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WO2025230927A1 - Anti-human dll3 antibody-camptothecin derivative conjugates and medical uses thereof - Google Patents

Anti-human dll3 antibody-camptothecin derivative conjugates and medical uses thereof

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Publication number
WO2025230927A1
WO2025230927A1 PCT/US2025/026726 US2025026726W WO2025230927A1 WO 2025230927 A1 WO2025230927 A1 WO 2025230927A1 US 2025026726 W US2025026726 W US 2025026726W WO 2025230927 A1 WO2025230927 A1 WO 2025230927A1
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Prior art keywords
compound
mmol
alkyl
antibody
reaction
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Application number
PCT/US2025/026726
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French (fr)
Inventor
Yi Zhu
Yong Zhang
Shi ZHUO
Weili WAN
Gangrui LI
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Systimmune Inc
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Systimmune Inc
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Publication of WO2025230927A1 publication Critical patent/WO2025230927A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • 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

Definitions

  • the application relates to the biopharmaceutical field, and in particular, to antibody-drug
  • Delta-Like Ligand 3 (or abbreviated as “DLL3”) is a transmembrane protein which is encoded by DLL3 gene and attached to cell surfaces, and it is one member of the Notch Ligand Family.
  • the Notch Ligand Family is a transmembrane protein family, and the Notch Ligand Family comprises members such as Delta-like Ligand 1 (DLL1), DLL3, DLL4, Jaggedl (JAG1) and Jagged2 (JAG 2). These ligands interact with Notch proteins, to trigger the activation of the Notch
  • the Notch signaling pathway is a highly conservative intercellular communication system that plays an important role in multiple development processes and tissue maintenances.
  • the activation of the Notch signaling pathway is dependent on normal cleavage and ligand binding of the Notch proteins.
  • the Notch protein Upon ligand binding, the Notch protein undergoes cleavages to release the Notch intercellular domain (NICD), and the NICD further enters nucleus to participate in gene transcription regulations.
  • the Notch signaling pathway plays important roles in processes such as embryonic developments, organ developments, cell proliferations and differentiations. Abnormal activities of the Notch signaling pathway are associated with the occurrence and progression of a variety of diseases, such as tumors, cardiovascular diseases, and neurological diseases.
  • DLL3 The expression of DLL3 is found both in embryonic development and in certain mature tissues. Studies have shown that the DLL3 plays an important role in the development of the nervous system. It participates in the processes such as neuronal directional migration, axon guidance, and branching of dendrite branching. In addition, the overexpression of DLL3 is also observed in cancers such as Small Cell Lung Cancer (SCLC), prostate cancer, pancreatic cancer, and esophageal squamous cell carcinoma. The DLL3 is highly expressed in SCLC cells, and the expression level is relevant to the invasiveness, metastatic ability, and treatment resistance of tumors.
  • SCLC Small Cell Lung Cancer
  • the DLL3 is highly expressed in SCLC cells, and the expression level is relevant to the invasiveness, metastatic ability, and treatment resistance of tumors.
  • antibody drugs targeting DLL3 mainly include Rovalpituzumab tesirine (Rova-T) from Stemcentrx Inc and AMG-757 from Amgen. Studies of Rova-T on SCLC indications have
  • AMG-757 is a bi specific antibody developed by Amgen using a new generation bi specific antibody platform, HLE BiTE, and is currently in phase III clinical trials.
  • the trispecific antibody HPN-328 from Harpoon Therapeutics is currently in phase II clinical trials.
  • clinical trials on DLLS include CAR-T therapy AMG-119 from Amgen, CAR-T therapy LB-2102 from Nanjing Legend Biotech, bispecific antibody OBT620 from Boehringer Ingelheim Gmbh/Oxford Biotherapeutics Ltd, monoclonal antibody 89Zr-DFO-SC16.56 from Memorial Sloan Kettering Cancer Center, bispecific antibody QLS-31904 from QILU PHARMACEUTICAL, bispecific antibody BL764532 from Boehringer-Ingelheim, and bispecific antibody PT-217 from Phanes Therapeutics Inc, all of which are in the clinical phase I stage.
  • ADCs Antibody-drug conjugates
  • ADCs are biological drugs that link antibodies or antibody fragments to small molecular drugs with biological activity through stable chemical linkers, which not only make the best of the high specificity of antibody-antigen binding and the high lethality of cytotoxins, but also effectively avoid shortcomings such as weak therapeutic effects of antibodies, and indiscriminate attacks of small molecular drugs.
  • Mylotarg is the first marketed ADC in worldwide, which was approved by the FDA in a speed to market in May 2000 for the treatment of patients aged 60 years and older with CD33 -positive Acute Myeloid Leukemia (AML) who have experienced a relapse for the first time and are not suitable candidates for cytotoxic chemotherapy. Later studies found that the treatment with Mylotarg resulted in severe fatal liver injury, and then Pfizer chose to withdraw from the market
  • ELAHERE is the first ADC drug targeting folate receptor alpha (FR-a) approved by FDA, for the treatment of patients with cervical cancer who have experienced a relapse or metastasis.
  • ADCs antibody-drug conjugates
  • drug-linker compounds such as ligand- camptothecin derivative conjugates, drug-linker compounds, pharmaceutical compositions or preparations comprising ADCs, compositions comprising drug-linker compounds, methods of making ADCs using drug-linker compounds, methods of using ADCs for treating diseases such as cancer or tumors.
  • the application discloses ligand-camptothecin derivative conjugates or its
  • the ligand-camptothecin derivative conjugate is
  • Ab is an antibody targeting human DLLS or an antigen-binding fragment thereof
  • Ln, L12 and LB are each independently selected from the group consisting of:
  • Formula A wherein Vis a skeleton selected from the group consisting of C 1 -C6 alkylene, substituted C 1 - C6 alkylene and C3-C8 cycloalkylene; Ac is a hydrophilic structural unit; the carbon atom at the
  • 3 position of 2 linked to Y has an absolute chirality of R-configuration or S-configuration
  • L 3 is present or absent, and when present, La is selected from a PEG hydrophilic unit
  • o is an integer selected from 1 to 10,
  • L4 is an enzyme digestible or cleavable unit
  • L5 is a linking unit
  • X is -C(O)-CRaRb-(CR 3 R4)m-O-, -C(O)-CR a R b -(CR 3 R 4 )m-NH- or -C(O)-CR a Rb-(CR 3 R 4 ) m -S-, in one embodiment, X is -C(O)-CRaRb-(CR 3 R4) m -O-, wherein
  • Ra and Rb each independently are selected from the group consisting of hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8
  • Ra, Rb and carbon atoms linked thereto form C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, 3- to 7-membered heterocyclyl or substituted 3- to 7-membered heterocyclyl;
  • R 3 , R4 are each independently hydrogen atom, deuterium atom, halogen, 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, 3- to 7-membered heterocyclyl, substituted 3- to 7- membered heterocyclyl; or
  • R 3 , R4 and carbon atoms linked thereto form C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, 3- to 7-membered heterocyclyl or substituted 3- to 7-membered heterocyclyl;
  • m is 0, 1, 2, 3 or 4; the carbon atom at the position of 1 linked to N has an absolute chirality of R- or S- configuration;
  • R is 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- to 10-membered heteroaryl, or substituted 5- to 10-membered heteroaryl;
  • Ri is hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl,
  • C1-C6 alkyl deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, or substituted 5- to 10-membered heteroaryl;
  • Ln, L12 and L13 are each independently:
  • Ln, L12 and L13 are each independently: o 0 o H H b , "'COOH and COOH
  • Ac has a structure of Formula B, wherein:
  • Z is carboxyl, phosphoryloxy or -(OCFLCH ⁇ iOCFL, wherein i is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • i 1, 2, 3, 4, 5, 6, 7 or 8;
  • Y’ is a structural unit linking amino group and Z; in one embodiment, Y is C1-C6 alkylene or carboxyl-substituted C1-C6 alkylene,; in one embodiment, Y is methylene, ethylidene, carboxylsubstituted methylene or carboxyl-substituted ethylidene; in one embodiment, Y is methylene, ethylidene, or carboxyl-substituted methylene.
  • Ac is a residue formed by removing one hydrogen atom from the amino terminus of an amino acid.
  • the amino acid may be glycine, (D/L) alanine, (D/L) leucine, (DL) 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, (DL) methionine, (D/L) asparagine, (D/L) glutamine, (D/L) threonine, (DL) aspartic
  • Ac is: o. OH
  • Ac is:
  • L4 is a peptide residue composed of amino acids, wherein the amino acids are optionally substituted by one or more substituents selected from the group consisting of
  • the peptide residue is a peptide residue formed by one, two or more amino acids.
  • the amino acids may be phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline (C), serine (S), glutamic acid (E) and aspartic acid (D).
  • the peptide residue is a tetrapeptide residue, - G-G-F-G-, derived from glycine (G) -glycine (G) - phenylalanine (F) - glycine (G).
  • Ls is -NRsCCReR? ⁇ - or a chemical bond, wherein q is 0, 1, 2, 3, 4, 5 or 6;
  • R 10 Rs, Re and R? are each independently hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted Cl -C 6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, or substituted 5- to 10-membered heteroaryl.
  • q is 0, 1, 2 or 3. In some embodiments, q is 0, 1 or 2. In one embodiment, q is 0 or 1.
  • Rs, Re and R? are each independently hydrogen atom or C1-C6 alkyl. In some embodiments, Rs, Re and R? are each independently hydrogen atom and C1-C4 alkyl. In some embodiments, Rs, Re and R? are each independently selected from the group consisting of
  • R 20 hydrogen atom, methyl, ethyl, n-propyl and n-butyl.
  • Rs, Re and R? are each independently hydrogen atom.
  • Ra and Rb are each independently hydrogen atom, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl or C6-C10 aryl C1-C6 alkyl; or, in one embodiment, Ra, Rb and carbon atoms linked thereto form C3-C8 cycloalkyl.
  • Ra is hydrogen atom or C1-C4 alkyl
  • Rb is hydrogen atom, C1-C4 alkyl, halogenated C1-C4 alkyl, C3-C6 cycloalkyl C1-C4 alkyl or phenyl C1-C4 alkyl; or, in one embodiment, Ra, Rb and carbon atoms linked thereto form C3-C6 cycloalkyl.
  • R a is hydrogen atom, methyl, ethyl, n-propyl or n-butyl
  • Rb is hydrogen atom, methyl, ethyl, n-propyl, n-butyl, halogenated methyl, halogenated ethyl, halogenated n-
  • Ra is hydrogen atom or methyl
  • Rb is hydrogen atom, methyl, ethyl, trifluoromethyl, cyclopropylmethyl, or phenylmethyl; or, in one embodiment, Ra, Rb and carbon atoms linked thereto form cyclopropyl, cyclobutyl or cyclopentyl.
  • the position shown by the right side wavy line is connected to Ls.
  • R3, R4 are each independently hydrogen atom or C1-C6 alkyl. In some embodiments, R3, R4 are each independently hydrogen atom or C1-C4 alkyl. In some embodiments, R3, R4 are each independently hydrogen atom.
  • n is 0, 1 or 2. In some embodiments, m is 0 or 1.
  • R is hydrogen atom or C1-C6 alkyl. In some embodiments, R is hydrogen atom or C1-C4 alkyl. In some embodiments, Ris hydrogen atom, methyl, ethyl, n-propyl or n-butyl. In some embodiments, R is hydrogen atom or methyl.
  • Ri is hydrogen atom or C1-C6 alkyl. In some embodiments, Ri is Cl- C6 alkyl. In some embodiments, Ri is C1-C4 alkyl. In some embodiments, Ri is methyl, ethyl, n- propyl or n-butyl. In some embodiments, Ri is methyl.
  • R2 is hydrogen atom, halogen or C1-C6 alkyl. In some embodiments, R2 is hydrogen atom, halogen or C1-C4 alkyl. In some embodiments, R2 is halogen. In some embodiments, R2 is fluorine, chlorine, or bromine. In some embodiments, R2 is fluorine.
  • o is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, o is 1, 2, 3, 4, 5, 6, 7 or 8.
  • Y is C1-C6 alkylene. In some embodiments, Y is C1-C4 alkylene. In some embodiments, Y is methylene, ethylidene, propylidene or butylidene. In some embodiments,
  • nl, n2 and n3 each independently are any integer of 0 to 8 or any decimal of 0 to 8, and nl, n2 and n3 are not 0 simultaneously, with I ⁇ nl+n2+n3 ⁇ 8. In some embodiments, 5 ⁇ nl+n2+n3 ⁇ 10. In some embodiments, 6 ⁇ nl+n2+n3 ⁇ 8. In some embodiments, 7 ⁇ nl+n2+n3 ⁇ 8.
  • the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises;
  • VH variable region
  • CDRs complementarity determining regions
  • VH CDR1 having a CDR1 sequence contained in the VH as set forth in SEQ ID NO: 1,
  • VH CDR2 having a CDR2 sequence contained in the VH as set forth in SEQ ID NO : 1, or having a sequence with substitution, deletion or addition of one or more amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR2 sequence contained in the VH;
  • VH CDR3 having a CDR3 sequence contained in the VH as set forth in SEQ ID NO: 1, or having a sequence with substitution, deletion or addition of one or more amino acids (e.g.,
  • VL CDR1 having a CDR1 sequence contained in the VL as set forth in SEQ ID NO:7, or having a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR1 sequence contained in the VL;
  • VL CDR2 having a CDR2 sequence contained in the VL as set forth in SEQ ID NO: 7,
  • the substitution described in any one of (i) to (vi) is a conservative substitution.
  • the CDR1, CDR2 and CDR3 contained in the heavy chain variable are identical to the CDR1, CDR2 and CDR3 contained in the heavy chain variable
  • VH 20 region (VH), and/or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by Rabat, Chothia or IMGT numbering system.
  • the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises:
  • the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH) and/or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by Rabat, Chothia or IMGT numbering system.
  • the antibody targeting human DLL3 or an antigen-binding fragment comprises
  • VH CDR1 which is composed of a sequence as set forth in SEQ ID NO:2,
  • VH CDR2 which is composed of a sequence as set forth in SEQ ID NO:3, and
  • VH CDR3 which is composed of a sequence as set forth in SEQ ID NON;
  • VL CDR1 which is composed of a sequence as set forth in SEQ ID NO: 8,
  • VL CDR2 which is composed of a sequence as set forth in SEQ ID NO:9
  • VL CDR3 which is composed of a sequence as set forth in SEQ ID NO: 10.
  • VH CDR1 as set forth in SEQ ID NO:2; VH CDR2 as set forth in SEQ ID NO:3; and, VH CDR3 as set forth in SEQ ID NO:4; and/or, the VL of the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO:8; VL CDR2 as set forth in SEQ ID NO:9; and VL CDR3 as set forth in SEQ ID NO:10.
  • the VH of the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: VH CDR1 as set forth in SEQ ID NO:2; VH CDR2 as set forth in SEQ ID NO:3; and VH CDR3 as set forth in SEQ ID NO:4; and the VL of the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO:2; VH CDR2 as set forth in SEQ ID NO:3; and VH CDR3 as set forth in SEQ ID NO:4; and the VL of the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO:2; VH CDR2 as set forth in SEQ ID NO:3; and VH CDR3 as set forth in SEQ ID NO:4; and the VL of the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO:2;
  • VL CDR2 as set forth in SEQ ID NO: 9
  • VL CDR3 as set forth in SEQ ID NO: 10.
  • the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • the substitution described in (ii) or (v) comprises a conservative substitution.
  • the antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region (VH), comprising a sequence as set forth in SEQ ID NO: 1 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO: 1, and a light chain variable region (VL), comprising a sequence as set forth in SEQ ID NO:7 or a sequence having a
  • the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises; a VH having a sequence as set forth in SEQ ID NO: 1 and a VL with a sequence as set forth in SEQ ID NO: 7.
  • the antibody targeting human DLL3 or an antigen-binding fragment thereof is humanized.
  • the antibody targeting human DLL3 or an antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin.
  • the antibody targeting human DLL3 or an antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline antibody gene), and/or, a light chain framework region of a human immunoglobulin (e.g., a light chain framework region contained in an amino acid sequence encoded by a human light chain germline antibody gene).
  • a heavy chain framework region of a human immunoglobulin e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline antibody gene
  • a light chain framework region of a human immunoglobulin e.g., a light chain framework region contained in an amino acid sequence encoded by a human light chain germline antibody gene
  • the heavy chain framework region and/or the light chain framework region optionally comprises one or more (such as, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to murine residues.
  • the antibody targeting human DLL3 or an antigen-binding fragment comprises
  • the antibody or the antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin.
  • the heave chain of the antibody or the antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (such as IgGl, IgG2, IgG3 or IgG4).
  • a human immunoglobulin such as IgGl, IgG2, IgG3 or IgG4.
  • the light chain of the antibody or an antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (such as K or X).
  • the antibody targeting human DLL3 or an antigen-binding fragment comprises
  • the antibody targeting human DLL3 or an antigen-binding fragment thereof are selected from the group consisting of monoclonal antibody, mouse antibody, rabbit antibody, humanized antibody, fully human antibody, chimeric antibody (e.g., human-mouse chimeric antibody), bispecific antibody, multi-specific antibody, single chain antibody, dAb, complementarity determining region fragment, Fv, single chain Fv (scfv), Fd, Fab, Fab', and F(ab') 2 .
  • the monoclonal antibody includes a non-CDR region, and the non- CDR region is derived from species other than murine. In one embodiment, the non-CDR region
  • -L11-L2-L3-L4-L5-, -L12-L2-L3-L4-L5- and -L13-L2-L3-L4- Ls- are each independently selected from the group comprising: wherein: Ac, o, Rs, Re and R? are as defined herein in the disclosure; the carbon atom at the position of 2 linked to N has an absolute chirality of R-configuration or S-configuration; the position shown by the left side wavy line is connected to the antibody or an antigenbinding fragment thereof, and the position shown by the right side wavy line is connected to X.
  • -L11-L2-L3-L4-L5-, -L12-L2-L3-L4-L5- and -L13-L2-L3-L4- Ls- each independently are selected from the group comprising:
  • the conjugate of Formula I has a structure represented by Formula II: wherein: Ab, Ln, L12, L13, Ac, L3, X, R, Ri, R2, nl, n2, n3 are as defined in any one embodiment of the application; the chiral atom at the position of 1, 2 or 3 has an absolute chirality of R-configuration or S- configuration.
  • the conjugate of Formula I is selected from the group consisting of:
  • 18M1 represents Ab; Ab, nl, n2 and n3 as defined in any one embodiment of the application.
  • Formula I is a salt formed by an acidic functional group in the structural formula with sodium, potassium, calcium or magnesium, or an acetate, a trifluoroacetate, a citrate, an oxalate, a tartrate, a malate, a nitrate, a chloride, a bromide, an iodide, a sulfate, a bisulfate, a phosphate, a lactate, an oleate, an ascorbate, a salicylate, a formate, a glutamate, a methanesulfonate, an ethanesulfonate, a benzenesulfonate or a p-toluenesulfonate formed by a basic functional group in the structural formula with an acid.
  • the application provides linker-drug compounds.
  • the compounds may be configured to conjugate with an antibody to provide antibody-drug conjugates.
  • the linker-drug compound is represented by Formula III-A or Formula III-B, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof,
  • L2, L4, L5, X, R, Ri, R2, Ac, L3, m, R a , Rb are as defined therein in this disclosure; the chiral carbon atom at the position of 1, 2 or 3 has an absolute chirality of R-configuration or S- configuration.
  • the linker-drug compound is represented by Formula III-A or Formula III-B compirsing:
  • o is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; in one embodiment, o isl, 2, 3, 4, 5, 6, 7 or 8.
  • the pharmaceutically acceptable salt of the linker-drug compound represented by Formula III-A or Formula III-B is a salt formed by an acidic functional group in the structural formula with sodium, potassium, calcium or magnesium, or an acetate, a trifluoroacetate, a citrate, an oxalate, a tartrate, a malate, a nitrate, a chloride, a bromide, an iodide,
  • the ligand-camptothecin derivative conjugate represented by Formula I comprises a camptothecin derivative having a structure represented by Formula d:
  • the compound represented by Formula d comprises:
  • the application provides methods of preparing, manufacturing or making the antibody-drug conjugates.
  • the antibody-drug conjugates disclosed herein are manufactured using the linker-drug compounds.
  • the linker-drug compounds may be represented by Formula III-A or Formula III-B, its stereoisomer or a
  • the antibody-drug conjugate may be represented by Formula I, its stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof.
  • the application provides compositions, formulations, or preparations comprising the antibody-drug conjugates or the linker-drug compounds.
  • the composition, formulations, or preparation may be a pharmaceutical composition, formulations, or preparation.
  • the composition, formulations, or preparation may, optionally, further include a pharmaceutically acceptable carrier.
  • the antibody-drug conjugate in the pharmaceutical composition, formulations, or preparations may be represented by Formula I, its stereoisomer or pharmaceutically acceptable salt or solvate thereof.
  • the composition, formulations, or preparations may be represented by Formula I, its stereoisomer or pharmaceutically acceptable salt or solvate thereof.
  • linker-drug compound in the composition may be represented by Formula III-A or Formula III-B, its stereoisomer thereof or pharmaceutically acceptable salt or solvate thereof.
  • the application provides the methods of using the antibody-drug
  • linker-drug compounds are used in manufacturing the antibody-drug conjugates.
  • the antibody-drug conjugates are used in formulating or manufacturing the pharmaceutical composition or a medicament for treating or preventing a disease.
  • the disease is a cancer or tumor.
  • the cancer or tumor expresses DLL3.
  • the cancer or tumor is selected from solid tumor and hematologic tumor.
  • Example cancer or tumor include without limitation adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urinary tract cancer, bladder cancer,
  • breast cancer 10 liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, breast cancer (e.g.,), rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma or leukemia.
  • the breast cancer may be triple-negative breast cancer.
  • the application provides methods or processes for making the antibodydrug conjugates or the pharmaceutical compositions, preparations or formulations comprising the antibody-drug conjugates thereof.
  • the antibody-drug conjugate comprises a ligand-camptothecin-derivative conjugate represented by Formula I or Formula II, its stereoisomer or pharmaceutically acceptable salt or solvate thereof.
  • the method comprise
  • C1-C6 alkyl and the “C1-C6 alkyl” in various complex groups involving “C1-C6 alkyl” may be replaced with “C1-C20 alkyl”, “C1-C12 alkyl” or “C1-C10 alkyl”; the “C3-C8 cycloalkyl” and the “C3-C8 cycloalkyl” in various complex groups involving
  • C3-C8 cycloalkyl may be replaced with “C3-C20 cycloalkyl” or “C3-C10 cycloalkyl”; the “C1-C6 alkoxy” and the “C1-C6 alkoxy” in various complex groups involving “C1-C6 alkoxy” may be replaced with “C1-C20 alkoxy”, “C1-C12 alkoxy” or “C1-C10 alkoxy”; the “C6-C10 aryl” and the “C6-C10 aryl” in various complex groups involving “C6-C10 aryl” may be replaced with “C6-C12 aryl”; the “3- to 7-membered heterocyclyl” and the “3- to 7-membered heterocyclyl” in various complex groups involving “3- to 7-membered heterocyclyl” may be replaced with a “3- to 20- membered heterocyclyl”, a “3- to 12-membere
  • the antibody-drug conjugates disclosed herein formed by conjugating a humanized anti-human DLL3 antibody with a camptothecin derivative, exhibit significantly enhanced molecular stability and improved preclinical therapeutic effects. They are expected to demonstrate superior clinical efficacy, offering substantial development value and therapeutic potential.
  • FIG. 1 shows a chromatogram of ADC-6 aggregation detected by SEC-HPLC
  • FIG. 2 shows a chromatogram of ADC-6 drug-antibody ratios (DAR) detected by RP-HPLC;
  • FIG. 3 shows in vitro tumor inhibitory activity of ADC-6 in an experimental model of the humanized cell strain NCI-H446 DLLS #B5C;
  • FIG. 4 shows in vitro tumor inhibitory activity of ADC-6 in an experimental model of the humanized cell strain A375 DLLS #B2G;
  • FIG. 5 shows in vitro tumor inhibitory activity of ADC-6 in an experimental model of the humanized cell strain NCI-H2286 DLLS #B6G;
  • FIG. 6 shows in vitro tumor inhibitory activity of ADC-6 in an experimental model of the humanized cell strain NCI-H1339 DLLS #C3G;
  • FIG. 7 shows in vitro tumor inhibitory activity of ADC-6 in an experimental model of the humanized cell strain SHP-77 Wild Type
  • FIG. 8 shows in vitro tumor inhibitory activity of ADC-6 in an experimental model of the humanized cell strain NCI-H460;
  • FIG. 9 shows in vitro tumor inhibitory activity of ADC-6, ADC- 107 and ADC- 108 in an experimental model of the humanized cell strain A375 DLLS #B2G;
  • FIG. 10 shows in vitro tumor inhibitory activity of ADC-6, ADC- 107 and ADC- 108 in an experimental model of the humanized cell strain NCI-H1339 DLLS ti-CSG;
  • FIG. 11 shows in vitro tumor inhibitory activity of ADC-6, ADC-107 and ADC-108 in an experimental model of the humanized cell strain NCI-H2286 DLLS #B6G;
  • FIG. 12 shows in vitro tumor inhibitory activity of ADC-6, ADC-107 and ADC-108 in an experimental model of the humanized cell strain SHP-77 Wild Type;
  • FIG. 13 shows in vitro tumor inhibitory activity of ADC-6, ADC- 107 and ADC- 108 in an experimental model of the humanized cell strain NCI-H460;
  • FIG. 14 shows in vivo efficacy test results of ADC-6 in a single tumor model of the SHP-77- DLL3 #B7G;
  • FIG. 15 shows in vivo efficacy test results of ADC-6 in a single tumor model of the A375- DLL3 #B2G;
  • FIG. 16 shows in vivo efficacy test results of ADC-6 in a single tumor model of the H446- DLL3 #B5C.
  • FIG. 17 shows in vivo efficacy test results of ADC-6, ADC- 107 and ADC- 108 in a single tumor model of the A375-DLL3 #B2G.
  • This disclosure is generally drawn, inter alia, to antibody-drug conjugates, linker-drug compounds, compositions comprising antibody-drug conjugates or linker-drug compounds, pharmaceutical preparations or formulations, methods of making and methods of using the conjugates, compounds, or pharmaceutical compositions as disclosed herein.
  • brand names are used herein, the brand names, unless otherwise indicated in the context, include the product formulations, the general drugs and the active ingredients of products with the brand names.
  • ligand is a macromolecular compound capable of identifying and binding to an antigen or receptor associated with a target cell.
  • the ligand functions to present a drug to a target cell population binding to the ligand, including, but not being limited to, protein hormone, lectin, growth factor, antibody, or other molecules capable of binding to cells.
  • the ligand, expressed as Ab may form a linking bond with a linking unit through a heteroatom in the ligand, preferably is an antibody or an antigen-binding fragment thereof, which is selected from the group consisting of a chimeric antibody, a humanized antibody, fully human antibody and a mouse antibody, preferably a monoclonal antibody.
  • the ligand unit is a targeting agent that specifically binds to a target.
  • the ligand can specifically bind to a cell component or to other target molecules of interest.
  • the target part or the target is usually on the surface of the cell.
  • the ligand unit functions to deliver a drug unit to a specific target cell population interacting with the ligand unit.
  • the ligands include, but are not limited to, proteins, polypeptides and peptides, as well as non-proteins such as sugars.
  • Suitable ligand units include, for example, antibodies such as full-length (intact) antibodies and antigen-binding fragments thereof.
  • the ligand unit is a non-antibody targeting agent
  • the ligand may be a peptide or a polypeptide, or a non-proteinaceous molecule.
  • targeting agents include interferons, lymphokines, hormones, growth factors and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell binding
  • a linker is covalently linked to the sulfur atom of
  • the sulfur atom is a sulfur atom of a cysteine residue, which forms an interchain disulfide bond of the antibody.
  • the sulfur atom is a sulfur atom of a cysteine residue which has been introduced into the ligand unit, which forms an interchain disulfide bond of the antibody.
  • the sulfur atom is a sulfur atom of a cysteine residue that has been introduced into the ligand unit e.g., by site-directed mutagenesis or chemical reaction.
  • the sulfur atom linked to the linker is selected from a cysteine residue that forms an interchain disulfide bond of the antibody and a cysteine residue that has been introduced into the ligand unit (e.g., by site-directed mutagenesis or chemical reaction).
  • numbering is performed according to the EU index numbering system in Kabat
  • the “antibody” or “antibody unit” includes within the scope thereof any one part of an antibody structure. This unit may bind, reactively associate, or complex with a receptor, an antigen or other receptor units possessed by the targeted cell population.
  • the antibody may be any protein or proteinaceous molecule, which can bind, complex, or react with a part of the cell population to be treated or to be biologically modified.
  • the antibody constituting the antibody-drug conjugate in the application retain its antigen-binding ability in the originally wild state.
  • the antibody of the application can specifically bind to an antigen.
  • Involved antigens include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules,
  • the tumor-associated factor may be a cluster of differentiation factor (e.g., a CD protein).
  • Antibodies used in the antibody drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the art and can be prepared by well-known methods and information for preparing antibodies in the art. To develop effective cell-level targets for cancer diagnosis and treatment,
  • tumor-associated polypeptides can be specifically expressed on the surface of one or more cancer cells, but less expressed or not expressed on the surface of one or more non-cancer cells. In general, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells than on the surface of noncancer cells.
  • the identification of the tumor-associated factors can greatly improve the specific targeting characteristic of the antibody-based cancer treatment.
  • information related to antigens well known in the art is indicated below, including name, other names, and GenBank accession numbers.
  • Nucleic acid and protein sequences correspondent to the tumor- associated antigens can refer to public databases, such as Genbank.
  • the tumor-associated antigens to which the antibody targets include all variants and congeners of amino acid sequences, having
  • inhibitor or “inhibition of’ refers to reducing detectable amount, or completely preventing.
  • cancer refers to the physiological condition or disease characterized by unregulated cell growth.
  • a “tumor” comprises cancerous cells.
  • autoimmune disease is a disease or disorder derived from tissues or proteins of an individual itself.
  • drug refers to a cytotoxic drug, expressed as “d”, being a chemical molecule
  • the cytotoxic drug can kill tumor cells in principle at a sufficiently high concentration, but due to a lack of the specificity, they will lead to the apoptosis of normal cells while killing tumor cells, resulting in serious side effects.
  • toxins such as small molecule toxins or enzymatically active toxins which are derived from bacterium, fungus, plant or animal, radioisotopes (e.g., radioactive isotopes of At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and Lu 176 ), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs.
  • radioisotopes e.g., radioactive isotopes of At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and Lu 176
  • toxic drugs chemotherapeutic drugs
  • antibiotics and nucleolytic enzymes preferably toxic drugs.
  • linker or “linking fragment” or “linking unit” refers to a chemical structural fragment or bond of which one end is linked to a ligand and the other end is linked to a drug directly or by other linkers.
  • the linker including an extender, a spacer and an amino acid unit, can be synthesized by methods known in the art, for example those described in US2005-0238649A1.
  • the linker may be a “cleavable linker” that facilitates the release of drugs in cells.
  • acid-labile linkers e.g., hydrazones
  • protease sensitive linkers e.g., peptidase-sensitive linkers
  • photo-labile linkers e.g., dimethyl linkers, or disulfide-containing linkers
  • disulfide-containing linkers can be used (Chari et al, Cancer Research 52: 127- 131, 1992; U.S. patent No.5, 208, 020.
  • the “linker” or the “linker of an antibody-drug conjugate” can be classified into two classes: non-cleavable linkers and cleavable linkers, according to the mechanisms of drug release in cells.
  • the antibody-drug conjugate containing a non-cleavable linker its drug release mechanism is that after the conjugate binds to an antigen and endocytosed by cells, the
  • the antibody-drug conjugate containing a cleavable linker its drug release mechanism is that after the conjugate binds to an antigen and endocytosed by cells, it is cleaved in the target cells and releases the active ingredient (the small molecule drug itself).
  • the cleavable linkers are mainly divided into: chemosensitive linkers and enzyme sensitive linkers. The chemosensitive linkers can be selectively cleaved due to
  • the pH sensitive linkers such as hydrazones, carbonates, acetals, and ketals, are relatively stable in neutral or weakly alkaline environment of blood (pH 7.3-7.5), but will be hydrolyzed in weakly acidic tumor microenvironment (pH of 5.0-6.5) and in lysosomes (pH of 4.5-5.0). Since the acid-cleavable linkers have a very limited plasma stability, the antibodydrug conjugates based on such linkers usually have a short half-life (2-3 days). This short half-life limits the use of the pH sensitive linkers in the new generation of antibody-drug conjugates to some extent.
  • Linkers sensitive to glutathione are also called as disulfide linkers. The release of drug is caused based on the difference between high glutathione concentrations (millimolar range) in cells and relatively low glutathione concentrations in blood (micromolar range). This is
  • the disulfide bond has a thermodynamical stability, and thus it can have a good stability in plasma.
  • the enzyme labile linkers e.g., a peptide linker
  • the peptide linkers can be efficiently cleaved by a protease in the lysosomes, e.g., cathepsin B. This peptide linkage is considered to be very stable in the plasma circulation due to the unsuitable extracellular pH value and the extracellular inactivity of protease caused by serum protease inhibitors.
  • the enzyme labile linkers are widely used as cleavable linkers for antibody-drug conjugates.
  • antibody-drug conjugate refers to an conjugate comprising an antibody linked to
  • the “ligand-drug conjugate” is preferably an antibody drug conjugate (ADC), which refers to a conjugate comprising a monoclonal antibody or an antibody fragment linked to a biologically active toxic drug via a stable linking unit.
  • ADC antibody drug conjugate
  • alkyl refer to a saturated aliphatic hydrocarbon group, which may be a linear or branched group containing 1 to 20 carbon atoms (i.e., “C1-C20 alkyl”), preferably an alkyl group containing 1 to 12 carbon atoms (i.e., “Cl -Cl 2 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
  • C1-C6 alkyl 30 6 carbon atoms (i.e., “C1-C6 alkyl”).
  • alkyl group include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, sec-butyl, n-pentyl, 1,1 -dimethylpropyl,
  • n-heptyl 2-methylhexyl, 3 -methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3- dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 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,
  • the more preferred alkyl group is a lower alkyl containing 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, sec-butyl, n-pentyl, 1,1 -dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1- ethylpropyl, 2-methylbutyl, 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,
  • the alkyl may be substituted or non- substituted, and when it is substituted, it may be substituted at any useful linking sites, and the substituents are preferably one or more independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen,
  • substituted alkyl refers to an alkyl of which hydrogen is substituted with substituents.
  • C6-C10 aryl 20 C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted with 1 to 3 halogens, unsubstituted Cl-8 alkyl, Cl-8 alkoxy or Cl-8 thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)-Cl-4 alkyl.
  • R’ and R” are linked to the same one nitrogen atom, they together with the nitrogen atom may form a 3-, 4-, 5-, 6- or 7-memebered ring.
  • -NR’R includes 1 -pyrrolidyl and 4- morpholinyl.
  • alkylene refers to a linear or branched aliphatic hydrocarbon group, which has two residues derived from the removal of two hydrogen atoms on the same carbon atom or on two different carbon atoms of the parent alkyl, and which is a linear or branched group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, and more preferably an alkylene group containing 1 to 6 carbon atoms.
  • 30 alkylene include, but not are limited to, methylene (-CH 2 -, l,l-ethylidene(-CH(CH3)-), 1,2- ethylidene (-CH 2 CH 2 )-, 1,1-propylidene (-CH(CH 2 CH3)-), 1,2-propylidene (-CH 2 CH(CH3)-), 1,3- propylidene (-CH 2 CH 2 CH 2 -), 1,4-butylidene (-CH 2 CH 2 CH 2 CH 2 -) and 1,5- pentylidene (- CH 2 CH 2 CH 2 CH 2 CH 2 -) and the like.
  • the alkylene may be substituted or unsubstituted, and when it is substituted, it may be substituted at any useful linking sites, and the substituents are preferably one or more independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.
  • alkoxy refers to -O-(alkyl) and -O-(cycloalkyl), wherein alkyl or cycloalkyl are as defined above.
  • Non-limiting examples of the C1 -C6 alkoxy include methoxy, ethoxy, propoxy,
  • alkoxy may be
  • the subsituents are preferably one or more independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, and the ring of the cycloalkyl contains 3 to 20 carbon atoms (i.e., “C3- C20 cycloalkyl”), preferably 3 to 12 carbon atoms (i.e., “C3-C12 cycloalkyl”), more preferably 3 to 10 carbon atoms (i.e., “C3-C10 cycloalkyl”), and most preferably 3 to 8 carbon atoms (i.e., “C3- C8 cycloalkyl”).
  • the monocyclic cycloalkyl e.g., “C3-C8 cycloalkyl”
  • the polycyclic cycloalkyl include spiro, fused and bridged cycloalkyl.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, which comprises 3 to 20 ring atoms (i.e., “3- to 20- membered heterocyclyls”), wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and S(O) m (where m is an integer of 0 to 2), but not include a cyclic moiety of -O-O-, -O-
  • the heterocyclyl contains 3 to 12 ring atoms (i.e., “3- to 12-membered heterocyclyls”), of which 1 to 4 atoms are heteroatoms. More preferably, the heterocyclyl contains 3 to 10 ring atoms (i.e., “3- to 10-membered
  • heterocyclyls include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.
  • Polycyclic heterocyclyls include spiro, fused, and bridged heterocyclyls.
  • cycloalkylalkyl refers to an alkyl group substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein the alkyl group is as defined above, and wherein the cycloalkyl group is as defined above, for example, C3-C8 cycloalkyl C1-C6 alkyl.
  • haloalkyl refers to an alkyl group substituted with one or more halogens, wherein the alkyl group is as defined above, for example, halogenated C1 -C6 alkyl.
  • deuterated alkyl refers to an alkyl group substituted with one or more deuterium
  • alkyl group is as defined above, for example, deuterated C1-C6 alkyl.
  • C6-C12 aryl refers to the group of a carbocyclic aromatic system having 6 to 12 carbon atoms.
  • C6-C10 aryl refers to the group of a carbocyclic aromatic system having 6-10 carbon atoms, such as phenyl and naphthyl.
  • 5-10 membered heteroaryl refers to an aromatic heterocyclic ring, usually is a 5-,
  • 6-, 7-, 8-, 9-, or 10-membered heterocyclic ring having 1 to 3 heteroatoms selected from the group consisting of N, O and S; the ring of the heteroaryl may optionally be further fused or linked to aromatic and non-aromatic carbocyclic rings and heterocyclic rings.
  • Non-limiting examples of the 5- to 10-membered heteroaryls are, for example, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,
  • substituted C6-C10 aryl or “substituted 5- to 10-membered heteroaryl” or “substituted 3- to 7-membered heterocyclyl” refers to an aryl or heteroaryl or heterocyclyl of which hydrogen is substituted by substituents.
  • the substituents of the aryl or heteroaryl or heterocyclyl may be one or more groups selected from the following group consisting of: -halogen, -OR’, -NR’R ”, -SR’, -SiR’R”R’”, -OC(O)R’, -C(O)R’, -CO 2 R’, -
  • R’, R” and R’ each independently represent hydrogen, unsubstituted Cl-8 alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted with 1 to 3 halogens, unsubstituted Cl-8 alkyl, Cl-8 alkoxy or Cl-8 thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)-Cl-4 alkyl.
  • R’ and R” are linked to the same one nitrogen atom, they together with the nitrogen atom may form a 3-, 4-, 5-, 6- or 7-memebered ring.
  • -NR’R includes 1 -pyrrolidyl and 4-morpholinyl.
  • hydroxyl refers to a -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH 2 .
  • nitro refers to -NO 2 .
  • amido refers to -C(O)N(alkyl) or -C(O)N(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.
  • carboxylate group refers to -C(O)O(alkyl) or -C(O)O(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.
  • the application also includes the compounds represented by Formula I in various deuterated forms. Each usable hydrogen atom linked to carbon atoms may be independently substituted with a deuterium atom. Those skilled in the art could synthesize the compounds represented by Formula I
  • deuterated starting materials can be used in the preparation of the compounds represented by Formula I in the deuterated form, or they can be synthesized with a deuterated reagent according to conventional techniques, and non-limiting examples of the deuterated reagent includes deuterated borane, trideuterated borane in tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane, and the like.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure formed by linking two identical heavy chains and two identical light chains via an interchain disulfide bond.
  • the amino acid composition and arrangement order in the heavy chain constant region of the immunoglobulin are different, and thus its antigenicity is also different. Accordingly,
  • the immunoglobulins can be classified into five classes, or called as the isotypes of
  • immunoglobulins namely IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being p, 8, y, a, and s chains, respectively.
  • the same class of Ig can be classified into different subclasses according to the differences in the amino acid composition of the hinge region and in the number and position of the heavy chain disulfide bonds, for example, IgG may be classified into IgGl, IgG2, IgG3 and IgG4.
  • the light chains are classified into K chains and X chains according to the differences in the constant regions.
  • Each class of Ig in the five classes of Ig may have either K chains or X chains.
  • the antibody of the application is preferably a specific antibody against cell surface antigens on target cells, preferably an anti-DLL-3 antibody.
  • solvate or “solvate compound” refer to a pharmaceutically acceptable solvate
  • Non-limiting examples of the solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.
  • drug loading rate refers to the average amount of cytotoxic drug loaded per antibody in Formula I, and it can also be expressed by a ratio of drug to antibody, and the drug loading rate can range from 0 to 12, preferably from 1 to 10, cytotoxic drugs (d) linked to per antibody (Ab).
  • the drug loading rate is expressed as n, which illustratively is a mean value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the average amount of drugs per ADC molecule after the conjugation reaction can be identified by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA test and HPLC characterization.
  • the cytotoxic drug is conjugated to a thiol group (-SH) of cysteine opened
  • the number of the drug molecules that can be conjugated to the antibody in the conjugation reaction will be less than or equal to the theoretical maximum.
  • the loading of the ligand cytotoxic drug conjugate can be controlled by the methods, including, without limitation, the following:
  • pharmaceutically acceptable salt or “pharmaceutically usable salt” refers to a salt
  • the ligand-drug conjugate compound has at least one carboxyl group and thus can form a salt with a base.
  • the pharmaceutically acceptable salts include without limitation sodium, potassium, calcium or magnesium salts, and the like.
  • the ligand-drug conjugate disclosed herein has at least one amino group, and thus it can form a salt with an acid.
  • Examples of the pharmaceutically acceptable salts include without limitation hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, biphosphate, dihydric phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, and p-toluenesulphonate.
  • amino acid refers to an amino acid having an isoelectric point of less than
  • the acidic amino acid molecule often has one or more acidic groups such as carboxyl group, which can be effectively ionized into a negative ion form in the structure to increase the hydrophilicity.
  • the acidic amino acid may be a natural amino acid or an unnatural amino acid.
  • natural amino acid refers to an amino acid synthesized in a living organism.
  • the natural amino acids are generally in a L-form, with a few exceptions, e.g., glycine, including natural and biosynthesized.
  • unnatural amino acid refers to an amino acid obtained by synthetic means.
  • reaction solution was cooled to under 50 °C and the solvent was removed under a reduced pressure.
  • the residue was dissolved with ethyl acetate (150 mL) and water (40 mL), and the obtained solution was adjusted with IM HC1 to have a pH of 2 to 3 under stirring in an ice bath, and separated.
  • IM HC1 IM HC1
  • the aqueous layer was additionally extracted with ethyl acetate once, and the organic layers are combined and dried under anhydrous sodium sulfate.
  • reaction solution 20 mmol was added in an ice bath, and continuously stirred for 30 min, the above obtained reaction solution was added. The resulting mixture was warmed up to room temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution
  • the resulting mixture was naturally warmed up to room temperature and reacted (for about 2 to 4 h), and monitored by TLC.
  • a saturated NaHCOa solution was added to the reaction solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica gel
  • reaction solution (1) To a 50 mL single-neck flask, Compound 7a (5 g, 9.2 mmol) and 15 mL of DMF were added and dissolved, then DBU (1.68 g, 11 mmol) were added in an ice bath, and the resulting mixture was reacted for 1 h, the resulting reaction solution was expressed as a reaction solution (1).
  • reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution.
  • the preparation solution was extracted with di chloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium
  • reaction solution was extracted with dichloromethane, washed with
  • the filtrate containing Compound 13c was placed in an ice bath, to which DIPEA (333 uL, 1.93 mmol) was added, and then Compound M3 (1.1 g, 1.87 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution.
  • reaction solution was cooled to room temperature, and filtered to remove insoluble substances.
  • reaction solution (1) To a 50 mL single-neck flask, Compound 15a(5 g, 9.95 mmol) and 15 mL ofDMF were added and dissolved, then DBU (1.68 g, 11 mmol) was added in an ice bath, and then the resulting mixture was reacted for 1 h, the reaction solution was expressed as the reaction solution (1);
  • reaction solution (1) To a 50 mL single-neck flask, Compound 17a (5 g, 8.86 mmol) and 15 mL of DMF were added and dissolved, then DBU (1.53 g, 10 mmol) were added in an ice bath, and then the resulting mixture was reacted for 1 h, the reaction solution was expressed as the reaction solution (1).
  • the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC.
  • reaction solution (1) To a 50 mL single-neck flask, Compound 19a (4 g, 7.8 mmol) and 10 mL of DMF were added and dissolved, then DBU (1.42 g, 9.3 mmol) was added in an ice bath, and then the resulting mixture was reacted for 1 h, the reaction solution was expressed as the reaction solution (1).
  • reaction solution was cooled to room temperature, and filtered to remove insoluble substances.
  • the filtrate was concentrated to obtain a crude product, and the crude product was purified by
  • reaction solution (1) To a 50 mL single-neck flask, Compound 20a (4 g, 7.6 mmol) and 10 mL of DMF were added and dissolved, then the DBU(1.39 g, 9.1 mmol) was added in an ice bath, and then the resulting mixture was reacted for 1 h, the reaction solution was expressed as the reaction solution (1).
  • reaction solution (1) was added.
  • the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored by HPLC.
  • the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution.
  • reaction solution was filtered to remove insoluble substances.
  • reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution.
  • reaction solution was directly purified by preparative liquid chromatography, and the preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound 48a (1.54 g), with a yield of
  • Compound 5c was rreeppllaacceedd wwiitthh CCoommppoouunndd 55cc’’,, aanndd CCoommppoouunndd 55dd’’ was prepared by referring to the synthetic route of Example 8.

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Abstract

The application relates to, among others, an antibody-drug conjugate formed from an anti-human DLL3 antibody and a camptothecin derivative, and a medicinal use of the antibody-drug conjugate. The application further relates to a linker-drug conjugate, which can be conjugated to an antibody to form the antibody-drug conjugate. Pharmaceutical compositions, preparations, and formulations comprising the antibody-drug conjugates are provided.

Description

ANTI-HUMAN DLL3 ANTIBODY-CAMPTOTHECIN DERIVATIVE CONJUGATES AND MEDICAL USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
The application claims the benefit of priority to Chinese Application No. 202410529362.3, filed on April 29, 2024, the disclosures of which are incorporated herein by reference in its entirety.
TECHNICAL FIELD
The application relates to the biopharmaceutical field, and in particular, to antibody-drug
10 conjugates, linker-drug compounds, and methods of preparation and uses thereof.
BACKGROUND
Unless otherwise indicated herein, the materials described in this section are not prior art to the claims in this application and are not admitted being prior art by inclusion in this section.
Delta-Like Ligand 3 (or abbreviated as “DLL3”) is a transmembrane protein which is encoded by DLL3 gene and attached to cell surfaces, and it is one member of the Notch Ligand Family.
The Notch Ligand Family is a transmembrane protein family, and the Notch Ligand Family comprises members such as Delta-like Ligand 1 (DLL1), DLL3, DLL4, Jaggedl (JAG1) and Jagged2 (JAG 2). These ligands interact with Notch proteins, to trigger the activation of the Notch
20 signaling pathway. The Notch signaling pathway is a highly conservative intercellular communication system that plays an important role in multiple development processes and tissue maintenances. The activation of the Notch signaling pathway is dependent on normal cleavage and ligand binding of the Notch proteins. Upon ligand binding, the Notch protein undergoes cleavages to release the Notch intercellular domain (NICD), and the NICD further enters nucleus to participate in gene transcription regulations. The Notch signaling pathway plays important roles in processes such as embryonic developments, organ developments, cell proliferations and differentiations. Abnormal activities of the Notch signaling pathway are associated with the occurrence and progression of a variety of diseases, such as tumors, cardiovascular diseases, and neurological diseases.
30 The expression of DLL3 is found both in embryonic development and in certain mature tissues. Studies have shown that the DLL3 plays an important role in the development of the nervous system. It participates in the processes such as neuronal directional migration, axon guidance, and branching of dendrite branching. In addition, the overexpression of DLL3 is also observed in cancers such as Small Cell Lung Cancer (SCLC), prostate cancer, pancreatic cancer, and esophageal squamous cell carcinoma. The DLL3 is highly expressed in SCLC cells, and the expression level is relevant to the invasiveness, metastatic ability, and treatment resistance of tumors.
Currently, antibody drugs targeting DLL3 mainly include Rovalpituzumab tesirine (Rova-T) from Stemcentrx Inc and AMG-757 from Amgen. Studies of Rova-T on SCLC indications have
40 progressed to phase III clinical trials and it is eventually declared that the trials are in failure.
1 AMG-757 is a bi specific antibody developed by Amgen using a new generation bi specific antibody platform, HLE BiTE, and is currently in phase III clinical trials. The trispecific antibody HPN-328 from Harpoon Therapeutics is currently in phase II clinical trials. In addition, clinical trials on DLLS include CAR-T therapy AMG-119 from Amgen, CAR-T therapy LB-2102 from Nanjing Legend Biotech, bispecific antibody OBT620 from Boehringer Ingelheim Gmbh/Oxford Biotherapeutics Ltd, monoclonal antibody 89Zr-DFO-SC16.56 from Memorial Sloan Kettering Cancer Center, bispecific antibody QLS-31904 from QILU PHARMACEUTICAL, bispecific antibody BL764532 from Boehringer-Ingelheim, and bispecific antibody PT-217 from Phanes Therapeutics Inc, all of which are in the clinical phase I stage.
10 Antibody-drug conjugates (ADCs) are biological drugs that link antibodies or antibody fragments to small molecular drugs with biological activity through stable chemical linkers, which not only make the best of the high specificity of antibody-antigen binding and the high lethality of cytotoxins, but also effectively avoid shortcomings such as weak therapeutic effects of antibodies, and indiscriminate attacks of small molecular drugs.
Mylotarg is the first marketed ADC in worldwide, which was approved by the FDA in a speed to market in May 2000 for the treatment of patients aged 60 years and older with CD33 -positive Acute Myeloid Leukemia (AML) who have experienced a relapse for the first time and are not suitable candidates for cytotoxic chemotherapy. Later studies found that the treatment with Mylotarg resulted in severe fatal liver injury, and then Pfizer chose to withdraw from the market
20 voluntarily in June 2010. Subsequently, in 2017, Mylotarg was approved by the FDA in a lower recommended dose for the treatment of children and adults aged 1 month and older with newly diagnosed CD33-positive AML, and for the treatment of children and adults aged 2 years and older with CD33-positive AML who have experienced a relapse or who is refractory. The recently approved ELAHERE is the first ADC drug targeting folate receptor alpha (FR-a) approved by FDA, for the treatment of patients with cervical cancer who have experienced a relapse or metastasis.
Therefore, there is a need for new ADCs with better targeting specificity, better therapeutic efficacies, broader treatment applications, or less side effect.
SUMMARY
30 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 application discloses, among others, antibody-drug conjugates (ADCs) such as ligand- camptothecin derivative conjugates, drug-linker compounds, pharmaceutical compositions or preparations comprising ADCs, compositions comprising drug-linker compounds, methods of making ADCs using drug-linker compounds, methods of using ADCs for treating diseases such as cancer or tumors.
In one aspect, the application discloses ligand-camptothecin derivative conjugates or its
40 derivatives thereof. In one embodiment, the ligand-camptothecin derivative conjugate is
2 represented by Formula I, a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof, wherein:
Ab is an antibody targeting human DLLS or an antigen-binding fragment thereof;
Ln, L12 and LB are each independently selected from the group consisting of:
10 Li has the structure of Formula A:
Y-AC
Formula A wherein Vis a skeleton selected from the group consisting of C 1 -C6 alkylene, substituted C 1 - C6 alkylene and C3-C8 cycloalkylene; Ac is a hydrophilic structural unit; the carbon atom at the
3 position of 2 linked to Y has an absolute chirality of R-configuration or S-configuration;
L3 is present or absent, and when present, La is selected from a PEG hydrophilic unit
0 , wherein o is an integer selected from 1 to 10,
L4 is an enzyme digestible or cleavable unit;
L5 is a linking unit;
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 -C(O)-CRaRb-(CR3R4)m-O-, wherein
Ra and Rb each independently are selected from the group consisting of hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8
10 cycloalkyl, C3-C8 cycloalkyl C1 -C6 alkyl, C6-C10 aryl C1-C6 alkyl, C1 -C6 alkoxy C1 -C6 alkyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl; or
Ra, Rb and carbon atoms linked thereto form C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, 3- to 7-membered heterocyclyl or substituted 3- to 7-membered heterocyclyl;
R3, R4 are each independently hydrogen atom, deuterium atom, halogen, 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, 3- to 7-membered heterocyclyl, substituted 3- to 7- membered heterocyclyl; or
20 R3, R4 and carbon atoms linked thereto form C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, 3- to 7-membered heterocyclyl or substituted 3- to 7-membered heterocyclyl; m is 0, 1, 2, 3 or 4; the carbon atom at the position of 1 linked to N has an absolute chirality of R- or S- configuration;
R is 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- to 10-membered heteroaryl, or substituted 5- to 10-membered heteroaryl;
Ri is hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted C1-C6 alkyl,
30 deuterated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, carboxyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, or substituted 5- to 10-membered heteroaryl;
R2 is 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, carboxyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, or substituted 5- to 10-membered heteroaryl; nl , n2 and n3 are each independently an integer from 0 to 10 or a decimal between 0 to 10, wherein nl, n2 and n3 are not 0 simultaneously, and l<nl+n2+n3<10.
4 In some embodiments, Ln, L12 and L13 are each independently:
In some embodiments, Ln, L12 and L13 are each independently: o 0 o H H b , "'COOH and COOH
In some embodiments, Ac has a structure of Formula B, wherein:
Z is carboxyl, phosphoryloxy or -(OCFLCH^iOCFL, wherein i is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
10 In one embodiment, i is 1, 2, 3, 4, 5, 6, 7 or 8;
Y’ is a structural unit linking amino group and Z; in one embodiment, Y is C1-C6 alkylene or carboxyl-substituted C1-C6 alkylene,; in one embodiment, Y is methylene, ethylidene, carboxylsubstituted methylene or carboxyl-substituted ethylidene; in one embodiment, Y is methylene, ethylidene, or carboxyl-substituted methylene.
In some embodiments, Ac is a residue formed by removing one hydrogen atom from the amino terminus of an amino acid. In one embodiment, the amino acid may be glycine, (D/L) alanine, (D/L) leucine, (DL) 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, (DL) methionine, (D/L) asparagine, (D/L) glutamine, (D/L) threonine, (DL) aspartic
20 acid or (D/L) glutamic acid.
In some embodiments, Ac is: o. OH
H
.o.
In some embodiments, Ac is:
In some embodiments, L4 is a peptide residue composed of amino acids, wherein the amino acids are optionally substituted by one or more substituents selected from the group consisting of
5 deuterium atom, halogen, hydroxyl, cyano, amino, nitro, carboxyl, C1 -C6 alkyl, substituted Cl - C6 alkyl, C1-C6 alkoxy and C3-C8 cycloalkyl and substituted C3-C8 cycloalkyl.
In some embodiments, the peptide residue is a peptide residue formed by one, two or more amino acids. In some embodiments, the amino acids may be phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline (C), serine (S), glutamic acid (E) and aspartic acid (D).
In some embodiments, the peptide residue is a tetrapeptide residue, - G-G-F-G-, derived from glycine (G) -glycine (G) - phenylalanine (F) - glycine (G).
In some embodiments, Ls is -NRsCCReR?^- or a chemical bond, wherein q is 0, 1, 2, 3, 4, 5 or 6;
10 Rs, Re and R? are each independently hydrogen atom, deuterium atom, halogen, C1-C6 alkyl, substituted Cl -C 6 alkyl, deuterated C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, or substituted 5- to 10-membered heteroaryl.
In some embodiments, q is 0, 1, 2 or 3. In some embodiments, q is 0, 1 or 2. In one embodiment, q is 0 or 1.
In some embodiments, Rs, Re and R? are each independently hydrogen atom or C1-C6 alkyl. In some embodiments, Rs, Re and R? are each independently hydrogen atom and C1-C4 alkyl. In some embodiments, Rs, Re and R? are each independently selected from the group consisting of
20 hydrogen atom, methyl, ethyl, n-propyl and n-butyl. In some embodiments, Rs, Re and R? are each independently hydrogen atom.
In some embodiments, Ra and Rb are each independently hydrogen atom, C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl or C6-C10 aryl C1-C6 alkyl; or, in one embodiment, Ra, Rb and carbon atoms linked thereto form C3-C8 cycloalkyl.
In some embodiments, Ra is hydrogen atom or C1-C4 alkyl, Rb is hydrogen atom, C1-C4 alkyl, halogenated C1-C4 alkyl, C3-C6 cycloalkyl C1-C4 alkyl or phenyl C1-C4 alkyl; or, in one embodiment, Ra, Rb and carbon atoms linked thereto form C3-C6 cycloalkyl.
In some embodiments, Ra is hydrogen atom, methyl, ethyl, n-propyl or n-butyl, Rb is hydrogen atom, methyl, ethyl, n-propyl, n-butyl, halogenated methyl, halogenated ethyl, halogenated n-
30 propyl, halogenated n-butyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclopropylethyl, cyclopentylethyl, cyclohexylethyl, phenylmethyl, phenylethyl or phenylpropyl; or, in one embodiment, Ra, Rb and carbon atoms linked thereto form cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
In some embodiments, Ra is hydrogen atom or methyl, Rb is hydrogen atom, methyl, ethyl, trifluoromethyl, cyclopropylmethyl, or phenylmethyl; or, in one embodiment, Ra, Rb and carbon atoms linked thereto form cyclopropyl, cyclobutyl or cyclopentyl.
In some embodiments,
6 some embodiments, the position shown by the right side wavy line is connected to Ls.
In some embodiments, R3, R4 are each independently hydrogen atom or C1-C6 alkyl. In some embodiments, R3, R4 are each independently hydrogen atom or C1-C4 alkyl. In some embodiments, R3, R4 are each independently hydrogen atom.
In some embodiments, m is 0, 1 or 2. In some embodiments, m is 0 or 1.
In some embodiments, R is hydrogen atom or C1-C6 alkyl. In some embodiments, R is hydrogen atom or C1-C4 alkyl. In some embodiments, Ris hydrogen atom, methyl, ethyl, n-propyl or n-butyl. In some embodiments, R is hydrogen atom or methyl.
10 In some embodiments, Ri is hydrogen atom or C1-C6 alkyl. In some embodiments, Ri is Cl- C6 alkyl. In some embodiments, Ri is C1-C4 alkyl. In some embodiments, Ri is methyl, ethyl, n- propyl or n-butyl. In some embodiments, Ri is methyl.
In some embodiments, R2 is hydrogen atom, halogen or C1-C6 alkyl. In some embodiments, R2 is hydrogen atom, halogen or C1-C4 alkyl. In some embodiments, R2 is halogen. In some embodiments, R2 is fluorine, chlorine, or bromine. In some embodiments, R2 is fluorine.
In some embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, o is 1, 2, 3, 4, 5, 6, 7 or 8.
In some embodiments, Y is C1-C6 alkylene. In some embodiments, Y is C1-C4 alkylene. In some embodiments, Y is methylene, ethylidene, propylidene or butylidene. In some embodiments,
20 Y is methylene.
In some embodiments, nl, n2 and n3 each independently are any integer of 0 to 8 or any decimal of 0 to 8, and nl, n2 and n3 are not 0 simultaneously, with I<nl+n2+n3<8. In some embodiments, 5<nl+n2+n3<10. In some embodiments, 6<nl+n2+n3<8. In some embodiments, 7<nl+n2+n3<8.
In some embodiments, the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises;
(a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs):
(i) VH CDR1, having a CDR1 sequence contained in the VH as set forth in SEQ ID NO: 1,
30 or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR1 sequence contained in the VH;
(ii) VH CDR2, having a CDR2 sequence contained in the VH as set forth in SEQ ID NO : 1, or having a sequence with substitution, deletion or addition of one or more amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR2 sequence contained in the VH; and
(iii) VH CDR3, having a CDR3 sequence contained in the VH as set forth in SEQ ID NO: 1, or having a sequence with substitution, deletion or addition of one or more amino acids (e.g.,
7 substitution, deletion or addition of one amino acid) as compared with the CDR3 sequence contained in the VH; and/or
(b) the following three light chain variable region (VL) CDRs;
(iv) VL CDR1, having a CDR1 sequence contained in the VL as set forth in SEQ ID NO:7, or having a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR1 sequence contained in the VL;
(v) VL CDR2, having a CDR2 sequence contained in the VL as set forth in SEQ ID NO: 7,
10 or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR2 sequence contained in the VL; and
(vi) VL CDR3, having a CDR3 sequence contained in the VL as set forth in SEQ ID NO: 7, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR3 sequence contained in the VL.
In some embodiments, the substitution described in any one of (i) to (vi) is a conservative substitution.
In some embodiments, the CDR1, CDR2 and CDR3 contained in the heavy chain variable
20 region (VH), and/or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by Rabat, Chothia or IMGT numbering system.
In some embodiments, the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises:
CDR1, CDR2 and CDR3 sequences contained in the VH as set forth in SEQ ID NO: 1; and/or CDR1, CDR2 and CDR3 sequences contained in the VL as set forth in SEQ ID NO:7.
In some embodiments, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH) and/or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by Rabat, Chothia or IMGT numbering system.
In some embodiments, the antibody targeting human DLL3 or an antigen-binding fragment
30 thereof comprises:
(a) the following three heavy chain variable region (VH) CDR;
(i) VH CDR1, which is composed of a sequence as set forth in SEQ ID NO:2,
(ii) VH CDR2, which is composed of a sequence as set forth in SEQ ID NO:3, and
(iii) VH CDR3, which is composed of a sequence as set forth in SEQ ID NON; and/or
(b) the following three light chain variable region (VL) CDR:
(iv) VL CDR1, which is composed of a sequence as set forth in SEQ ID NO: 8,
(v) VL CDR2, which is composed of a sequence as set forth in SEQ ID NO:9, and
(vi) VL CDR3, which is composed of a sequence as set forth in SEQ ID NO: 10.
40 In some embodiments, the VH of the antibody targeting human DLL3 or an antigen-binding
8 fragment thereof comprises: VH CDR1 as set forth in SEQ ID NO:2; VH CDR2 as set forth in SEQ ID NO:3; and, VH CDR3 as set forth in SEQ ID NO:4; and/or, the VL of the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO:8; VL CDR2 as set forth in SEQ ID NO:9; and VL CDR3 as set forth in SEQ ID NO:10.
In some embodiments, the VH of the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: VH CDR1 as set forth in SEQ ID NO:2; VH CDR2 as set forth in SEQ ID NO:3; and VH CDR3 as set forth in SEQ ID NO:4; and the VL of the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID
10 NO: 8; VL CDR2 as set forth in SEQ ID NO: 9; and VL CDR3 as set forth in SEQ ID NO: 10.
In some embodiments, the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises:
(a) a heavy chain variable region (VH), comprising an amino acid sequence selected from the following sequences:
(i) a sequence as set forth in SEQ ID NO: 1;
(ii) a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence as set forth in SEQ ID NO:1;
(iii) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at
20 least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO: 1; and
(b) a light chain variable region (VL), comprising an amino acid sequence selected from the following sequences:
(iv) a sequence as set forth in SEQ ID NO:7;
(v) a sequence with substitution, deletion or addition of one or several amino acids(e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence as set forth in SEQ ID NO:7;
(vi) a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least
30 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO:7.
In some embodiments, the substitution described in (ii) or (v) comprises a conservative substitution.
In some embodiments, the antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region (VH), comprising a sequence as set forth in SEQ ID NO: 1 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO: 1, and a light chain variable region (VL), comprising a sequence as set forth in SEQ ID NO:7 or a sequence having a
40 sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least
9 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO:7.
In some embodiments, the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises; a VH having a sequence as set forth in SEQ ID NO: 1 and a VL with a sequence as set forth in SEQ ID NO: 7.
In some embodiments, the antibody targeting human DLL3 or an antigen-binding fragment thereof is humanized.
In some embodiments, the antibody targeting human DLL3 or an antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin.
10 In some embodiments, the antibody targeting human DLL3 or an antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline antibody gene), and/or, a light chain framework region of a human immunoglobulin (e.g., a light chain framework region contained in an amino acid sequence encoded by a human light chain germline antibody gene).
In some embodiments, the heavy chain framework region and/or the light chain framework region optionally comprises one or more (such as, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to murine residues.
In some embodiments, the antibody targeting human DLL3 or an antigen-binding fragment
20 thereof comprises: a heavy chain with a sequence as set forth in SEQ ID NO:5 and a light chain with a sequence as set forth in SEQ ID NO: 11.
In some embodiments, the antibody or the antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin.
In some embodiments, the heave chain of the antibody or the antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (such as IgGl, IgG2, IgG3 or IgG4).
In some embodiments, the light chain of the antibody or an antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (such as K or X).
In some embodiments, the antibody targeting human DLL3 or an antigen-binding fragment
30 thereof comprises: a heavy chain constant region having a sequence as set forth in SEQ ID NO:6 and a light chain constant region having a sequence as set forth in SEQ ID NO: 12.
In some embodiments, the antibody targeting human DLL3 or an antigen-binding fragment thereof are selected from the group consisting of monoclonal antibody, mouse antibody, rabbit antibody, humanized antibody, fully human antibody, chimeric antibody (e.g., human-mouse chimeric antibody), bispecific antibody, multi-specific antibody, single chain antibody, dAb, complementarity determining region fragment, Fv, single chain Fv (scfv), Fd, Fab, Fab', and F(ab')2.
In some embodiments, the monoclonal antibody includes a non-CDR region, and the non- CDR region is derived from species other than murine. In one embodiment, the non-CDR region
40 is derived from a human antibody.
10 In some embodiments, in Formula I, -L11-L2-L3-L4-L5-, -L12-L2-L3-L4-L5- and -L13-L2-L3-L4- Ls- are each independently selected from the group comprising: wherein: Ac, o, Rs, Re and R? are as defined herein in the disclosure; the carbon atom at the position of 2 linked to N has an absolute chirality of R-configuration or S-configuration; the position shown by the left side wavy line is connected to the antibody or an antigenbinding fragment thereof, and the position shown by the right side wavy line is connected to X.
In some embodiments, in Formula I, -L11-L2-L3-L4-L5-, -L12-L2-L3-L4-L5- and -L13-L2-L3-L4- Ls- each independently are selected from the group comprising:
11 wherein: Ac, 0, R5, Re and R? are as defined herein in this disclosure; the carbon atom at the position of 2 linked to N has an absolute chirality of R-configuration or S-configuration; the position shown by the left side wavy line is connected to the antibody or an antigenbinding fragment thereof, and the position shown by the right-side wavy line is connected to X.
In some embodiments, the conjugate of Formula I has a structure represented by Formula II: wherein: Ab, Ln, L12, L13, Ac, L3, X, R, Ri, R2, nl, n2, n3 are as defined in any one embodiment of the application; the chiral atom at the position of 1, 2 or 3 has an absolute chirality of R-configuration or S- configuration.
In some embodiments, the conjugate of Formula I is selected from the group consisting of:
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64 wherein: 18M1 represents Ab; Ab, nl, n2 and n3 as defined in any one embodiment of the application.
In some embodiments, the pharmaceutically acceptable salt of the conjugate represented by
65 Formula I is a salt formed by an acidic functional group in the structural formula with sodium, potassium, calcium or magnesium, or an acetate, a trifluoroacetate, a citrate, an oxalate, a tartrate, a malate, a nitrate, a chloride, a bromide, an iodide, a sulfate, a bisulfate, a phosphate, a lactate, an oleate, an ascorbate, a salicylate, a formate, a glutamate, a methanesulfonate, an ethanesulfonate, a benzenesulfonate or a p-toluenesulfonate formed by a basic functional group in the structural formula with an acid.
In another aspect, the application provides linker-drug compounds. In some emboidments, the compounds may be configured to conjugate with an antibody to provide antibody-drug conjugates.
10 In one embodiment, the linker-drug compound is represented by Formula III-A or Formula III-B, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof,
? H o R
H ? H H a Rb
AXO
O=K R X J .-I _ NN. N
L^L4"L3"L2"L N 'm |
H o H o titr — V, R-
Ac' N r 1
1 o. CH3
J J i*r F
R1 Q
R2 'OH o
III-A III-B
0
0 0 o^rA Br- Cl
H 'V N wherein L is 0 0 H H A o or o o 5
L2, L4, L5, X, R, Ri, R2, Ac, L3, m, Ra, Rb are as defined therein in this disclosure; the chiral carbon atom at the position of 1, 2 or 3 has an absolute chirality of R-configuration or S- configuration.
In some embodiments, the linker-drug compound is represented by Formula III-A or Formula III-B compirsing:
66
67
68
69
70 £Z
74
75 wherein: o is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; in one embodiment, o isl, 2, 3, 4, 5, 6, 7 or 8.
In some embodiments, the pharmaceutically acceptable salt of the linker-drug compound represented by Formula III-A or Formula III-B is a salt formed by an acidic functional group in the structural formula with sodium, potassium, calcium or magnesium, or an acetate, a trifluoroacetate, a citrate, an oxalate, a tartrate, a malate, a nitrate, a chloride, a bromide, an iodide,
10 a sulfate, a bisulfate, a phosphate, a lactate, an oleate, an ascorbate, a salicylate, a formate, a glutamate, a methanesulfonate, an ethanesulfonate, a benzenesulfonate or a p-toluenesulfonate formed by a basic functional group in the structural formula with an acid.
In some embodiments, the ligand-camptothecin derivative conjugate represented by Formula I comprises a camptothecin derivative having a structure represented by Formula d:
76 wherein R, Ri, R2, m, Ra, Rb are as disclosed herein in the disclosure.
In some embodiments, the compound represented by Formula d comprises:
77
In a further aspect, the application provides methods of preparing, manufacturing or making the antibody-drug conjugates. In one embodiment, the antibody-drug conjugates disclosed herein are manufactured using the linker-drug compounds. In one embodiment, the linker-drug compounds may be represented by Formula III-A or Formula III-B, its stereoisomer or a
10 pharmaceutically acceptable salt or solvate thereof. In one embodiment, the antibody-drug conjugate may be represented by Formula I, its stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof.
In a further aspect, the application provides compositions, formulations, or preparations comprising the antibody-drug conjugates or the linker-drug compounds. In one embodiment, the composition, formulations, or preparation may be a pharmaceutical composition, formulations, or preparation. The composition, formulations, or preparation may, optionally, further include a pharmaceutically acceptable carrier. In one embodiment, the antibody-drug conjugate in the pharmaceutical composition, formulations, or preparations may be represented by Formula I, its stereoisomer or pharmaceutically acceptable salt or solvate thereof. In one embodiment, the
20 linker-drug compound in the composition may be represented by Formula III-A or Formula III-B, its stereoisomer thereof or pharmaceutically acceptable salt or solvate thereof.
In a further aspect, the application provides the methods of using the antibody-drug
78 conjugates or the linker-drug compounds as disclosed herein. In one embodiment, the linker-drug compounds are used in manufacturing the antibody-drug conjugates.
In one embodiment, the antibody-drug conjugates are used in formulating or manufacturing the pharmaceutical composition or a medicament for treating or preventing a disease. In one embodiment, the disease is a cancer or tumor. In some embodiments, the cancer or tumor expresses DLL3.
In some embodiments, the cancer or tumor is selected from solid tumor and hematologic tumor. Example cancer or tumor include without limitation adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urinary tract cancer, bladder cancer,
10 liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, breast cancer (e.g.,), rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma or leukemia. In one embodiment, the breast cancer may be triple-negative breast cancer.
In a further aspect, the application provides methods or processes for making the antibodydrug conjugates or the pharmaceutical compositions, preparations or formulations comprising the antibody-drug conjugates thereof. In one embodiment, the antibody-drug conjugate comprises a ligand-camptothecin-derivative conjugate represented by Formula I or Formula II, its stereoisomer or pharmaceutically acceptable salt or solvate thereof. In one embodiment, the method comprise
20 the steps of: conjugating a reduced antibody or an antigen-binding fragment thereof with a linker-drug compound, to obtain the ligand-camptothecin derivative conjugate represented by Formula I or Formula II,
79 wherein the chiral carbon atom at the position of 1, 2 or 3 has an absolute chirality of R- configuration or S-configuration;
Ab, L, Ln, L12, LB, L2, L3, L4, L5, X, R, Ri, R2, nl, n2 or n3 are as described herein.
In the application and embodiments disclosed thereof, the “C1-C6 alkyl” and the “C1-C6 alkyl” in various complex groups involving “C1-C6 alkyl” (such as, “substituted C1-C6 alkyl”, or “deuterated C1-C6 alkyl”) may be replaced with “C1-C20 alkyl”, “C1-C12 alkyl” or “C1-C10 alkyl”; the “C3-C8 cycloalkyl” and the “C3-C8 cycloalkyl” in various complex groups involving
10 “C3-C8 cycloalkyl” may be replaced with “C3-C20 cycloalkyl” or “C3-C10 cycloalkyl”; the “C1-C6 alkoxy” and the “C1-C6 alkoxy” in various complex groups involving “C1-C6 alkoxy” may be replaced with “C1-C20 alkoxy”, “C1-C12 alkoxy” or “C1-C10 alkoxy”; the “C6-C10 aryl” and the “C6-C10 aryl” in various complex groups involving “C6-C10 aryl” may be replaced with “C6-C12 aryl”; the “3- to 7-membered heterocyclyl” and the “3- to 7-membered heterocyclyl” in various complex groups involving “3- to 7-membered heterocyclyl” may be replaced with a “3- to 20- membered heterocyclyl”, a “3- to 12-membered heterocyclyl” or a “3- to 10-membered heterocyclyl”.
Beneficial Effects
20 Compared to existing drugs of the same class, the antibody-drug conjugates disclosed herein, formed by conjugating a humanized anti-human DLL3 antibody with a camptothecin derivative, exhibit significantly enhanced molecular stability and improved preclinical therapeutic effects. They are expected to demonstrate superior clinical efficacy, offering substantial development value and therapeutic potential.
80 BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other features of this disclosure will 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 will be described with additional specificity and detail through use of the accompanying drawings, in which:
FIG. 1 shows a chromatogram of ADC-6 aggregation detected by SEC-HPLC;
FIG. 2 shows a chromatogram of ADC-6 drug-antibody ratios (DAR) detected by RP-HPLC;
10 FIG. 3 shows in vitro tumor inhibitory activity of ADC-6 in an experimental model of the humanized cell strain NCI-H446 DLLS #B5C;
FIG. 4 shows in vitro tumor inhibitory activity of ADC-6 in an experimental model of the humanized cell strain A375 DLLS #B2G;
FIG. 5 shows in vitro tumor inhibitory activity of ADC-6 in an experimental model of the humanized cell strain NCI-H2286 DLLS #B6G;
FIG. 6 shows in vitro tumor inhibitory activity of ADC-6 in an experimental model of the humanized cell strain NCI-H1339 DLLS #C3G;
FIG. 7 shows in vitro tumor inhibitory activity of ADC-6 in an experimental model of the humanized cell strain SHP-77 Wild Type;
20 FIG. 8 shows in vitro tumor inhibitory activity of ADC-6 in an experimental model of the humanized cell strain NCI-H460;
FIG. 9 shows in vitro tumor inhibitory activity of ADC-6, ADC- 107 and ADC- 108 in an experimental model of the humanized cell strain A375 DLLS #B2G;
FIG. 10 shows in vitro tumor inhibitory activity of ADC-6, ADC- 107 and ADC- 108 in an experimental model of the humanized cell strain NCI-H1339 DLLS ti-CSG;
FIG. 11 shows in vitro tumor inhibitory activity of ADC-6, ADC-107 and ADC-108 in an experimental model of the humanized cell strain NCI-H2286 DLLS #B6G;
FIG. 12 shows in vitro tumor inhibitory activity of ADC-6, ADC-107 and ADC-108 in an experimental model of the humanized cell strain SHP-77 Wild Type;
30 FIG. 13 shows in vitro tumor inhibitory activity of ADC-6, ADC- 107 and ADC- 108 in an experimental model of the humanized cell strain NCI-H460;
FIG. 14 shows in vivo efficacy test results of ADC-6 in a single tumor model of the SHP-77- DLL3 #B7G;
FIG. 15 shows in vivo efficacy test results of ADC-6 in a single tumor model of the A375- DLL3 #B2G;
FIG. 16 shows in vivo efficacy test results of ADC-6 in a single tumor model of the H446- DLL3 #B5C; and
FIG. 17 shows in vivo efficacy test results of ADC-6, ADC- 107 and ADC- 108 in a single tumor model of the A375-DLL3 #B2G.
40
81 DETAILED DESCRIPTION
In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated
10 herein.
This disclosure is generally drawn, inter alia, to antibody-drug conjugates, linker-drug compounds, compositions comprising antibody-drug conjugates or linker-drug compounds, pharmaceutical preparations or formulations, methods of making and methods of using the conjugates, compounds, or pharmaceutical compositions as disclosed herein.
Abbreviations and Definitions
Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings. When brand names are used herein, the brand names, unless otherwise indicated in the context, include the product formulations, the general drugs and the active ingredients of products with the brand names.
20 Unless otherwise indicated, the terms used in the claims and specification herein have the following meanings.
The term “ligand” is a macromolecular compound capable of identifying and binding to an antigen or receptor associated with a target cell. The ligand functions to present a drug to a target cell population binding to the ligand, including, but not being limited to, protein hormone, lectin, growth factor, antibody, or other molecules capable of binding to cells. In one embodiment, the ligand, expressed as Ab, may form a linking bond with a linking unit through a heteroatom in the ligand, preferably is an antibody or an antigen-binding fragment thereof, which is selected from the group consisting of a chimeric antibody, a humanized antibody, fully human antibody and a mouse antibody, preferably a monoclonal antibody.
30 The ligand unit is a targeting agent that specifically binds to a target. The ligand can specifically bind to a cell component or to other target molecules of interest. The target part or the target is usually on the surface of the cell. In some embodiments, the ligand unit functions to deliver a drug unit to a specific target cell population interacting with the ligand unit. The ligands include, but are not limited to, proteins, polypeptides and peptides, as well as non-proteins such as sugars. Suitable ligand units include, for example, antibodies such as full-length (intact) antibodies and antigen-binding fragments thereof. In embodiments where the ligand unit is a non-antibody targeting agent, the ligand may be a peptide or a polypeptide, or a non-proteinaceous molecule. Examples of such targeting agents include interferons, lymphokines, hormones, growth factors and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell binding
40 molecules or substances. In some embodiments, a linker is covalently linked to the sulfur atom of
82 the ligand. In one aspect, the sulfur atom is a sulfur atom of a cysteine residue, which forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine residue which has been introduced into the ligand unit, which 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 introduced into the ligand unit e.g., by site-directed mutagenesis or chemical reaction. In other aspects, the sulfur atom linked to the linker is selected from a cysteine residue that forms an interchain disulfide bond of the antibody and a cysteine residue that has been introduced into the ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In some embodiments, numbering is performed according to the EU index numbering system in Kabat
10 {[Kabat E.A et al, (1991)], Sequences of Proteins of Immunological Interest, fifth edition, NIH publication 91-3242}.
The “antibody” or “antibody unit” includes within the scope thereof any one part of an antibody structure. This unit may bind, reactively associate, or complex with a receptor, an antigen or other receptor units possessed by the targeted cell population. The antibody may be any protein or proteinaceous molecule, which can bind, complex, or react with a part of the cell population to be treated or to be biologically modified. The antibody constituting the antibody-drug conjugate in the application retain its antigen-binding ability in the originally wild state. Thus, the antibody of the application can specifically bind to an antigen. Involved antigens include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules,
20 cell survival regulatory factors, cell proliferation regulatory factors, molecules associated with tissue growth and differentiation (e.g., known or foreseen to be functional), lymphokines, cytokines, molecules involved in the regulation of cell circulation, molecules involved in angiogenesis, and molecules associated with angiogenesis (e.g., known or foreseen to be functional). The tumor-associated factor may be a cluster of differentiation factor (e.g., a CD protein).
Antibodies used in the antibody drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the art and can be prepared by well-known methods and information for preparing antibodies in the art. To develop effective cell-level targets for cancer diagnosis and treatment,
30 researchers seek to find transmembrane or other tumor-associated polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells, but less expressed or not expressed on the surface of one or more non-cancer cells. In general, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells than on the surface of noncancer cells. The identification of the tumor-associated factors can greatly improve the specific targeting characteristic of the antibody-based cancer treatment. For convenience, information related to antigens well known in the art is indicated below, including name, other names, and GenBank accession numbers. Nucleic acid and protein sequences correspondent to the tumor- associated antigens can refer to public databases, such as Genbank. The tumor-associated antigens to which the antibody targets include all variants and congeners of amino acid sequences, having
40 a homology of at least 70%, 80%, 85%, 90% or 95% to the sequences identified in the references,
83 or having biological properties and characteristics completely consistent to the sequences of the tumor-associated antigen in the references.
The term “inhibit” or “inhibition of’ refers to reducing detectable amount, or completely preventing.
The term “cancer” refers to the physiological condition or disease characterized by unregulated cell growth. A “tumor” comprises cancerous cells.
The term “autoimmune disease” is a disease or disorder derived from tissues or proteins of an individual itself.
The term “drug” refers to a cytotoxic drug, expressed as “d”, being a chemical molecule
10 which, in tumor cells, has a strong ability to destroy normal growth the tumor cells. The cytotoxic drug can kill tumor cells in principle at a sufficiently high concentration, but due to a lack of the specificity, they will lead to the apoptosis of normal cells while killing tumor cells, resulting in serious side effects. The term includes toxins such as small molecule toxins or enzymatically active toxins which are derived from bacterium, fungus, plant or animal, radioisotopes (e.g., radioactive isotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and Lu176), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs.
The term “linker” or “linking fragment” or “linking unit” refers to a chemical structural fragment or bond of which one end is linked to a ligand and the other end is linked to a drug directly or by other linkers.
20 The linker, including an extender, a spacer and an amino acid unit, can be synthesized by methods known in the art, for example those described in US2005-0238649A1. The linker may be a “cleavable linker” that facilitates the release of drugs in cells. For example, acid-labile linkers (e.g., hydrazones), protease sensitive (e.g., peptidase-sensitive) linkers, photo-labile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Research 52: 127- 131, 1992; U.S. patent No.5, 208, 020.
As used herein, the “linker” or the “linker of an antibody-drug conjugate” can be classified into two classes: non-cleavable linkers and cleavable linkers, according to the mechanisms of drug release in cells. As to the antibody-drug conjugate containing a non-cleavable linker, its drug release mechanism is that after the conjugate binds to an antigen and endocytosed by cells, the
30 antibody is enzymolyzed in lysosomes, to release active molecules consisting of a small-molecule drug, a linker and antibody amino acid residues together. The resulted structural changes in the drug molecular do not reduce its cytotoxicity, but because the active molecules are charged (amino acid residues), they cannot permeate into neighboring cells. Thus, such active drugs cannot kill neighboring tumor cells that do not express the target antigen (antigen negative cells) (bystander effect) (Ducry et al, 2010, Bioconjugate Chem. 21: 5-13). As to the antibody-drug conjugate containing a cleavable linker, its drug release mechanism is that after the conjugate binds to an antigen and endocytosed by cells, it is cleaved in the target cells and releases the active ingredient (the small molecule drug itself). The cleavable linkers are mainly divided into: chemosensitive linkers and enzyme sensitive linkers. The chemosensitive linkers can be selectively cleaved due to
40 differences in the nature of plasma and cytoplasmic or tumor microenvironment, such as pH, and
84 glutathione concentration. The pH sensitive linkers, such as hydrazones, carbonates, acetals, and ketals, are relatively stable in neutral or weakly alkaline environment of blood (pH 7.3-7.5), but will be hydrolyzed in weakly acidic tumor microenvironment (pH of 5.0-6.5) and in lysosomes (pH of 4.5-5.0). Since the acid-cleavable linkers have a very limited plasma stability, the antibodydrug conjugates based on such linkers usually have a short half-life (2-3 days). This short half-life limits the use of the pH sensitive linkers in the new generation of antibody-drug conjugates to some extent. Linkers sensitive to glutathione are also called as disulfide linkers. The release of drug is caused based on the difference between high glutathione concentrations (millimolar range) in cells and relatively low glutathione concentrations in blood (micromolar range). This is
10 especially true for a tumor cell, where its low oxygen content leads to an enhanced activity of the reductase, thereby resulting in a higher glutathione concentration. The disulfide bond has a thermodynamical stability, and thus it can have a good stability in plasma. The enzyme labile linkers, e.g., a peptide linker, can better control drug release. The peptide linkers can be efficiently cleaved by a protease in the lysosomes, e.g., cathepsin B. This peptide linkage is considered to be very stable in the plasma circulation due to the unsuitable extracellular pH value and the extracellular inactivity of protease caused by serum protease inhibitors. In view of a high plasma stability and good selectivity and efficiency of intracellular cleavage, the enzyme labile linkers are widely used as cleavable linkers for antibody-drug conjugates.
The term “antibody-drug conjugate” refers to an conjugate comprising an antibody linked to
20 a biologically active drug via a stable linking unit. In the application, the “ligand-drug conjugate” is preferably an antibody drug conjugate (ADC), which refers to a conjugate comprising a monoclonal antibody or an antibody fragment linked to a biologically active toxic drug via a stable linking unit.
Three-letter codes and one-letter codes for amino acids used in the application are as described in J. boil. Chem. 1968, 243, 3558.
The term “alkyl” refer to a saturated aliphatic hydrocarbon group, which may be a linear or branched group containing 1 to 20 carbon atoms (i.e., “C1-C20 alkyl”), preferably an alkyl group containing 1 to 12 carbon atoms (i.e., “Cl -Cl 2 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
30 6 carbon atoms (i.e., “C1-C6 alkyl”). Non-limiting examples of the alkyl group include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, sec-butyl, n-pentyl, 1,1 -dimethylpropyl,
1.2-dimethylpropyl, 2,2-dimethylpropyl, 1 -ethylpropyl, 2-methylbutyl, 3 -methylbutyl, n-hexyl,
1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1 -dimethylbutyl, 1,2-dimethylbutyl, 2,2- dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl,
2.3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3 -methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3- dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 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, 2-methyl-2-ethylhexyl, 2-methyl-3-
40 ethylhexyl, 2,2-diethylpentyl, n-decyl, 3, 3 -di ethyl hexyl, 2,2-diethylhexyl, and various branched
85 isomers and the like. The more preferred alkyl group is a lower alkyl containing 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, sec-butyl, n-pentyl, 1,1 -dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1- ethylpropyl, 2-methylbutyl, 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 may be substituted or non- substituted, and when it is substituted, it may be substituted at any useful linking sites, and the substituents are preferably one or more independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen,
10 sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.
The term “substituted alkyl” refers to an alkyl of which hydrogen is substituted with substituents. Unless otherwise indicated herein, the substituents of the alkyl may be one or more groups selected from the following 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, and the number of the substituents may be 0 to (2m’+l), wherein m’ is the total number of the carbon atoms in the group. R’, R” and R’” each independently represent hydrogen, unsubstituted Cl-8 alkyl, unsubstituted C6-C12 aryl (or
20 C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted with 1 to 3 halogens, unsubstituted Cl-8 alkyl, Cl-8 alkoxy or Cl-8 thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)-Cl-4 alkyl. When R’ and R” are linked to the same one nitrogen atom, they together with the nitrogen atom may form a 3-, 4-, 5-, 6- or 7-memebered ring. For example, -NR’R” includes 1 -pyrrolidyl and 4- morpholinyl.
The term “alkylene” refers to a linear or branched aliphatic hydrocarbon group, which has two residues derived from the removal of two hydrogen atoms on the same carbon atom or on two different carbon atoms of the parent alkyl, and which is a linear or branched group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, and more preferably an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of the
30 alkylene include, but not are limited to, methylene (-CH2-, l,l-ethylidene(-CH(CH3)-), 1,2- ethylidene (-CH2CH2)-, 1,1-propylidene (-CH(CH2CH3)-), 1,2-propylidene (-CH2CH(CH3)-), 1,3- propylidene (-CH2CH2CH2-), 1,4-butylidene (-CH2CH2CH2CH2-) and 1,5- pentylidene (- CH2CH2CH2CH2CH2-) and the like. The alkylene may be substituted or unsubstituted, and when it is substituted, it may be substituted at any useful linking sites, and the substituents are preferably one or more independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.
The term “alkoxy” refers to -O-(alkyl) and -O-(cycloalkyl), wherein alkyl or cycloalkyl are as defined above. Non-limiting examples of the C1 -C6 alkoxy include methoxy, ethoxy, propoxy,
40 butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy. The alkoxy may be
86 optionally substituted or unsubstituted, and when it is substituted, the subsituents are preferably one or more independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.
The term “cycloalkyl” refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, and the ring of the cycloalkyl contains 3 to 20 carbon atoms (i.e., “C3- C20 cycloalkyl”), preferably 3 to 12 carbon atoms (i.e., “C3-C12 cycloalkyl”), more preferably 3 to 10 carbon atoms (i.e., “C3-C10 cycloalkyl”), and most preferably 3 to 8 carbon atoms (i.e., “C3- C8 cycloalkyl”). Non-limiting examples of the monocyclic cycloalkyl (e.g., “C3-C8 cycloalkyl”)
10 include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclopentatrienyl, cyclooctyl and the like; the polycyclic cycloalkyl include spiro, fused and bridged cycloalkyl.
The term “heterocyclyl” refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, which comprises 3 to 20 ring atoms (i.e., “3- to 20- membered heterocyclyls”), wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and S(O)m (where m is an integer of 0 to 2), but not include a cyclic moiety of -O-O-, -O-
5-, or -S-S-, with the remaining ring atoms being carbon. Preferably, the heterocyclyl contains 3 to 12 ring atoms (i.e., “3- to 12-membered heterocyclyls”), of which 1 to 4 atoms are heteroatoms. More preferably, the heterocyclyl contains 3 to 10 ring atoms (i.e., “3- to 10-membered
20 heterocyclyls”). Non-limiting examples of monocyclic heterocyclyls (e.g., 3- to 7-membered heterocyclyls) include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyls include spiro, fused, and bridged heterocyclyls.
The term “cycloalkylalkyl" refers to an alkyl group substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein the alkyl group is as defined above, and wherein the cycloalkyl group is as defined above, for example, C3-C8 cycloalkyl C1-C6 alkyl.
The term “haloalkyl” refers to an alkyl group substituted with one or more halogens, wherein the alkyl group is as defined above, for example, halogenated C1 -C6 alkyl.
The term “deuterated alkyl” refers to an alkyl group substituted with one or more deuterium
30 atoms, wherein the alkyl group is as defined above, for example, deuterated C1-C6 alkyl.
The term “C6-C12 aryl” refers to the group of a carbocyclic aromatic system having 6 to 12 carbon atoms.
The term “C6-C10 aryl” refers to the group of a carbocyclic aromatic system having 6-10 carbon atoms, such as phenyl and naphthyl.
The term “5-10 membered heteroaryl” refers to an aromatic heterocyclic ring, usually is a 5-,
6-, 7-, 8-, 9-, or 10-membered heterocyclic ring having 1 to 3 heteroatoms selected from the group consisting of N, O and S; the ring of the heteroaryl may optionally be further fused or linked to aromatic and non-aromatic carbocyclic rings and heterocyclic rings. Non-limiting examples of the 5- to 10-membered heteroaryls are, for example, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,
40 indolyl, imidazolyl, thiazolyl, isothiazolyl, thiaoxazolyl, pyrrolyl, phenyl -pyrrolyl, furyl, phenyl-
87 furyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, benzofuranyl, benzothienyl, 1,3-benzodioxolyl, isoindolinyl, benzimidazolyl, indazolyl, quinolinyl, isoquinolinyl, 1,2,3-triazolyl, 1 -phenyl- 1,2,3 - triazolyl, 2,3-indolinyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, benzopyranyl, 2,3- dihydrobenzoxazinyl, and 2,3-dihydroquinoxalinyl.
The term “substituted C6-C10 aryl” or “substituted 5- to 10-membered heteroaryl” or “substituted 3- to 7-membered heterocyclyl” refers to an aryl or heteroaryl or heterocyclyl of which hydrogen is substituted by substituents. Unless otherwise indicated herein, the substituents of the aryl or heteroaryl or heterocyclyl may be one or more groups selected from the following group consisting of: -halogen, -OR’, -NR’R ”, -SR’, -SiR’R”R’”, -OC(O)R’, -C(O)R’, -CO2R’, -
10 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, and the number of the substituents may be 0 to (2m’+l), wherein m’ is the total number of the carbon atom in the group. R’, R” and R’” each independently represent hydrogen, unsubstituted Cl-8 alkyl, unsubstituted C6-C12 aryl (or C6-C10 aryl), C6-C12 aryl (or C6-C10 aryl) substituted with 1 to 3 halogens, unsubstituted Cl-8 alkyl, Cl-8 alkoxy or Cl-8 thioalkoxy, or unsubstituted C6-C12 aryl (or C6-C10 aryl)-Cl-4 alkyl. When R’ and R” are linked to the same one nitrogen atom, they together with the nitrogen atom may form a 3-, 4-, 5-, 6- or 7-memebered ring. For example, -NR’R” includes 1 -pyrrolidyl and 4-morpholinyl.
The term “hydroxyl” refers to a -OH group.
20 The term “halogen” refers to fluorine, chlorine, bromine or iodine.
The term “amino” refers to -NH2.
The term “nitro” refers to -NO2.
The term “amido” refers to -C(O)N(alkyl) or -C(O)N(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.
The term “carboxylate group” refers to -C(O)O(alkyl) or -C(O)O(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.
The application also includes the compounds represented by Formula I in various deuterated forms. Each usable hydrogen atom linked to carbon atoms may be independently substituted with a deuterium atom. Those skilled in the art could synthesize the compounds represented by Formula
30 I in the deuterated form by reference to relevant literatures. Commercially available deuterated starting materials can be used in the preparation of the compounds represented by Formula I in the deuterated form, or they can be synthesized with a deuterated reagent according to conventional techniques, and non-limiting examples of the deuterated reagent includes deuterated borane, trideuterated borane in tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane, and the like.
The term “antibody” refers to an immunoglobulin, which is a tetrapeptide chain structure formed by linking two identical heavy chains and two identical light chains via an interchain disulfide bond. The amino acid composition and arrangement order in the heavy chain constant region of the immunoglobulin are different, and thus its antigenicity is also different. Accordingly,
40 the immunoglobulins can be classified into five classes, or called as the isotypes of
88 immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being p, 8, y, a, and s chains, respectively. The same class of Ig can be classified into different subclasses according to the differences in the amino acid composition of the hinge region and in the number and position of the heavy chain disulfide bonds, for example, IgG may be classified into IgGl, IgG2, IgG3 and IgG4. The light chains are classified into K chains and X chains according to the differences in the constant regions. Each class of Ig in the five classes of Ig may have either K chains or X chains. The antibody of the application is preferably a specific antibody against cell surface antigens on target cells, preferably an anti-DLL-3 antibody.
The term “solvate” or “solvate compound” refer to a pharmaceutically acceptable solvate
10 formed by the ligand-drug conjugate with one or more solvent molecules. Non-limiting examples of the solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.
The term “drug loading rate” refers to the average amount of cytotoxic drug loaded per antibody in Formula I, and it can also be expressed by a ratio of drug to antibody, and the drug loading rate can range from 0 to 12, preferably from 1 to 10, cytotoxic drugs (d) linked to per antibody (Ab). In one embodiment, the drug loading rate is expressed as n, which illustratively is a mean value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The average amount of drugs per ADC molecule after the conjugation reaction can be identified by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA test and HPLC characterization.
In one embodiment, the cytotoxic drug is conjugated to a thiol group (-SH) of cysteine opened
20 between antibody chains and/or a thiol group (-SH) of site-directed mutated cysteine residue in the antibody by a linking unit, and generally, the number of the drug molecules that can be conjugated to the antibody in the conjugation reaction will be less than or equal to the theoretical maximum.
The loading of the ligand cytotoxic drug conjugate can be controlled by the methods, including, without limitation, the following:
(1) controlling the molar ratio of linking reagent to monoclonal antibody,
(2) controlling the reaction time and temperature,
(3) selecting different reaction reagents.
For the preparation of conventional drug compositions, see the Chinese or US Pharmacopoeia.
The term “pharmaceutically acceptable salt” or “pharmaceutically usable salt” refers to a salt
30 of the ligand-drug conjugate, or a salt of the compound as disclosed herein, wherein the salt is safe use in a mammal with desired efficacy and biological activity. In some embodiments, the liganddrug conjugate compound has at least one carboxyl group and thus can form a salt with a base. Examples of the pharmaceutically acceptable salts include without limitation sodium, potassium, calcium or magnesium salts, and the like. In some embodiments, the ligand-drug conjugate disclosed herein has at least one amino group, and thus it can form a salt with an acid. Examples of the pharmaceutically acceptable salts include without limitation hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, biphosphate, dihydric phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, and p-toluenesulphonate.
40 The term “acidic amino acid” refers to an amino acid having an isoelectric point of less than
89 7. The acidic amino acid molecule often has one or more acidic groups such as carboxyl group, which can be effectively ionized into a negative ion form in the structure to increase the hydrophilicity. The acidic amino acid may be a natural amino acid or an unnatural amino acid.
The term “natural amino acid” refers to an amino acid synthesized in a living organism. The natural amino acids are generally in a L-form, with a few exceptions, e.g., glycine, including natural and biosynthesized.
The term “unnatural amino acid” refers to an amino acid obtained by synthetic means.
If the chemical name of the compound described in the application is inconsistent with the structural formula, the structural formula of the compound shall prevail.
10
EXAMPLES
The application is further described below with reference to the specific examples. It should be understood that these examples are only for illustrating the application but not for limiting the scope of the application. The test method of which specific conditions are not given in the examples is usually carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise indicated, all percentages, proportions, ratios or fractions are by weight.
Example 1 Synthesis of Compound Ml
H O
H o O
OH Pb(OAc) Fmoc N 4 /N H THF/ Fmoc N o
O Tol, 85 °C H
SM-1 M1
20 To a 5L single-neck flask, N-fluorenylmethyloxycarbonyl-glycine-glycine (100 g, 282 mmol), lead tetraacetate (175 g, 395 mmol), 2L of dry tetrahydrofuran and 670 mL of toluene were added, stirred well, and the resulting mixture was heated to 85 °C and reacted for 2.5 h under nitrogen protection, and the reaction was monitored with TLC. After the raw materials were reacted completely, the reaction solution was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by column chromatography to obtain Compound Ml (87 g); LC-MS: [M+NH4]+=386.0.
Example 2 Synthesis of Compound M3
To a 1000 mL single-neck flask, Compound SM-2 (synthesized according to the method as
30 disclosed in the patent application CN108452321A) (40 g, 96 mmol, 1.0 eq), trimethylamine (26.7 mL, 2.0 eq), and toluene (400 mL) were added, and the resulting mixture was heated to 120 °C, refluxed and reacted for 2 h, and the reaction was monitored with TLC. When the reaction was
90 substantially completed, the reaction solution was cooled to under 50 °C and the solvent was removed under a reduced pressure. The residue was dissolved with ethyl acetate (150 mL) and water (40 mL), and the obtained solution was adjusted with IM HC1 to have a pH of 2 to 3 under stirring in an ice bath, and separated. The aqueous layer was additionally extracted with ethyl acetate once, and the organic layers are combined and dried under anhydrous sodium sulfate. The resultant solution is filtrated, the resulting product was concentrated to obtain a pale yellow oily crude product, and the crude product was purified by column chromatography (DCM: MeOH=40: 1), to obtain Compound M2 (26.6 g); LC-MS: [M+H]+=399.3.
To a 1000 mL single-neck flask, Compound M2 (26.5 g, 60.5 mmol, l.Oeq),
10 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 reacted at room temperature for 30 min (monitored by TLC), and insoluble material was removed by filtration. The reaction solution was directly purified by preparative liquid chromatography, and the preparation solution was concentrated to remove acetonitrile under reduced pressure with a water pump in a water bath at 35 °C, and lyophilized to obtain Compound M3 (31.5g), with a yield of 64%; LC-MS: [M+H]+= 565.1.
Example 3 Synthesis of Compound e/it-M3 o
J %
‘BuO. N^ /
Q O. N
I Boc . O t ° BuO. O "O
OH TEA *BuO.
N Pentafluorophenol I U F. -F
O Boc COOHO /Tol,l20oC O Boc COOHO DCC I enr-SM-2 F I e«Z-M2 enZ-M3 p
By referring to the synthetic route of Example 2, Compound C///-M3 (27.8 g) was obtained;
LC-MS: [M+H]+=565.2.
20 Example 4 Synthesis of Compound 1
91
First Step: Compound la
To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL of THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl hydroxyacetate (5.4 g, 32.6 mmol) was dropwise added. Upon completion of the dropwise addition, the resulting mixture was naturally warmed up to room temperature and reacted (for about 2 to 4 h), and the reaction was monitored by TLC. Upon completion of the reaction, a saturated NaHCCL solution was added to the reaction solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium
10 sulfate, filtered, and concentrated, and the residue was purified by a silica gel column (PE: EA=10:l-5: l-l : l) to obtain Compound la (4 g), with a yield of 52%; EC -MS: [M+H]+=475.18.
Second Step: Compound lb
To a 25 mL single-neck flask, Compound la (2 g, 4.2 mmol), and 10 mL of DMF were added and stirred at 0 °C, and DBU (766 mg, 5.04 mmol) was added, and then the resulting mixture was reacted for 1 h, and the reaction was monitored with TLC. After the Fmoc de-protection was completed, a reaction solution was obtained, and set aside for use;
To another 25 mL single-neck flask, Compound M4 (prepared by referring to the method as disclosed in the patent application CN111 O5133OA) (1.73 g, 4.2 mmol), PyBOP (2.61 g, 5.04 mmol), HOBt (680 mg, 5.04 mmol) and 10 mL of DMF were added, and DIPEA (830 uL, 5.04
20 mmol) was added in an ice bath, and continuously stirred for 30 min, the above obtained reaction solution was added. The resulting mixture was warmed up to room temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution
92 was purified by preparative-HPLC, to obtain a preparation solution of product. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure, to obtain a solid compound lb (1.7 g), with a yield of 63%; LCMS: [M+H]+=648.26.
Third Step: Compound 1c
To a 25 mb single-neck flask, Compound lb (900 mg, 1.39 mmol), and 15 mb of DMF were added and dissolved, then 900 mg of 5% Pd/C was added, and then the hydrogenation reaction was carried out for 2 h. Upon completion of the reaction, the reaction solution was filtered to obtain a filtrate containing Compound 1c, which was directly used in the next step reaction without
10 purification.
Fourth Step: Compound Id
The filtrate containing Compound 1c was placed in an ice bath, to which DIPEA (235 uL, 1.39 mmol) was added, and then Compound M3 (784 mg, 1.39 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was lyophilized to obtain Compound Id (504 mg); LC-MS: [M+H]+=804.4.
Fifth Step: Compound le
20 To a 50 mb single-neck flask, Compound Id (500 mg, 0.62 mmol), Exatecan mesylate M5 (310 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mb of DMF were added, and then DIPEA (378 uL, 2.29 mmol) were added in an ice bath. The resulting mixture were heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound le, and the preparation solution was lyophilized to obtain Compound le (210 mg); LC-MS: [M+H]+=1221.6.
Sixth Step: Compound 1
To a 25 mL single-neck flask, Compound le (100 mg, 0.081 mmol), zinc bromide (368 mg, 1.63 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction
30 was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution of product, and the preparation solution was lyophilized to obtain Compound 1 (60 mg); LC-MS: [M+H]+=1065.3.
Example 5 Synthesis of Compound 2
93
Compound M3 was replaced with Compound C//Z-M3, and Compound 2 (51 mg) was prepared by referring to the synthetic route of Example 4; LC-MS: [M+H]+=1065.3.
Example 6 Synthesis of Compound 3
First Step: Compound 3a
To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL of THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 2-hydroxy-2-methylpropionate (6.3 g, 32.6 mmol) was added dropwise. Upon
10 completion of the dropwise addition, the resulting mixture was naturally warmed up to room temperature and reacted (for about 2 to 4 h), and monitored by TLC. Upon completion of the reaction, a saturated NaHCOa solution was added to the reaction solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica gel
94 column (PE: EA=10:l-5: l-2:l) to obtain Compound 3a (4.2 g), with a yield of 52%; LC-MS: [M+H]+=503.3.
Second Step: Compound 3b
To a 25 mL single-neck flask, Compound 3a (2 g, 4.0 mmol), and 10 mL of DMF were added and stirred at 0 °C, and then DBU (760 mg, 5.0 mmol) was added, The resulting mixture was reacted for 1 h, and the reaction was monitored with TEC. After the Fmoc de-protection was completed, a reaction solution was obtained, and set aside for use.
To another 25 mL single-neck flask, Compound 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 were added, and then DIPEA (823 uL, 5.04
10 mmol) was added in an ice bath, and continuously stirred for 30 min, the above obtained reaction solution was added. The resulting mixture was heated to room temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography, to obtain a preparation solution of product. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure, to obtain a solid compound 3b (1.4 g), with a yield of 53%; LC-MS: [M+H]+=676.2.
Third Step: Compound 3c
To a 25 mL single-neck flask, Compound 3b (700 mg, 1.04 mmol), and 10 mL of DMF were
20 added and dissolved, and then 700 mg of 5% Pd/C was added, then the hydrogenation reaction was carried out for 1.5 h. Upon completion of the reaction, the reaction solution was filtered to obtain a filtrate containing Compound 3c, which was directly used in the next step reaction without purification.
Fourth Step: Compound 3d
The filtrate containing Compound 3c was placed in an ice bath, to which DIPEA (210 uL, 1.25 mmol) was added, and then Compound M3 (704 mg, 1.25 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a
30 preparation solution, and the preparation solution was lyophilized to obtain Compound 3d (486 mg); LC-MS: [M-H]"=830.5.
Fifth Step: Compound 3e
To a 50 mL single-neck flask, Compound 3d (300 mg, 0.36 mmol), Exatecan mesylate 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, and then DIPEA (219.5 uL, 1.33 mmol) were added in an ice bath. The resulting mixture was heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 3e, and the preparation solution was lyophilized to obtain Compound 3e (157 mg); LC-MS: [M+H]+=l 249.6.
40 Sixth Step: Compound 3
95 To a 25 mL single-neck flask, Compound 3e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution of product, and the preparation solution was lyophilized to obtain Compound 3 (64 mg); LC-MS: [M+H]+=1093.1.
Example 7 Synthesis of Compound 4
10 Compound M3 was replaced with Compound C//Z-M3, and Compound 4 (60 mg) was prepared by referring to the synthetic route of Example 6; LC-MS: [M+H]+=1093.2.
Example 8 Synthesis of Compound 5A
First Step: Compound 5a
To a 25 mL single-neck flask, Compound Ml (500 mg, 1.4 mmol), p-toluenesulfonic acid monohydrate (26 mg, 0. Immol, 0. leq) and 10 mL of THF were added, stirred well, cooled to 0 °C, and then benzyl /.-lactate (benzyl (S)-(-)-lactate) (1.2 g, 7.0 mmol, 5 eq) was slowly added. After
96 the addition was completed, the resulting mixture was heated to room temperature and reacted, and the reaction was monitored with TLC. After the reaction was completed, a saturated NaHCOs solution was add to the reaction solution, the resulting mixture was extracted with ethyl acetate, dried with anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by a reverse phase column, to obtain Compound 5a (400 mg).
LC-MS: [M+NH4]+=506.2.
JH NMR(400 Mz, CDC13/CD3OD): 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, 1=8.0 Hz), 7.57 (2H, d, J = 8.0 Hz), 7.75 (2H,
10 d, J = 8.0 Hz).
Second Step: Compound 5b
To a 25 mL single-neck flask, Compound 5a (400 mg, 0.8 mmol, 1.0 eq) and 4mL of DMF were added, stirred well, cooled to 0 °C, and then DBU (137 mg, 0.9 mmol, 1.1 eq) was added slowly. After completion of the addition was completed, the resulting mixture was heated to room temperature and reacted, and the reation was monitored with TLC. The reaction was completed, the reaction solution was expressed as the reaction solution (1).
To another 25 mL single-neck flask, Compound 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, and stirred at room temperature for 5 minutes, and then the above reaction solution (1) was added. The resulting mixture was reacted at
20 room temperature, and the reaction was monitored with HPLC. The reaction was completed, and the reaction solution was purified by preparative high performance liquid chromatography, to obtain Compound 5b (326mg); LC-MS: [M+NH4]+=679.2.
Third Step: Compound 5c
To a 100 mL single-neck flask, Compound 5b (4.0 g, 6.05 mmol, 1.0 eq), and DMF (60 mL) were added and dissolved, then 5% Pd/C (4 g) was added, and then the hydrogenation reaction was carried out at room temperature for 4 h (the reaction was monitored by HPLC). The Pd/C was removed by filtration, and the filtrate containing Compound 5c are not concentrated and directly placed in an ice bath (about 0 °C), and set aside for use.
Fourth Step: Compound 5d
30 The filtrate containing Compound 5c was placed in an ice bath, to which DIPEA (1.1 mL, 1.1 eq) was added, and then Compound M3 (3.4 g, 6.05 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was lyophilized to obtain Compound 5d (3.15g) ; LC-MS : [M-H]"816.3.
Fifth Step: Compound 5e
To a 100 mL single-neck flask, Compound 5d (2.07 g, 2.53 mmol, 1.0 eq), Exatecan mesylate 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, and then DIPEA (1.05 mL, 1.5 eq) was added in an ice
40 bath. The resulting mixture was heated to room temperature and reacted for 2 h (the reaction was
97 monitored by HPLC). The reaction solution was directly purified by preparative liquid chromatography, and the resulting preparation solution was concentrated to remove acetonitrile under reduced pressure with a water pump in a water bath at 35 °C, and lyophilized to obtain Compound 5e (1.92 g), with a yield of 61%; LC-MS; [M+H]+=1235.4.
Sixth Step: Compound 5A
To a 100 mL single-neck flask, Compound 5e (1.0 g, 0.8 mmol, 1.0 eq), and 35 mL of nitromethane were added and dissolved, then zinc bromide (3.64 g, 16 mmol, 20.0 eq) was added, and the resulting mixture was reacted in an oil bath at 40 °C (pre-heated for stabilization in advance) for 30 min. The reaction solution was concentrated to remove nitromethane under reduced pressure
10 with a water pump in an oil bath at 45°C, to obtain a yellow residue solid (with monitoring by HPLC). The residue solid was purified by preparative liquid chromatography, 0.1% trifluoroacetic acid was added to the flowing phase, and a preparation solution of compound 5 A was obtained. The preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized, to obtain Compound 5 A (786 mg), with a yield of 90%.
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,
20 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, J =
9.6, 3.9 Hz, 1H), 4.73 (dd, 1 = 9.9, 6.9 Hz, 1H), 4.59 (dd, J = 10.1, 6.5 Hz, 1H), 4.49 (ddd, 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, J = 16.9, 5.7 Hz, 2H), 3.70 (d, 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
98
First Step: Compound 5d-l
To a 25 mL single-neck flask, Compound 5b (300 mg, 0.45 mmol, 1.0 eq), and DMF (3 mL) were added, stirred and dissolved, 5% Pd/C (300 mg) was added, three replacements of hydrogen gas were carried out, and the hydrogenation reaction was carried out for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was filtered to remove the Pd/C, to obtain a filtrate containing compound 5c. The filtrate was cooled to 0 °C to 5 °C, and then DIPEA (65 mg, 0.5 mmol, 1.1 eq) was added. Then, ent-M3 (255 mg, 0.45 mmol) was added to the filtrate. Upon completion of the addition, the resulting mixture was heated to
10 20±5 °C and reacted for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the purification was performed by preparative HPLC, and a preparation solution of product was collected and lyophilized to obtain Compound 5d- 1 (200 mg), with a yield of 54%; LC-MS: [M-H]"=816.3.
Second Step: Compound 5e-l
To a 25 mL single-neck flask, Compound 5d-l (200 mg, 0.24 mmol, 1.0 eq), Exatecan mesylate M5 (127 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2eq), HOBt (48 mg, 0.36 mmol, 1.2 eq) and DMF (6 mL) were added and cooled to 0 °C to 5°C in an ice bath, then DIPEA (62 mg, 0.48 mmol, 2.0 eq) was added. Upon completion of the addition, the resulting mixture was heated 20±5 °C and reacted for 2 h, and the reaction was monitored with HPLC until
20 the reaction was completed. The reaction solution was directly purified by preparative HPLC, and a preparation solution of product was collected and lyophilized to obtain Compound 5e-l (162.8 mg); LC-MS: [M+H]+=1235.4.
Third Step: Compound 5B
To a 25 mL single-neck flask, Compound 5e-l (110 mg, 0.089 mmol, 1.0 eq), ZnBn (400 mg, 1.78 mmol, 20.0 eq) and CH3NO2 (10 mL) were added in order, and upon completion of the addition, the resulting mixture was heated to 40 °C and reacted for 0.5 h, then the reaction was stopped. The reaction solution was directly dried under reduced pressure at 45 °C, to obtain a
99 yellow solid. By sampling, the reaction was monitored by HPLC. The dried solid was directly purified by preparative HPLC. The preparation solution of product was collected and lyophilized to obtain Compound 5B (73.4 mg), with the field 76.5%; LC-MS; [M+H]+= 1079.4.
Example 10 Preparation of Compound 6A
Benzyl (S)-(-)-lactate was replaced with benzyl (R)-(+)-lactate, and Compound 6 A (71 mg) was prepared by referring to the synthetic route of Example 8; LC-MS:[M+H]+=1079.4.
Example 11 Preparation of Compound 6B
10 Benzyl (S)-(-)-lactate was replaced with benzyl (R)-(+)-lactate, and Compound 6B (59 mg) was prepared by referring to the synthetic route of Example 9; LC-MS: [M+H]+=1079.4.
Example 12 Preparations of Compounds 7 A and 7B
100
First Step: Compound 7a
To a 250 mL single-neck flask, Compound Ml (10 g, 27.1 mmol), benzyl 3,3,3- trifluorolactate (prepared according to the method disclosed in the patent application W02020063673A1) (12.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added, and the resulting mixture was heated to 100 °C and reacted for 4 h. Upon completion of the reaction, the reaction solution was cooled to room temperature, and filtered to remove insoluble substances. The filtrate was concentrated to obtain a crude product. The crude product was purified by silica column chromatography (PE:EA=10: 1-5: 1-2: 1), to obtain 5.15 g of
10 Compound 7a, with a yield of 35.1%; LC-MS: [M+H]+=543.17.
Second Step: Compound 7b
To a 50 mL single-neck flask, Compound 7a (5 g, 9.2 mmol) and 15 mL of DMF were added and dissolved, then DBU (1.68 g, 11 mmol) were added in an ice bath, and the resulting mixture was reacted for 1 h, the resulting reaction solution was expressed as a reaction solution (1).
To another 50 mL single-neck flask, Compound M4 (3.8 g, 9.2 mmol), PyBOP (5.75 g, 11 mmol), HOBt (1.49 g, 11 mmol) and 10 mL of DMF were added and dissolved, then DIPEA (1.82 mL, 11 mmol) were added in an ice bath, the resulting mixture was reacted for 30 min, and then the reaction solution (1) was added. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction progress was monitored by HPLC After the reaction was
20 completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with di chloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium
101 sulfate, filtered, and concentrated to obtain 4.1 g of solid Compound 7b, with a yield of 62.3%; LC-MS: [M+H]+=716.25.
Third Step: Compound 7d
To a 25 mL single-neck flask, Compound 7b (900 mg, 1.26 mmol), and 15 mL of DMT were added and dissolved, then 900 mg of 5% Pd/C were added, the hydrogenation reaction was carried out for 2h. Upon completion of the reaction, the reaction solution was filtered to obtain a filtrate containing Compound 7c. The filtrate was placed in an ice bath, to which DIPEA (228 uL, 1.38 mmol) was added, and then Compound M3 (712 mg, 1.26 mmol) was add. Upon completion of the additions, the mixture was heated to room temperature and reacted for 1 h,
10 and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by preparative high performance liquid chromatography, to obtain a preparation solution. The preparation solution was lyophilized to obtain 525 mg of Compound 7d, with a yield of 47.9%; LC-MS: [M-H]"=870.33.
Fourth Step: Compound 7e
To a 50 mL single-neck flask, Compound 7d (500 mg, 0.57 mmol), Exatecan mesylate M5 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, and then DIPEA (378 uL, 2.29 mmol) were added in an ice bath. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by
20 preparative high-performance liquid chromatography, to obtain the preparation solutions of Compound 7e-l and Compound 7e-2. The preparation solutions were lyophilized respectively, to obtain 150 mg of Compound 7e-l, LC-MS: [M+H]+=1289.46; and 220mg of Compound 7e-2, LC- MS: [M+H]+=1289.46.
Fifth Step: Compound 7 A
To a 25 mL single-neck flask, Compound 7e-l (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude
30 product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 52 mg of Compound 7 A; TOP results: 1133.3613.
Sixth Step: Compound 7B
102
To a 25 mL single-neck flask, Compound 7e-2(100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added, and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 63 mg of Compound 7B; TOF result: 1133.3668.
Example 13 Synthesis of Compounds 8A and 8B
MeSO3H-H2N. lBuO. Ox / y—N r j
> BO&AQ O F rV
Fx J^F -O O J
O O
H H CF3 n \'ow N o ‘ x/N .OH
‘N x. ^N' 'o'S MS enf-M3 F O L H H H
7c O O O
' NBoc PyBOP, HOBt, DIPEA, DMF
DIPEA, DMF, rt f 8d
XOOfeu
First Step: Compound 8d
To a 25 mL single-neck flask, Compound 7c (900 mg, 1.83 mmol), and 20 mL of DMF were added and dissolved, then DIPEA (303 uL, 1.83 mmol) was added, and then ent-M3 (1034 mg, 1.83 mmol) were added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. After completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography, and the preparation solution was lyophilized to obtain 613 mg of Compound 8d, with a yield of 38.5%; LC-MS: [M-H]"=870.32.
Second Step: Compound 8e-l and Compound 8e-2
20 To a 50 mL single-neck flask, Compound 8d (500 mg, 0.57 mmol), Exatecan mesylate 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, and then DIPEA (378 uL, 2.29 mmol) was added in an ice bath. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by
103 preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 8e-l and Compound 8e-2, respectively. The preparation solutions were lyophilized respectively, to obtain 140 mg of Compound 8e-l, and 210 mg of Compound 8e-2. LC-MS of Compound 8e-l : [M+H]+=1289.47; LC-MS of Compound 8e-2: [M+H]+=1289.47.
Third Step: Compound 8A
To a 25 mL single-neck flask, Compound 8e-l (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was
10 concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 50 mg of Compound 8 A; TOP result: 1133.3623.
Fourth Step: Compound 8B
To a 25 mL single-neck flask, Compound 8e-2 (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude
20 product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 58 mg of Compound 8B; TOP result: 1133.3653.
Example 14 Synthesis of Compound 9A
104
First Step: Compound 9a
To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL of THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 1 -hydroxycyclopropanecarboxylate (prepared by referring to the method as disclosed in US20050020645 Al) (6.3 g, 32.6 mmol) was dropwise added. Upon completion of the dropwise addition, the resulting mixture was naturally heated to room temperature and reacted (for about 2h to 4 h), and the reaction was monitored by TLC. Upon completion of the reaction, a saturated NaHCCh solution was added to the reaction solution, the resulting mixture was extracted with
10 ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica column (PE:EA=10:l- 5 : 1-2: 1), to obtain Compound 9a (3.7 g), with a yield of 45%; LC-MS: [M+H]+=501.5.
Second Step: Compound 9b
To a 25 mL single-neck flask, Compound 9a (2 g, 4.0 mmol), and 10 mL of DMF were added and stirred at 0 °C, and then DBU (760 mg, 5.0 mmol) were added. The resulting mixture was reacted for 1 h, and the reaction was monitored with TLC. After the Fmoc deprotection was completed, the reaction solution was set aside for use.
To another 25 mL single-neck flask, Compound 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 were added, and then DIPEA (823 uL, 5.04
20 mmol) was added in an ice bath. The resulting mixture was continuously stirred for 30 min. The above obtained reaction solution was added to the flask, and the resulting mixture was heated to room temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with
105 a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and fdtered, and the filtrate was concentrated under reduced pressure, to obtain 1.5 g of solid Compound 9b, with a yield of 56%; LC-MS: [M+H]+=674.7.
Third Step: Compound 9c
To a 25 mb single-neck flask, Compound 9b (900 mg, 1.3 mmol), and 10 mL of DMF were added and dissolved, then 900 mg of 5% Pd/C was added, the hydrogenation reaction was carried out for 1.5 h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 9c, which was directly used in the next step reaction without purification.
Fourth Step: Compound 9d
10 The filtrate containing Compound 9c was placed in an ice bath, to which DIPEA (223 uL, 1.3 mmol) was added, and then Compound M3 (750 mg, 1.3 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by a preparative high-performance liquid chromatography, the preparation solution was lyophilized to obtain Compound 9d (529 mg); LC-MS; [M-H]"=828.4.
Fifth Step: Compound 9e
To a 50 mL single-neck flask, Compound 9d (500 mg, 0.6 mmol), Exatecan mesylate M5 (300 mg, 0.6 mmol), PyBOP (416 mg, 0.8 mmol), HOBt (108 mg, 0.5 mmol) and 15 mL of DMF were added, and then DIPEA (351 uL, 2.13 mmol) was added in an ice bath. The resulting mixture
20 was heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 9e, the preparation solution was lyophilized to obtain Compound 9e (257 mg); LC-MS: [M+H]+=1247.5.
Sixth Step: Compound 9A
To a 25 mL single-neck flask, Compound 9e (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a
30 preparation solution, and the preparation solution was lyophilized to obtain Compound 9 A (55 mg); LC-MS: [M+H]+=1091.3.
Example 15 Synthesis of Compound 9B
Compound M3 was replaced with Compound C///-M3, and Compound 9B (44 mg) was
106 prepared by referring to the synthetic route of Example 14; LC-MS: [M+H]+=1091 .3.
Example 16 Synthesis Compound 10A
First Step: Compound 10a
To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL of THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 1 -hydroxymethylpropanecarboxylate (prepared by referring to the method as disclosed in WO2013187496A1) (6.7 g, 32.6 mmol) was dropwise added. Upon completion of the dropwise addition, the resulting mixture was naturally warmed up to room temperature and reacted (for
10 about 2-4 h), and the reaction was monitored by TLC. Upon completion of the reaction, a saturated NaHCOg solution was added to the reaction solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica column (PE:EA=10:l- 5 : 1-2: 1) to obtain Compound 10a (4.9 g), with a yield of 58%; LC-MS: [M+H]+=515.4.
Second Step: Compound 10b
To a 25 mL single-neck flask, Compound 10a (4 g, 7.8 mmol), and 10 mL of DMF were added and stirred at 0 °C, and then DBU (1.2 g, 8.0 mmol) was added. The resulting mixture was reacted for 1 h, and the reaction was monitored with TLC. After the Fmoc de-protection was completed, the reaction solution was set aside for use.
20 To another 25 mL single-neck flask, Compound 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 were added, and the DIPEA (1.65 mL, 10.1 mmol) was added in an ice bath, and continuously stirred for 50 min. The above reaction solution
107 was added to the flask, and the resulting mixture was heated to room temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure, to obtain 2.3 g of solid Compound 10b, with a yield of 42%; LC-MS: [M+H]+=688.8.
Third Step: Compound 10c
To a 25 mL single-neck flask, Compound 10b (1.0 g, 1.45 mmol), and 15 mL of DMF was
10 added and dissolved, then 1.0 g of 5% Pd/C was added, and the hydrogenation reaction was carried out for 1.5 h. Upon completion of the reaction, the reaction solution was filtered to obtain a filtrate containing Compound 10c, which was directly used in the next step reaction without purification.
Fourth Step: Compound lOd
The filtration containing Compound 10c was placed in an ice bath, to which DIPEA (258 uL,
1.5 mmol) was added, and then Compound M3 (837 mg, 1.45 mmol) was added. Upon completion of the addition, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. After completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution,
20 and the preparation solution was lyophilized to obtain Compound lOd (499 mg); LC-MS; [M-H]" =842.4.
Fifth Step: Compound lOe
To a 50 mL single-neck flask, Compound lOd (400 mg, 0.48 mmol), Exatecan mesylate 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, and then DIPEA (330 uL, 2.0 mmol) was added in an ice bath, the resulting mixture were heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound l Oe. The preparation solution was lyophilized to obtain Compound lOe (188 mg); LC-MS: [M+H]+=1261.5.
30 Sixth Step: Compound 10A
To a 25 mL single-neck flask, Compound lOe (100 mg, 0.08 mmol), zinc bromide (360 mg,
1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h. , and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 10A (61 mg); LC-MS: [M+H]+=l 105.4.
Example 17 Synthesis of Compound 10B
108
Compound M3 was replaced with Compound ew/-M3, and Compound 10B (75 mg) was prepared by referring to the synthetic route of Example 16; LC-MS: [M+H]+=i 105.4.
Example 18 Synthesis of Compound 11A
First Step: Compound 11a
To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL of THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1 .63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 1 -hydroxy cyclobutanecarboxylate (synthesized according to the method as disclosed
10 in Journal of Medicinal Chemistry, 2013, 56 (13), 5541-5552) (6.7 g, 32.6 mmol) was dropwise added. Upon completion of the dropwise addition, the resulting mixture was naturally heated to room temperature and reacted (for about 2-4 h), the reaction was monitored by TLC. Upon
109 completion of the reaction, a saturated NaHCCh solution was added to the reaction solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, fdtered, and concentrated, and the residue was purified by a silica column (PE:EA=10: l-5:l-2: l), to obtain Compound Ila (5.1 g), with a yield of 62%; LC-MS: [M+H]+=515.7.
Second Step: Compound 11b
To a 25 mb single-neck flask, Compound Ila (4 g, 7.8 mmol), and 10 mL of DMF were added and stirred at 0 °C, then DBU (1.2 g, 8.0 mmol) was added, and the resulting mixture was reacted for 1 h. The reaction was monitored with TEC. After the Fmoc de-protection was completed, the
10 reaction solution was set aside for use.
To another 25 mL single-neck flask, Compound 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 were added, then DIPEA (1.63 mL, 10.0 mmol) was added in an ice bath, and the resulting mixture was continuously stirred for 40 min. The above reaction solution was added to the flask, and the resulting mixture was heated to room temperature and reacted. The reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure, to obtain 2.3 g of Compound 11b, with a yield
20 of 42%; LC-MS; [M+H]+=688.3.
Third Step: Compound 11c
To a 25 mL single-neck flask, Compound lib (2.0 g, 2.9 mmol), and 25 mL of DMF were added and dissolved, 2.0 g of 5% Pd/C were added, and the hydrogenation reaction was carried out for 3 h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 11c, which was directly used in the next step reaction without purification.
Fourth Step: Compound lid
The filtrate containing Compound 11c was placed in an ice bath, to which DIPEA (516 uL, 3.0 mmol) was added, and then Compound M3 (1 .7 g, 2.9 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 2 h, and the
30 reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was lyophilized to obtain Compound lid (934 mg); LC-MS: [M-H]" =842.4.
Fifth Step: Compound lie
To a 50 mL single-neck flask, Compound lid (800 mg, 0.96 mmol), Exatecan mesylate M5 (480 mg, 0.96 mmol), PyBOP (500 mg, 0.96 mmol), HOBt (208 mg, 0.96 mmol) and 30 mL of DMF were added, then DIPEA (660 uL, 4.0 mmol) was added in an ice bath, and the resulting mixture was heated to room temperature and reacted for 4 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high-
40 performance liquid chromatography, to obtain a preparation solution of Compound l ie. The
110 preparation solution was lyophilized to obtain Compound l ie (401 mg); LC-MS: [M+H]+=1261 .4.
Sixth Step: Compound 11A
To a 25 mL single-neck flask, Compound He (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol) and 10 mL of nitromethane were added and reacted at 40 °C 1 h. The reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound HA (86 mg); LC-MS: [M+H]+=l 105.4.
10 Example 19 Synthesis of Compound 11B
Compound M3 was replaced with Compound e?z/-M3, and Compound 11B (50 mg) was prepared by referring to the synthetic route of Example 18. LC-MS; [M+H]+=l 105.4.
Example 20 Synthesis of Compound 12A
First Step: Compound 12a
To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL THF, and p-
111 toluenesulfonic acid monohydrate (0.31 g, 1 .63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 1 -hydroxymethylcyclobutanecarboxylate (prepared by referring to the method as disclosed in the patent application W02009011285A1) (7.2 g, 32.6 mmol) was dropwise added. Upon completion of the dropwise addition, the resulting mixture was naturally heated to room temperature and reacted (for about 2 h to 4 h), the reaction was monitored by TLC. Upon completion of the reaction, a saturated NaHCOa solution was added to the reaction solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica column (PE:EA=10;l-5;l-2: l), to obtain Compound 12a (4.5 g), with a yield
10 of 52%; LC-MS: [M+H] ~529.4.
Second Step: Compound 12b
To a 25 mL single-neck flask, Compound 12a (4 g, 7.6 mmol), and 10 mL of DMF were added and stirred at 0 °C, then DBU (1.2 g, 8.0 mmol) was added, and the resulting mixture was reacted for 1 h. The reaction was monitored with TLC. After the Fmoc de-protection was completed, the reaction solution was set aside for use.
To another 25 mL single-neck flask, Compound 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 were added, and then DIPEA (1.49 mL, 0.9 mmol) was added in an ice bath, and the resulting mixture was continuously stirred for 30 min. The above reaction solution was added to the flask, and the resulting mixture was heated to room
20 temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure, to obtain 2.0 g of solid Compound 12b, with a yield of 37%; LC-MS: [M+H]+=702.8.
Third Step: Compound 12c
To a 25 mL single-neck flask, Compound 12b (1.0 g, 1.43 mmol), and 15 mL of DMF were added and dissolved, then 1 .0 g of 5% Pd/C were added, and then the hydrogenation reaction was carried out for 1.5 h. Upon completion of the reaction, the reaction solution was filtered, to obtain
30 a filtrate containing Compound 12c, which was directly used in the next step reaction without purification.
Fourth Step: Compound 12d
The filtrate containing Compound 12c was placed in an ice bath, to which DIPEA (258 uL, 1.5 mmol) was added, and Compound M3 (825 mg, 1.43 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 12d (522 mg); LC-MS: [M-H]" =856.4.
40 Fifth Step: Compound 12e
112 To a 50 mL single-neck flask, Compound 12d (400 mg, 0.47 mmol), Exatecan mesylate 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, then DIPEA (330 uL, 2.0 mmol) was added in an ice bath, and the resulting mixture was heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 12e. The preparation solution was lyophilized to obtain Compound 12e (198 mg); LC-MS: [M+H]+=1275.4.
Sixth Step: Compound 12A
To a 25 mL single-neck flask, Compound 12e (100 mg, 0.08 mmol), zinc bromide (360 mg,
10 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 12A (55 mg); LC-MS: [M+H]+=1119.4.
Example 21 Synthesis of Compound 12B
Compound M3 was replaced with Compound C///-M3, and Compound 12B (50 mg) was prepared by referring to the synthetic route of Example 20; LC-MS; [M+H]+=l 119.4.
20 Example 22 Synthesis of Compound 13A
113
First Step: Compound 13a
To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 1 -hydroxycyclopentanecarboxylate (synthesized according to the method as disclosed in the literation “Journal of Medicinal Chemistry”, 2013, 56(13), 5541-5552) (7.2 g, 32.6 mmol) was dropwise added. Upon completion of the dropwise addition, the resulting mixture was naturally heated to room temperature and reacted (for about 2 h to 4 h), the reaction was monitored by TLC. Upon completion of the reaction, a saturated NaHCOa solution was added to the reaction
10 solution, the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica column (PE:EA=10: l-5:l-2:l), to obtain Compound 13a (4.6 g), with a yield of 53%; LC-MS: [M+H]+=529.5.
Second Step: Compound 13b
To a 25 mL single-neck flask, Compound 13a (4 g, 7.6 mmol), and 10 mL of DMF were added and stirred at 0 °C, then DBU(1.17 g, 7.8 mmol) was added, and then the resulting mixture was reacted for 1 h. The reaction was monitored with TLC, after the Fmoc de-protection was completed, the reaction solution was set aside for use.
To another 25 mL single-neck flask, Compound M4 (3.14 g, 7.6 mmol), PyBOP (4.42 g, 8.5
20 mmol), HOBt (1.15 g, 8.5 mmol) and 10 mL of DMF were added, then DIPEA (1.39 mL, 0.85 mmol) was added in an ice bath, and then the resulting mixture was continuously stirred for 30
114 min. The above reaction solution was added to the flask, and the resulting mixture was heated to room temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure, to obtain 2.1 g of solid Compound 13b, with a yield of 39%; LC-MS: [M+H]+=702.8.
Third Step: Compound 13c
To a 25 mL single-neck flask, Compound 13b (1.5 g, 1.87 mmol), and 25 mb of DMF were
10 added and dissolved, then 1.5 g of 5% Pd/C were added, and then the hydrogenation reaction was carried out for 3 h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 13c, which was directly used in the next step reaction without purification.
Fourth Step: Compound 13d
The filtrate containing Compound 13c was placed in an ice bath, to which DIPEA (333 uL, 1.93 mmol) was added, and then Compound M3 (1.1 g, 1.87 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution.
20 The preparation solution was lyophilized to obtain Compound 13d (519 mg); LC-MS; [M-H]" =856.6.
Fifth Step: Compound 13e
To a 50 mL single-neck flask, Compound 13d (400 mg, 0.47 mmol), Exatecan mesylate M5 (240 mg, 0.48 mmol), PyBOP (250 mg, 0.48 mmol), HOBt (103 mg, 48 mmol) and 15 mL of DMF were added, then DIPEA (330 uL, 2.0 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 4 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 13e. The preparation solution was lyophilized to obtain Compound 13e (187 mg); LC-MS: [M+H]+=1275.5.
30 Sixth Step: Compound 13A
To a 25 mL single-neck flask, Compound 13e (100 mg, 0.08 mmol), zinc bromide (355 mg, 0.16 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 13A (60 mg); LC-MS: [M+H]+=l 119.6.
Example 23 Synthesis of Compound 13B
115
Compound M3 was replaced with Compound e/?Z-M3, and Compound 13B (51mg) was prepared by referring to the synthetic route of Example 22; LC-MS: [M+H]+=l 119.6.
Example 24 Synthesis of Compound 14A
First Step: Compound 14a
To a 250 mL single-neck flask, Compound Ml (6 g, 16.3 mmol), 100 mL of THF, and p- toluenesulfonic acid monohydrate (0.31 g, 1.63 mmol) were added, stirred and cooled to 0 °C, and then benzyl 1 -hydroxymethylcyclopentanecarboxylate (prepared by referring to the method as
10 disclosed in W02009011285A1) (7.6 g, 32.6 mmol) was dropwise added. Upon completion of the dropwise addition, the resulting mixture was naturally heated to room temperature and reacted (for about 2h to 4 h), the reaction was monitored by TLC. Upon completion of the reaction, a saturated NaHCOg solution was added to the reaction solution , the resulting mixture was extracted with ethyl acetate, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, and the residue was purified by a silica column (PE:EA=10:l- 5: 1-2:1), to obtain Compound 14a (4.4 g), with a yield of 49%; LC-MS: [M+H]+=543.6.
116 Second Step: Compound 14b
To a 25 mL single-neck flask, Compound 14a (4 g, 7.4 mmol), and 10 mL of DMF were added and stirred at 0 °C, then DBU (1.2 g, 8.0 mmol) was added, and then the resulting mixture was reacted for 1 h. The reaction was monitored with TLC, after the Fmoc de-protection was completed, the reaction solution was set aside for use.
To another 25 mL single-neck flask, Compound 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 were added, then DIPEA (1.49 mL, 9.0 mmol) was added in an ice bath, and then the resulting mixture was continuously stirred for 30 min. The above reaction solution was added to the flask, and the resulting mixture was heated to room
10 temperature and reacted, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and filtered, the filtrate was concentrated under reduced pressure, to obtain 2.6 g of solid Compound 14b, with a yield of 49%; LC-MS: [M+H]+=716.4.
Third Step: Compound 14c
To a 25 mL single-neck flask, Compound 14b (1.0 g, 1.4 mmol), and 15 mL of DMF were added and dissolved, then 1.0 g of 5% Pd/C were added, and then the hydrogenation reaction was carried out for 1.5 h. Upon completion of the reaction, the reaction solution was filtered, to obtain
20 a filtrate containing Compound 14c, which was directly used in the next step reaction without purification.
Fourth Step: Compound 14d
The filtrate containing Compound 14c was placed in an ice bath, to which DIPEA (248 uL,
1.5 mmol) was added, and then Compound M3 (808 mg, 1.4 mmol) was added. Upon completion of the additions, the mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 14d (500 mg); LC-MS: [M-H]"=870.5.
Fifth Step: Compound 14e
30 To a 50 mL single-neck flask, Compound 14d (400 mg, 0.46 mmol), Exatecan mesylate 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, then DIPEA (331 uL, 2.0 mmol) were added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 14e. The preparation solution was lyophilized to obtain Compound 14e (146 mg); LC-MS: [M+H]+=1289.5.
Sixth Step: Compound 14A
To a 25 mL single-neck flask, Compound 14e (100 mg, 0.08 mmol), zinc bromide (360 mg,
1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction
40 was monitored with HPLC. After the reaction was completed, the reaction solution was
117 concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a product preparation solution, and the preparation solution was lyophilized to obtain Compound 14A (52 mg); LC-MS: [M+H]+=l 133.4.
Example 25 Synthesis of Compound 14B
0,
NX F
Q
14B 'OH
0 Z
Compound M3 was replaced with Compound e/?Z-M3, and Compound 14B (48 mg) was prepared by referring to the synthetic route of Example 24; LC-MS: [M+H]+=1133.4.
Example 26 Synthesis of Compounds 15A and 15B
First Step: Compound 15a
To a 250 mL single-neck flask, Compound Ml (10 g, 27.1 mmol), benzyl 2-hydroxyl-butylate (synthesized by the method as disclosed in the literature “Chemical Communications”, 2019, 55(53), 7699-7702) (10.5 g, 54.3 mmol), and zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added, and the resulting mixture was heated and reacted at 100 °C for 4 h. Upon
118 completion of the reaction, the reaction solution was cooled to room temperature, and filtered to remove insoluble substances. The filtrate was concentrated to obtain a crude product, and the crude product was purified by silica column chromatography (PE:EA=10:l-5:l-2:l), to obtain 5.67 g of Compound 15a, with a yield of 42%; EC -MS; [M+H]+=5O3.5.
Second Step: Compound 15b
To a 50 mL single-neck flask, Compound 15a(5 g, 9.95 mmol) and 15 mL ofDMF were added and dissolved, then DBU (1.68 g, 11 mmol) was added in an ice bath, and then the resulting mixture was reacted for 1 h, the reaction solution was expressed as the reaction solution (1);
To another 50 mL single-neck flask, Compound M4 (4.1 g, 10.0 mmol), PyBOP (5.75 g, 11
10 mmol), HOBt (1.49 g, 11 mmol) and 10 mL ofDMF were added and dissolved, then DIPEA (1.82 mL, 11 mmol) was added in an ice bath, the resulting mixture was reacted for 40 min, and then the reaction solution (1) was added. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with di chloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, to obtain 4.6 g of solid Compound 15c, with a yield of 68%; LC-MS: [M+H]+ =676.7.
Third Step: Compound 15d
To a 25 mL single-neck flask, Compound 15b (2.0 g, 2.96 mmol), and 15 mL of DMF were
20 added and dissolved, then 2.0 g of 5% Pd/C was added, and then the hydrogenation reaction was carried out for 2h. Upon completion of the reaction, the reaction solution was filtered. The filtrate was placed in an ice bath, to which DIPEA (496 uL, 3.0 mmol) was added, and then Compound M3 (1.7 g, 2.96 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 1120.0 mg of Compound 15d, with a yield of 45%; LC-MS: [M-H]"=830.3.
Fourth Step: Compound 15e-l and Compound 15e-2
To a 50 mL single-neck flask, Compound 15d (500 mg, 0.60 mmol), Exatecan mesylate M5
30 (321 mg, 0.60 mmol), PyBOP (469 mg, 0.90 mmol), HOBt (121 mg, 0.90mmol) and 15 mL of DMF were added, then DIPEA (446 uL, 2.7 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 15e-l and Compound 15e-2, respectively. The preparation solutions were lyophilized respectively, 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.
Fifth Step: Compound 15A
119
To a 25 mL single-neck flask, Compound 15e- 1 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 59 mg of Compound 15A; LC-MS: [M+H]+=1093.4.
Sixth Step: Compound 15B
To a 25 mL single-neck flask, Compound 15e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 60 mg of Compound 15B; LC-MS: [M+H]+=1093.4.
Example 27 Synthesis of Compounds 16A and 16B
.0 ,Ph CH3
O
,O
N N *o v H H
O I HN
NH H
COOH o.
N j
O N F
Q
'OH
16A o
20 Compound M3 was replaced with Compound ez?/-M3, and Compound 16A (55 mg) was prepared by referring to the synthetic route of Example 26; LC-MS: [M+H]+=1093.4.
120
Compound M3 was replaced with Compound e«/-M3, and Compound 16B (54 mg) was prepared by referring to the synthetic route of Example 26; LC-MS: [M+H]+=1093.4.
Example 28 Synthesis of Compounds 17A and 17B
.0 ph Ph Ph ' O O O O
H H N ,0 N ;O
*< N O' N O' £ H o 1 X os H HN. H H H
O HN ^NBoc NBoc
^cooteu O, O, / y-N Xooteu V-N k J N F J
-O F
OK W Q
'OH " ["OH
17e-1 o 17e-2 O z
First Step: Compound 17a
To a 250 mL single-neck flask, Compound Ml (10 g, 27.1 mmol), benzyl 2-hydroxyl-3- phenylpropionate (synthesized by the method as disclosed in the literature “Nature Communications", 2020. 11 (1), 56) (14.7 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and
10 100 mL of toluene were added, and the resulting mixture was heated to 100 °C and reacted for 4 h. Upon completion of the reaction, the reaction solution was cooled to room temperature, and fdtered to remove insoluble substances. The fdtrate was concentrated to obtain a crude product, and the crude product was purified by silica column chromatography (PE:EA=10:l-5: l-2: l, to obtain 6.13 g of Compound 17a, with a yield of 40%; LC-MS: [M+H] =565.6.
Second Step: Compound 17b
121 To a 50 mL single-neck flask, Compound 17a (5 g, 8.86 mmol) and 15 mL of DMF were added and dissolved, then DBU (1.53 g, 10 mmol) were added in an ice bath, and then the resulting mixture was reacted for 1 h, the reaction solution was expressed as the reaction solution (1).
To another 50 mL single-neck flask, Compound M4 (3.6 g, 8.86 mmol), PyBOP (5.23 g, 10 mmol), HOBt (1.36 g, 10 mmol) and 10 mL of DMF were added and dissolved, then DIPEA (1.65 mL, 10 mmol) was added in an ice bath, and then the resulting mixture was continuously reacted for 30 min, and then the reaction solution (1) was added. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid
10 chromatography, to obtain a preparation solution. The preparation solution was extracted with di chloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 5.0 g of solid Compound 17b, with a yield of 77%; LC- MS: [M+H]+=738.3.
Third Step: Compound 17d
To a 25 mL single-neck flask, Compound 17b (3.0 g, 4.07 mmol), and 15 mL of DMF were added and dissolved, then 3.0 g of 5% Pd/C was added, and then the hydrogenation reaction was carried out for 2h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 17c. The filtrate was placed in an ice bath, to which DIPEA (744 uL, 4.5 mmol) was added, and then Compound M3 (2.34 g, 4.07 mmol) was added. Upon completion
20 of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was lyophilized to obtain 1.2 g of Compound 17d, with a yield of 33%; LC-MS: [M-H]‘=892.4.
Fourth Step: Compound 17e
To a 50 mL single-neck flask, Compound 17d (500 mg, 0.56 mmol), Exatecan mesylate 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, then DIPEA (330 uL, 2.0 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with
30 HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 17e-l and Compound 17e-2, respectively. The preparation solutions were lyophilized respectively to obtain 156 mg of Compound 17e-l, LC-MS: [M+H] =131 L4; and 150 mg of Compound 17e-2, LC-MS: [M+H]+=1311.7.
Fifth Step: Compound 17A
122
To a 25 mL single-neck flask, Compound 17e-l (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 43 mg of Compound 17A; LC-MS: [M+H]+=1155.4.
Sixth Step: Compound 17B
To a 25 mL single-neck flask, Compound 17e-2 (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 40 mg of Compound 17B; LC-MS: [M+H]+=1155.4.
Example 29 Synthesis of Compounds 18A and 18B yP _ /Ph Ph
Q u O o
H 9 V H
\ -N N N .0
N N N O y H H H o 0 O HN
NH
COOH o,
I
F
Q
18A 'OH
0 7
20 Compound M3 was replaced with Compound e/iLM3, and Compound 18A (54 mg) was prepared by referring to the synthetic route of Example 28; LC-MS: [M+H]+=l 155.4.
123 Ph o o
H N ,0
N o y H o HN o.
COOH I' F
O.
18B ''OH
0 ✓
Compound M3 was replaced with Compound e/zZ-M3, and Compound 18B (55 mg) was prepared by referring to the synthetic route of Example 28; LC-MS: [M+H]+=1155.4.
Example 30 Synthesis of Compounds 19A and 19B
First Step: Compound 19a
To a 250 mL single-neck flask, Compound Ml (10 g, 27.1 mmol), benzyl 2-cyclopropyl-2- hydroxyacetate (prepared by referring to the method as disclosed in WO2020244657A1) (11.2 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added, and the resulting
10 mixture was heated to 100 °C and reacted for 4 h. Upon completion of the reaction, the reaction solution was cooled to room temperature, and filtered to remove insoluble substances. The filtrate was concentrated to obtain a crude product, and the crude product was purified by silica column chromatography (PE:EA=10: l-5:l-2: l), to obtain 4.97 g of Compound 19a, with a yield of 36%; LC-MS: [M+H]+=515.2.
Second Step: Compound 19b
To a 50 mL single-neck flask, Compound 19a (4 g, 7.8 mmol) and 10 mL of DMF were added and dissolved, then DBU (1.42 g, 9.3 mmol) was added in an ice bath, and then the resulting mixture was reacted for 1 h, the reaction solution was expressed as the reaction solution (1).
To another 50 mL single-neck flask, Compound M4 (3.2 g, 7.8 mmol), PyBOP (4.5 g, 8.6
124 mmol), HOBt (1.16 g, 8.6 mmol) and 10 mL of DMF were added and dissolved, then DIPEA (1 .65 mL, 10 mmol) was added in an ice bath, and then the resulting mixture was reacted for 30 min, and then the reaction solution (1) was added. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, to obtain 4.2 g of solid Compound 19b, with a yield of 78%; LC-MS: [M+H]+=688.3.
10 Third Step: Compound 19d
To a 25 mL single-neck flask, Compound 19b (1000 mg, 1.45 mmol), and 15 mL of DMF were added and dissolved, then 1000 mg of 5% Pd/C was added, and then the hydrogenation reaction was carried out for 2 h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 19c. The filtrate was placed in an ice bath, to which DIPEA (248 uL, 1.5 mmol) was added, and then Compound M3 (720 mg, 1.45 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was lyophilized to obtain 503 mg of Compound
20 19d, with a yield of 41%; LC-MS: [M-H]"=842.3.
Fourth Step: Compounds 19e-l and 19e-2
To a 50 mL single-neck flask, Compound 19d (500 mg, 0.59 mmol), Exatecan mesylate 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, then DIPEA (292 uL, 1.77 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 19e-l and Compound 19e-2, respectively. The preparation solutions were lyophilized respectively to obtain L12mg of Compound 19e-l, LC-MS: [M+H]+=1261.5; and 131
30 mg of Compound 19e-2, LC-MS: [M+H]+=1261.5.
Fifth Step: Compound 19A
To a 25 mL single-neck flask, Compound 19e- 1 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude
125 product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 55 mg of Compound 19A; LC-MS: [M+H]+=l 105.4.
Sixth Step: Compound 19B
To a 25 mL single-neck flask, Compound 19e-2 (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude
10 product was purified by preparative high-performance liquid chromatography, to obtain a product preparation solution, and the preparation solution was lyophilized to obtain 58 mg of Compound 19B; LC-MS: [M+H]+=l 105.4.
Example 31 Synthesis of Compounds 20A and 20B o O H o r Ph
O O
H
V^
Fmoc A' X| X°Bn ^OBn o ■OAN^^ AN\OH H O H O H V OH _ Fmoc' K O' M4 H
Zn(OAc)2, PhMe, 100 °C O PyBOP, DIPEA, DMF, rt M1 20a o O x H N A ^OBn 5% Pd/C .OH
BnO N H2N^H N'
H H H H H
O O O o 0 2 O
20b 0c yO J
M3 O O
H H M5 N N.
DIPEA, DMF, rt P k *O' >/0H
PyBOP, HOBt, DIPEA, DMF o I H o H H
O O
NBoc
20d
COOteu
/~f° j? O ,o ' o O O
H ,0 H N N 0' G N ,O
* N ^N *0^
£ H H H HN. H H o O O O o HN
^NBoc NBoc
^cooteu o, y-N ^COO'BU Ox / y-N
J N F J
-O F
Ox W Ox
'OH L'OH
20e-1 O 20e-2 o z
First Step: Compound 20a
To a 250 mL single-neck flask, Compound Ml (10 g, 27.1 mmol), benzyl 2-hydroxyl-3- cyclopropylpropionate (prepared by referring to the method as disclosed in W02020063676A) (12.0 g, 54.3 mmol), zinc acetate (9.96 g, 54.3 mmol) and 100 mL of toluene were added, and the resulting mixture was heated 100 °C and reacted for 4 h. Upon completion of the reaction, the
20 reaction solution was cooled to room temperature, and filtered to remove insoluble substances. The filtrate was concentrated to obtain a crude product, and the crude product was purified by
126 silica column chromatography (PE:EA=10: l-5: l-2: l) to obtain 5.09 g of Compound 20a; LC-MS: [M+H]+=529.2.
Second Step: Compound 20b
To a 50 mL single-neck flask, Compound 20a (4 g, 7.6 mmol) and 10 mL of DMF were added and dissolved, then the DBU(1.39 g, 9.1 mmol) was added in an ice bath, and then the resulting mixture was reacted for 1 h, the reaction solution was expressed as the reaction solution (1).
To another 50 mL single-neck flask, Compound M4 (3.12 g, 7.6 mmol), PyBOP (4.5 g, 8.6 mmol), HOBt (1.16 g, 8.6 mmol) and 10 mL ofDMF were added and dissolved, then DIPEA (1.65 mL, 10 mmol) was added in an ice bath, and then the resulting mixture was reacted for 30 min,
10 and then the reaction solution (1) was added. The resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated, to obtain 4.5 g of solid Compound 20b, with a yield of 84%; LC-MS: [M+H]+=702.3.
Third Step: Compound 20d
To a 25 mL single-neck flask, Compound 20b (1000 mg, 1.42 mmol), and 15 mL of DMF were added and dissolved, then 1000 mg of 5% Pd/C were added, and then the hydrogenation
20 reaction was carried out for 2 h. Upon completion of the reaction, the reaction solution was filtered, to obtain a filtrate containing Compound 20c. The filtrate was placed in an ice bath, to which DIPEA (248 uL, 1.5 mmol) was added, and Exatecan mesylate M5 (708 mg, 1.42 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 443 mg of Compound 20d, with a yield of 36%; LC-MS: [M-H]"=856.4.
Fourth Step: Compounds 20e-1 and 20e-2
To a 50 mL single-neck flask, Compound 20d (400 mg, 0.47 mmol), Exatecan mesylate M5
30 (250 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol) and 10 mL of DMF were added, then DIPEA (248 uL, 1.5 mmol) were added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain preparation solutions of Compound 20e-l and Compound 20e-2, respectively. The preparation solutions were lyophilized respectively to obtain 103mg of Compound 20e-l, LC-MS: [M+H]+=1275.5; and 103 mg of Compound 20e-2, LC-MS: [M+H]+=1275.5.
Fifth Step: Compound 20A
127
To a 25 mL single-neck flask, Compound 20e-l (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 51 mg of Compound 20A; LC-MS: [M+H]+=1119.4.
Sixth Step: Compound 20B
To a 25 mL single-neck flask, Compound 20e-2(100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 47 mg of Compound 20B; LC-MS: [M+H]+=l 119.4.
Example 32 Synthesis of Compound 21
1 28
First Step: Compound SM3-1
To a 2000 mL single-neck flask, 77087-60-6 (100 g, 458 mmol), maleic acid (53.4 g, 460 mmol), TEA (64 mL, 460 mmol) and 1000 mL of toluene were added, and the resulting mixture was heated to 100 °C and reacted for 5 h. Upon completion of the reaction, the reaction solution was cooled to room temperature, and filtered to remove insoluble substances. The filtrate was concentrated to obtain a crude product, and the crude product was purified by silica column chromatography (PE:EA=100: l-50: l-20: l) to obtain 75.6 g of Compound SM3-1 ; LC-MS: [M+H]+=299.1.
10 Second Step: Compound /-butyl (7?)-2-hydroxyl-l,5-glutarate
To a 2000 mL single-neck flask, 172793-31-6 (100 g, 338 mmol) and 1000 mL water were added, then sodium nitrite (35 g, 507 mmol), concentrated sulfuric acid (32 mL, 35 mmol) were added in order, and then the resulting mixture was slowly heated to room temperature and reacted for 24 h. Upon completion of the reaction, the reaction solution was extracted with 500 mL of ethyl acetate three times, and the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by silica column chromatography (PE:EA=50: 1-30: 1-2: 1) to obtain 91.2 g of Compound /-butyl (7?)-2-hydroxyl-l, 5-glutarate; LC-MS: [M+H]+=261.4.
Third Step: Compound SMS
20 To a 2000 mL single-neck flask, /-butyl (R)-2-hydroxyl-l, 5-glutarate (50 g, 192 mmol) and 1000 mL of anhydrous tetrahydrofuran were added and cooled in an ice bath to 0 °C, then PPhg (87.7 g, 288 mmol), DEAD (50.2 g, 288 mmol) and Compound SM3-1 (57.3, 192 mmol) were added in order. The resulting mixture was slowly heated to room temperature and reacted for 13 h.
129 Upon completion of the reaction, the reaction solution was filtered to remove insoluble substances. The filtrate was concentrated to obtain a crude product, and the crude product was purified by silica column chromatography (PE:EA=50: 1-30: 1-1 : 1) to obtain 68.6 g of the product.
The above product was dissolved in 500 mL of methanol and cooled in an ice bath to 0 °C, at this temperature, NaOH (64 mL, 190 mmol, 3M/L) was dropwise added, and then the resulting mixture was reacted for 12 h at this temperature, then HC1 (6 M/L) was added to adjust the pH to be 3. The reaction solution was extracted with 500 mL of di chloromethane five times, dried with anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by column chromatography
10 (DCM/McOH-50/1 -20/1 -2/1), to obtain 50.4 g of Compound SM3; LC-MS: [M-H]"=525.5.
Fourth Step: Compound M6
To a 2000 mL single-neck flask, Compound SM3 (50 g, 95 mmol, l.Oeq), pentafluorophenol (19.2 g, 104.5 mmol, 1.1 eq), DCC (21.5 g, 104.5 mmol, 1.1 eq) and THE (600 mL) were added, and the resulting mixture was reacted at room temperature for 1 h (monitored by TLC), and then filtered to remove insoluble substances. The reaction solution was directly purified by preparative liquid chromatography, and the preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound M6 (51.9 g), with a yield of 79%; LC-MS: [M+H]+=693.3.
Fifth Step: Compound 21a
20 To a 25 mL single-neck flask, Compound 1c (1 g, 2.36 mmol), and 25 mL of DMF were added and dissolved, then DIPEA (430 uL, 2.6 mmol) was added, and then Compound M6 (1177 mg, 2.36 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution, and the preparation solution was lyophilized to obtain 555 mg of Compound 21a; LC-MS: [M-H]"=931.0.
Sixth Step: Compound 21b
To a 100 mL single-neck flask, Compound 21a (500 mg, 0.54 mmol), Exatecan mesylate M5 (285 mg, 0.54 mmol), PyBOP (239 mg, 0.6 mmol), HOBt (239 mg, 0.6 mmol) and 10 mL of DMF
30 were added, then DIPEA (248 uL, 1.5 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 21b, and the preparation solution was lyophilized to obtain 231 mg of Compound 21b; LC-MS: [M+H]+=1349.5.
Seventh Step: Compound 21
To a 25 mL single-neck flask, Compound 21b (200 mg, 0.1488 mmol), zinc bromide (665 mg, 2.96 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was
40 concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude
130 product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution of product, and the preparation solution was lyophilized to obtain 103 mg of Compound 21; LC-MS; [M+H]+=l 137.5.
Example 33 Synthesis of Compound 22
Compound M6 and Compound 3c were used as starting materials, and Compound 22 (91 mg) was prepared by referring to the synthetic route of Example 32; LC-MS: [M+H]+=l 165.5.
Example 34 Synthesis of Compound 23 and 24
Benzyl (S)-(-)-lactate was replaced with benzyl (R)-(+)-lactate, and Compound 5c’ was prepared by referring to the synthetic route of Example 8.
Compound M6 and Compound 5c were used as starting materials, and 102 mg of Compound
23 was prepared by referring to the synthetic route of Example 32, LC-MS: [M+H] 1 =1151.4.
Compound M6 and Compound 5c’ were used as starting materials, and 99 mg of Compound
24 was prepared by referring to the synthetic route of Example 32, LC-MS: [M+H]+=1151.4.
Example 35 Synthesis of Compounds 25 and 26
Compound M6 and 7c were used as starting materials, and by referring to the synthetic route
20 of Example 32, 83 mg of Compound 25 was prepared, LC-MS: [M+H]+=1205.7; and 80 mg of
131 Compound 26 was prepared, LC-MS: [M+H]+=1205.7.
Example 36 Synthesis of Compounds 27 and 28
Compound M6 and 19c were used as starting materials, and by referring to the synthetic route of Example 32, 100 mg of Compound 27 was prepared, LC-MS: [M+H]+=1177.5; 101 mg of Compound 28 was prepared, LC-MS: [M+H]+= 1177.5.
Example 37 Synthesis of Compound 29
First Step: Compound SM4-1
10 To a 5000 mL single-neck flask, maleic acid (50 g, 431 mmol, l.Oeq), 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 resulting mixture was heated, refluxed and reacted for 5 h (monitored by TLC), and filtered to remove insoluble substances. The reaction solution was directly evaporated under reduced pressure by a rotation evaporator to remove the solvent, and the residue was purified by a silica column chromatography (PE/EA=50/l-20/l-l/l, to obtain Compound SM4-1 (64.7 g), with a yield of 50%; LC-MS: [M+H]+=299.2.
Second Step: Compound SM4-2
132 To a 2000 mL single-neck flask, Compound SM4-1 (64 g, 215 mmol), and 1000 mL of DMF were added and dissolved, DIPEA (71 mL, 430 mmol) were added, and then nonaethylene glycol monomethyl ether mesylate (111.5 g, 220 mmol) was added. Upon completion of the addition, the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a silica column chromatography (PE/EA=50/l -20/1 -1/1), to obtain 59.9 g of the product; LC-MS: [M+H]+=709.4.
Third Step: Compound SM4
To a 2000 mL single-neck flask, Compound SM4-2 (59 g, 83 mmol), and 1000 mL of MeOH
10 were added and dissolved, thenK^CCL (11.75 g, 85 mmol) was added, and upon completion of the addition, the resulting mixture was reacted at room temperature for 4 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was filtered to remove insoluble substances, and purified by preparative liquid chromatography to obtain a preparation solution. The preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound SM4 (27 g); LC-MS: [M-H]"=693.5.
Fourth Step: Compound M7
To a 500 mL single-neck flask, Compound SM4 (25 g, 36 mmol, l.Oeq), 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
20 resulting mixture was reacted at room temperature for 1 h (monitored by TLC), and the reaction solution was filtered to remove insoluble substances, and directly purified by preparative liquid chromatography to obtain a preparation solution. The preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound M7 (23.3 g), with a yield of 93%; LC-MS: [M+H]+=695.8.
Fifth Step: Compound 29a
To a 25 mL single-neck flask, Compound 1c (1 g, 2.36 mmol), and 25 mL of DMF were added and dissolved, then DIPEA (430 uL, 2.6 mmol) was added, and then Compound M7 (1640 mg, 2.36 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon
30 completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution. The preparation solution was lyophilized to obtain 609 mg of the product; LC-MS: [M-H]"=1098.5.
Sixth Step: Compound 29b
To a 100 mL single-neck flask, Compound 29a (500 mg, 0.45 mmol), Exatecan mesylate 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, then DIPEA (248 uL, 1.5 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 29b, and the
40 preparation solution was lyophilized to obtain 187 mg of Compound 29b; LC-MS:
133 [M+H]+=1517.6.
Seventh Step: Compound 29
To a 25 mL single-neck flask, Compound 29b (150 mg, 0.988 mmol), zinc bromide (223 mg, 0.988 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution of product, and the preparation solution was lyophilized to obtain 114 mg of Compound 29; LC-MS; [M+H]+=1417.9.
10 Example 38 Synthesis of Compound 30
Compound M7 and Compound 3c were used as starting materials, Compound 30 (125 mg) was prepared by referring to the synthesis route of Example 37; LC-MS: [M+H]+=1445.6.
Example 39 Synthesis of Compounds 31 and 32 ,0 ^Ph > n fi f 9 u 9 C H 9 I fi — O p h O
H o
.0 H
N N' N .0
N O' ^N' *0' H H o H o 1 0 HN. H
O ■< HN.
NH n o 1 o NH
O. /A ' /8O' Ox / -O. o, y-N ro'
IF J '8 y-N o
F , s N F
(D AJ O j
.. " T'OH 'OH
31 o > 32 O
Compound M7 and Compound 5c were used as starting materials, and by referring to the synthesis route of Example 37, 61 mg of Compound 31 was prepared, LC-MS; [M+H]+=1431.7.
Compound M7 and Compound 5c’ were used as starting materials, and by referring to the synthesis route of Example 37, 63 mg of Compound 32 was prepared, LC-MS: [M+H]+=1431.7.
20 Example 40 Synthesis of Compounds 33 and 34
Compound M7 and Compound 7c were used as starting materials, and by referring to the synthesis route of Example 37, 60 mg of Compound 33 was prepared, LC-MS: [M+H] ' = 1485.6; and 58 mg of Compound 34 was prepared, LC-MS: [M+H]+=1485.6.
134 Example 41 Synthesis of Compound 35 and 36
Compound M7 and 19c were used as starting materials, and by referring to the synthesis route of Example 37, 102 mg of Compound 35 was prepared, LC-MS: [M+H]+=1457.8; and 102 mg of Compound 36 was prepared, LC-MS: [M+H]+=1457.8.
Example 42 Synthesis of Compound 37
First Step: Compound SM5-1
To a 2000 mL single-neck flask, Compound 16947-84-5 (100 g, 295 mmol, l.Oeq), DIPEA
10 (50 mL, 300 mmol), benzyl bromide (51.3 g, 300 mmol) and THE (1000 mL) were added and reacted at room temperature for 12 h (the reaction was monitored by TLC) The reaction solution was filtered to remove insoluble substances and directly evaporated under reduced pressure by a rotation evaporator to remove the solvent, and the residue was purified by silica column chromatography (PE/EA=50/1 -20/ 1-2/1), to obtain Compound SM5-1 (110.1 g), with a yield of 87%; LC-MS: [M+H]+=429.2.
Second Step: Compound SM5-2
To a 2000 mL single-neck flask, Compound SM5-1 (100 g, 233.4 mmol, 1.0 eq) and THF (1000 mL) were added, and cooled in an ice bath to 0 °C, and then NaH (37.4 g, 933.5 mmol), and
1 35 Mel (132.5 g, 933.5 mmol) were added in batch. The resulting mixture was reacted at 0 °C for 24 h (the reaction was monitored by TLC), and then 500 mL of a saturated NH4CI aqueous solution was added to quench the reaction. The reaction solution was extracted with 500 mL of ethyl acetate three times, and the organic phase was dried with anhydrous sodium sulfate and fdtered. The filtrate was directly evaporated under reduced pressure by a rotation evaporator to remove the solvent, and the residue was purified by silica column chromatography (PE/EA=100/l-50/l-10/l) to obtain Compound SM5-2 (37.1 g); LC-MS: [M+H]+=443.3.
Third Step: Compound SMS (referring to the literature Org. Lett., 2006, 8, 3387-3390.)
To a 1000 mL single-neck flask, Compound SM5-2 (35 g, 79 mmol, l.Oeq) and DCE (500
10 mL) was added, then palladium diacetate (180 mg, 0.8 mmol), I2 (20 g, 79 mmol), and iodobenzene diacetate (40.8 g, 126.4 mmol) were added in order. The resulting mixture was heated to 60 °C and reacted for 40 h (the reaction was monitored by TLC), then 500 mL of a sodium thiosulfate aqueous solution was added to quench the reaction. The reaction solution was extracted with 500 mL of ethyl acetate three times, and the organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was directly evaporated under reduced pressure by a rotation evaporator to remove the solvent, and the residue was purified by silica column chromatography (PE/EA=100/l- 50/1-10/1) to obtain Compound SM5 (28 g); LC-MS: [M+H]+=501.3.
Fourth Step: Compound SM6
To a 500 mL single-neck flask, Compound SM5 (25 g, 50 mmol, l.Oeq), potassium di-t-
20 butylphosphate (13.66 g, 55 mmol, 1.1 eq), p-toluenesulfonic acid monohydrate (951 mg, 5 mmol, 0.1 eq) and THE (200 mL) were added and reacted at room temperature for 1 h (the reaction was monitored by TLC). The reaction solution was filtered to remove insoluble substances, and directly purified by preparative liquid chromatography to obtain a preparation solution, and the preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound SM6 (15.1 g), with a yield of 46%; LC-MS: [M+H]+=651.4.
Fifth Step: Compound SM7
To a 250 mL single-neck flask, Compound SM6 (15 g, 23 mmol) and 100 mL of DMF were added and dissolved, then 15 g of 5% Pd/C was added in an ice bath, and then the atmosphere in
30 the system was replaced with hydrogen gas three time. The resulting mixture was reacted at room temperature for 12 h, and filtered to remove Pd/C, and the filtrate was evaporated under reduced pressure with an oil pump to remove the solvent, to obtain a crude product ready for use.
To another 250 mL single-neck flask, the above crude product, 100 mL of toluene, triethylamine (6.4 mL, 46 mmol), and maleic anhydride (2.4 g, 24 mmol) were added and dissolved, the resulting mixture was heated to 100 °C and reacted for 2 h. The reaction progress was monitored by HPLC, and after the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution. The preparation solution was extracted with dichloromethane, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 4.2 g of solid
40 Compound SM7, with a yield of 36%; LC-MS: [M+H]+=507.3.
136 Sixth Step: Compound M8
To a 100 mL single-neck flask, Compound SM7 (4 g, 7.9 mmol, l.Oeq), pentafluorophenol (1.6 g, 8.7 mmol, 1.1 eq), DCC (1.8 g, 8.7 mmol, 1.1 eq) and THE (60 mL) were added and reacted at room temperature for 1 h (the reaction was monitored by TEC), and the reaction solution was filtered to remove insoluble substances, and directly purified by preparative liquid chromatography to obtain a preparation solution, and the preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound M8 (3.7 g), with a yield of 70%; LC-MS: [M+H]+=673.2.
Seventh Step: Compound 37a
10 To a 25 mL single-neck flask, Compound 1c (1 g, 2.36 mmol), and 25 mL of DMF were added and dissolved, then DIPEA (430 uL, 2.6 mmol) was added, and then Compound M8 (1.2 g, 2.36 mmol) was added. Upon completion of the additions, the resulting mixture was heated to room temperature and reacted for 1 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was purified by a preparative high-performance liquid chromatography to obtain a preparation solution, the preparation solution was lyophilized to obtain 488 mg of Compound 37a; LC-MS: (M-H]’=91L0.
Eighth Step: Compound 37b
To a 100 mL single-neck flask, Compound 37a (400 mg, 0.44 mmol), Exatecan mesylate M5 (235 mg, 0.44 mmol), PyBOP (199 mg, 0.5 mmol), HOBt (69 mg, 0.5 mmol) and 10 mL of DMF
20 were added, then DIPEA (218 uL, 1.32 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 37b, and the preparation solution was lyophilized to obtain 201 mg of Compound 37b; LC-MS: [M+H]+=1329.6.
Ninth Step: Compound 37
To a 25 mL single-neck flask, Compound 37b (130 mg, 0.098 mmol), zinc bromide (221 mg, 0.98 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was
30 concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 96 mg of Compound 37; LC-MS: [M+H]+=1117.4.
Example 43 Synthesis of Compound 38
Compound M8 and Compound 3c were used as starting materials, and Compound 38 (51 mg)
137 was prepared by referring to the synthesis route of Example 42; LC-MS: [M+H]+=l 145.6.
Example 44 Synthesis of Compound 39 and 40
Compound M8 and Compound 5c were used as starting materials, and by referring to the synthesis route of Example 42, 57 mg of Compound 39 was prepared, LC-MS: [M+H]+=l 131.4.
Compound M8 and Compound 5c’ were used as starting materials, and 60 mg of Compound 40 was prepared by referring to the synthesis route of Example 42, LC-MS: [M+H]+=1131.4.
Example 45 Synthesis of Compound 41 and 42
10 Compound M8 and Compound 7c were used as starting materials, and by referring to the synthesis route of Example 42, 44 mg of Compound 41 was prepared, LC-MS: [M+H]+=l 185.3; and 44 mg of Compound 42 was prepared, LC-MS: [M+H]+=l 185.3.
Example 46 Synthesis of Compound 43 and 44
Ph
CL O o O
H H N N
^N' N N 1 X
H H H H H H o O O o HN*. .
'NH NH
<o <X / o, /
> ,OH y-N P O i .OH y-N 'N^ F u N F
O<P"OH
CD -yj O'AOH
CD -y) f'CDH f'OH
43 O Z 44 o
Compound M8 and Compound 19c were used as starting materials, and by referring to the synthesis route of Example 42, 62 mg of Compound 43 was prepared, LC-MS: [M+H]+=l 157.4; and 59 mg of Compound 44 was prepared, LC-MS: [M+H]+=l 157.4.
Example 47 Synthesis of Compound 46
138
First Step: Compound 46a
To a 50 mL single-neck flask, Compound Id (500 mg, 0.62 mmol), Compound 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, then DIPEA (378 uL, 2.29 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 46a (210 mg); LC-MS: [M+H]+=1221.6.
10 Second Step: Compound 46
To a 25 mL single-neck flask, Compound 46a (200 mg, 0.162 mmol), zinc bromide (736 mg, 3.26 mmol) and 10 mL of nitromethane were added and reacted 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 46 (120 mg); LC-MS: [M+H]+=1065.3.
Example 48 Synthesis of Compound 47
20 Compound M3 was replaced with Compound ent-M3, and Compound Id’ was prepared by referring to the synthetic route of Example 4.
Compound Id was replaced with Compound Id’, and Compound 47 (81 mg) was prepared by referring to the synthetic route of Example 47; LC-MS; [M+H]+=1065.3.
Example 49 Synthesis of Compound 48A
139
First Step: Compound 48a
To a 100 mL single-neck flask, Compound 5d (1.66 g, 2.02 mmol, 1.0 eq), Compound 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, then DIPEA (0.84 mL, 1.5 eq) was added in an ice bath, and the resulting mixture was heated to room temperature and reacted for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative liquid chromatography, and the preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to remove acetonitrile, and lyophilized to obtain Compound 48a (1.54 g), with a yield of
10 61%; LC-MS: [M+H]+=1235.4.
Second Step: Compound 48A
To a 100 mL single-neck flask, Compound 48a (1.0 g, 0.8 mmol, 1.0 eq), and 35 mL of nitromethane were added and dissolved, then zinc bromide (3.64 g, 16 mmol, 20.0 eq) was added, and then the resulting mixture was reacted in an oil bath at 40 °C (pre-heated for stabilization in advance) for 30 min. The reaction solution was concentrated to remove nitromethane under reduced pressure with a water pump in an water bath at 45°C, to obtain a yellow residue solid (monitored by HPLC). Through purification by preparative liquid chromatography, a preparation solution was obtained by adding 0.1% trifluoroacetic acid to the flowing phase, and the preparation solution was concentrated under reduced pressure with a water pump in a water bath at 35 °C to
20 remove acetonitrile, and lyophilized to obtain Compound 48A (786 mg) with a yield of 90%.
Example 50 Synthesis of Compound 48B
140
First Step: Compound 48b
To a 25 mL single-neck flask, Compound 5d- 1 (200 mg, 0.24 mmol, 1.0 eq), Compound M9 (127 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2eq), HOBt (48 mg, 0.36 mmol, 1.2 eq) and DMF (6 mL) were added and cooled to 0 °C to 5°C in an ice bath, then DIPEA (62 mg, 0.48 mmol, 2.0 eq) was added. Upon completion of the addition, the resulting mixture was heated to 20±5 °C and reacted for 2 h, and the reaction was monitored with HPLC Upon completion of the reaction, the reaction solution was directly purified by preparative HPLC. The preparation solution was collected and lyophilized to obtain Compound 48b (150.2 mg); LC-MS:
10 [M+H]+=1235.4.
Second Step: Compound 48B
To a 25 mL single-neck flask, Compound 48b (100 mg, 0.081 mmol, 1.0 eq), ZnBr2 (364 mg, 1.62 mmol, 20.0 eq) and CH3NO2 (10 mL) were added, and upon completion of the addition, the resulting mixture was heated to 40 °C and reacted for 0.5 h, then the reaction is stopped. The reaction solution was directly dried under reduced pressure at 45 °C, to obtain a yellow solid. By sampling, the reaction was monitored by HPLC. The dried solid was directly purified by preparative HPLC. The preparation solution was collected and lyophilized to obtain Compound 48B (70.0 mg); LC-MS: [M+H]+= 1079.4.
Example 51 Synthesis of Compound 49A
Compound 5c was rreeppllaacceedd wwiitthh CCoommppoouunndd 55cc’’,, aanndd CCoommppoouunndd 55dd’’ was prepared by referring to the synthetic route of Example 8. Compound 5d was replaced with Compound 5d’, and Compound 49A (71 mg) was prepared by referring to the synthetic route of Example 49; LC- MS: [M+H]+=1079.4.
141 Example
Compound 5c was replaced with Compound 5c-L, and Compound 5d-l ’ was prepared by referring to the synthetic route of Example 9. Compound 5d-l was replaced with Compound 5d- 1’, and Compound 49B (65 mg) was prepared by referring to the synthetic route of Example 50; LC-MS: [M+H]+=1079.4.
Example 53 Synthesis of Compound 50A and Compound SOB
First Step: Compound 50a and Compound 50b
10 To a 50 mL single-neck flask, Compound 7d (500 mg, 0.57 mmol), Compound M9 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, then DIPEA (378 uL, 2.29 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 50a and Compound 50b, respectively. The preparation solutions were lyophilized respectively 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.
Second Step: Compound 50A
142 To a 25 mL single-neck flask, Compound 50a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 44 mg of Compound 50A.
Third Step: Compound SOB
10 To a 25 mL single-neck flask, Compound 50b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 45 mg of Compound 50B.
Example 54 Synthesis of Compound 51A and Compound 51B
First Step: Compound 51a and Compound 51b
20 To a 50 mL single-neck flask, Compound 8d (500 mg, 0.57 mmol), Compound M9 (305 mg, 0.57 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, then DIPEA (378 uL, 2.29 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separate and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 51a and
143 Compound 51b, respectively. The preparation solutions were lyophilized respectively to obtain 190 mg of Compound 51a, and 186 mg of Compound 51b. LC-MS of Compound 51a: [M+H]+=1289.47; LC-MS of Compound 51b; [M+H]+=1289.47.
Second Step: Compound 51A
To a 25 mL single-neck flask, Compound 51a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude
10 product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 39 mg of Compound 51A.
Third Step: Compound 51B
To a 25 mL single-neck flask, Compound 51b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a
20 preparation solution, and the preparation solution was lyophilized to obtain 60 mg of Compound 51B.
Example 55 Synthesis of Compound 52A
144
First Step: Compound 52a
To a 50 mL single-neck flask, Compound l id (800 mg, 0.96 mmol), Compound 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, then DIPEA (660 uL, 4.0 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 4 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 52a, and the preparation solution was lyophilized to obtain Compound 52a (388 mg); LC-MS: [M+H]+=1261.4.
10 Second Step: Compound 52A
To a 25 mL single-neck flask, Compound 52a (150 mg, 0.12 mmol), zinc bromide (532 mg, 2.4 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 52A(79 mg); LC-MS: [M+H]+=l 105.4.
Example 56 Synthesis of Compound 52B
.o O o
H N ,O
N O Y" H H
O HN,„
O J NH
0 0
H H o.
N. N. OH ^COOH y— N j
C yr N N 'O’ H H ‘lA F
0 0 0 O
O,
NBoc
11d’ 52B V'OH
^COO'Bu O
Compound M3 was replaced with Compound C//Z-M3, and Compound lid’ was prepared by
20 referring to the synthetic route of Example 18.
Compound l id was replaced with Compound lid’, and Compound 52B (50 mg) was prepared by referring to the synthetic route of Example 55; LC-MS; [M+H]+= 1105.4.
Example 57 Synthesis of Compound 53A
145 MeSO3H*H2N,,.
0
O.
^■6- N' 'O' X)H y-N
S 1 H u ‘F PyBOP, HOBt, DIPE
12d A A, DMF NBoc ox s/J ^COOfeu n-"oH d M9
^COOfeu ^COOH -NBoc
0 O _NH
H H O O N. .0- •o H H N. N^,O.
A1 ^N' N' N’
H ,O
O 0 HN. H H Ph O O HN,
'" ZnBr2 A Ph
0. / Ox / y— N r j y-N
F AJ o F
53e ° j 53A o, yr'OH L"OH 0 / O
First Step: Compound 53a
To a 50 mL single-neck flask, Compound 12d (400 mg, 0.47 mmol), Compound 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, then DIPEA (330 uL, 2.0 mmol) was added in an ice bath, and the resulting mixture was heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution of Compound 53a, and the preparation solution was lyophilized to obtain Compound 53a (200 mg); LC-MS; [M+H]+=1275.4.
10 Second Step: Compound 53A
To a 25 mL single-neck flask, Compound 53a (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 53 A (51 mg); LC-MS: [M+H]+=l 119.4.
Example 58 Synthesis of Compound 53B
^COOH .NH O O : : u 0 H H N N .0. s^°
'N' H A
A O O HN, zo M xPh
H o H o o O,
■M N .N ._ y-N M
N N O' N F o L H H H
O O
NBoc O,
12d" 53B f'OH
^COO'Bu O
20 Compound M3 was replaced with Compound ent-M3, and Compound 12d’ was prepared by referring to the synthetic route of Example 20.
Compound 12d was replaced with Compound 12d’, and Compound 53B (50 mg) was prepared by referring to the synthetic route of Example 57; LC-MS: [M+H]+=l 119.4.
Example 59 Synthesis of Compound 54A and Compound 54B
146
First Step: Compound 54a and Compound 54b
To a 50 mL single-neck flask, Compound 19d (500 mg, 0.59 mmol), Compound 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, then DIPEA (292 uL, 1.77 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 54a and 54b, respectively. The preparation solutions were lyophilized respectively to obtain 103 mg of
10 Compound 54a, LC-MS: [M+H]+=1261.5; and 111 mg of Compound 54b, LC-MS: [M+H]+=1261.5.
Second Step: Compound 54A
To a 25 mL single-neck flask, Compound 54a (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 61 mg of Compound
20 54A; LC-MS: [M+H]+=l 105.4.
Third Step: Compound 54B
147
To a 25 mL single-neck flask, Compound 54b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 57 mg of Compound 54B; LC-MS: [M+H]+=l 105.4.
Example 60 Synthesis of Compound 55A and Compound 55B
O H o N. □ rPhH A /.o
N VrN o H H
O - 0 n 0 HN,
NBoc
O, / ^COOfeu y-N o
O
H N F -v) .N. .OH M9
"N C)
F A H H
O o O PyBOP, HOBt, "V'OH NBoc DIPEA, DMF 55a O
20d ^COOfeu
O o Ph O
H N. SN/ o ,0
•< 'N' y
£ H O ■« nv o H HN,
^NBoc
^COO'Bu o, y-N o
F O ov
'OH
10 55b O
First Step: Compound 55a and Compound 55b
To a 50 mL single-neck flask, Compound 20d (400 mg, 0.47 mmol), Compound 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, then DIPEA (248 uL, 1.5 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 55a and Compound 55b, respectively. The preparation solutions were lyophilized respectively to obtain 100 mg of Compound 55a, LC-MS: [M+H]+=1275.5; and 101 mg of Compound 55b, LC-MS:
20 [M+H] '=1275.5.
Second Step: Compound 55A
148
To a 25 mL single-neck flask, Compound 55a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 42 mg solid Compound 55A; LC-MS: [M+H]+=l 119.4.
Third Step: Compound 55B
To a 25 mL single-neck flask, Compound 55b (100 mg, 0.079 mmol), zinc bromide (357 mg, 1.59 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 45 mg of solid Compound 55B; LC-MS; [M+H]+=l 119.4.
Example 61 Synthesis of Compound 56
20 Compound M3 was replaced with Compound ent-M3, and Compound 20d’ was prepared by referring to the synthetic route of Example 31.
Compound 20d was replaced with Compound 20d’, and Compound 56 (50 mg) was prepared by referring to the synthetic route of Example 60; LC-MS: [M+H]+=l 119.3.
Example 62 Synthesis of Compound 57
149
Compound M3 was replaced with Compound ev?/-M3, and Compound 20d’ was prepared by referring to the synthetic route of Example 31.
Compound 20d was replaced with Compound 20d’, and Compound 57 (50 mg) was prepared by referring to the synthetic route of Example 60; LC-MS; [M+H]+=l 119.4.
Example 63 Synthesis of Compound 58
First Step: Synthesis of Compound 58a
To Exatecan mesylate M5 (15 g, 28 mol, prepared according to the method disclosed in
10 EP0737683A1), 400 mb of DMF was added, and the resulting mixture was cooled in an ice bath to 0°C, to which triethylamine was dropwise added to adjust pH to 7 to 8, then benzyl bromide (9.6 g, 56 mmol) was dropwise added in an ice bath, and then the resulting mixture was heated to room temperature (25 °C) and reacted for 1 h. The reaction was monitored with TEC, upon completion of the reaction, the reaction solution was concentrated under reduced pressure. The obtained crude product was purified by a preparative high performance liquid chromatography (acetonitrile /pure water system). The preparation solution at target peak was collected and concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain about 11 g of yellow solid Compound 58a, with a yield of about 74%, MS m/z: [M+H]+ 526.3.
Second Step: Synthesis of Compound 58b
20 At room temperature, to a 250 mb single-neck flask, Compound 58a (11 g, 21 mol), and 120 mb of formic acid were added in order and dissolved to obtain a bright yellow solution, to which 30 mb of 40% formaldehyde in water were added. The resulting mixture was heated to 50°C and reacted for 1 h, and the reaction was monitored with TEC. Upon completion of the reaction, the reaction solution was cooled to room temperature, and purified by preparative high performance
150 liquid chromatography (acetonitrile/pure water system). The preparation solution at target peak was collected and concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain about 4.5 g of Compound 58b, in a yellow powder solid, with a yield of about 40%, MS m/z: [M+H]+ 540.6.
Third Step: Synthesis of Compound 58:
At room temperature, to a 250 mL single-neck flask, Compound 58b (2.3 g, 4.3 mol) and 100 mL of DMF was added and dissolved to obtain a bright yellow solution, to which 2.3 g of 5% Pd/C were added. The atmosphere in the system was replaced with hydrogen gas. The resulting mixture was reacted for 1.5 at room temperature, and the reaction was monitored with HPLC. Upon
10 completion of the reaction, the reaction solution was filtered to remove Pd/C, concentrated, and purified by preparative high performance liquid chromatography (acetonitrile/pure water system). The preparation solution at target peak was collected and concentrated under reduced pressure to remove acetonitrile, and lyophilized to obtain about 1.0 g of Compound 58, in a yellow powder solid, with a yield of about 52%, MS m/z : [M+H]+ 450.5.
Example 64 Synthesis of Compound 59:
First Step: Compound 59a
To a 50 mL single-neck flask, Compound Id (500 mg, 0.62 mmol), Compound 58 (279 mg, 0.62 mmol), PyBOP (448 mg, 0.86 mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were
20 added, then DIPEA (378 uL, 2.29 mmol) was added in an ice bath, and the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 59a (166 mg); LC-MS: [M+H] ~ l 235.6.
Second Step: Compound 59
To a 25 mL single-neck flask, Compound 59a (100 mg, 0.081mmol), zinc bromide (368 mg, 1.63 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude
30 product was purified by preparative high-performance liquid chromatography, to obtain a
151 preparation solution, and the preparation solution was lyophilized to obtain Compound 59 (43 mg);
LC-MS: [M+H]+=1079.3.
Example 65 Synthesis of Compound 60
.0 O
H N ^O
A N 'O'
H H
>V= o N. NH o
H o H o Ov / N N .OH COOH y-N
N N O' AJ J N F
O H O H o o.
1d' 'OH
COOfBu 60 o
Compound M3 was replaced with Compound e/zZ-M3, and Compound Id’ was prepared by referring to the synthetic route of Example 4.
Compound Id was replaced with Compound Id’, and Compound 60 (40 mg) was prepared by referring to the synthetic route of Example 64; LC-MS: [M+H]+=1079.3.
Example 66 Synthesis of Compound 61
First Step: Compound 61a
To a 100 mL single-neck flask, Compound 5d (1.66 g, 2.02 mmol, 1.0 eq), Compound 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, then DIPEA (0.84 mL, 1.5 eq) was added in an ice bath, and the resulting mixture was heated to room temperature and reacted for 2 h (the reaction was monitored by HPLC). the reaction solution was directly purified by preparative liquid chromatography to obtain a preparation solution, and the preparation solution was concentrated to remove acetonitrile under reduced pressure with a water pump in a water bath at 35 °C, and lyophilized to obtain Compound 61a (1.21 g); LC-MS; [M+H]+=1249.4.
20 Second Step: Compound 61
To a lOOmLsingle-neck flask, Compound 61a (1.0 g, 0.8 mmol, 1.0 eq), 35 mL of nitromethane were added and dissolved, then zinc bromide (3.64 g, 16 mmol, 20.0 eq) was added, and then the resulting mixture was reacted for 30 min in an oil bath at 40 °C (pre-heated for stabilization in advance). The reaction solution was concentrated to remove nitromethane under
152 reduced pressure with a water pump in an water bath at 45 °C, to obtain a yellow residue solid (monitored by HPLC). Through purification by preparative liquid chromatography, a preparation solution was obtained by adding 0.1% trifluoroacetate to the flowing phase, and the preparation solution was concentrated to remove acetonitrile under reduced pressure with a water pump in a water bath at 35 °C , and lyophilized to obtain Compound 61 (786 mg), LC-MS: [M+H]+=1093.6.
Example 67 Synthesis of Compound 62
First Step: Compound 62a
To a 25 mL single-neck flask, Compound 5d-l (200 mg, 0.24 mmol, 1.0 eq), Compound 58
10 (110.3 mg, 0.24 mmol, 1.0 eq), PyBOP (187 mg, 0.36 mmol, 1.2eq), HOBt (48 mg, 0.36 mmol, 1.2 eq) and DMF (6 mL) were added and cooled to 0 °C to 5°C in an ice bath, then DIPEA (62 mg, 0.48 mmol, 2.0 eq) was added. Upon completion of the addition, the resulting mixture was heated to 20±5 °C and reacted from 2 h, and the reaction was monitored with HPLC. Upon completion of the reaction, the reaction solution was directly purified by preparative HPLC, and a preparation solution was collected and lyophilized to obtain Compound 62a (120.9 mg); LC-MS: [M+H]+=1249.4.
Second Step: Compound 62
To a 25 mL single-neck flask, Compound 62a (100 mg, 0.081 mmol, 1.0 eq), ZnBr2 (364 mg, 1.62 mmol, 20.0 eq) and CH3NO2 (10 mL) were added in order, and upon completion of the
20 addition, the resulting mixture was heated to 40 °C and reacted for 0.5 h, then the reaction was stopped. The reaction solution was directly dried under reduced pressure at 45 °C, to obtain a yellow solid, the reaction was monitored by HPLC by sampling. The dried solid was directly purified by preparative HPLC, and a preparation solution was collected and lyophilized to obtain Compound 62 (61 mg); LC-MS: [M+H]+=1093.4.
Example 68 Preparation of Compound 63
153
Compound 5d was replaced with Compound 5d’, and Compound 63 (60 mg) was prepared by referring to the synthetic route of Example 66; LC-MS: [M+H]+=1093.4.
Example 69 Preparation of Compound 64
Compound 5d-l was replaced with Compound 5d-l’, and Compound 64 (65 mg) was prepared by referring to the synthetic route of Example 67; LC-MS; [M+H]+=1093.4.
Example 70 Preparations of Compound 65A and Compound 65B
10 First Step: Compound 65a and Compound 65b
To a 50 mL single-neck flask, Compound 7d (500 mg, 0.57 mmol), Compound 58 (256.8 mg, 0.57 mmol), PyBOP (448 mg, 0.86mmol), HOBt (116 mg, 0.86 mmol) and 15 mL of DMF were added, then DIPEA (378 uL, 2.29 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC.
After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 65a and
154 65b, respectively. The preparation solutions were lyophilized respectively 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.
Second Step: Compound 65A
To a 25 mL single-neck flask, Compound 65a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude
10 product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 49 mg of solid Compound 65A.
Third Step: Compound 65B
To a 25 mL single-neck flask, 65b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the
20 preparation solution was lyophilized to obtain 47 mg of solid Compound 65B.
Example 71 Synthesis of Compound 66A and Compound 66B
155 H /N* /
O
H XJ O
H CF ";3 _ ,N OH Ox /
N N" 'O' ! V-N
A H H
O O O 20 -V) F PyBOP, HOBt, DIPEA, DMF NBoc
8d Ox ^COO'Bu ~"T'OH
O 58
O CF3 O O CF3
H N. «' V ? A H N YN N 0^° N O' H M H N. to NBoc o M o to »' O o NBoc
Ox / Ov / XoO'Bu y-N M] XoO*Bu y-N
F o F /"AJ /"V? 0 J o y w™ V 1''OH
66a o 7 66b O 7
First Step: Compound 66a and Compound 66b
To a 50 mL single-neck flask, Compound 8d (500 mg, 0.57 mmol), Compound 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, then DIPEA (378 uL, 2.29 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 66a and Compound 66b, respectively. The preparation solutions were lyophilized respectively to obtain
10 160 mg of Compound 66a, and 160 mg of Compound 66b. LC-MS of Compound 66a: [M+H]+=1303.7; LC-MS of Compound 66b: [M+H]+=1303.6.
Second Step: Compound 66A
To a 25 mL single-neck flask, Compound 66a (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 57 mg of Compound
20 66A, LC-MS: [M+H]+=l 147.5.
Third Step: Compound 66B
156
To a 25 mL single-neck flask, Compound 66b (100 mg, 0.077 mmol), zinc bromide (349 mg, 1.55 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 57 mg of Compound 66B, LC-MS: [M+H]+=l 147.5.
Example 72 Synthesis of Compound 67A
First Step: Compound 67a
To a 50 mL single-neck flask, Compound lid (800 mg, 0.96 mmol), Compound 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, then DIPEA (660 uL, 4.0 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 4 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 67a, and the preparation solution was lyophilized to obtain Compound 67a (402 mg); LC-MS: [M+H]+=1275.4.
Second Step: Compound 67A
20 To a 25 mL single-neck flask, Compound 67a (100 mg, 0.78 mmol), zinc bromide (356 mg, 1.57 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 67A (47 mg); LC-MS: [M+H]+=l 119.5.
1 57 Example 73 Synth
Compound lid was replaced with Compound lid’, and Compound 67B (50 mg) was prepared by referring to the synthetic route of Example 72; LC-MS: [M+H]+ 1119.4.
Example 74 Synthesis of Compound 68A
First Step: Compound 68a
To a 50 mL single-neck flask, Compound 12d (400 mg, 0.47 mmol), Compound 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
10 added, then DIPEA (330 uL, 2.0 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 3 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution of Compound 68a, and the preparation solution was lyophilized to obtain Compound 68a (177 mg); LC-MS: [M+H]+=1289.4.
Second Step: Compound 68A
To a 25 mL single-neck flask, Compound 68a (100 mg, 0.08 mmol), zinc bromide (360 mg, 1.6 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude
20 product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 68A (45 mg); LC-MS: [M+H]+=1133.4.
Example 75 Synthesis of Compound 68B
1 58
Compound 12d was replaced with Compound 12d’, and Compound 68B (50 mg) was prepared by referring to the synthetic route of Example 74; LC-MS; [M+H]+=1133.4.
Example 76 Synthesis of Compound 69A and Compound 69B
First Step: Compound 69a and Compound 69b
To a 50 mL single-neck flask, Compound 19d (500 mg, 0.59 mmol), Compound 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, then DIPEA (292 uL, 1.77 mmol) was added in an ice bath, and then the resulting mixture
10 was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 69a and Compound 69b, respectively. The preparation solutions were lyophilized respectively 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.
Second Step: Compound 69A
To a 25 mL single-neck flask, Compound 69a (100 mg, 0.078 mmol), zinc bromide (352 mg,
159 1 .56 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 53 mg of Compound 69A; LC-MS: [M+H]+=1119.4.
Third Step: Compound 69B
To a 25 mL single-neck flask, Compound 69b (100 mg, 0.078 mmol), zinc bromide (352 mg,
10 1.56 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 54 mg of Compound 69B; LC-MS: [M+H]+=1119.4.
Example 77 Synthesis of Compound 70A and Compound 70B ph
O
H N
N XN N O' H O H 1 H o N,
NBoc
O, / ^COO'Bu y—N
H 0 A JM F N .OH 58 O J
PA v 0 k H H H
O PyBOP, HOBt,
NBoc o O "T'OH DIPEA, DMF 70a O /
20d
^COO'BU Ph
O 0 rY f H N YNA' -^ V N O' i H O \ O 1 0 Z|IL NBoc ^cooteu o. y-N r j
F o
0,
'OH
70b O
First Step: Compound 70a and Compound 70b
To a 50 mL single-neck flask, Compound 20d (400 mg, 0.47 mmol), Compound 58 (211.7
20 mg, 0.47 mmol), PyBOP (223 mg, 0.56 mmol), HOBt (83 mg, 0.56 mmol) and 10 mL of DMF were added, then DIPEA (248 uL, 1.5 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by
160 preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 70a and Compound 70b, respectively, respectively. The preparation solutions were lyophilized respectively to obtain 106 mg of Compound 70a, LC-MS: [M+H]+=1289.5; and 101 mg of Compound 70b, LC-MS; [M+H]+=1289.4.
Second Step: Compound 70A
To a 25 mL single-neck flask, Compound 70a (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.57 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was
10 concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 39 mg of Compound 70A; LC-MS: [M+H]+=1133.4.
Third Step: Compound 70B
To a 25 mL single-neck flask, Compound 70b (100 mg, 0.078 mmol), zinc bromide (352 mg, 1.56 mmol) and 5 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude
20 product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 35 mg of Compound 70B; LC-MS: [M+H]+=1133.4.
Example 78 Synthesis of Compound 71
.0 Ph
0
H
N -^N
H H
O O
NH
O, /
^COOH y-N r
S, j *N/ F
CD
"T'CH
71 O /
Compound 20d was replaced with Compound 20d’, and Compound 71 (30 mg) was prepared
161 by referring to the synthetic route of Example 77; LC-MS: [M+H]+=l 133.3.
Example 79 Synthesis of Compound 72 o Ph
O
H H N ,N
N
H o o H O /N*v
NH
Ox / ^COOH V-N
A.J J N F
(D
"'V'OH
72 O
Compound 20d was replaced with Compound 20d’, and Compound 72 (33 mg) was prepared by referring to the synthetic route of Example 77; LC-MS: [M+H]+=l 133.4.
Example 80 Synthesis of Compound Mil
To a 100 mL single-neck flask, Compound M3 (11.0 g, 19.5 mmol, l.Oeq), DIPEA (2.8 g, 21.4 mmol, 1.1 eq), 27-amino-4,7,10,13,16,19,22,25-octaoxaheptacosanoic acid (9.7 g, 20.5 mmol,
10 1.05 eq) and DMF (60 mL) were added and reacted at room temperature for 20 min (the reaction was monitored by TLC). The reaction solution was directly purified by preparative liquid chromatography to obtain a preparation solution, and the preparation solution was concentrated to remove acetonitrile under reduced pressure with a water pump in a water bath at 35 °C, and lyophilized to obtain Compound M10 (13.2 g), with a yield of 78%; LC-MS: [M+H]+=866.5.
To a 100 mL single-neck flask, Compound M10 (13.0 g, 15 mmol, l.Oeq), pentafluorophenol (3 g, 16.5 mmol, 1.1 eq), DCC (3.4 g, 16.5 mmol, 1.1 eq) and THE (30 mL) were added and reacted at room temperature for 30 min (the reaction was monitored by TLC), and then the reaction solution was filtered to remove insoluble substances, and directly purified by preparative liquid chromatography to obtain a preparation solution, and the preparation solution was concentrated to
20 remove acetonitrile under reduced pressure with a water pump in a water bath at 35 °C, and lyophilized to obtain Compound Mi l (14.2g), with a yield of 92%; LC-MS: [M+H]+=1032.5.
Example 81 Synthesis of Compound 73
1 62 H o o
^ .OH
O M11
O H2N/'YN "Al ' o H s H o DIPEA, DMF, rt
'BuO\ _
N 1c
O Bo&1 6 ph
NH 0 u O < u 0
.OH MS
N O'
H
0 PyBOP, HOBt, DIPEA, DMF
73a
.O o «/FI O ' O '
'A "o
V' N
H H H HN* o BocN ZnBr2, CH3NO2 o,
'BUO2C' N L N J o F
73b
CD
'OH
0 /
.O ,Ph
O ' 0
.0. .O
■^N
N o'
6 H
H 8 H HN.
O Y, N / J
'|A F
73 0 -vJ
[ 'OH
0
First Step: Synthesis of Compound 73a
To Compound Mil (1 g, 0.79 mol), 10 mL of DMF was added, then the resulting mixture was cooled in an ice bath to 0°C, to which Compound 1c (334 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were added, and at this condition, the resulting mixture was reacted for 1 h. The reaction was monitored with TEC, upon completion of the reaction, the reaction solution was purified by preparative high performance liquid chromatography (acetonitrile/pure water system). The preparation solution at target peak was collected and concentrated to move acetonitrile under reduced pressure, and lyophilized to obtain about 1.2 g of Compound 73a, MS m/z:
10 [M+H]+=1271.9.
Second Step: Synthesis of Compound 73b
To a 25 mL single-neck flask, Compound 73a (1.2 g, 0.94 mmol), Exatecan mesylate M5 (500 mg, 0.94 mmol), PyBOP (625 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol) and 15 mL ofDMF were added, then DIPEA (310 mg, 2.4 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 73b (709 mg); LC-MS: [M+H]+=1720.8.
Third Step: Synthesis of Compound 73:
20 To a 25 mL single-neck flask, Compound 73b (200 mg, 0.116 mmol), zinc bromide (523 mg, 2.32 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction
163 was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 73 (88 mg); LC-MS: [M+H]+= 1532.6.
Example 82 Synthesis of Compound 74
Compound M3 was replaced with Compound C///-M3, and Compound MU’ was prepared by
10 referring to the synthetic route of Example 80.
Compound Mi l was replaced with Compound MU’, and Compound 74 (90 mg) was prepared by referring to the synthetic route of Example 81; LC-MS; [M+H]+=1532.6.
Example 83 Synthesis of Compound 75
164 rs /Ph O CH3
H
.OH H2N/Y N O’ M11 o. N H
I o h s O DIPEA, DMF, rt fBuCNx ,
N •'
O NV 5c
L o ph
A NH O U O < " O CH3
.OH M5
N 'O TT H 6 PyBOP, HOBt, DIPEA, DMF
75a
.0 .Ph ' 0 o CH3 -L /O
V-N -^N O'
N H o H HN. BocN ZnBr2, CH3NO2 - *- o,
'BUO2C" y— N I]
F
75b vi
Q
'OH
0 /
,O .Ph o o O CH ' O 3
N .N /L
•O.
N
H A N N Q y
H o 6 HISk
0x / y-N
N F
75
0, Aj
'OH
O
First Step: Synthesis of Compound 75a
To Compound Mil (1 g, 0.79 mol), 10 mL of DMF was added, then the resulting mixture was cooled in an ice bath to 0°C, to which Compound 5c (345 mg, 0.79 mol) and DIPEA (154 mg, 1.19 mol) were added, and then the resulting mixture was reacted for 1 h at this condition. The reaction was monitored with TEC, upon completion of the reaction, the reaction solution was purified by preparative high performance liquid chromatography (acetonitrile/pure water system). The preparation solution at target peak was collected and concentrated to remove acetonitrile under reduced pressure, and lyophilized to obtain 0.9 g of Compound 75a, MS m/z: [M+H]+ =1285.6.
10 Second Step: Synthesis of Compound 75b
To a 25 mL single-neck flask, Compound 75a (700 mg, 0.54 mmol), Exatecan mesylate M5 (289 mg, 0.54 mmol), PyBOP (313 mg, 0.6 mmol), HOBt (81 mg, 0.6 mmol) and 10 mL of DMF were added, then DIPEA (155 mg, 1.2 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was purified by preparative high- performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain 75b (304 mg); LC-MS: [M+H]+=1734.8.
Third Step: Synthesis of Compound 75
To a 25 mL single-neck flask, Compound 75b (200 mg, 0.116 mmol), zinc bromide (523 mg,
20 2.32 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction
165 was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 75 (96 mg); LC-MS: [M+H]+=l 546.6.
E
Compound Mi l was replaced with Compound MU’, and Compound 76 (92 mg) was prepared by referring to the synthetic route of Example 83; LC-MS: [M+H]+=1546.5.
10 E
Compound Mi l was replaced with Compound MU ’, Compound 5c was replaced with Compound 5c’, and Compound 77 (87 mg) was prepared by referring to the synthetic route of Example 83; LC-MS: [M+H]+= 1546.5.
Example 86 Synthesis of Compound 78 Q xPh O QH3
■O
X /N. N 'O/A H HN o H HN
O,
HO2C y— N
A J F
78 Q
'OH
0 /
Compound 5c was replaced with Compound 5c’, and Compound 78 (94 mg) was prepared by referring to the synthetic route of Example 83; LC-MS; [M+H]+=l 546.7.
Example 87 Synthesis of Compound 79 and Compound 80
166
First Step: Synthesis of Compound 79a
To Compound Mi l (1 g, 0.79 mol), 10 mL of DMF was added, then the mixture was cooled in an ice bath to 0°C, to which Compound 20c (377 mg, 0.79 mol) and DIPEA(154 mg, 1.19 mol) were added, and then at this condition, the resulting mixture was reacted for 1 h. The reaction was monitored with TLC, upon completion of the reaction, the reaction solution was purified by a preparative high performance liquid chromatography (acetonitrile/pure water system). The preparation solution at target peak was collected and concentrated to remove acetonitrile under reduced pressure, and lyophilized to obtain about 783 mg of Compound 79a, MS m/z:
10 [M+H]+=1325.8.
Second Step: Synthesis of Compound 79b-l and Compound 79b-2
To a 25 mL single-neck flask, Compound 79a (600 mg, 0.45 mmol), Exatecan mesylate 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, then DIPEA (130 mg, 1 mmol) was added in an ice bath, and then the resulting mixture was heated to room temperature and reacted for 2 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was separated and purified by preparative high-performance liquid chromatography, to obtain preparation solutions of Compound 79b-l and Compound 79b-2, respectively, and the preparation solutions were lyophilized respectively to obtain Compound 79b-l (124 mg); LC-MS: [M+H]+= 1743.0; and Compound 79b-l (122 mg);
167 LC-MS: [M+H]+= 1743.0.
Third Step: Synthesis of Compound 79
.0 Ph
O ' o
.0
AN N o y
N H H d HN
O BocN o, fBuO2C I tA F
79b-1 Q
ZnBr2 'OH
O
.0 Ph
O ' 0 o <' ,0
■\/N N O'
N H
H HN
O HN
O.
HO2C f
N/ F
79 Q
'OH
0 /
To a 25 mL single-neck flask, Compound 79b-l (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 79 (30 mg);
10 LC-MS: [M+H]+=l 586.9.
Fourth Step: Synthesis of Compound 80
168
To a 25 mL single-neck flask, Compound 79b-2 (100 mg, 0.057 mmol), zinc bromide (258 mg, 1.15 mmol) and 10 mL of nitromethane were added and reacted at 40 °C for 1 h, and the reaction was monitored with HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography, to obtain a preparation solution, and the preparation solution was lyophilized to obtain Compound 80 (33 mg); LC-MS: [M+H]+= 1587.0.
Exampl
10
Compound Mi l was replaced with Compound MU’, and Compound 81 (24 mg) was prepared by referring to the synthetic route of Example 87; LC-MS: [M+H]+=1586.9.
Example 89 Synthesis of Compound 82
169
Compound Mil was replaced with Compound MU’, and Compound 82 (29 mg) was prepared by referring to the synthetic route of Example 87; LC-MS: [M+H]+=1586.9.
Example 90 Expression and purification of antibody
Expi293 suspension cells were cultured. On the day before transfection, the cells were seeded on OPM-293 CD05 Medium at a certain density and cultured overnight under the conditions of 37 °C, 5% CO2 and 120 rpm in a cell culture shaker. On the next day, the transfection of antibody- expressed plasmid was performed with PEI-MAX. On the first day and third day after the transfection, OPM-293 Profeed was added, and on the sixth day after the transfection, the
10 supernatant was collected by centrifugation.
The supernatant was purified primarily by using a Protein A affinity chromatography column and then finely purified with hydroxyapatite (CHT) to remove impurities such as multimers, to obtain antibody 18M1, whose heavy and light chain variable regions have the sequences shown below:
18M1
SEQ ID No: 1 (variable region of heavy chain)
QVQLVESGGGLVQPGGSLRLSCAASGFDFNTYYMTWVRQAPGKGLEWVGVIYASGGT
YYATWAKGRFTISKSKNTMYLQMNSLRAEDTAVYYCARAYPDSGDGLDIWGQGTLVTV
20 SS
SEQ ID No: 2 (VH CDR1)
TYYMT
SEQ ID No: 3 (VH CDR2)
VIYASGGTYYATWAKG
SEQ ID No: 4 (VH CDR3) AYPDSGDGLDI
30
SEQ ID No: 5 (heavy chain)
QVQLVESGGGLVQPGGSLRLSCAASGFDFNTYYMTWVRQAPGKGLEWVGVIYASGGT
YYATWAKGRFTISKSKNTMYLQMNSLRAEDTAVYYCARAYPDSGDGLDIWGQGTLVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
170 SGLYSL S S VVT VP S S SLGTQTYICNVNHKP SNTK VDKRVEPK SCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID No: 6 (constant region of heavy chain )
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG
10 PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID No: 7 (variable region of light chain)
DVVMTQSPSSVSASVGDRVTITCQASEDISGWLAWYQQKPGQAPKLLIYWASNLASGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQSTFYGTSDVAAFGGGTKVEIK
SEQ ID No: 8 (VL CDR1)
20 QASEDISGWLA
SEQ ID No: 9 (VL CDR2) WASNLAS
SEQ ID No: 10 (VL CDR3) QSTFYGTSDVAA
SEQ ID No: 11 (light chain)
DVVMTQSPSSVSASVGDRVTITCQASEDISGWLAWYQQKPGQAPKLLIYWASNLASGVP
30 SRFSGSGSGTDFTLTISSLQPEDFATYYCQSTFYGTSDVAAFGGGTKVEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID No: 12 (constant region of light chain)
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID No: 13 (nucleic acid encoding sequence of heavy chain variable region)
CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAG
40 GCTGTCCTGCGCCGCCTCCGGCTTCGACTTCAACACCTACTACATGACCTGGGTGAG
171 GCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCGTGATCTACGCCTCCGGCGGCA
CCTACTACGCCACCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACACCA
TGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCA
GGGCCTACCCCGACAGCGGCGACGGCCTGGACATCTGGGGCCAGGGCACCCTGGTG ACAGTGTCCTCC
SEQ ID No: 14 (nucleic acid encoding sequence of VH CDR1)
ACCTACTACATGACC
10 SEQ ID No: 15 (nucleic acid encoding sequence of VH CDR2)
GTGATCTACGCCTCCGGCGGCACCTACTACGCCACCTGGGCCAAGGGC
SEQ ID No: 16 (nucleic acid encoding sequence of VH CDR3)
GCCTACCCCGACAGCGGCGACGGCCTGGACATC
SEQ ID No: 17 (nucleic acid encoding sequence of heavy chain)
CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAG
GCTGTCCTGCGCCGCCTCCGGCTTCGACTTCAACACCTACTACATGACCTGGGTGAG
GCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCGTGATCTACGCCTCCGGCGGCA
20 CCTACTACGCCACCTGGGCCAAGGGCAGGTTCACCATCTCCAAGTCCAAGAACACCA
TGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCA
GGGCCTACCCCGACAGCGGCGACGGCCTGGACATCTGGGGCCAGGGCACCCTGGTG
ACAGTGTCCTCCGCCTCTACCAAGGGCCCCAGCGTGTTTCCTCTGGCTCCCTCTTCTA
AGTCCACCTCGGGCGGCACAGCCGCGCTGGGTTGCCTGGTGAAGGACTACTTCCCTG
AACCCGTCACCGTGTCCTGGAACTCCGGCGCCTTAACATCTGGCGTGCACACCTTTCC
TGCCGTCCTGCAGAGCTCCGGACTGTACTCTCTTAGTAGCGTGGTGACAGTGCCTAGC
TCATCTCTGGGCACCCAGACCTACATCTGCAACGTCAACCACAAGCCCTCTAACACC
AAGGTGGACAAGCGGGTTGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCACC
TTGCCCCGCCCCTGAACTGCTGGGCGGCCCTTCCGTTTTTCTGTTCCCCCCTAAGCCT
30 AAGGACACCCTGATGATCTCTCGGACACCCGAAGTGACCTGCGTGGTGGTGGACGTG
TCCCATGAAGATCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCAC
AACGCCAAGACCAAGCCAAGAGAAGAACAGTACAACTCCACTTACAGAGTTGTGTC
CGTGCTGACCGTGCTGCATCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGG
TGAGTAACAAGGCTCTGCCTGCCCCTATCGAGAAAACCATCTCTAAGGCTAAGGGAC
AACCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTTCCCGGGACGAGCTGACCAAGA
ACCAGGTGTCCCTGACCTGTCTGGTGAAAGGCTTCTACCCCTCTGACATCGCCGTGG
AATGGGAGTCGAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTG
GACAGCGACGGATCTTTCTTCCTGTACTCCAAGCTGACCGTCGACAAGTCTAGATGG
CAGCAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCTCTGCACAATCACTAC
40 ACCCAGAAGTCCCTGTCCCTATCGCCAGGCTGA
172 SEQ ID No: 18 (nucleic acid encoding sequence of heavy chain constant region)
GCCTCTACCAAGGGCCCCAGCGTGTTTCCTCTGGCTCCCTCTTCTAAGTCCACCTCGG
GCGGCACAGCCGCGCTGGGTTGCCTGGTGAAGGACTACTTCCCTGAACCCGTCACCG
TGTCCTGGAACTCCGGCGCCTTAACATCTGGCGTGCACACCTTTCCTGCCGTCCTGCA
GAGCTCCGGACTGTACTCTCTTAGTAGCGTGGTGACAGTGCCTAGCTCATCTCTGGGC
ACCCAGACCTACATCTGCAACGTCAACCACAAGCCCTCTAACACCAAGGTGGACAAG
CGGGTTGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCACCTTGCCCCGCCCCT
GAACTGCTGGGCGGCCCTTCCGTTTTTCTGTTCCCCCCTAAGCCTAAGGACACCCTGA
10 TGATCTCTCGGACACCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCATGAAGATC
CTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACC
AAGCCAAGAGAAGAACAGTACAACTCCACTTACAGAGTTGTGTCCGTGCTGACCGT
GCTGCATCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGAGTAACAAGG
CTCTGCCTGCCCCTATCGAGAAAACCATCTCTAAGGCTAAGGGACAACCTAGAGAGC
CTCAGGTGTACACCCTGCCTCCTTCCCGGGACGAGCTGACCAAGAACCAGGTGTCCC
TGACCTGTCTGGTGAAAGGCTTCTACCCCTCTGACATCGCCGTGGAATGGGAGTCGA
ACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGA
TCTTTCTTCCTGTACTCCAAGCTGACCGTCGACAAGTCTAGATGGCAGCAGGGCAAC
GTGTTCTCCTGCTCTGTGATGCACGAGGCTCTGCACAATCACTACACCCAGAAGTCCC
20 TGTCCCTATCGCCAGGCTGA
SEQ ID No: 19 (nucleic acid encoding sequence of light chain variable region)
GACGTGGTGATGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTG
ACCATCACCTGCCAGGCCTCCGAGGACATCTCCGGCTGGCTGGCCTGGTACCAGCAG
AAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACTGGGCCTCCAACCTGGCCTCCGGC
GTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCT
CCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGTCCACCTTCTACGGCACCTC
CGACGTGGCCGCCTTCGGCGGCGGCACCAAGGTGGAGATCAAG
30 SEQ ID No: 20 (nucleic acid encoding sequence of VL CDR1)
CAGGCCTCCGAGGACATCTCCGGCTGGCTGGCC
SEQ ID No: 21 (nucleic acid encoding sequence of VL CDR2)
TGGGCCTCCAACCTGGCCTCC
SEQ ID No: 22 (nucleic acid encoding sequence of VL CDR3)
CAGTCCACCTTCTACGGCACCTCCGACGTGGCCGCC
SEQ ID No: 23 (nucleic acid encoding sequence of light chain)
40 GACGTGGTGATGACCCAGTCCCCCTCCTCCGTGTCCGCCTCCGTGGGCGACAGGGTG
173 ACCATCACCTGCCAGGCCTCCGAGGACATCTCCGGCTGGCTGGCCTGGTACCAGCAG
AAGCCCGGCCAGGCCCCCAAGCTGCTGATCTACTGGGCCTCCAACCTGGCCTCCGGC
GTGCCCTCCAGGTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCT
CCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGTCCACCTTCTACGGCACCTC
CGACGTGGCCGCCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCG
CTCCTAGCGTGTTCATCTTCCCCCCTTCCGACGAACAGCTGAAGTCCGGAACCGCTTC
TGTCGTGTGCCTGCTGAACAACTTCTACCCTAGAGAGGCCAAGGTGCAGTGGAAGGT
GGACAACGCCCTGCAATCGGGCAATTCCCAAGAGTCAGTCACTGAGCAGGACTCCA
AGGATTCCACCTACTCCCTGTCTTCTACCCTGACCTTGTCCAAGGCCGACTACGAGAA
10 GCACAAGGTGTACGCCTGCGAGGTGACCCATCAGGGCCTGTCCTCTCCTGTGACAAA
GAGCTTCAACCGGGGCGAATGTTGA
SEQ ID No: 24 (nucleic acid encoding sequence of light chain constant region)
CGGACCGTGGCCGCTCCTAGCGTGTTCATCTTCCCCCCTTCCGACGAACAGCTGAAG
TCCGGAACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTAGAGAGGCCAAG
GTGCAGTGGAAGGTGGACAACGCCCTGCAATCGGGCAATTCCCAAGAGTCAGTCAC
TGAGCAGGACTCCAAGGATTCCACCTACTCCCTGTCTTCTACCCTGACCTTGTCCAAG
GCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCATCAGGGCCTGTC
CTCTCCTGTGACAAAGAGCTTCAACCGGGGCGAATGTTGA
20
Example 91 Preparation of Antibody-Drug Conjugates
The antibody was transferred to an acetate buffer by using an ultrafiltration centrifuge tube, and the concentration was adjusted to 5 mg/mL. TCEP was added in an amount of 25 times of the molar molecular number of the antibody, and at room temperature, the resulting mixture was reacted for 2 h to open the interchain disulfide bonds of the antibody. A linker-payload was added in an amount of 20 times of the molar molecular number of the antibody, and the resulting mixture was reacted at room temperature for 2 hours. After the reaction was completed, the reaction solution was transferred into a histidine buffer by using an ultrafiltration centrifugal tube, while removing unconjugated linker-payload, to obtain a corresponding Antibody Drug Conjugate (ADC)
30 sample.
ADC-1 to ADC- 106 of the following Examples 92 to 197 were prepared and obtained by conjugating the compounds prepared in the examples of the application to antibody 18M1, with reference to this preparation method, in which the sum of nl, n2 and n3 represents the drug to antibody ratio (DAR) of the ADC.
Moreover, it should be noted that when the ADC of the application is prepared, the compound (linker-drug) in the ADC is easy to interact with water molecules under the condition of easy hydrolysis when it is conjugated to the antibody, and a hydrolytic ring-opening reaction occurs, and the hydrolysis site is at the maleimide. When multiple linker-drugs are conjugated to the antibody, depending on different hydrolysis degrees, the following cases may occur: (1) the
174 o maleimides are not hydrolyzed, i.e., each maleimide is in a closed-ring form o ; (2) the maleimides are not completely hydrolyzed, i.e., a part of the maleimides are in a ring-closed form o
^COOH O o , and another part of the maleimides are in a ring-opening form b or X-COOH ■ (3)
^COOH
Xr the maleimides are fully hydrolyzed, i.e. all the maleimides are in the ring-opening form o o
JAH |- or X COOH Hence, in the ADCs prepared in the following Examples 92 to 197, nl, n2, n3 are not 0 at the same time, and the sum of nl, n2 and n3 represents the DAR, the specific value of which is determined in Example 199. Meanwhile, those skilled in the art would understand that, as described above, the number of the linker-drugs conjugated to each antibody may be either the same or different, and therefore, the average number of the linker-drugs conjugated to each
10 antibody may be either an integer or a decimal. Accordingly, when the linker-drug is conjugated to the antibody, the maleimide may be hydrolyzed under the condition of easy hydrolysis, and the hydrolysis degrees may be either the same or different, therefore, nl, n2, and n3 may be either integers or decimals.
Example 92
According to the general conjugation method of Example 91, Compound 2 was conjugated
175 to antibody 18M 1 , to prepare and obtain ADC- 1 .
Example 93
According to the general conjugation method of Example 91, Compound 1 was conjugated to antibody 18M1, to prepare and obtain ADC-2.
Example 94
1 76
According to the general conjugation method of Example 91, Compound 4 was conjugated to antibody 18M1, to prepare and obtain ADC-3.
Example 95
According to the general conjugation method of Example 91, Compound 3 was conjugated
177 to antibody 18M1, to prepare and obtain ADC-4.
Example 96
According to the general conjugation method of Example 91, Compound 5B was conjugated to antibody 18M1, to prepare and obtain ADC-5.
Example 97
178
According to the general conjugation method of Example 91, Compound 5A was conjugated to antibody 18M1, to prepare and obtain ADC-6.
Example 98
179
According to the general conjugation method of Example 91, Compound 6B was conjugated to antibody 18M1, to prepare and obtain ADC-7.
Example 99
180 According to the general conjugation method of Example 91, Compound 6A was conjugated to antibody 18M1, to prepare and obtain ADC-8.
Example 100
According to the general conjugation method of Example 91, Compound 8B was conjugated to antibody 18M1, to prepare and obtain ADC-9.
Example 101
181
According to the general conjugation method of Example 91, Compound 7B was conjugated to antibody 18M1, to prepare and obtain ADC-10.
Example 102
According to the general conjugation method of Example 91, Compound 8A was conjugated
182 to antibody 18M1 , to prepare and obtain ADC-11 .
Example 103
According to the general conjugation method of Example 91, Compound 7A was conjugated to antibody 18M1, to prepare and obtain ADC-12.
Example 104
1 83
According to the general conjugation method of Example 91, Compound 9B was conjugated to antibody 18M1, to prepare and obtain ADC-13.
Example 105
According to the general conjugation method of Example 91, Compound 9A was conjugated
184 to antibody 18M1, to prepare and obtain ADC- 14.
Example 106
According to the general conjugation method of Example 91, Compound 10B was conjugated to antibody 18M1, to prepare and obtain ADC-15.
Example 107
185
According to the general conjugation method of Example 91 , Compound 1 OA was conjugated to antibody 18M1, to prepare and obtain ADC-16.
Example 108
186 According to the general conjugation method of Example 91 , Compound 1 IB was conjugated to antibody 18M1, to prepare and obtain ADC-17.
Example 109
According to the general conjugation method of Example 91, Compound 11 A was conjugated to antibody 18M1, to prepare and obtain ADC-18.
Example 110
187
According to the general conjugation method of Example 91 , Compound 12B was conjugated to antibody 18M1, to prepare and obtain ADC-19.
Example 111
188 According to the general conjugation method of Example 91 , Compound 12A was conjugated to antibody 18M1, to prepare and obtain ADC-20.
Example 112
According to the general conjugation method of Example 91, Compound 13B was conjugated to antibody 18M1, to prepare and obtain ADC-21.
Example 113
1 89
According to the general conjugation method of Example 91, Compound 13 A was conjugated to antibody 18M1, to prepare and obtain ADC-22.
Example 114
190 According to the general conjugation method of Example 91 , Compound 14B was conjugated to antibody 18M1, to prepare and obtain ADC-23.
Example 115
According to the general conjugation method of Example 91, Compound 14A was conjugated to antibody 18M1, to prepare and obtain ADC-24.
Example 116
191
According to the general conjugation method of Example 91, Compound 16A was conjugated to antibody 18M1, to prepare and obtain ADC-25.
Example 117
According to the general conjugation method of Example 91, Compound 15A was conjugated
1 92 to antibody 18M1, to prepare and obtain ADC-26.
Example 118
According to the general conjugation method of Example 91, Compound 16B was conjugated to antibody 18M1, to prepare and obtain ADC-27.
Example 119
193
According to the general conjugation method of Example 91, Compound 15B was conjugated to antibody 18M1, to prepare and obtain ADC-28.
Example 120
According to the general conjugation method of Example 91, Compound 18A was conjugated
194 to antibody 18M1, to prepare and obtain ADC-29.
Example 121
According to the general conjugation method of Example 91, Compound 17A was conjugated to antibody 18M1, to prepare and obtain ADC-30.
Example 122
195
According to the general conjugation method of Example 91, Compound 18B was conjugated to antibody 18M1, to prepare and obtain ADC-31.
Example 123
196 According to the general conjugation method of Example 91 , Compound 17B was conjugated to antibody 18M1, to prepare and obtain ADC-32.
Example 124
According to the general conjugation method of Example 91, Compound 20A was conjugated to antibody 18M1, to prepare and obtain ADC-33.
Example 125
1 97
According to the general conjugation method of Example 91, Compound 19B was conjugated to antibody 18M1, to prepare and obtain ADC-34.
Example 126
According to the general conjugation method of Example 91, Compound 20B was conjugated
1 98 to antibody 18M1, to prepare and obtain ADC-35.
Example 127
According to the general conjugation method of Example 91, Compound 19A was conjugated to antibody 18M1, to prepare and obtain ADC-36.
Example 128
199
According to the general conjugation method of Example 91, Compound 21 was conjugated to antibody 18M1, to prepare and obtain ADC-37.
Example 129
According to the general conjugation method of Example 91, Compound 22 was conjugated
200 to antibody 18M1, to prepare and obtain ADC-38.
Example 130
According to the general conjugation method of Example 91, Compound 23 was conjugated to antibody 18M1, to prepare and obtain ADC-39.
Example 131
201
According to the general conjugation method of Example 91, Compound 24 was conjugated to antibody 18M1, to prepare and obtain ADC-40.
Example 132
According to the general conjugation method of Example 91, Compound 25 was conjugated
202 to antibody 18M1, to prepare and obtain ADC-41 .
Example 133
According to the general conjugation method of Example 91, Compound 26 was conjugated to antibody 18M1, to prepare and obtain ADC-42.
Example 134
203
According to the general conjugation method of Example 91, Compound 27 was conjugated to antibody 18M1, to prepare and obtain ADC-43.
Example 135
According to the general conjugation method of Example 91, Compound 28 was conjugated
204 to antibody 18M1, to prepare and obtain ADC-44.
Example 136
According to the general conjugation method of Example 91, Compound 29 was conjugated to antibody 18M1, to prepare and obtain ADC-45.
Example 137
205
According to the general conjugation method of Example 91, Compound 30 was conjugated to antibody 18M1, to prepare and obtain ADC-46.
Example 138
206
According to the general conjugation method of Example 91, Compound 31 was conjugated to antibody 18M1, to prepare and obtain ADC-47.
Example 139
207
According to the general conjugation method of Example 91, Compound 32 was conjugated to antibody 18M1, to prepare and obtain ADC-48.
Example 140
208
According to the general conjugation method of Example 91, Compound 34 was conjugated to antibody 18M1, to prepare and obtain ADC-49.
Example 141
209
According to the general conjugation method of Example 91, Compound 33 was conjugated to antibody 18M1, to prepare and obtain ADC-50.
Example 142
210
According to the general conjugation method of Example 91, Compound 35 was conjugated to antibody 18M1, to prepare and obtain ADC-51.
Example 143
211
According to the general conjugation method of Example 91, Compound 36 was conjugated to antibody 18M1, to prepare and obtain ADC-52.
Example 144
212
According to the general conjugation method of Example 91, Compound 37 was conjugated to antibody 18M1, to prepare and obtain ADC-53.
Example 145
213
According to the general conjugation method of Example 91, Compound 38 was conjugated to antibody 18M1, to prepare and obtain ADC-54.
Example 146
214
According to the general conjugation method of Example 91, Compound 39 was conjugated to antibody 18M1, to prepare and obtain ADC-55.
Example 147
215
According to the general conjugation method of Example 91, Compound 40 was conjugated to antibody 18M1, to prepare and obtain ADC-56.
Example 148
216
According to the general conjugation method of Example 91, Compound 42 was conjugated to antibody 18M1, to prepare and obtain ADC-57.
Example 149
217
According to the general conjugation method of Example 91, Compound 41 was conjugated to antibody 18M1, to prepare and obtain ADC-58.
Example 150
218
According to the general conjugation method of Example 91, Compound 43 was conjugated to antibody 18M1, to prepare and obtain ADC-59.
Example 151
219 According to the general conjugation method of Example 91 , Compound 44 was conjugated to antibody 18M1, to prepare and obtain ADC-60.
Example 152
According to the general conjugation method of Example 91, Compound 47 was conjugated to antibody 18M1, to prepare and obtain ADC-61.
Example 153
220
According to the general conjugation method of Example 91, Compound 46 was conjugated to antibody 18M1, to prepare and obtain ADC-62.
Example 154
According to the general conjugation method of Example 91, Compound 48B was conjugated
221 to antibody 18M1, to prepare and obtain ADC-63.
Example 155
According to the general conjugation method of Example 91, Compound 48A was conjugated to antibody 18M1, to prepare and obtain ADC-64.
Example 156
222
According to the general conjugation method of Example 91, Compound 49B was conjugated to antibody 18M1, to prepare and obtain ADC-65.
Example 157
According to the general conjugation method of Example 91, Compound 49A was conjugated
223 to antibody 18M1, to prepare and obtain ADC-66.
Example 158
According to the general conjugation method of Example 91, Compound 5 IB was conjugated to antibody 18M1, to prepare and obtain ADC-67.
Example 159
224
According to the general conjugation method of Example 91, Compound SOB was conjugated to antibody 18M1, to prepare and obtain ADC-68.
Example 160
According to the general conjugation method of Example 91, Compound 51A was conjugated
225 to antibody 18M1, to prepare and obtain ADC-69.
Example 161
According to the general conjugation method of Example 91, Compound 50A was conjugated to antibody 18M1, to prepare and obtain ADC-70.
Example 162
226
According to the general conjugation method of Example 91 , Compound 53B was conjugated to antibody 18M1, to prepare and obtain ADC-71.
Example 163
227 According to the general conjugation method of Example 91 , Compound 53 A was conjugated to antibody 18M1, to prepare and obtain ADC-72.
Example 164
According to the general conjugation method of Example 91, Compound 54A was conjugated to antibody 18M1, to prepare and obtain ADC-73.
Example 165
228
According to the general conjugation method of Example 91, Compound 54B was conjugated to antibody 18M1, to prepare and obtain ADC-74.
Example 166
According to the general conjugation method of Example 91, Compound 55B was conjugated
229 to antibody 18M1, to prepare and obtain ADC-75.
Example 167
According to the general conjugation method of Example 91, Compound 55 A was conjugated to antibody 18M1, to prepare and obtain ADC-76.
Example 168
230
According to the general conjugation method of Example 91, Compound 57 was conjugated to antibody 18M1, to prepare and obtain ADC-77.
Example 169
According to the general conjugation method of Example 91, the compound was conjugated
231 to antibody 18M1, to prepare and obtain ADC-78.
Example 170
According to the general conjugation method of Example 91, Compound 60 was conjugated to antibody 18M1, to prepare and obtain ADC-79.
Example 171
232
According to the general conjugation method of Example 91, Compound 59 was conjugated to antibody 18M1, to prepare and obtain ADC-80.
Example 172
According to the general conjugation method of Example 91, Compound 62 was conjugated
233 to antibody 18M1, to prepare and obtain ADC-81 .
Example 173
According to the general conjugation method of Example 91, Compound 61 was conjugated to antibody 18M1, to prepare and obtain ADC-82.
Example 174
234
According to the general conjugation method of Example 91, Compound 64 was conjugated to antibody 18M1, to prepare and obtain ADC-83.
Example 175
According to the general conjugation method of Example 91, Compound 63 was conjugated
235 to antibody 18M1, to prepare and obtain ADC-84.
Example 176
According to the general conjugation method of Example 91, Compound 66B was conjugated to antibody 18M1, to prepare and obtain ADC-85.
Example 177
236
According to the general conjugation method of Example 91, Compound 65B was conjugated to antibody 18M1, to prepare and obtain ADC-86.
Example 178
According to the general conjugation method of Example 91, Compound 66A was conjugated
237 to antibody 18M1, to prepare and obtain ADC-87.
Example 179
According to the general conjugation method of Example 91, Compound 65 A was conjugated to antibody 18M1, to prepare and obtain ADC-88.
Example 180
238
According to the general conjugation method of Example 91 , Compound 68B was conjugated to antibody 18M1, to prepare and obtain ADC-89.
Example 181
239 According to the general conjugation method of Example 91 , Compound 68 A was conjugated to antibody 18M1, to prepare and obtain ADC-90.
Example 182
According to the general conjugation method of Example 91, Compound 69A was conjugated to antibody 18M1, to prepare and obtain ADC-91.
Example 183
240
According to the general conjugation method of Example 91, Compound 69B was conjugated to antibody 18M1, to prepare and obtain ADC-92.
Example 184
According to the general conjugation method of Example 91, Compound 70B was conjugated
241 to antibody 18M1, to prepare and obtain ADC-93.
Example 185
According to the general conjugation method of Example 91, Compound 70A was conjugated to antibody 18M1, to prepare and obtain ADC-94.
Example 186
242
According to the general conjugation method of Example 91, Compound 72 was conjugated to antibody 18M1, to prepare and obtain ADC-95.
Example 187
According to the general conjugation method of Example 91, Compound 71 was conjugated
243 to antibody 18M1, to prepare and obtain ADC-96.
Example 188
According to the general conjugation method of Example 91, Compound 74 was conjugated to antibody 18M1, to prepare and obtain ADC-97.
Example 189
244
According to the general conjugation method of Example 91, Compound 73 was conjugated to antibody 18M1, to prepare and obtain ADC-98.
Example 190
245 According to the general conjugation method of Example 91 , Compound 76 was conjugated to antibody 18M1, to prepare and obtain ADC-99.
Example 191
According to the general conjugation method of Example 91, Compound 77 was conjugated to antibody 18M1, to prepare and obtain ADC-100.
Example 192
246
According to the general conjugation method of Example 91, Compound 75 was conjugated to antibody 18M1, to prepare and obtain ADC-101.
Example 193
247 According to the general conjugation method of Example 91 , Compound 78 was conjugated to antibody 18M1, to prepare and obtain ADC-102.
Example 194
According to the general conjugation method of Example 91, Compound 82 was conjugated to antibody 18M1, to prepare and obtain ADC- 103.
Example 195
248
According to the general conjugation method of Example 91, Compound 81 was conjugated to antibody 18M1, to prepare and obtain ADC- 104.
Example 196
249 According to the general conjugation method of Example 91 , Compound 79 was conjugated to antibody 18M1, to prepare and obtain ADC- 105.
Example 197
According to the general conjugation method of Example 91, Compound 80 was conjugated to antibody 18M1, to prepare and obtain ADC- 106.
Example 198
The genes encoding the following variable regions of the light chain and the heavy chain (sequences of which were from WO2024012523A1) were constructed into an expression vector
10 containing the constant region of 18M1. The antibody was prepared according to the general antibody preparation method described in Example 90. According to the general conjugation method of Example 91, Compound 5 A was conjugated to the prepared antibody, to prepare and obtain ADC- 107, which is equivalent in quality and stability to ADC-6.
SEQ ID No: 25 (ADC-107-VH)
EVQLVQSGAEVKKPGASVKVSCKASGFTFTDYYMHWVRQAPGQSLEWMGLVYPY NGDISYAQKFQGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARQGNYVNNAIDYWGQG TLVTVSS
SEQ ID No: 26 (ADC-107-VL)
20 DIQMTQSPSSLSASVGDRVTITCKASQNVGNIIAWYQQKPGKSPKALIYLASYRYSG
VPSRFSGSGSGTDFTLTISSLQPEDVATYFCQQYSDSPYTFGQGTKVEIK
Example 199
The genes encoding the following variable regions of the light chain and the heavy chain
250 (sequences of which were from WO2024012523A1) were constructed into an expression vector containing the constant region of 18M1. The antibody was prepared according to the general antibody preparation method described in Example 90. According to the general conjugation method of Example 91, Compound 5 A was conjugated to the prepared antibody, to prepare and obtain ADC- 108, which is equivalent in quality and stability to ADC-6.
SEQ ID No: 27 (ADC-108-VH)
EVQLVQSGAEVKKPGASVKVSCKASGFTFTDYYMHWVRQAPGQSLEWMGLVYPY SGDISYAQKFQGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARQGNYVNNAIDYWGQG TLVTVSS
10
SEQ ID No: 28 (ADC-108-VL)
DIQMTQSPSSLSASVGDRVTITCKASQNVGNIIAWYQQKPGKSPKALIYLASYRYSG
VPSRFSGSGSGTDFTLTISSLQPEDVATYFCQQYSDSPYTFGQGTKVEIK
Example 200 Determination of monomer ratio of ADC
An SEC-HPLC method was used to determine the monomer rate.
Chromatographic column: Biocore SEC-300 5pm, 4.6x300 mm
Manufacturer: NanoChrom, Item No.: B213-050030-04630S
Flowing phase: 50 mM PB+300 mM NaCl+200 mM Arg+5% IPA, pH=6.5
The specific parameters were shown in Table 1.
20 Table 1. Method parameters
Parameters Settings
Flow rates 0.3 mL/min
Wavelength 280 nm
Column temperature 30 °C
Temperature of sample tray Room temperature
Injection volume 20 pg
Maximum pressure 150 bar/15 MPa/2175 PSI
Gradient Constant gradient
Running time 20 minutes
Table 2. Monomer rate data of the ligand-drug conjugates (ADC) disclosed in the application
Monomer
Molecule name Batch No. Aggregate % Degradation% rate %
ADC-6 C230714D101A 1.384 975 1.12
The determination results were shown in Table 2 and FIG. 1. The results showed that the ADCs disclosed in the examples of the application, such as ADC-1 to ADC-106, especially ADC- 6, had the excellent property of low degradation rate and low aggregation rate, and high monomer rate.
251 Example 201 Determination of drug to antibody rate (DAR) of ADC
A RP-HPLC method was used to determine the drug to antibody rate (DAR).
Chromatographic column; Proteomix RP-1000 4.6* 100mm 5pm 1000A
Manufacturer: Sepax Item No. : 465950-4610
The specific parameters were shown in Table 3.
Table 3 : Method parameters
Parameters Settings
Mobile phase A: 0.1%TFAin water; B: 0.1%TFA in acetonitrile
Flow rate 0.5 mL/min wavelength 214 nm and 280 nm
Column temperature 65 °C
Temperature of sample
Room temperature tray
Injection amount 25 pg
Maximum pressure 100bar/10MPa/1450PSI
Flowing
Flow rate Flowing
Time (min) phase B (mL/min) phase A (%) (%)
0.0 0.5 70 30
"T" 04 70 30
Gradient 04 55 45
"CT 05 50 50
~40~ 05 5 95
"43” 05 5 95
434 05 70 30
"Co05 70 30
Table 4: Detailed data of drug to antibody rate (DAR) of ADC Name of Samples Batch No. RP-DAR
ADC-6 C230714D101A 7.47
10 The Determination results were shown in Table 4 and FIG. 2. The results showed that the ADCs disclosed in the examples of the application, such as ADC-1 to ADC-106, especially ADC- 6, had the excellent property of high DAR value, and the drug concentration at the target site would be significantly improved under the same administration dose of the ADC.
Example 202
Plasma stability data of ADC;
A mixed solution of ADC and IgG-depleted plasma was formulated to make the final concentration of the ADC be 0.6 mg/mL, and incubated in a water bath in a 37 °C incubator. The incubation time was set to be 0 day, 3 days, and 7 days, and a non-incubated plasma was set as a
252 control. After incubation, samples were purified and extracted for determination of the drug to antibody ratio (DAR) to evaluate the stability of the ADC in plasma. The results were shown in the following table.
Plasma
Plasma Plasma Plasma without with with with
Name Batch No. incubation incubation incubation incubation and for 0 day for 3 days for 7 days extraction
ADC-6 C230714D101A 747 7.43 7.44 7.45
The results showed that the ADCs as disclosed in the examples of the application, such as ADC-1 to ADC-106, especially ADC-6, exhibited an excellent plasma stability.
Example 203 In Vitro Efficacy Tests 1
In the application, various human tumor cell lines NCI-H446 DLLS #B5C, A375 DLLS #B2G, NCLH1339 DLLS #C3G, NCI-H2286 DLLS #B6G, SHP-77 Wild Type and NCI-H460 were used
10 as in vitro cell models to evaluate the killing effects of bioactive molecules such as antibodies or antibody drug conjugates (ADC) on in vitro cells.
A certain number of tumor cells were seeded into a 96-well culture plate. After the cells adhere to the wall, a sample to be tested diluted in a gradient was added into the well plate with an initial concentration of the antibody or ADC of 500 nM and a 7-fold dilution, with three replicates of each test concentration for a total of 8 test concentration. The sample was incubated at 37 °C with 5% CO2 for 5 days, and the cell viability was detected by using CellTiter-Glo® Luminescence Cell Viability Assay (CTG). The cell viability was calculated using the formula: Cell viability = (Test group - blank group)/(Negative control group - blank group) x 100%, and Graph Pad Prism 9.0 statistical software was used to fit the curve by means of a four-parameter model, with the X
20 axis as the molar concentration (nM) of the tested sample and the ¥ axis as the cell viability (%), then the half-maximal inhibitory concentration (ICso) values of the tested samples were calculate to evaluate the in-vitro tumor cell killing activity of the tested samples.
Table 5. Statistic Table of the IC50 (nM) of the tested samples in an in-vitro cell model
Cell Lines 18M1 ADC-6
NCI-H446 DLLS #B5C >500 0.18
A375 DLLS B2G >500 0.16
NCI-H1339 DZZ5 #C3G >500 0.31
NCI-H2286 DLLS #B6G >500 0.12
SHP-77 Wild Type >500 108.57
NCI-H460 >500 >500
253 The test results were shown in Table 5 and FIG 3 to FIG. 8.
The test results showed that in the above in-vitro cell model, the bioactive molecule 18M1 of the application did not exhibit killing activity on tumor cells; the ADCs disclosed by the examples of the application, such as ADC-1 to ADC- 106, especially ADC-6, had the killing effect on tumor cells, especially the IC5o on NCI-H446 DLLS #B5C, A375 DLLS #B2G, NCI-H1339 DLLS #C3G, NCI-H2286 DLLS #B6G exhibited excellent killing activity.
Example 204 In Vitro Efficacy Tests 2
In the application, various human tumor cell lines A375 DLLS #B2G, NCI-H1339 DLLS #C3G, NCI-H2286 DLLS #B6G, SHP-77 and NCI-H460 were used as in vitro cell models to
10 evaluate the killing effects of bioactive molecules such as antibodies or antibody drug conjugates (ADC) on in vitro cells.
A certain number of tumor cells were seeded into a 96-well culture plate. After the cells adhere to the wall, a sample to be tested diluted in a gradient was added into the well plate with an initial concentration of the antibody or ADC of 500 nM and a 7-fold dilution, with three replicates of each test concentration for a total of 8 test concentration. The sample was incubated at 37 °C with 5% CO2 for 5 days, and the cell viability was detected by using CellTiter-Glo® Luminescence Cell Viability Assay (CTG). The cell viability was calculated using the formula: Cell viability = (Test group - blank group )/(Negative control group - blank group) >100%, and Graph Pad Prism 9.0 statistical software was used to fit the curve by means of a four-parameter model, with the X
20 axis as the molar concentration (nM) of the tested sample and the ¥ axis as the cell viability (%), then the half-maximal inhibitory concentration (ICso) values of the tested samples were calculate to evaluate the in-vitro tumor cell killing activity of the tested samples.
Table 6. Statistic Table of the IC50 (nM) of the tested samples in an in-vitro cell model
Cell Lines ADC-6 ADC-107 ADC-108
A3 75 DLL3#B2G 0.16 0.46 0.47
H1339 DLL3HC3G 0.45 0.83 0.84
H2286 DLL3 B6G 0.09 0.14 0.14
SHP-77 89.92 426.03 290.68
NCI-H460 >500 >500 >500
The test results were shown in Table 9 and FIG. 9 to FIG. 13.
The test results showed that in the above in-vitro cell model, the ADC-6 disclosed by the examples of the application exhibited specific killing activity on /9£/.3-positive tumor cells, and its efficacy is superior to that of ADC-107 and ADC-108. No significant killing activity were observed for the tested ADCs on the /)£/J-negative cell strains (H460).
30 Example 205 In Vivo Efficacy Test 1
The application established the human small cell lung cancer cell line SHP-77-DLL3 #B7G xenograft model in NOD Scid mice, in which the target was positively expressed, to evaluate the
254 in vivo efficacy of the ADCs, ADC-1 to ADC-106, as disclosed by the application, especially ADC- 6. SHP-77-DLL3 #B7G cells (5x l06 cells/mouse in 0.1 mL) were inoculated subcutaneously into the right scapular region of NOD Scid mice aged 6-7 weeks. When the average tumor volume reached around 200 mm3, the mice were randomly divided into a vehicle control group (Vehicle), a 18M1 antibody control group (2 mg/kg) and ADC-6 treatment groups (1 mg/kg, 1.5 mg/kg, 2 mg/kg), with 6 mice per group, and treatment was initiated (DO). Each group was administered by tail vein injection at 10 mL/kg body weight, once per week for 4 consecutive weeks (QW x4).
All groups were observed until 28st day (D28) after grouping and administration. The two- way statistical analysis showed that ADC-6 at doses of 1 mg/kg, 1.5 mg/kg, and 2 mg/kg exhibited
10 significant tumor growth inhibition effect as compared to the vehicle control group (P < 0.05) (see FIG. 14). The one-way statistical analysis of the average tumor volume on 21st day (D21) after grouping and administration showed that ADC-6 at doses of 1.5 mg/kg and 2 mg/kg exhibited significant tumor growth inhibition effect as compared to the vehicle control group QP<0.05) (see Table 7).
Table 7. Pharmacodynamic analysis table of each group in the human small cell lung cancer cell line SHP-77-DLL3 #B7G xenograft model in NOD Scid mice
21st day (D21) after administration
Experiment group Average tumor Relative tumor T/C TGI
P value volume/mm3 volume/mm3 (%) (%)
Vehicle 22446611..4444±±114499..0077 13.02±1.63
18M1 (2 mg/kg) 22440044..3311±±119955..6644 12.24±1.37 98.56 1.44 >0.9999
ADC-6 (1 mg/kg) 22000055..0066±±114455..3300 9.99±0.72 81.50 18.50 0.5261
ADC-6 (1.5 mg/kg) 11006677..3300±±111111..7755 5.33±0.45 43.67 56.33 0.0011
ADC-6 (2 mg/kg) 446677..6600±±6633..7755 2.30±0.24 19.12 80.88 0.0008
Note: 1. Data was expressed as “average value ± standard error”;
2. T/C%=TRTV/CRTVX 100%; TGI%=(1-T/C)X 100%;
3. P value was obtained by comparing the tumor volume of the treatment group and the
20 tumor volume of the vehicle group on D21 (21st day after administration).
Example 206 In Vivo Efficacy Test 2
The application established the human malignant melanoma cell line A375-DLL3 #B2G xenograft model in NOD Scid mice, in which the target was positively expressed, to evaluate the in vivo efficacy of the ADCs, ADC-1 to ADC- 106, as disclosed by the application, especially ADC-
6. A375-DLL3 #B2G cells (5x l06 cells/mouse in 0.1 mL) were inoculated subcutaneously into the right scapular region of NOD Scid mice aged 6-7 weeks. When the average tumor volume reached around 170 mm3, the mice were randomly divided into a vehicle control group (Vehicle), a 18M1 antibody control group (2 mg/kg) and ADC-6 treatment groups (1 mg/kg, 1.5 mg/kg, 2 mg/kg), with 6 mice per group, and the treatment was initiated (DO). Each group was administered by tail
30 vein injection at 10 mL/kg body weight, once per week for 4 consecutive weeks (QW x4).
All groups were observed until 28th day (D28) after grouping and administration. The two-
255 way statistical analysis showed that ADC-6 at doses of 1 mg/kg, 1 .5 mg/kg, and 2 mg/kg exhibited significant tumor growth inhibition effect as compared to the vehicle control group (P < 0.05) (see FIG. 15). The one-way statistical analysis of the average tumor volume on 28th day (D28) after grouping and administration showed that ADC-6 at doses of 1.5 mg/kg and 2 mg/kg exhibited significant tumor growth inhibition effect as compared to the vehicle control group (P<0.05) (see Table 8).
Table 8. Pharmacodynamic analysis table of each group in the human malignant melanoma cell line A375-DLL3 #B2G xenograft model in NOD Scid mice
28th day (D28) after administration
Experiment group Average tumor RReellaattiivvee ttuummoorr T/C TGI
P value volume/mm3 vvoolluummee//mmmm33 (%) (%)
Vehicle 2039.62±152.55 12.46=1=1.37
>0.999
18M1 (2 mg/kg) 1990.71=1=183.66 12.10=1=1.13 97.56 2.44
9
DC-6 (1 mg/kg) 1491.18=1=101.46 8.91±0.66 3.15 26.85 0.1922
ADC-6 (1.5 mg/kg) 449.10±80.95 2.63±0.37 22.07 77.93 0.0003
ADC-6 (2 mg/kg) 2.80±17.08 0.47±0.07 4.03 95.97 0.0006
Note: 1. Data was expressed as “average value ± standard error";
10 2. T/C%=TRTV/CRTV>< 100%; TGI%=(1-T/C)X 100%;
3. P value was obtained by comparing the tumor volume of the treatment group and the tumor volume of the vehicle group on D28 (28th day after administration).
Example 207 In Vivo Efficacy Test 3
The application established the human small cell lung cancer cell line H446-DLL3 #B5C xenograft model in NOD Scid mice, in which the target was positively expressed, to evaluate the in vivo efficacy of the ADCs, ADC-1 to ADC-106, as disclosed by the application, especially ADC- 6. H446-DLL3 #B5C cells (5x 106 cells/mouse in 0.1 mb) were inoculated subcutaneously into the right scapular region of NOD Scid mice aged 6-7 weeks. When the average tumor volume reached around 170 mm3, the mice were randomly divided into a vehicle control group (Vehicle), a 18M1
20 antibody control group (2 mg/kg) and ADC-6 treatment groups (1 mg/kg, 1.5 mg/kg, 2 mg/kg), with 5 mice per group, and the treatment was initiated (DO). Each group was administered by tail vein injection at 10 mL/kg body weight, once per week for 4 consecutive weeks (QW><4).
All groups were observed until 28th day (D28) after grouping and administration. The two- way statistical analysis showed that ADC-6 at doses of 1 mg/kg, 1.5 mg/kg, and 2 mg/kg exhibited significant tumor growth inhibition effect as compared to the vehicle control group (P < 0.05) (see FIG. 16). The one-way statistical analysis of the average tumor volume on 28th day (D28) after grouping and administration showed that ADC-6 at doses of 1.5 mg/kg and 2 mg/kg exhibited significant tumor growth inhibition effect as compared to the vehicle control group, but without statistical significance (P>0.05) (see Table 9).
30 Table 9. Pharmacodynamic analysis table of each group in the human small cell lung cancer
256 cell line H446-DLL3 #B5C xenograft model in NOD Scid mice
28th day (D28) after administration
Experiment group Relative
Average tumor T/C TGI tumor P value volume/mm3 volume/mm3 (%) (%)
Vehicle 22221155..9922±±445511..6688 15.36±3.64
ADC-6 (1 mg/kg) 1776.82±144.29 ) 7.44±1.77 78.37 21.63 0.9851
ADC-6 (1.5 mg/kg) 677.53±104.44 3.93±0.59 30.05 69.95 0.2451
ADC-6 (2 mg/kg) 229911.,2299±±113388..0033 1.52±0.54 13.07 86.93 0.1082
Note: 1. Data was expressed as “average value ± standard error”;
2. T/C %=TRTV/CRTV>< 100%; TGI%=(1-T/C)X 100%;
3. P value was obtained by comparing the tumor volume of the treatment group and the tumor volume of the vehicle group on D28 (28th day after administration).
Example 208 In Vivo Efficacy Test 4
The application established the human small cell lung cancer cell line SHP-77-DLL3 #B7G xenograft model in NOD Scid mice, in which the target was positively expressed, to evaluate the in vivo efficacy of ADC-6, ADC-107 and ADC-108, as disclosed by the application. X315-DLL3
10 #B2G cells (5>< 106 cells/mouse in 0.1 mL) were inoculated subcutaneously into the right scapular region of NOD Scid mice aged 6-7 weeks. When the average tumor volume reached around 200 mm3, the mice were randomly divided into a vehicle control group (Vehicle), ADC-6 treatment groups (1 mg/kg, 2 mg/kg), ADC-107 treatment groups (1 mg/kg) and ADC-108 treatment groups (1 mg/kg), with 5 mice per group, and the treatment was initiated (DO). Each group was administered by tail vein injection at 10 mL/kg body weight, once per week for 3 consecutive weeks (QWx3).
All groups were observed until 21th day (D21) after grouping and administration. The statistical analysis of the average tumor volume on 21th day (D21) after grouping and administration showed that ADC-6 treatment groups (1 mg/kg, 2 mg/kg) exhibited significantly
20 superior tumor growth inhibition effect as compared to the vehicle control group (E<0.05) (see Table 11); the ADC-106 treatment group (1 mg/kg) exhibited significantly better tumor growth inhibition effect as compared to the ADC- 107 treatment group (1 mg/kg) and ADC- 108 treatment group (1 mg/kg) (see FIG. 17).
Table 10. Pharmacodynamic analysis table of each group in the human small cell lung cancer cell line SHP-77-DLL3 #B7G xenograft model in NOD Scid mice
21th day (D21) after administration
Experiment group Average tumor RReellaattiivvee ttuummoorr T/C TGI
P value volume/mm3 vvoolluummee//mmmm33 (%) (%)
Vehicle 2944.72±279.62 15.44±1.58
ADC-6 (1 mg/kg) 965.26±122.45 5.0H0.51 32.46 67.54 0.0009
ADC-6 (2 mg/kg) 57.44±10.04 0.29±0.04 1.9 98.1 0.0031
257 ADC-107 (1 mg/kg) 1308.49±208.32 6.79±0.97 44 56 0.0175
ADC- 108 (1 mg/kg) 1626.12±89.04 8.54±0.39 55.31 44.69 0.0425
Note: 1. Data was expressed as “average value ± standard error”;
2. T/C%=TRTV/CRTVX 100%; TGI%=(1-T/C)x 100%;
3. P value was obtained by comparing the tumor volume of the treatment group and the tumor volume of the vehicle group on D21 (21th day after administration).
258

Claims

What is claimed is:
1. A ligand-camptothecin derivative conjugate represented by Formula I, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof, wherein:
Ab is an antibody targeting human DLL3 or an antigen-binding fragment thereof;
Ln, L12 and L13 each independently are selected from the group consisting of:
L2 has the structure represented by Formula A,
Formula A wherein ¥ is structural unit selected from the group consisting of C1-C6 alkylene, substituted
259 C1-C6 alkylene and C3-C8 cycloalkylene; Ac is a hydrophilic structural unit; the carbon atom at the position of 2 linked to Y has an absolute chirality of R-configuration or S-configuration;
L3 is present or absent, and when present, L3 is a PEG hydrophilic unit o wherein 0 is an integer selected from 1 to 10,
L4 is an enzyme cleavable unit;
L5 is a linking unit;
X is -C(O)-CRaRb-(CR3R4)m-O-, -C(O)-CRaRb-(CR3R4)m-NH- or -C(O)-CRaRb-(CR3R4)m-S-, preferably -C(O)-CRaRb-(CR3R4)m-O-, wherein
Ra and Rb each independently are selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C8 cycloalkyl, C3- C8 cycloalkyl C1-C6 alkyl, C6-C10 aryl C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, 3- to 7- membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6- C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl; or
Ra, Rb and carbon atoms linked thereto form C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, 3- to 7-membered heterocyclyl or substituted 3- to 7-membered heterocyclyl;
R3, R4 each independently are hydrogen, deuterium, halogen, 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- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl; or
R3, R4 and carbon atoms linked thereto form C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, 3- to 7-membered heterocyclyl or substituted 3- to 7-membered heterocyclyl; m is selected from the group consisting of 0, 1, 2, 3 and 4; the carbon atom at the position of 1 linked to N has an absolute chirality of R-configuration or S-configuration;
R is selected from the group consisting of 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- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl;
Ri is selected from the group consisting of 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- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10- membered heteroaryl;
R2 is selected from the group consisting of 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- to 7-membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10- membered heteroaryl; nl, n2 and n3 each independently are any integer of 0 to 10 or any decimal of 0 to 10, and nl,
260 n2 and n3 are not simultaneously 0, with l<nl+n2+n3<10.
2. The ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein Ln, Ln and L13 each independently are selected from the group consisting of: o o , COOK, and COOK; or
Ln, Ln and L13 each independently are selected from the group consisting of:
3. The ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to claim 1 or 2, wherein Ac has a structure of Formula B, wherein:
Z is carboxyl, phosphoryloxy or -(OCFBCFbjiOClL, wherein i is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and preferably i is 1, 2, 3, 4, 5, 6, 7 or 8;
Y’ is a structural unit linking amino group and Z, and is C1-C6 alkylene or carboxylsubstituted C1-C6 alkylene, preferably methylene, ethylidene, carboxyl-substituted methylene or carboxyl-substituted ethylidene, and further preferably methylene, ethylidene, or carboxylsubstituted methylene; or
Ac is a residue formed by removing one hydrogen from the amino terminus of 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, (DI) 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 or (D/L) glutamic acid; preferably, Ac is:
261 further preferably, Ac is:
4. The ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-3, wherein L4 is a peptide residue composed of amino acids, wherein the amino acids are optionally substituted by one or more substituents selected from the group consisting of deuterium, halogen, hydroxyl, cyano, amino, nitro, carboxyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy and C3-C8 cycloalkyl and substituted C3-C8 cycloalkyl; preferably, the peptide residue is a peptide residue formed by one, two or more amino acids selected from the group consisting of phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline (C), serine (S), glutamic acid (E) and aspartic acid (D); more preferably, the peptide residue is a tetrapeptide residue formed by glycine (G) -glycine (G) - phenylalanine (F) - glycine (G).
5. The ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-4, wherein Ls is -NRsfCReRvjq- or a chemical bond, wherein q is 0, 1, 2, 3, 4, 5 or 6, preferably 0, 1, 2 or 3, further preferably 0, 1 or 2, and even further preferably 0 or 1 ;
R5, Re and R? each independently are selected from the group consisting of 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- to 7 -membered heterocyclyl, substituted 3- to 7-membered heterocyclyl, C6-C10 aryl, substituted C6-C10 aryl, 5- to 10-membered heteroaryl, and substituted 5- to 10-membered heteroaryl; preferably, Rs, Re and R? each independently are selected from the group consisting of hydrogen and C1-C6 alkyl; further preferably, Rs, Re and R? each independently are selected from the group consisting of hydrogen and C1-C4 alkyl; further preferably, Rs, Re and R? each independently are selected from the group consisting of hydrogen, methyl, ethyl, n-propyl and n-butyl; more preferably, Rs, Re and R? each independently are hydrogen.
6. The ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-5, characterized by one or more of the following items: i) Ra and Rb each independently are selected from hydrogen, C1-C6 alkyl, halogenated Cl- C6 alkyl, C3-C8 cycloalkyl C1-C6 alkyl or C6-C10 aryl C1-C6 alkyl; or Ra, Rb and carbon atoms
262 linked thereto form C3-C8 cycloalkyl; preferably, Ra is hydrogen or C1-C4 alkyl, Rb is hydrogen, C1-C4 alkyl, halogenated C1-C4 alkyl, C3-C6 cycloalkyl C1-C4 alkyl or phenyl C1-C4 alkyl, or Ra, Rb and carbon atoms linked thereto form C3-C6 cycloalkyl; preferably, Ra is hydrogen, methyl, ethyl, n-propyl or n-butyl, Rb is hydrogen, methyl, ethyl, n-propyl, n-butyl, halogenated methyl, halogenated ethyl, halogenated n-propyl, halogenated n- butyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclopropylethyl, cyclopentylethyl, cyclohexylethyl, phenylmethyl, phenylethyl or phenylpropyl, or Ra, Rb and carbon atoms linked thereto form cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; preferably, Ra is hydrogen or methyl, Rb is hydrogen, methyl, ethyl, trifluoromethyl, cyclopropylmethyl, or phenylmethyl, or Ra, Rb and carbon atoms linked thereto form cyclopropyl, cyclobutyl or cyclopentyl; preferably, preferably, the position shown by the right side wavy line is connected to Ls; ii) Ra, R4 each independently are hydrogen or C1-C6 alkyl; iii) m is 0, 1 or 2, further preferably 0 or 1; iv) R is hydrogen or C1-C6 alkyl; preferably, R is hydrogen or C1-C4 alkyl; preferably, R is hydrogen, methyl, ethyl, n-propyl or n-butyl; preferably, R is hydrogen or methyl; v) Ri is hydrogen or C1-C6 alkyl; preferably, Ri is C1-C6 alkyl; preferably, Ri is C1-C4 alkyl; preferably, Ri is methyl, ethyl, n-propyl or n-butyl; preferably, Ri is methyl; vi) R2 is hydrogen, halogen or C1-C6 alkyl; preferably, R2 is hydrogen, halogen or C1-C4 alkyl; preferably, R2 is halogen; preferably, R2 is fluorine, chlorine, or bromine; preferably, R2 is fluorine; vii) 0 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably 0 is 1, 2, 3, 4, 5, 6, 7 or 8; viii) Y is C1-C6 alkylene; preferably, Y is C1-C4 alkylene; preferably, Y is methylene, ethylidene, propylidene or butylidene; preferably, Y is methylene; ix) nl, n2 and n3 each independently are any integer of 0 to 8 or any decimal of 0 to 8, and nl, n2 and n3 are not simultaneously 0, with I<nl+n2+n3<8; preferably, 5<nl+n2+n3<10; preferably, 6<nl+n2+n3<8; preferably, 7<nl+n2+n3<8.
7. The ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-6, wherein the antibody targeting human DLL3 or the antigen-binding fragment thereof comprises:
(a) the following three heavy chain variable region (VH) complementarity determining
263 regions (CDRs):
(i) VH CDR1, having a CDR1 sequence contained in the VH as set forth in SEQ ID NO: 1, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR1 sequence contained in the VH;
(ii) VH CDR2, having a CDR2 sequence contained in the VH as set forth in SEQ ID NO: 1, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR2 sequence contained in the VH; and
(iii) VH CDR3, having a CDR3 sequence contained in the VH as set forth in SEQ ID NO: 1, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one amino acid) as compared with the CDR3 sequence contained in the VH; and/or
(b) the following three light chain variable region (VL) CDRs:
(iv) VL CDR1, having a CDR1 sequence contained in the VL as set forth in SEQ ID NOV, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR1 sequence contained in the VL;
(v) VL CDR2, having a CDR2 sequence contained in the VL as set forth in SEQ ID NOV, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR2 sequence contained in the VL; and
(vi) VL CDR3, having a CDR3 sequence contained in the VL as set forth in SEQ ID NOV, or having a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of one or two amino acids) as compared with the CDR3 sequence contained in the VL; preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; preferably, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and/or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by Rabat, Chothia or IMGT numbering system.
8. The ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-7, wherein the antibody targeting human DLL3 or the antigen-binding fragment thereof comprises:
CDR1, CDR2 and CDR3 sequences contained in the VH as set forth in SEQ ID NO: 1; and/or CDR1, CDR2 and CDR3 sequences contained in the VL as set forth in SEQ ID NOV; preferably, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH) and/or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by Rabat, Chothia or IMGT numbering system;
264 preferably, the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises:
(a) the following three heavy chain variable region (VH) CDR:
(i) VH CDR1, which is composed of a sequence as set forth in SEQ ID NO:2,
(ii) VH CDR2, which is composed of a sequence as set forth in SEQ ID NON, and
(iii) VH CDR3, which is composed of a sequence as set forth in SEQ ID NON; and/or
(b) the following three light chain variable region (VL) CDR:
(iv) VL CDR1, which is composed of a sequence as set forth in SEQ ID NON,
(v) VL CDR2, which is composed of a sequence as set forth in SEQ ID NO: 9, and
(vi) VL CDR3, which is composed of a sequence as set forth in SEQ ID NO: 10; preferably, the VH of the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: VH CDR1 as set forth in SEQ ID NO:2; VH CDR2 as set forth in SEQ ID NON; and, VH CDR3 as set forth in SEQ ID NON; and/or, the VL of the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO:8; VL CDR2 as set forth in SEQ ID NO: 9; and, VL CDR3 as set forth in SEQ ID NO: 10; preferably, the VH of the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: VH CDR1 as set forth in SEQ ID NON; VH CDR2 as set forth in SEQ ID NON; and VH CDR3 as set forth in SEQ ID NON; and, the VL of the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO:8; VL CDR2 as set forth in SEQ ID NO: 9; and VL CDR3 as set forth in SEQ ID NO: 10; preferably, the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises:
(a) a heavy chain variable region (VH), comprising an amino acid sequence selected from the following sequences:
(i) a sequence as set forth in SEQ ID NO: 1;
(ii) a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence as set forth in SEQ ID NO: 1 ;
(iii) a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO: 1; and
(b) a light chain variable region (VL), comprising an amino acid sequence selected from the following sequences:
(iv) a sequence as set forth in SEQ ID NO: 7;
(v) a sequence with substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence as set forth in SEQ ID NO: 7;
(vi) a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least
265 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO: 7; preferably, the substitution described in (ii) or (v) is a conservative substitution; preferably, the antibody or the antigen-binding fragment thereof comprises; a heavy chain variable region (VH), comprising a sequence as set forth in SEQ ID NO: 1 or a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO: 1, and a light chain variable region (VL), comprising a sequence as set forth in SEQ ID NO:7 or a sequence with a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence as set forth in SEQ ID NO: 7; preferably, the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: a VH with a sequence as set forth in SEQ ID NO:1 and a VL with a sequence as set forth in SEQ ID NO:7.
9. The ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-8, wherein the antibody targeting human DLL3 or an antigen-binding fragment thereof is humanized; preferably, the antibody targeting human DLL3 or an antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin; preferably, the antibody targeting human DLL3 or an antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline antibody gene), and/or, a light chain framework region of a human immunoglobulin (e.g., a light chain framework region contained in an amino acid sequence encoded by a human light chain germline antibody gene); preferably, the heavy chain framework region and/or the light chain framework region optionally comprises one or more (such as, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to murine residues; preferably, the antibody targeting human DLL3 or an antigen-binding fragment thereof comprises: a heavy chain with a sequence as set forth in SEQ ID NO:5 and a light chain with a sequence as set forth in SEQ ID NO: 11; preferably, the antibody or the antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin; preferably, the heave chain of the antibody or the antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (such as IgGl, IgG2, IgG3 or IgG4);
266 preferably, the light chain of the antibody or the antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (such as K or X); preferably, the antibody targeting human DLLS or an antigen-binding fragment thereof comprises; a heavy chain constant region with a sequence as set forth in SEQ ID NO: 6 and a light chain constant region with a sequence as set forth in SEQ ID NO: 12; preferably, the antibody targeting human DLLS or an antigen-binding fragment thereof is selected from the group consisting of monoclonal antibody, mouse antibody, rabbit antibody, humanized antibody, fully human antibody, chimeric antibody (e.g., human-mouse chimeric antibody), bispecific antibody, multi-specific antibody, single chain antibody, dAb, complementarity determining region fragment, Fv, single chain Fv (scfv), Fd, Fab, Fab', and F(ab')2; preferably, the monoclonal antibody includes a non-CDR region, and the non-CDR region is derived from species other than murine, e.g., from a human antibody.
10. The ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-9, wherein
-L11-L2-L3-L4-L5-, -L12-L2-L3-L4-L5- and -L13-L2-L3-L4-L5- each independently are selected from the following structures:
267 preferably, each independently comprise: wherein: Ac, 0, Rs, Re and R? are as defined in any one of claims 1-9; the carbon atom at the position of 2 linked to N has an absolute chirality of R-configuration or S-configuration; the position shown by the left side wavy line is connected to the antibody or an antigenbinding fragment thereof, and the position shown by the right side wavy line is connected to X.
11. The ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-10, wherein the conjugate has a structure represented by Formula II:
268 wherein: Ab, Ln, L12, L13, Ac, L3, X, R, Ri, R2, nl, n2, n3 are as defined in any one of claims 1 to 10; the chiral atom at the position of 1, 2 or 3 has an absolute chirality of R-configuration or S- configuration.
12. The ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-11, wherein the conjugate is selected from the group consisting of:
269
270
271
272
273
274
275 9ZZ LL7,
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308 60S
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329 wherein 18M1 represents Ab; Ab, nl, n2 and n3 are as defined in any one of claims 1-11.
13. A linker-drug compound represented by Formula III-A or IIIB, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof, p— ' 0 O H o H o H Ra ,Rb o L4-L3-L2-L N N N ■Os. X /O r-V N~ R'N'X' N 'm [
H H o o
ACT R'N
1 1
H ,CH3 N o.
II
1 N/ F
R1 Q
R2 'OH
O
III-A m-B
O
F 0 wherein L is selected from the group consisting of 0 H H o 0
L2, L4, L5, X, R, Ri, R2, Ac, L3, m, Ra, Rb are as defined in any one of claims 1-11; the chiral carbon atom at the position of 1, 2 or 3 has an absolute chirality of R-configuration or S-configuration.
14. The linker-drug compound, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to claim 13, wherein the linker-drug compound is selected from the group consisting of:
330 l££ SEE
334
337
338 wherein: o is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, o is 1 , 2, 3, 4, 5, 6, 7 or 8.
15. Use of the linker-drug compound, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13-14 in the manufacture of an antibody-drug conjugate.
16. The ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-12 or the linker-drug compound, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13-14, wherein the pharmaceutically acceptable salt is a salt formed by an acidic functional group in the structural formula with sodium, potassium, calcium or magnesium, or an acetate, a trifluoroacetate, a citrate, an oxalate, a tartrate, a malate, a nitrate, a chloride, a bromide, an iodide, a sulfate, a bisulfate, a phosphate, a lactate, an oleate, an ascorbate, a salicylate, a formate, a glutamate, a methanesulfonate, an ethanesulfonate, a benzenesulfonate or a p-toluenesulfonate formed by a basic functional group in the structural formula with an acid.
340
17. A pharmaceutical composition, comprising the ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-12 or the linker-drug compound, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13-14, and optionally a pharmaceutically acceptable carrier.
18. A pharmaceutical preparation, comprising the ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-12 or the linker-drug compound, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13-14.
19. Use of the ligand-camptothecin derivative conjugate, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-12 or the linker-drug compound, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13-14 or the pharmaceutical composition according to claim 17 or the pharmaceutical preparation according to claim 18 in the manufacture of a medicament for treating or preventing a cancer or tumor; preferably, the cancer or tumor expresses DLL3; more preferably, the cancer or tumor is selected from the group consisting of solid tumor and hematologic tumor, wherein the cancer or tumor comprises adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urinary tract cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, breast cancer (e g., triple-negative breast cancer), rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma, and leukemia.
341
PCT/US2025/026726 2024-04-29 2025-04-29 Anti-human dll3 antibody-camptothecin derivative conjugates and medical uses thereof Pending WO2025230927A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018162724A1 (en) * 2017-03-09 2018-09-13 Mab Discovery Gmbh Antibodies specifically binding to human il-1r7
US20190201542A1 (en) * 2016-05-20 2019-07-04 Abbvie Stemcentrx Llc Anti-dll3 drug conjugates for treating tumors at risk of neuroendocrine transition
WO2023288241A1 (en) * 2021-07-14 2023-01-19 Genentech, Inc. Anti-c-c motif chemokine receptor 8 (ccr8) antibodies and methods of use
US20230241241A1 (en) * 2020-06-19 2023-08-03 Hangzhou Dac Biotech Co., Ltd. Conjugates of a cell-binding molecule with camptothecin analogs

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190201542A1 (en) * 2016-05-20 2019-07-04 Abbvie Stemcentrx Llc Anti-dll3 drug conjugates for treating tumors at risk of neuroendocrine transition
WO2018162724A1 (en) * 2017-03-09 2018-09-13 Mab Discovery Gmbh Antibodies specifically binding to human il-1r7
US20230241241A1 (en) * 2020-06-19 2023-08-03 Hangzhou Dac Biotech Co., Ltd. Conjugates of a cell-binding molecule with camptothecin analogs
WO2023288241A1 (en) * 2021-07-14 2023-01-19 Genentech, Inc. Anti-c-c motif chemokine receptor 8 (ccr8) antibodies and methods of use

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