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WO2025199666A1 - Lieurs multivalents pour conjugués anticorps-médicament à charge élevée, et compositions et procédés associés - Google Patents

Lieurs multivalents pour conjugués anticorps-médicament à charge élevée, et compositions et procédés associés

Info

Publication number
WO2025199666A1
WO2025199666A1 PCT/CN2024/083437 CN2024083437W WO2025199666A1 WO 2025199666 A1 WO2025199666 A1 WO 2025199666A1 CN 2024083437 W CN2024083437 W CN 2024083437W WO 2025199666 A1 WO2025199666 A1 WO 2025199666A1
Authority
WO
WIPO (PCT)
Prior art keywords
drug conjugate
antibody
linker
compound
independently
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/083437
Other languages
English (en)
Inventor
Ninghui YU
Rongliang Lou
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Canwell Biotech Ltd
Original Assignee
Canwell Biotech Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Canwell Biotech Ltd filed Critical Canwell Biotech Ltd
Priority to PCT/CN2024/083437 priority Critical patent/WO2025199666A1/fr
Priority to PCT/CN2025/084562 priority patent/WO2025201281A1/fr
Publication of WO2025199666A1 publication Critical patent/WO2025199666A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/54—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 organic compound
    • A61K47/55—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 organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
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    • 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
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    • 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
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
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    • 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
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy
    • 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
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    • A61P37/02—Immunomodulators
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    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the invention generally relates to novel compounds and therapeutic uses thereof. More particularly, the invention provides novel multivalent linkers with increased drug loading capabilities, antibody-drug conjugates thereof, and pharmaceutical compositions and methods of treatment for certain diseases or conditions.
  • ADCs Antibody-drug conjugates
  • ADCs are an important class of highly potent biopharmaceutical drugs designed as a targeted therapy for the treatment of people with cancer or other diseases. Unlike the nonspecific killing properties of chemotherapy, ADCs are designed to target and kill the cancer cells only and spare healthy cells. The focus of ADC development has increasingly been placed on reduction of systemic toxicity while increasing the therapeutic benefit.
  • ADCs with dual cytotoxic agents based on distinct MOAs with synergistic effects.
  • hydrophilic linkers with low molecular weight would benefit ADCs with improved properties of drug-likeness, such as reduced aggregation, improved solubility, improved ADC plasma clearance and pharmacokinetics, reduced immunogenicity, and improved therapeutic window.
  • novel linkers are hydrophilic by design and can help improve properties of ADCs to be more suitable drug, for example with reduced aggregation, improved solubility, improved ADC plasma clearance and pharmacokinetics, improved efficacy, reduced immunogenicity, and finally improved therapeutic windows.
  • the invention generally relates to an antibody-drug conjugate having the structural formula (I) :
  • Ab is an antigen binding moiety
  • each of L 1D , L 2D , L 3D and L 4D is independently a linker group covalently attached to D 1 , D 2 , D 3 and D 4 , respectively, wherein optionally each of L 1D , L 2D , L 3D and L 4D independently comprises a hydrophilic group;
  • L 5Ab is a linker group attached to Ab, wherein L 5Ab optionally comprises 1, 2, 3, 4, 5, 6, 7 or 8 hydrophilic groups;
  • Z is O or N, wherein when Z is N, D 2 is absent and L 2D represents L 2D A and L 2D B , each is independently a hydrophilic group covalently bound to said N;
  • n is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8,
  • L 1D -D 1 , L 2D -D 2 , L 3D -D 3 and L 4D -D 4 is optionally H, a C 1-3 alkyl or a hydrophilic group.
  • the invention generally relates to a compound having the structural formula (VI) :
  • each of L 1 , L 2 , L 3 and L 4 is independently selected from: H, C 1-3 alkyl, or (CH 2 ) k -S-R L A , wherein R L A is a linker group comprising a reactive functional group and optionally comprises a hydrophilic group;
  • L 5 is selected from: N 3 or R L B , wherein R L B is a linker group comprising a reactive functional group and optionally comprises a hydrophilic group; and
  • Z is O or N, wherein when Z is N, L 2 represents L 2 A and L 2 B , each is independently a hydrophilic group covalently bound to said N.
  • L 1D , L 2D , L 3D and L 4D optionally comprises a hydrophilic group.
  • the invention generally relates to a linker-drug conjugate having the structural formula (VIII) :
  • each of D 1 , D 2 , D 3 and D 4 is independently a drug moiety or absent;
  • each of L 1D , L 2D , L 3D and L 4D is independently a linker group
  • L 5 is selected from: N 3 or R L B , wherein R L B is a linker group comprising a reactive functional group and optionally comprises a hydrophilic group; and
  • Z is O or N, wherein when Z is N, D 2 is absent and L 2D represents L 2D A and L 2D B , each is independently a hydrophilic group covalently bound to said N.
  • the invention generally relates to an antibody-drug conjugate having the structural formula (X) :
  • Ab is an antigen binding moiety
  • each of D 1 , D 3 , D 4 , D 6 , D 7 , D 8 and D 9 is independently a drug moiety or absent;
  • each of L 1D , L 3D , L 4D , L 6D , L 7D , L 8D and L 9D is independently a linker group covalently attached to D 1 , D 3 , D 4 , D 6 , D 7 , D 8 and D 9 , respectively, wherein optionally each of L 1D , L 2D , L 3D and L 4D independently comprises a hydrophilic group;
  • L 5Ab is a linker group attached to Ab, wherein L 5Ab optionally comprises 1, 2, 3, 4, 5, 6, 7 or 8 hydrophilic groups;
  • each of X and Z is independently a triazole group
  • Y is a hydrophilic spacer group
  • n 1-8
  • L 1D -D 1 , L 3D -D 3 , L 4D -D 4 , L 6D -D 6 , L 7D -D 7 , L 8D -D 8 and L 9D -D 9 is optionally H, a C 1-3 alkyl or a hydrophilic group.
  • the invention generally relates to linker-drug conjugate having the structural formula (XI) :
  • each of D 1 , D 3 , D 4 , D 6 , D 7 , D 8 and D 9 is independently a drug moiety or absent;
  • each of L 1D , L 3D , L 4D , L 6D , L 7D , L 8D and L 9D is independently a linker group covalently attached to D 1 , D 3 , D 4 , D 6 , D 7 , D 8 and D 9 , respectively, wherein optionally each of L 1D , L 2D , L 3D and L 4D independently comprises a hydrophilic group;
  • L 5 is selected from: N 3 or R L B , wherein R L B is a linker group comprising a reactive functional group and optionally comprises a hydrophilic group; and
  • each of X and Z is independently a triazole group
  • Y is a hydrophilic spacer group
  • L 1D -D 1 , L 3D -D 3 , L 4D -D 4 , L 6D -D 6 , L 7D -D 7 , L 8D -D 8 and L 9D -D 9 is optionally H, a C 1-3 alkyl or a hydrophilic group.
  • the invention generally relates to a pharmaceutical composition comprising an antibody-drug conjugate disclosed herein.
  • the invention generally relates to a unit dosage form comprising a pharmaceutical composition of the invention.
  • the invention generally relates to a method for treating, reducing, or preventing a disease or disorder, comprising administering to a subject in need thereof an antibody-drug conjugate disclosed herein.
  • the invention generally relates to a method for modulating immune response, comprising administering to a subject in need thereof an antibody-drug conjugate disclosed herein.
  • the invention generally relates to use of an antibody-drug conjugate disclosed herein for treating or reducing a disease or disorder.
  • the invention generally relates to use of an antibody-drug conjugate disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent, in preparation of a medicament for treating or reducing a disease or disorder.
  • the invention generally relates to a method for making a compound, a linker-drug conjugate or an antibody-drug conjugate disclosed herein.
  • FIG. 1 shows exemplary HIC and SEC-HPLC chromatogram of ADC-10.
  • FIG. 2 shows exemplary HIC and SEC-HPLC chromatogram of ADC-11.
  • FIG. 3 shows exemplary HIC and SEC-HPLC chromatogram of ADC-3.
  • FIG. 4 shows exemplary HIC and SEC-HPLC chromatogram of ADC-12.
  • FIG. 5 shows exemplary HIC and SEC-HPLC chromatogram of ADC-13.
  • FIG. 6 shows exemplary HIC and SEC-HPLC chromatogram of ADC-14.
  • FIG. 7 shows exemplary HIC and SEC-HPLC chromatogram of ADC-17
  • FIG. 8 shows exemplary HIC and SEC-HPLC chromatogram of ADC-18.
  • FIG. 9 shows exemplary HIC and SEC-HPLC chromatogram of ADC-19.
  • FIG. 10 shows exemplary HIC and SEC-HPLC chromatogram of ADC-20.
  • FIG. 11 shows exemplary HIC and SEC-HPLC chromatogram of ADC-22.
  • FIG. 12 shows exemplary Mouse PK profile of ADC-7.
  • FIG. 15 shows exemplary Mouse PK profile of ADC-16.
  • FIG. 16 shows exemplary Mouse PK profile of ADC-17.
  • compositions and methods when used to define compositions and methods, is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements.
  • the term “consisting essentially of” when used to define compositions and methods, shall mean that the compositions and methods include the recited elements and exclude other elements of any essential significance to the compositions and methods.
  • “consisting essentially of” refers to administration of the pharmacologically active agents expressly recited and excludes pharmacologically active agents not expressly recited.
  • consisting essentially of does not exclude pharmacologically inactive or inert agents, e.g., pharmaceutically acceptable excipients, carriers or diluents.
  • the term “consisting of” when used to define compositions and methods, shall mean excluding trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.
  • Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis-and trans-isomers, R-and S-enantiomers, diastereomers, (D) -isomers, (L) -isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
  • Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50: 50, 60: 40, 70: 30, 80: 20, 90: 10, 95: 5, 96: 4, 97: 3, 98: 2, 99: 1, or 100: 0 isomer ratios are contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures.
  • Solvates and polymorphs of the compounds of the invention are also contemplated herein.
  • Solvates of the compounds of the present invention include, for example, hydrates.
  • alkyl refers to a straight, branched or cyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to ten carbon atoms (e.g., C 1-10 alkyl) .
  • a numerical range such as “1 to 10” refers to each integer in the given range; e.g., "1 to 10 carbon atoms” means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the present definition also covers the occurrence of the term "alkyl” where no numerical range is designated.
  • “alkyl” can be a C 1-6 alkyl group.
  • alkyl groups have 1 to 10, 1 to 8, 1 to 6, or 1 to 3 carbon atoms.
  • an alkyl group is optionally substituted by one or more of substituents which independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo (F, Cl, Br, I) , haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sul
  • substituents which independently
  • a substituted alkyl can be selected from fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, and phenethyl.
  • heteroatom means one or more of oxygen, sulfur, nitrogen, or phosphorus (including, any oxidized form of nitrogen, sulfur, or phosphorus; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3, 4-dihydro-2H-pyrrolyl) , NH (as in pyrrolidinyl) or NR + (as in N-substituted pyrrolidinyl) ) .
  • halogen refers to fluorine (F) , chlorine (Cl) , bromine (Br) , or iodine (I) .
  • halide or “halo” , means fluoro, chloro, bromo or iodo.
  • haloalkyl, “ “haloalkenyl, “ “haloalkynyl” and “haloalkoxy” include alkyl, alkenyl, alkynyl and alkoxy structures that are substituted with one or more halo groups or with combinations thereof.
  • fluoroalkyl and fluoroalkoxy include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine, such as, but not limited to, trifluoromethyl, difluoromethyl, 2, 2, 2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like.
  • halo is fluorine, such as, but not limited to, trifluoromethyl, difluoromethyl, 2, 2, 2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like.
  • alkyl, alkenyl, alkynyl and alkoxy groups are as defined herein and can be optionally further substituted as defined herein.
  • aromatic refers to a radical with 6 to 14 ring atoms (e.g., C 6-14 aromatic or C 6-14 aryl) that has at least one ring having a conjugated pi electron system which is carbocyclic (e.g., phenyl, fluorenyl, and naphthyl) .
  • the aryl is a C 6-10 aryl group.
  • bivalent radicals formed from substituted benzene derivatives and having the free valences at ring atoms are named as substituted phenylene radicals.
  • bivalent radicals derived from univalent polycyclic hydrocarbon radicals whose names end in"-yl" by removal of one hydrogen atom from the carbon atom with the free valence are named by adding "-idene” to the name of the corresponding univalent radical, e.g., a naphthyl group with two points of attachment is termed naphthylidene.
  • a numerical range such as "6 to 14 aryl” refers to each integer in the given range; e.g., "6 to 14 ring atoms” means that the aryl group can consist of 6 ring atoms, 7 ring atoms, etc., up to and including 14 ring atoms.
  • the term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of ring atoms) groups.
  • Polycyclic aryl groups include bicycles, tricycles, tetracycles, and the like. In a multi-ring group, only one ring is required to be aromatic, so groups such as indanyl are encompassed by the aryl definition.
  • Non-limiting examples of aryl groups include phenyl, phenalenyl, naphthalenyl, tetrahydronaphthyl, phenanthrenyl, anthracenyl, fluorenyl, indolyl, indanyl, and the like.
  • TLR7 and/or TLR8 ligand refers to a molecule, other than a compound disclosed herein, that interacts directly or indirectly with TLR7 and/or TLR8 through a TLR7 and/or TLR8 domain other than a TLR8 domain, and induces TLR7-and/or TLR8-mediated signaling.
  • a TLR7 and/or TLR8 ligand is a natural ligand, i.e., a TLR7 and/or TLR8 ligand that is found in nature.
  • a TLR7 and/or TLR8 ligand refers to a molecule other than a natural ligand of TLR7 and/or TLR8, e.g., a molecule prepared by human activity.
  • an antagonist refers to a compound that competes with an agonist or inverse agonist for binding to a receptor, thereby blocking the action of an agonist or inverse agonist on the receptor.
  • an antagonist has no effect on constitutive receptor activity. More specifically, an antagonist is a compound that inhibits the activity of TLR7 or TLR8 at the TLR7 or TLR8 receptor, respectively.
  • An antigen will preferably react, typically in a highly selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be evoked by other antigens.
  • Antigens as used herein may also be mixtures of several individual antigens.
  • antibody refers to molecules that are capable of binding an epitope or antigenic determinant.
  • the term is meant to include whole antibodies and antigen-binding fragments thereof.
  • the term encompasses a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F (ab') 2 , Fv, single chain Fv ( "scFv” ) , diabody, linear antibody, bispecific antibody, multi-specific antibody, chimeric antibody, humanized antibody, human antibody, or fusion protein comprising the antigen-binding portion of an antibody.
  • Antibodies can be from any animal origin.
  • the antibodies are mammalian, e.g., human, murine, rabbit, goat, guinea pig, camel, horse and the like, or other suitable animals.
  • Antibodies may recognize polypeptide or polynucleotide antigens.
  • the term includes active fragments, including for example, an antigen binding fragment of an immunoglobulin, a variable and/or constant region of a heavy chain, a variable and/or constant region of a light chain, a complementarity determining region (cdr) , and a framework region.
  • the terms include polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, chimeric antibodies, hybrid antibody molecules, F (ab) 2 and F (ab) fragments; Fv molecules (for example, noncovalent heterodimers) , dimeric and trimeric antibody fragment constructs; minibodies, humanized antibody molecules, and any functional fragments obtained from such molecules, wherein such fragments retain specific binding.
  • antigen binding fragment refers to one or more portions of an antibody that retain the ability to specifically interact with, e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution, an epitope of an antigen.
  • the two domains of the Fv fragment, V L and V H can be joined using recombinant methods by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules.
  • a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules.
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen binding fragment. ”
  • antigen binding fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • Antigen binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv. (See, e.g., Hollinger and Hudson, 2005 Nature Biotechnology 23: 1 126-1136. ) Antigen binding fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) . (See, e.g., U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies.
  • Fn3 fibronectin type III
  • Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (V H -C H 1-V H -C H 1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. (Zapata et al., 1995 Protein Eng. 8: 1057-1062; U.S. Pat. No. 5,641,870. )
  • bispecific antibody refers to an antibody, typically a monoclonal antibody, having binding specificities for at least two different antigenic epitopes.
  • the epitopes can be from the same antigen or from two different antigens.
  • Methods for making bispecific antibodies are known in the art.
  • bispecific antibodies can be produced recombinantly using the co-expression of two immunoglobulin heavy chain/light chain pairs.
  • bispecific antibodies can be prepared using chemical linkage.
  • Bispecific antibodies include bispecific antibody fragments. (See, e.g., Milstein et al. 1983 Nature 305: 537-39; Brennan et al. 1985 Science 229: 81; Hollinger et al. 1994 Proc. Natl. Acad. Sci. U.S.A. 90: 6444-48; Gruber et al. 1994 J. Immunol. 152: 5368-74. )
  • chimeric antibody refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind the target antigen and/or exhibit the desired biological activity.
  • human antibody refers to antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al. 2000 J. Mol. Biol. 296: 57-86) . Human antibodies may include amino acid residues not encoded by human sequences, e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a substitution to promote stability or manufacturing.
  • humanized antibody refers to antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes. “Monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by various methods known in the art, including the hybridoma method first described by Kohler et al. 1975 Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567) . “Monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. 1991 Nature 352: 624-628 and Marks et al. 1991 J. Mol. Biol. 222: 581-597, for example. These monoclonal antibodies will usually bind with at least a Kd of about 1 ⁇ M, more usually at least about 300 nM, typically at least about 30 nM, preferably at least about 10 nM.
  • cleavable linker refers to a linker or linker component that connects two moieties by covalent connections, but breaks down to sever the covalent connection between the moieties under physiologically relevant conditions.
  • a cleavable linker is severed in vivo more rapidly in an intracellular environment than when outside a cell, causing release of a payload to preferentially occur inside the targeted cell.
  • Cleavage may be enzymatic or non-enzymatic.
  • a payload is typically released from an antibody without degrading the antibody. Cleavage may leave some portion of a linker or linker component attached to the payload, or it may release the payload without any residual part or component of the linker (i.e., traceless release) .
  • non-cleavable linker refers to a linker or linker component that is not especially susceptible to breaking down under physiological conditions, i.e., it is at least as stable as the antibody or antigen binding fragment portion of the immunoconjugate.
  • linkers are sometimes referred to as “stable, ” meaning they are sufficiently resistant to degradation to keep the payload connected to the antigen binding moiety until the antigen binding moiety is itself at least partially degraded. In such a case, the degradation of Ab precedes cleavage of the linker in vivo.
  • Degradation of the antibody portion of an immunoconjugate having a stable or non-cleavable linker may leave some or all of the linker, and one or more amino acid groups from an antibody, attached to the payload or drug moiety that is delivered in vivo.
  • disease As used herein, the terms “disease” , “condition” or “disorder” are used interchangeably herein and refer to a pathological condition, for example, one that can be identified by symptoms or other identifying factors as diverging from a healthy or a normal state.
  • disease includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic diseases.
  • the term “in need of” a treatment refers to a subject that would benefit biologically, medically or in quality of life from such a treatment.
  • the term “effective amount” of an active agent refers to an amount sufficient to elicit the desired biological response.
  • the effective amount of a compound of the invention may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the patient.
  • treatment refers to a method of reducing, delaying or ameliorating such a condition before or after it has occurred. Treatment may be directed at one or more effects or symptoms of a disease and/or the underlying pathology.
  • the treatment can be any reduction and can be, but is not limited to, the complete ablation of the disease or the symptoms of the disease. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%as measured by any standard technique.
  • the terms “prevent” , “preventing” , or “prevention” refer to a method for precluding, delaying, averting, or stopping the onset, incidence, severity, or recurrence of a disease or condition.
  • a method is considered to be a prevention if there is a reduction or delay in onset, incidence, severity, or recurrence of a disease or condition or one or more symptoms thereof in a subject susceptible to the disease or condition as compared to a subject not receiving the method.
  • the disclosed method is also considered to be a prevention if there is a reduction or delay in onset, incidence, severity, or recurrence of osteoporosis or one or more symptoms of a disease or condition in a subject susceptible to the disease or condition after receiving the method as compared to the subject's progression prior to receiving treatment.
  • the reduction or delay in onset, incidence, severity, or recurrence of osteoporosis can be about a 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between.
  • a "pharmaceutically acceptable form” of a disclosed compound includes, but is not limited to, pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, isomers, prodrugs, and isotopically labeled derivatives thereof.
  • a "pharmaceutically acceptable form” includes, but is not limited to, pharmaceutically acceptable salts, esters, prodrugs and isotopically labeled derivatives thereof.
  • a "pharmaceutically acceptable form” includes, but is not limited to, pharmaceutically acceptable isomers and stereoisomers, prodrugs and isotopically labeled derivatives thereof.
  • the pharmaceutically acceptable form is a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19.
  • Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchioric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchioric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate,
  • organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, lactic acid, trifluoracetic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • compositions include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • the pharmaceutically acceptable base addition salt can be chosen from ammonium, potassium, sodium, calcium, and magnesium salts.
  • the pharmaceutically acceptable form is a prodrug.
  • prodrug refers to compounds that are transformed in vivo to yield a disclosed compound or a pharmaceutically acceptable form of the compound.
  • a prodrug can be inactive when administered to a subject, but is converted in vivo to an active compound, for example, by hydrolysis (e.g., hydrolysis in blood) .
  • hydrolysis e.g., hydrolysis in blood
  • a prodrug has improved physical and/or delivery properties over the parent compound.
  • Prodrugs can increase the bioavailability of the compound when administered to a subject (e.g., by permitting enhanced absorption into the blood following oral administration) or which enhance delivery to a biological compartment of interest (e.g., the brain or lymphatic system) relative to the parent compound.
  • exemplary prodrugs include derivatives of a disclosed compound with enhanced aqueous solubility or active transport through the gut membrane, relative to the parent compound.
  • Exemplary advantages of a prodrug can include, but are not limited to, its physical properties, such as enhanced water solubility for parenteral administration at physiological pH compared to the parent compound, or it can enhance absorption from the digestive tract, or it can enhance drug stability for long-term storage.
  • Prodrugs commonly known in the art include well-known acid derivatives, such as, for example, esters prepared by reaction of the parent acids with a suitable alcohol, amides prepared by reaction of the parent acid compound with an amine, basic groups reacted to form an acylated base derivative, etc.
  • acid derivatives such as, for example, esters prepared by reaction of the parent acids with a suitable alcohol, amides prepared by reaction of the parent acid compound with an amine, basic groups reacted to form an acylated base derivative, etc.
  • other prodrug derivatives may be combined with other features disclosed herein to enhance bioavailability.
  • those of skill in the art will appreciate that certain of the presently disclosed compounds having free amino, arnido, hydroxy or carboxylic groups can be converted into prodrugs.
  • Prodrugs include compounds having an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues which are covalently joined through peptide bonds to free amino, hydroxy or carboxylic acid groups of the presently disclosed compounds.
  • the amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid, citrulline homocysteine, homoserine, ornithine and methionine sulfone.
  • Prodrugs also include compounds having a carbonate, carbamate, amide or alkyl ester moiety covalently bonded to any of the above substituents disclosed herein.
  • the term “pharmaceutically acceptable” excipient, carrier, or diluent refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically acceptable material, composition or vehicle such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ring
  • wetting agents such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymer as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • isolated or “purified” refer to a material that is substantially or essentially free from components that normally accompany it in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography.
  • the term “subject” refers to any animal (e.g., a mammal) , including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.
  • the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • the term “low dosage” refers to at least 5%less (e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) than the lowest standard recommended dosage of a particular compound formulated for a given route of administration for treatment of any human disease or condition.
  • a low dosage of an agent that is formulated for administration by inhalation will differ from a low dosage of the same agent formulated for oral administration.
  • high dosage is meant at least 5% (e.g., at least 10%, 20%, 50%, 100%, 200%, or even 300%) more than the highest standard recommended dosage of a particular compound for treatment of any human disease or condition.
  • Isotopically-labeled compounds are also within the scope of the present disclosure.
  • an “isotopically-labeled compound” or “isotope derivative” refers to a presently disclosed compound including pharmaceutical salts and prodrugs thereof, each as described herein, in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes that can be incorporated into compounds presently disclosed include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 Cl, respectively.
  • the compounds may be useful in drug and/or substrate tissue distribution assays. Tritiated ( 3 H) and carbon-14 ( 14 C) labeled compounds are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium ( 2 H) can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds presently disclosed, including pharmaceutical salts, esters, and prodrugs thereof, can be prepared by any means known in the art. Benefits may also be obtained from replacement of normally abundant 12 C with 13 C. (See, WO 2007/005643, WO 2007/005644, WO 2007/016361, and WO 2007/016431. )
  • deuterium ( 2 H) can be incorporated into a compound disclosed herein for the purpose in order to manipulate the oxidative metabolism of the compound by way of the primary kinetic isotope effect.
  • the primary kinetic isotope effect is a change of the rate for a chemical reaction that results from exchange of isotopic nuclei, which in turn is caused by the change in ground state energies necessary for covalent bond formation after this isotopic exchange.
  • Exchange of a heavier isotope usually results in a lowering of the ground state energy for a chemical bond and thus causes a reduction in the rate in rate-limiting bond breakage. If the bond breakage occurs in or in the vicinity of a saddle-point region along the coordinate of a multi-product reaction, the product distribution ratios can be altered substantially.
  • a compound which has multiple potential sites of attack for oxidative metabolism for example benzylic hydrogen atoms and hydrogen atoms bonded to a nitrogen atom, is prepared as a series of analogues in which various combinations of hydrogen atoms are replaced by deuterium atoms, so that some, most or all of these hydrogen atoms have been replaced by deuterium atoms.
  • Half-life determinations enable favorable and accurate determination of the extent of the extent to which the improvement in resistance to oxidative metabolism has improved. In this way, it is determined that the half-life of the parent compound can be extended by up to 100%as the result of deuterium-hydrogen exchange of this type.
  • Deuterium-hydrogen exchange in a compound disclosed herein can also be used to achieve a favorable modification of the metabolite spectrum of the starting compound in order to diminish or eliminate undesired toxic metabolites. For example, if a toxic metabolite arises through oxidative carbon-hydrogen (C-H) bond cleavage, it can reasonably be assumed that the deuterated analogue will greatly diminish or eliminate production of the unwanted metabolite, even if the particular oxidation is not a rate-determining step. Further information on the state of the art with respect to deuterium-hydrogen exchange may be found, for example in Hanzlik et al., J. Org. Chem. 55, 3992-3997, 1990, Reider et al., J. Org.
  • Compounds of the present invention are, subsequent to their preparation, preferably isolated and purified to obtain a composition containing an amount by weight equal to or greater than 95% ( “substantially pure” ) , which is then used or formulated as described herein. In certain embodiments, the compounds of the present invention are more than 99%pure.
  • stable refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject) .
  • the invention provides novel multivalent linkers that allow improved drug loading and fine-tune drug of DAR and multiplexed loading of two or more distinct bioactive agents in an ADC.
  • the invention additionally provides pharmaceutical compositions and methods of therapeutic treatment for certain diseases or conditions using ADCs built on the multivalent linker platform disclosed herein.
  • the invention further provides methods of making such multivalent linkers, linker-drug conjugates, and ADCs, as well as methods for modifying or fine-tuning DARs thereof.
  • 2-azido-2-deoxy-D-glucose was utilized as a scaffold, the 1-, 3-, 4-, 6-OH’s were derivatized and each arm attached to a spacer.
  • a drug payload
  • SN-38 was used as payload as a proof of concept.
  • the PEG group is introduced to mask the hydrophobic payloads and increase solubility.
  • an ADC can be fine-tuned to have 4 to 32 drugs per antibody.
  • the linkers of the invention are hydrophilic by design and can help improve ADC properties such that they exhibit reduced aggregation, improved solubility, improved ADC plasma clearance and pharmacokinetics, improved efficacy, reduced immunogenicity, and finally improved therapeutic windows.
  • the invention generally relates to an antibody-drug conjugate having the structural formula (I) :
  • Ab is an antigen binding moiety
  • each of D 1 , D 2 , D 3 and D 4 is independently a drug moiety or absent;
  • each of L 1D , L 2D , L 3D and L 4D is independently a linker group covalently attached to D 1 , D 2 , D 3 and D 4 , respectively, wherein optionally each of L 1D , L 2D , L 3D and L 4D independently comprises a hydrophilic group;
  • L 5Ab is a linker group attached to Ab, wherein L 5Ab optionally comprises 1, 2, 3, 4, 5, 6, 7 or 8 hydrophilic groups;
  • Z is O or N, wherein when Z is N, D 2 is absent and L 2D represents L 2D A and L 2D B , each is independently a hydrophilic group covalently bound to said N;
  • n is an integer selected from 1, 2, 3, 4, 5, 6, 7 or 8,
  • L 1D -D 1 , L 2D -D 2 , L 3D -D 3 and L 4D -D 4 is optionally H, a C 1-3 alkyl or a hydrophilic group.
  • Z is O and the antibody-drug conjugate has the structural formula (I A ) :
  • none of L 1D -D 1 , L 2D -D 2 , L 3D -D 3 and L 4D -D 4 is optionally H or a C 1-3 alkyl.
  • L 1D -D 1 is R, wherein R is H or a C 1-3 alkyl, having the structural formula (I B ) :
  • L 4D -D 4 is R’ , wherein R’ is H or a C 1-3 alkyl, having the structural formula (I C ) :
  • each of L 1D , L 2D , L 3D and L 4D comprises a branching point and, respectively, a hydrophilic group R 1h , R 2h , R 3h or R 4h , having the structural formula (II) :
  • L 1d , L 2d , L 3d and L 4d represents the remaining portion of L 1D , L 2D , L 3D and L 4D , respectively.
  • L 1D -D 1 is R, wherein R is H or a C 1-3 alkyl, having the structural formula (II A ) :
  • L 4D -D 4 is R’ , wherein R’ is H or a C 1-3 alkyl, having the structural formula (II B ) :
  • L 2D -D 2 is R” , wherein R” is H or a C 1-3 alkyl, having the structural formula (II C ) :
  • each of R 1h , R 2h , R 3h and R 4h comprises a polyethylene glycol group having i units of ethylene glycol, wherein i is an integer in the range of about 2 to about 16.
  • one or more of L 1d , L 2d , L 3d and L 4d independently comprises a di-, tri-or tetra-peptide linker.
  • one or more of L 1d , L 2d , L 3d and L 4d is independently:
  • k is an integer selected from 1 and 4.
  • x is 1, 2, 3 or 4.
  • one or more of L 1d , L 2d , L 3d and L 4d independently comprises a lysine unit.
  • one or more of L 1d , L 2d , L 3d and L 4d independently is:
  • x is 1, 2, 3 or 4.
  • one or more of L 1D , L 2D , L 3D and L 4D comprises:
  • none of L 1D -D 1 , L 3D -D 3 and L 4D -D 4 is optionally H or a C 1-3 alkyl.
  • L 1D -D 1 is R, wherein R is H or a C 1-3 alkyl, having the structural formula (IV A ) :
  • each of R 1h , R 2h , R 3h and R 4h comprises a polyethylene glycol group having i units of ethylene glycol, wherein i is an integer in the range of about 2 to about 16.
  • one or more of L 1d , L 2d , L 3d and L 4d independently comprises a di-, tri-or tetra-peptide linker.
  • k is an integer selected from 1 and 4.
  • x is 1, 2, 3 or 4.
  • one or more of L 1d , L 3d and L 4d independently comprises a lysine unit.
  • one or more of L 1d , L 3d and L 4d independently is:
  • k is an integer selected from 1 and 4.
  • x is 1, 2, 3 or 4.
  • one or more of L 1D , L 3D and L 4D comprises:
  • k is an integer selected from 1 and 4.
  • one or more of L 1D , L 2D , L 3D and L 4D comprises:
  • k is an integer selected from 1 and 4.
  • one or more of L 1D , L 2D , L 3D and L 4D comprises:
  • k is an integer selected from 1 and 4.
  • each of D 1 , D 3 and D 4 is covalently attached respectively to L 1d , L 3d and L 4d via a carbonate linkage, a carbamate linkage, or an amide linkage.
  • L 5Ab comprises a group selected from:
  • L 5Ab is covalently attached to Ab via a thioether linkage.
  • the thioether linkage comprises maleimide thioether.
  • L 5Ab comprises 1, 2, 3, 4, 5, 6, 7 or 8 hydrophilic groups.
  • L 5Ab is:
  • each of R 1h , R 2h , R 3h and R 4h is independently (CH 2 -CH 2 O) p CH 3 , wherein p is an integer selected from about 4 to about 12.
  • Ab is an antibody.
  • Z is O or N, wherein when Z is N, L 2 represents L 2 A and L 2 B , each is independently a hydrophilic group covalently bound to said N.
  • one or more of L 1 , L 2 , L 3 and L 4 comprises:
  • one or more of L 1 , L 2 , L 3 and L 4 comprises:
  • one or more of L 1 , L 2 , L 3 and L 4 comprises:
  • Z is N, D 2 is absent and L 2 represents L 2 A and L 2 B , having the structural formula (VII) :
  • L 1 is R, wherein R is H or C 1-3 alkyl, having the structural formula (VII A ) :
  • L 4 is R’ , wherein R’ is H or C 1-3 alkyl, having the structural formula (VII B ) :
  • L 4 is N 3 .
  • one or more of L 1 , L 3 and L 4 if present independently comprise:
  • k is an integer selected from 1 and 4.
  • x is 1, 2, 3 or 4.
  • one or more of L 1 , L 3 and L 4 if present independently comprises a lysine unit.
  • one or more of L 1 , L 3 and L 4 independently comprises:
  • k is an integer selected from 1 and 4.
  • x is 1, 2, 3 or 4.
  • one or more of L 1 , L 3 and L 4 if present, comprises:
  • k is an integer selected from 1 and 4.
  • one or more of L 1 , L 3 and L 4 if present, comprises:
  • k is an integer selected from 1 and 4.
  • Non-limiting examples of compounds include:
  • k is an integer selected from 1 and 4.
  • x is 1, 2, 3 or 4.
  • one or more of L 1d , L 2d , L 3d and L 4d independently comprises a lysine unit.
  • one or more of L 1d , L 2d , L 3d and L 4d independently is:
  • k is an integer selected from 1 and 4.
  • x is 1, 2, 3 or 4.
  • one or more of L 1D , L 2D , L 3D and L 4D comprises:
  • k is an integer selected from 1 and 4.
  • one or more of L 1D , L 2D , L 3D and L 4D comprises:
  • k is an integer selected from 1 and 4.
  • one or more of L 1D , L 2D , L 3D and L 4D comprises:
  • k is an integer selected from 1 and 4.
  • Z is N, D 2 is absent and L 2D represents L 2D A and L 2D B , each is independently a hydrophilic group covalently bound to said N, having the structural formula (X) :
  • L 1D -D 1 is R, wherein R is H or a C 1-3 alkyl, having the structural formula (X A ) :
  • L 4D -D 4 is R’ , wherein R’ is H or a C 1-3 alkyl, having the structural formula (X B ) :
  • each of L 1D , L 3D and L 4D comprises a branching point and, respectively, a hydrophilic group R 1h , R 3h or R 4h , having the structural formula (XI) :
  • L 4D -D 4 is R’ , wherein R’ is H or a C 1-3 alkyl, having the structural formula (XI B ) :
  • each of R 1h , R 2h , R 3h and R 4h comprises a polyethylene glycol group having i units of ethylene glycol, wherein i is an integer in the range of about 2 to about 16.
  • one or more of L 1d , L 3d and L 4d if present independently is:
  • k is an integer selected from 1 and 4.
  • x is 1, 2, 3 or 4.
  • one or more of L 1d , L 3d and L 4d independently comprises a lysine unit.
  • one or more of L 1d , L 2d , L 3d and L 4d independently is:
  • one or more of L 1D , L 2D , L 3D and L 4D comprises:
  • k is an integer selected from 1 and 4.
  • one or more of L 1D , L 2D , L 3D and L 4D comprises:
  • one or more of L 1D , L 2D , L 3D and L 4D comprises:
  • k is an integer selected from 1 and 4.
  • L 5 comprises a group selected from:
  • L 5 comprises a group selected from:
  • L 5 comprises 1-4 hydrophilic groups.
  • the invention generally relates to an antibody-drug conjugate having the structural formula (X) :
  • Ab is an antigen binding moiety
  • each of D 1 , D 3 , D 4 , D 6 , D 7 , D 8 and D 9 is independently a drug moiety or absent;
  • each of L 1D , L 3D , L 4D , L 6D , L 7D , L 8D and L 9D is independently a linker group covalently attached to D 1 , D 3 , D 4 , D 6 , D 7 , D 8 and D 9 , respectively, wherein optionally each of L 1D , L 2D , L 3D and L 4D independently comprises a hydrophilic group;
  • L 5Ab is a linker group attached to Ab, wherein L 5Ab optionally comprises 1, 2, 3, 4, 5, 6, 7 or 8 hydrophilic groups;
  • each of X and Z is independently a triazole group
  • Y is a hydrophilic spacer group
  • L 1D -D 1 , L 3D -D 3 , L 4D -D 4 , L 6D -D 6 , L 7D -D 7 , L 8D -D 8 and L 9D -D 9 is optionally H, a C 1-3 alkyl or a hydrophilic group.
  • the invention generally relates to a pharmaceutical composition comprising an antibody-drug conjugate of formula (XII) .
  • the invention generally relates to a method for treating, reducing, or preventing a disease or disorder, comprising administering to a subject in need thereof an antibody-drug conjugate of formula (XII) .
  • the invention generally relates to linker-drug conjugate having the structural formula (XI) :
  • each of D 1 , D 3 , D 4 , D 6 , D 7 , D 8 and D 9 is independently a drug moiety or absent;
  • each of L 1D , L 3D , L 4D , L 6D , L 7D , L 8D and L 9D is independently a linker group covalently attached to D 1 , D 3 , D 4 , D 6 , D 7 , D 8 and D 9 , respectively, wherein optionally each of L 1D , L 2D , L 3D and L 4D independently comprises a hydrophilic group;
  • each of X and Z is independently a triazole group
  • Y is a hydrophilic spacer group
  • L 1D -D 1 , L 3D -D 3 , L 4D -D 4 , L 6D -D 6 , L 7D -D 7 , L 8D -D 8 and L 9D -D 9 is optionally H, a C 1-3 alkyl or a hydrophilic group.
  • Y comprises a polyethylene glycol group having i units of ethylene glycol, wherein i is an integer in the range of about 2 to about 16.
  • drug or “drug moiety” (D) , as used herein, refers to a biologically active agent and can be selected from chemotherapeutic agents and toxins. Each “drug” , “drug moiety” or “D” maybe independently selected from:
  • drug of particular interest in this invention are the camptothecin derivatives and metabolites (SN-38, Deruxtecan (Dxd) , Exatecan, MMAE, etc) .
  • Others includes diphtheria toxin, botulium toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, o, -amanitin, amanitin derivatives, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, tubulysin, geldanamycin, maytansinoid, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, a dolastatin analog, auristatin (such as monomethylauristatin E and monomethylauristatin
  • affinity ligand is a substrate, an inhibitor, a stimulating agent, a neurotransmitter, a radioisotope, or a combination of any of the foregoing;
  • radioactive label "P, "S, a fluorescent dye, an electron dense reagent, an enzyme, biotin, streptavidin, dioxigenin, a hapten, an immunogenic protein, a nucleic acid molecule with a sequence complementary to a target, or a combination of any of the foregoing;
  • the drug to antibody ratio (DAR) or drug loading may be characterized by conventional means such as UV, mass spectroscopy, ELISA assay, HIC, HPLC or electrophoresis.
  • DAR ranges from about 1 to 32, 1 to 16, 1 to 12, 1 to 8, 1 to 4, 1 to 2, 2 to 32, 4 to 32, 8 to 32, 1 to 4, 4 to 8, 8 to 12, 12 to 16, 16 to 32 or about 1.
  • compositions of this invention are formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, pharmaceutically acceptable compositions of this invention are administered without food. In other embodiments, pharmaceutically acceptable compositions of this invention are administered with food.
  • compositions of the present invention that are optionally combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration.
  • provided compositions should be formulated so that a dosage of between 0.01 -100 mg/kg body weight/day of the compound can be administered to a patient receiving these compositions.
  • a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated.
  • the amount of a compound of the present invention in the composition will also depend upon the particular compound in the composition.
  • Step 1 A suspension of sodium azide (4.22 g, 69.91 mmol) in acetonitrile (77 mL) was cooled to 0 °C in an ice-bath. Triflic anhydride (15.7 g, 55.65 mmol) was added to the mixture by a syringe during 0.5 h while stirring. After the reaction was maintained for 2 h at 0 °C, the acetonitrile solution of trifluoromethanesulfonyl azide was filtered and used directly to next reaction.
  • Step 5 A solution of compound 1-5 (600 mg, 1.727 mmol) and methyl thioglycolate (1.7 mL, 17.24 mmol) in dichloromethane (6 mL) was irradiated with UV 365 nm at room temperature for 1 h. After completion of the reaction, the mixture was concentrated. The residue was purified by silica gel (eluted with 15%EtOAc in petroleum ether) to afford the title compound 1-6 (400 mg, yield 51%) as a white solid.
  • Step 7 To a solution of compound 1-8 (1 g, 2.60 mmol) in dichloromethane (20 mL) was added dess-martin periodinane (1.43 g, 3.37 mmol) in several portions at 0 °C. The mixture was warmed to room temperature and stirred for 16 h. After completion of the reaction, the mixture was diluted with petroleum ether (20 mL) and stirred for additional 10 min. the mixture was filtered and concentrated. The residue was re-dissolved in dichloromethane (10 mL) and petroleum ether (10 mL) , and the undissolved impurity was filtered.
  • Step 11 A solution of compound 1-14 (1 g, 2.13 mmol) , 4-aminobenzyl alcohol (340 mg, 2.76 mmol) and EEDQ (930 mg, 3.76 mmol) in dichloromethane (20 mL) was stirred at room temperature in the dark for 16 h. After completion of the reaction, the mixture was diluted with dichloromethane (30 mL) , washed with 1 N HCl, water and brine. The organic layer was dried over anhydrous Na 2 SO 4 , filtered and concentrated. The crude product was triturated with petroleum ether for 1 h, filter and dried to give the title compound 1-15 (1.1 g, yield 91%) as a white solid.
  • Step 14 A mixture of compound 1-18 (500 mg, 1.27 mmol) , DIPEA (650 mg, 6.43 mmol) , DMAP (15 mg, 0.123 mmol) and t-butyldimethylsilyl chloride (580 mg, 3.85 mmol) in DMF (5 mL) was stirred at room temperature for 16 h. After completion of the reaction, the mixture was partitioned with DCM (50 mL) and water (50 mL) . The organic layer was washed with 0.5 N HCl and brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated.
  • Step 15 Under nitrogen atmosphere, to a solution of compound 1-19 (200 mg, 0.395 mmol) in anhydrous dichloromethane (5 mL) was added triphosgene (58 mg, 0.195 mmol) and DMAP (192 mg, 1.574 mmol) at 0 °C. After the mixture was stirred at 0 °C for 30 min, a solution of compound 1-17 (183 mg, 0.355 mmol) in anhydrous dichloromethane (2 mL) was added thereto. The resulting reaction was warmed to room temperature and stirred for 1 h. After completion of the reaction, the mixture was diluted with dichloromethane (20 mL) and washed with 0.5 N HCl.
  • Step 16 To a solution of compound 1-20 (230 mg, 0.219 mmol) in EtOAc (3 mL) was added dropwise a solution of HCl in EtOAc (4 N, 1 mL) at room temperature. After stirring for 10 min, a yellow precipitation was formed completely. The resulting mixture was centrifuged with EtOAc for three times and dried in vacuum to afford the title compound 1-21 (hydrochloride, 150 mg, yield 69%) as a yellow solid.
  • LCMS m/z calculated for C 52 H 65 N 5 O 10 Si: 948.2; found: 948.78 [M+H] + .
  • Step 19 A mixture of compound 1-24 (108 mg, 0.057 mmol) , TBAF (1 M in THF, 0.085 mL) and AcOH (10 mg, 0.167 mmol) in THF (2 mL) was stirred at 0 °C for 0.5 h. After completion of the reaction, the mixture was diluted with dichloromethane (10 mL) and washed with water. The organic was washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated to give the title compound 1-25 (82 mg, yield 78%) as a yellow oil, which was used at next step without purification.
  • LCMS m/z calculated for C 88 H 118 N 10 O 27 S: 1780.0; found: 1781.14 [M+H] + .
  • Step 21 To a solution of compound 1-27 (500 mg, 1.66 mmol) and DIPEA (540 mg, 4.18 mmol) in DCM (5 mL) was added compound 1-28 (615 mg, 1.99 mmol) at 0 °C. The mixture was warmed to room temperature and stirred for 15 h. After completion of the reaction, the mixture was concentrated. The residue was purified by reverse phase flash chromatography (C18 column, eluted with acetonitrile and water, HCl condition) . The desired components were lyophilized to afford the title compound 1-29 (730 mg, yield 95%) as a yellow oil.
  • LCMS m/z calculated for C 21 H 34 N 2 O 9 : 458.51; found: 459.90 [M+H] + .
  • Step 24 To a solution of compound 1-26 (30 mg, 0.018 mmol) and compound 1-31 (11 mg, 0.022 mmol) in degassed DMSO (1 mL) was added a fresh aqueous solution of ascorbic acid (1 M, 0.162 mL, 0.162 mmol) and a fresh aqueous solution of Cu 2 SO 4 (1 M, 0.054 mL, 0.054 mmol) . The mixture was purged with nitrogen for three times and stirred at room temperature for 1 h. After completion of the reaction, the mixture was filtered and purified directly by reverse phase flash chromatography (C18 column, eluted with acetonitrile and water, TFA condition) .
  • Step 25 A solution of 1-32 (32 mg, 0.015 mmol) and camphor sulfonic acid (21 mg, 0.090 mmol) in methanol (1 mL) was stirred at room temperature for 48 h. After completion of the reaction, the mixture was concentrated. The residue was purified by pre-HPLC (C18 column, eluted with acetonitrile and water, TFA condition) . The desired components were lyophilized to afford the title compound 1 (trifluoroacetate, 9.6 mg, yield 31%) as a yellow solid.
  • LCMS m/z calculated for C 95 H1 39 N 13 O 33 S: 2023.3; found: 1012.13 [M+2H] 2+ .
  • Step 1 Under nitrogen atmosphere, TBAF (1 M THF solution, 2.6 mL, 2.6 mmol) was added to a solution of compound 1-20 (1.8 g, 1.717 mmol) and acetic acid (206 mg, 3.43 mmol) in THF (18 mL) at 0 °C. The mixture was stirred at 0 °C for 1 h and diluted with dichloromethane (100 mL) , washed with water (100 mL x 2) and brine (100 mL x 2) . The organic layer was dried by anhydrous Na 2 SO 4 , filtered and concentrated. The residue was redissolved in dichloromethane (20 mL) and methanol (1 mL) .
  • Step 2 Under nitrogen atmosphere, NaH (60%in mineral oil, 730 mg, 18.25 mmol) was added in portions to a solution of compound 1-3 (800 mg, 3.65 mmol) in DMF (20 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h, to this was added slowly allyl bromide (2.2 g, 18.18 mmol) . The reaction was warmed slowly to room temperature and stirred for additional 16 h. After completion of the reaction, the mixture was quenched by addition of cold water (100 mL) . The mixture was extracted with ethyl acetate (50 mL x 2) and washed with brine (100 mL) .
  • Step 3 A mixture of compound 2-2 (900 mg, 2.65 mmol) and methyl thioglycolate (8 g, 75.3 mmol) was irradiated with UV 365 nm at room temperature for 2 h. After completion of the reaction, the mixture was concentrated. The residue was purified by silica gel column (eluted with 50%EtOAc in petroleum ether) to afford the title compound 2-3 (820 mg, yield 47%) as a colorless oil.
  • Step 4 2 N LiOH solution (6 mL, 12 mmol) was added at 0 °C to a solution of compound 2-3 (1 g, 1.52 mmol) in THF (10 mL) . The reaction was stirred at room temperature for 24 h. After completion of the reaction, the mixture was concentrated and adjusted pH to 3 with 1 N HCl at 0 °C. The aqueous solution was filtered and purified directly by reverse phase flash chromatography (C18 column, eluted with acetonitrile and water, HCl condition) . The desired components were lyophilized to afford the title compound 2-4 (670 mg, yield 71%) as a colorless oil.
  • Step 5 A solution of compound 2-4 (350 mg, 0.568 mmol) , HOBt (460 mg, 3.41 mmol) , DIPEA (880 mg, 6.82 mmol) and EDCI (650mg, 3.40 mmol) in dichloromethane (5 mL) was stirred at room temperature under nitrogen atmosphere for 10 min, a solution of compound 1-11 (1.6 g, 2.46 mmol) in dichloromethane (5 mL) was added thereto. The resulting reaction mixture was stirred at room temperature for 24 h. The mixture was diluted with dichloromethane (20 mL) and washed with 0.5 N HCl (20 mL) and water (20 mL) .
  • Step 9 To a solution of compound 2-8 (105 mg, 0.0226 mmol) in DMF (1 mL) was added hydroxylamine hydrochloride (47 mg, 0.676 mmol) and imidazole (46 mg, 0.676 mmol) . The mixture was stirred at room temperature for 8 h. After completion of the reaction, the mixture was filtered and purified directly by reverse phase flash chromatography (C18 column, eluted with acetonitrile and water, TFA condition) . The desired components were lyophilized to afford the title compound 2-9 (trifluoroacetate, 67 mg, yield 66%) as a yellow solid.
  • LCMS m/z calculated for C 199 H 280 N 24 O 65 S 3 : 4144.7; found: 1382.85 [M+3H] 3+ .
  • Step 1 Under nitrogen atmosphere, DMSO (1.5 g, 19.20 mmol) was added dropwise to a stirred solution of oxalyl chloride (2 g, 15.76 mmol) in anhydrous dichloromethane (50 mL) at -78 °C. After stirring for 15 min at -78 °C, to this was added dropwise a solution of alcohol 1-8 (3 g, 7.80 mmol) in anhydrous dichloromethane (30 mL) . After stirring at -78 °C for 30 min, TEA (3.9 g, 38.61 mmol) was added dropwise. After stirring at that temperature for 1 h, the mixture was warmed slowly to room temperature over 1 h.
  • DMSO 1.5 g, 19.20 mmol
  • Step 2 Under nitrogen atmosphere, a solution of compound 1-9 (2.46 g, 6.43 mmol) and compound 1-10 (1.6 g, 5.02 mmol) in MeOH (20 mL) was stirred at room temperature for 2 h.Then the mixture was cooled to 0 °C, and NaBH 3 CN (1 g, 15.87 mmol) was added in three portions. The resulting mixture was warmed to room temperature and stirred for 15 h. After completion of the reaction, the mixture was concentrated and redissolved in dichloromethane (50 mL) . The solution was washed with water and brine.
  • Step 3 Under nitrogen atmosphere, NaH (60%in mineral oil, 1.61 g, 40.25 mmol) was added in portions to a solution of compound 1-2 (1.38 g, 6.73 mmol) in DMF (30 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h, to this was added slowly allyl bromide (4.88 g, 40.33mmol) . The reaction was warmed slowly to room temperature and stirred for additional 16 h. After completion of the reaction, the mixture was quenched by addition of cold water (200 mL) . The mixture was extracted with ethyl acetate (100 mL x 3) and washed with brine (100 mL x 2) .
  • Step 4 A mixture of compound 3-1 (1.05 g, 2.87 mmol) and methyl thioglycolate (12 g, 113 mmol) was irradiated with UV 365 nm at room temperature for 2 h. After completion of the reaction, the mixture was concentrated. The residue was purified by silica gel column (eluted with 50%EtOAc in petroleum ether) to afford the title compound 3-2 (780 mg, yield 34%) as a colorless oil.
  • Step 5 2 N LiOH solution (7.6 mL, 15.2 mmol) was added at 0 °C to a solution of compound 3-2 (1 g, 1.26 mmol) in THF (10 mL) . The reaction was stirred at room temperature for 24 h. After completion of the reaction, the mixture was concentrated and adjusted pH to 3 with 1 N HCl at 0 °C. The aqueous solution was filtered and purified directly by reverse phase flash chromatography (C18 column, eluted with acetonitrile and water, HCl condition) . The desired components were lyophilized to afford the title compound 3-3 (720 mg, yield 77%) as a colorless oil.
  • LCMS m/z calculated for C 26 H 43 N 3 O 13 S 4 : 733.9; found: 756.41 [M+Na] + .
  • Step 6 A solution of compound 3-3 (250 mg, 0.341 mmol) , HOBt (370 mg, 2.74 mmol) , DIPEA (700 mg, 5.42 mmol) and EDCI (520 mg, 2.72 mmol) in dichloromethane (5 mL) was stirred at room temperature under nitrogen atmosphere for 10 min, a solution of compound 1-11 (hydrochloride, 1.2 g, 1.75 mmol) in dichloromethane (5 mL) was added thereto. The resulting reaction mixture was stirred at room temperature for 24 h. The mixture concentrated and the residue was purified by reverse phase flash chromatography (C18 column, eluted with acetonitrile and water, HCl condition) .
  • Step 8 To a solution of compound 3-5 (hydrochloride, 86 mg, 0.0342 mmol) and TEA (56 mg, 0.554 mmol) in dichloromethane (3 mL) was added diglycolic anhydride 1-22 (32 mg, 0.275 mmol) . The reaction was stirred at room temperature for 32 h. After completion of the reaction, the mixture was concentrated. The residue was purified by reverse phase flash chromatography (C18 column, eluted with acetonitrile and water, HCl condition) . The desired components were lyophilized to afford the title compound 3-6 (78 mg, yield 80%) as a colorless oil.
  • LCMS m/z calculated for C 118 H 219 N 11 O 57 S 4 : 2832.3; found: 1417.70 [M+2H] 2+ .
  • Step 9 To a solution of compound 3-6 (90mg, 0.0318 mmol) , compound 2-1 (hydrochloride, 190 mg, 0.218 mmol) and DIPEA (98mg, 0.760 mmol) in DMF (2 mL) was added T3P (50%in DMF solution, 200 mg, 0.314 mmol) . The mixture was stirred at room temperature for 24 h, LC-MS showed desired product was formed but not completely converted. Compound 2-1 (hydrochloride, 190 mg, 0.218 mmol) , DIPEA (98mg, 0.760 mmol) and T3P (50%in DMF solution, 200 mg, 0.314 mmol) were added again.
  • Step 10 To a solution of compound 3-7 (80 mg, 0.0131 mmol) in DMF (1 mL) was added hydroxylamine hydrochloride (36 mg, 0.518 mmol) and imidazole (36 mg, 0.529mmol) . The mixture was stirred at room temperature for 20 h. After completion of the reaction, the mixture was filtered and purified directly by reverse phase flash chromatography (C18 column, eluted with acetonitrile and water, TFA condition) . The desired components were lyophilized to afford the title compound 3-8 (trifluoroacetate, 50 mg, yield 64%) as a yellow solid.
  • LCMS m/z calculated for C 262 H 367 N 31 O 85 S 4 : 5439.2; found: 1361.10 [M+4H] 4+ .
  • Step 11 To a solution of compound 3-8 (trifluoroacetate, 80 mg, 0.0136 mmol) and compound 1-31 (20 mg, 0.0404 mmol) in degassed DMSO (3 mL) was added a fresh aqueous solution of ascorbic acid (1 M, 0.122 mL, 0.122 mmol) and a fresh aqueous solution of Cu 2 SO 4 (1 M, 0.041 mL, 0.041 mmol) . The mixture was purged with nitrogen for three times and stirred at room temperature for 1 h. After completion of the reaction, the resulting mixture was filtered and purified directly by pre-HPLC (C18 column, eluted with acetonitrile and water, TFA condition) .
  • pre-HPLC C18 column, eluted with acetonitrile and water, TFA condition
  • Step 1 Under nitrogen, to a solution of compound 1-4 (1.8 g, 5.85 mmol) in DMF (15 mL) at 0 °C was added NaH (60%in mineral oil, 586 mg, 14.65 mmol) in several portions. After stirring for 30 min at 0 °C, MeI (1.66 g, 11.69 mmol) was added thereto. The mixture was warmed to room temperature and stirred for 16 h. After completion of the reaction, the mixture was quenched by adding NH 4 Cl solution and extracted with EtOAc (100 mL x 2) . The combined organic layer was washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated. The crude product was purified by silica gel column (eluted with 10%EtOAc in Petroleum ether) to afford the title compound 4-1 (1.7 g, yield 90%) as a colorless oil.
  • Step 2 To a solution of compound 4-1 (2.49 g, 7.72 mmol) in MeOH (20 mL) was added D- (+) -10-Camphorsulfonic Acid (5.39 g, 23.2 mmol) at room temperature. The reaction mixture was stirred for 16 h. After completion of the reaction, the resulting mixture was concentrated. The residue was purified by silica gel column (eluted with 25%EtOAc in Petroleum ether) to afford the title compound 4-2 (1.7 g, yield 94%) as a colorless oil.
  • Step 3 Under nitrogen, to a solution of compound 4-2 (2.4 g, 10.29 mmol) in DMF (30 mL) at 0 °C was added NaH (60%in mineral oil, 1.64 g, 41.0 mmol) in several portions. After stirring for 1 h at 0 °C, allyl bromide (3.73 g, 30.83 mmol) was added dropwise thereto. The mixture was warmed to room temperature and stirred for 24 h. After completion of the reaction, the mixture was quenched by adding water and extracted with EtOAc (100 mL x 2) . The combined organic layer was washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated. The crude product was purified by silica gel column (eluted with 5%EtOAc in Petroleum ether) to afford the title compound 4-3 (2.55 g, yield 79%) as a colorless oil.
  • Step 4 A mixture of compound 4-3 (1.9 g, 6.06 mmol) and methyl thioglycolate (6 mL, 67.1 mmol) was irradiated with UV 365 nm at room temperature for 1 h. After completion of the reaction, the mixture was concentrated under high vacuum. The residue was purified by silica gel (eluted with 25%EtOAc in petroleum ether) to afford the title compound 4-4 (2.7 g, yield 80%) as a colorless oil.
  • LCMS m/z calculated for C 18 H 31 N 3 O 9 S 2 : 497.58; found: 520.82 [M+Na] + .
  • Step 6 A solution of compound 4-5 (350 mg, 0.703 mmol) , HOBt (190 mg, 1.41 mmol) , DIPEA (362 mg, 2.80 mmol) and EDCI (537 mg, 2.81 mmol) in anhydrous dichloromethane (5 mL) was stirred at room temperature for 10 min. To this was added a solution of compound 1-11 (hydrochloride, 1.49 g, 2.17 mmol) and DIPEA (260 mg, 2.01 mmol) in anhydrous dichloromethane (5 mL) . The resulting mixture was stirred at room temperature for 24 h.
  • LCMS m/z calculated for C 56 H 111 N 7 O 23 S 2 : 1314.65; found: 1315.68 [M+H] + .
  • LCMS m/z calculated for C 64 H 119 N 7 O 31 S 2 : 1546.79; found: 1547.98 [M+H] + .
  • Step 9 To a solution of compound 4-8 (110 mg, 0.0711) , compound 2-11 (hydrochloride, 210 mg, 0.241 mmol) and DIPEA (96 mg, 0.742 mmol) in anhydrous DMF (3 mL) was added dropwise T3P (50%in DMF, 200 mg, 0.314 mmol) . The mixture was stirred at room temperature for 24 h. After completion of the reaction, the mixture was quenched by adding water (1 mL) and filtered. The filtrate was purified by reverse phase flash chromatography (C18 column, eluted with acetonitrile and water, TFA condition) .
  • Step 11 To a solution of compound 4-10 (trifluoroacetate, 90 mg, 0.0292 mmol) and compound 1-31 (24 mg, 0.0484 mmol) in degassed DMSO (3 mL) was added a fresh aqueous solution of ascorbic acid (1 M, 0.285 mL, 0.285 mmol) and a fresh aqueous solution of Cu 2 SO 4 (1 M, 0.095 mL, 0.095 mmol) . The mixture was purged with nitrogen for three times and stirred at room temperature for 1 h. After completion of the reaction, the mixture was filtered and diluted with water (1 mL) .
  • Step 1 To a mixture of compound 5-1 (20.0 g, 56.4 mmol) in tetrahydrofuran (400 mL) and toluene (100 mL) was added lead tetraacetate (30.0 g, 69.2 mmol) in several portions at room temperature with stirring. The mixture was stirred at 85 °C for 4 h under nitrogen atmosphere. After completion of the reaction, the mixture was cooled to room temperature and the insoluble white solid was removed by filtration through diatomite. The filtrate was concentrated and the residue was re-dissolved in ethyl acetate (200 mL) , washed with water (200 mL) and brine (200 mL) .
  • Step 2 A mixture of compound 5-2 (10.0 g, 27.1 mmol) , compound 5-3 (45.1 g, 271 mmol) and PPTS (680 mg, 2.71 mmol) in anhydrous dichloromethane (100 mL) was stirred at 50 °C for 20 h under nitrogen atmosphere. After completion of the reaction, the mixture was cooled and diluted with ethyl acetate (400 mL) , washed with water (300 mL) and brine (300 mL) . The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated.
  • the next amino acids were added by using the following coupling condition: For coupling conditions: Fmoc-AA-OH/HBTU/DIPEA (15.60 : 14.82 : 31.20 mmol) in DMF for 30 min. The following amino acids were coupled one by one: Fmoc-Gly-OH, Fmoc-Phe-OH and Cbz-Gly-OH. The final resin was washed with DMF and MeOH. At last, desired linear peptide was cleaved from the resin with 20%hexafluoroisopropanol in DCM for 1 h. The mixture was filtered and the filtrate was concentrated under reduced pressure at 40 °C to give a residue.
  • Step 6 A mixture of compound 5-8 (625 mg, 0.68 mmol) , Pd (OH) 2 /C (5 w%, 150 mg) and Pd/C (5 w%, 150 mg) in MeOH (30 mL) was stirred at room temperature under hydrogen atmosphere (0.8 MPa) for 16 h. After completion of the reaction, the mixture was filtered through diatomite and concentrated to afford the title compound 5-9 (460 mg, yield 86%) as a yellow solid.
  • LCMS m/z calculated for C 40 H 42 FN 7 O 9 : 783.30; found: 784.76 [M+H] + .
  • Step 7 To a solution of compound 1-8 (2.7 g, 7.02 mmol) and DMAP (170 mg, 1.39 mmol) in DCM (30 mL) was added Et 3 N (2.84 g, 28.06 mmol) and tosyl chloride (4.69 g, 24.60 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 14 h. After completion of the reaction, the mixture was diluted with DCM (100 mL) and washed with H 2 O (100 mL) , 1 N HCl (100 mL) and brine (100 mL) . The organic layer was dried over anhydrous Na 2 SO 4 , filtered and concentrated to give a residue.
  • Step 8 Under nitrogen atmosphere, a solution of compound 5-11 (1.6 g, 7.87 mmol) in anhydrous THF (6 mL) was stirred and cooled to -75 °C over 10 min. To this was added slowly KHMDS (1 M in THF, 10 mL, 10.0 mmol) . The mixture was stirred at -75 °C for 1 h before a solution of compound 5-10 (3.0 g, 5.57 mmol) in anhydrous THF (8 mL) was added dropwise. The resulting mixture was stirred at -75 °C for 4 h and warmed to room temperature with further stirring for 12 h.
  • Step 9 4 N HCl in EtOAc (1.6 mL) was added to a solution of compound 5-12 (1.6 g, 2.81 mmol) in EtOAc (3 mL) . The mixture was stirred at room temperature for 3 h. After completion of the reaction, the mixture was concentrated to give the title compound 5-13 (hydrochloride, 1.4 g, yield 98%) as a yellow oil.
  • LCMS m/z calculated for C 21 H 43 NO 10 : 469.57; found: 470.81 [M+H] + .
  • Step 10 To a solution of compound 4-5 (460 mg, 0.92 mmol) and DIPEA (716 mg, 5.53 mmol) in anhydrous DMF (5 mL) was added HATU (1.4 g, 3.68 mmol) . The mixture was stirred at room temperature for 10 min before a solution of compound 5-13 (1.4 g, 2.75 mmol) and DIPEA (716 mg, 5.53 mmol) in anhydrous DMF (5 mL) was added thereto. The reaction mixture was stirred at room temperature for 12 h, additional HATU (702 mg, 1.85 mmol) and DIPEA (240 mg, 1.86 mmol) was added. The resulting mixture was stirred at room temperature for further 12 h.
  • LCMS m/z calculated for C 56 H 105 N 5 O 27 S 2 : 1344.58; found: 1345.87 [M+H] + .
  • Step 11 To a mixture of compound 10-12 (130 mg, 0.07 mmol) and N-Hydroxysuccinimide (25 mg, 0.22 mmol) in anhydrous DCM (5mL) was added EDCI (28 mg, 0.15 mmol) . The reaction mixture was stirred at room temperature for 4 h under nitrogen atmosphere. After completion of the reaction, the mixture was concentrated. The residue was purified by reverse phase flash chromatography (C18 column, eluted with acetonitrile and water, TFA condition) . The desired components were lyophilized to afford the title compound 10-13 (108 mg, yield 79%) as a white solid.
  • LCMS m/z calculated for C 93 H 137 N 15 O 24 S 2 : 1913.32; found: 957.04 [M+2H] 2+ .
  • Step 14 Under nitrogen atmosphere, to a solution of compound 10-15 (30 mg, 0.012 mmol) and compound 1-31 (9 mg, 0.018 mmol) in DMSO (2 mL) was added CuI (5 mg, 0.026 mmol) and DIPEA (13 mg, 0.10 mmol) . The reaction mixture was stirred at room temperature for 1 h. After completion of the reaction, the mixture was filtered and the filtrate was purified directly by pre-HPLC (C18 column, eluted with acetonitrile and water, TFA condition) . The desired components were lyophilized to afford the title compound 10 (14.8 mg, yield 41%) as a yellow solid.
  • LCMS m/z calculated for C 146 H 205 FN 22 O 43 S 2 : 3039.48; found: 1520.34 [M+2H] 2+ .
  • Step 1 To a solution of compound 1-4 (3.80 g, 12.36 mmol) in anhydrous DMF (30 mL) was added NaH (60%in mineral oil, 1.48 g, 37.00 mmol) in several portions under nitrogen atmosphere. After the mixture was stirred at 0 °C for 1 h, a solution of compound 10-3 (4.70 g, 15.56 mmol) in anhydrous DMF (5 mL) was added thereto. The resulting mixture was stirred at room temperature for 1 h. After completion of the reaction, the mixture was partitioned with EtOAc (200 mL) and water (300 mL) . The organic layer was washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated.
  • EtOAc 200 mL
  • water 300 mL
  • Step 3 Under nitrogen atmosphere, to a mixture of compound 12-2 (4.09 g, 11.20 mmol) and triphenylphosphine (3.23 g, 12.31 mmol) in anhydrous DCM (50 mL) was added N-bromosuccinimide (2.19 g, 12.30 mmol) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 14 h. After completion of the reaction, the mixture diluted with EtOAc (100 mL) and washed with water (100 mL) . The organic phase was washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated. The residue was purified by silica gel column chromatography (eluted with 15%EtOAc in petroleum ether) to afford the title compound 12-3 (3.4 g, yield 70%) as a colorless oil.
  • Step 4 A mixture of compound 12-3 (990 mg, 2.31 mmol) and potassium thioacetate (528 mg, 4.62 mmol) in anhydrous DMF (7 mL) was stirred at room temperature for 6 h. After completion of the reaction, the mixture was extracted with EtOAc (50 mL x 2) and H 2 O (70 mL) . The combined organic layers were washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated. The crude product was purified by silica gel column chromatography (eluted with 15%EtOAc in petroleum ether) to afford the title compound 12-4 (670 mg, yield 68%) as a colorless oil.
  • Step 7 To a solution of compound 12-6 (127 mg, 0.075 mmol) in THF (1 mL) and DCM (2 mL) was added 80%aqueous trifluoroacetic acid solution (0.5 mL) at 0 °C. The reaction was stirred at room temperature for 5 h. After completion of the reaction, the mixture was diluted with DCM and concentrated. The crude product was purified by reverse phase flash chromatography (C18 column, eluted with acetonitrile and water, TFA condition) . The desired components were lyophilized to afford the title compound 12-7 (58 mg, yield 48%) as a white solid.
  • LCMS m/z calculated for C 78 H 124 N 14 O 20 S: 1609.99; found: 1610.34 [M+H] + .
  • Step 8 Under nitrogen atmosphere, to a solution of compound 12-7 (50 mg, 0.031 mmol) and compound 1-31 (31 mg, 0.062 mmol) in DMSO (2 mL) was added CuI (6 mg, 0.031 mmol) and DIPEA (16 mg, 0.124 mmol) . The reaction mixture was stirred at room temperature for 1 h. After completion of the reaction, the mixture was filtered and the filtrate was purified directly by pre-HPLC (C18 column, eluted with acetonitrile and water, TFA condition) . The desired components were lyophilized to afford the title compound 12 (41.9 mg, yield 64%) as a white solid.
  • LCMS m/z calculated for C 102 H 161 N 17 O 28 S: 2105.56; found: 1053.79 [M+2H] 2+ .
  • the general conjugates of SL’s and monoclonal antibodies were formed using reported conjugation condition (Doronina, et al. 2003 Nature Biotechnology 21 (7) : 778-84) .
  • the mAbs were reduced with TCEP and then alkylated with the maleimido-containing SL’s forming the corresponding ADC conjugates (Table 1) .
  • the hydrophobic interaction chromatography (HIC) chromatographs and size exclusion high-performance liquid chromatography (SEC-HPLC) chromatographs were shown in FIGs. 14-18.
  • Example 24 Hydrophilicity measure by high-performance liquid chromatography (HPLC)
  • test compound spike aliquots of stock solution into 1x PBS to achieve the concentration (200 ⁇ g/mL) of working solution and mix thoroughly.
  • T0 sample plates For T0 sample plates, firstly, add 150 ⁇ L stop solution to the corresponding wells. Secondly, add 50.0 ⁇ L (experimental group) or 20 ⁇ L (positive control group) of the compound working solution to achieve final concentration Propantheline bromide aliquots 1.00 ⁇ M (positive control group) and the testing ADC 10 ⁇ g/mL (experimental group) and shake well. Finally take aliquots of 50 ⁇ L serum samples to T0 sample plate.
  • reaction for positive control group
  • 0.5h, 1h, 4h, 8h, 24h, 2d, 4d and 7d of reaction for experimental group, take aliquots of 50 ⁇ L serum samples to the corresponding wells of the sample plate (add 150 ⁇ L stop solution) to terminate the reaction.
  • ADCs’ serum stability data is shown in Table 2.
  • test article lyophilized powder was redissolved with sterile water to obtain the stock solution.
  • the stock solution was diluted into 0.2 mg/mL using the solvent PBS (pH 7.2-7.4) or Trehalose Polysorbate 80 pH 6.5 for dosing.
  • the dose level of the test article in this study was set at 2 mg/kg.
  • mice Nine mice were selected according to body weight and randomly divided into 3 groups (G1-G3) with 3 mice per group. All animals were administered by a single caudal intravenous injection. Animals do not fast and drink freely before administration. The dosage volume was 10 mL/kg.
  • Blood collection time points of PK in G1 group were: 5 min ⁇ 30s after administration, D1 ⁇ 20 min a day, D4 ⁇ 30 min a day, a total of 3 points. If it is not collected within the specified time, it should be recorded.
  • Blood collection time points of PK in G2 group were as follows: 2 hr ⁇ 5 min after administration, D2 ⁇ 30 min and D7 ⁇ 1 hr. If it is not collected within the specified time, it should be recorded.
  • Blood collection time points of PK in G3 group were: 7 hr ⁇ 10 min after administration, D3 ⁇ 30 min and D14 ⁇ 1 hr. If it is not collected within the specified time, it should be recorded.
  • PK blood collection time point before administration Another 3 spare mice were selected for blood collection as PK samples before administration.
  • Blood was collected from the mice through orbital venous plexus into a coagulation stimulating tube, and about 0.12 mL whole blood was collected at each time point. After blood collection, the coagulation stimulating tube was left for at least 10 min to completely coagulate the samples, and the samples were centrifuged at 4°C within 0.5 hr after blood collection, rotating speed was 3000 g, and centrifuged for 5 min. Serum was taken immediately after centrifugation and each blood sample was placed into a new labeled tube.
  • the freezing tube is placed on ice, and the serum sample is frozen to the -60 ⁇ -90°C refrigerator as soon as possible within 1 hr (if the serum sample can not be stored in the -60 ⁇ -90°C refrigerator in time, it can be temporarily stored in the -10 ⁇ -30°C refrigerator within 1 hr, and the temporary storage time is not more than 24 h) .
  • Blood was collected from the mice through the orbital venous plexus into the EDTA-K2 anticoagulant tube, about 0.06 mL whole blood was collected at each time point, and 20 ⁇ L whole blood was immediately added into the tube containing 60 ⁇ L acetonitrile (duplicated) , vortex mixed, and within 1 hr, freeze the whole blood sample to the -60 ⁇ -90°C refrigerator as soon as possible (if the whole blood sample can not be stored in the -60 ⁇ -90°C refrigerator in time, it can be temporarily stored in the -10 ⁇ -30°C refrigerator within 1 h, and the temporary storage time is not more than 24 h) . Samples were collected, processed and stored away from light.
  • Labels for whole blood and serum samples contain information such as topic number, animal number, group, substrate type, and time point.
  • ADC and total antibody were detected by ELISA, in which ADC was detected by anti-MMAE and/or anti-DXD and/or anti-SN-38 antibodies respectively.
  • a standard curve containing at least 6 concentration levels of the substance to be tested is prepared for each batch and accompanied by one or two sets of 3 low, medium and high concentration quality control (QC) samples. Calibration standard samples and quality control samples were measured by double and double holes, and their average values were taken, and other samples were measured by single holes.
  • the concentrations of free MMAE ⁇ SN-38 ⁇ DXD and/or CAN3010 (MsOH) in whole blood were detected by LC-MS/MS method.
  • Test articles with different solvents used their own vehicle control for analyses.
  • IR (%) [1– (RLU test article –RLU blank) / (RLU control –RLU blank) ] *100%.
  • the inhibitions of different doses of test articles were calculated in Excel file, and then were used to plot inhibition curve and evaluate related parameters, such as Bottom, Top and IC50. The data were interpreted by GraphPad Prism.
  • IR (%) [1– (RLU test article –RLU blank) / (RLU control –RLU blank) ] *100%.
  • the inhibitions of different doses of test articles were calculated in Excel file, and then were used to plot inhibition curve and evaluate related parameters, such as Bottom, Top and IC50. The data were interpreted by GraphPad Prism.

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Abstract

L'invention concerne de nouveaux lieurs multivalents, des conjugués anticorps-médicament et des compositions pharmaceutiques et des méthodes de traitement associées contre certaines maladies ou affections.
PCT/CN2024/083437 2024-03-25 2024-03-25 Lieurs multivalents pour conjugués anticorps-médicament à charge élevée, et compositions et procédés associés Pending WO2025199666A1 (fr)

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PCT/CN2025/084562 WO2025201281A1 (fr) 2024-03-25 2025-03-25 Agents de liaison multivalents pour conjugués anticorps-médicament à charge élevée, et compositions et méthodes associées

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WO2021174113A1 (fr) * 2020-02-28 2021-09-02 Regeneron Pharmaceuticals, Inc. Molécules bispécifiques de liaison à l'antigène qui se lient à her2, et leurs procédés d'utilisation
WO2022015656A1 (fr) * 2020-07-13 2022-01-20 Regeneron Pharmaceuticals, Inc. Analogues de camptothécine conjugués à un résidu de glutamine dans une protéine et leur utilisation
WO2022253035A1 (fr) * 2021-06-02 2022-12-08 四川科伦博泰生物医药股份有限公司 Conjugué anticorps-médicament, sa méthode de préparation et utilisation associée
WO2023137026A1 (fr) * 2022-01-12 2023-07-20 Regeneron Pharmaceuticals, Inc. Conjugués protéine-médicament comprenant des analogues de camptothécine et procédés d'utilisation associés

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CN109562185A (zh) * 2016-06-02 2019-04-02 赛诺菲 药剂与能够结合葡萄糖感应蛋白的部分的新颖缀合物
WO2023288033A1 (fr) * 2021-07-14 2023-01-19 Lycia Therapeutics, Inc. Composés et conjugués de liaison au récepteur de surface cellulaire asgpr
JP2024178476A (ja) * 2021-09-29 2024-12-25 国立大学法人大阪大学 α線放出抗体薬物複合体
EP4588489A1 (fr) * 2022-09-16 2025-07-23 Shanghai Junshi Biosciences Co., Ltd. Lieur pour conjugué anticorps-médicament et son utilisation

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Publication number Priority date Publication date Assignee Title
WO2021174113A1 (fr) * 2020-02-28 2021-09-02 Regeneron Pharmaceuticals, Inc. Molécules bispécifiques de liaison à l'antigène qui se lient à her2, et leurs procédés d'utilisation
WO2022015656A1 (fr) * 2020-07-13 2022-01-20 Regeneron Pharmaceuticals, Inc. Analogues de camptothécine conjugués à un résidu de glutamine dans une protéine et leur utilisation
WO2022253035A1 (fr) * 2021-06-02 2022-12-08 四川科伦博泰生物医药股份有限公司 Conjugué anticorps-médicament, sa méthode de préparation et utilisation associée
WO2023137026A1 (fr) * 2022-01-12 2023-07-20 Regeneron Pharmaceuticals, Inc. Conjugués protéine-médicament comprenant des analogues de camptothécine et procédés d'utilisation associés

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