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WO2025128837A1 - Anti-claudin18.2 antibody-camptothecin drug conjugate and pharmaceutical use thereof - Google Patents

Anti-claudin18.2 antibody-camptothecin drug conjugate and pharmaceutical use thereof Download PDF

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Publication number
WO2025128837A1
WO2025128837A1 PCT/US2024/059794 US2024059794W WO2025128837A1 WO 2025128837 A1 WO2025128837 A1 WO 2025128837A1 US 2024059794 W US2024059794 W US 2024059794W WO 2025128837 A1 WO2025128837 A1 WO 2025128837A1
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alkyl
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compound
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Yi Zhu
Shi ZHUO
Weili WAN
Yong Zhang
Gangrui LI
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Systimmune Inc
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Systimmune Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • 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
    • A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant
    • 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
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to the field of biopharmaceuticals, and specifically to an antibody-drug conjugate formed by an anti-Claudin!8.2 antibody and a camptothecin toxin, and to methods of preparation and use of the antibody-drug conjugate.
  • the present application also relates to linker-drug compounds that can be coupled with an antibody to form an antibody-drug conjugate.
  • Claudinl8.2 (or simply CLDN18.2) is a protein with tight junction function encoded by the Claudinl8 gene and is a member of the Claudin protein family.
  • Claudin family proteins are four-times transmembrane proteins containing two extracellular loops, N-terminal and C-terminal present in the cytoplasm, and four transmembrane regions. Claudin family proteins are important members involved in tight junctions, which play a role in connectivity between cells, and the aberrant expression of the proteins induces structural disruptions such as those of epithelial cells, leading to altered function and the triggering of a wide range of diseases.
  • Current research indicates that the mammalian Claudin family of proteins has 27 members, and humans contain all Claudin proteins except Claudinl3. The functions of each member are highly conserved, and each member is differentially expressed in different tissues and mediates different tumor types.
  • Claudinl8.2 The normal expression of Claudinl8.2 is limited to gastric mucosal cells and is not expressed in other normal tissues, and its overexpression leads to gastric, pancreatic, esophageal, and nonsmall-cell lung cancers, etc.
  • Claudinl8.1 a shear variant of Claudinl8.2, is selectively expressed in lung cells, although there is only an 8 amino acid difference between Claudinl8.1 and Claudinl8.1 in the first extracellular loop of the normal tissues.
  • Claudinl8.1 is selectively expressed in lung cells of normal tissues. Therefore, the highly restricted expression of Claudinl8.2 in normal tissues and its specific expression in tumor tissues make Claudinl8.2 an important potential target for tumor immunotherapy.
  • IMAB362 a monoclonal antibody drug developed by Ganymed Company of Germany
  • Ganymed's anti- Claudinl8.2 and CD3 dual antibody is in the preclinical stage, with more than 60% of Claudinl8.2 and CD3 dual targets in development, and dual antibodies targeting Claudinl8.2 and CD47/PD- L1/4-1BB are also in development.
  • the fastest progressing anti-Claudinl8.2 ADCs are in clinical phase l/II, including LM-302 from Lixin Pharmaceuticals, RC118 from Rongchang Biologies, and GMG901 from Konoia/Leep Bio.
  • the majority of ADCs targeting Claudinl8.2 are in clinical phase I or preclinical stage.
  • the present application discloses a safer and effective ADC drug targeting Claudinl8.2 with camptothecin as the toxin molecule with great developmental value and therapeutic potential.
  • Lu, Ln, and L13 are different from each other and are independently:
  • L2 has the structure shown in Formula A below.
  • L3 is present or absent, and when present, L3 is selected from PEG hydrophilic units: 0 , o is selected from an integer of 1-10; in one embodiment, o may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; in one embodiment, o is an integer of 2-8, such as 2, 3, 4, 5, 6, 7, or 8;
  • L4 is the enzymatically cleavable unit.
  • L5 is the connection unit.
  • the chiral carbon atom No. 1 attached to N in formula I has an absolute chirality of R or S;
  • R is selected from a hydrogen atom or a C1-C6 alkyl group.
  • Ri is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted Cl- C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3-7 meta-heterocyclic, substituted 3-7 meta-heterocyclic, C6-C10 aryl, substituted C6- C10 aryl, 5-10 meta-heteroaryl, substituted 5-10 heteroaryl.
  • Ri is selected from C1-C6 alkyl.
  • R2 is selected from a hydrogen atom, a halogen or a C1-C6 alkyl group. In one embodiment, R2 is selected from halogen.
  • X selected from -C(O)-CRaR b -(CR 3 R4)m-O-, -C(O)-CR a R b -(CR3R 4 ) m -NH- or -C(O)-CRaRb- (CR 3 R4)m-S-. In one embodiment, X is selected from -C(O)-CRaRb-(CR 3 R4)m-O-.
  • R a and R b are each independently selected from a hydrogen atom, a Cl- C6 alkyl, a halo-Cl-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl or a C6-C10 aryl C1-C6 alkyl.
  • R 3 , R 4 are the same or different and are independently hydrogen atoms, deuterium atoms, halogens, C1-C6 alkyl, halogenated Cl -C6 alkyl, deuterated Cl -C 6 alkyl, C1-C6 alkoxy, hydroxyl, amino, cyano, nitro, hydroxy C1-C6 alkyl, C3-C8 cycloalkyl, a 3-7-membered heterocyclic group, a substituted 3-7-membered heterocyclic group, respectively.
  • m may be 0 or 1 ; nl, n2, and n3 are each independently selected from any integer or any decimal number from 0-10, nl, n2, and n3 are not simultaneously 0, and 1 ⁇ n l + n2 + n3 ⁇ 10.
  • the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is characterized in that Ab is an antibody and comprises an IgGl heavy chain and a light chain.
  • the antibody comprising said IgGl heavy chain and K light chain specifically recognizes a human Claudinl8.2 protein.
  • the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I is characterized in that the antibody comprises a heavy chain and a light chain.
  • the light chain comprises CDRL1, CDRL2 and CDRL3 each having an amino acid sequence encoded by a nucleic acid coding sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, said heavy chain comprising CDRH1, CDRH2 and CDRH3 each having an amino acid sequence encoded by a nucleic acid sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.
  • the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I is characterized in that the antibody comprises a heavy chain and a light chain.
  • the heavy chain comprises a heavy-chain variable region having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, and said light chain comprising a light-chain variable region having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.
  • the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I is characterized in that the antibody comprises a heavy chain and a light chain.
  • the heavy chain variable region comprising a heavy chain variable region encoded by a nucleic acid sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13, and said light chain variable region encoded by a nucleic acid sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 19.
  • the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I is characterized in that Ab comprises a heavy chain having an amino acid sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, and a light chain having an amino acid sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11.
  • the ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof as shown in general formula I is characterized in that said X may be the following structures or isomers thereof:
  • the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I is characterized in that L4 may be peptide residues comprising amino acids.
  • said amino acid is further substituted with one or more substituents selected from one or more of deuterium atoms, halogens, hydroxyl, cyano, amino, nitro, carboxyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy and C3-C8 cycloalkyl or substituted C3-C8 cycloalkyl.
  • the ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof as shown in general formula I are characterized as follows:
  • the ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof are characterized in that said linking units - Ln -L2 -L3 -L4 -L5 -, -L12 -L2 -L3 -L4 -L5 - or -L13 -L2 -L3 -L4 -L5 - are not identical to each other and are independently and may be the following structures:
  • -Ln -L2 -L3 -L4 -L5 -L12 -L2 -L3 -L4 -L5 - or -L13 -L2 -L3 -L4 -L5 - are not the same as each other and are each independently and non-limitingly selected from: wherein:
  • R5, Rs and R7 are each independently selected from a hydrogen atom
  • the carbon atom #2 attached to N has absolute chirality of R or the S configuration
  • nl, n2, and n3 is independently chosen from any integer or any decimal number from 0 to 10, nl, n2, and n3 are not simultaneously 0, and 1 ⁇ nl + n2 + n3 ⁇ 10.
  • the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof is characterized in that said Ac may be glycine, (D/L) alanine, (D/E) leucine, (D/E) isoleucine, (D E) valine, (D/E) phenylalanine, (D/E) proline, (D/E) tryptophan, (D/E) serine, (D/E) tyrosine, (D/E) cysteine, (D/L) cystine, (D/E) arginine, (D E) histidine, (D/E) methionine, (D/E) asparagine, (D/L) glutamine, (D/E) threonine, (D/E) aspartic acid, (D/L) glutamic acid, natural or non-natural amino acid derivatives or the following structures or isomers thereof.
  • said Ac may be glycine, (D/L
  • R is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated Cl -C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, substituted 5-10 heteroaryl; In one embodiment R is selected from a hydrogen atom or a C1-C6 alkyl group;
  • Ri is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted Cl- C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3-7 meta-heterocyclic, substituted 3-7 meta-heterocyclic, C6-C10 aryl, substituted C6- C10 aryl, 5-10 meta-heteroaryl, or substituted 5-10 heteroaryl;
  • Ri is selected from C1-C6 alkyl
  • R2 is selected from hydrogen atom, deuterium atom, halogen, C1 -C6 alkyl, substituted Cl - C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3-7 meta-heterocyclic, substituted 3-7 meta-heterocyclic, C6-C10 aryl, substituted C6- CIO aryl, 5-10 meta-heteroaryl, or substituted 5-10 heteroaryl;
  • R a and Rb are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl group C1-C6 alkyl group, a C1-C6 alkoxy group C1-C6 alkyl group, a 3-7 metaheterocyclic group, a substituted 3-7 metaheterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl, substituted 5-10 heteroaryl;
  • Ra and Rb are each independently selected from a hydrogen atom, a C1-C6 alkyl, a halo-Cl-C6 alkyl, a C1
  • the 1 -position chiral carbon atom may be in R or S configuration; m is selected from 0 or 1.
  • the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is characterized in that: said structural formula d may be the following compounds:
  • linker-drug compound or a pharmaceutically acceptable salt or solvate thereof having the structure shown in formula III below:
  • R is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated Cl -C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, substituted 5-10 heteroaryl;
  • R a is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3-7 meta-heterocyclic group, substituted 3-7 meta-heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 meta-heteroaryl, substituted 5-10 heteroaryl;
  • Rb is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3-7 meta-heterocyclic group, substituted 3-7 meta-heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 meta-heteroaryl, substituted 5-10 heteroaryl;
  • R a and Rb are each independently selected from a hydrogen atom, a Cl- C6 alkyl, a halo-Cl-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl, a C6-C10 aryl; In one embodiment, R a and Rb are each independently selected from a hydrogen atom, a methyl group, an ethyl group, a trifluoromethyl group, a cyclopropylmethyl group, a phenyl group;
  • R a , Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocycloalkyl group, a substituted 3-7 heterocycloalkyl group;
  • Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group (e.g., a C3-C5 cycloalkyl group);
  • L3 is absent or present, and when L3 is present, it is selected from 0 , o is selected from an integer from 1 to 10; in one embodiment, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • the 1 -position or 2-position chiral carbon atom each independently has R or S configuration
  • Ac is a hydrophilic structural unit; m is selected from 0 or 1;
  • said linker-drug compound or a pharmaceutically acceptable salt or solvate thereof is used to couple with ligand Ab to form said ligand-camptothecin derivative conjugates of formula I or formula II.
  • L may comprise a succinimide group.
  • the ligand-drug conjugate may undergo hydrolysis under readily hydrolyzable conditions, with the site of hydrolysis being the butanediimide moiety of the linker unit.
  • the site of hydrolysis being the butanediimide moiety of the linker unit.
  • the succinimide groups are completely non-hydrolyzed, i.e., the succinimide o 0 groups are all in the closed ring form o or 0 ;
  • all or some of these succinimide moieties may be in a closed-loop form, partially open-loop form, or all or some of them may be in an open-loop form.
  • linker-drug compounds or pharmaceutically acceptable salts or solvates thereof as disclosed herein are characterized in that: said Ac has the structure shown in formula B below, the
  • Z may be the group consisting of one or more of a hydrophilic structural carboxyl group, a phosphoric acid, a polyphosphoric acid, a phosphite, a sulfonic acid, a sulfmic acid, or a polyethylene glycol (PEG);
  • Y' is optionally a scaffold connecting the amino group to Z;
  • Y' is a Cl- C6 alkylene group (e.g., methylene); Ac is connected to the 2-position carbon that has been labeled in structural formula I by means of a bracket Y.
  • the linker-drug compounds or pharmaceutically acceptable salts or solvates thereof is characterized in that said Ac may be glycine, (DA) alanine, (D L) leucine, (D L) isoleucine, (D/L) valine, (DI) phenylalanine, (D/L) proline, (D/L) tryptophan, (D/L) serine, (D/L) tyrosine, (D/L) cysteine, (D/L) cystine, (D/L) arginine, (D/L) histidine, (D/L) methionine, (D/L) asparagine, (D/L) glutamine, (D/L) threonine, (D/L) aspartic acid, (D/L) glutamic acid, natural or non-natural amino acid derivatives, or the following structures.
  • the linker-drug compounds or pharmaceutically acceptable salts or solvates thereof are characterized in that said Ac may be
  • linker-drug compounds or pharmaceutically acceptable salts or solvates thereof is characterized in that said linker-drug compounds are non-limitingly selected from the following structures or isomers thereof.
  • o is selected from integers of 1-10; for example, o may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • linker-drug compounds or pharmaceutically acceptable salts or solvates thereof disclosed herein can be used as intermediates for coupling with the ligand Ab to form ligand-camptothecin derivatives conjugates of formula I or formula II.
  • the application discloses methods of preparing a ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I or general formula II.
  • the method comprises the steps of conjugating the reduced antibody or antigen-binding fragment thereof with the linker-drug compound by the coupling reaction to provide the ligand-camptothecin derivative conjugates as shown in general formula I or general formula II:
  • the 1-position, 2-position, or 3-position chiral carbon atoms have absolute chirality in R- or S-configuration;
  • the ligand-camptothecin derivative conjugates, the linker-drug compounds, or pharmaceutically acceptable salt or solvate thereof is characterized in that said pharmaceutically acceptable salt comprises a sodium salt, a potassium salt, a calcium salt, or a magnesium salt formed with an acidic functional group in the structural formula, and acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate, formed with the basic functional group in the structure.
  • said pharmaceutically acceptable salt comprises a sodium salt, a potassium salt, a calcium salt, or a magnesium salt formed with an acidic functional group in the structural formula, and
  • the application discloses the ligand-camptothecin derivative conjugates, and the linker-drug compounds as described herein and their a pharmaceutically acceptable salts or solvates thereof, pharmaceutical compositions and pharmaceutical formulations for use in the treatment or prevention of cancer or tumors.
  • the application discloses methods of treating or preventing cancer or tumors.
  • the method comprises the step of administering to a subject in need thereof a prophylactically or therapeutically effective amount of a ligand-camptothecin derivative conjugate, a linker-drug compound, or their pharmaceutically acceptable salt or solvate thereof, as described herein, the pharmaceutical composition or pharmaceutical formulations.
  • the cancer or tumor expresses Claudinl8.2.
  • the cancer or tumor may be adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urothelial cancer, bladder cancer, hepatocellular carcinoma, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, breast cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, or sarcoma.
  • the cancer or tumor are solid tumors. In one embodiment, the cancer or tumor are hematological tumors such as lymphoma and leukemia.
  • Figure 1 shows the SEC-HPLC detection of ADC-6 aggregation (A) and ADC-107 aggregation (B);
  • Figure 2 shows the RP-HPLC detection of drug-antibody coupling ratio (DAR) for ADC-6 (A) and ADC- 107 (B);
  • FIG 3 shows that ADC-6 maintains an equal affinity of the 51O3F3-BSM antibody to the antigen Claudinl8.2 (A) and ADC-107 also maintains an equal affinity of the 64C9 antibody to the antigen Claudinl8.2 (B);
  • Figure 5 shows the in vitro activity of tumor suppression by ADC-6 and ADC- 107 in the experimental model of a human embryonic kidney cell line expressing Claudinl8.2, HEK293T- 18.2 (A); a human pancreatic cancer cell line expressing Claudinl 8.2, BxPC-3 #A9F (B); a human gastric cancer cell line expressing Claudinl8.2, SNU-5 (C); and another human gastric cancer cell line expressing Claudinl 8.2, SNU-16 (D);
  • a ligand unit is a targeting agent that binds specifically to a target component.
  • Said ligand is capable of binding specifically to a cellular fraction or to a cellular component or to other target molecules of interest.
  • the target portion or target is typically on the surface of the cell.
  • the ligand unit serves 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, and 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 reagent
  • it may be a peptide or polypeptide, or a non-protein molecule.
  • targeting reagents include interferons, lymphokines, hormones, growth and colony stimulating factors, vitamins, nutrient transporter 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 a sulfur atom of a cysteine residue that forms an interchain disulfide bond of the antibody.
  • the sulfur atom is a sulfur atom of a cysteine residue that has been imported into the ligand unit, which forms an interchain disulfide bond of the antibody.
  • the sulfur atom is a sulfur atom that has been introduced into a cysteine residue of the ligand unit (e.g., by targeted mutagenesis or chemical reaction).
  • the linker-bound sulfur atom is selected from a cysteine residue that forms the interchain disulfide bond of the antibody or a cysteine residue that has been introduced into the ligand unit (e g., by targeted mutagenesis or chemical reaction).
  • the EU index numbering system in accordance with Kabat ⁇ [Kabat E.Aet al, (1991)] Sequences of proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242 ⁇ .
  • Antigens involved include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (such as are known or foreseen to have functionality), lymphokines, cytokines, molecules involved in the regulation of cellular cycling, molecules involved in angiogenic molecules (if known or predicted to be functional).
  • TAA tumor-associated antigens
  • cell survival regulators such as are known or foreseen to have functionality
  • lymphokines such as are known or foreseen to have functionality
  • lymphokines such as are known or foreseen to have functionality
  • cytokines molecules involved in the regulation of cellular cycling
  • angiogenic molecules if known or predicted to be functional
  • Tumor- associated factors may be cluster differentiation factors (e.g., CD proteins).
  • Antibodies applied in antibody drug couplings include, but are not limited to, antibodies directed against cell surface receptors and tumor-associated antigens.
  • tumor-associated antigens are well known in the industry and can be prepared by methods and information for antibody preparation well known in the industry.
  • 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.
  • tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancer cells. Identification of such tumor-associated factors can greatly enhance the specific targeting properties of antibody -based cancer therapies.
  • Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in publicly available databases, such as Genbank, and antibody-targeted tumor-associated antigens include all amino acid sequence variants and isoforms having at least 70%, 80%, 85%, 90%, or 95% homology to sequences identified in the references or having biological properties and characteristics that are identical to the sequences of the tumor-associated antigens identified in the cited documents.
  • cancer refers to a physiological condition or disease characterized by dysregulated cell growth.
  • Tumor includes cancer cells.
  • autoimmune disease refers to diseases or disorders that originate in tissues or proteins that target an individual's own body.
  • d refers to cytotoxic drugs, denoted by d, which are chemical molecules that have a strong ability to disrupt normal growth in tumor cells. Cytotoxic drugs can in principle kill tumor cells at sufficiently high concentrations, but due to a lack of specificity, they can kill tumor cells while causing apoptosis of normal cells, leading to serious side effects.
  • toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g., radioisotopes of At 211 , 1 131 , 1 125 , Y 90 , R 6 186 , R 6 188 , Sm 153 , Bi 212 , P 32 and Lu 176 ), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs.
  • radioisotopes e.g., radioisotopes of At 211 , 1 131 , 1 125 , Y 90 , R 6 186 , R 6 188 , Sm 153 , Bi 212 , P 32 and Lu 176
  • toxic drugs e.g., chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs.
  • junctions including extensions, spacers and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1.
  • the junctions can be "cleavable junctions" that facilitate the release of the drug in the cell.
  • acid-unstable junctions e g., hydrazone
  • protease-sensitive e.g., peptidase-sensitive
  • photo-unstable junctions e.g., dimethyl junctions, or disulfide-containing junctions
  • U.S. Patent No. 5, 208, 020 can be used.
  • linkers or “linkers of antibody-drug couplers” can be categorized into two types: unbreakable linkers and breakable linkers.
  • unbreakable linkers For antibody-drug couplers containing an unbreakable linker, the mechanism of drug release is as follows: after the coupler binds to the antigen and is endocytosed by the cell, the antibody is enzymatically cleaved in the lysosome, releasing an active molecule consisting of the small drug molecule, the linker, and the amino acid residues of the antibody.
  • Chemically sensitive linkers can be selectively broken due to differences in the nature of the plasma and cytoplasm or tumor microenvironment. Such properties include pH, glutathione concentration, etc. pH- sensitive linkers, which are relatively stable in the neutral or weakly alkaline environment of blood (pH 7.3-7.5), will be hydrolyzed within the weakly acidic tumor microenvironment (pH 5.0-6.5) and lysosomes (pH 4.5-5.0), e.g., stilbenes, carbonates acetals, and ketones. Antibody-drug couplings based on such linkers typically have a short half-life (2-3 days) due to the limited plasma stability of acid breakage linkers.
  • Peptide linkers can be efficiently severed by lysosomal proteases such as tissue protease (Cathepsin B). This peptide linkage is thought to be very stable in the plasma circulation due to the unfavorable extracellular pH and serum protease inhibitors resulting in proteases that are normally inactive outside the cell. In view of the high plasma stability and good intracellular break selectivity and effectiveness, enzyme-unstable linkers are widely used as breakable linkers for antibody-drug couplings.
  • tissue protease Cathepsin B
  • antibody-drug conjugate refers to the attachment of an antibody to a biologically active drug by means of a stabilized linkage unit.
  • ligand-drug coupling preferably antibody-drug conjugate (ADC) refers to the attachment of a monoclonal antibody or antibody fragment to a biologically active toxic drug via a stabilized linkage unit.
  • 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-m ethylpentyl, 3 -methylpentyl, 4-methylpentyl, 2, 3 -dimethylbutyl, n-heptyl, 2-methylhexyl, 3 -methylhexyl, 4-
  • substituted alkyl refers to saturated straight-chain or branched-chain aliphatic hydrocarbon radicals having two residues derived from the removal of two hydrogen atoms from the same carbon atom of the parent alkyl or from two different carbon atoms, which are straightchain or branched-chain groups containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, and more preferably containing 1 to 6 carbon atoms.
  • alkoxy refers to -O-(alkyl) and -O-(cycloalkyl), where alkyl or cycloalkyl is as defined above.
  • C1-C6 alkoxides include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexoxy.
  • the alkoxy group may be optionally substituted or unsubstituted.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptatrienyl, cyclooctyl, and the like; and multi-cyclic cycloalkyl groups include cycloalkyl groups of spirocycles, thickened rings, and bridged rings.
  • R' and R" When R' and R" are attached to the same nitrogen atom, they may form a 3-, 4-, 5-, 6- or 7-membered ring with that nitrogen atom, for example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.
  • hydroxyl refers to the -OH group.
  • halogen means fluorine, chlorine, bromine or iodine.
  • amino means -NH 2 .
  • nitro means -NO 2 .
  • amide group means -C(O)N(alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl is as defined above.
  • carboxylate group means -C(O)O(alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl is as defined above.
  • the present application also includes various deuterated forms of Formula I.
  • Each of the available hydrogen atoms attached to the carbon atom may be independently replaced by a deuterium atom.
  • a person skilled in the art can synthesize the deuterated form of Formula I with reference to the relevant literature.
  • Commercially available deuterated starting materials may be used in the preparation of the deuterated form of Formula I, or they may be synthesized by conventional techniques using deuterated reagents, non-limiting examples of deuterated reagents include deuteroborane, tri deuteroborane tetrahydrofuran solution, lithium-aluminum deuterohydride, ethyl iodine deuteride, methylene iodide deuteride, and the like.
  • antibody refers to immunoglobulins, which are tetrapeptide chains consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in the composition and arrangement of the amino acids in the constant region of the heavy chain, and therefore differ in their antigenicity. Accordingly, immunoglobulins can be categorized into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, with corresponding heavy chains of p-chain, 8-chain, '/-chain, a-chain and s-chain, respectively.
  • the same class of Ig can be further divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of disulfide bonds of the heavy chain, such as IgG can be divided into IgGl, IgG2, IgG3, IgG4.
  • the light chain is divided into K-chain or Z-chain through the differences in the constant region.
  • Each of the five classes of Ig may have either a K chain or a z chain.
  • solvates refers to the formation of a pharmaceutically usable solvent compound of the ligand-drug conjugate of the present application with one or more solvent molecules.
  • solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.
  • drug load refers to the average amount of cytotoxic drug loaded on each antibody in Formula I. It can also be expressed as a ratio of drug amount to antibody amount, and the drug load can range from 0-12, preferably 1-10 cytotoxic drugs (d) per antibody (Ab).
  • the drug load is denoted as n, which exemplarily may be the mean of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, for example.
  • the average amount of drug per ADC molecule after coupling reaction can be identified by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA, and HPLC.
  • the cytotoxic drug is coupled to the open cysteine sulfhydryl-SH and/or the sulfhydryl-SH of the cysteine residue of site-specific mutation between the antibody chains through a linker unit, and in general, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than or equal to a theoretical maximum.
  • the loading of ligand-cytotoxic drug conjugates can be controlled by using the following non-limiting methods, including:
  • pharmaceutically acceptable salt refers to the salt of the ligand-drug conjugate of the present application, or the salt of the compound described in the present application, which is safe and effective when used in mammals and is biologically active as desired.
  • the ligand-drug conjugate compounds of the present application contain at least one carboxyl group and can therefore form salts with bases.
  • Non-limiting examples of pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts or magnesium salts.
  • pharmaceutically acceptable salt refers to the salt of the ligand-drug conjugate of the present application, or the salt of the compound described in the present application, which is safe and effective when used in mammals and is biologically active as desired.
  • the ligand-drug conjugate of the invention contains at least one amino group and can therefore form salts with acids.
  • Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydriodate, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pyrite, hydrophosphate, dihydrophosphate, salicylate, citric acid hydrochloride, tartaric acid salt, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.
  • acidic amino acid refers to amino acids with an isoelectric point of less than 7. Acidic amino acid molecules often contain one or more acidic groups, such as carboxyl groups, which can be effectively ionized into negative ionic forms in the structure to increase hydrophilicity. Acidic amino acids can be natural or unnatural.
  • Natural amino acids refers to biologically synthesized amino acids. Natural amino acids are generally L-types, but there are a few exceptions, such as glycine, that comes naturally and biosynthesized by living organisms.
  • amino acids refers to amino acids obtained by synthetic means.
  • compound SM-2 (synthesized according to the method published in CN108452321 A) (40 g, 96 mmol, 1.0 eq), tri ethylamine (26.7 mL, 2.0 eq), toluene (400 mL) were added, and the reaction was carried out by refluxing the compound at a temperature of 120 °C for 2 h. The reaction was monitored to be almost completely reacted by TLC, and then the solvent was spun off by reducing the temperature to 50 °C and decompression. The solvent was removed by spinning under reduced pressure at 50 °C.
  • Step 3 Compound 1c lb (900 mg, 1.39 mmol) was added to a 25 mL flask, and after 15 mL of DMF was dissolved, 900 mg of 5% Pd/C was added, and the reaction was hydrogenated for 2 h. The reaction was completed, and the filtrate was filtered to obtain the filtrate, which was used directly for the next step of the reaction without purification.
  • reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound le, which was lyophilized to obtain le (210 mg); LC-MS: [M+H] + 1221.6.
  • reaction solution (T) 7a and 15 mL of DMF in a 50 mL flask, dissolve and clear, and then add DBU (1.68 g, 11 mmol) in an ice-water bath and react for 1 h. This was recorded as reaction solution (T);
  • Step 2 Compound 8e-l and Compound 8e-2
  • Step 1
  • Step 5 Compound lOe lOd (400 mg, 0.48 mmol), M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (104 mg, 0.48 mmol) and 15 mL of DMF were added to a 50 mb flask in an ice-water bath and DIPEA (330 uL, 2.0 mmol) was added, and the reaction was allowed to proceed for 3 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to give the preparative solution of compound lOe. The reaction was carried out at room temperature for 3 h.
  • Step 1 Compound Ila
  • Step 3 Compound 11c lib (2.0 g, 2.9 mmol) was added to a 25 mL flask, and after 25 mL of DMF was dissolved and cleared, 2.0 g of 5% Pd/C was added, and the hydrogenation reaction was carried out for 3 h. After completion of the reaction, it was filtered to obtain the filtrate, which was used directly for the next step of the reaction without purification.
  • Step 5 Compound lie lid (800 mg, 0.96 mmol), M5 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol) and 30 mb of DMF were added to a 50 mL flask in an ice-water bath and DIPEA (660 uL, 4.0 mmol) was added, and the reaction was allowed to proceed for 4 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to give the preparative solution of compound lie. The reaction was carried out at room temperature for 4 h.
  • Step 5 Compound 12e 12d (400 mg, 0.47 mmol), M5 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt
  • Step 5 Compound 14e 14d (400 mg, 0.46 mmol), M5 (235 mg, 0.46 mmol), PyBOP (245 mg, 0.46 mmol), HOBt
  • reaction solution (T) 15a and 15 mL of DMF in a 50 mL flask, dissolve the solution, and then add DBU (1.68 g, 11 mmol) in an ice-water bath and react for 1 h. The reaction was recorded as reaction solution (T);
  • reaction solution (T) In a 50 mL flask, 17a (5 g, 8.86 mmol) and 15 mL ofDMF were added, dissolved and cleared, and then, in an ice-water bath, DBU (1.53 g, 10 mmol) was added and the reaction was carried out for 1 h, which was recorded as reaction solution (T);
  • HPLC high performance liquid chromatography
  • reaction solution (T) 1 mL of DMF in a 50 mL flask, dissolve the solution, and then add DBU (1.42 g, 9.3 mmol) in an ice-water bath and react for 1 h. The reaction was recorded as reaction solution (T);
  • reaction solution (T) 20a (4 g, 7.6 mmol) and 10 mL of DMF were added to a 50 mL flask, and after dissolving and clearing, DBU (1.39 g, 9.1 mmol) was added in an ice-water bath and the reaction was carried out for 1 h, which was recorded as reaction solution (T);
  • Step 4 Compounds 20e-l and 20e-2 20d (400 mg, 0.47 mmol), ezetimibe methanesulfonate (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF were added to a 50 mL vial, and DIPEA (248 uL, 1.5 mmol) was added to the vial under an ice-water bath. DIPEA (248 uL, 1.5 mmol) was added in an ice-water bath, and the reaction was brought to room temperature for 2 h.
  • Step 5 Compound 20A
  • 8A 100 mg, 0.078 mmol
  • zinc bromide 352 mg, 1.57 mmol
  • 5 mL of nitromethane 5 mL
  • the solvent was concentrated under reduced pressure and the crude product was obtained.
  • Step 1 Compound SM3-1
  • Step 2 Compound (A)-tert-butyl 2-hydroxy-l, 5-pentanedioate
  • Step 1 Compound SM4-1
  • Step 3 Compound SM4 SM4-2 (59 g, 83 mmol) was added into a 2000 mL flask, and after 1000 mL MeOH was dissolved, K2CO3 (11.75 g, 85 mmol) was added, and the reaction was carried out at room temperature for 4 h.
  • Step 3 Compound SM5 (see Org. Lett, 2006, 8, 3387-3390.)
  • Compound 45 was synthesized according to the method provided in Example 58 of CN104755494A.
  • compound 48a (1.0 g, 0.8 mmol, 1.0 eq) was added, 35 mL of nitromethane, dissolved and then added zinc bromide (3.64 g, 16 mmol, 20.0 eq), the reaction was carried out in an oil bath at 40 °C (stabilized in advance by preheating) for 30 min, and then the pump was concentrated at 45 °C under reduced pressure in a water bath to remove the nitromethane, resulting in a yellow residue solid (monitored by HPLC). After acid preparation, the prepared solution was obtained, and the prepared solution was concentrated by pump decompression water bath at 35 °C to remove acetonitrile and lyophilized to obtain compound 48A (786 mg) in 90% yield.
  • Step 1
  • HPLC high performance liquid chromatography
  • Step 3 Compound 54B
  • 54b 100 mg, 0.079 mmol
  • zinc bromide 357 mg, 1.59 mmol
  • 5 mL of nitromethane 5 mL
  • the reaction was carried out at 40 °C for 1 h.
  • the solvent was concentrated under reduced pressure and removed to obtain the crude product.
  • Step 1 Compounds 55a and 55b 20d (400 mg, 0.47 mmol), M9 (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt
  • Step 1 Synthesis of compound 58a
  • compound 58a (11 g, 21 mol) was added sequentially in a 250 mL flask, 120 mL of formic acid was dissolved, 30 mL of formaldehyde (40% aqueous solution) was added to the resulting bright yellow solution, and the reaction was heated up to 50 °C for 1 h.
  • the reaction was monitored by TLC to see if the reaction was complete, and then the reaction solution was cooled down to room temperature, and then the reaction solution was purified by preparative high- performance liquid chromatography (acetonitrile/water), and the target peak was collected.
  • the target peak after removing acetonitrile under reduced pressure, lyophilized to give compound 58b about 4.5 g, yellow powdery solid, yield about 40%, MS m/z : [M+H] + 540.6.
  • Step 1 Compound 59a
  • Step 1 Compound 61a
  • Step 2 Compound 62
  • Compound 62a 100 mg, 0.081 mmol, 1.0 eq
  • ZnBr? (364 mg, 1.62 mmol, 20.0 eq)
  • CH3 NO2 10 mL
  • the reaction was raised to 40 °C for 0.5 h, then the reaction was stopped, and the reaction solution was dried directly at 45 °C by spindrying under reduced pressure to obtain a yellow solid, which was sampled for HPLC monitoring.
  • the reaction was monitored by HPLC.
  • the spin-dried solid was directly purified by HPLC preparation, and the product preparation was collected and lyophilized to obtain compound 62 (61 mg);
  • LC-MS: [M+H] 1 1093.4.
  • Step 1 Compounds 65a and 65b
  • HPLC high performance liquid chromatography
  • Step 1 Compound 68a 12d (400 mg, 0.47 mmol), 58 (211.7 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt
  • Step 1
  • Step 1 Compounds 70a and 70b
  • Step 1 Synthesis of compound 73a
  • Step 1 Synthesis of compound 79a
  • Expi293 (Shanghai 0PM Biotechnology Co., Ltd.) suspension cells were used to express 5103F3-BSM antibody.
  • cells were inoculated at a density of 0.9* 10 6 cells/mLin 1 L shake flasks containing 300 mL ofOPM-293 CD05 Medium (81075-001, Shanghai Oppermax Bio-technology Co., Ltd.), and cultured overnight at 37 °C, 5% CO2, and 120 rpm on a cell culture shaker.
  • PEI-MAX was used to transfect the antibody expression plasmid, in which the mass ratio of plasmid:PEI-MAX was 1:3.
  • OPM-293 ProFeed supplement was added at 5% (v/v) on the first day after transfection, and then at 5% (v/v) on the third day after transfection, and then centrifuged to collect the supernatant on the sixth day after transfection.
  • the cell expression supernatants obtained were collected and eluted with 0.05 M sodium acetate buffer (pH 3.6) by a Protein A affinity chromatography column (UniMab 50, Suzhou Nano
  • the captured antibody was adjusted to pH 7.0 with 1 M Tris-HCl (pH 8.8) at 0.7/10 (v/v), and then passed through a gel filtration chromatography column SEC (Superdex 200 Increase, Cytiva) to remove impurities such as polymers, while the antibody buffer was replaced with 20 mM sodium acetate buffer (pH 6.0).
  • the anti-Claudin 18.2 antibody was replaced in 20 mM NaAc-HAc, pH 6.0 buffer, concentrated or diluted, and the anti-Claudin 18.2 antibody was concentrated to a protein concentration of 5 mg/mL.
  • the white powder of linker plus the drug was dissolved in DMA to 10 mg/mL and set aside.
  • 15- fold TCEP was added according to the ratio of molecules and reacted at room temperature for 2 h.
  • 16-fold ligand plus drug solution was added according to the ratio of molecules and reacted at room temperature for 2 h.
  • the ligand plus drug that had not been coupled with the anti-Claudinl8.2 antibody was removed by ultrafiltration using a 30 KDa ultrafiltration centrifuge tube. After the reaction, use 30 KDa ultrafiltration centrifuge tube to remove the linker plus drug that is not coupled with anti-Claudinl8.2 antibody.
  • Samples of the antibody 64C9-drug conjugate were prepared by coupling 64C9 antibody with payload after the cell expression and purification by Protein A affinity chromatography and molecular sieve chromatography, the anti-Claudinl 8.2 antibody was replaced in 20 mM NaAc- HAc, pH 6.0 buffer, and the anti-Claudin 18.2 antibody was concentrated or diluted to a protein concentration of 5 mg/mL.
  • the white powder linker plus drug was dissolved in DMA to 10 mg/mL for backup.
  • 8-12 times of TCEP was added according to the molecular ratio, and the reaction was carried out at room temperature for 1-2 h.
  • linker plus drug solution was added according to the molecular ratio, and the reaction was carried out at room temperature for 1-2 h.
  • the linker plus drug that had not been coupled with anti-Claudin 18.2 antibody was removed at the end of the reaction by ultrafiltration using 30 KDa ultrafiltration centrifuge tube.
  • a 30 KDa ultrafiltration centrifuge tube was used to remove the linker plus drug that is not coupled with anti-Claudinl 8.2 antibody, and then the sample of anti-Claudinl 8.2 antibody-drug conjugate was obtained.
  • the resulting samples of anti-Claudinl 8.2 antibody-drug conjugate will be used to determine the monomer rate by SEC-HPLC and the drug load by RP-HPLC or HIC-HPLC.
  • ADC-1 was prepared according to the generalized coupling method of Example 92:
  • ADC-2 was prepared according to the generalized coupling method of Example 92:
  • ADC-3 was prepared according to the generalized coupling method of Example 92:
  • ADC-4 was prepared according to the generalized coupling method of Example 92 : Example 98
  • ADC-5 was prepared according to the generalized coupling method of Example 92:
  • ADC-6 was prepared according to the generalized coupling method of Example 92: Example 100
  • ADC-7 was prepared according to the generalized coupling method of Example 92:
  • ADC- 12 was prepared according to the generalized coupling method of Example 92: Example 106
  • ADC- 19 was prepared according to the generalized coupling method of Example 92:
  • ADC-21 was prepared according to the generalized coupling method of Example 92:
  • ADC-22 was prepared according to the generalized coupling method of Example 92: Example 116
  • ADC-23 was prepared according to the generalized coupling method of Example 92:
  • ADC-24 was prepared according to the generalized coupling method of Example 92: Example 118
  • ADC-25 was prepared according to the generalized coupling method of Example 92:
  • ADC-26 was prepared according to the generalized coupling method of Example 92: Example 120
  • ADC-27 was prepared according to the generalized coupling method of Example 92:
  • ADC-28 was prepared according to the generalized coupling method of Example 92: Example 122
  • ADC-29 was prepared according to the generalized coupling method of Example 92.
  • ADC-30 was prepared according to the generalized coupling method of Example 92: Example 124
  • ADC-31 was prepared according to the generalized coupling method of Example 92:
  • ADC-32 was prepared according to the generalized coupling method of Example 92: Example 126
  • ADC-33 was prepared according to the generalized coupling method of Example 92:
  • ADC-34 was prepared according to the generalized coupling method of Example 92: Example 128
  • ADC-35 was prepared according to the generalized coupling method of Example 92:
  • ADC-36 was prepared according to the generalized coupling method of Example 92: Example 130
  • ADC-37 was prepared according to the generalized coupling method of Example 92:
  • ADC-38 was prepared according to the generalized coupling method of Example 92: Example 132
  • ADC-39 was prepared according to the generalized coupling method of Example 92:
  • ADC-40 was prepared according to the generalized coupling method of Example 92: Example 134
  • ADC-41 was prepared according to the generalized coupling method of Example 92:
  • ADC-42 was prepared according to the generalized coupling method of Example 92: Example 136
  • ADC-43 was prepared according to the generalized coupling method of Example 92:
  • ADC-44 was prepared according to the generalized coupling method of Example 92: Example 138
  • ADC-45 was prepared according to the generalized coupling method of Example 92:
  • ADC-46 was prepared according to the generalized coupling method of Example 92: Example 140
  • ADC-47 was prepared according to the generalized coupling method of Example 92:
  • ADC-48 was prepared according to the generalized coupling method of Example 92: Example 142
  • ADC-49 was prepared according to the generalized coupling method of Example 92:
  • ADC-50 was prepared according to the generalized coupling method of Example 92: Example 144
  • ADC-51 was prepared according to the generalized coupling method of Example 92:
  • ADC-52 was prepared according to the generalized coupling method of Example 92: Example 146
  • ADC-53 was prepared according to the generalized coupling method of Example 92:
  • ADC-54 was prepared according to the generalized coupling method of Example 92: Example 148
  • ADC-55 was prepared according to the generalized coupling method of Example 92:
  • ADC-58 was prepared according to the generalized coupling method of Example 92: Example 152
  • ADC-59 was prepared according to the generalized coupling method of Example 92:
  • ADC-60 was prepared according to the generalized coupling method of Example 92: Example 154
  • ADC-62 was prepared according to the generalized coupling method of Example 92: Example 156
  • ADC-63 was prepared according to the generalized coupling method of Example 92:
  • ADC-64 was prepared according to the generalized coupling method of Example 92: Example 158
  • ADC-65 was prepared according to the generalized coupling method of Example 92:
  • ADC-66 was prepared according to the generalized coupling method of Example 92
  • Example 160
  • ADC-67 was prepared according to the generalized coupling method of Example 92:
  • ADC-68 was prepared according to the generalized coupling method of Example 92: Example 162
  • ADC-69 was prepared according to the generalized coupling method of Example 92:
  • ADC-70 was prepared according to the generalized coupling method of Example 92: Example 164
  • ADC-71 was prepared according to the generalized coupling method of Example 92:
  • ADC-72 was prepared according to the generalized coupling method of Example 92: Example 166
  • ADC-73 was prepared according to the generalized coupling method of Example 92:
  • ADC-74 was prepared according to the generalized coupling method of Example 92: Example 168
  • ADC-75 was prepared according to the generalized coupling method of Example 92:
  • ADC-76 was prepared according to the generalized coupling method of Example 92.
  • Example 170 ADC-76 was prepared according to the generalized coupling method of Example 92.
  • ADC-77 was prepared according to the generalized coupling method of Example 92:
  • ADC-78 was prepared according to the generalized coupling method of Example 92: Example 172
  • ADC-79 was prepared according to the generalized coupling method of Example 92:
  • ADC-80 was prepared according to the generalized coupling method of Example 92: Example 174
  • ADC-81 was prepared according to the generalized coupling method of Example 92:
  • ADC-82 was prepared according to the generalized coupling method of Example 92: Example 176
  • ADC-83 was prepared according to the generalized coupling method of Example 92:
  • ADC-84 was prepared according to the generalized coupling method of Example 92: Example 178
  • ADC-85 was prepared according to the generalized coupling method of Example 92:
  • ADC-86 was prepared according to the generalized coupling method of Example 92: Example 180
  • ADC-87 was prepared according to the generalized coupling method of Example 92:
  • ADC-88 was prepared according to the generalized coupling method of Example 92: Example 182
  • ADC-89 was prepared according to the generalized coupling method of Example 92:
  • ADC-90 was prepared according to the generalized coupling method of Example 92: Example 184
  • ADC-91 was prepared according to the generalized coupling method of Example 92:
  • ADC-92 was prepared according to the generalized coupling method of Example 92: Example 186
  • ADC-93 was prepared according to the generalized coupling method of Example 92:
  • ADC-94 was prepared according to the generalized coupling method of Example 92: Example 188
  • ADC-95 was prepared according to the generalized coupling method of Example 92:
  • ADC-96 was prepared according to the generalized coupling method of Example 92: Example 190
  • ADC-97 was prepared according to the generalized coupling method of Example 92:
  • ADC-98 was prepared according to the generalized coupling method of Example 92: Example 192
  • ADC-99 was prepared according to the generalized coupling method of Example 92:
  • ADC- 100 was prepared according to the generalized coupling method of Example 92: Example 194
  • ADC- 101 was prepared according to the generalized coupling method of Example 92:
  • ADC- 102 was prepared according to the generalized coupling method of Example 92: Example 196
  • ADC- 103 was prepared according to the generalized coupling method of Example 92:
  • ADC- 104 was prepared according to the generalized coupling method of Example 92: Example 198
  • ADC- 105 was prepared according to the generalized coupling method of Example 92:
  • ADC- 106 was prepared according to the generalized coupling method of Example 92: Example 200
  • ADC- 107 was prepared according to the general coupling method of Example 93: wherein 64C9 is an anti-Claudinl8.2 antibody.
  • the antibody-drug conjugates (ADCs) disclosed in the present application has the characteristics of low degradation rate and low aggregation rate, and the excellent property of high monomer rate.
  • the ADCs in the present application have the excellent property of high DAR value, which may significantly increase the ADC drug concentration at the target site when the same dose of ADC is administered.
  • ADCs were mixed with IgG-free plasma to make the final concentration of ADC at 0.6 mg/mL, incubated in a 37 °C water bath for a period of 0, 3, and 7 days. A unincubated plasma was used as a control. After incubation, the sample was purified and extracted, and the drug antibody ratio DAR was measured to reflect the plasma stability of ADCs.
  • the ADCs disclosed in the present disclosure have good plasma stability, and there is no significant change in the DAR value during plasma incubation.
  • ADC maintains the affinity of the corresponding original anti-Claudinl8.2 antibody 51O3F3- BSM for Claudinl8.2.
  • the relative affinities of 5103F3-BSM versus ADC-6; 64C9 versus ADC- 107 for Claudinl8.2 were compared by using double antigen sandwich ELISA.
  • the specific steps of the Claudinl8.2 relative binding activity assay are as follows: the antigen was diluted with PBS to the appropriate concentration, spiked into the plate and incubated at 4°C overnight. After coating, wash the plate and seal it at 37°C for 1 h. After sealing, wash the plate, dilute the test material in a gradient according to the appropriate concentration, add the sample into the sealed enzyme plate and incubate. Dilute the rabbit anti-human antibody with buffer to the appropriate concentration, add the sample and incubate. Add the homemade color development solution to develop the color for 7min and then add the termination solution to terminate the reaction. 450nm read the plate, four-parameter fit to the reading results, calculate the EC50 value, and then get the relative binding activity results.
  • HEK293T-18.1 and HEK293T-18.2 which are two human embryonic kidney cell lines overexpressing Claudinl8.1 and Claudinl8.2, respectively, were used as experimental models, and the specific binding of each naked antibody of ADC to Claudinl8.2 was analyzed by flow cytometry.
  • HEK293T-18.1/18.2 in logarithmic growth phase were taken, trypsinized and washed twice with pre-cooled 1 X PBS, and resuspended to 1 x 10 7 cells/mL with 1 *PBS containing 3% BSA.
  • the set-up gate strategy was as follows: the cells to be analyzed were circled with the SSC/FSC scatter plot; single cells were circled with the FSC-H/FSC-A scatter plot to exclude adhesions and debris; the single-cell gate was applied to the PB450-Ato obtain the live cells, and finally the cell population to be tested was identified in the FITC-A and the fluorescence values corresponding to different antibody concentrations were derived by statistics.
  • the 4-parameter model of Graph Pad Prism 9.0 data analysis software was utilized for data statistics and analysis.
  • the starting concentration of the antibody drug was 500 nM, and the dilution was 7-fold, totaling 8 concentration points, and treated for 5 days.
  • the present application establishes a subcutaneous graft tumor model of target-positively expressing human gastric cancer cells SNU-5 NOD Scid mice to evaluate the in vivo efficacy of ADC-6.
  • 5* 10 6 SNU-5 cells (0.1 mL/each) were injected subcutaneously into the right scapula of NOD Scid mice at 6 ⁇ 7 weeks of age.
  • the mice were randomly divided into a lysate control group (Vehicle) and an ADC-6 treatment group (5 mg/kg) of 5 mice each, and the administration of ADC-6 was started (DO).
  • T/C % TRTV / CRTV x 100%;
  • TGI% (1-T/C) x 100%;
  • p value was obtained by comparing the tumor volume of the treatment group with that of the lysate control group at D 28 (day 28 after administration).
  • the present application establishes a target (i.e., Claudinl8.2)-overexpressing human gastric cancer cells (e.g., MKN-45) in BALB/c Nude mouse subcutaneous tumor graft model to evaluate the in vivo efficacy of ADC-6 drug.
  • MKN-45 cells (2x l0 6 ) were injected subcutaneously (0. ImL/mouse) into the right scapula of BALB/c Nude mice aged 6 to 7 weeks.
  • the mice were randomly divided into vehicle control group (Vehicle) and ADC-6 treatment group (2mg/kg, 5mg/kg), 5 mice in each group, and drug administration was started (DO).
  • Each group was administered 10 mL/kg body weight via tail vein injection once a week for 4 consecutive weeks (QW X 4). All groups were observed on the 28th day after group administration (D28) (Table 9, Figure 7), and statistical analysis of the mean tumor volume on day 28 (D28) after subgroup administration showed that ADC-6 administered caudal- venously at the doses of 2 mg/kg and 5 mg/kg (QW*4) had a significant tumor-suppressing effect (P ⁇ 0.05). Table 9. Efficacy analysis of each group in the subcutaneous tumor graft model of human gastric cancer cells, MKN-45, in BALB/c Nude mice
  • T/C % TRTV / CRTV x 100%;
  • TGI% (1-T/C) x 100%;
  • p value was obtained by comparing the tumor volume of the treatment group with that of the lysate control group at D28 (day 28 after administration).

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Abstract

An antibody targeting human Claudin18.2 was coupled with a camptothecin analog to form a stable and homogeneous antibody-drug conjugate with a drug-to-antibody ratio (DAR) of 6.0-8.0. This antibody-drug conjugate has the structure shown in general formula I, where Ab refers to an antibody targeting Claudin18.2 coupled with a linker-camptothecin analog. The present application also discloses methods of preparation and purification of conjugates, their applications in cancer therapy, and linker-drug compounds that can be coupled with Ab to form antibody-drug conjugates.

Description

ANTI-CLAUDIN18.2 ANTIBODY-CAMPTOTHECIN DRUG CONJUGATE AND PHARMACEUTICAL USE THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of the filing date of CN 202311712634.5, filed November 15, 2023, the entire disclosures of which are incorporated by reference herein.
TECHNICAL FIELD
The present invention relates to the field of biopharmaceuticals, and specifically to an antibody-drug conjugate formed by an anti-Claudin!8.2 antibody and a camptothecin toxin, and to methods of preparation and use of the antibody-drug conjugate. The present application also relates to linker-drug compounds that can be coupled with an antibody to form an antibody-drug conjugate.
BACKGROUND
Claudinl8.2 (or simply CLDN18.2) is a protein with tight junction function encoded by the Claudinl8 gene and is a member of the Claudin protein family.
Claudin family proteins are four-times transmembrane proteins containing two extracellular loops, N-terminal and C-terminal present in the cytoplasm, and four transmembrane regions. Claudin family proteins are important members involved in tight junctions, which play a role in connectivity between cells, and the aberrant expression of the proteins induces structural disruptions such as those of epithelial cells, leading to altered function and the triggering of a wide range of diseases. Current research indicates that the mammalian Claudin family of proteins has 27 members, and humans contain all Claudin proteins except Claudinl3. The functions of each member are highly conserved, and each member is differentially expressed in different tissues and mediates different tumor types.
The normal expression of Claudinl8.2 is limited to gastric mucosal cells and is not expressed in other normal tissues, and its overexpression leads to gastric, pancreatic, esophageal, and nonsmall-cell lung cancers, etc. Claudinl8.1, a shear variant of Claudinl8.2, is selectively expressed in lung cells, although there is only an 8 amino acid difference between Claudinl8.1 and Claudinl8.1 in the first extracellular loop of the normal tissues. Claudinl8.1 is selectively expressed in lung cells of normal tissues. Therefore, the highly restricted expression of Claudinl8.2 in normal tissues and its specific expression in tumor tissues make Claudinl8.2 an important potential target for tumor immunotherapy.
At present, for antibody drugs targeting Claudinl8.2, Zolbetuximab (IMAB362), a monoclonal antibody drug developed by Ganymed Company of Germany, is in the lead, and the research on the indications of gastric and gastroesophageal cancers has progressed to phase III clinic trial, and that of pancreatic cancer has progressed to phase II clinic trial. Ganymed's anti- Claudinl8.2 and CD3 dual antibody is in the preclinical stage, with more than 60% of Claudinl8.2 and CD3 dual targets in development, and dual antibodies targeting Claudinl8.2 and CD47/PD- L1/4-1BB are also in development. The fastest progressing anti-Claudinl8.2 ADCs are in clinical phase l/II, including LM-302 from Lixin Pharmaceuticals, RC118 from Rongchang Biologies, and GMG901 from Konoia/Leep Bio. The majority of ADCs targeting Claudinl8.2 are in clinical phase I or preclinical stage.
Antibody-drug conjugate (ADC) is a class of biological drugs that connect antibodies or antibody fragments with biologically active small molecule toxins through stable chemical linkers, which not only make full use of the high specificity of antibody-antigen binding and the high lethality of cytotoxins, but also effectively circumvents the disadvantages of weak therapeutic effect of antibodies and indiscriminate attack of small molecule toxins. At the same time, it effectively avoids the disadvantages of weak therapeutic effect of antibody and indiscriminate attack of small molecule toxin.
There are currently 14 approved ADC drugs on the market worldwide.
Figure imgf000004_0001
Mylotarg is one of the first ADCs to be marketed globally, which was approved for accelerated marketing by the FDA in May 2000 for the treatment of patients with acute myelogenous leukemia (AML) who are in first relapse, over the age of 60, CD33 -positive, and who are not candidates for cytotoxic chemotherapy. Pfizer chose to withdraw the drug from the market on its own in June 2010 after later studies found that treatment with Mylotarg led to severe and fatal liver injury. Subsequently in 2017, Mylotarg was approved for re-launch by the FDA at a reduced dose for the treatment of newly diagnosed CD33 -positive AML (children and adults >1 month of age), relapsed or refractory CD33 -positive AML (children and adults >2 years of age). Recently approved for marketing, Tivdak is the first FDA-approved ADC targeting tissue factor (TF) for the treatment of patients with recurrent or metastatic cervical cancer.
The present application discloses a safer and effective ADC drug targeting Claudinl8.2 with camptothecin as the toxin molecule with great developmental value and therapeutic potential. SUMMARY
The following summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.
The disclosure provides, among others, the ADC class of drugs including anti-Claudinl8.2 antibody-drug conjugates, compounds such as drug moieties and linker derivatives useful for forming part of the antibody-drug conjugates, methods of preparing the conjugates, pharmaceutical compositions comprising said conjugates or compounds, and methods of using said conjugates or pharmaceutical composition for treating diseases such as cancers. The disclosure further provides linker-drug compounds that can be coupled to an anti-Claudinl8.2 antibody to form the disclosed antibody-drug conjugates. In some embodiments, the anti-Claudinl8.2 antibody-drug conjugates have excellent clinical therapeutic effects with good molecular stability and significantly improved preclinical efficacy compared to existing drugs of the similar category.
In one aspect, the disclosure provides a ligand-camptothecin derivative conjugate as shown in general formula I or a pharmaceutically acceptable salt or solvate thereof:
Figure imgf000005_0001
wherein, Ab is an antibody having a binding affinity to human Claudinl8.2 or an antigen-binding fragment thereof;
Lu, Ln, and L13 are different from each other and are independently:
Figure imgf000006_0001
L2 has the structure shown in Formula A below.
Figure imgf000006_0002
Formula A wherein Y is a scaffold selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl; In one embodiment Y is C1-C6 alkyl; Ac is a hydrophilic structural unit; and the carbon 2 attached to Y is absolutely chiral in R or S configuration.
In one embodiment, L3 is present or absent, and when present, L3 is selected from PEG
Figure imgf000006_0003
hydrophilic units: 0 , o is selected from an integer of 1-10; in one embodiment, o may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; in one embodiment, o is an integer of 2-8, such as 2, 3, 4, 5, 6, 7, or 8;
In one embodiment, L4 is the enzymatically cleavable unit.
In one embodiment, L5 is the connection unit.
In one embodiment, the chiral carbon atom No. 1 attached to N in formula I has an absolute chirality of R or S;
R is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10-membered heteroaryl, substituted 5-10-membered heteroaryl.
In one embodiment, R is selected from a hydrogen atom or a C1-C6 alkyl group.
Ri is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted Cl- C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3-7 meta-heterocyclic, substituted 3-7 meta-heterocyclic, C6-C10 aryl, substituted C6- C10 aryl, 5-10 meta-heteroaryl, substituted 5-10 heteroaryl.
In one embodiment, Ri is selected from a hydrogen atom or a C1-C6 alkyl group.
In one embodiment, Ri is selected from C1-C6 alkyl.
R2 is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted Cl- C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3-7 meta-heterocyclic, substituted 3-7 meta-heterocyclic, C6-C10 aryl, substituted C6- C10 aryl, 5-10 meta-heteroaryl, substituted 5-10 heteroaryl.
In one embodiment, R2 is selected from a hydrogen atom, a halogen or a C1-C6 alkyl group. In one embodiment, R2 is selected from halogen. X selected from -C(O)-CRaRb-(CR3R4)m-O-, -C(O)-CRaRb-(CR3R4)m-NH- or -C(O)-CRaRb- (CR3R4)m-S-. In one embodiment, X is selected from -C(O)-CRaRb-(CR3R4)m-O-.
Ra and Rb are each independently selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl, a deuterated C1-C6 alkyl, a halogenated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3- C8 cycloalkyl C1-C6 alkyl, a C6-C10 aryl C1-C6 alkyl, a C1-C6 alkoxy C1-C6 alkyl, a 3-7 metaheterocyclic group, a substituted 3-7 metaheterocyclic group, a C6-C10 aryl, substituted C6- C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl.
In one embodiment, Ra and Rb are each independently selected from a hydrogen atom, a Cl- C6 alkyl, a halo-Cl-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl or a C6-C10 aryl C1-C6 alkyl.
Alternatively, Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C 1-C6 alkyl group, a 3-7-membered heterocycloalkyl group, a substituted 3-7-membered heterocycloalkyl group. In one embodiment Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group.
R3, R4 are the same or different and are independently hydrogen atoms, deuterium atoms, halogens, C1-C6 alkyl, halogenated Cl -C6 alkyl, deuterated Cl -C 6 alkyl, C1-C6 alkoxy, hydroxyl, amino, cyano, nitro, hydroxy C1-C6 alkyl, C3-C8 cycloalkyl, a 3-7-membered heterocyclic group, a substituted 3-7-membered heterocyclic group, respectively.
In one embodiment, R3, R4 are independently hydrogen atoms or C1-C6 alkyl groups, respectively. Alternatively, R3, R4 and the carbon atoms attached thereto constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7-membered heterocyclic group, a substituted 3-7-membered heterocyclic group. m is selected from an integer from 0-4. In one embodiment, m may be 0, 1, 2, 3, or 4. In one embodiment, m may be 0 or 1 ; nl, n2, and n3 are each independently selected from any integer or any decimal number from 0-10, nl, n2, and n3 are not simultaneously 0, and 1 < n l + n2 + n3 < 10.
In some embodiment, ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof as shown in general formula I, is characterized in that Ab is an antibody targeting Claudinl8.2 or an antigen-binding fragment thereof.
In one embodiment, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is characterized in that Ab is an antibody and comprises an IgGl heavy chain and a light chain. In one embodiment, the antibody comprising said IgGl heavy chain and K light chain specifically recognizes a human Claudinl8.2 protein.
In one embodiment, the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, is characterized in that the K light chain of the antibody comprises CDRs having amino acid sequences with at least 80%, 90%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and the IgGl heavy chain comprises CDRs having amino acid sequences with at least 80%, 90%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4.
In one embodiment, the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I, is characterized in that the antibody comprises a heavy chain and a light chain. In one embodiment, the light chain comprises CDRL1, CDRL2 and CDRL3 each having an amino acid sequence encoded by a nucleic acid coding sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, said heavy chain comprising CDRH1, CDRH2 and CDRH3 each having an amino acid sequence encoded by a nucleic acid sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.
In one embodiment, the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I, is characterized in that the antibody comprises a heavy chain and a light chain. In one embodiment, the heavy chain comprises a heavy-chain variable region having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, and said light chain comprising a light-chain variable region having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.
In one embodiment, the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I, is characterized in that the antibody comprises a heavy chain and a light chain. In one embodiment, the heavy chain variable region comprising a heavy chain variable region encoded by a nucleic acid sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13, and said light chain variable region encoded by a nucleic acid sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 19.
In one embodiment, the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I, is characterized in that Ab comprises a heavy chain having an amino acid sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, and a light chain having an amino acid sequence having at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11.
In one embodiment, the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I, is characterized in that the nucleic acid coding sequence for the antibody heavy chain has at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17, and the nucleic acid coding sequence for the antibody light chain has at least 80%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23.
In one embodiment, the ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof as shown in general formula I is characterized in that said X may be the following structures or isomers thereof:
Figure imgf000008_0001
Figure imgf000009_0001
The position shown by the left wavy line is connected to the derivative part, and the position shown by the right wavy line is connected to L5.
In one embodiment, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I, is characterized in that L4 may be peptide residues comprising amino acids. In one embodiment, optionally, said amino acid is further substituted with one or more substituents selected from one or more of deuterium atoms, halogens, hydroxyl, cyano, amino, nitro, carboxyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy and C3-C8 cycloalkyl or substituted C3-C8 cycloalkyl. In one embodiment, said peptide residue is a peptide residue formed from one, two or more amino acids selected from among phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline (C), serine (S), glutamic acid (E) or aspartic acid (D). In one embodiment, said peptide residue is a tetrapeptide residue comprising glycine (G) -glycine (G) -phenylalanine (F) -glycine (G). In one embodiment, said peptide residue is -GGFG-.
In one embodiment, the ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof as shown in general formula I are characterized as follows:
L5 is non-limitingly selected from -NRffCFLFDq- or a chemical bond, q is selected from an integer from 0-6 (e.g., 0, 1, 2, 3, 4, 5, or 6);
R5, R6 and R7 are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a 3-7 meta heterocyclic group, a substituted 3-7 meta heterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl, 5-10 heteroaryl, substituted 5-10 heteroaryl. In one embodiment, R5, R6 and R7 are each independently selected from a hydrogen atom or a C1-C6 alkyl group. In one embodiment, R5, R6 and R7 are each independently selected from a hydrogen atom.
In certain embodiments, Li may be:
Figure imgf000009_0002
In one embodiment, the ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof, are characterized in that said linking units - Ln -L2 -L3 -L4 -L5 -, -L12 -L2 -L3 -L4 -L5 - or -L13 -L2 -L3 -L4 -L5 - are not identical to each other and are independently and may be the following structures:
Figure imgf000009_0003
Figure imgf000010_0001
In one embodiment, -Ln -L2 -L3 -L4 -L5 -L12 -L2 -L3 -L4 -L5 - or -L13 -L2 -L3 -L4 -L5 - are not the same as each other and are each independently and non-limitingly selected from:
Figure imgf000010_0002
wherein:
Ac is a hydrophilic structural unit;
R5, R6 and R7 are the same or different and are each independently selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3-7-membered heterocyclic, substituted 3-7-membered heterocyclic, C6-C10 aryl, substituted C6-C10 aryl, 5-10- membered heteroaryl, substituted 5-10-membered heteroaryl. In one embodiment, R5, Rs and R7 are each independently selected from a hydrogen atom or a C1-C6 alkyl group;
In one embodiment, R5, Rs and R7 are each independently selected from a hydrogen atom;
The carbon atom #2 attached to N has absolute chirality of R or the S configuration;
The position shown by the left wavy line is connected to the antibody or its antigen-binding fragment portion, and the position shown by the right wavy line is connected to X; o is an integer selected from 1-10; for example, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In a further aspect, the application discloses a ligand-camptothecin derivative conjugate as shown in general formula II, or a pharmaceutically acceptable salt or solvate thereof;
Figure imgf000011_0001
Among them:
Ab is an antibody having a binding affinity (or specificity) to human Claudinl8.2 or an antigen-binding fragment thereof;
Ln, L12, and L13 are connecting units, and Ln, L12, and L13 are not identical to each other and are independently and non-limitingly selected from:
Figure imgf000011_0002
L3 is present or absent, and when L3 is present, L3 is selected from 0 , o is selected from an integer from 1-10; for example, o many be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment from an integer from 2-8;
Ac is a hydrophilic structural unit;
The 1 -position, 2-position and 3-position chiral carbon atoms each independently has a chiral configuration of R or S;
R is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated Cl -C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10-membered heteroaryl, substituted 5-10-membered heteroaryl; In one embodiment R is selected from a hydrogen atom or a C1-C6 alkyl group;
Ri is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted Cl- C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3-7 meta-heterocyclic, substituted 3-7 meta-heterocyclic, C6-C10 aryl, substituted C6- C10 aryl, 5-10 meta-heteroaryl, substituted 5-10 heteroaryl. In one embodiment Ri is selected from a hydrogen atom or a C1-C6 alkyl group. In one embodiment Ri is selected from C1-C6 alkyl;
R2 is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted Cl- C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3-7 meta-heterocyclic, substituted 3-7 meta-heterocyclic, C6-C10 aryl, substituted C6- C10 aryl, 5-10 meta-heteroaryl, or substituted 5-10 heteroaryl. In one embodiment, R2 is selected from a hydrogen atom, a halogen or a C1-C6 alkyl group. In one embodiment, R2 is selected from halogen;
X is -C(O)-CRaRb-(CR3R4)m-O-, -C(O)-CRaRb-(CR3R4)m-NH- or -C(O)-CRaRb-(CR3R4)m-S-.
In one embodiment, X is selected from -C(O)-CRaRb-(CR3R4)m-O-;
Ra and Rb are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1 -C6 alkyl group, a deuterated C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl group C1-C6 alkyl group, a C1-C6 alkoxy group C1-C6 alkyl group, a 3-7 metaheterocyclic group, a substituted 3-7 metaheterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5- 10-membered heteroaryl, or substituted 5-10-membered heteroaryl; In one embodiment, Ra and Rb are each independently selected from a hydrogen atom, a C1-C6 alkyl, a halo-Cl-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl or a C6-C10 aryl C1-C6 alkyl; Alternatively, Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocycloalkyl group, a substituted 3-7 heterocycloalkyl group; In one embodiment Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group;
R3, R4 are the same or different and are independently hydrogen atoms, deuterium atoms, halogens, C1-C6 alkyl, halogenated Cl -C6 alkyl, deuterated C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, amino, cyano, nitro, hydroxy C1-C6 alkyl, C3-C8 cycloalkyl, 3-7 heterocyclic, substituted 3-7 heterocyclic, respectively; In one embodiment, R3, R4 are independently hydrogen atoms or Cl- C6 alkyl groups, respectively; Alternatively, R3, R4 and the carbon atoms attached thereto constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group; m is selected from integers 0-4; for example, m may be 0, 1, 2, 3 or 4. In one embodiment, m is 0 or 1;
Each of nl, n2, and n3 is independently chosen from any integer or any decimal number from 0 to 10, nl, n2, and n3 are not simultaneously 0, and 1 < nl + n2 + n3 < 10.
In one embodiment, the ligand-camptothecin derivative conjugate as shown in general formula II, or a pharmaceutically acceptable salt or solvate thereof, is characterized in that said Ac has the structure shown in formula B:
Figure imgf000013_0001
Among them: Z may be the group consisting of one or more of a hydrophilic structural carboxyl group, a phosphoric acid, a polyphosphoric acid, a phosphite, a sulfonic acid, a sulfinic acid, or a polyethylene glycol (PEG);
In one embodiment Z is selected from hydrophilic structural carboxylates, phosphoric acid or polyethylene glycol (PEG);
Y1 is optionally a scaffold connecting the amino group to Z; In one embodiment Y1 is a Cl- C6 alkylene group (e g. methylene); and
Ac is connected to the 2-position carbon that has been labeled in structural formula I by means of a bracket Y.
In some embodiments, the ligand-camptothecin derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof, is characterized in that said Ac may be glycine, (D/L) alanine, (D/E) leucine, (D/E) isoleucine, (D E) valine, (D/E) phenylalanine, (D/E) proline, (D/E) tryptophan, (D/E) serine, (D/E) tyrosine, (D/E) cysteine, (D/L) cystine, (D/E) arginine, (D E) histidine, (D/E) methionine, (D/E) asparagine, (D/L) glutamine, (D/E) threonine, (D/E) aspartic acid, (D/L) glutamic acid, natural or non-natural amino acid derivatives or the following structures or isomers thereof.
Figure imgf000013_0002
In some embodiments, the ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof is characterized in that said Ac may be a glycine, phosphoric acid, (D/E) glutamic acid, or poly(ethylene glycol) hydrophilic structure. In some embodiments, the ligand-camptothecin derivatives conjugates or pharmaceutically acceptable salts or solvates thereof are characterized in that said camptothecin derivatives have the structure shown in formula d below;
Figure imgf000014_0001
Among them :
R is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated Cl -C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, substituted 5-10 heteroaryl; In one embodiment R is selected from a hydrogen atom or a C1-C6 alkyl group;
Ri is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted Cl- C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3-7 meta-heterocyclic, substituted 3-7 meta-heterocyclic, C6-C10 aryl, substituted C6- C10 aryl, 5-10 meta-heteroaryl, or substituted 5-10 heteroaryl;
In one embodiment, Ri is selected from a hydrogen atom or a C1-C6 alkyl group;
In one embodiment, Ri is selected from C1-C6 alkyl;
R2 is selected from hydrogen atom, deuterium atom, halogen, C1 -C6 alkyl, substituted Cl - C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3-7 meta-heterocyclic, substituted 3-7 meta-heterocyclic, C6-C10 aryl, substituted C6- CIO aryl, 5-10 meta-heteroaryl, or substituted 5-10 heteroaryl;
In one embodiment, R2 is selected from a hydrogen atom, a halogen or a C1-C6 alkyl group; In one embodiment, R2 is selected from halogen;
Ra and Rb are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C1-C6 alkyl group, a deuterated C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl group C1-C6 alkyl group, a C1-C6 alkoxy group C1-C6 alkyl group, a 3-7 metaheterocyclic group, a substituted 3-7 metaheterocyclic group, a C6-C10 aryl group, a substituted C6-C10 aryl group, a 5-10 heteroaryl, substituted 5-10 heteroaryl; In one embodiment, Ra and Rb are each independently selected from a hydrogen atom, a C1-C6 alkyl, a halo-Cl-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl or a C6- CIO aryl C1-C6 alkyl; In one embodiment, Ra and Rb are each independently selected from a hydrogen atom, a C1-C6 alkyl, a halo-Cl-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl, a C6-C10 aryl; In one embodiment Ra and Rb are each independently selected from a hydrogen atom, a methyl group, an ethyl group, a trifluoromethyl group, a cyclopropylmethyl group, a phenyl group; Alternatively, Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocycloalkyl group, a substituted 3-7 heterocycloalkyl group; In one embodiment, Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group (e.g., a C3-C5 cycloalkyl group);
The 1 -position chiral carbon atom may be in R or S configuration; m is selected from 0 or 1.
In some embodiments, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof is characterized in that: said structural formula d may be the following compounds:
Figure imgf000015_0001
Figure imgf000016_0001
In a further aspect, the application discloses a linker-drug compound or a pharmaceutically acceptable salt or solvate thereof, having the structure shown in formula III below:
Figure imgf000016_0002
Among them:
Figure imgf000016_0003
R is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated Cl -C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, substituted 5-10 heteroaryl;
Ra is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3-7 meta-heterocyclic group, substituted 3-7 meta-heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 meta-heteroaryl, substituted 5-10 heteroaryl;
Rb is selected from hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3-7 meta-heterocyclic group, substituted 3-7 meta-heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 meta-heteroaryl, substituted 5-10 heteroaryl;
Alternatively, Ra, Rb and the carbon atoms attached thereto constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group;
In one embodiment, Ra and Rb are each independently selected from a hydrogen atom, a Cl- C6 alkyl, a halo-Cl-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl, a C6-C10 aryl; In one embodiment, Ra and Rb are each independently selected from a hydrogen atom, a methyl group, an ethyl group, a trifluoromethyl group, a cyclopropylmethyl group, a phenyl group;
Alternatively, Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocycloalkyl group, a substituted 3-7 heterocycloalkyl group; In one embodiment, Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group (e.g., a C3-C5 cycloalkyl group);
Figure imgf000017_0001
L3 is absent or present, and when L3 is present, it is selected from 0 , o is selected from an integer from 1 to 10; in one embodiment, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
The 1 -position or 2-position chiral carbon atom each independently has R or S configuration;
Ac is a hydrophilic structural unit; m is selected from 0 or 1;
In one embodiment, said linker-drug compound or a pharmaceutically acceptable salt or solvate thereof is used to couple with ligand Ab to form said ligand-camptothecin derivative conjugates of formula I or formula II.
In some embodiments, L may comprise a succinimide group. In these embodiments, the ligand-drug conjugate may undergo hydrolysis under readily hydrolyzable conditions, with the site of hydrolysis being the butanediimide moiety of the linker unit. When the ligand contains multiple linker-drugs, the following scenarios can occur with varying degrees of hydrolysis:
(1) the succinimide groups are completely non-hydrolyzed, i.e., the succinimide o 0
Figure imgf000017_0002
groups are all in the closed ring form o or 0 ;
(2) incomplete hydrolysis of the butanediimide group, i.e., some of the butanediimide
Figure imgf000018_0004
(3) complete hydrolysis of the succinimide groups, i.e., the succinimide groups are all
Figure imgf000018_0001
Thus, when multiple L's containing a succinimide moiety are present in the ADC at the same time (i.e., Ab is connected to multiple drug-linkers containing a succinimide moiety), all or some of these succinimide moieties may be in a closed-loop form, partially open-loop form, or all or some of them may be in an open-loop form.
In some embodiments, the linker-drug compounds or pharmaceutically acceptable salts or solvates thereof as disclosed herein are characterized in that: said Ac has the structure shown in formula B below, the
Figure imgf000018_0002
Among them:
Z may be the group consisting of one or more of a hydrophilic structural carboxyl group, a phosphoric acid, a polyphosphoric acid, a phosphite, a sulfonic acid, a sulfmic acid, or a polyethylene glycol (PEG);
Y' is optionally a scaffold connecting the amino group to Z; In one embodiment Y' is a Cl- C6 alkylene group (e.g., methylene); Ac is connected to the 2-position carbon that has been labeled in structural formula I by means of a bracket Y.
In some embodiments, the linker-drug compounds or pharmaceutically acceptable salts or solvates thereof is characterized in that said Ac may be glycine, (DA) alanine, (D L) leucine, (D L) isoleucine, (D/L) valine, (DI) phenylalanine, (D/L) proline, (D/L) tryptophan, (D/L) serine, (D/L) tyrosine, (D/L) cysteine, (D/L) cystine, (D/L) arginine, (D/L) histidine, (D/L) methionine, (D/L) asparagine, (D/L) glutamine, (D/L) threonine, (D/L) aspartic acid, (D/L) glutamic acid, natural or non-natural amino acid derivatives, or the following structures.
Figure imgf000018_0003
In some embodiments, the linker-drug compounds or pharmaceutically acceptable salts or solvates thereof are characterized in that: Ac may be a glycine, phosphoric acid, (D/L) glutamic acid or polyethylene glycol hydrophilic structure.
In some embodiments, the linker-drug compounds or pharmaceutically acceptable salts or solvates thereof is characterized in that said linker-drug compounds are non-limitingly selected from the following structures or isomers thereof.
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
In one embodiment, o is selected from integers of 1-10; for example, o may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
The linker-drug compounds or pharmaceutically acceptable salts or solvates thereof disclosed herein can be used as intermediates for coupling with the ligand Ab to form ligand-camptothecin derivatives conjugates of formula I or formula II.
In a further aspect, the application discloses methods of preparing a ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I or general formula II. In one embodiment, the method comprises the steps of conjugating the reduced antibody or antigen-binding fragment thereof with the linker-drug compound by the coupling reaction to provide the ligand-camptothecin derivative conjugates as shown in general formula I or general formula II:
Figure imgf000023_0002
Figure imgf000024_0001
The 1-position, 2-position, or 3-position chiral carbon atoms have absolute chirality in R- or S-configuration;
Ab, L, Ln, L12, LB, L2, L3, L4, L5, X, R, Ri, R2, nl, n2, or n3 as previously described.
The present disclosure also relates to the use of the linker-drug compounds described herein, or pharmaceutically acceptable salts or solvates thereof, as intermediates in the preparation of ligand-camptothecin derivative conjugates or pharmaceutically acceptable salts or solvates thereof.
In certain embodiments, said preparation is carried out according to the method of preparation disclosed herein. In some embodiments, the ligand-camptothecin derivative conjugates, or their pharmaceutically acceptable salts or solvates thereof, are characterized in that said ligand- camptothecin derivative conjugates, or their pharmaceutically acceptable salt or solvate thereof, may be the following structure, or a butanediimide ring-opening structure thereof or an isomer thereof.
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Among them:
5103F3-BSM is an antibody targeting (i.e., having an affinity or specificity to) human Claudinl8.2 or an antigen-binding fragment thereof; Each of nl, n2, and n3 is independently chosen from any integer or any decimal number from 0 to 10, nl, n2, and n3 are not simultaneously 0, and 1 < nl + n2 + n3 < 10.
In some embodiments, the ligand-camptothecin derivative conjugates, the linker-drug compounds, or pharmaceutically acceptable salt or solvate thereof is characterized in that said pharmaceutically acceptable salt comprises a sodium salt, a potassium salt, a calcium salt, or a magnesium salt formed with an acidic functional group in the structural formula, and acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate, formed with the basic functional group in the structure.
In a further aspect, the application provides methods for making the conjugates, compounds, and their salts, solvates, or derivatives thereof. In one embodiment, the anti-Claudin!8.2 antibodydrug conjugates are obtained by coupling a humanized anti-Claudinl8.2 antibody with a camptothecin derivative.
In a further aspect, the application discloses a pharmaceutical composition comprising a ligand-camptothecin derivative conjugates and linker-drug compounds as described herein and their pharmaceutically acceptable salts or solvates thereof, and optionally a pharmaceutically acceptable carrier.
In a further aspect, the application discloses a pharmaceutical formulation comprising the ligand-camptothecin derivative conjugates and the linker-drug compounds as described herein and their a pharmaceutically acceptable salts or solvates thereof.
In a further aspect, the application discloses the ligand-camptothecin derivative conjugates, and the linker-drug compounds as described herein and their a pharmaceutically acceptable salts or solvates thereof, pharmaceutical compositions and pharmaceutical formulations in the preparation of medicines for use in the treatment or prevention of cancers or tumors.
In a further aspect, the application discloses the ligand-camptothecin derivative conjugates, and the linker-drug compounds as described herein and their a pharmaceutically acceptable salts or solvates thereof, pharmaceutical compositions and pharmaceutical formulations for use in the treatment or prevention of cancer or tumors.
In a further aspect, the application discloses methods of treating or preventing cancer or tumors. In one embodiment, the method comprises the step of administering to a subject in need thereof a prophylactically or therapeutically effective amount of a ligand-camptothecin derivative conjugate, a linker-drug compound, or their pharmaceutically acceptable salt or solvate thereof, as described herein, the pharmaceutical composition or pharmaceutical formulations.
In one embodiment, the cancer or tumor expresses Claudinl8.2.
In one embodiment, the cancer or tumor may be adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urothelial cancer, bladder cancer, hepatocellular carcinoma, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, breast cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, or sarcoma.
In one embodiment, the cancer or tumor are solid tumors. In one embodiment, the cancer or tumor are hematological tumors such as lymphoma and leukemia.
In the above aspects, the "C1-C6 alkyl" in "C1-C6 alkyl" and various structural moi eties comprising "C1-C6 alkyl" (such as "substituted C1-C6 alkyl," " deuterated C1-C6 alkyl") can be replaced with "C1-C20 alkyl", "C1-C12 alkyl " or "C1-C10 alkyl"; the C3-C8 cycloalkyl" in "C3-C8 cycloalkyl" and various structural moieties comprising "C3- C8 cycloalkyl" may be replaced by "C3-C20 cycloalkyl" or "C3-C10 cycloalkyl"; the "C1-C6 alkoxy" in "C1-C6 alkoxy" and in various structural moieties comprising "C1-C6 alkoxy" may be replaced with "C1-C20 alkoxy," "C1-C12 alkoxy" or "C1-C10 alkoxy"; the "C6-C10 aryl" in "C6-C10 aryl" and various structural moieties comprising "C6-C10 aryl" may be replaced with "C6-C12 aryl"; the "3-7-membered heterocyclic group" in "3-7 heterocyclic group" and various structural moieties comprising "3-7-membered heterocyclic group" may be replaced with "3-20-membered heterocyclic group", "3-12-membered heterocyclic group" or "3-10-membered heterocyclic group".
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other features of this disclosure may become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments arranged in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure may be described with additional specificity and detail through use of the accompanying drawings, in which:
Figure 1 shows the SEC-HPLC detection of ADC-6 aggregation (A) and ADC-107 aggregation (B);
Figure 2 shows the RP-HPLC detection of drug-antibody coupling ratio (DAR) for ADC-6 (A) and ADC- 107 (B);
Figure 3 shows that ADC-6 maintains an equal affinity of the 51O3F3-BSM antibody to the antigen Claudinl8.2 (A) and ADC-107 also maintains an equal affinity of the 64C9 antibody to the antigen Claudinl8.2 (B);
Figure 4 shows the binding curve of antibody to Claudinl 8.1 -expressing cells (A) and to Claudinl8.2 expressing cells (B);
Figure 5 shows the in vitro activity of tumor suppression by ADC-6 and ADC- 107 in the experimental model of a human embryonic kidney cell line expressing Claudinl8.2, HEK293T- 18.2 (A); a human pancreatic cancer cell line expressing Claudinl 8.2, BxPC-3 #A9F (B); a human gastric cancer cell line expressing Claudinl8.2, SNU-5 (C); and another human gastric cancer cell line expressing Claudinl 8.2, SNU-16 (D);
Figure 6 shows the results of the in vivo efficacy of ADC-6 in SNU-5 monoma model; and Figure 7 shows the results of the in vivo efficacy of ADC-6 in MKN-45 single tumor model.
DETAILED DESCRIPTION
Abbreviations and Definitions
Unless otherwise indicated, the following terms and phrases, as used herein, are intended to have the meanings set forth below. When a trade name is used herein, unless otherwise indicated in the context, the trade name includes the product formulation, generic drug and active ingredient of said trade name product.
Unless stated to the contrary, terms used in the claims and specification herein have the meanings set forth below.
The term "ligand" refers to a macromolecular compound that recognizes and binds to an antigen or receptor associated with a target cell. The ligand serves to present a drug to a target cell population bound to a ligand, which includes, but is not limited to, a protein-like hormone, a lectin, a growth factor, an antibody, or other molecule that binds to the cell. In this embodiment of the invention, the ligand is denoted as Ab, and the ligand may form a linkage bond with a linkage unit via a heteroatom on the ligand, preferably an antibody or an antigen-binding fragment thereof, said antibody being selected from chimeric, humanized, fully human, or murine antibodies; preferably monoclonal antibodies.
A ligand unit is a targeting agent that binds specifically to a target component. Said ligand is capable of binding specifically to a cellular fraction or to a cellular component or to other target molecules of interest. The target portion or target is typically on the surface of the cell. In some aspects, the ligand unit serves 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, and non-proteins such as sugars. Suitable ligand units include, for example, antibodies, such as full-length (intact) antibodies and antigen-binding fragments thereof. In embodiments where the ligand unit is a non-antibody targeting reagent, it may be a peptide or polypeptide, or a non-protein molecule. Examples of such targeting reagents include interferons, lymphokines, hormones, growth and colony stimulating factors, vitamins, nutrient transporter molecules, or any other cell-binding molecule or substance. In some embodiments, the linker is covalently attached to the sulfur atom of the ligand. In some aspects, the sulfur atom is a sulfur atom of a cysteine residue that forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine residue that has been imported into the ligand unit, which forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom that has been introduced into a cysteine residue of the ligand unit (e.g., by targeted mutagenesis or chemical reaction). In other aspects, the linker-bound sulfur atom is selected from a cysteine residue that forms the interchain disulfide bond of the antibody or a cysteine residue that has been introduced into the ligand unit (e g., by targeted mutagenesis or chemical reaction). In some embodiments, the EU index numbering system in accordance with Kabat {[Kabat E.Aet al, (1991)] Sequences of proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242}.
As used herein, "antibody" or "antibody unit" includes, to the extent thereof, any part of an antibody structure. This unit may bind, reactively associate, or complex with a receptor, antigen, or other receptor unit possessed by the target cell population. The antibody may be any protein or protein-like molecule that binds, complexes, or reacts with a portion of the cell population to be treated or biologically modified. The antibodies comprising the antibody-drug coupling compounds of the present invention maintain their original antigen-binding capacity in the wild. Thus, the antibodies of the present invention are capable of binding exclusively to antigens. Antigens involved include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (such as are known or foreseen to have functionality), lymphokines, cytokines, molecules involved in the regulation of cellular cycling, molecules involved in angiogenic molecules (if known or predicted to be functional). Tumor- associated factors may be cluster differentiation factors (e.g., CD proteins).
Antibodies applied in antibody drug couplings 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 industry and can be prepared by methods and information for antibody preparation well known in the industry. In order to develop effective cellular level targets that can be used in cancer diagnosis and therapy, researchers seek to find transmembrane or other tumor-associated peptides. 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. Identification of such tumor-associated factors can greatly enhance the specific targeting properties of antibody -based cancer therapies. For convenience, information related to antigens known to the industry is labeled below, including name, other names, and genebank accession number. Nucleic acid and protein sequences corresponding to tumor-associated antigens can be found in publicly available databases, such as Genbank, and antibody-targeted tumor-associated antigens include all amino acid sequence variants and isoforms having at least 70%, 80%, 85%, 90%, or 95% homology to sequences identified in the references or having biological properties and characteristics that are identical to the sequences of the tumor-associated antigens identified in the cited documents.
The term "inhibition" or "inhibiting" means that a detectable amount is reduced or completely prevented.
The term "cancer" refers to a physiological condition or disease characterized by dysregulated cell growth. "Tumor" includes cancer cells.
The term "autoimmune disease" refers to diseases or disorders that originate in tissues or proteins that target an individual's own body.
The term "drug" refers to cytotoxic drugs, denoted by d, which are chemical molecules that have a strong ability to disrupt normal growth in tumor cells. Cytotoxic drugs can in principle kill tumor cells at sufficiently high concentrations, but due to a lack of specificity, they can kill tumor cells while causing apoptosis of normal cells, leading to serious side effects. The term includes toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g., radioisotopes of At211, 1131, 1125, Y90, R6 186, R6 188, Sm153, Bi212, P32 and Lu176), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs.
The term "linker" or "linker fragment" or "linker unit" refers to a fragment or bond of a chemical structure that is attached to a ligand at one end and to the drug at the other end or may be attached to other connectors and then to the drug, or to other connectors and then to the drug.
Junctions, including extensions, spacers and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The junctions can be "cleavable junctions" that facilitate the release of the drug in the cell. For example, acid-unstable junctions (e g., hydrazone), protease-sensitive (e.g., peptidase-sensitive)junctions, photo-unstable junctions, dimethyl junctions, or disulfide-containing junctions can be used (Chari et al. Cancer Research 52: 127-131, 1992); U.S. Patent No. 5, 208, 020.
According to the mechanism of intracellular drug release, as used herein, "linkers" or "linkers of antibody-drug couplers" can be categorized into two types: unbreakable linkers and breakable linkers. For antibody-drug couplers containing an unbreakable linker, the mechanism of drug release is as follows: after the coupler binds to the antigen and is endocytosed by the cell, the antibody is enzymatically cleaved in the lysosome, releasing an active molecule consisting of the small drug molecule, the linker, and the amino acid residues of the antibody. The resulting change in the structure of the drug molecule does not diminish its cytotoxicity, but because the active molecule is electrically charged (amino acid residues), it cannot penetrate into neighboring cells. Therefore, such active drugs cannot kill neighboring tumor cells that do not express the targeted antigen (antigen-negative cells) (bystander effect) (Ducry et al., 2010, Bioconjugate Chem. 21 : 5- 13). For antibody-drug couplings containing a breakable linker, the mechanism of drug release is that the coupling binds to the antigen and is endocytosed by the cell, then breaks and releases the active ingredient (the small-molecule drug itself) in the target cell. Breakable linkers are mainly categorized into chemical-sensitive linkers and enzyme-sensitive linkers. Chemically sensitive linkers can be selectively broken due to differences in the nature of the plasma and cytoplasm or tumor microenvironment. Such properties include pH, glutathione concentration, etc. pH- sensitive linkers, which are relatively stable in the neutral or weakly alkaline environment of blood (pH 7.3-7.5), will be hydrolyzed within the weakly acidic tumor microenvironment (pH 5.0-6.5) and lysosomes (pH 4.5-5.0), e.g., stilbenes, carbonates acetals, and ketones. Antibody-drug couplings based on such linkers typically have a short half-life (2-3 days) due to the limited plasma stability of acid breakage linkers. This short half-life has somewhat limited the use of pH-sensitive linkers in the new generation of antibody-drug couplers. For glutathione-sensitive linkers, also known as disulfide-bonded linkers. Drug release is based on the difference between the high intracellular glutathione concentration (millimolar range) and the relatively low glutathione concentration in the blood (micromolar range). This is particularly true for tumor cells, whose low oxygen content leads to enhanced reductase activity and thus to higher glutathione concentrations. Disulfide bonds are thermodynamically stable and therefore have better stability in plasma. Enzyme destabilizing linkers, such as peptide linkers, provide better control of drug release. Peptide linkers can be efficiently severed by lysosomal proteases such as tissue protease (Cathepsin B). This peptide linkage is thought to be very stable in the plasma circulation due to the unfavorable extracellular pH and serum protease inhibitors resulting in proteases that are normally inactive outside the cell. In view of the high plasma stability and good intracellular break selectivity and effectiveness, enzyme-unstable linkers are widely used as breakable linkers for antibody-drug couplings.
The term "antibody-drug conjugate" refers to the attachment of an antibody to a biologically active drug by means of a stabilized linkage unit. In the present application, "ligand-drug coupling", preferably antibody-drug conjugate (ADC), refers to the attachment of a monoclonal antibody or antibody fragment to a biologically active toxic drug via a stabilized linkage unit.
The three-letter codes and single-letter codes for amino acids used in this disclosure are as described in J. boil. chem. 1968, 243, 3558.
The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms (i.e., "C1-C20 alkyl"), preferably an alkyl group containing 1 to 12 carbon atoms (i.e., "C1-C12 alkyl"), more preferably an alkyl group containing 1 to 10 carbon atoms (i.e., "C1-C10 alkyl"), and most preferably an alkyl group containing 1 to 6 carbon atoms (i.e., "C1-C6 alkyl"). 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-m ethylpentyl, 3 -methylpentyl, 4-methylpentyl, 2, 3 -dimethylbutyl, n-heptyl, 2-methylhexyl, 3 -methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3- di methyl pentyl, 2, 4-dimethylpentyl, 2, 2-dimethylpentyl 2, 3 -di methylpentyl, 3, 3-dimethylpentyl, 2-ethylpentyl, 3 -ethylpentyl, n-octyl, 2, 3 -dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 2, 2-dimethylhexyl, 3, 3 -dimethylhexyl, 4, 4-dimethylhexyl, 2-ethylhexyl, 3 -ethylhexyl, 4- ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3 -ethylpentyl, n-nonyl, n-ethylpentyl, 2, 2- dimethylpentyl, n-nonyl, 2, 2-dimethylpentyl, n-nonyl, 2, 2-dimethylpentyl, 2, 2-methylpentyl, 2, 2-methylpentyl ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-m ethyl-3 -ethylhexyl, 2, 2- diethylpentyl, n-decyl, 3, 3 -di ethylhexyl, 2, 2-diethylhexyl, and their various branched isomers, and the like. More preferred are lower alkyl groups containing from 1 to 6 carbon atoms, and nonlimiting embodiments include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, secbutyl, n-pentyl, 1, 1 -dimethylpropyl, 1, 2-dimethylpropyl, 2, 2-dimethylpropyl, 1 -ethylpropyl, 2- m ethylbutyl, 3-methylbutyl, n-hexyl, l-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, and the like. The alkyl group may be substituted or non- substituted, and when substituted, the substituent group may be substituted at any available point of attachment, said substituent group preferably being one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkane thio, heterocycloalkylthio, oxo. The term "substituted alkyl" means that the hydrogen in the alkyl group is replaced by a substituent group which, unless otherwise indicated in the text, may be a variety of groups selected from the following group: -halogen, -OR', -NR'R' ', -SR', -SiR'R"R'", -OC(O)R ', -C(O)R', -CO2 R', -CONR'R", -OC(O)NR'R'", -NR"C(O)R", -NR'-C(O)NR"R"", -NR"C(O)2 R", -NH- C(NH2 )=NH, -NR'C(NH2 )=NH, -NH- C(NH2 )=NR', -S(O)R', -S(O)2 R', -S(O)2 NR'R", -NR'S(O) 2R", -CN and -NO2, the number of substituents is from 0 to (2m'+l), where m' is the total number of carbon atoms in the group. R', R" and R'" each independently refer to hydrogen, unsubstituted C1-8 alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted by 1-3 halogens, unsubstituted Ci-salkyl, C1-8 alkoxy or C1-8 thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)-Ci-4 alkyl. When R' and R" are attached to the same nitrogen atom, they may form a 3-, 4-, 5-, 6-, or 7-metacyclic ring together with that nitrogen atom. For example, - NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.
The term “substituted alkyl” refers to saturated straight-chain or branched-chain aliphatic hydrocarbon radicals having two residues derived from the removal of two hydrogen atoms from the same carbon atom of the parent alkyl or from two different carbon atoms, which are straightchain or branched-chain groups containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, and more preferably containing 1 to 6 carbon atoms. Non-limiting examples of alkanyl groups include, but are not limited to, methylene (-CH2-, 1, 1 -ethene (-CH(CH3)-), 1, 2- ethene (-CH2CH2)-, 1, 1 -propene (-CH(CH2CH3)-), 1, 2- propylidene (-CH2CH(CH3)-), 1, 3- propylidene (-CH2CH2CH2-), 1, 4-butylidene (-CH2CH2CH2CH2-) and 1, 5-butylidene (- CH2CH2CH2CH2CH2-). The alkylidene group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available attachment point, said substituent being independently selected preferably from the group consisting of alkyl, alkynyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycle alkoxy, cycloalkyl sulfide, heterocycle alkyl sulfide and one or more substituents selected independently of one another from the group consisting of oxygencontaining substituents.
The term “alkoxy” refers to -O-(alkyl) and -O-(cycloalkyl), where alkyl or cycloalkyl is as defined above. Non-limiting examples of C1-C6 alkoxides include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexoxy. The alkoxy group may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkynyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycle alkyl, aryl, heteroaryl, cycloalkoxy, heterocycle alkoxy, cycloalkylthio, heterocycle alkylthio.
The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring comprises from 3 to 20 carbon atoms (i.e. "C3-C20 cycloalkyl"), preferably from 3 to 12 carbon atoms (i.e. "C3-C12 cycloalkyl"), more preferably comprising 3 to 10 carbon atoms (i.e., "C3-C10 cycloalkyl"), most preferably comprising 3 to 8 carbon atoms (i.e., "C3-C8 cycloalkyl "). Non-limiting examples of monocyclic cycloalkyl groups (e.g., "C3-C8 cycloalkyl") include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptatrienyl, cyclooctyl, and the like; and multi-cyclic cycloalkyl groups include cycloalkyl groups of spirocycles, thickened rings, and bridged rings.
The term “heterocyclic group” means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent group that contains 3 to 20 ring atoms (i.e., a “3-20 membered heterocyclic group”), wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O)m (where m is an integer of 0 to 2), but excluding a ring portion of -O-O-, -O-S- or -S-S-, and the remaining ring atoms are carbon. Preferably comprising 3 to 12 ring atoms (i.e., a “3-12 membered heterocyclic group”), wherein 1 to 4 are heteroatoms; more preferably the heterocyclic group comprises 3 to 10 ring atoms (i.e., a “3-10 membered heterocyclic group”). Non-limiting examples of monocyclic heterocyclic groups (e.g., 3-7 membered heterocyclic groups) include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl. Polycyclic heterocyclic groups include heterocyclic groups with spiro, fused, or bridge rings.
The term “cycloalkylalkyl” means an alkyl group substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein the alkyl group is as defined above and the cycloalkyl group is as defined above, for example, C3-C8 cycloalkyl C1-C6 alkyl.
The term “haloalkyl” means an alkyl group substituted with one or more halogens, wherein the alkyl group is as defined above, for example, halo C1-C6 alkyl.
The term “deuterated alkyl” means an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above, for example, deuterated C1-C6 alkyl.
The term "C6-C12 aryl" refers to a group of carbocyclic aromatic systems having 6-12 carbon atoms.
The term "C6-C10 aryl" refers to a group of carbocyclic aromatic systems having 6-10 carbon atoms, such as phenyl, naphthyl, etc.
The term "5-10-membered heteroaryl" refers to an aromatic heterocyclic ring, typically a 5-, 6-, 7-, 8-, 9-, 10-membered heterocyclic ring having 1 to 3 heteroatoms selected from N, O, or S; the heteroaryl ring may optionally be further consolidated or attached to aromatic and non-aromatic carbon and heterocyclic rings. Non-limiting examples of said 5- to 10- membered heteroaryl ring are, for example, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, isothiazolyl, thioxazolyl, pyrrolyloxy, phenyl-pyrrolyloxy, furanyl, phenyl-furyl, phenyl-furanyl, oxazolyl, isoxazolyl, pyrazolyl, thiophenyi, benzo- furanyl, benzothiopheneyl, benzo 1,3-dioxolane (benzodioxolyl), isodihydroindolyl, benzimidazolyl, indazolyl, quinolinyl, isoquinolinyl, 1,2,3 -tri azolyl, 1 -phenyl- 1,2,3 - triazolyl, 2,3-dihydroindolyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothiophene, benzylpyranyl, 2,3-dihydrobenzoxazinyl, 2,3-dihydroquinoxalinyl, and others.
The terms “substituted C6-C10 aryl,” “substituted 5-10-membered heteroaryl,” or “substituted 3-7-membered heterocyclic” mean that the hydrogen in the aryl or heteroaryl or heterocyclic group has been replaced by a substituent group, unless otherwise specified in the text. The substituent group of the aryl or heteroaryl or heterocyclic group may be a group selected from the group consisting of - halogen, -OR', -NR R"', -SR', -SiR'R"R'", -OC(O)R', -C(O)R', -CO2R', - CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NH-C(NH2)=NH, - NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R", -NR'S(O)2R", -CN and - NO2, with 0 to (2m'+l) substituents, where m' is the total number of carbon atoms in the group. R', R" and R'" each independently refer to hydrogen, an unsubstituted C1-8 alkyl group, an unsubstituted C6-C12 aryl group (or C6-C10 aryl group), a C6-C12 aryl group (or C6-C10 aryl group) substituted by 1-3 halogens, an unsubstituted C i-s alkyl, Cns alkoxy or Ci-s thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl) -C1-4 alkyl. When R' and R" are attached to the same nitrogen atom, they may form a 3-, 4-, 5-, 6- or 7-membered ring with that nitrogen atom, for example, -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.
The term "hydroxyl" refers to the -OH group.
The term "halogen" means fluorine, chlorine, bromine or iodine.
The term "amino" means -NH2. The term "nitro" means -NO2.
The term "amide group" means -C(O)N(alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl is as defined above.
The term "carboxylate group" means -C(O)O(alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl is as defined above.
The present application also includes various deuterated forms of Formula I. Each of the available hydrogen atoms attached to the carbon atom may be independently replaced by a deuterium atom. A person skilled in the art can synthesize the deuterated form of Formula I with reference to the relevant literature. Commercially available deuterated starting materials may be used in the preparation of the deuterated form of Formula I, or they may be synthesized by conventional techniques using deuterated reagents, non-limiting examples of deuterated reagents include deuteroborane, tri deuteroborane tetrahydrofuran solution, lithium-aluminum deuterohydride, ethyl iodine deuteride, methylene iodide deuteride, and the like.
The term "antibody" refers to immunoglobulins, which are tetrapeptide chains consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in the composition and arrangement of the amino acids in the constant region of the heavy chain, and therefore differ in their antigenicity. Accordingly, immunoglobulins can be categorized into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, with corresponding heavy chains of p-chain, 8-chain, '/-chain, a-chain and s-chain, respectively. The same class of Ig can be further divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of disulfide bonds of the heavy chain, such as IgG can be divided into IgGl, IgG2, IgG3, IgG4. The light chain is divided into K-chain or Z-chain through the differences in the constant region. Each of the five classes of Ig may have either a K chain or a z chain. The antibodies described in the present invention are preferably specific antibodies against cell surface antigens on target cells, and non-limiting embodiments are the following antibodies: anti-EGFRvIII antibody, anti -DLL-3 antibody, anti -PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGFl antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti- LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg/AFP antibody, anti-Claudinl8.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT/CD117 antibody, anti-STEAPl antibody, anti- SLAMF7/CS1 antibody, anti-NaPi2B/SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUCl/CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti- B7-H3 (CD276) antibody, anti-PTK7/CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-N0TCH3 antibody, anti-Nectin 4 antibody, anti-TROP-2 antibody, antiCD 142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRa antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-Integrin Av antibody, anti- CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6 antibody, anti- LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti- CD30 antibody, anti-CD37 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody or anti-CD123 antibody.
The term “solvates” or “solvated compound” refers to the formation of a pharmaceutically usable solvent compound of the ligand-drug conjugate of the present application with one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.
The term "drug load" refers to the average amount of cytotoxic drug loaded on each antibody in Formula I. It can also be expressed as a ratio of drug amount to antibody amount, and the drug load can range from 0-12, preferably 1-10 cytotoxic drugs (d) per antibody (Ab). In embodiments of the present application, the drug load is denoted as n, which exemplarily may be the mean of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, for example. The average amount of drug per ADC molecule after coupling reaction can be identified by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA, and HPLC.
In one embodiment of the present application, the cytotoxic drug is coupled to the open cysteine sulfhydryl-SH and/or the sulfhydryl-SH of the cysteine residue of site-specific mutation between the antibody chains through a linker unit, and in general, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than or equal to a theoretical maximum.
The loading of ligand-cytotoxic drug conjugates can be controlled by using the following non-limiting methods, including:
(1) controlling the molar ratio of the linker reagent and the monoclonal antibody,
(2) controlling the reaction time and temperature,
(3) selecting different reaction reagents.
For the preparation of conventional pharmaceutical compositions, see the pharmacopeia such as the Chinese Pharmacopoeia or USP.
The term “pharmaceutically acceptable salt” or “pharmaceutically acceptable salt” refers to the salt of the ligand-drug conjugate of the present application, or the salt of the compound described in the present application, which is safe and effective when used in mammals and is biologically active as desired. The ligand-drug conjugate compounds of the present application contain at least one carboxyl group and can therefore form salts with bases. Non-limiting examples of pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts or magnesium salts.
The term “pharmaceutically acceptable salt” or “pharmaceutically acceptable salt” refers to the salt of the ligand-drug conjugate of the present application, or the salt of the compound described in the present application, which is safe and effective when used in mammals and is biologically active as desired. The ligand-drug conjugate of the invention contains at least one amino group and can therefore form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydriodate, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pyrite, hydrophosphate, dihydrophosphate, salicylate, citric acid hydrochloride, tartaric acid salt, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.
The term "acidic amino acid" refers to amino acids with an isoelectric point of less than 7. Acidic amino acid molecules often contain one or more acidic groups, such as carboxyl groups, which can be effectively ionized into negative ionic forms in the structure to increase hydrophilicity. Acidic amino acids can be natural or unnatural.
The term "natural amino acids" refers to biologically synthesized amino acids. Natural amino acids are generally L-types, but there are a few exceptions, such as glycine, that comes naturally and biosynthesized by living organisms.
The term "unnatural amino acids" refers to amino acids obtained by synthetic means.
The present application may be understood more readily by reference to the following detailed description of specific embodiments and examples included herein. Although the present application has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the disclosure. Experimental methods without specific conditions indicated in the following embodiments are generally carried out under normal conditions or under conditions recommended by the manufacturer. Unless otherwise indicated, all percentages, proportions, ratios, or parts are by weight.
Example 1
Synthesis of compound
Figure imgf000088_0001
In a 5000 mL flask, N-fluorenylmethoxycarbonyl-glycine-glycine (100 g, 282 mmol, 1.0 eq), lead tetraacetate (175 g, 395 mmol, 1.4 eq), 2000 mL of dry tetrahydrofuran and 670 mL of toluene were added, stirred uniformly, protected by nitrogen, and the reaction was heated to 85 °C for 2.5 h. The reaction was monitored by TLC, and the raw materials were finished reacting. After the reaction, cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure, the residue was purified by column chromatography to obtain compound Ml (87 g); LC-MS: [M+NH ]4 + =386.0.
Example 2
Synthesis of compound M3:
Figure imgf000089_0001
In a 1000 mL flask, compound SM-2 (synthesized according to the method published in CN108452321 A) (40 g, 96 mmol, 1.0 eq), tri ethylamine (26.7 mL, 2.0 eq), toluene (400 mL) were added, and the reaction was carried out by refluxing the compound at a temperature of 120 °C for 2 h. The reaction was monitored to be almost completely reacted by TLC, and then the solvent was spun off by reducing the temperature to 50 °C and decompression. The solvent was removed by spinning under reduced pressure at 50 °C. Dissolve with ethyl acetate (150 mL), water (40 mL), adjust pH to 2-3 with IM HC1 under ice bath stirring, and partition. The aqueous layer was extracted once more with ethyl acetate, the organic layers were combined and dried by adding anhydrous sodium sulfate. After filtration, a light yellow oily crude was obtained by concentration, and the crude was purified by column chromatography (DCM:MeOH=40: l) to obtain compound M2 (26.6 g); LC-MS: [M+H]+ =399.3.
In a 1000 mL flask, compound M2 (26.5 g, 60.5 mmol, 1.0 eq), pentafluorophenol (12.2 g, 66.5 mmol, 1.1 eq), DCC (13.7 g, 66.5 mmol, 1.1 eq), and THF (300 mL) were added, and the reaction was carried out at room temperature for 30 min (monitored by TLC), the insoluble material was filtered out. The reaction solution was purified directly by preparation, and the prepared solution was concentrated at 35 °C in a reduced pressure water bath to remove acetonitrile, and lyophilized to obtain compound M3 (31.5 g) in 64% yield; LC-MS: [M+H]+ =565.1.
Example 3
Synthesis of the compound ent-M3:
Figure imgf000089_0002
Referring to the synthetic route of Example 2, compound ent-M3 (27.8 g) was obtained; LC- MS: [M+H]+ =565.2.
Example 4
Synthesis of compound 1:
Figure imgf000090_0001
Step 1 : Compound la
In a 250 mL flask, add Ml (6 g, 16.3 mmol), 100 mL THF, p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol), stirring and cooled to 0 °C, dropwise addition of benzyl hydroxyacetate (5.4 g, 32.6 mmol), dropwise natural warming to room temperature reaction (the reaction is about 2-4 h), TLC monitoring. At the end of the reaction, saturated NaHCOa solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, fdtered, concentrated, and the residue was purified by silica gel column (PE:EA=10:l-5:l-l:l) to obtain la (4 g) in 52% yield; LC-MS: [M+H]+ =475.18. Step 2: Compound lb
In a 25 mL flask, add la (2 g, 4.2 mmol), 10 mL DMF, stir at 0 °C, add DBU (766 mg, 5.04 mmol), and react for 1 h. After completion of Fmoc deprotection was monitored by TLC, the reaction was set aside;
Another 25 mL flask was filled with M4 (prepared by the method published in CN111051330 A) (1.73 g, 4.2 mmol), PyBOP (2.61 g, 5.04 mmol), HOBt (680 mg, 5.04 mmol), and 10 mL of DMF, and DIPEA (830 uL, 5.04 mmol) was added to the reaction vial under ice-water bath, and stirring was continued for 30 min, the above reaction solution was added to the reaction flask, and the reaction was raised to room temperature. HPLC monitored the end of the reaction, the reaction solution was purified by preparative liquid phase to obtain the product preparation, the preparation was extracted by dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain solid lb (1.7 g) in 63% yield; LCMS: [M+H]+ =648.26.
Step 3: Compound 1c lb (900 mg, 1.39 mmol) was added to a 25 mL flask, and after 15 mL of DMF was dissolved, 900 mg of 5% Pd/C was added, and the reaction was hydrogenated for 2 h. The reaction was completed, and the filtrate was filtered to obtain the filtrate, which was used directly for the next step of the reaction without purification.
Step 4: Compound Id
The crude product 1c was placed in an ice-water bath, DIPEA (235 uL, 1.39 mmol) was added, and compound M3 (784 mg, 1.39 mmol) was added, and the reaction was brought to room temperature for 1 h. The reaction was monitored for completion by HPLC, and the reaction solution was purified by high-performance liquid phase purification to obtain the preparative solution, which was lyophilized to obtain Id (504 mg); LC-MS: [M+H]+ = 804.4. Step 5: Compound le
Id (500 mg, 0.62 mmol), M5 (310 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added to a 50 mb single-necked vial, DIPEA (378 uL, 2.29 mmol) was added to the vial under an ice-water bath, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to give the preparative solution of compound le, and the preparative solution was lyophilized to give le (210 mg); LCLC monitored the completion of the reaction, and the preparative solution was lyophilized to give le (210 mg). The reaction was carried out at room temperature for 2 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound le, which was lyophilized to obtain le (210 mg); LC-MS: [M+H]+ 1221.6.
Step 6: Compound 1 le (100 mg, 0.081 mmol), zinc bromide (368 mg, 1 .63 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 1 (60 mg); LC-MS: [M+H]+ =1065.3.
Example 5 Synthesis of compound 2:
Figure imgf000091_0001
Referring to the synthetic route of Example 4, compound 2 (51 mg) was obtained; LC-MS: [M+H]+ =1065.3.
Example 6
Synthesis of compound 3:
Figure imgf000092_0001
Step 1 : Compound 3a
In a 250 mL flask, add Ml (6 g, 16.3 mmol), 100 mL THF, p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol), stirring and cooled to 0 °C, dropwise addition of benzyl 2- hydroxy-2-methylpropionate (6.3 g, 32.6 mmol), after dropwise natural warming to room temperature reaction (reaction about 2-4 h), TLC monitoring. At the end of the reaction, saturated NaHCOa solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, fdtered, concentrated, and the residue was purified by silica gel column (PE:EA=10: 1-5: 1-2: 1) to give 3a (4.2 g) in 52% yield; LC-MS: [M+H]+ =503.3.
Step 2: Compound 3b
In a 25 mL flask, add 3a (2 g, 4.0 mmol), 10 mL DMF, stir at 0 °C, add DBU (760 mg, 5.0 mmol), and react for 1 h. After the completion of Fmoc deprotection was monitored by TLC, the reaction was set aside;
Add M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol) and 10 mL of DMF into another 25 mL flask, and add DIPEA (823 uL, 5.04 mmol) in an ice-water bath with continued stirring for 30 min, and then add the above reaction solution to the reaction flask and raise the reaction to room temperature. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain the product preparation solution, which was extracted by dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the solid 3b (1.4 g) in 53% yield; LC-MS: [M+H]+ =676.2.
Step 3: Compound 3c
3b (700 mg, 1.04 mmol) was added to a 25 mL flask, and after 10 mL of DMF was dissolved, 700 mg of 5% Pd/C was added, and the reaction was hydrogenated for 1.5 h. The reaction was completed, and the filtrate was filtered to obtain the filtrate, which was used directly for the next step of the reaction without purification.
Step 4: Compound 3d
The crude product 3c was placed in an ice-water bath, DIPEA (210 uL, 1.25 mmol) was added, and compound M3 (704 mg, 1.25 mmol) was added, and the reaction was brought to room temperature for 1 h. The completion of the reaction was monitored by HPLC, and the reaction solution was purified by high-performance liquid phase purification to give the preparation, which was lyophilized to give 3d (486 mg); LC-MS: [M-H]' = 830.5. Step 5: Compound 3e
3d (300 mg, 0.36 mmol), M5 (180 mg, 0.36 mmol), PyBOP (260 mg, 0.5 mmol), HOBt (67 mg, 0.5 mmol), and 10 mL of DMF were added to a 50 mb flask, DIPEA (219.5 uL, 1.33 mmol) was added to the vial under an ice-water bath, and the reaction was carried out at room temperature for 3 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to give the preparative solution of compound 3e. The reaction was carried out at room temperature for 3 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound 3e, which was lyophilized to obtain 3e (157 mg); LC-MS: [M+H]+ =1249.6. Step 6: Compound 3 3e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL single-necked vial and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 3 (64 mg); LC-MS: [M+H]+ = 1093.1.
Example 7
Synthesis of compound 4:
Figure imgf000093_0001
Referring to the synthetic route of Example 6, compound 4 (60 mg) was obtained; LC-MS: [M+H]+ =1093.2.
Example 8
Synthesis of compound 5A:
Figure imgf000094_0001
Step 1 : Compound 5a
Add Ml (500 mg, 1.4 mmol, 1.0 eq), p-toluenesulfonic acid monohydrate (26 mg, 0.1 mmol, 0.1 eq) and 10 mL of THF in a 25 mL flask, stirred well, and then lowered to 0 °C, and then slowly added L-lactic acid benzyl ester (1.2 g, 7.0 mmol, 5 eq), and then raised to room temperature after the addition of the reaction, TLC monitoring. At the end of the reaction, saturated NaHCCh solution was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by reversed-phase column to give 5a (400 mg);
LC-MS: [M+NH ]4 + = 506.2.
'H NMR (400 Mz, CDC13 /CD3 OD):1.39 (3H, d, J= 6.8 Hz), 3.78 (2H, t, J= 4.0 Hz), 4.17- 4.27 (2H, m), 4.42 (2H, d, J = 4.0 Hz), 4.72-4.85 (2H, m), 5.11-5.58 (2H, m), 5.43 (1H, s), 7.06 (1H, t, J= 8.0 Hz), 7.25-7.33 (6H, m), 7.38 (2H, t„ 7.38 (2H, t, 7.06 (1H, t, 7.06), 7.25-7.33 (6H, m), 7.25-7.33 (7.25-7.33) 5.43 (1H, s), 7.06 (1H, t, J= 8.0 Hz), 7.25-7.33 (6H, m), 7.38 (2H, t, J = 8.0 Hz), 7.57 (2H, d, J= 8.0 Hz), 7.75 (2H, d, J= 8.0 Hz).
Step 2: Compound 5b
Compound 5a (400 mg, 0.8 mmol, 1.0 eq) and 4 mL of DMF were added to a 25 mL flask, stirred well, and then lowered to 0 °C before slowly adding the DBU (137 mg, 0.9 mmol, 1.1 eq), and after addition, the reaction was brought to room temperature. The reaction was monitored by TLC and the end of the reaction was recorded as reaction solution (T);
Another 25 mL single-necked vial was taken and M4 (372 mg, 0.9 mmol, 1.1 eq), PyBOP (852 mg, 1.6 mmol, 2.0 eq) and 3 mL of DMF were added, stirred at room temperature for 5 min, and reaction solution (T) was added, the reaction was carried out at room temperature, and monitored by HPLC. The reaction was completed, and the reaction solution was purified by HPLC to yield compound 5b (326 mg); LC-MS: [M+NH ]4 =679.2.
Step 3: Compound 5c
5b (4.0 g, 6.05 mmol, 1.0 eq) was added to a 100 mL flask, DMF (60 mL) was dissolved, then 5% Pd/C (4 g) was added, and the hydrogenation reaction was carried out at room temperature for 4 h (HPLC was used to monitor the progress of the reaction). The Pd/C was filtered, and the filtrate was placed directly in an ice-water bath (ca. 0 °C) without concentration and left for use. Step 4: Compound 5d
The crude product 5c was placed in an ice-water bath, DIPEA (1.1 mL, 1.1 eq) was added, and compound M3 (3.4 g, 6.05 mmol) was added, and the reaction was brought to room temperature for 2 h. The completion of the reaction was detected by HPLC, and the reaction solution was purified by high performance liquid phase purification to obtain the preparative solution, which was lyophilized to obtain 5d (3.15 g); LC-MS: [M-H]' =816.3.
Step 5: Compound 5e
5d (2.07 g, 2.53 mmol, 1.0 eq), M5 (1.35 g, 2.53 mmol, 1.0 eq), PyBOP (1.98 g, 3.79 mmol,
1.5 eq), HOBt (0.51 g, 3.79 mmol, 1.5 eq) and DMF (40 mL) were added to a 100 mL singlenecked vial, and the reaction was brought to room temperature for 2 h. The reaction was monitored by HPLC. DIPEA (1.05 mL, 1.5 eq) was added, and the reaction was brought to room temperature for 2 h (monitored by HPLC). The reaction solution was purified directly by preparation, and the preparative solution was concentrated by pumping water under reduced pressure at 35 °C to remove acetonitrile, and lyophilized to obtain compound 5e (1.92 g) in 61% yield; LC-MS: [M+H]+ =1235.4.
Step 6: Compound 5A
Compound 5e (1.0 g, 0.8 mmol, 1.0 eq), 35 mL nitromethane was added in a 100 mL flask, dissolved and then added zinc bromide (3.64 g, 16 mmol, 20.0 eq), reacted for 30 min in an oil bath at 40 °C (stabilized in advance by preheating), and pumped to concentrate at 45 °C under reduced pressure in a water bath to remove the nitromethane, resulting in a solid yellow residue (monitored by HPLC). After acid preparation, the preparative solution of compound 5A was obtained, and the preparative solution was concentrated and spun off acetonitrile by pump decompression water bath at 35 °C and lyophilized to obtain compound 5A (786 mg) in 90% yield.
LC-MS: [M+H]+ = 1079.4;
'H NMR (400 MHz, DMSO-d6) 5 9.39 - 9.02 (m, 1H), 8.70 (t, J= 6.5 Hz, 1H), 8.64 (t, J= 5.7 Hz, 1H), 8.56 (d, J= 8.8 Hz, 1H), 8.34 (t, J= 5.7 Hz, 1H), 8.16 ( d, J = 8.2 Hz, 1H), 8.01 (t, J =
5.5 Hz, 1H), 7.71 (d, J= 10.9 Hz, 1H), 7.30 (s, 1H), 7.28 - 7.15 (m, 4H), 7.14 (s, 2H), 5.53 (dd, J = 14.5, 6.4 Hz, 1H), 5.49 - 5.34 (m, 2H), 5.22 (d, J= 18.8 Hz, 1H), 5.09 (d, J= 18.7 Hz, 1H), 5.03 (dd, .7= 9.6, 3.9 Hz, 1H), 4.73 (dd, J= 9.9, 6.9 Hz, 1H), 4.59 (dd, J= 10.1 , 6.5 Hz, 1H), 4.49 (dd, J= 13.2, 8.6, 4.4 Hz, 1H), 4.14 (dd, J= 13.3, 6.6 Hz, 2H), 3.93 (s, 2H), 3.84 (dd, J= 16.5, 6.3 Hz, 1H), 3.76 (dd, .7 = 16.9, 5.7 Hz, 2H), 3.70 (d, J = 9.9, 6.9 Hz, 2H) 3.70 (dd, J = 5.2 Hz, 2H), 3.60 (dd, J= 16.7, 5.4 Hz, 1H), 3.52 (dd, J= 16.4, 5.1 Hz, 1H), 3.45 (dd, J= 12.8, 10.1 Hz, 1H), 3.25 - 3.15 (m, 1H), 3.14 - 3.05 (m, 1H), 3.01 (dd, J = 13.7, 4.1 Hz, 1H), 2.73 (dd, J = 13.5, 9.8 Hz, 1H), 2.54 - 2.47 (m, 1H), 2.33 (s, 2H), 2.17 (d, J= 5.5 Hz, 2H ), 1.91 - 1.79 (m, 2H), 1.33 (d, J =
6.6 Hz, 2H), 0.87 (t, J= 7.3 Hz, 2H).
Example 9
Synthesis of compound 5B:
Figure imgf000096_0001
Step 1 : Compound 5d-l
Compound 5b (300 mg, 0.45 mmol, 1 .0 eq), DMF (3 mL) was added in a 25 mL flask with stirring to dissolve the clear, 5% Pd/C (300 mg) was added, hydrogen displacement was performed three times, and the hydrogenation reaction was carried out for 2 h. The end of the reaction was monitored by HPLC. At the end of the reaction, the Pd/C was removed by filtration, the filtrate was cooled down to 0-5 °C, DIPEA (65 mg, 0.5 mmol, 1.1 eq) was added, and then ent-M3 (255 mg, 0.45 mmol) was added to the filtrate, and then the reaction was raised to 20±5 °C for 1 h. The end of the reaction was monitored by HPLC. At the end of the reaction, it was purified by HPLC preparation, and the product preparation was collected and lyophilized to obtain compound 5d-l (200 mg) in 54% yield; LC-MS: [M-H]' =816.3.
Step 2: Compound 5e-l
Compound 5d-l (200 mg, 0.24 mmol, 1.0 eq), M5 (127 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2 eq), HOBt (48 mg, 0.36 mmol, 1.2 eq), and DMF (6 mL) were added to a 25 mL flask. Water bath was lowered to 0-5 °C, DIPEA (62 mg, 0.48 mmol, 2.0 eq) was added, and the reaction was raised to 20 ± 5 °C for 2 h. The end of the reaction was monitored by HPLC. The reaction solution was purified directly by HPLC preparation, and the product preparation was collected and lyophilized to obtain compound 5e-l (162.8 mg); LC-MS: [M+H]+ =1235.4. Step 3: Compound 5B
Compound 5e-l (110 mg, 0.089 mmol, 1.0 eq), ZnBr? (400 mg, 1.78 mmol, 20.0 eq) and CH3 NO2 (10 mL) were sequentially added into a 25 mL flask. After addition, the reaction was raised to 40 °C for 0.5 h, and then the reaction was stopped, and the reaction solution was dried directly at 45 °C by spin-drying under reduced pressure to obtain a yellow solid. The reaction was monitored by HPLC. The spin-dried solid was directly purified by HPLC preparation, and the product preparation was collected and lyophilized to obtain compound 5B (73.4 mg) in 76.5% yield; LC-MS: [M+H] 1 =1079.4.
Example 10
Preparation of compound 6A:
Figure imgf000097_0001
Referring to the synthetic route of Example 8, compound 6A (71 mg) was obtained; LC-MS: [M+H]+ =1079.4.
Example 11
Figure imgf000097_0002
Referring to the synthetic route of Example 9, compound 6B (59 mg) was obtained; LC-MS: [M+H]+ =1079.4.
Example 12 Preparation of compounds 7A and 7B:
Figure imgf000097_0003
Step 1 : Compound 7a
In a 250 mL flask, add Ml (10 g, 27.1 mmol), benzyl 3, 3, 3 -trifluorolactate (prepared according to the method published in Patent W02020063673 Al) (12.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol), and 100 mL of toluene, and react at 100 °C for 4 h. The reaction was completed. The reaction was completed, reduced to room temperature, filtered to remove insoluble material, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA=10: 1-5: 1-2: 1) to obtain 5.15 g of the target material, yield 35.1%; LC-MS: [M+H]+ =543.17.
Step 2: Compound 7b
Add 7a (5 g, 9.2 mmol) and 15 mL of DMF in a 50 mL flask, dissolve and clear, and then add DBU (1.68 g, 11 mmol) in an ice-water bath and react for 1 h. This was recorded as reaction solution (T);
Another 50 mL flask was taken, M4 (3.8 g, 9.2 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol) and 10 mL DMF were added, and after dissolved, DIPEA (1.82 mL, 11 mmol) was added in an ice-water bath, and the reaction was continued for 30 min, then reaction solution (1) was added, and the reaction was brought to room temperature for 2 h. The reaction was monitored by HPLC. The reaction was monitored by HPLC for 2 h. After completion of the reaction, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution. The prepared solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 4.1 g of solid, with a yield of 62.3%; LC-MS: [M+H]+ =716.25.
Step 3: Compound 7d
Add 7b (900 mg, 1 .26 mmol) in a 25 mL flask, after 15 mL of DMF was dissolved, add 900 mg 5% Pd/C, hydrogenation reaction for 2 h, the reaction was completed, filtration, the filtrate was placed in an ice-water bath, add DIPEA (228 uL, 1.38 mmol), and then add M3 (712 mg, 1.26 mmol), and then add the solution to the room temperature. The reaction was monitored by HPLC for 1 h. The reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution, which was lyophilized to obtain 525 mg of the product in 47.9% yield; LC-MS: [M-H]' =870.33.
Step 4: Compound 7e
7d (500 mg, 0.57 mmol), M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added to a 50 mL flask, DIPEA (378 uL, 2.29 mmol) was added to the flask under an ice-water bath, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to obtain the preparative solution of compound 7e-l and compound 7e-2, and the preparative solution was lyophilized to obtain 150 mg of compound 7e-2. The reaction was carried out at room temperature for 2 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solutions of compound 7e-l and compound 7e-2, which were lyophilized to obtain 150 mg of compound 7e-l, LC-MS: [M+H]+ =1289.46, and 220 mg of compound 7e-2, LC-MS: [M+H]+ =1289.46, respectively. Step 5: Compound 7A
Figure imgf000099_0001
7e-l (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 52 mg of solid; TOF result: 1133.3613.
Step 6: Compound 7B
Figure imgf000099_0002
7e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 63 mg of solid; TOF result: 1133.3668.
Example 13
Synthesis of compounds 8A
Figure imgf000099_0004
Figure imgf000099_0003
Figure imgf000099_0005
Step 1 : Compound 8d
In a 25 mL flask, 7c (900 mg, 1.83 mmol) was added, 20 mL of DMF was dissolved, then DIPEA (303 uL, 1.83 mmol) was added, and ent-M3 (1034 mg, 1.83 mmol) was added, and the reaction was brought to room temperature for 1 h. The completion of the reaction was detected by HPLC, and the reaction solution was purified by high-performance liquid chromatography (HPLC) to obtain the preparative solution, which was lyophilized to give 613 mg of product in 38.5% yield. The prepared solution was lyophilized to obtain 613 mg of product with 38.5% yield; LC-MS: [M- H]' =870.32.
Step 2: Compound 8e-l and Compound 8e-2
8d (500 mg, 0.57 mmol), M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mb of DMF were added into a 50 mL flask, DIPEA (378 uL, 2.29 mmol) was added into the vial in an ice-water bath, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to obtain the preparation of compound 8e-l and compound 8e-2. After the reaction was completed by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solutions of compound 8e-l and compound 8e- 2, which were lyophilized to obtain 140 mg of compound 8e-l and 210 mg of compound 8e-2, respectively. The LC-MS of compound 8e-l : [M+H]+ =1289.47; the LC-MS of compound 8e-2: [M +H]+ =1289.47.
Step 3:
Figure imgf000100_0001
Compound 8e-l (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added to a 25 mL flask and reacted at 40 °C for 1 h. After the reaction was completed by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 50 mg of solid; TOF result: 1133.3623.
Step 4
Figure imgf000100_0002
Compound 8e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added to a 25 mL single-necked vial and reacted at 40 °C for 1 h. After the reaction was completed by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain a solid 58 mg; TOF result: 1133.3653.
Example 14 Synthesis of compound 9A:
Figure imgf000101_0001
Step 1 :
Figure imgf000101_0002
In a 250 mL flask, add Ml (6 g, 16.3 mmol), 100 mL THF, p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol), stir and cool to 0 °C, dropwise add benzyl 2-hydroxy-2- cyclopropyl acetate (prepared according to the method published in US20050020645 Al) (6.3 g, 32.6 mmol), the reaction was naturally warmed up to room temperature after dropping (the reaction was about 2-4 h) and monitored by TLC. At the end of the reaction, saturated NaHCCh solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA=10:l-5: 1-2: 1) to give 9a (3.7 g) in 45% yield; LC-MS: [M+H]+ =501.5.
Step 2: Compound 9b
In a 25 mL flask, add 9a (2 g, 4.0 mmol), 10 mL DMF, stir at 0 °C, add DBU (760 mg, 5.0 mmol), and react for 1 h. After the completion of Fmoc deprotection was monitored by TLC, the reaction was set aside;
Add M4 (1.65 g, 4.0 mmol), PyBOP (2.59 g, 5.0 mmol), HOBt (675 mg, 5.0 mmol) and 10 mL of DMF into another 25 mL flask, and add DIPEA (823 uL, 5.04 mmol) in an ice-water bath with continued stirring for 30 min, and then add the above reaction solution to the reaction flask and raise the reaction to room temperature. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain the product preparation solution, which was extracted by di chloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a solid of 1.5 g in 56% yield; LC-MS: [M+H]+ =674.7.
Step 3: Compound 9c
9b (900 mg, 1.3 mmol) was added to a 25 mL flask, and after 10 mL of DMF was dissolved and cleared, 900 mg of 5% Pd/C was added, and the hydrogenation reaction was carried out for 1.5 h. After completion of the reaction, it was filtered to obtain the filtrate, which was used directly for the next step of the reaction without purification.
Step 4: Compound 9d
The crude product 9c was placed in an ice-water bath, DIPEA (223 uL, 1.3 mmol) was added, and compound M3 (750 mg, 1.3 mmol) was added, and the reaction was brought to room temperature for 1 h. The reaction was monitored for completion by HPLC, and the reaction solution was purified by high-performance liquid phase purification to give the preparation, which was lyophilized to give 9d (529 mg); LC-MS: [M-H]' = 828.4. Step 5: Compound 9e
9d (500 mg, 0.6 mmol), M5 (300 mg, 0.6 mmol), PyBOP (416 mg, 0.8 mmol), HOBt (108 mg, 0.5 mmol) and 15 mb of DMF were added into a 50 mL vial, DIPEA (351 uL, 2.13 mmol) was added into the flask under an ice-water bath, and the reaction was carried out for 3 h. After the reaction was completed by HPLC, the reaction solution was purified by HPLC to obtain the preparative solution of compound 9e. After the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound 9e, which was lyophilized to obtain 9e (257 mg); LC-MS: [M+H]+ =1247.5.
Step 6: Compound 9A 9e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 9A (55 mg); LC-MS: [M+H]+ =1091.3.
Example 15
Synthesis of compound 9B:
Figure imgf000102_0001
Referring to the synthetic route of Example 14, compound 9B (44 mg) was obtained; LC-MS: [M+H]+ =1091.3.
Example 16
Synthesis of compound 10A:
Figure imgf000103_0001
Step 1 : Compound 10a
In a 250 mL flask, add Ml (6 g, 16.3 mmol), 100 mL THF, p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol), stirring and cooling to 0 °C, dropwise add benzyl 3-hydroxy-2- cyclopropylpropionate (prepared with reference to the method published in WO2013187496A1) (6.7 g, 32.6 mmol). The reaction was naturally warmed to room temperature after dropping (about
2-4 h) and monitored by TLC. At the end of the reaction, saturated NaHCCL solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA=10:l-5:l-2:l) to give 10a (4.9 g) in 58% yield; LC-MS: [M+H]+ =515.4.
Step 2: Compound 10b
In a 25 mL flask, add 10a (4 g, 7.8 mmol), 10 mL DMF, stir at 0 °C, add DBU (1.2 g, 8.0 mmol), react for 1 h. After the completion of Fmoc deprotection was monitored by TLC, the reaction was set aside;
Another 25 mL flask was filled with M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol) and 10 mL of DMF, and DIPEA (1.65 mL, 10.1 mmol) was added in an icewater bath with continued stirring for 50 min, and then the above reaction solution was added to the flask and raised to room temperature. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain the product preparation solution, which was extracted by dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a solid of 2.3 g in 42% yield; LC-MS: [M+H]+ =688.8.
Step 3: Compound 10c
10b (1.0 g, 1.45 mmol) was added to a 25 mL flask, and after 15 mL of DMF was dissolved and cleared, 1.0 g of 5% Pd/C was added, and the reaction was hydrogenated for 1.5 h. The reaction was completed, and the fdtrate was filtered to obtain the filtrate, which was used directly for the next step of the reaction without purification.
Step 4: Compound lOd
The crude product 10c was placed in an ice- water bath, DIPEA (258 uL, 1.5 mmol) was added, and compound M3 (837 mg, 1.45 mmol) was added, and the reaction was brought to room temperature for 1 h. The completion of the reaction was monitored by HPLC, and the reaction solution was purified by high-performance liquid phase purification to obtain the preparative solution, which was lyophilized to obtain lOd (499 mg); LC-MS: [M-H]’ = 842.4.
Step 5: Compound lOe lOd (400 mg, 0.48 mmol), M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (104 mg, 0.48 mmol) and 15 mL of DMF were added to a 50 mb flask in an ice-water bath and DIPEA (330 uL, 2.0 mmol) was added, and the reaction was allowed to proceed for 3 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to give the preparative solution of compound lOe. The reaction was carried out at room temperature for 3 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound lOe, which was lyophilized to obtain lOe (188 mg); LC-MS: [M+H]+ =1261.5.
Step 6: Compound 10A lOe (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 10A (61 mg); LC-MS: [M+H]+ =1105.4.
Example 17
Figure imgf000104_0001
Referring to the synthetic route of Example 16, compound 10B (75 mg) was obtained; LC- MS: [M+H]+ =1105.4. Example 18
Synthesis of compound HA:
Figure imgf000105_0001
Step 1 : Compound Ila
In a 250 mL flask, add Ml (6 g, 16.3 mmol), 100 mL THF, p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol), stir and cool to 0 °C, dropwise addition of benzyl 2-hydroxy- 2-cyclobutylacetate (sec Journal of Medicinal Chemistry, 2013, 56(13), 5541-5552. The method of synthesis) (6.7 g, 32.6 mmol), the reaction was naturally warmed up to room temperature after dropwise (the reaction was carried out for about 2-4 h) and monitored by TLC. At the end of the reaction, saturated NaHCCL solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA=10: 1-5: 1-2:1) to give Ila (5.1 g) in 62% yield; LC-MS: [M+H]+ =515.7.
Step 2: Compound lib
In a 25 mL flask, add Ila (4 g, 7.8 mmol), 10 mL DMF, stir at 0 °C, add DBU (1.2 g, 8.0 mmol), react for 1 h. After the completion of Fmoc deprotection was monitored by TLC, the reaction was set aside;
Add M4 (3.3 g, 8.0 mmol), PyBOP (5.2 g, 10.0 mmol), HOBt (1.35 g, 10.0 mmol) and 10 mL of DMF into another 25 mL single-necked vial, and add DIPEA (1.63 mL, 10.0 mmol) in an ice-water bath with continued stirring for 40 min, and then add the above reaction solution to the flask. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain the product preparation solution, which was extracted by dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a solid of 2.3 g in 42% yield; LC- MS: [M+H]+ =688.3.
Step 3: Compound 11c lib (2.0 g, 2.9 mmol) was added to a 25 mL flask, and after 25 mL of DMF was dissolved and cleared, 2.0 g of 5% Pd/C was added, and the hydrogenation reaction was carried out for 3 h. After completion of the reaction, it was filtered to obtain the filtrate, which was used directly for the next step of the reaction without purification.
Step 4: Compound lid
The crude product 11c was placed in an ice-water bath, DIPEA (516 uL, 3.0 mmol) was added, and compound M3 (1.7 g, 2.9 mmol) was added, and the reaction was brought to room temperature for 2 h. The completion of the reaction was monitored by HPLC, and the reaction solution was purified by high-performance liquid phase purification to obtain the preparative solution, which was lyophilized to give lid (934 mg); LC-MS: [M-H]' = 842.4. Step 5: Compound lie lid (800 mg, 0.96 mmol), M5 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol) and 30 mb of DMF were added to a 50 mL flask in an ice-water bath and DIPEA (660 uL, 4.0 mmol) was added, and the reaction was allowed to proceed for 4 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to give the preparative solution of compound lie. The reaction was carried out at room temperature for 4 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound lie, which was lyophilized to obtain lie (401 mg); LC-MS: [M+H]+ =1261.4.
Step 6: Compound HA lie (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound HA (86 mg); LC-MS: [M+H]+ =1105.4.
Example 19
Synthesis of compound 11B
Figure imgf000106_0001
Referring to the synthetic route of Example 18, compound HB (50 mg) was obtained. LC- MS: [M+H]+ 1105.4.
Example 20
Synthesis of compound 12A:
Figure imgf000107_0001
Step 1 : Compound 12a
In a 250 mL flask, add Ml (6 g, 16.3 mmol), 100 mL THF, p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol), stirring and cooled to 0 °C, dropwise add benzyl 3-hydroxy-2- cyclobutylpropionate (prepared according to the method published in W02009011285A1) (7.2 g, 32.6 mmol). The reaction was allowed to warm up to room temperature (about 2-4 h) and monitored by TLC. At the end of the reaction, saturated NaHCCh solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA=10: 1-5:1- 2: 1) to give 12a (4.5 g) in 52% yield; LC-MS: [M+H]+ =529.4.
Step 2: Compound 12b
In a 25 mL flask, add 12a (4 g, 7.6 mmol), 10 mL DMF, stir at 0 °C, add DBU (1.2 g, 8.0 mmol), react for 1 h. After the completion of Fmoc deprotection was monitored by TLC, the reaction was set aside;
Add M4 (3.2 g, 7.6 mmol), PyBOP (4.7 g, 9.0 mmol), HOBt (1.22 g, 9.0 mmol) and 10 mL of DMF in another 25 mL flask, and add DIPEA (1.49 mL, 0.9 mmol) under ice-water bath, and continue stirring for 30 min, then add the above reaction solution to the reaction flask and raise the temperature to room temperature. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain the product preparation solution, which was extracted by dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a solid of 2.0 g in 37% yield; LC-MS: [M+H]+ =702.8.
Step 3: Compound 12c
12b (1.0 g, 1.43 mmol) was added to a 25 mL flask, and after 15 mL of DMF was dissolved and cleared, 1.0 g of 5% Pd/C was added, and the hydrogenation reaction was carried out for 1.5 h. After completion of the reaction, it was filtered to obtain the filtrate, which was used directly for the next step of the reaction without purification.
Step 4: Compound 12d
The crude product 12c was placed in an ice- water bath, DIPEA (258 uL, 1.5 mmol) was added, and compound M3 (825 mg, 1.43 mmol) was added, and the reaction was brought to room temperature for 1 h. The completion of the reaction was monitored by HPLC, and the reaction solution was purified by high-performance liquid phase purification to obtain the preparative solution, which was lyophilized to obtain 12d (522 mg); LC-MS: [M-H]’ = 856.4.
Step 5: Compound 12e 12d (400 mg, 0.47 mmol), M5 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt
(101 mg, 0.47 mmol) and 15 mL of DMF were added to a 50 mb flask in an ice-water bath and DIPEA (330 uL, 2.0 mmol) was added, and the reaction was allowed to proceed for 3 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to give the preparative solution of compound 12e. The reaction was carried out at room temperature for 3 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound 12e, which was lyophilized to obtain 12e (198 mg); LC-MS: [M+H]+ =1275.4.
Step 6: Compound 12A
12e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 12A (55 mg); LC-MS: [M+H]+ =1119.4.
Example 21
Figure imgf000108_0001
Referring to the synthetic route of Example 20, compound 12B (50 mg) was obtained; LC-MS: [M+H]+ =1119.4. Example 22
Synthesis of compound 13A:
Figure imgf000109_0001
Step 1 : Compound 13a
In a 250 mL flask, add Ml (6 g, 16.3 mmol), 100 mL THF, p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol), stir and cool to 0 °C, dropwise addition of benzyl 2-hydroxy- 2-cyclopentylacetate (see Journal of Medicinal Chemistry, 2013, 56(13), 5541-5552. For the method of synthesis) (7.2 g, 32.6 mmol), the reaction was naturally warmed up to room temperature after dropwise (the reaction was carried out for about 2-4 h) and monitored by TLC. At the end of the reaction, saturated NaHCCh solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA=10: 1-5: 1-2: 1) to give 13a (4.6 g) in 53% yield; LC-MS: [M+H]+ =529.5.
Step 2: Compound 13b
In a 25 mL flask, add 13a (4 g, 7.6 mmol), 10 mL DMF, stir at 0 °C, add DBU (1.17 g, 7.8 mmol), and react for 1 h. After the completion of Fmoc deprotection was monitored by TLC, the reaction was set aside;
Add M4 (3.14 g, 7.6 mmol), PyBOP (4.42 g, 8.5 mmol), HOBt (1.15 g, 8.5 mmol) and 10 mL of DMF into another 25 mL flask, and add DIPEA (1.39 mL, 0.85 mmol) in an ice-water bath with continued stirring for 30 min, and then add the above reaction solution to the flask. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain the product preparation solution, which was extracted by dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a solid of 2.1 g in 39% yield; LC-MS: [M+H]+ =702.8.
Step 3: Compound 13c
13b (1.5 g, 1.87 mmol) was added to a 25 mL flask, and after 25 mL of DMF was dissolved and cleared, 1.5 g of 5% Pd/C was added, and the hydrogenation reaction was carried out for 3 h. After completion of the reaction, it was filtered, and the filtrate was obtained, and was used directly for the next step of the reaction without purification.
Step 4: Compound 13d
The crude product 13c was placed in an ice-water bath, DIPEA (333 uL, 1.93 mmol) was added, and compound M3 (1.1 g, 1.87 mmol) was added, and the reaction was brought to room temperature for 1 h. The completion of the reaction was monitored by HPLC, and the reaction solution was subjected to high-performance liquid-phase purification to obtain the preparative solution, which was lyophilized to give 13d (519 mg); LC-MS: [M-H] '=856.6. Step 5: Compound 13e
13d (400 mg, 0.47 mmol), M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (103 mg, 48 mmol) and 15 mb of DMF were added to a 50 mL flask in an ice-water bath and DIPEA (330 uL, 2.0 mmol) was added, and the reaction was carried out for 4 h. After HPLC monitoring, the reaction solution was purified by HPLC to obtain the preparative solution of compound 13e, and the preparative solution was lyophilized to obtain 13e (187 mg); LC-LLC was performed to monitor the reaction completion. The reaction was carried out for 4 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound 13e, which was lyophilized to obtain 13e (187 mg); LC-MS: [M+H] 1 =1275.5. Step 6: Compound 13A
13e (100 mg, 0.08 mmol), zinc bromide (355 mg, 0.16 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 13A (60 mg); LC-MS: [M+H]+ =1119.6.
Example 23
Synthesis of compound 13B:
Figure imgf000110_0001
Referring to the synthetic route of Example 22, compound 13B (51 mg) was obtained; LC- MS: [M+H] 1 =1119.6.
Example 24
Synthesis of compound 14A:
Figure imgf000111_0001
Step 1 : Compound 14a
In a 250 mL flask, add Ml (6 g, 16.3 mmol), 100 mL THF, p-toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol), stirring and cooling to 0 °C, dropwise add benzyl 3-hydroxy-2- cyclopentylpropionate (synthesized by reference to the method published in W02009011285A1) (7.6 g, 32.6 mmol). The reaction was carried out at room temperature (about 2-4 h) after dropwise natural warming and monitored by TLC. At the end of the reaction, saturated NaHCCh solution was added, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column (PE:EA=10:l-5: 1-2:1) to give 14a (4.4 g) in 49% yield; LC-MS: [M+H]+ =543.6.
Step 2: Compound 14b
In a 25 mL flask, add 14a (4 g, 7.4 mmol), 10 mL DMF, stir at 0 °C, add DBU (1.2 g, 8.0 mmol), react for 1 h. After the completion of Fmoc deprotection was monitored by TLC, the reaction was set aside;
Add M4 (3.1 g, 7.4 mmol), PyBOP (4.6 g, 8.8 mmol), HOBt (1.19 g, 8.8 mmol) and 10 mL of DMF in another 25 mL flask, and add DIPEA (1 .49 mL, 9.0 mmol) under ice-water bath, and continue stirring for 30 min, then add the above reaction solution to the reaction flask and raise the temperature to room temperature. After the reaction was monitored by HPLC, the reaction solution was purified by preparative liquid phase to obtain the product preparation solution, which was extracted by dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a solid of 2.6 g in 49% yield; LC-MS: [M+H]+ =716.4.
Step 3: Compound 14c
In a 25 mL flask, 14b (1.0 g, 1.4 mmol) was added, and after 15 mL of DMF was dissolved and cleared, 1.0 g of 5% Pd/C was added, and the hydrogenation reaction was carried out for 1.5 h. After completion of the reaction, it was filtered, and the filtrate was obtained, and was used directly for the next step of the reaction without purification.
Step 4: Compound 14d
The crude product 14c was placed in an ice- water bath, DIPEA (248 uL, 1.5 mmol) was added, then compound M3 (808 mg, 1.4 mmol) was added, and the reaction was brought to room temperature for 1 h. The reaction was monitored for completion by HPLC, and the reaction solution was purified by high-performance liquid phase purification to obtain the preparative solution, which was lyophilized to obtain 14d (500 mg); LC-MS: [M-H]’ = 870.5.
Step 5: Compound 14e 14d (400 mg, 0.46 mmol), M5 (235 mg, 0.46 mmol), PyBOP (245 mg, 0.46 mmol), HOBt
(99 mg, 0.46 mmol) and 15 mL of DMF were added to a 50 mL flask in an ice-water bath and DIPEA (331 uL, 2.0 mmol) was added, and the reaction was allowed to proceed for 3 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to give the preparative solution of compound 14e. The reaction was carried out at room temperature for 3 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound 14e, which was lyophilized to obtain 14e (146 mg); LC-MS: [M+H]+ =1289.5.
Step 6: Compound 14A
14e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 14A (52 mg); LC-MS: [M+H]+ =1133.4.
Example 25
Figure imgf000112_0001
Referring to the synthetic route of Example 24, compound 14B (48 mg) was obtained; LC- MS: [M+H]+ =1133.4. Example 26
Synthesis of compounds 15A and 15B:
Figure imgf000113_0001
Step 1 : Compound 15a
In a 250 mL flask, Ml (10 g, 27.1 mmol), benzyl 2-hydroxy-butyrate (prepared by the method published in Chemical Communications, 2019, 55(53), 7699-7702) (10.5 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added, heated to 100 °C reaction for 4 h. After the reaction was completed, reduced to room temperature, filtration to remove insoluble material, the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA=10: l -5: l-2: l) to obtain 5.67 g of the target material in 42% yield; LC-MS: [M+H]+ =503.5.
Step 2: Compound 15b
Add 15a (5 g, 9.95 mmol) and 15 mL of DMF in a 50 mL flask, dissolve the solution, and then add DBU (1.68 g, 11 mmol) in an ice-water bath and react for 1 h. The reaction was recorded as reaction solution (T);
Another 50 mL flask was taken, M4 (4.1 g, 10.0 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol) and 10 mL of DMF were added, and after dissolved, DIPEA (1.82 mL, 11 mmol) was added in an ice-water bath, and the reaction was continued for 40 min, then reaction solution (1) was added and the reaction was brought to room temperature for 2 h. The reaction was monitored by HPLC. The reaction was monitored by HPLC for 2 h. After completion of the reaction, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution. The prepared solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 4.6 g of solid, with a yield of 68%; LC-MS: [M+H]+ =676.7. Step 3: Compound 15d
Add 15b (2.0 g, 2.96 mmol) in a 25 mL flask, 15 mL of DMF was dissolved, then 2.0 g of 5% Pd/C was added, and the hydrogenation reaction was carried out for 2 h. After completion of the
Ill reaction, filtration was carried out, and the filtrate was placed in an ice-water bath, and DIPEA (496 uL, 3.0 mmol) was added, followed by M3 (1.7 g, 2.96 mmol), and then the reaction was brought to room temperature after completion of the reaction. The reaction was monitored by HPLC for 1 h. The reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution, which was lyophilized to give 1120.0 mg of the product in 45% yield; LC-MS: [M-H]’ =830.3.
Step 4: Compound 15e
15d (500 mg, 0.60 mmol), M5 (321 mg, 0.60 mmol), PyBOP (469 mg, 0.90 mmol), HOBt (121 mg, 0.90 mmol) and 15 mb ofDMF were added to a 50 mL flask, DIPEA (446 uL, 2.7 mmol) was added to the vial in an ice-water bath, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to obtain the preparative solution of compound 15e-l and compound 15e-2, and the preparative solution was lyophilized to obtain 138 mg of compound 15e-2 respectively. The reaction was carried out at room temperature for 2 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solutions of compound 15e-l and compound 15e-2, which were lyophilized to obtain 138 mg of compound 15e-l, LC-MS: [M+H]+ =1249.5, and 140 mg of compound 15e-2, LC-MS: [M+H]+ =1249.5, respectively.
Step
Figure imgf000114_0001
15e-l (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain a solid of 59 mg; LC-MS: [M+H]+ =1093.4.
Step 6: Compound 15B
Figure imgf000114_0002
15e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 60 mg of solid; LC-MS: [M+H]+ =1093.4.
Example 27
Synthesis of compounds
Figure imgf000115_0001
Referring to the synthetic route of Example 26, compound 16A (55 mg) was obtained; LC- MS: [M+H]+ =1093.4.
Figure imgf000115_0002
Referring to the synthetic route of Example 26, compound 16B (54 mg) was obtained; LC- MS: [M+H]+ =1093.4.
Example 28
Synthesis of compounds 17A and 17B:
Figure imgf000115_0003
Step 1 : Compound 17a
Add Ml (10 g, 27.1 mmol), benzyl 2-hydroxy-phenylpropionate (synthesized by the method published in Nature Communications, 2020. //(I), 56 (14.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene to a 250 mL flask. Toluene was heated to 100 °C for 4 h. After the reaction was completed, it was lowered to room temperature, fdtered to remove insoluble material, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA=10: 1-5: 1-2: 1) to obtain 6.13 g of the target material in 40% yield; LC-MS: [M+H]+ =565.6.
Step 2: Compound 17b
In a 50 mL flask, 17a (5 g, 8.86 mmol) and 15 mL ofDMF were added, dissolved and cleared, and then, in an ice-water bath, DBU (1.53 g, 10 mmol) was added and the reaction was carried out for 1 h, which was recorded as reaction solution (T);
Another 50 mL flask was taken, M4 (3.6 g, 8.86 mmol), PyBOP (5.23 g, 10 mmol), HOBt (1.36 g, 10 mmol) and 10 mL ofDMF were added, and after dissolved, DIPEA(1.65 mL, 10 mmol) was added in an ice-water bath, and the reaction was continued for 30 min, then reaction solution (T) was added, and the reaction was brought to room temperature for 2 h. The reaction was monitored by HPLC. The reaction was monitored by HPLC for 2 h. After completion of the reaction, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution. The prepared solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 5.0 g of solid in 77% yield; LC-MS: [M+H]+ =738.3.
Step 3: Compound 17d
Add 17b (3.0 g, 4.07 mmol) in a 25 mL flask, after 15 mL ofDMF was dissolved, 3.0 g of 5% Pd/C was added, and the reaction was completed for 2 h. The reaction was completed by filtration, and the filtrate was placed in an ice-water bath, and DIPEA (744 uL, 4.5 mmol) was added, followed by M3 (2.34 g, 4.07 mmol), and the reaction was brought to room temperature after addition. The reaction was monitored by HPLC for 1 h. The reaction solution was purified by HPLC to obtain the preparative solution, which was lyophilized to give 1.2 g of product in 33% yield; LC-MS: [M-H]’ =892.4.
Step 4: Compound 17e
17d (500 mg, 0.56 mmol), M5 (300 mg, 0.56 mmol), PyBOP (438 mg, 0.84 mmol), HOBt (113 mg, 0.84 mmol) and 15 mL of DMF were added to a 50 mL flask, and DIPEA (330 uL, 2.0 mmol) was added to the flask under an ice-water bath, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to obtain the preparative solution of compound 17e-l and compound 17e-2, and the preparative solution was lyophilized to obtain 156 mg of compound 17e-2 respectively. The reaction was carried out at room temperature for 2 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solutions of compound 17e-l and compound 17e-2, which were lyophilized to obtain 156 mg of compound 17e-l, LC-MS: [M+H]+ =1311.4, and 150 mg of compound 17e-2, LC-MS: [M+H]+ = 1311.7, respectively.
Step 5: Compound 17A
Figure imgf000117_0001
17e-l (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 43 mg of solid; LC-MS: [M+H]+ =1155.4.
Step 6: Compound 17B
Figure imgf000117_0002
17e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 40 mg of solid; LC-MS: [M+H]+ =1155.4.
Example 29
Synthesis of compounds
Figure imgf000117_0003
Referring to the synthetic route of Example 28, compound 18A (54 mg) was obtained; LC- MS: [M+H]+ =1155.4.
Figure imgf000117_0004
Referring to the synthetic route of Example 28, compound 18B (55 mg) was obtained; LC- MS: [M+H]+ =1155.4.
Example 30 Synthesis of compounds 19A and 19B:
Figure imgf000118_0001
Step 1 : Compound 19a
Add Ml (10 g, 27.1 mmol), benzyl 2-cyclopropyl-2-hydroxyacetate (prepared according to the method published in patent WO2020244657A1) (11.2 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene in a 250 mL flask and heat the reaction to 100 °C for 4 h. The reaction was completed. The reaction was completed, reduced to room temperature, filtered to remove insoluble material, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA= 10: 1-5: 1 -2: 1) to obtain 4.97 g of the target material with 36% yield; LC-MS: [M+H]+ =515.2.
Step 2: Compound 19b
Add 19a (4 g, 7.8 mmol) and 10 mL of DMF in a 50 mL flask, dissolve the solution, and then add DBU (1.42 g, 9.3 mmol) in an ice-water bath and react for 1 h. The reaction was recorded as reaction solution (T);
Another 50 mL single-necked vial was taken, and M4 (3.2 g, 7.8 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol) and 10 mL of DMF were added, and after dissolved, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath, and the reaction was continued for 30 min, then reaction solution (T) was added, and the reaction was brought to room temperature for 2 h. The reaction process was monitored by HPLC, and the preparation was purified by HPLC. The reaction was monitored by HPLC for 2 h. After completion of the reaction, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution. The prepared solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 4.2 g of solid, with a yield of 78%; LC-MS: [M+H]+ =688.3.
Step 3: Compound 19d
In a 25 mL flask, 19b (1000 mg, 1.45 mmol) was added, 15 mL of DMF was dissolved, then 1000 mg of 5% Pd/C was added, and the reaction was hydrogenated for 2 h. The reaction was completed, filtered, and the filtrate was placed in an ice-water bath, and DIPEA (248 uL, 1.5 mmol) was added, followed by M3 (720 mg, 1.45 mmol), and then the reaction was brought to room temperature for 1 h. The reaction was monitored by HPLC, and the reaction solution was purified by HPLC to obtain the preparative solution, and the preparative solution was lyophilized to obtain 503 mg of product in 41% yield. The reaction was monitored by HPLC, and the reaction solution was purified by HPLC to obtain the preparative solution, which was lyophilized to obtain 503 mg of the product in 41% yield; LC-MS: [M-H]' =842.3.
Step 4: Compounds 19e-l and 19e-2
19d (500 mg, 0.59 mmol), M5 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol) and 10 mL of DMF were added to a 50 mL flask, and DIPEA (292 uL, 1.77 mmol) was added to the flask under an ice-water bath, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to obtain the preparative solution of compound 19e-l and compound 19e-2, and the preparative solution was lyophilized to obtain 112 mM of compound 19e-2, respectively. The reaction was carried out at room temperature for 2 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solutions of compound 19e-l and compound 19e-2, which were lyophilized to obtain 112 mg of compound 19e-l, LC-MS: [M+H]+ =1261.5, and 131 mg of compound 19e-2, LC-MS: [M+H]+ = 1261.5, respectively.
Step 5: Compound 19A
Figure imgf000119_0001
19e-l (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the completion of the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure and removed to obtain the crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 55 mg of solid; LC-MS: [M+H]+ =1105.4.
Step 6: Compound 19B
Figure imgf000119_0002
19e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was completed by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain a solid 58 mg; LC-MS: [M+H]+ =1105.4.
Example 31
Synthesis of compounds 20A and 20B:
Figure imgf000120_0001
Step 1 : Compound 20a
Add Ml (10 g, 27.1 mmol), benzyl 2-hydroxy-cyclopropylpropionate (synthesized by the method published in W02020063676A) (12.0 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mb of toluene to a 250 mL flask, and react at 100 °C for 4 h. When the reaction was completed, reduce to room temperature. The reaction was completed, lowered to room temperature, filtered to remove insoluble material, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA=10: 1-5: 1-2: 1) to obtain 5.09 g of the target material; LC-MS: [M+H]+ =529.2.
Step 2: Compound 20b
20a (4 g, 7.6 mmol) and 10 mL of DMF were added to a 50 mL flask, and after dissolving and clearing, DBU (1.39 g, 9.1 mmol) was added in an ice-water bath and the reaction was carried out for 1 h, which was recorded as reaction solution (T);
Another 50 mL flask was taken, and M4 (3.12 g, 7.6 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol) and 10 mL of DMF were added, and after dissolved, DIPEA (1.65 mL, 10 mmol) was added in an ice-water bath and the reaction was continued for 30 min, and then the reaction solution (1) was added to the reaction solution and the reaction was brought to room temperature for 2 h. HPLC was performed to monitor the reaction progress, and the reaction solution was purified by high performance liquid phase purification to obtain the preparative solution. The reaction was monitored by HPLC, and the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution. The prepared solution was extracted with di chloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 4.5 g of solid, with a yield of 84%; LC-MS: [M+H]+ =702.3.
Step 3 : Compounds 20d
Add 20b (1000 mg, 1.42 mmol) in a 25 mL flask, 15 mL of DMF was dissolved, then 1000 mg of 5% Pd/C was added, and hydrogenation reaction was carried out for 2 h. After the reaction was completed, filtration was performed, and the filtrate was placed in an ice-water bath, and then DIPEA (248 uL, 1.5 mmol) was added, followed by M5 (708 mg, 1.42 mmol), and the reaction was brought to room temperature for 1 h. The reaction was monitored by HPLC, and the reaction solution was purified by HPLC to obtain the preparative solution, and the preparative solution was lyophilized to obtain 443 mg of product in 36% yield. The reaction was monitored by HPLC and the reaction solution was purified by HPLC to obtain the preparative solution, which was lyophilized to obtain 443 mg of product in 36% yield; LC-MS: [M-H]’ =856.4.
Step 4: Compounds 20e-l and 20e-2 20d (400 mg, 0.47 mmol), ezetimibe methanesulfonate (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol), and 10 mL of DMF were added to a 50 mL vial, and DIPEA (248 uL, 1.5 mmol) was added to the vial under an ice-water bath. DIPEA (248 uL, 1.5 mmol) was added in an ice-water bath, and the reaction was brought to room temperature for 2 h. After the reaction was monitored by HPLC, the reaction solution was subjected to high- performance liquid-phase purification to obtain the preparative solutions of compound 20e-l and compound 20e-2, which were lyophilized to obtain 103 mg of compound 20e-l, LC-MS: [M+H]+ = 1275.5, and 103 mg of compound 20e-2, LC-MS: [M+H]+ = 1275.5.
Step 5: Compound 20A
Figure imgf000121_0001
In a 25 mL flask, 8A (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40 °C for 1 h. After the completion of the reaction was detected by HPLC, the solvent was concentrated under reduced pressure and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 51 mg of solid; LC-MS: [M+H]+ =1119.4.
Step 6: Compound 20B
Figure imgf000121_0002
20e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added to a 25 mL single-necked vial and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain a solid of 47 mg; LC-MS: [M+H]+ =1119.4. Example 32
Synthesis of compound 21:
Figure imgf000122_0001
Step 1 : Compound SM3-1
Add 77087-60-6 (100 g, 458 mmol), maleic acid (53.4 g, 460 mmol), TEA (64 mL, 460 mmol) and 1000 mL toluene in a 2000 mL flask and heat to 100 °C for 5 h. After the reaction was completed, the reaction was brought down to room temperature, insoluble material was removed by fdtration and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA=100: 1-50: 1-20:1) to obtain 75.6 g of the target; LC-MS: [M+H]+ =299.1.
Step 2: Compound (A)-tert-butyl 2-hydroxy-l, 5-pentanedioate
Add 172793-31-6 (100 g, 338 mmol) and 1000 mL water in 2000 mL flask, then add sodium nitrite (35 g, 507 mmol) and concentrated sulfuric acid (32 mL, 35 mmol), and slowly raise the temperature to room temperature for 24 h. After the reaction was completed, the product was extracted with 500 mL of ethyl acetate for three times, and the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The organic phase was dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by silica gel column chromatography (PE:EA=50: 1-30: 1-2: 1) to obtain 91.2 g of the target material; LC-MS: [M+H]+ =261.4.
Step 3: Compound SM3
Add (7?)-tert-butyl 2-hydroxy-l, 5-glutarate (50 g, 192 mmol) and 1000 mL of anhydrous tetrahydrofuran into a 2000 mL flask, cool down the temperature to 0 °C, then add PPhs (87.7 g, 288 mmol), DEAD (50.2 g, 288 mmol), and SM3-1 (57.3, 192 mmol) in sequence. The reaction was slowly warmed up to room temperature for 13 h. After completion of the reaction, the insoluble material was removed by filtration and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (PE:EA=50: 1-30: 1- 1 : 1) to obtain 68.6 g of product;
The above product was dissolved in 500 mL of methanol, cooled to 0 °C in an ice-water bath, NaOH (64 mL, 190 mmol, 3M/L) was added dropwise at this temperature, and the reaction was maintained at this temperature for 12 h. After the reaction, the pH was adjusted to 3 by the addition of HC1 (6 M/L), dichloromethane was extracted for five times with 500 mL of dichloromethane, and the mixture was dried with anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the crude product obtained was purified by column chromatography ( DCM/MeOH=50/1 -20/ 1-2/1) purification, obtained SM3 50.4 g; LC-MS: [M-H]' =525.5. Step 4: Compound M6
In a 2000 mL flask, compound SM3 (50 g, 95 mmol, 1.0 eq), pentafluorophenol (19.2 g,
104.5 mmol, 1.1 eq), DCC (21.5 g, 104.5 mmol, 1.1 eq) and THF (600 mL) were added, and the reaction was carried out at room temperature for 1 h (monitored by TLC), and insoluble material was filtered off. The reaction solution was purified directly by preparation, and the preparative solution was concentrated by pumping water under reduced pressure at 35 °C to remove acetonitrile, and lyophilized to obtain compound M6 (51.9 g) in 79% yield; LC-MS: [M+H]+ =693.3.
Step 5: Compound 21a
In a 25 mL flask, 1c (1 g, 2.36 mmol) was added, 25 mL DMF was dissolved, then DIPEA (430 uL, 2.6 mmol) was added, then M6 (1177 mg, 2.36 mmol) was added, and then the reaction was brought to room temperature for 1 h. The completion of the reaction was monitored by HPLC, and the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution, which was lyophilized to yield the following product 555 mg; LC- MS: [M-H]- =931.0.
Step 6: Compound 21b
In a 100 mL flask, 21a (500 mg, 0.54 mmol), ezetimibe methanesulfonate M5 (285 mg, 0.54 mmol), PyBOP (239 mg, 0.6 mmol), HOBt (239 mg, 0.6 mmol), and 10 mL of DMF were added, and DIPEA (248 uL, 1.5 mmol) was added to the reaction in an ice-water bath. DIPEA (248 uL,
1.5 mmol) was added in an ice- water bath, and the reaction was brought to room temperature for 2 h. After the completion of the reaction was monitored by HPLC, the reaction solution was subjected to high-performance liquid-phase purification to obtain the preparative solution of compound 21b, which was lyophilized to give 231 mg of the compound; LC-MS: [M+H]+ =1349.5. Step 7: Compound 21
Compound 21b (200 mg, 0.1488 mmol), zinc bromide (665 mg, 2.96 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 103 mg of solid; LC-MS: [M+H]+ =1137.5. Example 33
Synthesis of compound 22:
Figure imgf000124_0001
Using compounds M6 and 3c as starting materials and referring to the synthetic route of Example 32, compound 22 (91 mg) was obtained; LC-MS: [M+H]+ =1165.5.
Example 34
Synthesis of compounds 23 and 24:
Figure imgf000124_0002
Using compounds M6 and 5c as starting materials and referring to the synthetic route of Example 32, 102 mg of compound 23 was obtained, LC-MS: [M+H]+ =1151.4; 99 mg of compound 24 was obtained, LC-MS: [M+H]+ =1151.4.
Example 35
Synthesis of compounds 25 and 26:
Figure imgf000124_0003
Using compounds M6 and 7c as starting materials and referring to the synthetic route of Example 32, 83 mg of compound 25 was obtained, LC-MS: [M+H]+ =1205.7; 80 mg of compound 26 was obtained, LC-MS: [M+H]+ =1205.7.
Example 36
Synthesis of compounds 27 and 28:
Figure imgf000124_0004
Using compounds M6 and 19c as starting materials and referring to the synthetic route of Example 32, 100 mg of compound 27 was obtained, LC-MS: [M+H]+ =1177.5; 101 mg of compound 28 was obtained, LC-MS: [M+H]+ =1177.5. Example 37
Synthesis of compound 29:
Figure imgf000125_0001
Step 1 : Compound SM4-1
In a 5000 mL flask, maleic acid (50 g, 431 mmol, 1.0 eq), 114559-25-0 (110 g, 431 mmol, 1 eq), TEA (263 g, 2.16 mol, 5 eq), and toluene (2000 mL) were added, and the reaction was heated to reflux for 5 h (monitored by TLC), and insoluble material was filtered off. The reaction solution was directly removed from the solvent by rotary distillation under reduced pressure, and the residue was subjected to silica gel column chromatography (PE/EA=50/l -20/1-1 -1/1) to obtain SM4-1 (64.7 g) in 50% yield; LC-MS: [M+H]+ =299.2.
Step 2: Compound SM4-2
SM4-1 (64 g, 215 mmol) was added into a 2000 mL flask, and after 1000 mL of DMF was dissolved, DIPEA (71 mL, 430 mmol) was added, and then nona-ethylene glycol monomethyl ether methanesulfonate (111.5 g, 220 mmol) was added, and then the reaction was brought to room temperature for 2 h. The completion of the reaction was monitored by HPLC, and the reaction solution was purified by silica gel column chromatography (PE/EA=50/l-20/l-l/l) to obtain 59.9 g; LC-MS: [M+H]=709.4. The reaction solution was purified by silica gel column chromatography (PE/EA=50/l -20/1 -1/1), and 59.9 g of the product was obtained; LC-MS: [M+H] 1 =709.4. Step 3 : Compound SM4 SM4-2 (59 g, 83 mmol) was added into a 2000 mL flask, and after 1000 mL MeOH was dissolved, K2CO3 (11.75 g, 85 mmol) was added, and the reaction was carried out at room temperature for 4 h. The reaction was monitored by HPLC, and the insoluble material was filtered out, and the reaction solution was purified directly by the preparation, and the prepared solution was pumped to concentrate at 35 °C under reduced pressure in a water bath to remove acetonitrile, and then lyophilized to obtain compound SM4 (27 g); LC-MS: [M-I I] =693.5. Step 4: Compound M7
In a 500 mL flask, compound SM4 (25 g, 36 mmol, 1.0 eq), pentafluorophenol (7.3 g, 40 mmol, 1.1 eq), DCC (8.2 g, 40 mmol, 1.1 eq) and THF (200 mL) were added, and the reaction was carried out at room temperature for 1 h (monitored by TLC), and insoluble material was filtered off. The reaction solution was purified directly by preparation, and the preparative solution was concentrated by pumping water under reduced pressure at 35 °C to remove acetonitrile, and lyophilized to obtain compound M7 (23.3 g) in 93% yield; LC-MS: [M+H]+ =695.8.
Step 5: Compound 29a
In a 25 mL flask, 1c (1 g, 2.36 mmol) was added, 25 mL DMF was dissolved, then DIPEA (430 uL, 2.6 mmol) was added, then M7 (1640 mg, 2.36 mmol) was added, and then the reaction was brought to room temperature for 1 h. The completion of the reaction was monitored by HPLC, and the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution, which was lyophilized to obtain 609 mg of product; [M-H] = 1098.5 by LC-MS. The product was 609 mg; LC-MS: [M-H]- =1098.5.
Step 6: Compound 29b
29a (500 mg, 0.45 mmol), ezetimibe methanesulfonate M5 (240 mg, 0.45 mmol), PyBOP (215 mg, 0.54 mmol), HOBt (215 mg, 0.54 mmol), and 10 mL of DMF were added to a 100 mL vial, and DIPEA (248 uL, 1.5 mmol) was added to the flask under an ice-water bath. 1.5 mmol) in an ice water bath, and the reaction was brought to room temperature for 2 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound 29b, and lyophilization of the preparative solution yielded 187 mg of the compound; LC-MS: [M+H]+ = 1517.6.
Step 7: Compound 29
Compound 29b (150 mg, 0.988 mmol), zinc bromide (223 mg, 0.988 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 114 mg of solid; LC-MS: [M+H]+ =1517.9.
Example 38
Synthesis of compound 30:
Figure imgf000126_0001
Using compounds M7 and 3c as starting materials and referring to the synthetic route of Example 37, compound 30 (125 mg) was obtained; LC-MS: [M+H]+ =1445.6.
Example 39 Synthesis of compounds 31 and 32:
Figure imgf000127_0001
Using compounds M7 and 5c as starting materials and referring to the synthetic route of Example 37, 61 mg of compound 31 was obtained, LC-MS: [M+H]+ =1431.7; 63 mg of compound 32 was obtained, LC-MS: [M+H]+ =1431.7.
Example 40
Synthesis of compounds 33 and 34:
Figure imgf000127_0002
Using compounds M7 and 7c as starting materials, referring to the synthetic route of Example 37, 60 mg of compound 33 was obtained, LC-MS: [M+H]+ =1485.6; 58 mg of compound 34 was obtained, LC-MS: [M+H]+ =1485.6.
Example 41
Synthesis of compounds 35 and 36:
Figure imgf000127_0003
Using compounds M7 and 19c as starting materials and referring to the synthetic route of Example 37, 102 mg of compound 35 was obtained, LC-MS: [M+H]+ =1457.8; 102 mg of compound 36 was obtained, LC-MS: [M+H]+ =1457.8.
Example 42
Synthesis of compound 37:
Figure imgf000128_0001
Step 1 : Compound SM5-1
In a 2000 mL flask, compound 16947-84-5 (100 g, 295 mmol, 1.0 eq), DIPEA (50 mL, 300 mmol), benzyl bromide (51.3 g, 300 mmol) and THF (1000 mL) were added and reacted at room temperature for 12 h (monitored by TLC), and the insoluble material was filtered off. The reaction solution was directly removed from the solvent by rotary distillation under reduced pressure, and the residue was subjected to silica gel column chromatography (PE/EA=50/1 -20/ 1-2/1) to obtain SM5-1 (110.1 g) in 87% yield; LC-MS: [M+H]+ =429.2.
Step 2: Compound SM5-2
In a 2000 mL flask, compound SM5-1 (100 g, 233.4 mmol, l.Oeq) and THF (1000 mL) were added, cooled to 0 °C in an ice-water bath, NaH (37.4 g, 933.5 mmol) and Mel (132.5 g, 933.5 mmol) were added in batches, and the reaction was maintained at 0 °C for 24 h (monitored by TLC), the reaction was quenched by adding 500 mL of saturated NH4CI aqueous solution, extracted three times with 500 mL of ethyl acetate, dried the organic phase with anhydrous sodium sulfate, and filtered. The filtrate was directly removed from the solvent by rotary distillation under reduced pressure, and the residue was analyzed by silica gel column chromatography (PE/EA=100/l-50/l -10/1) to obtain SM5-2 (37.1 g); LC-MS: [M+H]+ =443.3.
Step 3: Compound SM5 (see Org. Lett, 2006, 8, 3387-3390.)
In a 1000 mL flask, compound SM5-2 (35 g, 79 mmol, l.Oeq) and DCE (500 mL) were added, palladium diacetate (180 mg, 0.8 mmol), I2 (20 g, 79 mmol), and iodobenzene diacetate (40.8 g, 126.4 mmol) were added, and the reaction was carried out at 60 °C for 40 h (monitored by TLC). The reaction was quenched by adding 500 mL of saturated aqueous sodium thiosulfate, extracted three times with 500 mL of dichloromethane, dried the organic phase with anhydrous sodium sulfate, and filtered. The filtrate was directly removed from the solvent by rotary distillation under reduced pressure, and the residue was analyzed by silica gel column chromatography (PE/EA=100/l-50/l-10/l) to obtain SM5 (28 g); LC-MS: [M+H]+ =501.3. Step 4: Compound SM6
In a 500 mL flask, compound SM5 (25 g, 50 mmol, 1.0 eq), potassium di-tert-butyl phosphate (13.66 g, 55 mmol, 1.1 eq), p-toluenesulfonic acid monohydrate (951 mg, 5 mmol, 0.1 eq) and THF (200 mL) were added, and the reaction was carried out at room temperature for 1 h (monitored by TLC), and insoluble material was filtered off. The reaction solution was purified directly by preparation, and the preparative solution was concentrated by pumping water under reduced pressure at 35 °C to remove acetonitrile, and lyophilized to obtain compound SM6 (15.1 g) in 46% yield; LC-MS: [M+H]+ =651.4.
Step 5: Compound SM7
SM6 (15 g, 23 mmol) and 100 mL of DMF were added into a 250 mL flask, and after dissolving, 15 g of 5% Pd/C was added in an ice-water bath, and the atmosphere in the system was replaced by hydrogen for three times, and the reaction was carried out at room temperature for 12 h. The Pd/C was removed by filtration, and the solvent was removed by decompression evaporation using an oil pump, and the solvent was set aside for use.
Another 250 mL flask, add the above crude product and 100 mL of toluene, tri ethylamine (6.4 mL, 46 mmol), maleic anhydride (2.4 g, 24 mmol), dissolved clear, rise to 100 °C reaction for 2 h. HPLC to monitor the progress of the reaction, the reaction is completed, the reaction solution by high-performance liquid chromatography purification, to obtain the preparative solution. The prepared solution was extracted with dichloromethane, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a solid of 4.2 g in 36% yield; LC-MS: [M+H]+ =507.3.
Step 6: Compound M8
In a 100 mL flask, compound SM7 (4 g, 7.9 mmol, 1.0 eq), pentafluorophenol (1.6 g, 8.7 mmol, 1. 1 eq), DCC (1.8 g, 8.7 mmol, 1.1 eq) and THF (60 mL) were added, and the reaction was carried out at room temperature for 1 h (monitored by TLC), and insoluble material was filtered off. The reaction solution was purified directly by preparation, and the preparative solution was concentrated by pumping water under reduced pressure at 35 °C to remove acetonitrile, and lyophilized to obtain compound M8 (3.7 g) in 70% yield; LC-MS: [M+H]+ =673.2.
Step 7: Compound 37a
In a 25 mL flask, 1c (1 g, 2.36 mmol) was added, 25 mL of DMF was dissolved, then DIPEA (430 uL, 2.6 mmol) was added, then M8 (1.2 g, 2.36 mmol) was added, and the reaction was brought to room temperature for 1 h. The completion of the reaction was detected by HPLC, and the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution, which was lyophilized to obtain the following products 488 mg; LC-MS: [M-H]- =911.0.
Step 8: Compound 37b
37a (400 mg, 0.44 mmol), ezetimibe methanesulfonate M5 (235 mg, 0.44 mmol), PyBOP (199 mg, 0.5 mmol), HOBt (69 mg, 0.5 mmol), and 10 mL of DMF were added to a 100 mL flask, and DIPEA (218 uL, 1.32 mmol) was added to the reaction in an ice-water bath. DIPEA (218 uL, 1.32 mmol) was added in an ice-water bath, and the reaction was brought to room temperature for 2 h. After the reaction was monitored by HPLC, the reaction solution was subjected to high- performance liquid-phase purification to obtain the preparative solution of compound 37b, which was lyophilized to give 201 mg of the compound; LC-MS: [M+H]+ =1329.6.
Step 9: Compound 37
Compound 37b (130 mg, 0.098 mmol), zinc bromide (221 mg, 0.98 mmol) and 10 mb of nitromethane were added to a 25 mb flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 96 mg of solid; LC-MS: [M+H]+ =1117.4.
Example 43
Synthesis of compound 38:
Figure imgf000130_0001
Using compounds M8 and 3c as starting materials and referring to the synthetic route of Example 42, compound 38 (51 mg) was obtained; LC-MS: [M+H]+ =1145.6.
Example 44
Synthesis of compounds 39 and 40:
Figure imgf000130_0002
Using compounds M8 and 5c as starting materials and referring to the synthetic route of Example 42, 57 mg of compound 39 was obtained, LC-MS: [M+H]+ =1131.4; 60 mg of compound 40 was obtained, LC-MS: [M+H]+ =1131.4.
Example 45
Synthesis of compounds 41 and 42:
Figure imgf000130_0003
Using compounds M7 and 7c as starting materials, referring to the synthetic route of Example 42, 44 mg of compound 41 was obtained, LC-MS: [M+H]+ =1185.3; 44 mg of compound 42 was obtained, LC-MS: [M+H]+ =1185.3.
Example 46 Synthesis of compounds 43 and 44:
Figure imgf000131_0001
Using compounds M8 and 19c as starting materials and referring to the synthetic route of Example 42, 62 mg of compound 43 was obtained, LC-MS: [M+H]+ =1157.4; 59 mg of compound 44 was obtained, LC-MS: [M+H]+ =1157.4.
Example 47 (control example)
Figure imgf000131_0002
Compound 45 was synthesized according to the method provided in Example 58 of CN104755494A.
Example 48
Synthesis of compound 46:
Figure imgf000131_0003
Step 1 : Compound 46a
Id (500 mg, 0.62 mmol), M9 (310 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added to a 50 mL flask, and DIPEA (378 uL, 2.29 mmol) was added to the vial under an ice-water bath, and the reaction was allowed to proceed for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to obtain the preparative solution, which was lyophilized to obtain 46a (210 mg); LC-MS: the preparative solution was lyophilized to obtain 46a (210 mg); LC-MS: the preparative solution was purified by HPLC. The reaction was carried out at room temperature for 2 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution, which was lyophilized to give 46a (210 mg); LC-MS: [M+H]+ =1221.6. Step 2: Compound 46
46a (200 mg, 0.162 mmol), zinc bromide (736 mg, 3.26 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain solid compound 46 (120 mg); LC-MS: [M+H]+ =1065.3.
Example 49
Synthesis of compound 47:
Figure imgf000132_0001
Referring to the synthetic route of Example 48, compound 47 (81 mg) was obtained; LC-MS: [M+H]+ =1065.3.
Example 50
Synthesis of compound 48A:
Figure imgf000132_0002
Step 1 : Compound 48a
5d (1.66 g, 2.02 mmol, 1.0 eq), M9 (1.08 g, 2.02 mmol, 1.0 eq), PyBOP (1.58 g, 3.03 mmol, 1.5 eq), HOBt (0.41 g, 3.03 mmol, 1.5 eq) and DMF (40 mL) were added to a 100 mL flask, and the reaction was brought to room temperature for 2 h. The reaction was monitored by HPLC. DIPEA (0.84 mL, 1.5 eq) was added, and the reaction was brought to room temperature for 2 h (monitored by HPLC). The reaction solution was purified directly by preparation, and the preparative solution was concentrated by pumping water under reduced pressure at 35 °C to remove acetonitrile, and lyophilized to obtain compound 48a (1.54 g) in 61% yield; LC-MS: [M+H]+ =1235.4.
Step 2: Compound 48A
In a 100 mL flask, compound 48a (1.0 g, 0.8 mmol, 1.0 eq) was added, 35 mL of nitromethane, dissolved and then added zinc bromide (3.64 g, 16 mmol, 20.0 eq), the reaction was carried out in an oil bath at 40 °C (stabilized in advance by preheating) for 30 min, and then the pump was concentrated at 45 °C under reduced pressure in a water bath to remove the nitromethane, resulting in a yellow residue solid (monitored by HPLC). After acid preparation, the prepared solution was obtained, and the prepared solution was concentrated by pump decompression water bath at 35 °C to remove acetonitrile and lyophilized to obtain compound 48A (786 mg) in 90% yield.
Example 51
Synthesis of compound 48B:
Figure imgf000133_0001
Step 1 : Compound 48b
Compound 5d-l (200 mg, 0.24 mmol, 1.0 eq), M9 (127 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2 eq), HOBt (48 mg, 0.36 mmol, 1.2 eq), and DMF (6 mL) were added to a 25 mL flask. Water bath was lowered to 0-5 °C, DIPEA (62 mg, 0.48 mmol, 2.0 eq) was added, and the reaction was raised to 20 ± 5 °C for 2 h. The end of the reaction was monitored by HPLC. The reaction solution was purified directly by HPLC preparation, and the product preparation was collected and lyophilized to obtain compound 48b (150.2 mg); LC-MS: [M+H]+ =1235.4. Step 2: Compound 48B
Compound 48b (100 mg, 0.081 mmol, 1.0 eq), ZnBn (364 mg, 1.62 mmol, 20.0 eq) and CH3NO2 (10 mL) were added to a 25 mL flask in sequence, and then the reaction was raised to 40 °C for 0.5 h. The reaction was stopped, and the reaction solution was directly spun-dried at 45 °C under reduced pressure to obtain a yellow solid, which was monitored by sampling HPLC. The reaction was monitored by HPLC. The spin-dried solid was directly purified by HPLC preparation, and the product preparation was collected and lyophilized to obtain compound 48B (70.0 mg); LC-MS: [M+H]~ =1079.4.
Example 52
Synthesis of compound 49A:
Figure imgf000133_0002
Referring to the route of Example 50, compound 49A (71 mg) was obtained; LC-MS: [M+H]+ = 1079.4. Example 53
Preparation of compound 49B:
Figure imgf000134_0001
Referring to the synthetic route of Example 51, compound 49B (65 mg) was obtained; LC- MS: [M+H]+ =1079.4.
Example 54
Synthesis of compounds 50A and SOB:
Figure imgf000134_0002
Step 1 : Compounds 50a and 50b 7d (500 mg, 0.57 mmol), M9 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL ofDMF were added to a 50 mL flask, DIPEA (378 uL, 2.29 mmol) was added to the flask under an ice-water bath, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to obtain compound 50a and compound 50b, and the preparation was lyophilized to obtain 170 mg of compound 50a. The reaction was carried out at room temperature for 2 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solutions of compound 50a and compound 50b, which were lyophilized to obtain 170 mg of compound 50a, LC-MS: [M+H]+ =1289.46, and 202 mg of compound 50b, LC-MS: [M+H]+ =1289.46, respectively. Step 2: Compound 50A
Figure imgf000134_0003
In a 25 mL flask, 50a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 44 mg of solid.
Step 3 : Compound 50B
Figure imgf000135_0001
In a 25 mL trial, 50b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mb of nitromethane were added, and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 45 mg of solid.
Example 55
Synthesis of compounds 51A and 51B:
Figure imgf000135_0002
Step 1 : Compound 51a and Compound 51b
8d (500 mg, 0.57 mmol), M9 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL ofDMF were added to a 50 mL flask, DIPEA (378 uL, 2.29 mmol) was added to the flask in an ice-water bath, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to obtain the preparations of compound 51a and compound 51b. After the reaction was completed by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solutions of compound 51a and compound 51b, which were lyophilized to obtain 190 mg of compound 51a and 186 mg of compound 51b, respectively. The LC-MS of compound 51a: [M+H]+ =1289.47; LC-MS of compound 51b: [M+H]+ = 1289.47.
Step 2: Compound 51 A
Figure imgf000136_0001
Compound 51a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 39 mg of solid.
Step 3: Compound 51 B
Figure imgf000136_0002
Compound 51b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 60 mg of solid.
Example 56
Synthesis of compound 52A:
Figure imgf000136_0003
Step 1 : Compound 52a lid (800 mg, 0.96 mmol), M9 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol) and 30 mL of DMF were added to a 50 mL flask in an ice-water bath and DIPEA (660 uL, 4.0 mmol) was added, and the reaction was allowed to proceed for 4 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to give the preparative solution of compound 52a. The reaction was carried out at room temperature for 4 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound 52a, which was lyophilized to obtain 52a (388 mg); LC-MS: [M+H]+ =1261.4.
Step 2: Compound 52A
52A (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 52A (79 mg); LC-MS: [M+H]+ =1105.4.
Example 57
Figure imgf000137_0001
Referring to the synthetic route of Example 56, compound 52B (50 mg) was obtained. LC- MS: [M+H]+ 1105.4.
Example 58
Synthesis of compound 53A:
Figure imgf000137_0002
Step 1 : Compound 53a 12d (400 mg, 0.47 mmol), M9 (240 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt
(101 mg, 0.47 mmol) and 15 mL of DMF were added to a 50 mL flask with an ice-water bath and DIPEA (330 uL, 2.0 mmol), and the reaction was carried out at room temperature for 3 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC. The reaction was carried out at room temperature for 3 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound 53a, which was lyophilized to obtain 53a (200 mg); LC-MS: [M+H]+ =1275.4.
Step 2: Compound 53A 53A (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 53A (51 mg); LC-MS: [M+H]+ =1119.4.
Example 59
Figure imgf000138_0001
Referring to the synthetic route of Example 58, compound 53B (50 mg) was obtained; LC- MS: [M+H]+ =1119.4.
Example 60
Synthes
Step 1 :
Figure imgf000138_0002
19d (500 mg, 0.59 mmol), M9 (317 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol) and 10 mL of DMF were added to a 50 mL flask with an ice-water bath and DIPEA (292 uL, 1.77 mmol) was added, the reaction was raised to room temperature and carried out for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solutions of compounds 54a and 54b, which were lyophilized to obtain 103 mg of compound 54a, LC-MS: [M+H]+ =1261.5, and 111 mg of compound 54b, LC-MS: [M+H]+ =1261.5, respectively.
Step 2: Compound 54A
Figure imgf000139_0001
In a 25 mL flask, 54a (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain a solid of 61 mg; LC-MS: [M+H]+ =1105.4.
Step 3: Compound 54B
Figure imgf000139_0002
In a 25 mL flask, 54b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure and removed to obtain the crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 57 mg of solid; LC-MS: [M+H]+ =1105.4.
Example 61
Synthesis of compounds 55A and 55B:
Figure imgf000139_0003
Step 1 : Compounds 55a and 55b 20d (400 mg, 0.47 mmol), M9 (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt
(83 mg, 0.56 mmol) and 10 mL of DMF were added to a 50 mL flask in an ice-water bath and DIPEA (248 uL, 1.5 mmol) was added, and the reaction was brought to room temperature and carried out for 2 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solutions of compounds 55a and 55b, which were lyophilized to obtain 100 mg of compound 55a, LC-MS: [M+H]+ =1275.5, and 101 mg of compound 55b, LC-MS: [M+H]+ = 1275.5, respectively.
Step 2: Compound 55A
Figure imgf000140_0001
55a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 42 mg of solid; LC-MS: [M+H]+ =1119.4.
Step 3: Compound 55B
Figure imgf000140_0002
55b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 45 mg of solid; LC-MS: [M+H]+ =1119.4.
Example 62
Synthesis of compound 56:
Figure imgf000140_0003
Referring to the synthetic route of Example 60, compound 56 (50 mg) was obtained; LC-MS: [M+H]+ =1119.3.
Example 63
Synthesis of compound 57:
Figure imgf000141_0001
Referring to the synthetic route of Example 61, compound 57 (50 mg) was obtained; LC-MS: [M+H]+ =1119.4.
Example 64
Synthesis of compound 58:
Figure imgf000141_0002
Step 1 : Synthesis of compound 58a
To Exatecan mesylate M5 (15 g, 28 mol, prepared by the method disclosed in EP0737683A1, 400 mL of DMF was added, the reaction was cooled to 0°C in an ice-water bath, tri ethylamine was added dropwise, the pH was adjusted to 7-8, and benzyl bromide (9.6 g, 56 mmol) was added dropwise in an ice-water bath, the reaction was warmed to room temperature (25°C) for 1 hour, TLC to monitor the reaction was complete, the reaction solution was concentrated under reduced pressure, the resulting crude product was purified by preparative-grade high-performance liquid chromatography (acetonitrile/pure water system), the target peak was collected, and after removing the acetonitrile under reduced pressure, lyophilization, to obtain the compound 58a of about 11 g, a yellow solid, the yield was about 74%, MS m/z : [M + H]+ 526.3.
Step 2: Synthesis of compound 58b
At room temperature, compound 58a (11 g, 21 mol) was added sequentially in a 250 mL flask, 120 mL of formic acid was dissolved, 30 mL of formaldehyde (40% aqueous solution) was added to the resulting bright yellow solution, and the reaction was heated up to 50 °C for 1 h. The reaction was monitored by TLC to see if the reaction was complete, and then the reaction solution was cooled down to room temperature, and then the reaction solution was purified by preparative high- performance liquid chromatography (acetonitrile/water), and the target peak was collected. The target peak, after removing acetonitrile under reduced pressure, lyophilized to give compound 58b about 4.5 g, yellow powdery solid, yield about 40%, MS m/z : [M+H]+ 540.6.
Step 3: Synthesis of compound 58:
At room temperature, add compound 58b (2.3 g, 4.3 mol) in a 250 mL flask, add 100 mL of DMF to dissolve, add 2.3 g of 5% Pd/C to the resulting bright yellow solution, hydrogen balloon to replace the atmosphere in the system, and maintain the reaction at room temperature for 1.5 hours, the reaction was completely detected by HPLC, and then the reaction was filtered to remove the Pd/C, and the resulting reaction solution was concentrated, and then purified by preparative grade HPLC (acetonitrile/pure water system). After the obtained reaction solution was concentrated, purified by preparative grade high performance liquid chromatography (acetonitrile/water system), the target peaks were collected, and after removing acetonitrile under reduced pressure, lyophilized to obtain about 1.0 g of Compound 58, a yellow powdery solid with about 52% yield, MS m/z : [M+H]+ 450.5.
Example 65
Synthesis of compound 59:
Figure imgf000142_0001
Step 1 : Compound 59a
Id (500 mg, 0.62 mmol), 58 (279 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL ofDMF were added to a 50 mL flask, and DIPEA (378 uL, 2.29 mmol) was added to the flask under an ice-water bath, and the reaction was allowed to proceed for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by HPLC to obtain the preparative solution, and the preparative solution was lyophilized to obtain 59a (166 mg); LC- MS was used to obtain the preparative solution. The reaction was carried out at room temperature for 2 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution, which was lyophilized to give 59a (166 mg); LC-MS: [M+H]+ =1235.6.
Step 2: Compound 59
59a (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 59 (43 mg); LC-MS: [M+H]+ =1079.3.
Example 66
Synthesis of compound 60:
Figure imgf000143_0001
Referring to the synthetic route of Example 65, compound 60 (40 mg) was obtained; LC-MS: [M+H]+ - 1079.3.
Example 67
Synthesis of compound 61:
Figure imgf000143_0002
Step 1 : Compound 61a
5d (1.66 g, 2.02 mmol, 1.0 eq), 58 (0.91 g, 2.02 mmol, 1.0 eq), PyBOP (1 .58 g, 3.03 mmol, 1.5 eq), HOBt (0.41 g, 3.03 mmol, 1.5 eq), and DMF (40 mL) were added to a 100 mL flask, and the reaction was brought to room temperature for 2 h. The reaction was monitored by HPLC. DIPEA (0.84 mL, 1.5 eq) was added, and the reaction was brought to room temperature for 2 h (monitored by HPLC). The reaction solution was purified directly by preparation, and the preparative solution was concentrated by pumping water under reduced pressure at 35 °C to remove acetonitrile, and lyophilized to obtain compound 61a (1.21 g); LC-MS: [M+H]+ =1249.4. Step 2: Compound 61
In a 100 mL flask, compound 61a (1.0 g, 0.8 mmol, 1.0 eq) was added, 35 mL of nitromethane, dissolved and then added zinc bromide (3.64 g, 16 mmol, 20.0 eq), the reaction was carried out in an oil bath at 40 °C (stabilized in advance by preheating) for 30 min, and then the pump was concentrated at 45 °C under reduced pressure in a water bath to remove the nitromethane, resulting in a yellow residue solid (monitored by HPLC). After acid preparation, the preparation liquid was obtained, the preparation liquid was concentrated by pump decompression water bath at 35 °C to spin off acetonitrile, lyophilized to obtain compound 61 (786 mg), LC-MS: [M+H]+ =1093.6.
Example 68
Synthesis of compound 62:
Figure imgf000144_0001
Compounds 5d-l (200 mg, 0.24 mmol, 1.0 eq), 58 (110.3 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2 eq), HOBt (48 mg, 0.36 mmol, 1.2 eq), and DMF (6 mL) were added to a 25 mL flask. The reaction was carried out at 20±5 °C for 2 h. The reaction was monitored by HPLC for completion. The reaction solution was purified by direct HPLC preparation, and the product preparation was collected and lyophilized to obtain compound 62a (120.9 mg); LC-MS: [M+H]+ =1249.4.
Step 2: Compound 62 Compound 62a (100 mg, 0.081 mmol, 1.0 eq), ZnBr? (364 mg, 1.62 mmol, 20.0 eq) and CH3 NO2 (10 mL) were added to a 25 mL flask. After addition, the reaction was raised to 40 °C for 0.5 h, then the reaction was stopped, and the reaction solution was dried directly at 45 °C by spindrying under reduced pressure to obtain a yellow solid, which was sampled for HPLC monitoring. The reaction was monitored by HPLC. The spin-dried solid was directly purified by HPLC preparation, and the product preparation was collected and lyophilized to obtain compound 62 (61 mg); LC-MS: [M+H] 1 =1093.4.
Example 69
Figure imgf000144_0002
Referring to the route of Example 67, compound 63 (60 mg) was obtained; LC-MS: [M+H]+ = 1093.4.
Example 70
Preparation of compound 64:
Figure imgf000145_0001
Referring to the synthetic route of Example 68, compound 64 (65 mg) was obtained; LC-MS: [M+H] 1 =1093.4.
Example 71
Preparation of compounds 65A and 65B:
Figure imgf000145_0002
Step 1 : Compounds 65a and 65b
7d (500 mg, 0.57 mmol), 58 (256.8 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL DMF were added into a 50 mL flask and DIPEA (378 uL, 2.29 mmol) was added into the vial in an ice bath. The reaction was carried out at room temperature for 2 h. After the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solutions of compounds 65a and 65b, which were lyophilized to obtain 155 mg of compound 65a, LC-MS: [M+H]+ =1303.4, and 158 mg of compound 65b, LC-MS: [M+H]+ =1303.6, respectively.
Step 2: Compound 65A
Figure imgf000145_0003
In a 25 mL flask, 50a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution, which was lyophilized to obtain 49 mg as a solid.
Step 3: Compound 65B
Figure imgf000146_0001
In a 25 mL flask, 65b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40 °C for 1 h. After the reaction was completed by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution, which was lyophilized to obtain a solid of 47 mg.
Example 72
Synthesis of compounds 66A and 66B:
Figure imgf000146_0002
Step 1 : Compound 66a and Compound 66b
In a 50 mL flask, 8d (500 mg, 0.57 mmol), 58 (256.8 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, and DIPEA (378 uL, 2.29 mmol) was added to the flask in an ice bath. After the reaction was completed by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solutions of compound 66a and compound 66b, which were lyophilized to obtain 160 mg of compound 66a and 160 mg of compound 66b, respectively.LC-MS of compound 66a: [M+H]+ =1303.7, and LC-MS of compound 66b: [M+H]+ = 1303.6.
Step 2:
Figure imgf000146_0003
Compound 66a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 57 mg of solid, LC-MS: [M+H]+ =1147.5.
Step 3 : Compound 66B
Figure imgf000147_0001
Compound 66b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mb of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure and the product was obtained as a crude. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 57 mg of solid, LC-MS: [M+H]+ =1147.5.
Example 73
Synthesis of compound 67A:
Figure imgf000147_0002
Step 1 : Compound 67a lid (800 mg, 0.96 mmol), 58 (432.5 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol) and 30 mL of DMF were added to a 50 mL flask, and DIPEA (660 uL, 4.0 mmol) was added to the vial under an ice-water bath. The reaction was brought to room temperature for 4 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound 67a, which was lyophilized to obtain 67a (402 mg); LC-MS: [M+H]+ =1275.4.
Step 2: Compound 67A
67A (100 mg, 0.78 mmol), zinc bromide (356 mg, 1.57 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 67A (47 mg); LC-MS: [M+H]+ =1119.5. Example 74
Synthesis of compound 67B:
Figure imgf000148_0001
Referring to the synthetic route of Example 73, compound 67B (50 mg) was obtained. LC- MS: [M+H]+ 1119.4.
Example 75
Synthesis of compound 68A:
Figure imgf000148_0002
Step 1 : Compound 68a 12d (400 mg, 0.47 mmol), 58 (211.7 mg, 0.47 mmol), PyBOP (250 mg, 0.47 mmol), HOBt
(101 mg, 0.47 mmol) and 15 mL of DMF were added to a 50 mL flask, and DIPEA (330 uL, 2.0 mmol) was added to the flask under an ice-water bath. The reaction was brought to room temperature for 3 h. After the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compound 68a, which was lyophilized to give 68a (177 mg); LC-MS: [M+H]+ =1289.4.
Step 2: Compound 68A
68A (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 68A (45 mg); LC-MS: [M+H]+ =1133.4.
Example 76
Synthesis of compound 68B:
Figure imgf000149_0001
Referring to the synthetic route of Example 75, compound 68B (50 mg) was obtained; LC- MS: [M+H] 1 =1133.4.
Example 77
Synthes
Step 1 :
Figure imgf000149_0002
19d (500 mg, 0.59 mmol), 58 (266 mg, 0.59 mmol), PyBOP (339 mg, 0.65 mmol), HOBt (88 mg, 0.86 mmol) and 10 mL of DMF were added to a 50 mL flask with an ice-water bath and
DIPEA (292 uL, 1.77 mmol) was added, and the reaction was raised to room temperature and the reaction was carried out for 2 h. After the completion of the reaction was monitored by EIPLC, the reaction solution was purified by high performance liquid chromatography (EIPLC) to obtain the preparative solutions of compounds 69a and 69b, which were lyophilized to obtain 109 mg of compound 69a, LC-MS: [M+H]+ =1275.5, and 111 mg of compound 69b, LC-MS: [M+H]+ = 1275.7, respectively.
Step 2: Compound 69A
Figure imgf000149_0003
69a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by EIPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (EIPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 53 mg of solid; LC-MS: [M+H] 1 =1119.4. Step 3: Compound 69B
Figure imgf000150_0001
69b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol) and 5 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 54 mg of solid; LC-MS: [M+H]+ =1119.4.
Example 78 Synthesis of compounds 70A and 70B:
Figure imgf000150_0002
Step 1 : Compounds 70a and 70b
20d (400 mg, 0.47 mmol), 58 (211.7 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol) and 10 mL of DMF were added to a 50 mL flask, and DIPEA (248 uL, 1.5 mmol) was added to the flask under an ice-water bath. The reaction was brought to room temperature for 2 h. After HPLC monitoring, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solutions of compounds 70a and 70b, which were lyophilized to obtain 106 mg of compound 70a, LC-MS: [M+H] 1 =1289.5, and 101 mg of compound 70b, LC-MS: [M+H]+ =1289.4, respectively. Step 2: Compound 70A
Figure imgf000150_0003
In a 25 mL flask, 70a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were added, and the reaction was carried out at 40 °C for 1 h. After the completion of the reaction was detected by HPLC, the solvent was concentrated under reduced pressure and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain a solid of 39 mg; LC-MS: [M+H]+ =1133.4.
Step 3: Compound 70B
Figure imgf000151_0001
In a 25 mL flask, 70b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol) and 5 mb of nitromethane were added and the reaction was carried out at 40 °C for 1 h. After the completion of the reaction was detected by HPLC, the solvent was concentrated under reduced pressure and removed to obtain the crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain 35 mg of solid; LC-MS: [M+H]+ =1133.4.
Example 79
Synthesis of compound 71:
Figure imgf000151_0002
Referring to the synthetic route of Example 78, compound 71 (30 mg) was obtained; LC-MS: [M+H]+ =1133.3.
Example 80
Synthesis of compound 72:
Figure imgf000151_0003
Referring to the synthetic route of Example 78, compound 72 (33 mg) was obtained; LC-MS: [M+H]+ =1133.4.
Example 81
Figure imgf000152_0001
Synthesis of compound Mil:
In a 100 mL flask, compound M3 (11.0 g, 19.5 mmol, 1.0 eq), DIPEA (2.8 g, 21.4 mmol, 1.1 eq), 27-amino-4, 7, 10, 13, 16, 19, 22, 25 -octaoxaheptadecanoic acid (9.7 g, 20.5 mmol, 1.05 eq) and DMF (60 mL) and reacted for 20 min at room temperature (monitored by TLC). The reaction solution was purified directly by preparation, and the preparative solution was concentrated by pumping water under reduced pressure at 35 °C to remove acetonitrile, and lyophilized to obtain compound M10 (13.2 g) in 78% yield; LC-MS: [M+H]+ =866.5.
In a 100 mL flask, compound M10 (13.0 g, 15 mmol, 1.0 eq), pentafluorophenol (3 g, 16.5 mmol, 1.1 eq), DCC (3.4 g, 16.5 mmol, 1.1 eq) and THF (30 mL) were added, and the reaction was carried out at room temperature for 30 min (monitored by TLC), and insoluble material was filtered off. The reaction solution was purified directly by preparation, and the preparative solution was concentrated by pumping water under reduced pressure at 35 °C to remove acetonitrile, and lyophilized to obtain compound Mil (14.2 g) in 92% yield; LC-MS: [M+H]+ =1032.5.
Example 82
Figure imgf000152_0002
Step 1 : Synthesis of compound 73a
To Mil (1 g, 0.79 mol), 10 mL of DMF was added, cooled to 0 °C in an ice-water bath, compound lc (334 mg, 0.79 mol), DIPEA (154 mg, 1.19 mol) was added, and the reaction was maintained under these conditions for 1 h. The reaction was monitored by TLC for complete reaction, and the reaction solution was purified by preparative grade HPLC (acetonitrile/water system), and the target peak was collected. The target peak was collected, and after removing acetonitrile under reduced pressure, lyophilized to give compound 73a about 1.2 g, MS m/z : [M+H]+ =1271.9.
Step 2: Synthesis of compound 73b
73a (1.2 g, 0.94 mmol), M5 (500 mg, 0.94 mmol), PyBOP (625 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol) and 15 mL of DMF were added to a 25 mL flask, and DIPEA (310 mg, 2.4 mmol) was added to the vial under an ice- water bath, and the reaction was carried out for 2 h. After the completion of the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution, which was lyophilized to give 73b (709 mg); LC-MS: [M+H]+ =1720.8.
Step 3: Synthesis of compound 73:
73b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 73 (88 mg); LC-MS: [M+H]+ =1532.6.
Example 83
Synthesis of compound 74:
Figure imgf000153_0001
Referring to the synthetic route of Example 82, compound 74 (90 mg) was obtained; LC-MS: [M+H]+ =1532.6.
Example 84
Synthesis of compound 75:
Figure imgf000154_0001
Step 1 : Synthesis of compound 75a
To Mil (1 g, 0.79 mol), 10 mL of DMF was added, cooled to 0 °C in an ice-water bath, compound 5c (345 mg, 0.79 mol), DIPEA (154 mg, 1.19 mol) was added, and the reaction was maintained under these conditions for 1 h. The reaction was monitored by TLC for complete reaction, and the reaction solution was purified by preparative grade HPLC (acetonitrile/water system), and the target peak was collected. The target peak was collected, and after removing acetonitrile under reduced pressure, lyophilized to give compound 75a 0.9 g, MS m/z: [M+H]+ = 1285.6. Step 2: Synthesis of compound 75b
75a (700 mg, 0.54 mmol), M5 (289 mg, 0.54 mmol), PyBOP (313 mg, 0.6 mmol), HOBt (81 mg, 0.6 mmol) and 10 mL DMF were added to a 25 mL flask, and DIPEA (155 mg, 1.2 mmol) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h. After the reaction was completed, the reaction solution was purified by HPLC to obtain a preparative solution, which was lyophilized to obtain 75b (304 mg); LC-MS: [M+H]+=1734.8.
Step 3: Synthesis of compound 75:
75b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure to remove the solvent, and the crude product was obtained. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 75 (96 mg); LC-MS: [M+H]+ =1546.6.
Example 85
Synthesis of compound 76:
Figure imgf000155_0001
Referring to the synthetic route of Example 84, compound 76 (92 mg) was obtained; LC-MS: [M+H]+ =1546.5.
Example 86
Synthesis of compound 77:
Figure imgf000155_0002
Referring to the synthetic route of Example 84, compound 77 (87 mg) was obtained; LC-MS: [M+H]+ =1546.5.
Example 87
Synthesis of compound 78:
Figure imgf000155_0003
Referring to the synthetic route of Example 84, compound 78 (94 mg) was obtained; LC-MS: [M+H]+ =1546.7.
Example 88
Synthesis of compounds 79 and 80:
Figure imgf000156_0001
Step 1 : Synthesis of compound 79a
To Mi l (1 g, 0.79 mol), 10 mL of DMF was added, cooled to 0 °C in an ice-water bath, compound 20c (377 mg, 0.79 mol), DIPEA (154 mg, 1.19 mol) was added, and the reaction was maintained under these conditions for 1 h. The reaction was monitored for completeness by TLC, and the reaction solution was purified by preparative-grade high-performance liquid chromatography (acetonitrile/water system), and the target peak was collected. The target peak was collected and after removing acetonitrile under reduced pressure, lyophilized to give 783 mg of compound 79a, MS m/z: [M+H]+ =1325.8. Step 2: Synthesis of compounds 79b-l and 79b-2
79a (600 mg, 0.45 mmol), M5 (240 mg, 0.45 mmol), PyBOP (261 mg, 0.5 mmol), HOBt (68 mg, 0.5 mmol) and 10 mL of DMF were added to a 25 mL flask, and DIPEA (130 mg, 1 mmol) was added to the flask under an ice-water bath, and the reaction was carried out for 2 h. The reaction was monitored by HPLC. After the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography (HPLC) to obtain the preparative solution of compounds 79b-l and 79b-2, and the preparative solution was lyophilized to obtain 79b-l (124 mg); LC-MS: [M+H]+ = 1743.0; 79b-l (122 mg) was obtained; LC-MS: [M+H] 1 = 1743.0.
Step 3: Synthesis of compound 79
Figure imgf000157_0001
79b-l (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure and the product was obtained as a crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 79 (30 mg); LC-MS: [M+H]+ =1586.9.
Step 4: Synthesis of Compound 80
Figure imgf000157_0002
79b-2 (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol) and 10 mL of nitromethane were added to a 25 mL flask and the reaction was carried out at 40 °C for 1 h. After the completion of the reaction was monitored by HPLC, the solvent was concentrated under reduced pressure and removed to obtain the crude product. The crude product was purified by high performance liquid chromatography (HPLC) to obtain the product preparation solution, and the preparation solution was lyophilized to obtain the solid compound 80 (33 mg); LC-MS: [M+H]+ =1587.0.
Example 89
Synthesis of compound 81:
Figure imgf000158_0001
Referring to the synthetic route of Example 88, compound 81 (24 mg) was obtained; LC-MS: [M+H]+ =1586.9.
Example 90
Synthesis of compound 82:
Figure imgf000158_0002
Referring to the synthetic route of Example 88, compound 82 (29 mg) was obtained; LC-MS: [M+H] 1 =1586.9.
Example 91 1) Expression and purification of a humanized anti-Claudinl8.2 antibody, 5103F3-BSM:
Expi293 (Shanghai 0PM Biotechnology Co., Ltd.) suspension cells were used to express 5103F3-BSM antibody. One day before transfection, cells were inoculated at a density of 0.9* 106 cells/mLin 1 L shake flasks containing 300 mL ofOPM-293 CD05 Medium (81075-001, Shanghai Oppermax Bio-technology Co., Ltd.), and cultured overnight at 37 °C, 5% CO2, and 120 rpm on a cell culture shaker. On the next day, PEI-MAX was used to transfect the antibody expression plasmid, in which the mass ratio of plasmid:PEI-MAX was 1:3. OPM-293 ProFeed supplement was added at 5% (v/v) on the first day after transfection, and then at 5% (v/v) on the third day after transfection, and then centrifuged to collect the supernatant on the sixth day after transfection.
The cell expression supernatants obtained were collected and eluted with 0.05 M sodium acetate buffer (pH 3.6) by a Protein A affinity chromatography column (UniMab 50, Suzhou Nano
Technology Co., Ltd.), and the captured antibody was adjusted to pH 7.0 with 1 M Tris-HCl (pH 8.8) at 0.7/10 (v/v), and then passed through a gel filtration chromatography column SEC (Superdex 200 Increase, Cytiva) to remove impurities such as polymers, while the antibody buffer was replaced with 20 mM sodium acetate buffer (pH 6.0).
Example 92
Preparing the samples of 51O3F3-BSM antibody-drug conjugate by coupling 51O3F3-BSM antibody with payload:
After the cell expression and purification by Protein A affinity chromatography and molecular sieve chromatography, the anti-Claudin 18.2 antibody was replaced in 20 mM NaAc-HAc, pH 6.0 buffer, concentrated or diluted, and the anti-Claudin 18.2 antibody was concentrated to a protein concentration of 5 mg/mL. The white powder of linker plus the drug was dissolved in DMA to 10 mg/mL and set aside. To open the interchain disulfide bonds of anti-Claudinl 8.2 antibody, 15- fold TCEP was added according to the ratio of molecules and reacted at room temperature for 2 h. Then 16-fold ligand plus drug solution was added according to the ratio of molecules and reacted at room temperature for 2 h. At the end of the reaction, the ligand plus drug that had not been coupled with the anti-Claudinl8.2 antibody was removed by ultrafiltration using a 30 KDa ultrafiltration centrifuge tube. After the reaction, use 30 KDa ultrafiltration centrifuge tube to remove the linker plus drug that is not coupled with anti-Claudinl8.2 antibody.
The resulting samples of anti-Claudin 18.2 antibody-drug conjugate will be used to determine the monomer rate by SEC-HPLC and the drug load by RP-HPLC or HIC-HPLC. Example 93
Samples of the antibody 64C9-drug conjugate were prepared by coupling 64C9 antibody with payload after the cell expression and purification by Protein A affinity chromatography and molecular sieve chromatography, the anti-Claudinl 8.2 antibody was replaced in 20 mM NaAc- HAc, pH 6.0 buffer, and the anti-Claudin 18.2 antibody was concentrated or diluted to a protein concentration of 5 mg/mL. The white powder linker plus drug was dissolved in DMA to 10 mg/mL for backup. To open the interchain disulfide bonds of anti-Claudinl8.2 antibody, 8-12 times of TCEP was added according to the molecular ratio, and the reaction was carried out at room temperature for 1-2 h. Then 5-8 times of the linker plus drug solution was added according to the molecular ratio, and the reaction was carried out at room temperature for 1-2 h. The linker plus drug that had not been coupled with anti-Claudin 18.2 antibody was removed at the end of the reaction by ultrafiltration using 30 KDa ultrafiltration centrifuge tube. At the end of the reaction, a 30 KDa ultrafiltration centrifuge tube was used to remove the linker plus drug that is not coupled with anti-Claudinl 8.2 antibody, and then the sample of anti-Claudinl 8.2 antibody-drug conjugate was obtained.
The resulting samples of anti-Claudinl 8.2 antibody-drug conjugate will be used to determine the monomer rate by SEC-HPLC and the drug load by RP-HPLC or HIC-HPLC.
Example 94
ADC-1 was prepared according to the generalized coupling method of Example 92:
Figure imgf000160_0001
ADC-2 was prepared according to the generalized coupling method of Example 92:
Figure imgf000160_0002
ADC-3 was prepared according to the generalized coupling method of Example 92:
Figure imgf000161_0001
ADC-4 was prepared according to the generalized coupling method of Example 92 :
Figure imgf000161_0002
Example 98
ADC-5 was prepared according to the generalized coupling method of Example 92:
Figure imgf000162_0001
ADC-6 was prepared according to the generalized coupling method of Example 92:
Figure imgf000162_0002
Example 100
ADC-7 was prepared according to the generalized coupling method of Example 92:
Figure imgf000163_0001
ADC-8 was prepared according to the generalized coupling method of Example 92:
Figure imgf000163_0002
Example 102
ADC-9 was prepared according to the generalized coupling method of Example 92.
Figure imgf000164_0001
ADC- 10 was prepared according to the generalized coupling method of Example 92:
Figure imgf000164_0002
Example 104
ADC- 11 was prepared according to the generalized coupling method of Example 92:
Figure imgf000165_0001
ADC- 12 was prepared according to the generalized coupling method of Example 92:
Figure imgf000165_0002
Example 106
ADC- 13 was prepared according to the generalized coupling method of Example 92:
Figure imgf000166_0001
ADC- 14 was prepared according to the generalized coupling method of Example 92:
Figure imgf000166_0002
Example 108
ADC- 15 was prepared according to the generalized coupling method of Example 92:
Figure imgf000167_0001
Example 109
ADC- 16 was prepared according to the generalized coupling method of Example 92:
Figure imgf000167_0002
Example 110
ADC- 17 was prepared according to the generalized coupling method of Example 92:
Figure imgf000168_0001
ADC- 18 was prepared according to the generalized coupling method of Example 92:
Figure imgf000168_0002
Example 112
ADC- 19 was prepared according to the generalized coupling method of Example 92:
Figure imgf000169_0001
Example 113
ADC-20 was prepared according to the generalized coupling method of Example 92:
Figure imgf000169_0002
Example 114
ADC-21 was prepared according to the generalized coupling method of Example 92:
Figure imgf000170_0001
ADC-22 was prepared according to the generalized coupling method of Example 92:
Figure imgf000170_0002
Example 116
ADC-23 was prepared according to the generalized coupling method of Example 92:
Figure imgf000171_0001
Example 117
ADC-24 was prepared according to the generalized coupling method of Example 92:
Figure imgf000171_0002
Example 118
ADC-25 was prepared according to the generalized coupling method of Example 92:
Figure imgf000172_0001
ADC-26 was prepared according to the generalized coupling method of Example 92:
Figure imgf000172_0002
Example 120
ADC-27 was prepared according to the generalized coupling method of Example 92:
Figure imgf000173_0001
ADC-28 was prepared according to the generalized coupling method of Example 92:
Figure imgf000173_0002
Example 122
ADC-29 was prepared according to the generalized coupling method of Example 92.
Figure imgf000174_0001
ADC-30 was prepared according to the generalized coupling method of Example 92:
Figure imgf000174_0002
Example 124
ADC-31 was prepared according to the generalized coupling method of Example 92:
Figure imgf000175_0001
ADC-32 was prepared according to the generalized coupling method of Example 92:
Figure imgf000175_0002
Example 126
ADC-33 was prepared according to the generalized coupling method of Example 92:
Figure imgf000176_0001
ADC-34 was prepared according to the generalized coupling method of Example 92:
Figure imgf000176_0002
Example 128
ADC-35 was prepared according to the generalized coupling method of Example 92:
Figure imgf000177_0001
ADC-36 was prepared according to the generalized coupling method of Example 92:
Figure imgf000177_0002
Example 130
ADC-37 was prepared according to the generalized coupling method of Example 92:
Figure imgf000178_0001
ADC-38 was prepared according to the generalized coupling method of Example 92:
Figure imgf000178_0002
Example 132
ADC-39 was prepared according to the generalized coupling method of Example 92:
Figure imgf000179_0001
ADC-40 was prepared according to the generalized coupling method of Example 92:
Figure imgf000179_0002
Example 134
ADC-41 was prepared according to the generalized coupling method of Example 92:
Figure imgf000180_0001
ADC-42 was prepared according to the generalized coupling method of Example 92:
Figure imgf000180_0002
Example 136
ADC-43 was prepared according to the generalized coupling method of Example 92:
Figure imgf000181_0001
ADC-44 was prepared according to the generalized coupling method of Example 92:
Figure imgf000181_0002
Example 138
ADC-45 was prepared according to the generalized coupling method of Example 92:
Figure imgf000182_0001
ADC-46 was prepared according to the generalized coupling method of Example 92:
Figure imgf000182_0002
Example 140
ADC-47 was prepared according to the generalized coupling method of Example 92:
Figure imgf000183_0001
ADC-48 was prepared according to the generalized coupling method of Example 92:
Figure imgf000183_0002
Example 142
ADC-49 was prepared according to the generalized coupling method of Example 92:
Figure imgf000184_0001
ADC-50 was prepared according to the generalized coupling method of Example 92:
Figure imgf000184_0002
Example 144
ADC-51 was prepared according to the generalized coupling method of Example 92:
Figure imgf000185_0001
ADC-52 was prepared according to the generalized coupling method of Example 92:
Figure imgf000185_0002
Example 146
ADC-53 was prepared according to the generalized coupling method of Example 92:
Figure imgf000186_0001
ADC-54 was prepared according to the generalized coupling method of Example 92:
Figure imgf000186_0002
Example 148
ADC-55 was prepared according to the generalized coupling method of Example 92:
Figure imgf000187_0001
ADC-56 was prepared according to the generalized coupling method of Example 92:
Figure imgf000187_0002
Example 150
ADC-57 was prepared according to the generalized coupling method of Example 92:
Figure imgf000188_0001
ADC-58 was prepared according to the generalized coupling method of Example 92:
Figure imgf000188_0002
Example 152
ADC-59 was prepared according to the generalized coupling method of Example 92:
Figure imgf000189_0001
ADC-60 was prepared according to the generalized coupling method of Example 92:
Figure imgf000189_0002
Example 154
ADC-61 was prepared according to the generalized coupling method of Example 92:
Figure imgf000190_0001
ADC-62 was prepared according to the generalized coupling method of Example 92:
Figure imgf000190_0002
Example 156
ADC-63 was prepared according to the generalized coupling method of Example 92:
Figure imgf000191_0001
ADC-64 was prepared according to the generalized coupling method of Example 92:
Figure imgf000191_0002
Example 158
ADC-65 was prepared according to the generalized coupling method of Example 92:
Figure imgf000192_0001
ADC-66 was prepared according to the generalized coupling method of Example 92
Figure imgf000192_0002
Example 160
ADC-67 was prepared according to the generalized coupling method of Example 92:
Figure imgf000193_0001
ADC-68 was prepared according to the generalized coupling method of Example 92:
Figure imgf000193_0002
Example 162
ADC-69 was prepared according to the generalized coupling method of Example 92:
Figure imgf000194_0001
ADC-70 was prepared according to the generalized coupling method of Example 92:
Figure imgf000194_0002
Example 164
ADC-71 was prepared according to the generalized coupling method of Example 92:
Figure imgf000195_0001
Example 165
ADC-72 was prepared according to the generalized coupling method of Example 92:
Figure imgf000195_0002
Example 166
ADC-73 was prepared according to the generalized coupling method of Example 92:
Figure imgf000196_0001
ADC-74 was prepared according to the generalized coupling method of Example 92:
Figure imgf000196_0002
Example 168
ADC-75 was prepared according to the generalized coupling method of Example 92:
Figure imgf000197_0001
ADC-76 was prepared according to the generalized coupling method of Example 92.
Figure imgf000197_0002
Example 170
ADC-77 was prepared according to the generalized coupling method of Example 92:
Figure imgf000198_0001
ADC-78 was prepared according to the generalized coupling method of Example 92:
Figure imgf000198_0002
Example 172
ADC-79 was prepared according to the generalized coupling method of Example 92:
Figure imgf000199_0001
ADC-80 was prepared according to the generalized coupling method of Example 92:
Figure imgf000199_0002
Example 174
ADC-81 was prepared according to the generalized coupling method of Example 92:
Figure imgf000200_0001
ADC-82 was prepared according to the generalized coupling method of Example 92:
Figure imgf000200_0002
Example 176
ADC-83 was prepared according to the generalized coupling method of Example 92:
Figure imgf000201_0001
ADC-84 was prepared according to the generalized coupling method of Example 92:
Figure imgf000201_0002
Example 178
ADC-85 was prepared according to the generalized coupling method of Example 92:
Figure imgf000202_0001
ADC-86 was prepared according to the generalized coupling method of Example 92:
Figure imgf000202_0002
Example 180
ADC-87 was prepared according to the generalized coupling method of Example 92:
Figure imgf000203_0001
ADC-88 was prepared according to the generalized coupling method of Example 92:
Figure imgf000203_0002
Example 182
ADC-89 was prepared according to the generalized coupling method of Example 92:
Figure imgf000204_0001
Example 183
ADC-90 was prepared according to the generalized coupling method of Example 92:
Figure imgf000204_0002
Example 184
ADC-91 was prepared according to the generalized coupling method of Example 92:
Figure imgf000205_0001
ADC-92 was prepared according to the generalized coupling method of Example 92:
Figure imgf000205_0002
Example 186
ADC-93 was prepared according to the generalized coupling method of Example 92:
Figure imgf000206_0001
ADC-94 was prepared according to the generalized coupling method of Example 92:
Figure imgf000206_0002
Example 188
ADC-95 was prepared according to the generalized coupling method of Example 92:
Figure imgf000207_0001
ADC-96 was prepared according to the generalized coupling method of Example 92:
Figure imgf000207_0002
Example 190
ADC-97 was prepared according to the generalized coupling method of Example 92:
Figure imgf000208_0001
Example 191
ADC-98 was prepared according to the generalized coupling method of Example 92:
Figure imgf000208_0002
Example 192
ADC-99 was prepared according to the generalized coupling method of Example 92:
Figure imgf000209_0001
Example 193
ADC- 100 was prepared according to the generalized coupling method of Example 92:
Figure imgf000209_0002
Example 194
ADC- 101 was prepared according to the generalized coupling method of Example 92:
Figure imgf000210_0001
Example 195
ADC- 102 was prepared according to the generalized coupling method of Example 92:
Figure imgf000210_0002
Example 196
ADC- 103 was prepared according to the generalized coupling method of Example 92:
Figure imgf000211_0001
Example 197
ADC- 104 was prepared according to the generalized coupling method of Example 92:
Figure imgf000211_0002
Example 198
ADC- 105 was prepared according to the generalized coupling method of Example 92:
Figure imgf000212_0001
Example 199
ADC- 106 was prepared according to the generalized coupling method of Example 92:
Figure imgf000212_0002
Example 200
ADC- 107 was prepared according to the general coupling method of Example 93:
Figure imgf000213_0001
wherein 64C9 is an anti-Claudinl8.2 antibody.
Example 309
To determine the monomer rate, the SEC-HPLC method below was used:
Column: Biocore SEC-300 5 pm, 4.6x300 mm
Manufacturer: NanoChrom, Item No.: B213-050030-04630S
Mobile phase: 50 mM PB+300 mM NaCl+200 mM Arg+5% IPA, pH=6.5
Table 1. Methods and parameters
Figure imgf000213_0002
Table 2. The ADC monomer rate
Figure imgf000213_0003
Figure imgf000214_0001
CONCLUSION: The antibody-drug conjugates (ADCs) disclosed in the present application has the characteristics of low degradation rate and low aggregation rate, and the excellent property of high monomer rate.
Example 310
To determine the drug antibody ratio (DAR), the RP-HPLC method below was used:
Column name: Proteomix RP-1000 4.6* 100mm 5pm 1000A
Manufacturer: Sepax Item No.: 465950-4610 Table 3. Methods and parameters
Figure imgf000214_0002
Table 4. ADC drug-antibody coupling ratio (DAR)
Figure imgf000214_0003
CONCLUSION: The ADCs in the present application have the excellent property of high DAR value, which may significantly increase the ADC drug concentration at the target site when the same dose of ADC is administered.
Example 311 Plasma stability of ADC:
ADCs were mixed with IgG-free plasma to make the final concentration of ADC at 0.6 mg/mL, incubated in a 37 °C water bath for a period of 0, 3, and 7 days. A unincubated plasma was used as a control. After incubation, the sample was purified and extracted, and the drug antibody ratio DAR was measured to reflect the plasma stability of ADCs.
Table 5. Plasma stability of ADCs
Figure imgf000215_0001
CONCLUSION: The ADCs disclosed in the present disclosure have good plasma stability, and there is no significant change in the DAR value during plasma incubation.
Example 312
ADC maintains the affinity of the corresponding original anti-Claudinl8.2 antibody 51O3F3- BSM for Claudinl8.2. The relative affinities of 5103F3-BSM versus ADC-6; 64C9 versus ADC- 107 for Claudinl8.2 were compared by using double antigen sandwich ELISA.
The specific steps of the Claudinl8.2 relative binding activity assay are as follows: the antigen was diluted with PBS to the appropriate concentration, spiked into the plate and incubated at 4°C overnight. After coating, wash the plate and seal it at 37°C for 1 h. After sealing, wash the plate, dilute the test material in a gradient according to the appropriate concentration, add the sample into the sealed enzyme plate and incubate. Dilute the rabbit anti-human antibody with buffer to the appropriate concentration, add the sample and incubate. Add the homemade color development solution to develop the color for 7min and then add the termination solution to terminate the reaction. 450nm read the plate, four-parameter fit to the reading results, calculate the EC50 value, and then get the relative binding activity results.
CONCLUSION: As shown in Figures 3A and 3B, the coupled ADCs maintained a similar affinity with the corresponding antibodies, with no significant difference in the EC50 values, indicating that the coupling of antibodies to the toxin did not affect their affinity for the antigen.
Example 313
Analysis of antibody binding specificity:
In the present application, HEK293T-18.1 and HEK293T-18.2, which are two human embryonic kidney cell lines overexpressing Claudinl8.1 and Claudinl8.2, respectively, were used as experimental models, and the specific binding of each naked antibody of ADC to Claudinl8.2 was analyzed by flow cytometry. HEK293T-18.1/18.2 in logarithmic growth phase were taken, trypsinized and washed twice with pre-cooled 1 XPBS, and resuspended to 1 x 107 cells/mL with 1 *PBS containing 3% BSA. 2* 105 cells were taken in 96-well plates, and a gradient dilution of the test antibody was added to the cells for 1 hour of incubation on ice. At the end of the incubation pre-cooled PBS was used to wash the cells, followed by the addition of FITC-mouse anti -Human fluorescent secondary antibody for 30 min on ice, washed, resuspended, and analyzed using a Beckman Coulter CytoFlex flow cytometer. The set-up gate strategy was as follows: the cells to be analyzed were circled with the SSC/FSC scatter plot; single cells were circled with the FSC-H/FSC-A scatter plot to exclude adhesions and debris; the single-cell gate was applied to the PB450-Ato obtain the live cells, and finally the cell population to be tested was identified in the FITC-A and the fluorescence values corresponding to different antibody concentrations were derived by statistics. The 4-parameter model of Graph Pad Prism 9.0 data analysis software was utilized for data statistics and analysis.
Table 6. Specific binding of Claudinl8.2 by subject ADC- 107 naked antibody ADC-6 naked antibody (also see Figure 4A and 4B).
Figure imgf000216_0001
Remarks: ND stands for not detected.
CONCLUSION: The naked antibodies, 64C9 and 5103F3-BSM, while did not recognize Claudinl8.1, display high specific recognition of Claudinl8.2. The binding affinity of 51O3F3- BSM was characteristically better than that of 64C9. Example 314
In vitro pharmacodynamic assays:
In the present application, a variety of human gastric adenocarcinoma tumor cell lines, polyclonal and monoclonal cell lines overexpressing Claudinl8.2, including HEK293T-18.2, BxPC-3 #A9F, SNU-5, and SNU-16, were used as experimental models to evaluate the efficacy of ADC drugs in vitro. Several tumor cell lines were inoculated in 96-well plates, and a gradient dilution of the subject antibody and the corresponding ADC drug were added to the cells, which were treated for 5 days, and the cell viability was detected by MTS, and the inhibitory effects of the subject antibody and ADC on the tumor cell lines were evaluated by calculating IC50. The starting concentration of the antibody drug was 500 nM, and the dilution was 7-fold, totaling 8 concentration points, and treated for 5 days. The final algorithm was based on survival rate = (experimental group - blank) / (control group - blank group) x 100%, followed by fitting the curve using Graph Pad Prism (Figure 5) and calculating the half inhibitory concentration (IC50).
Table 7. The results of the in vitro efficacy of naked antibody 64C9 versus ADC- 107, naked antibody 5103F3-BSM versus ADC-6 at cellular level (also see Figure 5A, 5B, 5C, and 5D).
Figure imgf000217_0001
CONCLUSION: In HE293T-18.2, BxPC-3#A9F, SNU-5 and SNU-16 cell models, no significant difference in tumor cell killing activity was seen between 64C9 and 5103F3-BSM, however, ADC- 6 (DAR=8) exhibits the significantly higher tumor cell killing activity in vitro, i.e., efficacy and potency, than ADC- 107 (DAR=8).
Example 315
The present application establishes a subcutaneous graft tumor model of target-positively expressing human gastric cancer cells SNU-5 NOD Scid mice to evaluate the in vivo efficacy of ADC-6. 5* 106 SNU-5 cells (0.1 mL/each) were injected subcutaneously into the right scapula of NOD Scid mice at 6~7 weeks of age. When the average tumor size of the mice grew to about 180 mm3, the mice were randomly divided into a lysate control group (Vehicle) and an ADC-6 treatment group (5 mg/kg) of 5 mice each, and the administration of ADC-6 was started (DO). All groups were administered by tail vein injection at 10 mL/kg body weight once a week for 4 weeks (QW><4). All groups were observed up to day 28 (D28) after subgroup administration (Table 8, Figure 6), and statistical analysis of the mean tumor volume on day 28 (D28) after subgroup administration showed that ADC-6 administered caudal-venously at a dose of 5 mg/kg (QWx4) had a significant tumor-suppressing effect (P<0.05). Table 8. Efficacy analysis of each group in the subcutaneous tumor graft model of human gastric cancer cells (SNU-5) in NOD Scid mice. Day 0 after Day 28 after administration administration
Experimental _ . _ . Relative
Mean tumor Mean tumor group . . Tumor TGI T/C P volume volume
- volume (%) (%) value
( x ±S) mm3 ( x ±S) mm3 -
( X ±b)
Gr°Up l 177.98±8.91 1493.75il90.25 8.34±0.87
Vehicle
Group 2
ADC-6 177.7H10.43 53.60i3.17 0.30i0.01 96.38 3.62 <0.001
(5 mg/kg)
Note: 1. Data are expressed as "mean ± standard error";
2. T/C % = TRTV / CRTV x 100%; TGI% = (1-T/C) x 100%;
3. p value was obtained by comparing the tumor volume of the treatment group with that of the lysate control group at D 28 (day 28 after administration).
Example 316
The present application establishes a target (i.e., Claudinl8.2)-overexpressing human gastric cancer cells (e.g., MKN-45) in BALB/c Nude mouse subcutaneous tumor graft model to evaluate the in vivo efficacy of ADC-6 drug. MKN-45 cells (2x l06) were injected subcutaneously (0. ImL/mouse) into the right scapula of BALB/c Nude mice aged 6 to 7 weeks. When the average tumor size of the mice grew to about 160 mm3, the mice were randomly divided into vehicle control group (Vehicle) and ADC-6 treatment group (2mg/kg, 5mg/kg), 5 mice in each group, and drug administration was started (DO). Each group was administered 10 mL/kg body weight via tail vein injection once a week for 4 consecutive weeks (QWX4). All groups were observed on the 28th day after group administration (D28) (Table 9, Figure 7), and statistical analysis of the mean tumor volume on day 28 (D28) after subgroup administration showed that ADC-6 administered caudal- venously at the doses of 2 mg/kg and 5 mg/kg (QW*4) had a significant tumor-suppressing effect (P<0.05). Table 9. Efficacy analysis of each group in the subcutaneous tumor graft model of human gastric cancer cells, MKN-45, in BALB/c Nude mice
Day 0 after Day 28 after administration administration
Experimental _ . _ . Relative
Mean tumor Mean tumor group . . Tumor TGI T/C P volume volume
- volume (%) (%) value
( x ±S) mm3 ( x ±S) mm3 -
( x ±S)
Group 1 167.08il2.44 1240.62i274.11 7.98i2.40 Vehicle
Group 2 ADC-6 166.79±16.44 72.00±27.44 0.43±0.17 94.55 5.45 <0.001
(2 mg/kg) _
Group 3 ADC-6 167.76±14.39 6.12±0.95 0.04±0.00 99.55 0.45 <0.001
(5 mg/kg) _
Note: 1. Data are expressed as "mean ± standard error";
2. T/C % = TRTV / CRTV x 100%; TGI% = (1-T/C) x 100%;
3. p value was obtained by comparing the tumor volume of the treatment group with that of the lysate control group at D28 (day 28 after administration).
Figure imgf000220_0001
Figure imgf000221_0001
Figure imgf000222_0001
Figure imgf000223_0001
Figure imgf000224_0001

Claims

1. A ligand-camptothecin derivative conjugate as shown in General Formula I, or a pharmaceutically acceptable salt or solvate thereof;
Figure imgf000225_0001
wherein
Ab is an antibody targeting human Claudinl8.2 or an antigen-binding fragment thereof;
Ln, L12, and L13 are different and are independently selected from a group comprising:
Figure imgf000225_0002
L2 has the structure shown in Formula A below.
Figure imgf000225_0003
Formula A wherein Y is a scaffold selected from C1-C6 alkyl, substituted C1-C6 alkyl, or C3-C8 cycloalkyl; preferably Y is C1-C6 alkyl; Ac is a hydrophilic structural unit; and the carbon 2 attached to Y has an absolute chirality in R or S configuration; Ls is present or absent, and when present, L3 comprises PEG hydrophilic units:
Figure imgf000226_0001
0 , o is an integer of 1-10, preferably an integer of 2-8;
L4 is an enzymatically cleavable unit;
L5 is a linking unit; the chiral carbon atom No. 1 attached to N in formula I has an absolute chirality in R or S configuration;
R is hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, C6-C10 aryl, substituted C6- C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; preferably R is hydrogen or a C1-C6 alkyl group;
Ri is hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, carboxylic acid, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; preferably Ri is hydrogen or a C1-C6 alkyl group; more preferably Ri is C1-C6 alkyl;
R? is hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, carboxylic acid, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10-membered heteroaryl, or substituted 5-10 heteroaryl; preferably R2 is hydrogen, halogen or C1-C6 alkyl; more preferably R2 is halogen;
X is -C(O)-CRaRb -(CR3R4)m -O-, -C(O)-CRaRb -(CR3R4)m-NH- or -C(O)-CRaRb-(CR3R4)m- S-. preferably X is -C(O)-CRaRb-(CR3R4) m-O-;
Ra and Rb are each independently hydrogen, deuterium, halogen, C1-C6 alkyl, deuteriumsubstituted C1-C6 alkyl, halogen- substituted C1-C6 alkyl, C3-C8 cycloalkyl, a C3-C8 cycloalkyl C1-C6 alkyl group, a C6-C10 aryl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5- 10 heteroaryl, or substituted 5-10 heteroaryl; preferably, Ra and Rb are each independently hydrogen, C1-C6 alkyl, halo-Cl-C6 alkyl, a C3- C8 cycloalkyl C1-C6 alkyl group, or a C6-C10 aryl C1-C6 alkyl group; alternatively, Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl, a 3-7 heterocycloalkyl, or a substituted 3-7 heterocycloalkyl; preferably Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl;
R3, R4 are the same or different and are independently hydrogen, deuterium, halogens, C1-C6 alkyl, halogenated C1-C6 alkyl, deuterated C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, amino, cyano, nitro, hydroxy C1-C6 alkyl, C3-C8 cycloalkyl, a 3-7 heterocyclic group, or a substituted 3-7 heterocyclic group, respectively; preferably, R3, R4 are independently hydrogen or C1-C6 alkyl, respectively;
Alternatively, R3, R4 and the carbon atoms attached thereto constitute a C3-C8 cycloalkyl, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocyclic group, or a substituted 3-7 heterocyclic group; m is an integer between 0-4, preferably 0, 1; nl, n2, n3 are each independently selected from any integer or any decimal number from 0-10, nl, n2, n3 are not simultaneously 0, and 1 < nl + n2 + n3 < 10.
2. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to Claim 1, wherein Ab is an antibody having a binding affinity to human Claudinl8.2.
3. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to Claim 2, wherein the antibody comprises an IgG heavy chain and a light chain.
4. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to Claims 3, wherein the light chain comprises CDRs of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and the IgG heavy chain comprises CDRs of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.
5. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to Claim 1, wherein Ab comprises a light chain, comprising CDRL1, CDRL2 and CDRL3 each having an amino acid sequence encoded by a nucleic acid sequence of SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and a heavy chain comprising CDRH1, CDRH2 and CDRH3 each having an amino acid sequence encoded by a nucleic acid sequence of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.
6. The ligand-camptothecin derivative conjugate of Claim 1 as shown in general formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein Ab comprises a heavy chain comprising a heavy chain variable region of SEQ ID NO: 1, and a light chain comprising a light chain variable region of SEQ ID NO: 7.
7. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to Claim 1, wherein Ab comprises a heavy chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence of SEQ ID NO: 13, and a light chain variable region comprising an amino acid sequence encoded by a nucleic acid sequence of SEQ ID NO: 19.
8. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to Claim 1 , comprising: a heavy chain having an amino acid sequence of SEQ ID NO: 5, and a light chain having an amino acid sequence of SEQ ID NO: 11 .
9. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to Claim 1, comprising, a heavy chain having an amino acid sequence encoded by a nucleic acid sequence of SEQ ID NO: 17, and a light chain having an amino acid sequence encoded by a nucleic acid sequence of SEQ ID NO: 23.
10. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 1-9, wherein said X comprises the following structures or isomers thereof:
Figure imgf000228_0001
wherein the position indicated by the wavy line on the left is connected to the camptothecin derivative, and the position indicated by the wavy line on the right is connected to L5.
11. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to Claim 10, wherein L4 comprises peptide residues consisting of amino acids. wherein optionally, said amino acid is further substituted with one or more substituents selected from one or more of deuterium, halogens, hydroxyl, cyano, amino, nitro, carboxyl, Cl- C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, or substituted C3-C8 cycloalkyl; preferably, said peptide residue is a peptide residue formed from one, two or more amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline (C), serine (S), glutamic acid (E) or aspartic acid (D); more preferably, said peptide residue is a tetrapeptide residue comprising glycine (G) -glycine (G) -phenylalanine (F) -glycine (G).
12. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to Claim 11, wherein
L5 is -NR.5 (CRelGjq- or a chemical bond, and q is an integer of 0-6;
R5, Rs and R7 are the same or different and are each independently hydrogen, deuterium, halogen, a C1-C6 alkyl, a substituted C1-C6 alkyl, a deuterated C1-C6 alkyl, a C3-C8 cycloalkyl, a C3-C8 cycloalkyl C1-C6 alkyl, a C1-C6 alkoxy C1-C6 alkyl, a 3-7 heterocycloheteroaryl, a substituted 3-7 heterocycloheteroaryl, a C6-C10 aryl, a substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; preferably, R5, Rs and R7 are each independently hydrogen or a C1-C6 alkyl group; more preferably, R5, Rs and R7 are each independently hydrogen.
13. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of Claim 1, wherein said linking units -L11-L2-L3-L4-L5-, - L12-L2-L3-L4-L5- or -L13-L2-L3-L4-L5- are different from each other and each independently: pre
Figure imgf000229_0001
m each other and are each independently:
Figure imgf000229_0002
Figure imgf000230_0001
wherein
Ac is a hydrophilic structural unit;
Rs, Re and R? are the same or different and are each independently hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3-7 heterocyclic, substituted 3-7 heterocyclic, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl. preferably, Rs, Re and R? are each independently hydrogen or a C1-C6 alkyl; more preferably, Rs, Re and R? are each independently hydrogen; the carbon atom #2 attached to N has an absolute chirality in either R or S configuration; the position shown by the left wavy line is connected to Ab, and the position shown by the right wavy line is connected to X; o is an integer of 1-10.
14. A ligand-camptothecin derivative conjugate as shown in general formula II, or a pharmaceutically acceptable salt or solvate thereof;
Figure imgf000230_0002
wherein
Ab is an antibody having a binding affinity to human Claudinl8.2 or an antigen-binding fragment thereof; Ln, L12, and Ln are connecting units, and Ln, L12, and L13 are different from each other and are independently:
L3 is pre
Figure imgf000231_0001
of 1-10, preferably an integer of 2-8;
Ac is a hydrophilic structural unit; the 1 -position, 2-position and 3 -position chiral carbon atoms are each independently in R or S configuration;
R is hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; preferably R is hydrogen or C1-C6 alkyl;
Ri is hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, carboxyl, a 3-7 heterocyclic group, a substituted 3-7 meta-heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 meta-heteroaryl, or substituted 5-10 heteroaryl; preferably Ri is hydrogen or C1-C6 alkyl; more preferably Ri is C1-C6 alkyl;
R2 is hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, carboxyl, 3-7 meta-heterocyclic, substituted 3-7 meta-heterocyclic, C6-C10 aryl, substituted C6-C10 aryl, 5-10 meta-heteroaryl, or substituted 5-10 heteroaryl; preferably R2 is hydrogen, halogen or C1-C6 alkyl; more preferably R2 is halogen;
X is -C(O)-CRaRb-(CR3R4)m-O-, -C(O)-CRaRb-(CR3R4)m-NH- or -C(O)-CRaRb-(CR3R4)m-S-. preferably X is -C(O)-CRaRb-(CR3R4)m-O-;
Ra and Rb are each independently hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated Cl- C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, 3-7 heterocyclyl, substituted 3-7 heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; preferably, Ra and Rb are each independently hydrogen, C1-C6 alkyl, halo-Cl-C6 alkyl, a C3- C8 cycloalkyl C1-C6 alkyl group, or a C6-C10 aryl C1 -C6 alkyl group; alternatively, Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocycloalkyl group, a substituted 3-7 heterocycloalkyl group; preferably Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group;
R3, R4 are the same or different and are independently hydrogen, deuterium, halogens, C1-C6 alkyl, halogenated C1-C6 alkyl, deuterated C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, amino, cyano, nitro, hydroxy C1-C6 alkyl, C3-C8 cycloalkyl, a 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, respectively; preferably R3, R4, are independently hydrogen or C1-C6 alkyl, respectively; alternatively, R3, R4 and the carbon atoms attached thereto constitute C3-C8 cycloalkyl, a C3- C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocyclic group, or a substituted 3-7 heterocyclic group; m is an integer of 0-4, preferably 0, 1; nl, n2, and n3 are each independently an integer or a decimal number from 0 to 10, nl, n2, and n3 are not simultaneously 0, and l < nl + n2 + n3 < 10.
15. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 1 or 14, wherein said Ac has the structure shown in formula B:
Figure imgf000232_0001
wherein
Z comprises a hydrophilic structural carboxyl group, a phosphoric acid, a polyphosphoric acid, a phosphite, a sulfonic acid, a sulfmic acid, or a polyethylene glycol (PEG); preferably Z comprises hydrophilic structural carboxylates, phosphoric acid or polyethylene glycol (PEG);
Y' is optionally a scaffold connecting the amino group to Z; preferably Y' is C1-C6 alkyl; positions shown by wavy lines are connection sites.
16. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims lor 14, wherein said Ac comprises glycine, (D/L) alanine, (DI) leucine, (D/L) isoleucine, (D'l) valine, (D/L) phenylalanine, (D/L) proline, (D/L) tryptophan, (D/L) serine, (D 'L) tyrosine, (D 'L) cysteine, (D/L) cystine, (D/L) arginine, (D/L) histidine, (D/L) methionine, (D/L) asparagine, (D/L) glutamine, (D/L) threonine, (D/L) aspartic acid, (D/L) glutamic acid, natural or non-natural amino acid derivatives, or the following structures or isomers thereof.
Figure imgf000232_0002
preferably
Figure imgf000233_0001
17. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 1 or 14, wherein said Ac comprises glycine, phosphoric acid, (D/L) glutamic acid, or polyethylene glycol hydrophilic structure.
18. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 1 or 14, wherein said camptothecin derivative has the structure shown in formula d below;
Figure imgf000233_0002
wherein
R comprises hydrogen, deuterium, halogen, C1-C6 alkyl, substituted Cl -C6 alkyl, deuterated C1-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; preferably R is hydrogen or C1-C6 alkyl;
Ri comprises hydrogen, deuterium, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3- 7 meta-heterocyclic, substituted 3-7 meta-heterocyclic, C6-C10 aryl, substituted C6-C10 aryl, 5- 10 meta-heteroaryl, or substituted 5-10 heteroaryl; preferably Ri is hydrogen or C1-C6 alkyl; more preferably Ri comprises C1-C6 alkyl;
R2 comprises hydrogen, deuterium, halogen, C1-C6 alkyl, substituted Cl -C6 alkyl, deuterated C1-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, carboxylic acid, 3-7 heterocyclic group, a substituted 3-7 heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; preferably R2 is hydrogen, halogen or C1-C6 alkyl; more preferably R2 is halogen;
Ra and Rb are each independently hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C 1 - C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a 3-7 meta-heterocyclic group, a substituted 3-7 meta- heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl; preferably, Ra and Rb are each independently hydrogen, C1-C6 alkyl, halo-Cl-C6 alkyl, a C3- C8 cycloalkyl C1-C6 alkyl group, C6-C10 aryl; alternatively, Ra, Rb and the carbon atoms to which they are attached constitute C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl group, 3-7 heterocycloalkyl, substituted 3-7 heterocycloalkyl; preferably Ra, Rb and the carbon atoms to which they are attached constitute a C3-C8 cycloalkyl group; the 1 -position chiral carbon atom is in R or the S configuration; m is 0 or 1.
19. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 1 or 14, wherein said structural formula d comprises the following:
Figure imgf000234_0001
Figure imgf000235_0001
20. A linker-drug compound or a pharmaceutically acceptable salt or solvate thereof, having the structure shown in formula III below.
Figure imgf000235_0002
wherein
Figure imgf000236_0001
R comprises hydrogen, deuterium, halogen, C1-C6 alkyl, substituted Cl -C6 alkyl, deuterated C1-C6 alkyl, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 heteroaryl, or substituted 5-10 heteroaryl;
Ra comprises hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, a C3-C8 cycloalkyl C1-C6 alkyl group, a C1-C6 alkoxy C1-C6 alkyl group, a 3-7 meta-heterocyclic group, a substituted 3-7 meta-heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 meta-heteroaryl, or substituted 5-10-membered heteroaryl;
Rb comprises hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated Cl -C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, a 3-7 meta-heterocyclic group, a substituted 3-7 meta-heterocyclic group, C6-C10 aryl, substituted C6-C10 aryl, 5-10 meta-heteroaryl, or substituted 5-10 heteroaryl; alternatively, Ra, Rb and the carbon atoms attached thereto constitute a C3-C8 cycloalkyl group, a C3-C8 cycloalkyl C1-C6 alkyl group, a 3-7 heterocyclic group, or a substituted 3-7- membered heterocyclic group;
Figure imgf000236_0002
L3 present or absent; when L3 is present, L3 is 0 , o is an integer of 1 to 10; the chiral carbon atom at the 1 -position, 2-position or 3 -position is in R or S configuration;
Ac is a hydrophilic structural unit; m comprises 0 or 1; preferably, said linker-drug compound or a pharmaceutically acceptable salt or solvate thereof is configured to couple with the ligand Ab to form a ligand-camptothecin derivative conjugate of formula I or formula II according to any one of Claims 1-19.
21. The linker-drug compound or a pharmaceutically acceptable salt or solvate thereof according to Claim 20, wherein said Ac has the structure shown in formula B below.
Figure imgf000236_0003
wherein:
Z comprises a hydrophilic structural carboxyl group, a phosphoric acid, a polyphosphoric acid, a phosphite, a sulfonic acid, a sulfinic acid, or a polyethylene glycol (PEG);
Y1 is an optional unit connecting the amino group and Z; positions shown by wavy lines are connection sites.
22. The linker-drug compound or a pharmaceutically acceptable salt or solvate thereof according to Claim 20, wherein said Ac comprises glycine, (D/L) alanine, (D/L) leucine, (D/L) isoleucine, (D/L) valine, (D/L) phenylalanine, (D/L) proline, (D/L) tryptophan , (D/L) serine, (D/L) tyrosine, (D/L) cysteine, (D/L) cystine, (D/L) arginine, (DI) histidine, (D/L) methionine, (D/L) asparagine, (D/L) glutamine, (D/L) threonine, (D/L) aspartic acid, (D/L) glutamic acid, natural or unnatural amino acid derivatives, or the following structures:
Figure imgf000237_0001
23. The linker-drug compound or a pharmaceutically acceptable salt or solvate thereof according to Claim 20, wherein Ac comprises glycine, phosphoric acid, (D/L) glutamic acid, or polyethylene glycol hydrophilic structure.
24. The linker-drug compound or a pharmaceutically acceptable salt or solvate thereof according to Claim 20, wherein said linker-drug compound comprises the following structures or isomers thereof:
Figure imgf000237_0002
Figure imgf000238_0001
Figure imgf000239_0001
Figure imgf000240_0001
Figure imgf000241_0001
wherein o is an integer from 1-10.
25. A method for preparing a ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof as shown in general formula I or general formula II, wherein it comprises the following steps: obtaining the ligand-camptothecin derivative conjugates as shown in general formula I or general formula II by the coupling the reduced antibody or antigen-binding fragment thereof with the linker-drug compound;
Figure imgf000241_0002
Figure imgf000242_0001
the 1-position, 2-position, or 3-position chiral carbon atoms have absolute chirality in the R- or S-configuration;
Ab, L, Ln, L12, LB, L2, L3, L4 , L5 , X, R, Ri , R2 , nl , n2 or n3 are as claimed in any one of claims 1-24.
26. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 1 or 14, or the method according to claim 25, wherein said ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof comprises the following structure or a butanediimide ring-opening structure thereof or an isomer thereof.
Figure imgf000243_0001
Figure imgf000244_0001
Figure imgf000245_0001
Figure imgf000246_0001
Figure imgf000247_0001
Figure imgf000248_0001
Figure imgf000249_0001
Figure imgf000250_0001
Figure imgf000251_0001
Figure imgf000252_0001
Figure imgf000253_0001
Figure imgf000254_0001
Figure imgf000255_0001
Figure imgf000256_0001
Figure imgf000257_0001
Figure imgf000258_0001
Figure imgf000259_0001
Figure imgf000260_0001
Figure imgf000261_0001
Figure imgf000262_0001
Figure imgf000263_0001
Figure imgf000264_0001
Figure imgf000265_0001
Figure imgf000266_0001
Figure imgf000267_0001
Figure imgf000268_0001
Figure imgf000269_0001
Figure imgf000270_0001
Figure imgf000271_0001
Figure imgf000272_0001
Figure imgf000273_0001
Figure imgf000274_0001
Figure imgf000275_0001
Figure imgf000276_0001
Figure imgf000277_0001
Figure imgf000278_0001
Figure imgf000279_0001
Figure imgf000280_0001
Figure imgf000281_0001
Figure imgf000282_0001
Figure imgf000283_0001
Figure imgf000284_0001
Figure imgf000285_0001
Figure imgf000286_0001
Figure imgf000287_0001
Figure imgf000288_0001
Figure imgf000289_0001
Figure imgf000290_0001
Figure imgf000291_0001
Figure imgf000292_0001
Figure imgf000293_0001
Figure imgf000294_0001
Figure imgf000295_0001
wherein
5103F3-BSM is an antibody or an antigen-binding fragment thereof having a binding affinityman Claudinl8.2; nl , n2, and n3 are each independently selected from any integer or any decimal number from 0 to 10, nl, n2, and n3 are not simultaneously 0, and 1 < nl + n2 + n3 < 10.
27. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 1, 14, or 20, or a linker-drug compound or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 20-24, wherein said pharmaceutically acceptable salts comprise sodium salts, potassium salts, calcium salts, calcium salts or magnesium salts formed with the acidic functional group in the structural formula and acetates, trifluoroacetates, citrates, oxalates, tartrates, bromides, iodides, malates, nitrates, chlorides, iodates, iodines, and so on. salt or magnesium salt and acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate formed with the basic functional group in the structure.
28. A pharmaceutical composition comprising a ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 1-19 or 26- 27, or a linker-drug compound or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 20-24, and optionally a pharmaceutically acceptable carrier.
29. A pharmaceutical preparation comprising a ligand-camptothecin derivative conjugate compound or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 1-19 or 26-28, or a linker-drug compound or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 20-24.
30. The ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 1-19 or 26-27, the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 20-24, the pharmaceutical composition of Claim 28, or the pharmaceutical formulation of Claim 29, in the preparation of a drug for the treatment of or prevention of cancer or tumors; alternatively, the ligand-camptothecin derivative conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 1-19 or 26-27, the linker-drug compound or a pharmaceutically acceptable salt or solvate thereof according to any one of Claims 20-24, the pharmaceutical composition of Claim 27, or the pharmaceutical formulation of Claim 29, for the treatment or prevention of cancer or tumors; preferably, the cancer or tumor expresses Claudin 18.2; more preferably, the cancer or tumor comprises adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urothelial cancer, bladder cancer, hepatocellular carcinoma, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, triple-negative breast cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, solid or hematologic tumors such as sarcoma, lymphoma, and leukemia.
31. Use of a prophylactically or therapeutically effective amount of the ligand-camptothecin derivative conjugate compound or the pharmaceutically acceptable salt or solvate thereof according to any one of Claims 1 or 14, a linker-drug compound or the pharmaceutically acceptable salt or solvate thereof according to Claim 20, the pharmaceutical composition of Claim 28, or the pharmaceutical formulation of Claim 29 in a manufacture of medicament for treating or preventing cancer or tumor comprising administering to a subject in need thereof; preferably, the cancer or tumor expresses Claudinl8.2; more preferably, the cancer or tumor comprises adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urothelial cancer, bladder cancer, hepatocellular carcinoma, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, triple-negative breast cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, solid or hematologic tumors such as sarcoma, lymphoma, and leukemia.
PCT/US2024/059794 2023-12-13 2024-12-12 Anti-claudin18.2 antibody-camptothecin drug conjugate and pharmaceutical use thereof Pending WO2025128837A1 (en)

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