitro, carboxyl, alkyl, substituted alkyl, alkoxy and substituted by one or more substituents in cycloalkyl or substituted cycloalkyl; preferably by one, two or more selected from phenylalanine (F), glycine (G), valine (V), Peptide residues formed from amino acids of lysine (K), citrulline, serine (S), glutamic acid (E) or aspartic acid (D).
  • phenylalanine F
  • G glycine
  • V valine
  • citrulline serine
  • S serine
  • E glutamic acid
  • D aspartic acid
  • L 4 is selected from: -NR 8 (CR 9 R 10 ) q -, -C(O)NR 8 -, -C(O)NR 8 (CH 2 ) q - or chemical bond, q is selected from 0- an integer of 6;
  • R 8 , R 9 and R 10 are the same or different, and are each independently selected from hydrogen atom, deuterium atom, halogen, alkyl, substituted alkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxy alkyl, heterocyclyl, aryl, substituted aryl or heteroaryl.
  • the linking unit -L- is non-limitingly selected from the following structures;
  • the left wavy line is connected with the ligand part
  • the right wavy line is connected with X.
  • linker-drug conjugate is non-limitingly selected from the following structures:
  • the 1-position carbon atom has absolute chirality in either the R or S configuration.
  • the present invention also provides a ligand-drug conjugate comprising a linker-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the ligand-drug conjugate comprising formula I structure:
  • R is selected from hydrogen atom, deuterium atom, halogen, alkyl, substituted alkyl, deuterated alkyl, cycloalkylalkyl, alkoxyalkyl, aryl, substituted aryl or heteroaryl;
  • R 1 is selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a substituted alkyl group, a deuterated alkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a carboxyl group, a heterocyclic group, an aryl group, a substituted aryl group or a heterocyclic group Aryl;
  • R 2 is selected from hydrogen atom, deuterium atom, halogen, alkyl group, substituted alkyl group, deuterated alkyl group, cycloalkylalkyl group, alkoxyalkyl group, carboxyl group, heterocyclic group, aryl group, substituted aryl group or heterocyclic group Aryl;
  • X is selected from -C(O) -CRaRb- ( CR3R4 ) m - O-, -C(O) -CRaRb- ( CR3R4 ) m - NH- or -C( O)-CR a R b -(CR 3 R 4 ) m -S-;
  • Ra is selected from hydrogen atom, deuterium atom, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl;
  • R b is selected from hydrogen atom, deuterium atom, halogen, alkyl, deuterated alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, substituted aryl or Heteroaryl;
  • R a , R b and the carbon atoms to which they are attached constitute C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclyl;
  • R 3 and R 4 are the same or different, and are each independently a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, and a hydroxyalkyl group , cycloalkyl or heterocyclyl;
  • R 3 , R 4 and the carbon atoms to which they are attached constitute C 3-6 cycloalkyl, cycloalkylalkyl or heterocyclyl;
  • Ab is a ligand unit selected from antibodies, antibody fragments, targeting proteins, Fc-fusion proteins, etc.;
  • L is the linking unit with Ab
  • X is the modification unit of the drug part
  • n is selected from an integer or a decimal of 1-20.
  • the ligand is selected from the group consisting of murine antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies and multispecific antibodies.
  • the antibody or its antigen-binding fragment is selected from, without limitation: anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, Anti-ETBR Antibody, Anti-MSLN Antibody, Anti-TIM-1 Antibody, Anti-LRRC15 Antibody, Anti-LIV-1 Antibody, Anti-CanAg/AFP Antibody, Anti-cladin 18.2 Antibody, Anti-Mesothelin Antibody, Anti-HER2(ErbB2) Antibody, Anti-EGFR Antibody, Anti-c-MET Antibody, Anti-SLITRK6 Antibody, Anti-KIT/CD117 Antibody, Anti-STEAP1 Antibody, Anti-SLAMF7/CS1 Antibody, Anti-NaPi2B/SLC34A2 Antibody, Anti-GPNMB Antibody, Anti-HER3(ErbB3) Antibody, Anti-MUC1/CD227 Antibody, Antibody
  • the ligand-drug conjugate is selected from the following structures without limitation:
  • Ab is a ligand unit; n is selected from the integer or decimal of 1-20;
  • the 1-position carbon atom has absolute chirality in either the R or S configuration.
  • the present invention also provides a method for preparing linker-drug conjugates or tautomers, mesomers, racemates, enantiomers, diastereomers or mixtures thereof, or The method for its pharmaceutically acceptable salt or solvate, comprising the steps of:
  • L 2 , L 3 , R, R 8 , R 9 , R 10 , q and X are as described in the general formula LXD.
  • the present invention further provides a preparation of ligand-drug conjugates or tautomers, mesomers, racemates, enantiomers, diastereomers or mixtures thereof, or
  • the method for its pharmaceutically acceptable salt or solvate comprising the steps of:
  • the pharmaceutically acceptable salts include compounds formed with acidic functional groups in the structural formula Sodium, potassium, calcium or magnesium salts, etc.; or acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, Chloride, Bromide, Iodide, Sulfate, Bisulfate, Phosphate, Lactate, Oleate, Ascorbate, Salicylate, Formate, Glutamate, Mesylate, Ethyl Sulfonate, benzenesulfonate or p-toluenesulfonate.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the camptothecin derivative, linker-drug conjugate or ligand-drug conjugate according to any one of claims 1-23 or a tautomer, meso, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable acceptable carrier, diluent or excipient.
  • the present invention provides a camptothecin derivative, linker-drug conjugate or ligand-drug conjugate or its tautomer, meso, racemate, enantiomer Forms of isomers, diastereomers or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof, and pharmaceutically acceptable carriers, diluents or excipients, in the preparation of medicaments for the treatment or prevention of tumors use in.
  • the tumor is breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophagus cancer, lung cancer, Colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia, etc. Solid tumors or hematological tumors.
  • ligand is a macromolecular compound capable of recognizing and binding to an antigen or receptor associated with a target cell.
  • the role of the ligand is to present the drug to the target cell population to which the ligand binds, including but not limited to protein hormones, lectins, growth factors, antibodies or other molecules that can bind to cells.
  • the ligand is represented as Ab, and the ligand can form a linking bond with the connecting unit through the heteroatom on the ligand, preferably an antibody or an antigen-binding fragment thereof, the antibody is selected from chimeric antibodies, human-derived antibody, fully human antibody or murine antibody; preferably a monoclonal antibody.
  • Ligand units are targeting agents that specifically bind to the target moiety.
  • the ligands are capable of specifically binding to cellular components or to cellular components or to other target molecules of interest.
  • the target moiety or target is usually on the cell surface.
  • the function of the Ligand unit is to deliver the Drug unit to a specific target cell population with which the Ligand unit interacts.
  • Ligands include, but are not limited to, proteins, polypeptides and peptides, as well as non-proteins such as sugars.
  • Suitable Ligand units include, for example, antibodies, such as full-length (intact) antibodies and antigen-binding fragments thereof.
  • the Ligand unit is a non-antibody targeting agent
  • it may be a peptide or polypeptide, or a non-proteinaceous molecule.
  • targeting agents include interferons, lymphokines, hormones, growth and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell binding molecule or substance.
  • the linker is covalently attached to the sulfur atom of the ligand.
  • the sulfur atom is that of a cysteine residue, which forms an interchain disulfide bond of the antibody.
  • the sulfur atom is one that has been introduced into a cysteine residue of the Ligand unit, which forms an interchain disulfide bond of the antibody.
  • the sulfur atom is one that has been introduced into a cysteine residue of the Ligand unit (eg, by site-directed mutagenesis or chemical reaction).
  • the sulfur atom to which the linker binds is selected from cysteine residues that form interchain disulfide bonds of the antibody or frontal cysteine residues that have been introduced into the Ligand unit (eg, by site-directed mutagenesis or chemical reaction).
  • the EU index in Kabat Kabat (Kabat E.A et al., (1991) Sequences of proteins of Immunological Interest, Fifth Edition, NIH publication 91-3242) numbering system.
  • an "antibody” or “antibody unit” is within its scope to include any portion of an antibody structure. This unit may bind, reactively associate, or complex a receptor, antigen, or other receptor unit possessed by the targeted cell population.
  • An antibody can be any protein or protein-like molecule that can bind, complex, or otherwise react with a portion of the cell population to be treated or bioengineered.
  • the antibody constituting the antibody-drug conjugate in the present invention preferably maintains the antigen-binding ability of its original wild state. Therefore, the antibody of the present invention can specifically bind to the antigen.
  • Antigens involved include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, regulators of cell survival, regulators of cell proliferation, molecules associated with tissue growth and differentiation (as known or predicted) functional), lymphokines, cytokines, molecules involved in the regulation of the cell cycle, molecules involved in angiogenesis, and molecules involved in angiogenesis (as known or predicted to be functional).
  • TAAs tumor-associated antigens
  • cell surface receptor proteins and other cell surface molecules regulators of cell survival, regulators of cell proliferation, molecules associated with tissue growth and differentiation (as known or predicted) functional)
  • lymphokines cytokines
  • molecules involved in the regulation of the cell cycle molecules involved in angiogenesis
  • angiogenesis as known or predicted to be functional
  • the tumor-associated factor can be a cluster differentiation factor (eg, CD protein).
  • Antibodies for use in antibody drug conjugates include, but are not limited to, antibodies directed against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the art and can be prepared by methods and information well known in the art for preparing antibodies.
  • tumor-associated antigens are well known in the art and can be prepared by methods and information well known in the art for preparing antibodies.
  • To develop effective cellular-level targets for cancer diagnosis and therapy researchers seek to find transmembrane or other tumor-associated polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells with little or no expression on the surface of one or more non-cancer cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancer cells. Identifying such tumor-associated factors could greatly improve the specific targeting properties of antibody-based cancer therapy.
  • tumor-associated antigens For convenience, antigen-related information that is well known in the industry is indicated below, including name, other names, and GenBank accession numbers. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in public databases such as Genbank. Antibodies targeting corresponding tumor-associated antigens include all amino acid sequence variants and isotypes that are at least 70%, 80%, 85%, 90%, or 95% homologous to sequences identified in references, or have The tumor-associated antigen sequences in the literature have completely identical biological properties and characteristics.
  • inhibitor refers to reducing by a detectable amount, or preventing completely.
  • cancer refers to a physiological condition or disease characterized by unregulated cell growth.
  • Tumor includes cancer cells.
  • autoimmune disease is a disease or disorder that results from targeting an individual's own tissues or proteins.
  • cytotoxic drug refers to a cytotoxic drug, denoted D, a chemical molecule capable of strongly disrupting normal growth within tumor cells.
  • D cytotoxic drug
  • cytotoxic drugs can kill tumor cells at a high enough concentration, but due to the lack of specificity, when killing tumor cells, they can also lead to apoptosis of normal cells, resulting in serious side effects.
  • toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (eg At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 ) , P 32 and Lu radioisotopes), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolysins, preferably toxic drugs.
  • radioisotopes eg At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212
  • P 32 and Lu radioisotopes eg At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212
  • P 32 and Lu radioisotopes eg At 211 , I 131 , I 125 ,
  • linker or “linker fragment” or “linker unit” refers to a chemical structural fragment or bond that is linked to a ligand at one end and a drug at the other end, and can also be linked to a drug after other linkers are attached.
  • Linkers including stretchers, spacers and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1.
  • the linker can be a "cleavable linker" that facilitates the release of the drug in the cell.
  • acid-labile linkers eg, hydrazones
  • protease-sensitive linkers eg, peptidase-sensitive linkers
  • photolabile linkers eg, dimethyl linkers, or disulfide-containing linkers
  • linkers or “linkers of antibody drug conjugates” can be divided into two categories: non-cleavable linkers and cleavable linkers.
  • the drug release mechanism is: after the conjugate binds to the antigen and is endocytosed by the cell, the antibody is enzymatically hydrolyzed in the lysosome to release the small molecule drug, which is linked to the It is an active molecule composed of amino acid residues of antibodies.
  • the resulting changes in the molecular structure of the drug do not reduce its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into adjacent cells.
  • active drugs cannot kill adjacent tumor cells that do not express the target antigen (antigen-negative cells) (bystander effect) (Ducry et al., 2010, Bioconjugate Chem. 21:5-13).
  • ligand-drug conjugate refers to an antibody linked to a biologically active drug through a stable linking unit.
  • ligand-drug conjugate is preferably antibody-drug conjugate (antibody drug conjugate, ADC), which refers to linking a monoclonal antibody or antibody fragment with a biologically active toxic drug through a stable linking unit .
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably 1 to 10 carbon atoms atomic alkyl groups, most preferably those containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -Methylhexyl, 3-methylhexyl, 4-methylhe
  • lower alkyl groups containing 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl base, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-Methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylpropyl butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl base, 2,3-dimethylbutyl, etc.
  • Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from alkanes group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkane Oxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.
  • substituents may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from alkanes group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclo
  • R', R" and R"' each independently refers to hydrogen, unsubstituted C 1-8 alkyl, unsubstituted aryl, aryl substituted by 1-3 halogens, unsubstituted C 1-8 alkyl, C 1- 8 alkoxy or C 1-8 thioalkoxy, or unsubstituted aryl-C 1-4 alkyl.
  • R' and R" When R' and R" are attached to the same nitrogen atom, they may form together with the nitrogen atom 3-, 4-, 5-, 6- or 7-membered rings.
  • -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein alkyl is as defined above.
  • alkylene refers to a saturated straight or branched chain aliphatic hydrocarbon group having 2 residues derived by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkane, which are A straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms.
  • Non-limiting examples of alkylene groups include, but are not limited to, methylene ( -CH2- , 1,1-ethylene (-CH( CH3 )-), 1,2-ethylene (-CH2CH ) 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene ( -CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -) and 1,5-butylene (-CH 2 CH 2 CH 2 CH 2 CH 2 -), etc.
  • the alkylene groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, preferably independently optionally selected from alkyl, alkenyl, alkyne group, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy substituted with one or more of the substituents among the radicals, cycloalkylthio, heterocycloalkylthio and oxo.
  • alkoxy refers to -O-(alkyl) and -O-(cycloalkyl), wherein alkyl or cycloalkyl is as defined above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • Alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms carbon atoms, most preferably 3 to 8 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene
  • Polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2) heteroatoms excluding ring moieties of -O-O-, -O-S- or -S-S-, the remaining ring atoms being carbon.
  • it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably the cycloalkyl ring contains 3 to 10 ring atoms.
  • Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.
  • Polycyclic heterocyclyls include spiro, fused and bridged heterocyclyls.
  • spiroheterocyclyl refers to a 5- to 20-membered monocyclic polycyclic heterocyclic group sharing one atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen or S(O )m (where m is an integer from 0 to 2) heteroatoms and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings have a fully conjugated electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan.
  • spiroheterocyclyls are classified into mono-spiroheterocyclyl, bis-spiroheterocyclyl or poly-spiroheterocyclyl, preferably mono-spiroheterocyclyl and bis-spiroheterocyclyl. More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiroheterocyclyl group.
  • cycloalkylalkyl refers to an alkyl group substituted with one or more cycloalkyl groups, preferably one cycloalkyl group, wherein alkyl is as defined above, wherein cycloalkyl is as defined above.
  • haloalkyl refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.
  • deuterated alkyl refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.
  • hydroxy refers to the -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH2 .
  • nitro refers to -NO2 .
  • amido refers to -C(O)N(alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.
  • carboxylate refers to -C(O)O(alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.
  • the present invention also includes various deuterated forms. Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can refer to relevant literature to synthesize deuterated compounds. In preparing the deuterated compounds, commercially available deuterated starting materials can be used, or they can be synthesized using conventional techniques using deuterated reagents, non-limiting examples of deuterated reagents include: deuterated methanol, deuterated water, deuterated Borane, trideuteroborane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane, etc.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
  • the amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , alpha chains and epsilon chains.
  • IgG can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into ⁇ chains or ⁇ chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • the antibodies of the present invention are preferably specific antibodies against cell surface antigens on target cells, non-limiting examples are the following antibodies: anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody , Anti-ENPP3 Antibody, Anti-TDGF1 Antibody, Anti-ETBR Antibody, Anti-MSLN Antibody, Anti-TIM-1 Antibody, Anti-LRRC15 Antibody, Anti-LIV-1 Antibody, Anti-CanAg/AFP Antibody, Anti-cladin 18.2 Antibody, Anti-Mesothelin Antibody, Anti-HER2 (ErbB2) Antibody, Anti-EGFR Antibody, Anti-c-MET Antibody, Anti-SLITRK6 Antibody, Anti-KIT/CD117 Antibody, Anti-STEAP1 Antibody, Anti-SLAMF7/CS1 Antibody, Anti-NaPi2B/SLC34A2 Antibody, Anti-GPNMB Antibody, Anti-HER3(ErbB3) Antibody,
  • solvate refers to the ligand-drug conjugates of the invention forming pharmaceutically acceptable solvates with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile , isopropanol, DMSO, ethyl acetate.
  • drug load refers to the average amount of cytotoxic drug loaded on each antibody in the molecule of formula I, and can also be expressed as the ratio of the amount of drug to the amount of antibody, and the drug load can range from each antibody (Ab) linked. 0-12, preferably 1-10 cytotoxic drugs (D).
  • the drug load is expressed as n, and an exemplary value may be the mean value of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the average amount of drug product per ADC molecule after the coupling reaction can be characterized by conventional methods such as UV/Vis spectroscopy, mass spectrometry, ELISA assay and HPLC.
  • the cytotoxic drug is coupled to the N-terminal amino group of the ligand and/or the ⁇ -amino group of the lysine residue through the linking unit.
  • the cytotoxic drug can be coupled to the antibody in the coupling reaction.
  • the number of drug molecules will be less than the theoretical maximum.
  • Ligand cytotoxic drug conjugate loading can be controlled by the following non-limiting methods, including:
  • pharmaceutically acceptable salt refers to salts of the ligand-drug conjugates of the present invention, or salts of the compounds described in the present invention, when such salts are used in mammals With safety and efficacy, and with due biological activity, the ligand-drug conjugate compound of the present invention contains at least one carboxyl group, so it can form a salt with a base.
  • pharmaceutically acceptable salts include: sodium salt, potassium salt, calcium salt or magnesium salt, etc.
  • pharmaceutically acceptable salts or “pharmaceutically acceptable salts” refers to salts of the antibody-drug conjugates of the present invention, or salts of the compounds described in the present invention, which when used in mammals have Safe and effective, and have due biological activity, the ligand-drug conjugate compound of the present invention contains at least one amino group, so it can form a salt with an acid.
  • Non-limiting examples of pharmaceutically acceptable salts include: hydrochloric acid Salt, Hydrobromide, Hydroiodide, Sulfate, Hydrogen Sulfate, Citrate, Acetate, Succinate, Ascorbate, Oxalate, Nitrate, Pearate, Hydrogen Phosphate, Phosphoric Acid Dihydrogenate, Salicylate, Hydrogencitrate, Tartrate, Maleate, Fumarate, Formate, Benzoate, Mesylate, Ethanesulfonate, Besylate , p-toluenesulfonate.
  • Acid amino acid means that the isoelectric point of the amino acid is less than 7, and the acidic amino acid molecule often has one or more acid groups such as carboxyl groups, which can be effectively ionized into negative ions in the structure to increase hydrophilicity. Acidic amino acids may be natural or unnatural amino acids.
  • Natural amino acid refers to an amino acid that is biosynthesized. Natural amino acids are generally L-form, but there are a few exceptions, such as glycine, both natural and biosynthetic.
  • Unnatural amino acid refers to an amino acid obtained by synthetic means.
  • the reaction system is directly suction filtered, the filter cake is washed with methanol, the filtrate is collected, and the filter cake is dried to obtain an off-white solid compound, It is ixitecan, the isomer of compound 1, which is set aside for later use.
  • the filtrate was concentrated under reduced pressure, purified by liquid phase preparation, and lyophilized to obtain a light brown solid (5.07 g, 41%).
  • LC-MS obtained [M+H] + : 436.2.
  • reaction solution was separated and purified by liquid phase preparation, and the prepared solution was concentrated under vacuum at 35°C and lyophilized to obtain 49.7 mg of compound 5 as a pale yellow solid with a yield of 48.7%; and 60.5 mg of compound 6 as a pale yellow solid with a yield of 59.3%.
  • the reaction droplets in the No. 1 bottle were added to the No. 2 reaction bottle.
  • HPLC monitors whether the reaction raw materials disappear, and after the reaction is complete, HPLC preparation and separation are performed. Freeze-dried to obtain 4.83 g of a white solid product with a yield of 66.3%.
  • the reaction droplets in the No. 1 bottle were added to the No. 2 reaction bottle.
  • Example 18 General method for preparing ADC drug by coupling
  • the antibody Ab with a monomer rate greater than 95% after preliminary purification was changed into a phosphate buffer using an ultrafiltration centrifuge tube at a concentration of 10 mg/ml.
  • use an ultrafiltration centrifuge tube with a molecular weight cut-off of 30KDa to change the medium to PBS, and remove the uncoupled payload.
  • the ADC samples after the liquid change were filtered through a 0.22-micron sterile filter before use.
  • the average drug/antibody ratio (DAR) value of the conjugated product was determined by reversed-phase high performance liquid chromatography.
  • the obtained ADC drug molecule related information and the corresponding situation with the payload molecule are as follows:
  • the logP value is closely related to the water solubility, membrane permeability, ADME process in vivo and the affinity of the compound to the receptor, which plays a key role in the process of the compound passing through the biological membrane.
  • the logP value was obtained by testing the partition coefficient of the camptothecin drug in n-octane (oil) and water to study the drug, and the results are as follows:
  • Example 20 Plasma aggregation, degradation test of ADC
  • Three tubes of each ADC are prepared and placed in a 37°C water bath to incubate, respectively. After incubation for 0h, 72h, and 7d, the ADC samples were taken out, and 100 ⁇ L of ProteinA (MabSelect SuRe TMLX Lot: #10221479GE, washed with PBS) was added to each tube, and the vertical mixer was shaken for adsorption for 2 hours. After washing, elution and Tris-HCl and step, obtain the ADC after incubation, and perform SEC detection on the ADC sample incubated for a specific time to determine the aggregation degradation.
  • the payload of ADC-1 is consistent with that of the marketed anti-tumor drug Enhertu. From the test experiments on the aggregation and degradation of ADC drugs in plasma, it can be seen that the aggregation and degradation of the chiral derivative ADC of camptothecin is significantly lower than that of ADC-1, and has higher Good physical and chemical properties, more stable in plasma.
  • camptothecin chiral derivatives The cytotoxic activity of camptothecin chiral derivatives was determined by the following experimental procedure: Camptothecin chiral derivatives were added to A431, MDA-MB-468, SK-BR-3, Bxpc-3 and SW620 tumor cells, respectively , and cell viability was determined after 72 hours. Cell-based in vitro assays were used to determine cell viability, cytotoxicity, and apoptosis induced by the camptothecin drugs of the present invention.
  • the in vitro efficacy of camptothecin drugs was determined by cell proliferation assay.
  • CellTiter The Aqueous One Solution Cell Proliferation Assay is commercially available (Promega Corp., Madison, WI).
  • the One Solution Cell Proliferation Assay(a) is a colorimetric assay for the detection of viable cell numbers in cell proliferation and cytotoxicity assays.
  • This reagent contains a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and An electron coupling agent (phenazine ethosulfate; PES).
  • MTS Olet's reagent
  • NADPH NADPH
  • NADH NADH
  • the amount of formazan product detected at 490 nm is proportional to the number of viable cells in culture. Since the formazan product of MTS is soluble in tissue culture medium, CellTiter One Solution Assay has fewer steps than MTT or INT methods.
  • A431, MDA-MB-468, SK-BR-3, Bxpc-3 and SW620 are used as research systems for in vitro drug efficacy detection.
  • a 96-well plate evenly seed the appropriate cell density and incubate at 37°C in a carbon dioxide incubator. After 24 hours, the cells were confirmed to be in normal state under the microscope, and the camptothecin drug was added.
  • Diluted camptothecin drug in medium (initial concentration is 1uM, 7-fold dilution, 8 concentration points, the last two columns are control group (ie cells + medium, no drug treatment) and blank group (ie no cells, only Contains culture medium for background subtraction), after fully mixing the drug, add the drug to the corresponding cell well and put it into a carbon dioxide incubator for 3 days at 37°C. After 3 days, add 20ul MTS (Promegm, G3581) to each well to react for 2 hours , Microplate reader (Molecular Device, model: SpectraMAX190) 490nM reading. By detecting the activity of dehydrogenase in mitochondria, calculating IC50 to evaluate the inhibitory effect of camptothecin drugs on cell proliferation. Corresponding camptothecin chiral derivatives IC50 (nM) The test results are as follows:
  • Dxd is the active ingredient of the marketed cancer ADC drug Enhertu, a highly active camptothecin drug.
  • Enhertu a highly active camptothecin drug.
  • the inventors have proved that the chiral derivatives of camptothecin described in the present invention are all shown in representative tumor cells BXPC-3, A431, SW620, MDA-MB-468 and SK-BR-3. Cell viability comparable to or higher than Dxd.
  • ADC samples Aseptically take a certain amount of ADC samples and add them to sterile human plasma from which human IgG has been removed so that the final concentration of ADC is 0.6 mg/ml. Three tubes are prepared for each ADC, placed in a water bath at 37°C for incubation, and incubated separately.
  • the ADC sample was taken out, and 100 ⁇ L of ProteinA (MabSelect SuRe TMLX Lot: #10221479GE, washed with PBS) was added to each tube, the vertical mixer was shaken for adsorption for 2h, and after washing, elution and Tris-HCl neutralization In the step, the ADC after incubation is obtained, and the DAR value of the ADC sample incubated for a specific time is detected by RP-HPLC to determine the plasma stability of the sample.
  • the experimental results show that the ADC of the present invention has little or no loss during plasma incubation, and has better stability in plasma because the possibility of premature degradation to produce toxicity is low.
  • MDA-MB-468 and BT474 are used as research systems for in vitro drug efficacy detection.
  • An appropriate number of tumor cell lines were evenly seeded into a 96-well plate and incubated in a carbon dioxide incubator. After 24 hours, the cells were confirmed to be in normal state under the microscope, and drug addition treatment was performed. Dilute the drug with the medium (the initial concentration of the ADC drug is 500nM, the dilution factor is 7 times, there are 8 concentration points in total, the theoretical coupling ratio (DAR) of toxin and antibody is 8:1, and the actual coupling ratio is roughly 7.5: 1.
  • DAR theoretical coupling ratio
  • the initial concentration of the toxin is 4.0 ⁇ M, 7-fold concentration gradient dilution, 8 concentration points), after mixing, add it to the corresponding cell wells, where the last two columns are the control group (ie cells + medium, no drug) treatment) and blank group (ie, no cells, containing only medium, for background subtraction), put into a carbon dioxide incubator at 37°C for 5 days. After 5 days, 20 ⁇ L of MTS (Promega, G3581) was added to each well to react for 2 hours, and the absorbance value at a wavelength of 490 nm was read with a microplate reader (Molecular Device, model: SpectraMAX190). By detecting the activity of dehydrogenase in mitochondria, IC50 was calculated to evaluate the inhibitory effect of ADC drugs on tumor cell proliferation.
  • ADC-1 is the marketed antitumor drug Enhertu.
  • the camptothecin chiral derivative drug of the present invention showed good anti-tumor activity in multiple tumor cell lines after coupling with the antibody through the linking unit L, and was compatible with ADC- Compared with 1, some ADCs have better activity and have great clinical application value.
  • Example 24 ADC in vivo efficacy test
  • a single tumor NCI-N87 tumor-bearing nude mouse model is established to evaluate the in vivo efficacy of the ADC conjugated drug. That is, 5 ⁇ 10 6 NCI-N87 cells were inoculated subcutaneously on the right shoulder of BALB/c nude mice aged 4 to 6 weeks, and when the average tumor volume of the mice grew to 162 mm 3 , they were randomly divided into groups according to the tumor size. 5 rats were intravenously injected with blank control (buffer solution blank) and 3 mg/kg dose of antibody drug conjugates ADC-1, ADC-2, ADC-3, ADC-5, ADC-6, ADC-7. The tumor volume measurement data was displayed as the average tumor volume at the time of measurement, and the changes in the body weight of the mice were recorded to observe the in vivo efficacy and preliminary toxicity of ADC drugs.

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Abstract

一种喜树碱类衍生物及其配体-药物偶联物，提供了更优的抗肿瘤喜树碱类ADC药物，具有更高的安全性、有效性，更好满足临床需求。

Description

一种喜树碱类衍生物及其配体-药物偶联物 技术领域
本发明涉及一种喜树碱类衍生物及其配体-药物偶联物。
背景技术
配体-药物偶联物(ADC)作为新型的靶向药物，一般由三部分组成：抗体或抗体类配体，小分子药物以及将配体和药物偶联起来的连接子。抗体药物偶联物利用抗体对抗原的特异性识别，将药物分子运输至靶细胞附近并有效释放药物分子，达到治疗目的。2011年8月，美国食品药品监督管理局(FDA)批准西雅图基因公司研制的用于治疗霍奇金淋巴瘤以及复发性变性大细胞淋巴瘤(ALCL)的ADC新药Adecteis TM上市，临床应用已经证明了此类药物的安全性和有效性。
喜树碱类药物作为抗肿瘤性的小分子化合物，已知作为抑制DNA拓扑异构酶I而呈现抗肿瘤作用，包括伊立替康，依喜替康，SN38等。许多喜树碱类药物已在临床中广泛应用，主要适应症为骨癌、前列腺癌、乳腺癌、胰腺癌等。与目前临床使用的伊立替康不同，依喜替康不需要通过利用酶进行活化。另外，与作为伊立替康的药效本体的SN-38、同在临床中使用的拓扑替康相比，依喜替康的拓扑异构酶I抑制活性更强，在体外针对多种癌细胞具有更强的杀细胞活性。尤其是，对于通过P-糖蛋白的表达面对SN-38等显示耐性的癌细胞也显示效果。依喜替康作为单独化疗药物尚未成功上市，推测与其较高的细胞活性相关，导致治疗窗口窄。
抗体-药物偶联物(ADC)类药物的优势在于增加水溶性，提高靶向性，特异性抗体与抗原的结合，将药物携带至靶细胞周围，通过在靶细胞附近释放药物，有效杀灭肿瘤细胞，降低毒副作用。喜树碱类药物在ADC药物中具有可观的应用前景，目前以依喜替康为毒素的抗体偶联药物trastuzumab deruxtecan(商品名为Enhertu)已于2019年12月20日被美国FDA获批上市，作为第一个上市的喜树碱类ADC药物，很好证明了此类药物在ADC领域的成药能力和应用前景。
Enhertu为针对HER2靶点的ADC药物，单个抗体上连接了约8个依喜替康衍生物(DAR＝8)。但同时，其原研公司第一三共也意识到，因为安全性的原因，有必要降低DAR值，在其公布的在临床中的ADC：DS-1062已将DAR值降低至4,DAR值的降低可能带来治疗效果的降低。由此可见，开发更加安全的喜树碱衍生物是很有必要 的。
在ADC药物中，为保证安全性会选择降低药物的DAR值，从而导致治疗效果的降低。而药物的溶解性是影响DAR值的重要因素之一，同时也是影响ADC药物的旁观者效应的主要因素。喜树碱类药物虽已在临床中广泛应用，但其水溶性差仍是亟待解决的难题。对于水溶性差的药物，提高药物的水溶性，可以改善药物的物理性质，改变其在体内的代谢情况，从而提高药效，加强治疗效果，减少药物剂量，降低成本。差的水溶性会导致ADC聚集程度增加，从而易于被正常细胞内吞，产生毒副作用或更易于被代谢、缩短半衰期。
本公司在研究过程中，意外的发现依喜替康的氨基手性异构体化合物具有明显优于依喜替康的水溶性，依据其合成的衍生物同样具有更好的水溶性，可合成制备更优的抗肿瘤喜树碱类ADC药物，使其具有更高的安全性、有效性，更好满足临床需求。
发明内容
本发明提供一种如通式D所示的喜树碱类衍生物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物；
Figure PCTCN2021122823-appb-000001
其中：
与
Figure PCTCN2021122823-appb-000002
相连的手性碳原子具有R构型的绝对手性；
R选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基；
R 1选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
R 2选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
X选自-C(O)-CR aR b-(CR 3R 4) m-O-、-C(O)-CR aR b-(CR 3R 4) m-NH-或-C(O)-CR aR b-(CR 3R 4) m-S-；
R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R b选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
或者，R a、R b及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
或者，R 3、R 4及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
m选自0-4的整数。
作为优选方式，R 1选自C 1-3烷基。
作为优选方式，R 2选自C 1-3烷基或氟原子。
喜树碱衍生物具有如下式D 1所示结构：
Figure PCTCN2021122823-appb-000003
其中：
与
Figure PCTCN2021122823-appb-000004
相连的手性碳原子具有R构型的绝对手性；
R选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基；
X选自-C(O)-CR aR b-(CR 3R 4) m-O-、-C(O)-CR aR b-(CR 3R 4) m-NH-或-C(O)-CR aR b-(CR 3R 4) m-S-；
R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R b选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
或者，R a、R b及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
或者，R 3、R 4及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
m选自0-4的整数。
作为优选方式，所述R选自C 1-3烷基。
作为优选方式，所述X非限制性地选自以下结构：
Figure PCTCN2021122823-appb-000005
其中左侧波浪线与喜树碱衍生物部分相连，右侧波浪线与连接单元相连。
作为优选方式，所述化合物非限制性地选自以下所示的结构：
Figure PCTCN2021122823-appb-000006
Figure PCTCN2021122823-appb-000007
其中R选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基。
进一步优选，所述化合物非限制性地选自以下所示的结构：
Figure PCTCN2021122823-appb-000008
Figure PCTCN2021122823-appb-000009
本发明还提供了一种包括喜树碱类衍生物的连接子-药物偶联物或其药学上可接受的盐或溶剂化物、或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式：
Figure PCTCN2021122823-appb-000010
其中：
与
Figure PCTCN2021122823-appb-000011
相连的手性碳原子具有R构型的绝对手性；
R选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基；
R 1选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
R 2选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
X选自-C(O)-CR aR b-(CR 3R 4) m-O-、-C(O)-CR aR b-(CR 3R 4) m-NH-或-C(O)-CR aR b-(CR 3R 4) m-S-；
R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R b选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
或者，R a、R b及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
或者，R 3、R 4及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
m选自0-4的整数；
L为-L 1-L 2-L 3-L 4-。
作为优选方式，-L-为-L 1-L 2-L 3-L 4-，其中L 1端与配体Ab相连，L 4端与X相连。
进一步优选，L 1非限制性地选自：
Figure PCTCN2021122823-appb-000012
进一步优选，L 2选自：-NC(R 5R 6)C(O)、-NR 7(CH 2) oC(O)-、-NR 7(CH 2CH 2O) oCH 2C(O)-、-S(CH 2) pC(O)-或者化学键，其中o选自0-20的整数；p选自0-20的整数；
R 5、R 6相同或者不同，且各自独立地选自氢原子、氘原子、烷基、取代烷基、氘代烷基、杂烷基、羧基、氨基、取代氨基；
R 7选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基；
L 1与L 2共用N原子。
进一步优选，L 3选自由氨基酸构成的肽残基，其中任选氨基酸进一步被选自氘原子、卤素、羟基、氰基、氨基、硝基、羧基、烷基、取代烷基、烷氧基和环烷基或者取代环烷基中的一个或多个取代基所取代；优选由一个、两个或者多个选自苯丙氨酸 (F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸、丝氨酸(S)、谷氨酸(E)或者天冬氨酸(D)中的氨基酸形成的肽残基。
进一步优选，L 4选自：-NR 8(CR 9R 10) q-、-C(O)NR 8-、-C(O)NR 8(CH 2) q-或者化学键，q选自0-6的整数；
R 8、R 9和R 10相同或者不同，且各自独立地选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基。
作为优选方式，所述连接单元-L-非限制性地选自以下结构；
Figure PCTCN2021122823-appb-000013
Figure PCTCN2021122823-appb-000014
Figure PCTCN2021122823-appb-000015
Figure PCTCN2021122823-appb-000016
Figure PCTCN2021122823-appb-000017
其中：左侧波浪线与配体部分相连，右侧波浪线与X相连。
进一步优选，所述连接子-药物偶联物非限制性地选自以下结构：
Figure PCTCN2021122823-appb-000018
Figure PCTCN2021122823-appb-000019
Figure PCTCN2021122823-appb-000020
Figure PCTCN2021122823-appb-000021
Figure PCTCN2021122823-appb-000022
其中：
1位碳原子具有R或S两种构型的绝对手性。
本发明还提供了一种包括连接子-药物偶联物的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述配体-药物偶联物包含式Ⅰ所示的结构：
Figure PCTCN2021122823-appb-000023
其中：
与
Figure PCTCN2021122823-appb-000024
相连的手性碳原子具有R构型的绝对手性；
R选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基；
R 1选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
R 2选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
X选自-C(O)-CR aR b-(CR 3R 4) m-O-、-C(O)-CR aR b-(CR 3R 4) m-NH-或-C(O)-CR aR b-(CR 3R 4) m-S-；
R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
R b选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
或者，R a、R b及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
或者，R 3、R 4及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
Ab为配体单元，选自抗体、抗体片段、靶向蛋白、Fc-融合蛋白等；
L为与Ab连接单元；X为药物部分修饰单元；
m选自0-4的整数；n选自1-20的整数或小数。
作为优选方式，所述配体选自鼠源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体。
进一步优选，所述的抗体或其抗原结合片段，非限制性地选自：抗EGFRvIII抗体、抗DLL-3抗体、抗PSMA抗体、抗CD70抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM-1抗体、抗LRRC15抗体、抗LIV-1抗体、抗CanAg/AFP抗体、抗cladin 18.2抗体、抗Mesothelin抗体、抗HER2(ErbB2)抗体、抗EGFR抗体、抗c-MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3(ErbB3)抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7-H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4抗体、抗TROP-2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P-cadherin抗体、抗GD3抗体、抗Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b抗体、抗CD19抗体、抗CD33抗体、抗CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD47抗体、抗CD138抗体、抗CD352抗体、抗CD25抗体或抗CD123抗体。
更进一步优选，所述配体-药物偶联物非限制性地选自以下结构：
Figure PCTCN2021122823-appb-000025
Figure PCTCN2021122823-appb-000026
Figure PCTCN2021122823-appb-000027
Figure PCTCN2021122823-appb-000028
其中：
Ab为配体单元；n选自1-20的整数或小数；
1位碳原子具有R或S两种构型的绝对手性。
本发明同时提供了一种制备连接子-药物偶联物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物，或其药学上可接受的盐或溶剂化物的方法，包括如下步骤：
Figure PCTCN2021122823-appb-000029
通过连接单元L a与通式化合物D 1的取代反应，得到如通式L a-X-D 1所示的连接子-药物偶联物；
其中：
与
Figure PCTCN2021122823-appb-000030
相连的手性碳原子具有R构型的绝对手性；
L 2、L 3、R、R 8、R 9、R 10、q及X如通式L-X-D所述。
本发明又提供了一种制备配体-药物偶联物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物，或其药学上可接受的盐或溶剂化物的方法，包括如下步骤：
Figure PCTCN2021122823-appb-000031
通过还原的抗体、抗体片段或者其抗原结合片段与通式(L-X-D 1)偶联反应，得到如通式Ab-L-X-D 1所示配体-药物偶联物；
其中：
与
Figure PCTCN2021122823-appb-000032
相连的手性碳原子具有R构型的绝对手性；
Ab、L、X、R及n如通式Ⅰ所述。
喜树碱类衍生物、连接子-药物偶联物或配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述药学上可接受的盐包括与结构式中酸性官能团形成的钠盐、钾盐、钙盐或镁盐等；或与结构中碱性官能团形成的醋酸盐、三氟乙酸盐、柠檬酸盐、草酸盐、酒石酸盐、苹果酸盐、硝酸盐、氯化物、溴化物、碘化物、硫酸盐、硫酸氢盐、磷酸盐、乳酸盐、油酸盐、抗坏血酸盐、水杨酸盐、甲酸盐、谷氨酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐或对甲苯磺酸盐。
本发明提供了一种药物组合物，其含有治疗有效量的权利要求1-23中任一项所述的喜树碱类衍生物、连接子-药物偶联物或配体-药物偶联物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，以及药学上可接受的载体、稀释剂或赋形剂。
本发明提供了一种喜树碱类衍生物、连接子-药物偶联物或配体-药物偶联物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化合物，以及药学上可接受的载体、稀释剂或者赋形剂，在制备用于治疗或预防肿瘤的药物中的用途。
作为优选方式，所述的肿瘤为乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、肺癌、结肠癌、直肠癌、结直肠癌、骨癌、皮肤癌、甲状腺癌、胰腺癌、黑色素瘤、神经胶质 瘤、神经母细胞瘤、多形性胶质细胞瘤、肉瘤、淋巴瘤和白血病等实体瘤或血液瘤。
具体实施方式
发明的详细说明
除非另有限定，本文所用的所有技术和科学术语均与本公开所属领域普通技术人员的通常理解一致。虽然也可采用与本文所述相似或等同的任何方法和材料实施或测试本公开，但本文描述了优选的方法和材料。描述和要求保护本发明时，依据以下定义使用下列术语。
当本发明中使用商品名时，申请人旨在包括该商品名产品的制剂、该商品名产品的非专利药和活性药物部分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“配体”是能识别和结合目标细胞相关的抗原或受体的大分子化合物。配体的作用是将药物呈递给与配体结合的目标细胞群，这些配体包括但不限于蛋白类激素、凝集素、生长因子、抗体或其他能与细胞结合的分子。在本发明实施方式中，配体表示为Ab，配体可通过配体上的杂原子与连接单元形成连接键，优选为抗体或其抗原结合片段，所述抗体选自嵌合抗体、人源化抗体、全人抗体或鼠源抗体；优选为单克隆抗体。
配体单元是与靶标部分特异性结合的靶向剂。所述配体能够特异性结合至细胞组分或结合至细胞组分或结合至其他感兴趣的靶标分子。靶标部分或靶标通常在细胞表面上。在一些方面中，配体单元的作用是将药物单元递送至配体单元与之相互作用的特定靶细胞群。配体包括但不限于蛋白质、多肽和肽，以及非蛋白质如糖。合适的配体单元包括，例如，抗体，例如全长(完整)抗体及其抗原结合片段。在配体单元是非抗体靶向试剂的实施方式中，其可以是肽或多肽，或非蛋白质分子。这类靶向试剂的示例包括干扰素、淋巴因子、激素、生长因子和集落刺激因子、维生素、营养转运分子、或任何其他细胞结合分子或物质。在一些实施方式中，连接子共价连接至配体的硫原子。在一些方面中，硫原子是半胱氨酸残基的硫原子，其形成抗体的链间二硫键。在另一方面中，硫原子是已经导入配体单元的半胱氨酸残基的硫原子，其形成抗体的链间二硫键。在另一方面中，硫原子是已经导入配体单元的半胱氨酸残基的硫原子(例如，通过定点诱变或化学反应)。在其他方面中，连接子结合的硫原子选自形成抗体的链间二硫键的半胱氨酸残基或已经引入配体单元的额半胱氨酸残基(例如，通 过定点诱变或化学反应)。在一些实施方式中，按照Kabat(Kabat E.A等，(1991))《免疫学感兴趣的蛋白质序列》(Sequences of proteins of Immunological Interest),第五版，NIH出版物91-3242)中的EU索引编号系统。
如本文所用，“抗体”或“抗体单元”在其所属的范围内，包括抗体结构的任何部分。这一单元可以结合，反应性关联，或者络合一个受体，抗原，或者靶向细胞群体具有的其它受体单元。抗体可以是任何蛋白或蛋白类分子，它可以结合，络合，或者与待治疗或生物改造的细胞群体的一部分发生反应。本发明中组成抗体药物偶联物的抗体好保持其原有野生状态时的抗原结合能力。因此，本发明中的抗体能够，好专一性的与抗原结合。涉及的抗原包括，例如，肿瘤相关抗原(TAA)，细胞表面受体蛋白和其他细胞表面分子，细胞存活调节因子，细胞增殖调节因子，与组织生长与分化相关的分子(如已知或预知的具有功能性的)，淋巴因子，细胞因子，参与细胞循环调节的分子，参与血管生成的分子，以及与血管生成有关的分子(如已知或预知的具有功能性的)。肿瘤相关因子可以是簇分化因子(如CD蛋白)。
应用在抗体药物偶联物中的抗体包括，但不局限于，针对细胞表面受体和肿瘤相关抗原的抗体。这样的肿瘤相关抗原是业内所熟知的，可以通过业内熟知的抗体制备方法和信息来制备。为了开发可用于癌症诊断与治疗的有效的细胞水平目标物，研究人员力图找寻跨膜或其他肿瘤相关多肽。这些目标物能够特异性的表达在一种或多种癌细胞表面，而在一种或多种非癌细胞表面表达很少或不表达。通常，相对于非癌细胞表面而言，这样的肿瘤相关多肽在癌细胞表面更加过度表达。确认这样的肿瘤相关因子，可大大提高基于抗体治疗癌症的专一靶向特性。为方便起见，为业内所熟知的抗原相关信息标示如下，包括名称，其他名称，基因库登录号。与肿瘤相关抗原对应的核酸和蛋白序列可参见公开数据库，例如Genbank。抗体靶向对应的肿瘤相关抗原包括所有的氨基酸序列变种和同种，与参考文献中确认的序列具有至少70％,80％,85％,90％或者95％的同源性，或者具备与引用文献中的肿瘤相关抗原序列具有完全一致的生物性质和特征。
术语“抑制”或“的抑制”指，减少了可检测的量，或完全阻止。
术语“癌症”指的是以失调的细胞生长为特征的生理病症或疾病。“肿瘤”包括癌细胞。
术语“自身免疫疾病”是源自针对个体自身的组织或蛋白质的疾病或紊乱。
术语“药物”是指细胞毒性药物，药物表示为D，能在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。该术语包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素(例如At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32和Lu的放射性同位素)，毒性药物，化疗药物，抗生素和核溶酶，优选为毒性药物。
术语“连接子”或“连接片段”或“连接单元”是指一端与配体连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与药物相连。
接头，包括延伸物、间隔物和氨基酸单元，可以通过本领域己知方法合成，诸如US2005-0238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等Cancer Research 52:127-131,1992)；美国专利No.5,208,020。
按照在细胞内药物释放的机制，如本文所用，“连接子”或“抗体药物偶联物的连接子”可被分为两类：不可断裂连接子和可断裂连接子。对于含有不可断裂连接子的抗体药物偶联物，其药物释放机制为：偶联物与抗原结合并被细胞内吞后，抗体在溶酶体中被酶解，释放出由小分子药物，连接子，和抗体氨基酸残基共同组成的活性分子。由此带来的药物分子结构改变并不减弱其细胞毒性，但由于活性分子是带电荷的(氨基酸残基)，从而导致其不能渗入邻近细胞。因此，此类活性药物不能杀死邻近不表达靶向抗原(抗原阴性细胞)的肿瘤细胞(旁观者效应，bystander effect)(Ducry等，2010，Bioconjugate Chem.21:5-13)。
术语“配体-药物偶联物"，指抗体通过稳定的连接单元与具有生物活性的药物相连。在本发明中“配体-药物偶联物"优选为抗体-药物偶联物(antibody drug conjugate,ADC)，指把单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的毒性药物相连。
本公开所用氨基酸三字母代码和单字母代码如J.boil.Chem.1968,243,3558.中所述。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团， 优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“取代烷基”指烷基中的氢被取代基团取代，除非文中另有说明，烷基的取代基可以是选自下组的多种基团：-卤素、-OR’、-NR’R”、-SR’、-SiR’R”R”’、-OC(O)R’、-C(O)R’、-CO 2R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R”’、-NR”C(O) 2R’、-NH-C(NH 2)＝NH、-NR’C(NH 2)＝NH、-NH-C(NH 2)＝NR’、-S(O)R’、-S(O) 2R’、-S(O) 2NR’R”、-NR’S(O) 2R”、-CN和-NO 2，取代基数量为0至(2m’+1)，其中m’为该基团中碳原子的总数。R’、R”和R”’各自独立的指代氢、未取代的C 1-8烷基、未取代的芳基、由1-3个卤素取代的芳基、未取代的C 1-8烷基、C 1-8烷氧基或C 1-8硫代烷氧基、或未取代的芳基-C 1-4烷基。R’和R”连接于同一个 氮原子时，它们可与该氮原子一起形成3-，4-，5-，6-或7-元环。例如，-NR’R”包括1-吡咯烷基和4-吗啉基。
术语“杂烷基”指含有一个或多个选自N、O或S的杂原子的烷基，其中烷基如上所定义。
术语“亚烷基”指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子，更优选含有1至6个碳原子的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH 2-、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)和1,5-亚丁基(-CH 2CH 2CH 2CH 2CH 2-)等。亚烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基中的一个或多个取代基所取代。
术语“烷氧基"指-O-(烷基)和-O-(环烷基)，其中烷基或环烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选环烷基环包含3至10个环原子。单环杂环基的非 限制性实例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
术语“螺杂环基"指5至20元的单环之间共用一个原子(称螺原子)的多环杂环基团，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，其余环原子为碳。其可以含有一个或多个双键，但没有一个环具有完全共轭的兀电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺杂环基分为单螺杂环基、双螺杂环基或多螺杂环基，优选为单螺杂环基和双螺杂环基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环基。
术语“环烷基烷基”指烷基被一个或多个环烷基取代，优选被一个环烷基取代，其中烷基如上所定义，其中环烷基如上所定义。
术语“卤代烷基”指烷基被一个或多个卤素取代，其中烷基如上所定义。
术语“氘代烷基”指烷基被一个或多个氘原子取代，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH 2。术语“硝基”指-NO 2。
术语“酰胺基"指-C(O)N(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
术语“羧酸酯基"指-C(O)O(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
本发明还包括各种氘化形式。与碳原子连接的各个可用的氢原子可独立地被氘原子替换。本领域技术人员能够参考相关文献合成氘化化合物。在制备氘代化合物时，可使用市售的氘代起始物质，或它们可使用常规技术采用氘代试剂合成，氘代试剂的非限制性实例包括:氘代甲醇、氘代水、氘代硼烷、三氘代硼烷四氢呋喃溶液、氘代氢化锂铝、氘代碘乙烷和氘代碘甲烷等。
术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ 链。五类Ig中每类Ig都可以有κ链或λ链。本发明所述的抗体优选为针对靶细胞上细胞表面抗原的特异性抗体，非限制性实施例为以下抗体：抗EGFRvIII抗体、抗DLL-3抗体、抗PSMA抗体、抗CD70抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM-1抗体、抗LRRC15抗体、抗LIV-1抗体、抗CanAg/AFP抗体、抗cladin 18.2抗体、抗Mesothelin抗体、抗HER2(ErbB2)抗体、抗EGFR抗体、抗c-MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3(ErbB3)抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7-H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4抗体、抗TROP-2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P-cadherin抗体、抗GD3抗体、抗Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b抗体、抗CD19抗体、抗CD33抗体、抗CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD138抗体、抗CD352抗体、抗CD25抗体或抗CD123抗体中一个或多个；优选为曲妥珠单抗(Trastuzumab,商品名Herceptin)、帕妥珠单抗(Pertuzumab,也被称作2C4，商品名Perjeta)、尼妥珠单抗(Nimotuzumab，商品名泰欣生)、Enoblituzumab、Emibetuzumab、Inotuzumab、Pinatuzumab、Brentuximab、Gemtuzumab、Bivatuzumab、Lorvotuzumab、cBR96和Glematumamab。
术语“溶剂化物”或“溶剂化合物”指本发明的配体-药物偶联物与一种或多种溶剂分子形成可药用的溶剂化物，溶剂分子的非限制性实例包括水、乙醇、乙腈、异丙醇、DMSO、乙酸乙酯。
术语“载药量”是指式Ⅰ分子中每个抗体上加载的细胞毒性药物平均数量，也可以表示为药物量和抗体量的比值，药物载量的范围可以是每个抗体(Ab)连接0-12个，优选1-10个细胞毒性药物(D)。在本发明的实施方式中，载药量表示为n,示例性的可以为1,2,3,4,5,6,7,8,9,10的均值。可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的药物品均数量。
本发明的一个实施方式中，细胞毒性药物通过连接单元偶联在配体的N端氨基和/ 或赖氨酸残基的ε-氨基上，一般地，偶联反应中能与抗体偶联的药物分子数将小于理论上的最大值。
可以用以下非限制性方法控制配体细胞毒性药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
常规的药物组合物的制备见中国药典。
术语“药学上可接受的盐”或“可药用盐”是指本发明配体-药物偶联物的盐，或本发明中所述的化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性，本发明配体-药物偶联化合物至少含有一个羧基，因此可以与碱形成盐，药学上可接受的盐的非限制性实例包括：钠盐、钾盐、钙盐或镁盐等。
术语“药学上可接受的盐”或“可药用盐”是指本发明抗体-药物偶联物的盐，或本发明中所述的化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性，本发明配体-药物偶联化合物至少含有一个氨基，因此可以与酸形成盐，药学上可接受的盐的非限制性实例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
“酸性氨基酸”指氨基酸的等电点小于7，酸性氨基酸分子中往往带有一个或多个羧基等酸性基团，在结构中可有效电离为负离子形式而增加亲水性。酸性氨基酸可以为天然的，也可为非天然的氨基酸。
“天然氨基酸”指由生物合成的氨基酸。天然氨基酸一般情况下是L-型的，但也有少数例外，比如甘氨酸，包括天然的和生物体合成的。
“非天然氨基酸”指通过合成手段所获得的氨基酸。
下面结合具体实施例，进一步阐述本发明，应理解，这些实施例只用于说明本发明，而不用于限制本发明的范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另外说明，否则所有的百分数、比例、比率、或份数按重量计。
除非另行定义，文中所使用的所有专业和科学用于与本领域技术人员所熟悉的意义相同。
此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中
所述的较佳实施方法与材料仅作示范之用。
实施例1：依喜替康手性异构化合物1的合成
Figure PCTCN2021122823-appb-000033
向1L的圆底烧瓶中称入化合物SM-1(N-(8-氨基-6-氟-5-甲基-1-氧代-1,2,3,4-四氢萘-2-基)乙酰胺，外购)(6.25g,25.0mmol)、SM-2((S)-4-乙基-4-羟基-7,8-二氢-1H-吡喃O[3,4-F]吲哚嗪-3,6,10(4H)-酮，外购)(7.25g,27.5mmol)、对甲苯磺酸吡啶盐(1.26g,5.0mmol)，加入500mL甲苯，加热回流分水，反应5小时，HPLC监测反应至SM-2小于10％，停止反应。降温，减压浓缩至溶液体积只剩一半，冰水浴搅拌析晶2h，过滤，30mL甲苯分三次洗涤滤饼，真空烘干，得到化合物Ac-1:10.68g，89％。LC-MS得到[M+H] +:478.2。
将Ac-1(10.68g，22.5mmol)、甲磺酸(55mL)、水(110mL)和甲苯(55mL)混合于1L圆底烧瓶中，加热回流。4小时后，TLC监测反应，原料消失，将反应体系冷却至室温。4室温下向体系中缓慢滴加500mL甲醇，并持续搅拌，待滴加完毕，搅拌过夜，将反应体系直接抽滤，甲醇洗涤滤饼，收集滤液，将滤饼烘干，得到灰白色固体化合物，为化合物1的异构体依喜替康，放置备用。将滤液减压浓缩，液相制备纯化，冻干，得到浅棕色固体(5.07g，41％)，LC-MS得到[M+H] +:436.2。
实施例2：化合物2的合成
Figure PCTCN2021122823-appb-000034
25ml圆底烧瓶中加入化合物1(为三氟乙酸盐的形式)(100.0mg，0.188mmol)、羟基乙酸(43.0mg，0.565mmol)、HATU(107.2mg，0.282mmol)、HOBt(38.1mg，0.282mmol)和5ml超干DMF，瓶口液封。将烧瓶置于冰水浴下搅拌10分钟，然后加入DIEA(94uL，0.565mmol)。TLC检测反应进度，反应约3h。反应完全后，反应液送制备分离，制备液35℃真空浓缩，冻干，得白色固体化合物2：63.8mg，收率69.7％。LC-MS结果显示[M+H] +：493.9。 1H NMR(400MHz,DMSO-d6)δ8.39(d,J＝9.0Hz,1H),7.68(d,J＝10.9Hz,1H),7.29(s,1H),6.51(s,1H),5.53(dt,J＝9.1,6.0Hz,1H),5.41(s,2H),5.12(d,J＝19.1Hz,1H),4.92(d,J＝18.9Hz,1H),3.99(s,2H),3.10(qt,J＝16.7,6.1Hz,2H),2.29(d,J＝1.8Hz,3H),2.16(q,J＝6.4Hz,2H),2.00–1.81(m,2H),0.91(t,J＝7.3Hz,3H).
实施例3：化合物3的合成
Figure PCTCN2021122823-appb-000035
25ml圆底烧瓶中加入化合物1(为三氟乙酸盐的形式)(100.0mg，0.188mmol)、L-乳酸(56.6mg，0.565mmol)、HATU(107.2mg，0.282mmol)、HOBt(38.1mg，0.282mmol)和5ml超干DMF，瓶口液封。将烧瓶置于冰水浴下搅拌10分钟，然后加入DIEA(94uL，0.565mmol)。HPLC检测反应进度，反应约3h。反应完全后，反应液加入饱和氯化铵水溶液，抽滤得淡黄色粗品，粗品经TLC制备板刮板分离。得42.0mg 淡黄色固体化合物3，收率46.1％。
1H NMR(400MHz,DMSO-d6)δ8.46(d,J＝9.2Hz,1H),7.71(d,J＝10.9Hz,1H),7.29(s,1H),6.53(s,1H),5.68(d,J＝4.9Hz,1H),5.54(q,J＝7.3Hz,1H),5.42(s,2H),5.20(d,J＝19.0Hz,1H),4.93(d,J＝18.9Hz,1H),4.20–4.08(m,1H),3.25–3.01(m,2H),2.32(s,3H),2.14(q,J＝6.5Hz,2H),1.96–1.80(m,J＝7.1Hz,2H),1.42(d,J＝6.8Hz,3H),0.90(t,J＝7.2Hz,3H).
实施例4：化合物4的合成
Figure PCTCN2021122823-appb-000036
25ml圆底烧瓶中加入化合物1(为三氟乙酸盐的形式)(100.0mg，0.188mmol)、D-乳酸(56.6mg，0.565mmol)、HATU(107.2mg，0.282mmol)、HOBt(38.1mg，0.282mmol)和5ml超干DMF，瓶口液封。将烧瓶置于冰水浴下搅拌10分钟，然后加入DIEA(94uL，0.565mmol)。HPLC检测反应进度，反应约3h。反应完全后，反应液加入饱和氯化铵水溶液，抽滤得淡黄色粗品，粗品经TLC制备板刮板分离后，用二氯甲烷和甲醇对固体进行打浆纯化，得16.8mg淡黄色固体化合物4，收率18.4％。
LC-MS结果显示[M+H] +：508.2。
实施例5：化合物5和化合物6的合成
Figure PCTCN2021122823-appb-000037
25ml圆底烧瓶中加入化合物1(为三氟乙酸盐的形式)(200mg，0.377mmol)、三氟乳酸(162.8mg，1.13mmol)、HATU(214.8mg，0.565mmol)、HOBt(76.3mg，0.565mmol)和5ml超干DMF，瓶口液封。将烧瓶置于冰水浴下搅拌10分钟，然后加入DIEA(187uL，1.13mmol)。HPLC检测反应进度，反应约6h。反应完全后，反应液液相制备分离纯化，制备液35℃真空浓缩，冻干，得49.7mg淡黄色固体化合物5，收率48.7％；得60.5mg淡黄色固体化合物6，收率59.3％。
化合物5：
LC-MS结果显示[M+H] +：562.2。
1H NMR(400MHz,DMSO-d6)δ9.00(d,J＝8.7Hz,1H),7.78(d,J＝10.9Hz,1H),7.31(s,1H),7.19(d,J＝6.9Hz,1H),6.54(s,1H),5.59(dt,J＝10.1,5.3Hz,1H),5.43(s,2H),5.16(q,J＝19.2Hz,2H),4.63(p,J＝7.6Hz,1H),3.14(s,2H),2.38(s,3H),2.16(p,J＝7.1Hz,2H),1.87(hept,J＝7.1Hz,2H),0.88(t,J＝7.3Hz,3H).
化合物6：
LC-MS结果显示[M+H] +：562.2。
1H NMR(400MHz,DMSO-d6)δ8.97(d,J＝8.6Hz,1H),7.78(d,J＝11.0Hz,1H),7.31(s,1H),7.26(d,J＝6.7Hz,1H),6.54(s,1H),5.57(dt,J＝9.5,5.3Hz,1H),5.43(s,2H),5.24–5.03(m,2H),4.63(p,J＝7.4Hz,1H),3.14(t,J＝6.5Hz,2H),2.38(d,J＝1.9Hz,3H),2.28–2.07(m,2H),1.95–1.78(m,J＝7.1Hz,2H),0.88(t,J＝7.3Hz,3H).
实施例6：化合物7和化合物8的合成
Figure PCTCN2021122823-appb-000038
25ml圆底烧瓶中加入化合物1(为三氟乙酸盐的形式)(200mg，0.377mmol)、1-羟基环丙烷甲酸(131.2mg，1.13mmol)、HATU(214.8mg，0.565mmol)、HOBt(76.3mg，0.565mmol)和5ml超干DMF，瓶口液封。将烧瓶置于冰水浴下搅拌10 分钟，然后加入DIEA(187uL，1.13mmol)。HPLC检测反应进度，反应约6h。反应完全后，反应液加水析出固体，饱和氯化铵水溶液洗涤2次，纯水洗涤1次，抽干后二氯甲烷和甲醇溶解固体。TLC制备板刮板分离。得81.0mg淡黄色固体化合物7，收率82.4％；得60.0淡黄色固体化合物8，收率61.8％。
化合物7：LC-MS结果显示[M+H] +：534.2。
1H NMR(400MHz,d6DMSO)δ8.40(d,J＝9.0Hz,1H),7.78–7.62(m,1H),7.29(s,1H),6.52(s,1H),5.58(dt,J＝5.3,1.4Hz,1H),5.53(q,J＝7.5Hz,1H),5.42(s,2H),5.26–4.91(m,2H),3.62(t,J＝5.8Hz,1H),3.23–3.02(m,2H),2.30(d,J＝3.2Hz,3H),2.14(q,J＝7.9,7.0Hz,2H),1.90(p,J＝7.0Hz,2H),1.29–1.19(m,3H),0.91(t,J＝7.3Hz,3H),0.57–0.37(m,4H).
化合物8：LC-MS结果显示[M+H] +：534.2
实施例7：化合物9的合成
Figure PCTCN2021122823-appb-000039
25ml圆底烧瓶中加入化合物1(为三氟乙酸盐的形式)(100.0mg，0.188mmol)、1-(羟甲基)环丁烷甲酸(43.0mg，0.565mmol)、HATU(107.2mg，0.282mmol)、HOBt(38.1mg，0.282mmol)和5ml超干DMF，瓶口液封。将烧瓶置于冰水浴下搅拌10分钟，然后加入DIEA(94uL，0.565mmol)。HPLC检测反应进度，反应约6h。反应完全后，反应液加入饱和氯化铵水溶液析出固体，抽滤得到固体，固体经水洗2次，乙酸乙酯洗涤1次，二氯甲烷洗涤1次。得100.0mg灰白色固体化合物9，收率98.0％。
LC-MS结果显示[M+H] +：548.2。
1H NMR(400MHz,DMSO-d6)δ7.99(d,J＝8.6Hz,1H),7.69(d,J＝10.9Hz,1H),7.29(s, 1H),6.52(s,1H),5.53(dt,J＝8.7,5.5Hz,1H),5.42(s,2H),5.21–4.93(m,3H),3.63(d,J＝4.8Hz,2H),3.10(t,J＝6.2Hz,2H),2.39–2.24(m,5H),2.12(q,J＝6.3Hz,2H),1.99–1.70(m,6H),0.91(t,J＝7.3Hz,3H).
实施例8：化合物M1的合成
Figure PCTCN2021122823-appb-000040
于5000mL单口瓶中加入N-芴甲氧羰基-甘氨酸-甘氨酸(100g,282mmol,1.0eq，外购)，四乙酸铅(175g,553mmol,1.4eq)，2000mL干燥四氢呋喃和670mL甲苯，搅拌均匀，氮气保护，加热至85℃反应2.5h。TLC监控，原料反应完后，冷却至室温，过滤，滤液减压浓缩，残余物经柱色谱纯化，得化合物M1(91g)。LC-MS：[M+NH 4] +386.0。
实施例9：化合物M2的合成
Figure PCTCN2021122823-appb-000041
于1000mL单口瓶，加入化合物SM-3(49.9g，100.0mmol,1.0eq，参照本公司专利申请CN108452321公开的方法合成)、五氟苯酚(18.5g,110.0mmol,1.1eq)、DCC(20.64g,110.0mmol,1.1eq)及THF(500mL)，室温反应1h(采用TLC监测反应完全)，过滤滤去不溶物，将此滤液记为滤液A，2-8℃保存，以备后用(直接取用溶剂，按照0.2M计算使用)。LC-MS：[M+H] +565.2。
实施例10：化合物Ln-D1的合成
Figure PCTCN2021122823-appb-000042
Figure PCTCN2021122823-appb-000043
第一步：化合物1a的合成
500ml圆底烧瓶中加入M1(15.00g，40.75mmol)和150ml THF并置于冰水浴下搅拌15min。加入一水合对甲苯磺酸(775.0mg，4.08mmol)，继续搅拌10min。烧瓶上端安装恒压滴液漏斗，滴加羟基乙酸苄酯(11.6ml，81.50mmol)。滴加完毕后继续冰水浴下搅拌，约3h反应完全。TLC检测原料消失后，用饱和碳酸氢钠淬灭反应，混合液用乙酸乙酯萃取3次，无水硫酸钠干燥，浓缩后进行硅胶柱层析分离。分离得9.06g白色固体，收率46.9％。
第二步：化合物1b的合成
向1号50ml圆底烧瓶中加入1a(5.34g，11.26mmol)和25ml DMF，置于冰水浴下搅拌15min。滴加三乙胺(1854uL，12.39mmol)，反应约5h，TLC检测原料是否消失，反应完全后备用。向2号100ml圆底烧瓶中加入M4(4.65g，11.25mmol)、PyBop(7.03g，13.52mmol)、DIEA(2226uL，13.56mmo)和20ml DMF，置于冰水浴下搅拌15min，将反应完全的1号瓶中反应液滴加入2号反应瓶中。HPLC监测反应原料是否消失，待反应完全后，进行HPLC制备分离。冷冻干燥得产品4.83g白色固体，收率66.3％。
第三步：化合物1c的合成
于50mL单口瓶中加入1b(500.0mg，0.772mmol)、5％Pd/C(500.0mg，100％m)和10ml DCM，添加氢气球并置换氢气，室温下反应约3h。HPLC监测反应完全后。过滤，得滤液，得粗产品1c直接用于下一步反应。
第四步：化合物1d的合成
将粗产品1c置于冰水浴中，加入DIPEA(0.14mL,0.82mmol)，再加入化合物M2(4.0mL,0.8mmol)，加毕升至室温反应1h。HPLC监测反应完毕，液相纯化，得制备液，冻干得到370.2mg白色固体，收率59.7％。LC-MS：[M+H] +804.4。
第五步：化合物1e的合成
10ml单口瓶中加入1d(260.0mg，0.281mmol)、化合物1(149.3mg，0.281mmol)、HATU(160.4mg，0.422mmol)、HOBt(57.0mg，0.422mmol)和5ml DMF，置于冰水浴下搅拌10min，HPLC监测反应完全后制备分离，制备液冻干得368.0mg黄色固体,收率97.3％。LC-MS：[M+H] +1221.5；
第六步：化合物Ln-D1的合成
25ml单口瓶中加入1e(368mg，0.289mmol)、溴化锌(1301.0mg，5.78mmol)和15ml硝基甲烷，置于室温下搅拌。HPLC监测原料消失，反应液浓缩后HPLC制备纯化，制备液冻干，得120mg黄色固体，收率38.5％。LC-MS：[M+H] +1065.4。
实施例11：化合物Ln-D2的合成
Figure PCTCN2021122823-appb-000044
Figure PCTCN2021122823-appb-000045
第一步：化合物2a的合成
250ml圆底烧瓶中加入M1(7.45g，20.3mmol)和120ml THF并置于冰水浴下搅拌15min。加入一水合对甲苯磺酸(385.0mg，2.03mmol)，继续搅拌10min。烧瓶上端安装恒压滴液漏斗，滴加羟基乙酸苄酯(6.0ml，41.5mmol)，速度控制为2秒一滴。滴加完毕后继续冰水浴下搅拌，约3h反应完全。TLC检测原料消失后，用饱和碳酸氢钠淬灭反应，混合液用乙酸乙酯萃取3次，无水硫酸钠干燥，浓缩后进行硅胶柱层析分离。分离得4.82白色固体，收率49％。
第二步：化合物2b的合成
向1号25ml圆底烧瓶中加入2a(2.53g，5.20mmol)和10ml DMF，置于冰水浴下搅拌15min。滴加DBU(900uL，6.0mmol)，反应约0.5h，TLC检测原料是否消失，反应完全后备用。向2号50ml圆底烧瓶中加入M4(2.31g，5.6mmol)、PyBop(3.50g，6.8mmol)、DIEA(1115uL，6.8mmo)和10ml DMF，置于冰水浴下搅拌15min，将反应完全的1号瓶中反应液滴加入2号反应瓶中。HPLC监测反应原料是否消失，待反应完全后，进行HPLC制备分离。冷冻干燥得2.53g白色固体，收率74％。
第三步：化合物2c的合成
于50mL单口瓶中加入2b(500.0mg，0.76mmol)、5％Pd/C(500.0mg，100％m)和10ml DCM，添加氢气球并置换氢气，室温下反应约3h。HPLC监测反应完全后。过滤，得滤液，得粗产品2c直接用于下一步反应。
第四步：化合物2d的合成
将粗产品2c置于冰水浴中，加入DIPEA(0.12mL,0.70mmol)，再加入化合物M2(4.0mL,0.8mmol)，加毕升至室温反应1h。HPLC监测反应完毕，液相纯化，得制备液，冻干得到378.2mg白色固体，收率62％。LC-MS：[M+H] +818.3。
第五步：化合物2e的合成
10ml单口瓶中加入2d(228mg，0.28mmol)、化合物1(148.3mg，0.28mmol)、HATU(160.1mg，0.42mmol)、HOBt(57.2mg，0.42mmol)和5ml DMF，置于冰水浴下搅拌10min，滴加DIEA(140uL，0.84mmol)继续反应，HPLC监测反应完全后制备分离，制备液冻干得269.5mg黄色固体,收率78％。LC-MS：[M+H] +1235.5；
第六步：化合物Ln-D2的合成
25ml单口瓶中加入2e(269.5mg，0.22mmol)、溴化锌(1012.9mg，0.44mmol)和10ml硝基甲烷，置于室温下搅拌。HPLC监测原料消失，反应液浓缩后HPLC制备纯化，制备液冻干，得96.3mg黄色固体，收率41％。LC-MS：[M+H] +1079.4。
实施例12：化合物Ln-D3的合成
Figure PCTCN2021122823-appb-000046
参照实施例11合成路线和方法，得到化合物L-D3(93.8mg)。LC-MS：[M+H] +1079.4。
实施例13：化合物Ln-D4和Ln-D5的合成
Figure PCTCN2021122823-appb-000047
第一步：化合物3a的合成
100ml圆底烧瓶中加入M1(2.87g，7.79mmol)、三氟乳酸苄酯(3.65g，15.59mmol)、无水醋酸锌(2.86g，15.59mmol)和30ml甲苯，氮气保护外温100℃反应。TLC监测反应进度，反应约5h。反应完全后过滤，滤液浓缩得粗品油状物。粗品经硅胶柱层析纯化，得白色固体1.51g，收率35.7％。
第二步：化合物3b的合成
向1号50ml圆底烧瓶中加入3a(2.82g，5.20mmol)和10ml DMF，置于冰水浴下搅拌15min。滴加DB U(0.949g，6.24mmol)，反应约0.5h，TLC检测原料是否消失，反应完全后备用。向2号50ml圆底烧瓶中加入M4((2.36g，5.71mmol)、PyBop(3.25g，6.24mmol)、HOBt(0.84g，6.24mmol)、DIEA(1000uL，6.24mmol)和 10ml DMF，置于冰水浴下搅拌15min，将反应完全的1号瓶中反应液滴加入2号反应瓶中。HPLC监测反应原料是否消失，待反应完全后，进行HPLC制备分离。DCM萃取制备液3次，无水硫酸钠干燥，浓缩有机相，真空干燥得2.32g白色固体，收率62％。
第三步：化合物3c的合成
于50mL单口瓶中加入3b(500.0mg，0.70mmol)、5％Pd/C(500.0mg，100％m)和10ml DMF，氢化室温下反应约3h。HPLC监测反应完全后。过滤，得滤液，得粗产品3c直接用于下一步反应。
第四步：化合物3d的合成
将粗产品3c置于冰水浴中，加入DIPEA(0.12mL,0.70mmol)，再加入化合物M2(4.0mL,0.8mmol)，加毕升至室温反应1h。HPLC监测反应完毕，液相纯化，得制备液，冻干得到280.0mg白色固体，收率46％。LC-MS：[M+H] +872.3。
第五步：化合物3e的合成
10ml单口瓶中加入3d((280.0mg，0.241mmol)、化合物1(142.2mg，0.241mmol)、HATU(137.6mg，0.362mmol)、HOBt(38.9mg，0.362mmol)和5ml DMF，置于冰水浴下搅拌10min，滴加DIEA(120uL，0.723mmol)继续反应，HPLC监测反应完全后制备分离，制备液冻干得154.1mg黄色固体3e，收率73.6％，LC-MS：[M+H] +1289.5；得62.5mg黄色固体iso-3e，收率29.8％，LC-MS：[M+H] +1289.5。
第六步：化合物Ln-D4和Ln-D5的合成
25ml单口瓶中加入3e(154.1mg，0.118mmol)、溴化锌(532.5mg，2.36mmol)和10ml硝基甲烷，置于室温下搅拌。HPLC监测原料消失，反应液浓缩后HPLC制备纯化，制备液冻干，得82.0mg黄色固体，收率60％。LC-MS：[M+H] +1133.4。
25ml单口瓶中加入iso-3e(62.5mg，0.048mmol)、溴化锌(216.0mg，0.959mmol)和7ml硝基甲烷，置于室温下搅拌。HPLC监测原料消失，反应液浓缩后HPLC制备纯化，制备液冻干，得96.3mg黄色固体，收率41％。LC-MS：[M+H] +1133.4。
实施例14：化合物Ln-D6和Ln-D7的合成
Figure PCTCN2021122823-appb-000048
第一步：化合物4a的合成
250ml圆底烧瓶中加入M1(3.75g，10.19mmol)和50ml THF并置于冰水浴下搅拌15min。加入一水合对甲苯磺酸(194.0mg，1.02mmol)，继续搅拌10min。烧瓶上端安装恒压滴液漏斗，滴加2-环丙基-2-羟基乙酸苄酯(4.34g，20.4mmol)约6h反应完全。TLC检测原料消失后，用饱和碳酸氢钠淬灭反应，混合液用乙酸乙酯萃取3次，无水硫酸钠干燥，浓缩后进行硅胶柱层析分离。分离得2.13g白色固体，收率41％。
第二步：化合物4b的合成
向1号50ml圆底烧瓶中加入4a(2.80g，5.38mmol)和10ml DMF，置于冰水浴下搅拌15min。滴加DIEA(0.981g，6.45mmol)，反应约0.5h，TLC检测原料是否消失，反应完全后备用。向2号100ml圆底烧瓶中加入M4(2.22g，5.38mmol)、PyBop (3.35g，6.45mmol)、HOBt(0.87g，6.45mmol)、DIEA(1000uL，6.45mmol)和10ml DMF，置于冰水浴下搅拌15min，将反应完全的1号瓶中反应液滴加入2号反应瓶中。HPLC监测反应原料是否消失，待反应完全后，进行HPLC制备分离。冷冻干燥得2.63g白色固体，收率61％。
第三步：化合物4c的合成
于50mL单口瓶中加入4b(1003.1mg，1.46mmol)、5％Pd/C(1004.2mg，100％m)和15ml DMF，添加氢气球并置换氢气，室温下反应约3h。HPLC监测反应完全后。过滤，得滤液，得粗产品4c直接用于下一步反应。
第四步：化合物4d的合成
将粗产品4c置于冰水浴中，加入DIPEA(0.26mL,1.6mmol)，再加入化合物M2(8.0mL,1.6mmol)，加毕升至室温反应2h。HPLC监测反应完毕，液相纯化，得制备液，冻干得到945.1mg白色固体，收率76％。LC-MS：[M+H] +844.4。
第五步：化合物4e的合成
10ml单口瓶中加入4d(422.0mg，0.50mmol)、化合物1(265.5mg，0.50mmol)、HATU(285.1mg，0.75mmol)、HOBt(101.3mg，0.75mmol)和5ml DMF，置于冰水浴下搅拌10min，滴加DIEA(248uL，1.50mmol)继续反应，HPLC监测反应完全后制备分离，制备液冻干得268.0mg黄色固体，收率42％，LC-MS：[M+H] +1261.5。
第六步：化合物Ln-D6和Ln-D7的合成
50ml单口瓶中加入4d((268.0mg，0.204mmol)、溴化锌(1168.1mg，5.187mmol)和10ml硝基甲烷，置于室温下搅拌。HPLC监测，待反应完全，反应液浓缩后HPLC制备纯化，制备液冻干，得Ln-D6黄色固体68.0mg，收率28％，LC-MS：[M+H] +1105.4；得Ln-D7黄色固体82.0mg，收率35％，LC-MS：[M+H] +1105.4。
实施例15：化合物Ln-D8的合成
Figure PCTCN2021122823-appb-000049
第一步：化合物5a的合成
250ml圆底烧瓶中加入M1(3.74g，10.16mmol)和50ml THF并置于冰水浴下搅拌15min。加入一水合对甲苯磺酸(193.1mg，1.02mmol)，继续搅拌10min。烧瓶上端安装恒压滴液漏斗，滴加1-(羟甲基)环丁烷甲酸苄酯(4.59g，20.3mmol)。滴加完毕后继续冰水浴下搅拌，约5h反应完全。TLC检测原料消失后，用饱和碳酸氢钠淬灭反应，混合液用乙酸乙酯萃取3次，无水硫酸钠干燥，浓缩后进行硅胶柱层析分离。分离得4.60g白色固体，收率82％。
第二步：化合物5b的合成
向1号50ml圆底烧瓶中加入5a(4.38g，8.29mmol)和20ml DMF，置于冰水浴下 搅拌15min。滴加DBU(1363uL，9.11mmol)，反应约0.5h，TLC检测原料是否消失，反应完全后备用。向2号50ml圆底烧瓶中加入M4(3.42g，8.29mmol)、PyBop(5.17g，9.95mmol)、HOBt(1.34g，9.95mmol)、DIEA(1640uL，9.95mmol)和30ml DMF，置于冰水浴下搅拌15min，将反应完全的1号瓶中反应液滴加入2号反应瓶中。HPLC监测反应原料是否消失，待反应完全后，进行HPLC制备分离。冷冻干燥得4.60g白色固体，收率68％。
第三步：化合物5c的合成
于50mL单口瓶中加入5b(1041.5mg，1.26mmol)、5％Pd/C(1056.7mg，100％m)和10ml DMF，添加氢气球并置换氢气，室温下反应约3h。HPLC监测反应完全后，超声除氢气。过滤，得滤液，得粗产品5c直接用于下一步反应。
第四步：化合物5d的合成
将粗产品5c置于冰水浴中，加入DIPEA(0.24mL,1.4mmol)，再加入化合物M2(7.0mL,1.4mmol)，加毕升至室温反应1h。HPLC监测反应完毕，液相纯化，得制备液，冻干得到803.5mg白色固体，收率64％。LC-MS：[M+H] +858.4。
第五步：化合物5e的合成
10ml单口瓶中加入5d(201.3mg，0.203mmol)、M2氨基异构(100.0mg，0.203mmol)、HATU(107.4mg，0.304mmol)、HOBt(38.2mg，0.304mmol)和5ml DMF，置于冰水浴下搅拌10min，滴加DIEA(248uL，1.50mmol)继续反应，HPLC监测反应完全后制备分离，制备液冻干得219.1mg黄色固体,收率84％，LC-MS：[M+H] +1275.5。
第六步：化合物Ln-D8的合成
50ml单口瓶中加入5d(219.1mg，0.170mmol)、溴化锌(765.3mg，3.40mmol)和20ml硝基甲烷，置于室温下搅拌。HPLC监测，待反应完全，反应液浓缩后HPLC制备纯化，制备液冻干，得黄色固体120.2mg，收率62％，LC-MS：[M+H] +1119.4。
实施例16：化合物Ln-D9的合成
Figure PCTCN2021122823-appb-000050
第一步：化合物1f的合成
于50mL单口瓶中加入1b(500.0mg，0.772mmol)、5％Pd/C(500.0mg，100％m)和10ml DMF，添加氢气球并置换氢气，室温下反应约3h。HPLC监测反应完全后，超声除氢气。过滤，得滤液，得粗产品1c。冰水浴下，向滤液中依次加入MC(280.1mg，0.9mmol)，DIEA(235mg，1.8mmol)，氮气保护，加完后升至室温反应1h，HPLC监控，液相制备纯化，冻干得化合物1f(268.9mg，86％)，MS：[M+H] +617.2。
第二步：化合物Ln-D9的合成
10ml单口瓶中加入1f(268.9mg，0.44mmol)、依喜替康甲磺酸盐(233.8mg，0.44mmol，外购)、HATU(190.5mg，0.50mmol)、HOBt(67.8mg，0.50mmol)和5ml DMF，置于冰水浴下搅拌10min，滴加DIEA(140uL，0.84mmol)继续反应，HPLC监测反应完全后制备分离，制备液冻干得254.3mg黄色固体,收率56％。LC-MS：[M+H] +1034.4。
实施例17：用于对照的化合物合成
参照专利CN111689980和WO2020063676中的方法合成得到下列化合物：
Figure PCTCN2021122823-appb-000051
Figure PCTCN2021122823-appb-000052
实施例18：偶联制备ADC药物通法
将通过初步的纯化后单体率大于95％的抗体Ab，使用超滤离心管换液至磷酸盐缓冲液中，浓度10mg/ml。加入20倍于抗体摩尔分子数的TCEP，室温下反应10h以打开抗体链间二硫键。加入20倍于抗体摩尔分子数的连接子-毒素，室温下反应2h。反应结束后，使用截留分子量为30KDa的超滤离心管换液至PBS中，并去除未偶联的payload。换液后的ADC样品使用0.22微米除菌过滤器过滤后备用。
将偶联用payload化合物Ln-D1、Ln-D2、Ln-D4、Ln-D5、Ln-D6、Ln-D7、Ln-D8和Ln-D9采用本实施例18所述偶联通法，与抗体分子Ab偶联(抗体分子Ab分别可为A：Trastuzumab抗体；B：Cetuximab抗体)。偶联产物经反相高效液相色谱测定其平均药物/抗体比(DAR)值。所得ADC药物分子相关信息及与payload分子的对应情况如下：
Figure PCTCN2021122823-appb-000053
Figure PCTCN2021122823-appb-000054
Figure PCTCN2021122823-appb-000055
实施例19：喜树碱手性衍生物的logP值测试
logP值与化合物的水溶性、透膜性、体内ADME过程以及化合物与受体的亲和力均存在密切的关系，在化合物透过生物膜的过程中具有关键作用。通过测试喜树碱药物在正辛烷(油)和水中的分配系数来得到logP值，以对药物进行研究，结果如下：
Figure PCTCN2021122823-appb-000056
Figure PCTCN2021122823-appb-000057
Figure PCTCN2021122823-appb-000058
Figure PCTCN2021122823-appb-000059
以上LogP结果可见，本发明的化合物具有更低的LogP值，水溶性更好。
实施例20：ADC的血浆聚集、降解测试
无菌取一定量的ADC样品，加入到已去除人IgG的人血浆中，使得ADC在血浆中的终浓度是0.6mg/ml,每种ADC制备三管，放置在37℃水浴中孵育，分别孵育0h、72h、7d后，取出ADC样品，每管加入ProteinA(MabSelect SuRe TMLX  Lot:#10221479GE，取PBS洗涤过的)100μL，垂直混合仪晃动吸附2h，经过洗涤、洗脱和Tris-HCl中和步骤，获得孵育后的ADC，对孵育特定时间的ADC样品进行SEC检测，测定聚集降解情况。
Figure PCTCN2021122823-appb-000060
Figure PCTCN2021122823-appb-000061
ADC-1的payload与已上市抗肿瘤药物Enhertu的一致，从ADC药物在血浆中聚集和降解的测试实验可知，喜树碱手性衍生物ADC的聚集和降解明显低于ADC-1，具有更好的物理性质和化学性质，能够在血浆中更稳定的存在。
实施例21：喜树碱手性衍生物的细胞活性测试
通过下列实验过程测定喜树碱手性衍生物的细胞毒性活性：将喜树碱手性衍生药物分别加入到A431、MDA-MB-468、SK-BR-3、Bxpc-3和SW620肿瘤细胞中，72小时后测定细胞存活率。基于细胞的体外实验用于测定细胞存活率、细胞毒性和本发明喜树碱药物诱导的细胞程序性死亡。
通过细胞增殖试验测定喜树碱药物的体外药效。CellTiter
Figure PCTCN2021122823-appb-000062
Aqueous One Solution Cell Proliferation Assay为商购的(Promega Corp.，Madison,WI)。
Figure PCTCN2021122823-appb-000063
One Solution Cell Proliferation Assay(a)是一种用比色法来检测细胞增殖和细胞毒性实验中的活细胞数量的检测试剂。此试剂含有一个新型的四唑化合物[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt；MTS]和一种电子偶联剂(phenazine ethosulfate；PES)。PES具有增强的化学稳定性，这使它可与MTS混合形成稳定的溶液。试剂中的MTS(Owen’s reagent)被细胞生物还原成为一种有色的甲臜产物，可直接溶解于培养基中。这种转化很可能是在代谢活跃的细胞中的脱氢酶产生的NADPH或NADH的作用下完成的。检测时，只需将适量的
Figure PCTCN2021122823-appb-000064
One Solution Reagent直接加入培养板孔的培养基中，孵育1–4小时，以酶标仪读取490nm的吸光度值。
在490nm处检测到的甲臜产物的量与培养中的活细胞数成正比。由于MTS的甲臜产物在组织培养基中可溶，CellTiter
Figure PCTCN2021122823-appb-000065
One Solution Assay与MTT或INT法相比操作步骤更少。
本发明中采用A431、MDA-MB-468、SK-BR-3、Bxpc-3和SW620作为体外药效检测的研究体系。在96孔板中，均匀接种适当的细胞密度，二氧化碳培养箱37℃孵育。24小时后，显微镜下确认细胞状态正常，进行喜树碱药物加药。培养基稀释喜树碱药物(起始浓度为1uM，7倍稀释，8个浓度点，最后两列分别为对照组(即细胞+培养基，无药物处理)和空白组(即无细胞，仅含有培养基，用于扣除背景)，充分混匀药物后，将药物加入对应的细胞孔中放入二氧化碳培养箱37℃孵育3天。3天后每孔加入20ul MTS(Promegm，G3581)反应2小时，酶标仪(Molecular Device，型号：SpectraMAX190)490nM读数。通过检测线粒体内的脱氢酶的活性，计算IC50评价喜树碱药物对细胞的增殖抑制作用。相应的喜树碱手性衍生物IC50(nM)测试结果如下：
Figure PCTCN2021122823-appb-000066
Figure PCTCN2021122823-appb-000067
Dxd为已上市癌症ADC药物Enhertu的活性成分，是高活性的喜树碱类药物。发明人通过细胞活性实验，证明本发明所述的喜树碱手性衍生物在代表性的肿瘤细胞BXPC-3、A431、SW620、MDA-MB-468和SK-BR-3中均表现出了与Dxd相当或更高的细胞活性。
实施例22：ADC的体外血浆稳定性
无菌取一定量的ADC样品，加入到已去除人IgG的无菌人血浆中，使得ADC的终浓度是0.6mg/ml，每种ADC制备三管，放置在37℃水浴中孵育，分别孵育0h、72h、7d后，取出ADC样品，每管加入ProteinA(MabSelect SuRe TMLX Lot:#10221479GE，取PBS洗涤过的)100μL，垂直混合仪晃动吸附2h，经过洗涤、洗脱和Tris-HCl中和步骤，获得孵育后的ADC，对孵育特定时间的ADC样品进行RP-HPLC检测其DAR值，以判定样品的血浆稳定性。实验结果表明，本发明的ADC在血浆孵育过程中几乎没有损失或者损失很少，在血浆中具有较好的稳定性，因为提前降解产生毒性的可能性较低。
Figure PCTCN2021122823-appb-000068
实施例23：ADC抗肿瘤细胞活性测试
本发明中采用MDA-MB-468、BT474作为体外药效检测的研究体系。均匀接种适当数量肿瘤细胞系96孔板中，放入二氧化碳培养箱孵育。24小时后，镜下确认细胞状态正常，进行药物加药处理。用培养基稀释药物(ADC药物起始浓度为500nM，稀释倍数为7倍，共8个浓度点，毒素与抗体的理论偶联比例(DAR)为8:1，实际偶联比例大致为7.5:1，因此毒素的起始浓度4.0μM，7倍浓度梯度稀释，8个浓度点)，混匀后加入对应的细胞孔中，其中最后两列分别为对照组(即细胞+培养基，无药物处理)和空白组(即无细胞，仅含有培养基，用于扣除背景)，放入二氧化碳培养箱37℃孵育5天。5天后，每孔加入20μL MTS(Promega，G3581)反应2小时，用酶标仪(Molecular  Device，型号：SpectraMAX190)读取波长为490nm的吸光值读数。通过检测线粒体内的脱氢酶的活性，计算IC50评价ADC药物对肿瘤细胞的增殖抑制作用。
Figure PCTCN2021122823-appb-000069
ADC-1为已上市抗肿瘤药物Enhertu。经过以上ADC细胞活性测试，本发明所述的喜树碱手性衍生物药物在通过连接单元L与抗体偶联后，在多个肿瘤细胞系中均表现出良好的抗肿瘤活性，与ADC-1相比，部分ADC活性更优异，具有极大的临床应用价值。
实施例24：ADC体内药效实验
本发明中建立了单体瘤NCI-N87荷瘤裸鼠模型，以评价ADC偶联药物的体内药效。即以5×10 6个NCI-N87细胞通过接种至4～6周龄的BALB/c裸鼠右肩侧皮下，待小鼠肿瘤体积平均大小生长至162mm 3，根据肿瘤大小随机分组，每组5只，在第0、7、14、21天分别静脉注射空白对照(缓冲溶液空白)和3mg/kg剂量的抗体药物偶联物ADC-1、ADC-2、ADC-3、ADC-5、ADC-6、ADC-7。肿瘤体积测量数据显示为测量时肿瘤平均体积，同时记录小鼠体重变化情况，用以观察ADC药物的体内药效和初步毒性。
Figure PCTCN2021122823-appb-000070

Claims (28)

  1. 一种如通式D所示的喜树碱类衍生物或其药学上可接受的盐或溶剂化物；
    Figure PCTCN2021122823-appb-100001
    其中：
    与
    Figure PCTCN2021122823-appb-100002
    相连的手性碳原子具有R构型的绝对手性；
    R选自氢原子、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基；
    R 1选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
    R 2选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
    X选自-C(O)-CR aR b-(CR 3R 4) m-O-、-C(O)-CR aR b-(CR 3R 4) m-NH-或-C(O)-CR aR b-(CR 3R 4) m-S-；
    R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    R b选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    或者，R a、R b及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
    或者，R 3、R 4及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    m选自0-4的整数。
  2. 根据权利要求1所述的喜树碱类衍生物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，其特征在于：R 1选自C 1-3烷基。
  3. 根据权利要求1所述的喜树碱类衍生物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，其特征在于：R 2选自C 1-3烷基或氟原子。
  4. 根据权利要求1-3中任一项所述的喜树碱类衍生物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，其特征在于：所述喜树碱衍生物具有如下式D 1所示结构：
    Figure PCTCN2021122823-appb-100003
    其中：
    与
    Figure PCTCN2021122823-appb-100004
    相连的手性碳原子具有R构型的绝对手性；
    R选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基；
    X选自-C(O)-CR aR b-(CR 3R 4) m-O-、-C(O)-CR aR b-(CR 3R 4) m-NH-或-C(O)-CR aR b-(CR 3R 4) m-S-；
    R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    R b选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    或者，R a、R b及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
    或者，R 3、R 4及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    m选自0-4的整数。
  5. 根据权利要求4所述的喜树碱类衍生物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，其特征在于：所述R选自C 1-3烷基。
  6. 根据权利要求4所述的喜树碱类衍生物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，其特征在于，所述X非限制性地选自以下结构：
    Figure PCTCN2021122823-appb-100005
    其中左侧波浪线与喜树碱衍生物部分相连，右侧波浪线与连接单元相连。
  7. 根据权利要求1所述的喜树碱类衍生物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式、或其药学上可接受的盐或溶剂化物，其特征在于，所述化合物非限制性地选自以下所示的结构：
    Figure PCTCN2021122823-appb-100006
    其中R选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基。
  8. 根据权利要求7所述的喜树碱类衍生物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式、或其药学上可接受的盐或溶剂化物，其特征在于所述化合物非限制性地选自以下所示的结构：
    Figure PCTCN2021122823-appb-100007
  9. 一种包括权利要求1-8任一所述喜树碱类衍生物的连接子-药物偶联物或其药学上可接受的盐或溶剂化物、或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式：
    Figure PCTCN2021122823-appb-100008
    其中：
    与
    Figure PCTCN2021122823-appb-100009
    相连的手性碳原子具有R构型的绝对手性；
    R选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基；
    R 1选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
    R 2选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
    X选自-C(O)-CR aR b-(CR 3R 4) m-O-、-C(O)-CR aR b-(CR 3R 4) m-NH-或-C(O)-CR aR b-(CR 3R 4) m-S-；
    R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    R b选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    或者，R a、R b及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
    或者，R 3、R 4及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    m选自0-4的整数；
    L为-L 1-L 2-L 3-L 4-。
  10. 根据权利要求9所述的连接子-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于：-L-为-L 1-L 2-L 3-L 4-，其中L 1端与配体Ab相连，L 4端与X相连。
  11. 根据权利要求9或10中所述的连接子-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于L 1非限制性地选自：
    Figure PCTCN2021122823-appb-100010
  12. 根据权利要求9或10中所述的连接子-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于：
    L 2选自：-NC(R 5R 6)C(O)、-NR 7(CH 2) oC(O)-、-NR 7(CH 2CH 2O) oCH 2C(O)-、-S(CH 2) pC(O)-或者化学键，其中o选自0-20的整数；p选自0-20的整数；
    R 5、R 6相同或者不同，且各自独立地选自氢原子、氘原子、烷基、取代烷基、氘代烷基、杂烷基、羧基、氨基、取代氨基；
    R 7选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基；
    L 1与L 2共用N原子。
  13. 根据权利要求9或10所述的连接子-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于：L 3选自由氨基酸构成的肽残基，其中任选氨基酸进一步被选自氘原子、卤素、羟基、氰基、氨基、硝基、羧基、烷基、取代烷基、烷氧基和环烷基或者取代环烷基中的一个或多个取代基所取代；
  14. 根据权利要求9或10所述的连接子-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于：L3优选由一个、两个或者多个选自苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸、丝氨酸(S)、谷氨酸(E)或者天冬氨酸(D)中的氨基酸形成的肽残基。
  15. 根据权利要求9或10所述的连接子-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于：
    L 4选自：-NR 8(CR 9R 10) q-、-C(O)NR 8-、-C(O)NR 8(CH 2) q-或者化学键，q选自0-6的整数；
    R 8、R 9和R 10相同或者不同，且各自独立地选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基。
  16. 根据权利要求9所述的连接子-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于：所述连接单元-L-非限制性地选自以下结构；
    Figure PCTCN2021122823-appb-100011
    Figure PCTCN2021122823-appb-100012
    Figure PCTCN2021122823-appb-100013
    其中：左侧波浪线与配体部分相连，右侧波浪线与X相连。
  17. 根据权利要求16所述的连接子-药物偶联物或者其药学上可接受的盐或溶剂化物，其特征在于：所述连接子-药物偶联物非限制性地选自以下结构：
    Figure PCTCN2021122823-appb-100014
    Figure PCTCN2021122823-appb-100015
    Figure PCTCN2021122823-appb-100016
    Figure PCTCN2021122823-appb-100017
    Figure PCTCN2021122823-appb-100018
    其中：
    1位碳原子具有R或S两种构型的绝对手性。
  18. 一种包括权利要求9-17任一所述连接子-药物偶联物的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于：所述配体-药物偶联物包含式Ⅰ所示的结构：
    Figure PCTCN2021122823-appb-100019
    其中：
    与
    Figure PCTCN2021122823-appb-100020
    相连的手性碳原子具有R构型的绝对手性；
    R选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、芳基、取代芳基或杂芳基；
    R 1选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
    R 2选自氢原子、氘原子、卤素、烷基、取代烷基、氘代烷基、环烷基烷基、烷氧基烷基、羧基、杂环基、芳基、取代芳基或杂芳基；
    X选自-C(O)-CR aR b-(CR 3R 4) m-O-、-C(O)-CR aR b-(CR 3R 4) m-NH-或-C(O)-CR aR b-(CR 3R 4) m-S-；
    R a选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    R b选自氢原子、氘原子、卤素、烷基、氘代烷基、卤代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基、取代芳基或杂芳基；
    或者，R a、R b及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    R 3、R 4相同或者不同，且分别独立地为氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
    或者，R 3、R 4及其所连接碳原子构成C 3-6环烷基、环烷基烷基或杂环基；
    Ab为配体单元，选自抗体、抗体片段、靶向蛋白、Fc-融合蛋白等；
    L为与Ab连接单元；X为药物部分修饰单元；
    m选自0-4的整数；n选自1-20的整数或小数。
  19. 根据权利要求18所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于：Ab为抗体，可通过其杂原子与连接单元形成连接键，所述抗体选自鼠源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体。
  20. 根据权利要求19所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于，所述的抗体或其抗原结合片段，非限制性地选自：抗EGFRvIII抗体、抗DLL-3抗体、抗PSMA抗体、抗CD70抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM-1抗体、抗LRRC15抗体、抗LIV-1抗体、抗CanAg/AFP抗体、抗cladin 18.2抗体、抗Mesothelin抗体、抗HER2(ErbB2)抗体、抗EGFR抗体、抗c- MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3(ErbB3)抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7-H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4抗体、抗TROP-2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P-cadherin抗体、抗GD3抗体、抗Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b抗体、抗CD19抗体、抗CD33抗体、抗CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD47抗体、抗CD138抗体、抗CD352抗体、抗CD25抗体或抗CD123抗体。
  21. 根据权利要求19或20中所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，其特征在于，所述配体-药物偶联物非限制性地选自以下结构：
    Figure PCTCN2021122823-appb-100021
    Figure PCTCN2021122823-appb-100022
    Figure PCTCN2021122823-appb-100023
    Figure PCTCN2021122823-appb-100024
    Figure PCTCN2021122823-appb-100025
    其中：
    Ab为配体单元；n选自1-20的整数或小数；
    1位碳原子具有R或S两种构型的绝对手性。
  22. 一种制备如权利要求9-17任一所述连接子-药物偶联物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物，或其药学上可接受的盐或溶剂化物的方法，其特征在于，包括如下步骤：
    Figure PCTCN2021122823-appb-100026
    通过连接单元L a与通式化合物D 1的取代反应，得到如通式L a-X-D 1所示的连接子-药物偶联物；
    其中：
    与
    Figure PCTCN2021122823-appb-100027
    相连的手性碳原子具有R构型的绝对手性；
    L 2、L 3、R、R 8、R 9、R 10、q及X如通式L-X-D所述。
  23. 一种制备如权利要求18-21任一所述配体-药物偶联物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物，或其药学上可接受的盐或溶剂化物的方法，其特征在于，包括如下步骤：
    Figure PCTCN2021122823-appb-100028
    通过还原的抗体、抗体片段或者其抗原结合片段与通式(L-X-D 1)偶联反应，得到如通式Ab-L-X-D 1所示配体-药物偶联物；
    其中：
    与
    Figure PCTCN2021122823-appb-100029
    相连的手性碳原子具有R构型的绝对手性；
    Ab、L、X、R及n如通式Ⅰ所述。
  24. 如权利要求1-23中任一项所述的喜树碱类衍生物、连接子-药物偶联物或配体-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于：所述药学上可接受的盐包括与结构式中酸性官能团形成的钠盐、钾盐、钙盐或镁盐等；或与结构中碱性官能团形成的醋酸盐、三氟乙酸盐、柠檬酸盐、草酸盐、酒石酸盐、苹果酸盐、硝酸盐、氯化物、溴化物、碘化物、硫酸盐、硫酸氢盐、磷酸盐、乳酸盐、油酸盐、抗坏血酸盐、水杨酸盐、甲酸盐、谷氨酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐或对甲苯磺酸盐。
  25. 一种药物组合物，其含有治疗有效量的权利要求1-24中任一项所述的喜树碱类衍生物、连接子-药物偶联物或配体-药物偶联物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，以及药学上可接受的载体、稀释剂或赋形剂。
  26. 一种包括权利要求1-24中任一项所述的喜树碱类衍生物、连接子-药物偶联物或配体-药物偶联物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化合物，以及药学上可接受的载体、稀释剂或者赋形剂，在制备用于治疗或预防肿瘤的药 物中的用途。
  27. 根据权利要求26所述的用途，其中所述的肿瘤为实体瘤和血液瘤。
  28. 根据权利要求27所述的用途，其中所述的肿瘤为乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、肺癌、结肠癌、直肠癌、结直肠癌、骨癌、皮肤癌、甲状腺癌、胰腺癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、多形性胶质细胞瘤、肉瘤、淋巴瘤或白血病。
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