WO2024098066A1 - Exatecan derivatives and antibody-drug conjugates thereof - Google Patents

Exatecan derivatives and antibody-drug conjugates thereof Download PDF

Info

Publication number
WO2024098066A1
WO2024098066A1 PCT/US2023/078842 US2023078842W WO2024098066A1 WO 2024098066 A1 WO2024098066 A1 WO 2024098066A1 US 2023078842 W US2023078842 W US 2023078842W WO 2024098066 A1 WO2024098066 A1 WO 2024098066A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
mmol
equiv
antibody
linker
Prior art date
Application number
PCT/US2023/078842
Other languages
French (fr)
Inventor
Jaume Pons
Marija VRLJIC
Peter Strop
Janica Cheuk-ying WONG
Hanako DAINO-LAIZURE
Original Assignee
ALX Oncology Inc.
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 ALX Oncology Inc. filed Critical ALX Oncology Inc.
Publication of WO2024098066A1 publication Critical patent/WO2024098066A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings

Definitions

  • ADC Antibody-drug conjugates
  • Topoisomerase I plays a critical role in DNA replication in both normal and diseased conditions (e.g., cancer). As inhibition of topoisomerase I leads to cell death, compounds that bind to and inhibit topoisomerase I may be useful as therapeutic agents.
  • Camptothecin is a natural product with cytotoxic activity in a variety of cell lines. The binding of its active lactone ring to topoisomerase I inhibits DNA replication, thus causing cell apoptosis.
  • its limitations for drug development include, for example, poor water solubility and an equilibrium between its active, lactone form and its inactive, ring-opened form.
  • Exatecan is a water-soluble camptothecin derivative. As a chemotherapeutic agent, exatecan mesylate did not gain drug approval after several clinical trials due to lack of efficacy or high toxicity at tested doses.
  • exatecan Efforts to enable the clinical utility of exatecan have been made by converting exatecan into a prodrug form, where exatecan is covalently linked to a carboxymethyldextran polyalcohol polymer via a peptidyl spacer (a substrate for intracellular cathepsin proteases).
  • this prodrug did not succeed in clinical trials.
  • preferential delivery of topoisomerase I inhibitors to diseased tissues through antibody-drug conjugates could lead to improved safety and efficacy, thereby providing therapeutic options for a larger number of patients and types of cancers.
  • the present disclosure relates to compounds useful for the treatment of cancer.
  • the present disclosure is directed, in part, to linker-payload constructs useful for attaching payloads to antibodies and exatecan-based drug conjugates.
  • compounds representing a therapeutic payload, a linker-payload construct, or a drug conjugate are compounds representing a therapeutic payload, a linker-payload construct, or a drug conjugate.
  • the present disclosure provides linker-payload constructs and drug conjugates, each comprising a disclosed therapeutic payload.
  • disclosed compounds as medicinal agents, processes for their preparation, and pharmaceutical compositions containing them as an active ingredient both alone or in combination with other agents, as well as provides for their use as medicaments and/or in the manufacture of medicaments for the treatment of cancer.
  • a therapeutic payload represented by Formula II: or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: Y is hydrogen or -C 1-3 alkyl; R is selected from the group consisting of -C(O)-C 3 alkyl, -C(O)-C 3 cycloalkyl, -C(O)- C4cycloalkyl, -C1alkyl, -C(O)-(pyrrolidinyl), and -C(O)-CH2ONH-C(O)-(pyrrolidinyl); wherein: -C(O)-C 3 alkyl, -C(O)-C 3 cycloalkyl, -C(O)-C 4 cycloalkyl or -C 1 alkyl is substituted by one or two substituents each independently selected from the group consisting of hydroxyl, -NH2, -CHO, and -COOH; -C(O)-(pyrroli
  • Methods of treating cancer comprising administering to a patient in need thereof an effective amount of a disclosed compound.
  • a method of treating cancer in patient in need thereof comprising administering to the patient an effective amount of a disclosed therapeutic payload, a disclosed linker-payload construct, or a disclosed drug conjugate.
  • Pharmaceutical compositions comprising at least one disclosed compound and a pharmaceutically acceptable carrier are additionally described herein.
  • a pharmaceutically acceptable composition comprising a disclosed compound, e.g., a disclosed therapeutic payload, a disclosed linker-payload construct, or a disclosed drug conjugate and a pharmaceutically acceptable excipient.
  • FIG.1A, FIG.1B, and FIG.1C show tumor suppression effect of antibody drug conjugates 3031, 3036, and 3038 on lung carcinoma cell line NCI-H292 derived xenograft in athymic nude mice. Compounds exemplified here exhibit tumor suppression greater than control.
  • FIG.2A and FIG.2B show tumor suppression effect of antibody drug conjugates 3058A and 3053B on human throat cancer cell line FaDu derived xenograft in athymic nude mice. Compounds exemplified here exhibit tumor suppression greater than control.
  • FIG.3A, FIG.3B, FIG.3C, and FIG.3D show tumor suppression effect of antibody drug conjugates 3058A, 3058B, 3102, and 3053B on human lung cancer cell line H1975 derived xenograft in athymic nude mice. Compounds exemplified here exhibit tumor suppression greater than control.
  • FIG.4A, FIG.4B, and FIG.4C show tumor suppression effect of antibody drug conjugates 3053B, 3058A, and 3102 on human breast cancer cell line MDA-MB-468 derived xenograft in NOD-SCID mice. Compounds exemplified here exhibit tumor suppression greater than control.
  • FIG.5A, FIG.5B, FIG.5C, FIG 5D, FIG.5E, and FIG.5F show tumor suppression effect of antibody drug conjugates 3058A ,3102, 3059B, 3108, 3110, and 3053B on human throat cancer cell line FaDu derived xenograft in NOD-SCID mice. Compounds exemplified here exhibit tumor suppression greater than control.
  • FIG.6A, FIG.6B, FIG.6C, and FIG.6D show tumor suppression effect of antibody drug conjugates 3058A, 3053B, 3102, and 3059B on human breast cancer cell line MDA-MB-468 and colon cancer cell line SW620-luc co-inoculated xenograft in NOD-SCID mice.
  • FIG.7A, FIG.7B, FIG.7C, and FIG.7D show tumor suppression effect of antibody drug conjugates 3058A, 3053B, 3102, and 3059B on human breast cancer cell line MDA-MB-468 and colon cancer cell line SW620-luc co-inoculated xenograft in NOD-SCID mice. All four figures show luciferase signal expressed as total flux (photons per second) as a function of time after start of antibody drug conjugate administration compared to vehicle control group. Compounds exemplified here exhibit tumor suppression greater than control.
  • FIG.8A, FIG.8B, FIG.8C, and FIG.8D show tumor suppression effect of antibody drug conjugates 3111, 3112, 3110, and 3109 on human throat cancer cell line FaDu derived xenograft in NOD-SCID mice (10 mg per kg, 1 dose). Compounds exemplified here exhibit tumor suppression greater than control.
  • FIG.9A, FIG.9B, FIG.9C, and FIG.9D show tumor suppression effect of antibody drug conjugates 3111, 3112, 3110, and 3109 on human throat cancer cell line FaDu derived xenograft in NOD-SCID mice (3 mg per kg, 1 dose). Compounds exemplified here exhibit tumor suppression greater than control.
  • FIG.10A, FIG.10B, FIG.10C, and FIG.10D show tumor suppression effect of antibody drug conjugates 3111, 3112, 3110, and 3109 on human throat cancer cell line FaDu derived xenograft in NOD-SCID mice (1 mg per kg, 3 doses). Compounds exemplified here exhibit tumor suppression greater than control.
  • FIG.11A, FIG.11B, FIG.11C, and FIG.11D shows tumor suppression effect of antibody drug conjugates 3111, 3112, 3110, and 3109 on human breast cancer cell line MDA- MB-468 derived xenograft in NOD-SCID mice (3 mg per kg, 1 dose). Compounds exemplified here exhibit tumor suppression greater than control.
  • alkenyl refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond.
  • alkenyl groups include, but are not limited to, a straight or branched group of 2-6 or 3-4 carbon atoms, referred to herein as C2-6alkenyl, and C3-4alkenyl, respectively.
  • alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, etc.
  • alkoxy refers to a straight or branched alkyl group attached to oxygen (alkyl-O-).
  • alkoxy groups include, but are not limited to, alkoxy groups of 1-6 or 2-6 carbon atoms, referred to herein as C1-6alkoxy, and C2-6alkoxy, respectively.
  • Exemplary alkoxy groups include, but are not limited to methoxy, ethoxy, isopropoxy, etc.
  • alkoxyalkyl refers to a straight or branched alkyl group attached to oxygen, attached to a second straight or branched alkyl group (alkyl-O-alkyl-).
  • alkoxyalkyl groups include, but are not limited to, alkoxyalkyl groups in which each of the alkyl groups independently contains 1-6 carbon atoms, referred to herein as C 1-6 alkoxy-C 1- 6alkyl.
  • Exemplary alkoxyalkyl groups include, but are not limited to methoxymethyl, 2- methoxyethyl, 1-methoxyethyl, 2-methoxypropyl, ethoxymethyl, 2-isopropoxyethyl etc.
  • alkyoxycarbonyl refers to a straight or branched alkyl group attached to oxygen, attached to a carbonyl group (alkyl-O-C(O)-).
  • alkoxycarbonyl groups include, but are not limited to, alkoxycarbonyl groups of 1-6 carbon atoms, referred to herein as C1-6alkoxycarbonyl.
  • Exemplary alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, etc.
  • alkenyloxy used herein refers to a straight or branched alkenyl group attached to oxygen (alkenyl-O-).
  • alkenyloxy groups include, but are not limited to, groups with an alkenyl group of 3-6 carbon atoms, referred to herein as C3-6alkenyloxy.
  • alkenyloxy groups include, but are not limited to allyloxy, butenyloxy, etc.
  • alkynyloxy used herein refers to a straight or branched alkynyl group attached to oxygen (alkynyl-O).
  • exemplary alkynyloxy groups include, but are not limited to, groups with an alkynyl group of 3-6 carbon atoms, referred to herein as C3-6alkynyloxy.
  • Exemplary alkynyloxy groups include, but are not limited to, propynyloxy, butynyloxy, etc.
  • alkyl as used herein refers to a saturated straight or branched hydrocarbon.
  • Exemplary alkyl groups include, but are not limited to, straight or branched hydrocarbons of 1-6, 1-4, or 1-3 carbon atoms, referred to herein as C 1-6 alkyl, C 1-4 alkyl, and C 1- 3 alkyl, respectively.
  • Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-butyl, 3-methyl-2-butyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4- methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, etc.
  • alkylcarbonyl refers to a straight or branched alkyl group attached to a carbonyl group (alkyl-C(O)-).
  • exemplary alkylcarbonyl groups include, but are not limited to, alkylcarbonyl groups of 1-6 atoms, referred to herein as C1-6alkylcarbonyl groups.
  • exemplary alkylcarbonyl groups include, but are not limited to, acetyl, propanoyl, isopropanoyl, butanoyl, etc.
  • Alkylene means a straight or branched, saturated aliphatic divalent radical having the number of carbons indicated.
  • Cycloalkylene refers to a divalent radical of carbocyclic saturated hydrocarbon group having the number of carbons indicated.
  • alkynyl refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond.
  • Exemplary alkynyl groups include, but are not limited to, straight or branched groups of 2-6, or 3-6 carbon atoms, referred to herein as C2-6alkynyl, and C3-6alkynyl, respectively.
  • alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, etc.
  • carbonyl as used herein refers to the radical -C(O)-.
  • cyano as used herein refers to the radical -CN.
  • cycloalkoxy as used herein refers to a cycloalkyl group attached to oxygen (cycloalkyl-O-).
  • Exemplary cycloalkoxy groups include, but are not limited to, cycloalkoxy groups of 3-6 carbon atoms, referred to herein as C 3-6 cycloalkoxy groups. Exemplary cycloalkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclohexyloxy, etc.
  • the terms “cycloalkyl” or a “carbocyclic group” as used herein refers to a saturated or partially unsaturated hydrocarbon group of, for example, 3-6, or 4-6 carbons, referred to herein as C3-6cycloalkyl or C4-6cycloalkyl, respectively.
  • cycloalkyl groups include, but are not limited to, cyclohexyl, cyclopentyl, cyclopentenyl, cyclobutyl or cyclopropyl.
  • halo or halogen as used herein refer to F, Cl, Br, or I.
  • heteroaryl or “heteroaromatic group” as used herein refers to a monocyclic aromatic 5-6 membered ring system containing one or more heteroatoms, for example one to three heteroatoms, such as nitrogen, oxygen, and sulfur. Where possible, said heteroaryl ring may be linked to the adjacent radical though carbon or nitrogen.
  • heteroaryl rings include but are not limited to furan, thiophene, pyrrole, thiazole, oxazole, isothiazole, isoxazole, imidazole, pyrazole, triazole, pyridine or pyrimidine etc.
  • heterocyclyl or “heterocyclic group” are art-recognized and refer to e.g., saturated or partially unsaturated, 4-10 membered monocyclic or bicyclic ring structures, or e.g., 4-9 or 4-6 membered saturated ring structures, including bridged, fused or spirocyclic rings, and whose ring structures include one to three heteroatoms, such as nitrogen, oxygen, and sulfur. Where possible, heterocyclyl rings may be linked to the adjacent radical through carbon or nitrogen.
  • heterocyclyl groups include, but are not limited to, pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, oxetane, azetidine, tetrahydrofuran or dihydrofuran etc.
  • heterocyclyloxy refers to a heterocyclyl group attached to oxygen (heterocyclyl-O-).
  • heteroaryloxy refers to a heteroaryl group attached to oxygen (heteroaryl-O-).
  • hydroxy and “hydroxyl” as used herein refer to the radical -OH.
  • “Pharmaceutically or pharmacologically acceptable” include molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologics standards.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” as used herein refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration.
  • compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.
  • pharmaceutical composition refers to a composition comprising at least one compound as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.
  • “Individual,” “patient,” or “subject” are used interchangeably and include any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.
  • the compounds of the present disclosure can be administered to a mammal, such as a human, but can also be administered to other mammals such as an animal in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).
  • “Modulation” includes antagonism (e.g., inhibition), agonism, partial antagonism and/or partial agonism.
  • “Treating” includes any effect, e.g., lessening, reducing, modulating, or eliminating, that results in the improvement of the condition, disease, disorder and the like.
  • the term “therapeutically effective amount” or “effective amount” means the amount of the subject compound that will elicit the biological or medical response of a tissue, system or animal, (e.g., mammal or human) that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • the compounds of the present disclosure are administered in therapeutically effective amounts to treat a disease.
  • a therapeutically effective amount of a compound is the quantity required to achieve a desired therapeutic and/or prophylactic effect, such as an amount which results in weight loss.
  • pharmaceutically acceptable salt(s) refers to salts of acidic or basic groups that may be present in compounds used in the compositions.
  • compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids.
  • the acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including, but not limited to, malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulf
  • Compounds included in the present compositions that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations.
  • Examples of such salts include alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.
  • Compounds included in the present compositions that include a basic or acidic moiety may also form pharmaceutically acceptable salts with various amino acids.
  • the compounds of the disclosure may contain both acidic and basic groups; for example, one amino and one carboxylic acid group. In such a case, the compound can exist as an acid addition salt, a zwitterion, or a base salt.
  • the compounds of the disclosure may contain one or more double bonds and, therefore, exist as geometric isomers resulting from the arrangement of substituents around a carbon-carbon double bond.
  • Compounds of the disclosure may contain a carbocyclic or heterocyclic ring and therefore, exist as geometric isomers resulting from the arrangement of substituents around the ring.
  • the arrangement of substituents around a carbocyclic or heterocyclic ring are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting carbocyclic or heterocyclic rings encompass both “Z” and “E” isomers.
  • Substituents around a carbocyclic or heterocyclic rings may also be referred to as “cis” or “trans”, where the term “cis” represents substituents on the same side of the plane of the ring and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis/trans.”
  • Individual enantiomers and diastereomers of compounds of the present disclosure can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art.
  • Stereoselective syntheses a chemical or enzymatic reaction in which a single reactant forms an unequal mixture of stereoisomers during the creation of a new stereocenter or during the transformation of a pre-existing one, are well known in the art.
  • Stereoselective syntheses encompass both enantio- and diastereoselective transformations and may involve the use of chiral auxiliaries. For examples, see Carreira and Kvaerno, Classics in Stereoselective Synthesis, Wiley-VCH: Weinheim, 2009.
  • the compounds disclosed herein can exist in solvated as well as unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the present disclosure embrace both solvated and unsolvated forms.
  • the compound is amorphous.
  • the compound is a single polymorph.
  • the compound is a mixture of polymorphs.
  • the compound is in a crystalline form.
  • the present disclosure also embraces isotopically labeled compounds of the disclosure which are identical to those recited herein, except that 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 examples include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, 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.
  • a compound of the disclosure may have one or more H atom replaced with deuterium.
  • Certain isotopically labeled disclosed compounds e.g., those labeled with 3 H and 14 C are useful in compound and/or substrate tissue distribution assays.
  • Tritiated (i.e., 3 H) and carbon-14 (i.e., 14 C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., 2 H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances.
  • Isotopically labeled compounds of the present disclosure can generally be prepared by following procedures analogous to those disclosed in the examples herein by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.
  • prodrug refers to compounds that are transformed in vivo to yield a disclosed compound or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (such as by esterase, amidase, phosphatase, oxidative and or reductive metabolism) in various locations (such as in the intestinal lumen or upon transit of the intestine, blood or liver). Prodrugs are well known in the art (for example, see Rautio, Kumpulainen, et al, Nature Reviews Drug Discovery 2008, 7, 255).
  • a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as (C 1-8 )alkyl, (C 2-12 )alkylcarbonyloxymethyl, 1-(alkylcarbonyloxy)ethyl having from 4 to 9 carbon atoms, 1-methyl-1-(alkylcarbonyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)amino)e
  • a group such as (C 1-8 )alkyl, (C 2-12 )alkylcarbonyloxymethyl, 1-(alkylcarbon
  • a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as (C1-6)alkylcarbonyloxymethyl, 1-((C1-6)alkylcarbonyloxy)ethyl, 1-methyl-1-((C1- 6 )alkylcarbonyloxy)ethyl (C 1-6 )alkoxycarbonyloxymethyl, N-(C 1-6 )alkoxycarbonylaminomethyl, succinoyl, (C 1-6 )alkylcarbonyl, ⁇ -amino(C 1-4 )alkylcarbonyl, arylalkylcarbonyl and ⁇ - aminoalkylcarbonyl, or ⁇ -aminoalkylcarbonyl- ⁇ -aminoalkylcarbonyl, where each ⁇ - aminoalkylcarbonyl group is independently selected from the naturally occurring L-amino acids, P(O)(OH)2, -
  • a prodrug can be formed, for example, by creation of an amide or carbamate, an N- alkylcarbonyloxyalkyl derivative, an (oxodioxolenyl)methyl derivative, an N-Mannich base, imine or enamine.
  • a secondary amine can be metabolically cleaved to generate a bioactive primary amine, or a tertiary amine can be metabolically cleaved to generate a bioactive primary or secondary amine.
  • Salts of compounds disclosed herein can be prepared by the reaction of a compound disclosed herein with an appropriate acid or base in a suitable solvent, or mixture of solvents (such as an ether, for example, diethyl ether, or an alcohol, for example ethanol, or an aqueous solvent) using conventional procedures. Salts of a compound disclosed herein can be exchanged for other salts by treatment using conventional ion-exchange chromatography procedures.
  • a suitable solvent such as an ether, for example, diethyl ether, or an alcohol, for example ethanol, or an aqueous solvent
  • a disclosed drug conjugate of Formula I may be represented by Formula IA: or a pharmaceutically acceptable salt or stereoisomer thereof; wherein s is 1, 2, 3, 4, 5, 6, 7, or 8.
  • Lig is a monoclonal antibody.
  • Lig is selected from the group consisting of an anti-TROP2 antibody, an anti- EGRF antibody, an anti-HER2 antibody, an anti-B7-H3 antibody, an anti-CD30 antibody, an anti-CD33 antibody, and an anti-CD70 antibody.
  • Lig is selected from the group consisting of an anti-TROP2 antibody, an anti-EGRF antibody, and an anti-HER2 antibody.
  • Lig is an anti-TROP2 antibody. In certain other embodiments, Lig is an anti-EGRF antibody. In still other embodiments, Lig is an anti-HER2 antibody. For example, in certain embodiments Lig may be selected from an antibody disclosed in Table 4. In further embodiments, s is 1 or 8. For example, is some embodiments s is 1. [0070] In some embodiments, L is selected, for example, from the group consisting of:
  • R is selected from the group consisting of -CH 2 CH 2 O-, - C(O)-CH2ONH-, -C(O)-O-CH2CH2O-, -C(O)-NH-CH2CH2O-, -C(O)-C1alkyl-C(O)-NH- CH2CH2O-, -C(O)-C3alkyl-C(O)-NH-CH2CH2O-, and -C(O)-CH(CH3)-NH-.
  • R is selected from the group consisting of -C(O)- triazolyl-CH 2 CH 2 O-, -CH 2 -triazolyl-CH 2 CH 2 O-, and -C(O)-furanyl-CH 2 O-.
  • R is selected from the group consisting of -C(O)- C 3 cycloalkyl-CH 2 CH 2 O-, -C(S)-C 3 cycloalkyl-CH 2 CH 2 O-,-C(O)-C 3 cycloalkyl-NH-, and -C(O)- O-phenyl-NH-.
  • R is selected from the group consisting of -C(O)- pyrrolidinyl- and -C(O)-pyrrolidinyl-C(O)-CH(CH3)-NH-, wherein pyrrolidinyl may optionally be substituted by one or two fluoro atoms.
  • R is selected from the group consisting of
  • a disclosed drug conjugate may be selected from the group consisting of , ,
  • Lig is selected from the group consisting of an anti-TROP2 antibody, an anti-EGRF antibody, and an anti-HER2 antibody.
  • Lig is an anti-TROP2 antibody.
  • Lig is an anti-EGRF antibody, for example, Panitumumab, Nimotuzumab, Matuzumab, or Cetuximab.
  • Lig is an anti-HER2 antibody.
  • s is 1 or 8.
  • s is 1.
  • contemplated drug conjugates of the present disclosure are provided in Tables 1-3. Table 1. HER 2 Antibody Drug Conjugates
  • EGFR Antibody Drug Conjugates [0079] For compounds 3049-3107, Anti-EGFR IgG11 was used; for compound 3108, Anti-EGFR IgG12 was used; for compound 3109-3110, Anti-EGFR IgG13 was used, for compounds 3111-3112, Anti-EGFR IgG14 was used; and for compound 3113, Anti-EGFR IgG1 5 was used.
  • a therapeutic payload represented by Formula II: or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: Y is hydrogen or -C1-3alkyl; R is selected from the group consisting of -C(O)-C 3 alkyl, -C(O)O-C 3 alkyl, -C(O)- C3cycloalkyl, -C(O)-C4cycloalkyl, -C1alkyl, -C(O)-(pyrrolidinyl), and -C(O)-CH2ONH-C(O)- (pyrrolidinyl); wherein: -C(O)-C 3 alkyl, -C(O)-C 3 cycloalkyl, -C(O)O-C 3 alkyl, -C(O)-C 4 cycloalkyl or - C1alkyl is substituted by one or two substituents each independently selected from the group consisting of hydroxyl
  • a disclosed therapeutic payload of Formula II may be represented by Formula IIA: ; or a pharmaceutically acceptable salt or stereoisomer thereof.
  • Y is hydrogen.
  • Y is -C 1-3 alkyl.
  • R is selected, for example, from the group consisting of: ,
  • a disclosed therapeutic payload may be selected, for example, from any one of the compounds disclosed in Table 5, or a pharmaceutically acceptable salt or stereoisomer thereof. Table 5.
  • a therapeutic payload disclosed herein may be selected from the group consisting of:
  • linker-payload construct represented by Formula IIIA or Formula I or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L is selected from the group consisting of:
  • R is selected from the group consisting of:
  • a disclosed linker-payload construct of Formula III may be represented by Formula IIIC: or a pharmaceutically acceptable salt or stereoisomer thereof wherein s is 1, 2, 3, 4, 5, 6, 7, or 8.
  • R is selected, for example, from the group consisting of:
  • a disclosed linker-payload construct may be selected, for example, from any one of the compounds disclosed in Table 6, or a pharmaceutically acceptable salt or stereoisomer thereof. Table 6.
  • a linker-payload construct contemplated herein may be selected from the group consisting of:
  • Methods [0091] Disclosed herein, for example, is a method of treating cancer in patient in need thereof, comprising administering to the patient an effective amount of a therapeutic payload disclosed herein, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, and esophageal cancer.
  • the cancer is selected from the group consisting of lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, and esophageal cancer.
  • Also disclosed herein is a method of treating cancer in patient in need thereof, comprising administering to the patient an effective amount of a linker-payload construct disclosed herein, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, and esophageal cancer.
  • the cancer is selected from the group consisting of lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, and esophageal cancer.
  • a method of treating cancer in patient in need thereof comprising administering to the patient an effective amount of a drug conjugate comprising any of the payloads as disclosed herein, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, and esophageal cancer.
  • a method of delivering a therapeutically effective amount of a therapeutic payload moiety to a patient in need thereof, comprising administering to the patient any one of the drug conjugates disclosed herein.
  • administering a disclosed compound may comprise subcutaneous administration.
  • administering a disclosed compound may comprise intravenous administration.
  • administering a disclosed compound may comprise oral administration.
  • Provided methods of treatment may include administering a disclosed compound once, twice, or three times daily; about every other day (e.g., every 2 days); twice weekly (e.g., every 3 days, every 4 days, every 5 days, every 6 days, or e.g., administered with an interval of about 2 to about 3 days between doses); once weekly; three times weekly; every other week; twice monthly; once a month; every other month; or even less often.
  • the present disclosure provides a method of treating one or more of the above medical indications comprising administering to a subject in need thereof a therapeutically effective amount of a compound described herein.
  • the compound utilized by one or more of the methods disclosed herein is one of the generic, subgeneric, or specific compounds described herein.
  • the compounds of the present disclosure may be administered to patients (animals and humans) in need of such treatment in dosages that will provide optimal pharmaceutical efficacy.
  • a compound of the present disclosure may be administered orally, subcutaneously, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
  • Parenteral administration may include subcutaneous injections, intravenous or intramuscular injections or infusion techniques.
  • Treatment can be continued for as long or as short a period as desired.
  • a suitable treatment period can be, for example, at least about one week, at least about two weeks, at least about one month, at least about six months, at least about 1 year, or indefinitely.
  • a treatment period can terminate when a desired result is achieved.
  • Pharmaceutical Compositions and Kits [00102] Another aspect of the present disclosure provides pharmaceutical compositions comprising compounds as disclosed herein formulated together with a pharmaceutically acceptable carrier. In particular, the present disclosure provides pharmaceutical compositions comprising compounds as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.
  • compositions include those suitable for oral, rectal, topical, buccal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), vaginal, or aerosol administration, although the most suitable form of administration in any given case will depend on the degree and severity of the condition being treated and on the nature of the particular compound being used.
  • disclosed compositions may be formulated as a unit dose, and/or may be formulated for oral or subcutaneous administration.
  • a pharmaceutical composition comprising a therapeutic payload disclosed herein, and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising a linker-payload construct disclosed herein, and a pharmaceutically acceptable excipient.
  • compositions of this disclosure may be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains one or more disclosed compounds, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications.
  • the active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.
  • the active object compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of the disease.
  • the principal active ingredient may be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a disclosed compound, or a non-toxic pharmaceutically acceptable salt thereof.
  • a pharmaceutical carrier e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a
  • the subject composition is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alg
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent.
  • Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art.
  • Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, cyclodextrins and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate
  • Suspensions in addition to the subject composition, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a subject composition with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent.
  • suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent.
  • Dosage forms for transdermal administration of a subject composition include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • a pharmaceutically acceptable carrier such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays may contain, in addition to a subject composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Compositions and compounds of the present disclosure may alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A non-aqueous (e.g., fluorocarbon propellant) suspension could be used.
  • Sonic nebulizers may be used because they minimize exposing the agent to shear, which may result in degradation of the compounds contained in the subject compositions.
  • an aqueous aerosol is made by formulating an aqueous solution or suspension of a subject composition together with conventional pharmaceutically acceptable carriers and stabilizers.
  • the carriers and stabilizers vary with the requirements of the particular subject composition, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.
  • Aerosols generally are prepared from isotonic solutions.
  • compositions of this disclosure suitable for parenteral administration comprise a subject composition in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and non-aqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate and cyclodextrins.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate and cyclodextrins.
  • Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • enteral pharmaceutical formulations including a disclosed compound and an enteric material; and a pharmaceutically acceptable carrier or excipient thereof.
  • Enteric materials refer to polymers that are substantially insoluble in the acidic environment of the stomach, and that are predominantly soluble in intestinal fluids at specific pHs.
  • the small intestine is the part of the gastrointestinal tract (gut) between the stomach and the large intestine, and includes the duodenum, jejunum, and ileum.
  • the pH of the duodenum is about 5.5
  • the pH of the jejunum is about 6.5
  • the pH of the distal ileum is about 7.5.
  • enteric materials are not soluble, for example, until a pH of about 5.0, of about 5.2, of about 5.4, of about 5.6, of about 5.8, of about 6.0, of about 6.2, of about 6.4, of about 6.6, of about 6.8, of about 7.0, of about 7.2, of about 7.4, of about 7.6, of about 7.8, of about 8.0, of about 8.2, of about 8.4, of about 8.6, of about 8.8, of about 9.0, of about 9.2, of about 9.4, of about 9.6, of about 9.8, or of about 10.0.
  • Exemplary enteric materials include cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP), polyvinyl acetate phthalate (PVAP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), cellulose acetate trimellitate, hydroxypropyl methylcellulose succinate, cellulose acetate succinate, cellulose acetate hexahydrophthalate, cellulose propionate phthalate, cellulose acetate maleate, cellulose acetate butyrate, cellulose acetate propionate, copolymer of methylmethacrylic acid and methyl methacrylate, copolymer of methyl acrylate, methylmethacrylate and methacrylic acid, copolymer of methylvinyl ether and maleic anhydride (Gantrez ES series), ethyl methyacrylate-methylmethacrylate-chlorotrimethylammonium ethyl acrylate copolymer, natural resins
  • the present disclosure also provides kits for use by e.g., a consumer in need of treatment of cancer.
  • kits include a suitable dosage form such as those described herein and instructions describing the method of using such dosage form to mediate, reduce or prevent inflammation.
  • the instructions would direct the consumer or medical personnel to administer the dosage form according to administration modes known to those skilled in the art.
  • kits could advantageously be packaged and sold in single or multiple kit units.
  • An example of such a kit is a so-called blister pack.
  • Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process recesses are formed in the plastic foil. The recesses have the size and shape of the tablets or capsules to be packed.
  • the tablets or capsules are placed in the recesses and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed.
  • the tablets or capsules are sealed in the recesses between the plastic foil and the sheet.
  • the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.
  • a memory aid on the kit, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the tablets or capsules so specified should be ingested.
  • a memory aid is a calendar printed on the card, e.g., as follows “First Week, Monday, Tuesday, . .. etc.... Second Week, Monday, Tuesday, ...” etc.
  • a “daily dose” can be a single tablet or capsule or several pills or capsules to be taken on a given day.
  • a daily dose of a first compound can consist of one tablet or capsule while a daily dose of the second compound can consist of several tablets or capsules and vice versa.
  • the memory aid should reflect this.
  • EXAMPLES [00120] The compounds described herein can be prepared in a number of ways based on the teachings contained herein and synthetic procedures known in the art. In the description of the synthetic methods described below, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be chosen to be the conditions standard for that reaction, unless otherwise indicated. It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule should be compatible with the reagents and reactions proposed.
  • Example 1 General Methods [00122] Reactions were monitored by HPLC-MS analyses using a Shimadzu UFLC- MS-2020 system with ESI, and/or by thin-layer chromatography (TLC) using silica gel 60 F254 plates (Merck) and visualized by UV at 254 nm. Purifications were performed using automated flash chromatography system (ECOM), using prepacked column containing C18 or modified C18 silica gel (Interchim, PT-15C18AQ, 15 ⁇ m Puriflash 200, 5g, 12g, or 25g).
  • ECOM automated flash chromatography system
  • UV-Vis spectra were recorded with a Shimadzu SPD-M2OA Prominence diode array detector, in the range 200-800 nm.
  • NMR spectra were recorded using > 99% deuterated solvents, on a 400 MHz Bruker AVANCE III spectrometer (1H at 400 MHz) and/or on a Bruker AVANCE 500 (1H at 500.0 MHz). Chemical shifts (in ppm, ⁇ scale) were solvent signal in 1H spectra.
  • Intermediates and final products were freeze-dried using a Gregory instruments lyophilizer (model L4-110), from water or water mixtures of dioxane or acetonitrile.
  • Anti-HER2 antibody amino acid sequence was based on heavy and light chain variable domain trastuzumab amino acid sequence KEGG database entry D03257
  • anti-EGFR antibody was based on heavy and light chain variable domain panitumumab amino acid sequence KEGG database entry D05350, cetuximab amino acid sequence KEGG database entry D03255, nimotuzumab amino acid sequence DrugBank accession number DB06192, or matuzumab amino acid sequence DrugBank accession number DB06192.
  • Anti-HER2 light chain SEQ ID NO: 1 [00124] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-HER2 IgG1 heavy chain, SEQ ID NO: 2 [00125] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA MDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP
  • the heavy and light chain genes were cloned into separate mammalian expression vectors and then transiently co-transfected in EXPI293F cells (ThermoFisher).
  • Antibody expression was carried out in EXPI293F expression medium (ThermoFisher) and supernatant harvested 5 days post transfection.
  • Respective antibodies were purified using MABSELECT Sure Resin (Cytiva) and buffer exchanged into 1x phosphate buffer saline pH 7.4. Analytical size-exclusion chromatography (Cytiva, Superdex 20010/300) data indicated that respective antibodies were approximately 99% monomer.
  • the anti-HER2, anti-TROP2 and anti-EGFR antibody-drug conjugates were produced by reacting a linker-drug compound as described herein with an anti-HER2, anti-TROP2 or anti-EGFR antibody and forming a thioether bond at cysteine residues forming disulfide bond sites present between light and heavy chain antibody subunits and heavy and heavy chain antibody subunits respectively.
  • Deruxtecan (CAS No: 1599440-13-7) was obtained from ChemScene, Inc.
  • Respective linker-drug lyophilized powder was dissolved in 100% DMSO (weight per volume) and added to the reduced antibody reaction mixture at 6-37 molar equivalents relative to monoclonal antibody as 5-8 % v/v solution of DMSO, and the reaction solution was nutated for 2 hr at 20°C. Afterwards, 20 mM water solution of N-acetylcysteine (SigmaAldrich) was added at 9 molar equivalent relative to antibody and reaction mixture incubated for 20 min at 20°C.
  • Procedure C [00145] 2.4 to 10 mg/ml of respective anti-EGFR IgG1 monoclonal antibody 1, 2, 3, 4, or 5 in 1x PBS pH 7.4, 10% sucrose, 5 mM EDTA, was partially reduced by adding 40 molar equivalents of TCEP (ThermoFisher) relative to monoclonal antibody for 60 min at 20°C. Respective linker-drug lyophilized powder was dissolved in 100% DMSO (weight per volume) and added to the reduced antibody reaction mixture at 2-37 molar equivalents relative to monoclonal antibody as 5-8 % v/v solution of DMSO, and the reaction solution was nutated for 2 hr at 20°C.
  • TCEP ThermoFisher
  • N-acetylcysteine SigmaAldrich was added at 1 molar equivalent relative to linker-payload and reaction mixture incubated for 20 min at 20°C.
  • Antibody drug conjugate and unconjugated linker-drug mixture in 1x PBS pH 7.4 were diluted in 10% sucrose, 12.5 mM Na- acetate pH 5.0, 0 mM NaCl buffer and loaded onto HiTrap SP HP resin column (Cytiva). Respective non-conjugated linker-drugs did not bind to the column, while respective antibody- drug conjugates bound to the column and were eluted in 10% sucrose, 100-150 mM NaCl, 12.5 mM Na-acetate pH 5.0 buffer.
  • antibody drug conjugates were deglycosylated using PNGase F treatment (New England Biolabs Rapid PNGAse) and cysteine disulfide bridges reduced to separate heavy and light chain subunits and sample analyzed by reverse phase liquid chromatography coupled to mass spectrometry (RPLC-MS). Following reduction of cysteine disulfide bridges and removal of N- linked glycans, the heavy and light chains eluted in distinct peaks.
  • PNGase F treatment New England Biolabs Rapid PNGAse
  • RPLC-MS reverse phase liquid chromatography coupled to mass spectrometry
  • Electrospray ionization baseline subtracted spectra and deconvoluted mass spectra of RPLC eluted peaks were used to determine respective heavy and light chain masses and abundance (mass peak intensity) of antibody’s heavy and light chain subunit species conjugated to 0, 1, 2 or 3 linker-drug for heavy chain subunit (here referred to as H0, H1, H2, H3) and 0 or 1 linker-drug for light chain subunit (here referred to as L 0 , L 1 ).
  • Data was used to determine the overall average linker-drug to antibody conjugate ratio (DAR) of each antibody drug conjugate.
  • DAR linker-drug to antibody conjugate ratio
  • Reverse phase liquid chromatography condition was mobile phase A was 0.05% TFA in water, mobile phase B was 0.05% trifluoro acetic acid in acetonitrile.
  • Gradient program was 10-20% of mobile phase B in 1 min, 20-50% mobile phase B in 9 min, flow rate 0.5 mL/min, post-column split, 2.1x50 mm Halo Diphenyl 2.7 ⁇ m, 80 ⁇ C.
  • Mass spectrometer was Waters Xevo G2-XS QTof, with Acquity i-Class UPLC. Mass spectra data processing was performed by ProMass HR for MassLyxn software (Waters Limited).
  • Average linker payload conjugated to heavy chain (mass peak intensity of H0 x 0/ (0 x mass peak intensity of H 0 + 1 x mass peak intensity of H 1 + 2 x mass peak intensity of H 2 + 3 x mass peak intensity of H 3 )) + (mass peak intensity of H 1 x 1/ (0 x mass peak intensity of H0 + 1 x mass peak intensity of H1 + 2 x mass peak intensity of H2 + 3 x mass peak intensity of H 3 )) + (mass peak intensity of H 2 x 2/ (0 x mass peak intensity of H 0 + 1 x mass peak intensity of H 1 + 2 x mass peak intensity of H 2 + 3 x mass peak intensity of H 3 )) + (mass peak intensity of H3 x 3/ (0 x mass peak intensity of H0 + 1 x mass peak intensity of H1 + 2 x mass peak intensity of H2 + 3 x mass peak intensity of H3)).
  • Average number of conjugated drug molecules to antibody composed of two light chain and two heavy chain subunits (2x average linker payload conjugated to light chain + 2x average linker payload conjugated to heavy chain).
  • detected peaks in RP-HPLC chromatograms were assigned to L 0 , L 1 , H 0 , H 1 , H 2 , and H3 by comparison of retention times with L0 and H0 of parent monoclonal antibody, comparison of 280 nm and 370 nm detection signal (here described linker-payloads absorb at 370 nm) and comparison with known LC/MS DAR values of heavy and light chains.
  • the heavy and light chains were eluted in following order: L 0 , L 1 , H 0 , H 1 , H 2 , and H 3.
  • L1 molar extinction coefficient (molar extinction coefficient L 0 + 1x molar extinction coefficient linker-payload).
  • H 1 molar extinction coefficient (molar extinction coefficient H0 + 1x molar extinction coefficient linker-payload),
  • H2 molar extinction coefficient (molar extinction coefficient H0 + 2x molar extinction coefficient linker- payload),
  • H 3 molar extinction coefficient (molar extinction coefficient H 0 + 3x molar extinction coefficient linker-payload).
  • Average linker payload conjugated to heavy chain peak area of H0 x 0/ (0 x peak area of H0 + 1 x peak area of H1 + 2 x peak area of H2 + 3 x peak area of H 3 )) + (peak area of H 1 x 1/ (0 x peak area of H 0 + 1 x peak area of H 1 + 2 x peak area of H2 + 3 x area intensity of H3)) + (peak area of H2 x 2/ (0 x peak area of H0 + 1 x peak area of H1 + 2 x peak area of H2 + 3 x peak area of H3)) + (peak area of H3 x 3/ (0 x peak area of H 0 + 1 x peak area of H 1 + 2 x peak area of H 2 + 3 x peak area of H 3 )).
  • the crude reaction mixture was purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in water (0 ⁇ 50% ACN in H 2 O). The desired product was recovered as a white powder, after lyophilization from water (12 mg, 57 %).
  • Example 3 Synthesis of Compound 1001 [00153] Intermediate 1. Ethanolamine (100 mg, 1.637 mmol) and dimethoxysquarate (1.2 equiv., 1.964 mmol, 279 mg) were dissolved in 10 mL of 1 M borate buffer (pH 9). The reaction mixture was stirred at room temperature for 16 h. The solvent was evaporated under reduced pressure, the resulting solid was re-dissolved in DMF and directly loaded on column. The product was purified by reverse-phase flash chromatography, using a column containing 40 g of C18, and using a gradient of ACN in water (0 ⁇ 20% ACN in water). The desired product was recovered as a white solid, after lyophilization from water (205 mg, 73 %).
  • Example 4 Synthesis of Compound 3002 [00158] Compound 3002 was prepared according to Procedure A of the General Methods section in Example 1 using 13 molar equivalents of linker payload compound 1001 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 3.7 based on a molecular weight of linker-drug 1117 of Da.
  • Example 5 Synthesis of Compounds 3028A and 3028B [00159] Compound 3028A was prepared according to Procedure B of the General Methods section in Example 1 using 21 molar equivalents of linker payload compound 1001 to anti-TROP2 IgG1 monoclonal antibody.
  • DAR based on RPLC-MS was 4.21 based on a molecular weight of linker-drug of 1117 Da.
  • a DAR of 7.5 was achieved to afford compound 3028B.
  • Example 6 Synthesis of Compound 3050 [00160] Compound 3050 was prepared according to Procedure C of the General Methods section in Example 1 using 13 molar equivalents of linker payload compound 1001 to anti-EGFR IgG1 monoclonal antibody 1.
  • DAR based on RPLC-MS was 4.5 based on a molecular weight of linker-drug of 1117 Da.
  • Example 7 Synthesis of Compound 12 [00161] Intermediate 1.
  • Exatecan mesylate 39 mg, 0.0737 mmol
  • malonic acid 5 equiv., 0.3687 mmol, 38 mg
  • 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM, 5 equiv., 0.3687 mmol, 102 mg) were dissolved in a 5:1 mixture of DMF and water (6 mL).
  • Triethylamine 50 equiv., 3.6873 mmol, 514 ⁇ L was added and the reaction mixture was stirred for 3 hours at room temperature.
  • the reaction mixture was stirred at room temperature for 30 minutes. The mixture was then filtered through a 0.2 ⁇ m syringe filter and directly loaded on column.
  • the product was purified by reverse-phase flash HPLC, using a semipreparative column containing diol-modified C18, and using a gradient of ACN in water (0 ⁇ 80% ACN in water). The desired product was recovered as a yellow solid, after lyophilization from water - dioxane (11 mg, 90 %).
  • Example 9 Synthesis of compound 3003 [00168] Compound 3003 was prepared according to Procedure A of the General Methods section in Example 1 using 2 molar equivalents of linker payload compound 1005 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 2.1 based on a molecular weight of linker-drug 1107 of Da.
  • Example 10 Synthesis of Compounds 3029A and 3029B [00169] Compound 3029A was prepared according to Procedure B of the General Methods section in Example 1 using 2 molar equivalents of linker payload compound 1005 to anti-TROP2 IgG1 monoclonal antibody.
  • DAR based on RPLC-MS was 1.2 based on a molecular weight of linker-drug of 1107 Da.
  • a DAR of 4.4 was achieved to provide compound 3029B.
  • Example 11 Synthesis of Compounds 3051A and 3051B [00170]
  • Compound 3051A was prepared according to Procedure C of the General Methods section in Example 1 using 2 molar equivalents of linker payload compound 1005 to anti-EGFR IgG1 monoclonal antibody 1.
  • DAR based on RPLC-MS was 1.9 based on a molecular weight of linker-drug of 1107 Da.
  • Triethylamine 200 ⁇ L was added and the reaction mixture was stirred for 3 hours at room temperature. Solvents were evaporated under reduced pressure, and the crude reaction mixture was purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in 1% TFA (0 40% ACN in 1% TFA). The desired product was recovered as a brown powder, after lyophilization from water-dioxane (18 mg, 89 %). MS calc. for C 28 H 25 FN 3 O 7 : 534.17, found: 534.80 [M-H]-. [00172] Intermediate 2.
  • Example 14 Synthesis of Compound 3004 [00177] Compound 3004 was prepared according to Procedure A of the General Methods section in Example 1 using 17 molar equivalents of linker payload compound 1023 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 4.4 based on a molecular weight of linker-drug 1121 of Da.
  • Example 15 Synthesis of Compound 3030 [00178] Compound 3030 was prepared according to Procedure B of the General Methods section in Example 1 using 17 molar equivalents of linker payload compound 1023 to anti-TROP2 IgG1 monoclonal antibody.
  • Example 16 Synthesis of Compound 3052 [00179] Compound 3052 was prepared according to Procedure C of the General Methods section in Example 1 using 17 molar equivalents of linker payload compound 1023 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 6.6 based on a molecular weight of linker-drug of 1121 Da.
  • Example 17 Synthesis of Compound 22 [00180] Intermediate 1.
  • the desired product Sc-122 was re-purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in 1% TFA (0 ⁇ 40% ACN in 1% TFA), and recovered as a white powder, after lyophilization from water (16 mg, 81 % over 2 steps, calculated on Exatecan). MS calc. for C27H27FN3O7: 524.18, found: 524.20, [M+H] + .
  • Example 18 Synthesis of Compound 1007 [00182] Intermediate 1.
  • Compound 22 28 mg, 0.0535 mmol
  • FmocGGFG-OAc (2 equiv., 0.107 mmol, 67 mg) were dissolved in 1 mL of anhydrous DMF.
  • HCl 100 ⁇ L, 2 M in Et2O
  • the mixture was directly loaded on column.
  • the product was purified by reverse-phase flash chromatography, using a column containing 25 g of C18, and using a gradient of ACN in water ⁇ 70% ACN in water).
  • the desired product was recovered as a white solid, after lyophilization from water (23 mg, 39 %).
  • Example 19 Synthesis of Compound 3005 [00185]
  • Compound 3005 was prepared according to Procedure A of the General Methods section in Example 1 using 12 molar equivalents of linker payload compound 1007 to anti-HER2 IgG1 monoclonal antibody.
  • Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 4.7 based on a molecular weight of linker-drug 1066 of Da.
  • Example 20 Synthesis of Compound 3031 [00186] Compound 3031 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1007 to anti-TROP2 IgG1 monoclonal antibody.
  • DAR based on RPLC-MS was 6.2 based on a molecular weight of linker-drug of 1066 Da.
  • Example 21 Synthesis of Compounds 3053A and 3053B [00187] Compound 3053A was prepared according to Procedure C of the General Methods section in Example 1 using 12 molar equivalents of linker payload compound 1007 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 3.5 based on a molecular weight of linker-drug of 1066 Da. Molar ratios of mAb and linker-payload were adjusted to achieve DAR 7.7 (compound 3053B).
  • Example 22 Synthesis of Compound 42 [00188] Intermediate 1.
  • Exatecan mesylate (0.0602 mmol, 32 mg) and diisopropylethylamine (2 equiv., 0.120 mmol, 21 ⁇ L) were dissolved in 1 mL of anhydrous DMF, and the solution was cooled at 0 °C.
  • a dichloromethane solution of the previously prepared isocyanate intermediate 1 (2 mL, 0.1150 mmol) was added at 0 °C, and the reaction mixture was allowed to reach room temperature and stirred for 1 h.
  • the desired product was re-purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in 1% TFA (0 ⁇ 40% ACN in 1% TFA), and recovered as a white powder, after lyophilization from water (15 mg, 48% over 3 steps, calculated from exatecan).
  • Example 24 Synthesis of Compound 3007 [00193] Compound 3007 was prepared according to Procedure A of the General Methods section in Example 1 using 9 molar equivalents of linker payload compound 1008 to anti-HER2 IgG1 monoclonal antibody.
  • Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 4.9 based on a molecular weight of linker-drug 1065 of Da.
  • Example 25 Synthesis of Compound 3032 [00194] Compound 3032 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1008 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 6.6 based on a molecular weight of linker-drug of 1065 Da.
  • Example 26 Synthesis of Compound 3054 [00195] Compound 3054 was prepared according to Procedure C of the General Methods section in Example 1 using 9 molar equivalents of linker payload compound 1008 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 4.4 based on a molecular weight of linker-drug of 1065 Da.
  • Example 27 Synthesis of Compound 52
  • Example 29 Synthesis of Compound 3006 [00203]
  • Compound 3006 was prepared according to Procedure A of the General Methods section in Example 1 using 40 molar equivalents of linker payload compound 1010 to anti-HER2 IgG1 monoclonal antibody.
  • Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.9 based on a molecular weight of linker-drug 1117 of Da.
  • Example 30 Synthesis of Compound 103 [00204] Compound 103.
  • exatecan mesylate (10 mg, 0.019 mmol), 5-(hydroxymethyl)furan-2-carboxlic acid (3 equiv., 8 mg, 0.057 mmol), N-ethyl-N′-(3- dimethylaminopropyl)carbodiimide hydrochloride (2.5 equiv., 10 mg, 0.048 mmol) and 1- hydroxybenzotriazole (2.5 equiv., 7 mg, 0.048 mmol) in DMF (1 mL) was added diisopropylethylamine (5 equiv., 17 ⁇ L, 0.095 mmol) under an argon atmosphere and the mixture was stirred at room temperature for 40 minutes.
  • Example 31 Synthesis of Compound 1012 [00205] Intermediate 1. To the solution of FmocGGFGG-OAc (1.0 equiv., 50 mg, 0.079 mmol) and 5-(hydroxymethyl)furan-2-carboxylic acid (1.2 equiv., 14 mg, 0.095 mmol) in anhydrous DMF (0.5 ml) was added 2M HCl/Et 2 O (70 ⁇ L) and the resulting mixture was stirred at room temperature for 2 hours.
  • Example 32 Synthesis of Compound 3008 [00209] Compound 3008 was prepared according to Procedure A of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1012 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.7 based on a molecular weight of linker-drug 1102 of Da.
  • Example 33 Synthesis of Compound 3033 [00210] Compound 3033 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1012 to anti-TROP2 IgG1 monoclonal antibody.
  • DAR based on RPLC-MS was 4.5 based on a molecular weight of linker-drug of 1102 Da.
  • Example 34 Synthesis of Compound 3055 [00211] Compound 3055 was prepared according to Procedure C of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1012 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 7.3 based on a molecular weight of linker-drug of 1102 Da.
  • Example 35 Synthesis of Compound 105 [00212] Compound 105.
  • Example 36 Synthesis of Compound 1013 [00213] Intermediate 1.
  • the mixture of compound 105 (1.0 equiv., 20 mg, 0.034 mmol) and FmocGGFG-OAc (2.0 equiv., 0.068 mmol, 43 mg) was dissolved in anhydrous DMF (1 mL), followed by the addition of 2M HCl/Et2O (100 ⁇ L).
  • the reaction mixture was stirred at room temperature for 4 h and during that time, several portions (ca.0.5 equiv. each) of FmocGGFG-OAc were added to the reaction mixture.
  • Example 37 Synthesis of Compound 3009 [00216] Compound 3009 was prepared according to Procedure A of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1013 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.5 based on a molecular weight of linker-drug 1022 of Da.
  • DAR drug to antibody ratios
  • Example 38 Synthesis of Compound 3034 [00217] Compound 3034 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1013 to anti- TROP2 IgG1 monoclonal antibody.
  • DAR based on RPLC-MS was 6.2 based on a molecular weight of linker-drug of 1022 Da.
  • Example 39 Synthesis of Compound 3056 [00218] Compound 3056 was prepared according to Procedure C of the General Methods section in Example 1 using 4 molar equivalents of linker payload compound 1013 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 3.9 based on a molecular weight of linker-drug of 1022 Da.
  • Example 40 Synthesis of Compound 108 [00219] Diisopropylethylamine (2.5 equiv., 0.07 mmol, 9 mg, 13 ⁇ L) and propargyl bromide (2.5 equiv., 0.07 mmol, 8.5 mg, 9 ⁇ L 80% solution in toluene) were added to the suspension of exatecan mesylate (1.0 equiv., 15 mg, 0.028 mmol) in DMF (0.2 ml) and the resulting solution was stirred for 48 hours.
  • Diisopropylethylamine 2.5 equiv., 0.07 mmol, 9 mg, 13 ⁇ L
  • propargyl bromide 2.5 equiv., 0.07 mmol, 8.5 mg, 9 ⁇ L 80% solution in toluene
  • 2-azidoethanol 5.0 equiv., 0.14 mmol, 12 mg, 11 ⁇ L
  • tris(benzyltriazolylmethyl)amine 1.5 equiv., 0.042 mmol, 22 mg
  • CuSO4.5H2O 1.0 equiv., 0.028 mmol, 140 ⁇ L 2M aqueous solution
  • sodium ascorbate 2.0 equiv., 0.056 mmol, 56 ⁇ L 1M aqueous solution
  • Example 41 Synthesis of Compound 1015 F [00220] Intermediate 1.
  • Compound 108 1.0 equiv., 30 mg, 0.0536 mmol
  • FmocGGFG-OAc 2.0 equiv., 0.107 mmol, 67 mg
  • 2M HCl/Et2O 150 ⁇ L
  • the reaction mixture was stirred at room temperature for 4 h and during that time, several portions (ca.0.5 equiv. each) of FmocGGFG- OAc were added to the reaction mixture.
  • Example 42 Synthesis of Compound 3010 [00223] Compound 3010 was prepared according to Procedure A of the General Methods section in Example 1 using 15 molar equivalents of linker payload compound 1015 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.6 based on a molecular weight of linker-drug 1103 of Da.
  • Example 43 Synthesis of Compound 3035 [00224] Compound 3035 was prepared according to Procedure B of the General Methods section in Example 1 using 15 molar equivalents of linker payload compound 1015 to anti-TROP2 IgG1 monoclonal antibody.
  • DAR based on RPLC-MS was 6.3 based on a molecular weight of linker-drug of 1103 Da.
  • Example 44 Synthesis of Compound 3057 [00225] Compound 3057 was prepared according to Procedure C of the General Methods section in Example 1 using 15 molar equivalents of linker payload compound 1015 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 6.0 based on a molecular weight of linker-drug of 1103 Da.
  • Example 45 Synthesis of Compound 111 [00226] Compound 111.
  • Methyl 2-(hydroxymethyl)cyclopropane-1-carboxylate (25 mg, 0.175 mmol) was dissolved in 1 mL of methanol, and 870 ⁇ L of 1M NaOH (1 equiv.) were added. The mixture was stirred at room temperature for 5h, thus, the solvents were evaporated and the crude product was lyophilized from water. To the obtained solid were added exatecan mesylate (46 mg, 0.5 equiv., 0,874 mmol), DMTMM (48 mg, 1 equiv., 0.175 mmol), and 10 mL of a 4:1 DMF/water mixture. The mixture was stirred at room temperature for 30 min.
  • Example 46 Synthesis of Compound 1016 H [00229] Intermediate 1.2-((Benzyloxy)methyl)cyclopropane-1-carboxylic acid (27.6 mg, 0.1338 mmol) was dissolved in 3 mL of anhydrous dioxane. Pd/C (10%) was added and hydrogen was bubbled into the solution for 5 h, while stirring at room temperature. The solution was filtered with 0.2 ⁇ m syringe filters, and the flask washed with acetonitrile. The filtrate was evaporated, re-dissolved in dioxane and lyophilized overnight.
  • Example 47 Synthesis of Compound 3012 [00233] Compound 3012 was prepared according to Procedure A of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1016 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC was 2 based on a molecular weight of linker-drug 1076 of Da.
  • Example 48 Synthesis of Compound 3037 [00234] Compound 3037 was prepared according to Procedure B of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1016 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC was 1.45 based on a molecular weight of linker-drug of 1076 Da.
  • Example 49 Synthesis of Compound 1017
  • reaction mixture was purified by reverse-phase flash chromatography using a semipreparative column (diol-modified C18, 0 ⁇ 75% ACN/0.1% HCl). Fractions containing the product (co-eluting with impurity) were lyophilised to obtain 31 mg of impure intermediate 1, which was used directly into the next step. MS calc. for C 46 H 56 FN 7 O 12 : 898.40, found: 898.40, [M-H]-. [00236] Intermediate 2.
  • Example 50 Synthesis of Compound 3013 [00239] Compound 3013 was prepared according to Procedure A of the General Methods section in Example 1 using 37 molar equivalents of linker payload compound 1017 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.6 based on a molecular weight of linker-drug 1200 of Da.
  • Example 51 Synthesis of Compound 3038 [00240] Compound 3038 was prepared according to Procedure B of the General Methods section in Example 1 using 37 molar equivalents of linker payload compound 1017 to anti-TROP2 IgG1 monoclonal antibody.
  • DAR based on RPLC-MS was 6.6 based on a molecular weight of linker-drug of 1200 Da.
  • Example 52 Synthesis of Compound 3059 [00241] Compound 3059A was prepared according to Procedure C of the General Methods section in Example 1 using 37 molar equivalents of linker payload compound 1017 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 6.4 based on a molecular weight of linker-drug of 1200 Da. Molar ratios of mAb and linker-payload were adjusted to achieve DAR 7.7 (compound 3059B).
  • Example 53 Synthesis of Compound 1025
  • Example 54 Synthesis of Compound 3011 [00246] Compound 3011 was prepared according to Procedure A of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1025 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 5.4 based on a molecular weight of linker-drug 1076 of Da.
  • Example 55 Synthesis of Compound 3036 [00247] Compound 3036 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1025 to anti-TROP2 IgG1 monoclonal antibody.
  • DAR based on RPLC-MS was 6.4 based on a molecular weight of linker-drug of 1076 Da.
  • Example 56 Synthesis of Compounds 3058A and 3058B [00248] Compound 3058A was prepared according to Procedure C of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1025 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 7.1 based on a molecular weight of linker-drug of 1076 Da. Molar ratios of mAb and linker-payload were adjusted to achieve DAR 3.1 (compound 3058B).
  • Example 57 Synthesis of Compound 130 [00249] Intermediate 1.
  • Example 59 Synthesis of Compound 3015 [00254]
  • Compound 3015 was prepared according to Procedure A of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1018 to anti-HER2 IgG1 monoclonal antibody.
  • Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 5 based on a molecular weight of linker-drug 1136 of Da.
  • Example 60 Synthesis of Compound 3040 [00255]
  • Compound 3040 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1018 to anti-TROP2 IgG1 monoclonal antibody.
  • DAR based on RPLC-MS was 4.8 based on a molecular weight of linker-drug of 1136 Da.
  • E 3061 Compound 3061 was prepared according to Procedure C of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1018 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 6.3 based on a molecular weight of linker-drug of 1136 Da.
  • Example 62 Synthesis of Compound 1019 [00257] Intermediate 1.
  • Example 63 Synthesis of Compound 3014 [00260]
  • Compound 3014 was prepared according to Procedure A of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1019 to anti-HER2 IgG1 monoclonal antibody.
  • Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 5.2 based on a molecular weight of linker-drug 1176 of Da.
  • Example 64 Synthesis of Compound 3039 [00261] Compound 3039 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1019 to anti-TROP2 IgG1 monoclonal antibody.
  • Example 65 Synthesis of Compound 3060 [00262] Compound 3060 was prepared according to Procedure C of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1019 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 7.8 based on a molecular weight of linker-drug of 1176 Da.
  • Example 66 Synthesis of Compound 177 [00263] Intermediate 1.
  • Example 68 Synthesis of Compound 3100 [00269] Compound 3100 was prepared according to Procedure A of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1034 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC was 3.6 based on a molecular weight of linker-drug 1092 of Da.
  • DAR drug to antibody ratios
  • Example 69 Synthesis of Compound 3101 [00270] Compound 3101 was prepared according to Procedure B of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1034 to anti-TROP2 IgG1 monoclonal antibody.
  • reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ⁇ 60% ACN/0.1% TFA), giving compound 180 (15 mg, 57 %) as a white powder after lyophilization.
  • Example 71 Synthesis of Compound 193 [00273] Compound 193.
  • Compound 180 (10 mg, 0.018 mmol) and DIPEA (5 ⁇ L) were dissolved in DCM (2mL).
  • the Ac 2 O (2.6 ⁇ L, 0.027) was added, and the reaction was stirred over night at room temperature.
  • the mixture was evaporated, and residue was purified by reverse- phase HPLC chromatography (semipreparative, diol-modified C18, 0 ⁇ 50% ACN/H2O). Fractions containing the product were lyophilized from water (4.9 mg, 46%).
  • Example 72 Synthesis of Compound 194 [00274] Compound 194.
  • Formic acetic anhydride (2.2 equiv., 0.048, 4.2 ⁇ L) and diisopropylethylamine (2.5 equiv., 0.055 mmol, 10 ⁇ L) were added to the solution of compound 180 (1.0 equiv., 15 mg, 0.027 mmol) in DCM (2 ml) and the reaction was stirred over night at room temperature. Then, mixture was evaporated, and the residue was purified by reverse-phase HPLC chromatography (semipreparative, diol-modified C18, 0 ⁇ 50% ACN/H2O).
  • reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ⁇ 80 % ACN/0.1% aq. TFA), offering intermediate 1 (two separated isomers A and B), both as white solids after lyophilization (isomer A: 19 mg, isomer B: 21 mg, combined yield: 87 %).
  • Example 75 Synthesis of Compound 3016 [00280] Compound 3016 was prepared according to Procedure A of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1035 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.5 based on a molecular weight of linker-drug 1186 of Da.
  • DAR drug to antibody ratios
  • Example 76 Synthesis of Compound 3041 [00281] Compound 3041 was prepared according to Procedure B of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1035 to anti-TROP2 IgG1 monoclonal antibody.
  • DAR based on RPLC was 7.5 based on a molecular weight of linker-drug of 1186 Da.
  • Example 77 Synthesis of Compounds 3062A and 3062B [00282] Compound 3062A was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1035 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC was 7.5 based on a molecular weight of linker-drug of 1186 Da. When 25 molar equivalents of linker payload compound 1035 to anti-EGFR IgG1 monoclonal antibody 1 was used, a DAR of 4.7 was achieved to provide compound 3062B.
  • Example 78 Synthesis of Compound 3017 [00283]
  • Compound 3017 was prepared according to Procedure A of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1036 to anti-HER2 IgG1 monoclonal antibody.
  • Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.5 based on a molecular weight of linker-drug 1186 of Da.
  • Example 79 Synthesis of Compound 3042 [00284]
  • Compound 3042 was prepared according to Procedure B of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1036 to anti-TROP2 IgG1 monoclonal antibody.
  • DAR based on RPLC was 7.5 based on a molecular weight of linker-drug of 1186 Da.
  • Example 80 Synthesis of Compound 3063 [00285] Compound 3063 was prepared according to Procedure C of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1036 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC was 7.5 based on a molecular weight of linker-drug of 1186 Da.
  • Example 81 Synthesis of Compound 1037
  • Example 82 Synthesis of Compound 3018 [00288] Compound 3018 was prepared according to Procedure A of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1037 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.5 based on a molecular weight of linker-drug 1032 of Da.
  • Example 83 Synthesis of Compound 3043 [00289] Compound 3043 was prepared according to Procedure B of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1037 to anti-TROP2 IgG1 monoclonal antibody.
  • DAR based on RPLC was 7.5 based on a molecular weight of linker-drug of 1032 Da.
  • Example 84 Synthesis of Compound 3064 [00290] Compound 3064 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1037 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC was 7.5 based on a molecular weight of linker-drug of 1032 Da.
  • Example 85 Synthesis of Compound 196
  • Example 86 Synthesis of Compound 197 [00293] Intermediate 1. To the mixture of exatecan mesylate (1.0 equiv., 40 mg, 0.0753 mmol), Fmoc-L-proline (1.1 equiv., 28 mg, 0.0818 mmol) and DMTMM (1.1 equiv., 23 mg, 0.0818 mmol) was added DMF/water (4:1, 2 ml) and diisopropylethylamine (30 ⁇ l) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material.
  • Example 88 Synthesis of Compound 199 [00297] Intermediate 1. To the mixture of exatecan mesylate (1.1 equiv., 38 mg, 0.0711 mmol), Fmoc-3-fluoro-D-proline (1 equiv., 23 mg, 0.0647 mmol) and DMTMM (1.5 equiv., 27 mg, 0.0971 mmol) was added DMF/water (4:1, 2 ml) and diisopropylethylamine (30 ⁇ l) and the resulting mixture was stirred at room temperature for 30 minutes, as LC-MS analysis indicated the full consumption of the starting material.
  • DMF/water 4:1, 2 ml
  • diisopropylethylamine 30 ⁇ l
  • Example 89 Synthesis of Compounds 200 and 201 [00299] Intermediate 1.
  • exatecan mesylate (1 equiv., 40 mg, 0.0752 mmol)
  • Fmoc-3,3-difluoro-L-proline 1.2 equiv., 18 mg, 0.0903 mmol
  • DMTMM 1.2 equiv., 25 mg, 0.0903 mmol
  • Example 90 Synthesis of Compound 202 [00303] Compound 202. To a mixture of compound 197 (1 equiv., 10 mg, 0.0188 mmol), Boc-Gly-OH (1.1 equiv., 4 mg, 0.0207 mmol) and DMTMM (1.1 equiv., 6 mg, 0.0207 mmol) was added DMF/water (4:1, 2 ml) and diisopropylethylamine (10 ⁇ l) and the resulting mixture was stirred at room temperature for 30 minutes, as LC-MS analysis indicated the full consumption of the starting material.
  • Boc-Gly-OH 1.1 equiv., 4 mg, 0.0207 mmol
  • DMTMM 1.1 equiv., 6 mg, 0.0207 mmol
  • Example 91 Synthesis of Compound 203 [00304] Compound 203. To the mixture of exatecan mesylate (1 equiv., 19 mg, 0.0354 mmol), Boc-Glu(OtBu)-OH (1.5 equiv., 16 mg, 0.0531 mmol) and DMTMM (1.5 equiv., 15 mg, 0.0531 mmol) were added DMF/water (4:1, 2 ml) and diisopropylethylamine (10 ⁇ l) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The solvent was removed under reduced pressure and the solid was re-dissolved in 2 mL of dioxane.
  • Example 92 Synthesis of Compound 204 [00305] Compound 204. To a mixture of exatecan mesylate (20 mg, 0.038 mmol), L-pyroglutamic acid (7.3 mg, 0.056 mmol) and DMTMM (21 mg, 0.076 mmol) was added DMF/water (4:1, 2.5 ml) and diisopropylethylamine (50 ⁇ l) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material.
  • Example 93 Synthesis Compound 1038 [00306] Intermediate 1. To the mixture of exatecan mesylate (1 equiv., 40 mg, 0.0753 mmol), Boc-Glu(OBn)-OH (1.5 equiv., 38 mg, 0.113 mmol) and DMTMM (1.5 equiv., 31 mg, 0.113 mmol) were added DMF/water (3:1, 4 ml) and diisopropylethylamine (25 ⁇ l) and the resulting mixture was stirred at room temperature for 3 hours, as LC-MS analysis indicated the full consumption of the starting material. The solvent was removed under reduced pressure and the solid was re-dissolved in 3 mL of dioxane.
  • Example 94 Synthesis of Compound 205 [00310] N-Boc-GlyProOH.
  • N-Boc-Gly NHS ester (277 mg, 1.02 mmol) and L-Pro (117 mg, 1.02 mmol) in DMF (3 ml) was added DIPEA (300 ⁇ l), and the mixture was stirred for 1 hour at room temperature.
  • the reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ⁇ 100 % ACN), giving N-Boc- GlyProOH as a white solid after lyophilization (270 mg, 97 %).
  • the reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ⁇ 100% ACN, 0.1 % TFA) and fractions containing the product were evaporated. The residue was dissolved in dioxane (1 ml), and HCl in dioxane was added (4M; 2ml). The resulting mixture was stirred at room temperature for 3 hours. Then, the product was isolated by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ⁇ 100% ACN, 0.1 % TFA), giving 205 as a yellow solid after lyophilization (7.0 mg, 38%). MS calc.
  • Example 95 Synthesis of Compound 206 [00312] To a mixture of exatecan mesylate (1.0 equiv., 15 mg, 0.028 mmol), (R)-3- hydroxybutanoic acid (1.1 equiv., 3.2 mg, 0.030 mmol) and DMTMM (1.1 equiv., 8.5 mg, 0.030 mmol) was added DMF/water (5:1, 1.2 ml) and diisopropylethylamine (2.1 equiv., 10 ⁇ l, 0.059 mmol) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material.
  • DMF/water 5:1, 1.2 ml
  • diisopropylethylamine 2.1 equiv., 10 ⁇ l, 0.059 mmol
  • reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ⁇ 60 % ACN/H2O), giving 206 as a white solid after lyophilization (10 mg, 69 %).
  • Example 96 Synthesis of Compound 207 [00313] To a 4:1 DMF/water mixture (4 mL) were added exatecan mesylate (20 mg, 0.038 mmol), cis-3-hydroxycyclobutane-1-carboxylic acid (1.25 equiv., 6 mg, 0.048 mmol), DMTMM (2.0 equiv., 21 mg, 0.076 mmol) and diisopropylethylamine (20 ⁇ L). The resulting solution was stirred for 1 hour at room temperature, as LC-MS indicated the full consumption of the starting material.
  • reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ⁇ 75% ACN/H 2 O), offering intermediate 1 as a white solid after lyophilization (126 mg, 98 %). MS calc. for C 72 H 81 FN 11 O 15 : 1358.59, found: 1358.60, [M+H] + . [00315] Intermediate 2. To the solution of intermediate 1 (1.0 equiv., 126 mg, 0.092 mmol) in the mixture of DMF (1.0 ml) and dioxane (1.5 ml) was added 10% Pd/C (15 mg) and the reaction mixture was hydrogenated (balloon) for 3 hours at room temperature.
  • Example 98 Synthesis of Compound 3024 [00317]
  • Compound 3024 was prepared according to Procedure A of the General Methods section in Example 1 using 24 molar equivalents of linker payload compound 1021 to anti-HER2 IgG1 monoclonal antibody.
  • Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.3 based on a molecular weight of linker-drug 1241 of Da.
  • Example 99 Synthesis of Compound 3048 [00318]
  • Compound 3048 was prepared according to Procedure B of the General Methods section in Example 1 using 24 molar equivalents of linker payload compound 1021 to anti-TROP2 IgG1 monoclonal antibody.
  • Drug to antibody ratios (DAR) for anti-TROP2 antibody drug conjugate based on RPLC-MS was 7.2 based on a molecular weight of linker-drug 1241 of Da.
  • Example 100 Synthesis of Compound 1022 [00319] Intermediate 1. (S,S)-3-fluoropyrrolidine-2-carboxylic acid (62.5 mg, 0.47 mmol) was dissolved in 1,4-dioxane (1 mL) and H2O (3 mL) and cooled to 0°C. K2CO3 (162 mg, 1.18 mmol) was added, and then Fmoc-Cl (115 mg, 0.45 mmol) was added. The mixture was stirred at RT overnight and H 2 O (10 mL) was added. The mixture acidified with aqueous HCl (1 M) to pH 2–3 and extracted with DCM (2 ⁇ 10 mL).
  • Example 101 Synthesis of Compound 3021 [00325] Compound 3021 was prepared according to Procedure A of the General Methods section in Example 1 using 24 molar equivalents of linker payload compound 1022 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.9 based on a molecular weight of linker-drug 1121 of Da.
  • Example 102 Synthesis of Compound 3045 [00326] Compound 3045 was prepared according to Procedure B of the General Methods section in Example 1 using 24 molar equivalents of linker payload compound 1022 to anti-TROP2 IgG1 monoclonal antibody.
  • Example 103 Synthesis of Compound 3066 [00327] Compound 3066 was prepared according to Procedure C of the General Methods section in Example 1 using 24 molar equivalents of linker payload compound 1022 to anti-EGFR IgG1 monoclonal antibody 1. Drug to antibody ratios (DAR) for anti-EGFR antibody drug conjugate based on RPLC-MS was 7.4 based on a molecular weight of linker-drug 1121 of Da.
  • Example 104 Synthesis of Compound 1028
  • Example 105 Synthesis of Compound 3019 [00331]
  • Compound 3019 was prepared according to Procedure A of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1028 to anti-HER2 IgG1 monoclonal antibody.
  • Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.9 based on a molecular weight of linker-drug 1103 of Da.
  • Example 106 Synthesis of Compound 1029
  • Example 107 Synthesis of Compound 3025 [00335] Compound 3025 was prepared according to Procedure A of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1029 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC was 7 based on a molecular weight of linker-drug 1036 of Da.
  • Example 109 Synthesis of Compound 1030 and 1032
  • reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ⁇ 100% ACN/ 0.1% TFA).
  • the two L and D isomers 1030 and 1032 were separated and re-purified individually by a second flash chromatography using semipreparative column (diol-modified C18, 0 ⁇ 100 % ACN/ 0.1% TFA).
  • the two isomers were obtained as yellowish solids after lyophilization (16 and 15 mg, 52% total yield).
  • Example 110 Synthesis of Compound 3022 [00340] Compound 3022 was prepared according to Procedure A of the General Methods section in Example 1 using 23 molar equivalents of linker payload compound 1030 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.4 based on a molecular weight of linker-drug 1139 of Da.
  • Example 111 Synthesis of Compound 3046 [00341] Compound 3046 was prepared according to Procedure B of the General Methods section in Example 1 using 23 molar equivalents of linker payload compound 1030 to anti-TROP2 IgG1 monoclonal antibody.
  • Drug to antibody ratios (DAR) for anti-TROP2 antibody drug conjugate based on RPLC was 6.5 based on a molecular weight of linker-drug 1139 of Da.
  • Example 113 Synthesis of Compound 3023 [00343] Compound 3023 was prepared according to Procedure A of the General Methods section in Example 1 using 27 molar equivalents of linker payload compound 1032 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.4 based on a molecular weight of linker-drug 1139 of Da.
  • Example 114 Synthesis of Compound 3047 [00344] Compound 3047 was prepared according to Procedure B of the General Methods section in Example 1 using 27 molar equivalents of linker payload compound 1032 to anti-TROP2 IgG1 monoclonal antibody.
  • Example 115 Synthesis of Compound 3068 [00345] Compound 3068 was prepared according to Procedure C of the General Methods section in Example 1 using 27 molar equivalents of linker payload compound 1032 to anti-EGFR IgG1 monoclonal antibody 1. Drug to antibody ratios (DAR) for anti-EGFR antibody drug conjugate based on RPLC was 7.4 based on a molecular weight of linker-drug 1139 of Da.
  • Example 116 Synthesis of Compound 1031 [00346] Compound 1031.
  • reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ⁇ 70% ACN/H2O), offering 1031 as a white solid after lyophilization (29 mg, 71%).
  • Example 117 Synthesis of Compound 3020 [00347]
  • Compound 3020 was prepared according to Procedure A of the General Methods section in Example 1 using 31 molar equivalents of linker payload compound 1031 to anti-HER2 IgG1 monoclonal antibody.
  • Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.4 based on a molecular weight of linker-drug 1103 of Da.
  • Example 118 Synthesis of Compound 3044 [00348]
  • Compound 3044 was prepared according to Procedure B the General Methods section in Example 1 using 31 molar equivalents of linker payload compound 1031 to anti- TROP2 IgG1 monoclonal antibody.
  • Example 119 Synthesis of Compound 3065 [00349] Compound 3065 was prepared according to Procedure C of the General Methods section in Example 1 using 31 molar equivalents of linker payload compound 1031 to anti-EGFR IgG1 monoclonal antibody 1. Drug to antibody ratios (DAR) for anti-EGFR antibody drug conjugate based on RPLC was 7.4 based on a molecular weight of linker-drug 1103 of Da.
  • Example 120 Synthesis of Compound 1033 [00350] Intermediate 1.
  • Example 121 Synthesis of Compound 3026 [00353] Compound 3026 was prepared according to Procedure A of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1033 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.5 based on a molecular weight of linker-drug 1180 of Da.
  • Example 122 Synthesis of Compound 3070 [00354] Compound 3070 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1033 to anti-EGFR IgG1 monoclonal antibody 1. Drug to antibody ratios (DAR) for anti-EGFR antibody drug conjugate based on RPLC was 7.5 based on a molecular weight of linker-drug 1180 of Da.
  • Example 123 Synthesis of Compound 71
  • Exatecan mesylate 100 mg, 0.188 mmol
  • 2-azidoacetic acid 1.1 equiv., 0.207 mmol, 21 mg
  • DMTMM 1.3 equiv., 0.244 mmol, 68mg
  • DIPEA 50 ⁇ L
  • the reaction mixture was stirred at room temperature for 1 hour.
  • the mixture was directly purified by reverse-phase flash chromatography (diol-modified C18, 25g, 0 ⁇ 100% ACN in water).
  • the desired product was obtained as a white powder after lyophilization (79 mg, 74%). MS calc.
  • Example 126 Synthesis of Compound 74 [00360] Compound 74.
  • the previously prepared intermediate 2 of Example 63 (10 mg, 0.0193 mmol), 3-butyn-1-ol (1.2 equiv., 0.0232 mmol, 1.50 ⁇ L), sodium ascorbate (0.2 equiv., 0.00386 mmol, 2 M in water, 1.93 ⁇ L), copper sulphate pentahydrate (0.1 equiv., 0.00193 mmol, 1 M in water, 1.93 ⁇ L) and TBTA (0.15 equiv., 0.0029 mmol, 1.5 mg) were dissolved in 2 mL of a 4:1 mixture of DMF /water.
  • exatecan mesylate (20 mg, 0.0376 mmol)
  • 3-(1,3-dioxolan-2-yl)propanoic acid (2 equiv., 0.0753 mmol, 11 mg)
  • DMTMM 1.5 equiv., 0.0564 mmol, 16 mg
  • diisopropylethylamine (20 ⁇ L).
  • the resulting solution was stirred for 1 hour at room temperature, as LC-MS indicated the full consumption of the starting material.
  • the mixture was directly purified by reverse-phase HPLC chromatography using a semipreparative column (diol-modified C18, 0 ⁇ 100% ACN/H 2 O).
  • Example 128 Synthesis of Compound 209 [00363] Intermediate 1.
  • DMF diisopropylethylamine
  • Example 130 Synthesis of Compound 210 [00369] Intermediate 1. To the solution of (R)-1-(benzyloxy)propan-2-ol ( 100 mg, 0.6 mmol) and bis(4-nitrophenyl) carbonate (2.0 equiv., 366 mg, 1.2 mmol) in DMF (2 mL) was added diisopropylethylamine (1.1 equiv., 115 ⁇ L, 0.66 mmol) and the resulting mixture was stirred at room temperature for 2 hours.
  • DMF diisopropylethylamine
  • the product was purified by reverse-phase flash HPLC, using a semipreparative column containing diol-modified C18 (0 ⁇ 100% ACN/10mM aq. NH4OAc).
  • the product was re-purified by reverse-phase flash HPLC, using 0 ⁇ 100% ACN/0.1% TFA and subsequently using 0 ⁇ 100% ACN/10mM aq. NH4OAc.
  • the desired product 1040 was recovered as a white solid, after lyophilization from water (12 mg, 31 %), it contains 5% of compound 209. MS calc. for C 53 H 59 FN 9 O 15 : 1080.40, found: 1080.66, [M + H] + .
  • Example 132 Synthesis of Compound 211 [00375] Intermediate 1. To the solution of (S)-2-(benzyloxy)propan-1-ol (1.0 equiv., 100 mg, 0.6 mmol) and bis(4-nitrophenyl) carbonate (2.0 equiv., 366 mg, 1.2 mmol) in DMF (2 mL) was added diisopropylethylamine (1.5 equiv., 157 ⁇ L, 0.9 mmol) and the resulting mixture was stirred at room temperature for 2 hours.
  • DMF diisopropylethylamine
  • Example 134 Synthesis of Compound 1042 [00381] Intermediate 1.
  • Fmoc-GGFG-OAc 60 mg, 0.095 mmol
  • 2,2-difluoro-3- hydroxypropanoic acid 36 mg, 0.29 mmol
  • HCl 4M in dioxane, 30 ⁇ L
  • Example 135 Synthesis of Compound 1044 [00385] Intermediate 1.
  • the mixture of compound 130 (60 mg, 0.11 mmol), (2S,3S)1(Fmoc)3fluoropyrrolidine-2-carboxylic acid (40 mg, 0.11 mmol) and DMTMM (30 mg, 0.11 mmol) was added DMF/water (3:1, 2 ml) and diisopropylethylamine (60 ⁇ L) and the resulting mixture was stirred at room temperature for 90 min.
  • the reaction mixture was purified by reverse phase flash chromatography (25 g, diol-modified C18, 0 50% ACN/H2O), offering Intermediate 1 as a white solid after lyophilization (47 mg, 51 %).
  • Example 139 Synthesis of Compound 3113: [00399] Compound 3113 was prepared according to Procedure C of the General Methods section in Example 1 using 34 molar equivalents of linker payload compound 1025 to anti-EGFR IgG1 monoclonal antibody 5. DAR based on RP-HPLC was 7.6.
  • Example 140 Synthesis of compound 3053B [00400] Compound 3053B was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1007 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC was 7.6.
  • Example 141 Synthesis of compound 3102 [00401] Compound 3102 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1046 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC was 7.7.
  • Example 142 Synthesis of compound 3103 [00402] Compound 3103 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1047 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC was 3.6.
  • Example 143 Synthesis of compound 3104 [00403] Compound 3104 was prepared according to Procedure C of the General Methods section in Example 1 using 25 molar equivalents of linker payload of compound 1045 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC was 7.8.
  • Example 144 Synthesis of compound 3105 [00404] Compound 3105 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1044 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC-MS was 7.6 was based on molecular weight of the drug of 1194.17.
  • Example 145 Synthesis of compound 3107 [00405] Compound 3107 was prepared according to Procedure C of the General Methods section in Example 1 using 25 molar equivalents of linker payload of compound 1042 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC was 7.7.
  • Example 146 Synthesis of compound 3059B [00406] Compound 3059B was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1017 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC was 7.7.
  • Example 147 Synthesis of compound 3108 [00407] Compound 3108 was prepared according to Procedure C of the General Methods section in Example 1 using 22 molar equivalents of linker payload of compound 1025 to anti-EGFR IgG1 monoclonal antibody 2. DAR based on RP-HPLC was 7.5.
  • Example 148 Synthesis of compound 3110 [00408] Compound 3110 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1025 to anti-EGFR IgG1 monoclonal antibody 3. DAR based on RP-HPLC was 7.8.
  • Example 149 Synthesis of compound 3111 [00409] Compound 3111 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1025 to anti-EGFR IgG1 monoclonal antibody 4. DAR based on RP-HPLC was 7.6, while DAR based on RPHPLC-MS was 7.8.
  • Example 150 Synthesis of compound 3109 [00410] Compound 3109 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1007 to anti-EGFR IgG1 monoclonal antibody 3. DAR based on RP-HPLC was 7.7.
  • Example 151 Synthesis of compound 3112 [00411] Compound 3112 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1007 to anti-EGFR IgG1 monoclonal antibody 4. DAR based on RP-HPLC was 7.6, while DAR based on RPHPLC-MS was 7.8.
  • Example 152 Synthesis of Compound 1043: F [00412] Intermediate 1.
  • Exatecan mesylate (1.0 equiv., 0.049 mmol, 26 mg) and DIPEA (4.0 equiv., 0.049 mmol, 34 ⁇ L) were added to the solution of intermediate 2 (1.0 equiv., 50 mg, 0.049 mmol) in anhydrous DMF (3 ml).
  • the reaction mixture was stirred at room temperature for 40 hours, as LC-MS indicated the full consumption of starting material.
  • Purification by reverse-phase flash chromatography (diol-modified C18, 0 ⁇ 75% ACN/10mM aq. NH4OAc) offered intermediate 3 as a white solid after lyophilisation (48 mg, 75 %). MS calc.
  • Example 153 Antibody-Drug Conjugates Effectively Kill Cognate Cells expressing HER2, TROP2 and EGFR
  • DXD a similar ADC
  • cell-killing assays were run on multiple cells lines expressing HER2, TROP2 and EGFR.
  • anti-HER-2 ADCs NCI-N87 and SK-BR-3 cell lines were used.
  • anti-TROP2 ADCs MDA-MB-468 and FaDu cell lines were used.
  • MDA-MB-468, and HCC827, NCI-H292, FaDu, OVCAR3 cell lines were used.
  • NCI-N87, MDA-MB-468, HCC827, NCI-H292 and OVCAR3 were cultured in RPMI-1640 media (Gibco, Life Technologies) supplemented with 10% v/v heat inactivated FBS (Corning), SK-BR-3 cells were maintained in McCoys 5A media (Gibco, Life Technologies) supplemented with 10% v/v heat inactivated FBS (Corning) at 37 ⁇ C in a humidified incubator containing 5% CO2. FaDu cells were maintained in EMEM media (Gibco, Life Technologies) supplemented with 10% v/v heat inactivated FBS (Corning) at 37 ⁇ C in a humidified incubator containing 5% CO2.
  • ADCs The viability of cancer cells in the presence of ADCs was measured in a series of in vitro assays.
  • HER2 and TROP2-expressing cells cells were plated in 384-well white flat-bottomed plates (Corning) at 0.5 ⁇ 10 3 per well in 30 ⁇ L culture medium.
  • EGFR- expressing cells cells were plated in 96-well white flat-bottomed plates (Corning) at 2 ⁇ 10 3 per well in 100 ⁇ L culture medium.
  • ADCs were added at a range of concentrations as eight-point serial dilution as quadruplicates.
  • Luminescence was measured using the SpectraMax iD3 plate reader (Molecular Devices). Luminescence values were plotted against log concentration of test compounds, and cell viability was calculated by dividing luminescence values at different antibody drug conjugate concentrations by luminescence values at antibody concentration of zero. ADC dose- response IC50 values were calculated by GraphPad Prism as best-fit values using four parameter dose-response curve fit, with R squared ranging from 0.97-0.999.
  • Table 7A shows the percent cell viability of NCI-N87 cells after exposure to the different anti-HER2 ADCs of the instant disclosure.
  • Table 7B shows the percent viability of SK- BR-3 cells after exposure to the different anti-HER2 ADCs of the instant disclosure.
  • Table 7C shows the percent cell viability of NCI-N87 cells after exposure to the various exatecan release anti-HER2 ADCs described herein.
  • Table 7D shows the percent cell viability of SK-BR-3 cells after exposure to the various exatecan release anti-HER2 ADCs described herein.
  • IC50 Linker-Payload (nM) was calculated from concentration of linker-payload delivered by the ADC (calculated as DAR multiplied by ADC (nM)).
  • Table 7C Table 7D [00421] Table 8A shows the percent cell viability of FaDu cells after exposure to the different anti-TROP2 ADCs of the instant disclosure.
  • Table 8B shows the percent viability of MDA-MB-468 cells after exposure to the different anti-TROP2 ADCs of the instant disclosure.
  • Table 8C shows the percent cell viability of FaDu cells after exposure to the various exatecan release anti-TROP2 ADCs described herein.
  • Table 8D shows the percent cell viability of MDA- MB-468 cells after exposure to the various exatecan release anti-TROP2 ADCs described herein.
  • IC50 Linker-Payload was calculated from concentration of linker-payload delivered by the ADC (calculated as DAR multiplied by ADC (nM)). Table 8A
  • Table 8C Table 8D [00423] Table 9A shows the percent cell viability of MDA-MB-468 cells after exposure to the different anti-EGFR ADCs of the instant disclosure.
  • Table 9B shows the percent viability of HCC827 cells after exposure to the different anti-EGFR ADCs of the instant disclosure.
  • Table 9C shows the percent cell viability of MDA-MB-468 cells after exposure to the various exatecan release anti-EGFR ADCs described herein.
  • Table 9D shows the percent cell viability of HCC827 cells after exposure to the various exatecan release anti-EGFR ADCs described herein.
  • Table 9E shows the percent cell viability of cell lines NCI-H292, OVCAR3 and FaDu cells after exposure to the different anti-EGFR ADCs of the present disclosure.
  • NCI-H292, HT-29, MCF-7, NCI-N87 cells MDA-MB-468, and HCC827 were cultured in RPMI-1640 media (Gibco, Life Technologies) supplemented with 10% v/v heat inactivated FBS (Corning), FaDu cells were maintained in EMEM media (Gibco, Life Technologies) supplemented with 10% v/v heat inactivated FBS (Corning) at 37 ⁇ C in a humidified incubator containing 5% CO 2 .
  • SK-BR-3 cells were maintained in McCoys 5A medium (Gibco, Life Technologies) supplemented with 10% v/w heat inactivated FBS (Corning) at 37 °C in a humidified incubator containing 5% CO2.
  • test compounds were added at a range of concentrations as ten-point serial dilution as duplicates or triplicates. Following further incubation for 6 days 37 o C, 5% CO2, cell viability was assessed with the use of a CellTiter-Glo Luminescent Cell Viability Assay (Promega). Luminescence was measured using the GloMax instrument (Promega). Luminescence values were plotted against log concentration of test compounds, and the IC50 values were calculated by GraphPad Prism 9 as best-fit values using four parameter dose-response curve fit, with R squared values ranging from 0.97-0.999. For a subset of payloads, each treatment was independently repeated two to eight times, and IC50 values were averaged.
  • Table 10 shows IC50 cytotoxicity values (nmol/L) of therapeutic payloads described herein for multiple tumor cell lines. Repeated treatments as two to eight independent experiments were used to determine the means of IC50 values and standard deviations (stdev). Standard deviation is shown as (N/A) for treatment performed once.
  • Table 10 Example 155: Tumor growth suppression effect on human lung carcinoma NCI-H292 of anti-TROP2 antibody drug conjugates 3038, 3036, and 3031 [00428] NCI-H292 human lung carcinoma cell line was purchased from ATCC.
  • mice (Jackson Labs NU/J # 2019), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x10 6 NCI-H292 cells suspended 1:1 with Matrigel in serum-free medium. After tumor size reached ⁇ 100 to 200 mm 3 mice were randomized into groups (5 animals per group, day 8). Respective anti-TRO2 antibody-drug conjugate was intravenously administered as a single dose of 3 mg/kg to the tail of each mouse (Start of dosing is referred to as Day 0 in the figures. Arrows indicate timing and frequency of dose administration). Phosphate buffer saline (PBS) was administered to the control group. Mean tumor volume was plotted until either the first death per group or end of the study.
  • PBS Phosphate buffer saline
  • FIG.1A shows mean tumor volume as a function of time after start of antibody drug conjugate 3031 administration compared to vehicle control group.
  • FIG.1B shows mean tumor volume as a function of time after start of antibody drug conjugate 3036 administration.
  • FIG. 1C shows mean tumor volume as a function of time after start of antibody drug conjugate 3038 administration.
  • Example 156 Tumor growth suppression effect on human throat cancer cell line FaDu of anti-EGFR antibody drug conjugates 3058A and 3053B [00429] FaDu human throat carcinoma cell line was purchased from ATCC.
  • mice (Jackson Labs NU/J # 2019), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x10 6 NCI-H292 cells suspended 1:1 with Matrigel in serum-free medium. After tumor size reached ⁇ 100 to 200 mm 3 mice were randomized into groups (5 animals per group, day 10). Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 3 mg/kg to the tail of each mouse for total of 3 doses, 1 week apart (start of dosing is referred to as Day 0 in the figures. Arrows indicate timing and frequency of dose administration). Phosphate buffer saline (PBS) was administered to the control group.
  • PBS Phosphate buffer saline
  • FIG.2A shows mean tumor volume as a function of time after start of antibody drug conjugate 3058A administration compared to vehicle control group. 5/5 mice were tumor free in antibody drug conjugate 3058A group.
  • FIG.2B shows mean tumor volume as a function of time after start of antibody drug conjugate 3053B administration. 5/5 mice were tumor free in antibody drug conjugate 3053B group.
  • Example 157 Tumor growth suppression effect on human lung cancer cell line NCI- H1975 of anti-EGFR antibody drug conjugates 3058A, 3058B, 3102, and 3053B [00430] NCI-H1975 human lung carcinoma cell line was purchased from ATCC.
  • mice (Jackson Labs NU/J # 2019), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x10 6 NCI-H1975 cells suspended 1:1 with Matrigel in serum-free medium. After tumor size reached ⁇ 100 to 200 mm 3 mice were randomized into groups (5 animals per group, day 15). Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 10 mg/kg to the tail of each mouse for total of 3 doses, 1 week apart (start of dosing is referred to as Day 0 in the figures. Arrows indicate timing and frequency of dose administration). Phosphate buffer saline (PBS) was administered to the control group.
  • PBS Phosphate buffer saline
  • FIG.3A shows mean tumor volume as a function of time after start of antibody drug conjugate 3058A administration compared to vehicle control.3/5 mice were tumor free in antibody drug conjugate 3058A group.
  • FIG.3B shows mean tumor volume as a function of time after start of antibody drug conjugate 3058B administration compared to vehicle control.1/5 mice were tumor free in antibody drug conjugate 3058B group.
  • FIG.3C shows mean tumor volume as a function of time after start of antibody drug conjugate 3102 administration compared to vehicle control.3/5 mice were tumor free in antibody drug conjugate 3102 group.
  • FIG.3D shows mean tumor volume as a function of time after start of antibody drug conjugate 3053B administration compared to vehicle control.2/5 mice were tumor free in antibody drug conjugate 3053B group.
  • Example 158 Tumor growth suppression effect on human breast cancer cell line MDA- MB-468 of anti-EGFR antibody drug conjugates 3053B, 3058A, and 3102 [00431] MDA-MB-468 human breast cancer cell line was purchased from Accegen. NOD-SCID mice (Charles River Labs, strain code #394), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x10 6 MDA-MB-468 cells suspended in 10% Matrigel in serum-free medium.
  • mice were randomized into groups (5 animals per group, day 12). Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 3 mg/kg to the tail of each mouse for 1 dose, start of dosing is referred to as Day 0 in the figures as indicated by the arrow. Phosphate buffer saline (PBS) was administered to the control group. Mean tumor volume was plotted until either the first death per group or end of the study. Error bars represent the standard error of the mean.
  • FIG. 4A shows mean tumor volume as a function of time after start of antibody drug conjugate 3053B, administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3053B group.
  • FIG.4B shows mean tumor volume as a function of time after start of antibody drug conjugate 3058A administration compared to vehicle control.5/5 mice were tumor free in antibody drug conjugate 3058A group.
  • FIG.4C shows mean tumor volume as a function of time after start of antibody drug conjugate 3102 administration compared to vehicle control. 3/5 mice were tumor free in antibody drug conjugate 3102 group.
  • Example 159 Tumor growth suppression effect on human throat cancer cell line FaDu of anti-EGFR antibody drug conjugates 3058A, 3102, 3059B, 3108, 3110, and 3053B [00432] FaDu human throat carcinoma cell line was purchased from ATCC.
  • NOD-SCID mice (Charles River Labs, strain code #394), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x10 6 FaDu cells suspended in 10% Matrigel in serum- free medium. After tumor size reached ⁇ 100 to 200 mm 3 mice were randomized into groups (5 animals per group, day 9). Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 1 mg/kg to the tail of each mouse for total of 3 doses, 1 week apart (start of dosing is referred to as Day 0 in the figures. Arrows indicate timing and frequency of dose administration). Phosphate buffer saline (PBS) was administered to the control group.
  • PBS Phosphate buffer saline
  • FIG.5A shows mean tumor volume as a function of time after start of antibody drug conjugate 3058A administration compared to vehicle control.
  • FIG.5B shows mean tumor volume as a function of time after start of antibody drug conjugate 3102 administration compared to vehicle control.
  • FIG.5C shows mean tumor volume as a function of time after start of antibody drug conjugate 3059B administration compared to vehicle control.
  • FIG.5D shows mean tumor volume as a function of time after start of antibody drug conjugate 3108 administration compared to vehicle control.
  • FIG.5E shows mean tumor volume as a function of time after start of antibody drug conjugate 3110 administration compared to vehicle control.
  • FIG.5F shows mean tumor volume as a function of time after start of antibody drug conjugate 3053B administration compared to vehicle control.
  • Compounds exemplified here exhibit tumor suppression greater than control.
  • Example 160 Tumor growth suppression effect on in vivo bystander model using human breast cancer cell line MDA-MB-468 co-inoculated with human colon adenocarcinoma cell line SW620-luc of anti-EGFR antibody drug conjugates 3058A, 3053B, 3102, and 3059B [00433]
  • MDA-MB-468 human breast cancer cell line was purchased from Accegen.
  • SW620-luc (luciferase) cells was purchased from FenicsBIO.
  • NOD-SCID mice (Charles River Labs, strain code #394), female, 6-8 weeks old were inoculated subcutaneously in the right hind flank with 5 x10 6 MDA-MB-468 cells and 5 x10 6 SW620-luc cells suspended in 10% Matrigel in serum-free medium.5 x10 6 SW620-luc cells suspended in 10% Matrigel in serum-free medium was inoculated subcutaneously in the left hind flank. After the right hind tumor size reached ⁇ 100 to 200 mm 3 mice were randomized into groups (5 animals per group, day 9).
  • Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 3 mg/kg to the tail of each mouse for 1 dose, start of dosing is referred to as Day 0 in the figures as indicated by the arrow.
  • Phosphate buffer saline (PBS) was administered to the control group.
  • Mean tumor volume was measured by caliper and plotted until either the first death per group or end of the study. Error bars represent the standard error of the mean.
  • Bioluminescent imaging was performed once a week from start of dosing. Each mouse received 15 mg of VivoGlo luciferin (Promega) by intraperitoneal injection and imaged by the IVIS Lumina S5 System (Perkin Elmer).
  • FIG.6A shows mean tumor volume as a function of time after start of antibody drug conjugate 3058A administration compared to vehicle control.5/5 mice were tumor free in antibody drug conjugate 3058A group.
  • FIG.6B shows mean tumor volume as a function of time after start of antibody drug conjugate 3053B administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3053B group.
  • FIG.6C shows mean tumor volume as a function of time after start of antibody drug conjugate 3102 administration compared to vehicle control.5/5 mice were tumor free in antibody drug conjugate 3102 group.
  • FIG.6D shows mean tumor volume as a function of time after start of antibody drug conjugate 3059B administration compared to vehicle control.3/5 mice were tumor free in antibody drug conjugate 3059B group.
  • FIG.7A shows change in luciferase activity as a function of time after start of antibody drug conjugate 3058A administration compared to vehicle control.
  • FIG.7B shows change in luciferase activity as a function of time after start of antibody drug conjugate 3053B administration compared to vehicle control.
  • FIG.7C shows change in luciferase activity as a function of time after start of antibody drug conjugate 3102 administration compared to vehicle control.
  • FIG.7D shows change in luciferase activity as a function of time after start of antibody drug conjugate 3059B administration compared to vehicle control.
  • Example 161 Tumor growth suppression effect on human throat cancer cell line FaDu of anti-EGFR antibody drug conjugates 3111, 3112, 3110, and 3109 [00435] FaDu human throat carcinoma cell line was purchased from ATCC. NOD-SCID mice (Charles River Labs, strain code #394), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x10 6 FaDu cells suspended in 10% Matrigel in serum- free medium. After tumor size reached ⁇ 100 to 200 mm 3 mice were randomized into groups (5 animals per group, day 9).
  • Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 10, 3, or 1 mg/kg to the tail of each mouse for total of 1 or 3 doses, 1 week apart (start of dosing is referred to as Day 0 in the figures. Arrows indicate timing and frequency of dose administration). Phosphate buffer saline (PBS) was administered to the control group. Mean tumor volume was plotted until either the first death per group or end of the study. Error bars represent the standard error of the mean. [00436] FIG, 8A shows mean tumor volume as a function of time after start of antibody drug conjugate 3111 administration compared to vehicle control.5/5 mice were tumor free in antibody drug conjugate 3111 group.
  • FIG.8B shows mean tumor volume as a function of time after start of antibody drug conjugate 3112 administration compared to vehicle control.5/5 mice were tumor free in antibody drug conjugate 3112 group.
  • FIG.8C shows mean tumor volume as a function of time after start of antibody drug conjugate 3110 administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3110 group.
  • FIG.8D shows mean tumor volume as a function of time after start of antibody drug conjugate 3109 administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3109 group.
  • FIG.9A shows mean tumor volume as a function of time after start of antibody drug conjugate 3111 administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3111 group.
  • FIG.9B shows mean tumor volume as a function of time after start of antibody drug conjugate 3112 administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3112 group.
  • FIG.9C shows mean tumor volume as a function of time after start of antibody drug conjugate 3110 administration compared to vehicle control.2/5 mice were tumor free in antibody drug conjugate 3110 group.
  • FIG.9D shows mean tumor volume as a function of time after start of antibody drug conjugate 3109 administration compared to vehicle control.5/5 mice were tumor free in antibody drug conjugate 3109 group.
  • FIG.10A shows mean tumor volume as a function of time after start of antibody drug conjugate 3111 administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3111 group.
  • FIG.10B shows mean tumor volume as a function of time after start of antibody drug conjugate 3112 administration compared to vehicle control.3/5 mice were tumor free in antibody drug conjugate 3112 group.
  • FIG.10C shows mean tumor volume as a function of time after start of antibody drug conjugate 3110 administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3110 group.
  • FIG.10D shows mean tumor volume as a function of time after start of antibody drug conjugate 3109 administration compared to vehicle control.3/5 mice were tumor free in antibody drug conjugate 3109 group.
  • MDA-MB-468 Tumor growth suppression effect on human breast cancer cell line MDA- MB-468 of anti-EGFR antibody drug conjugates 3111, 3112, 3110, and 3109 [00439]
  • MDA-MB-468 human breast cancer cell line was purchased from Accegen.
  • NOD-SCID mice (Charles River Labs, strain code #394), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x10 6 MDA-MB-468 cells suspended in 10% Matrigel in serum-free medium. After tumor size reached ⁇ 100 to 200 mm 3 mice were randomized into groups (5 animals per group, day 20).
  • Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 3 mg/kg to the tail of each mouse for 1 dose, start of dosing is referred to as Day 0 in the figures as indicated by the arrow.
  • Phosphate buffer saline (PBS) was administered to the control group.
  • Mean tumor volume was plotted until either the first death per group or end of the study. Error bars represent the standard error of the mean.
  • FIG.11A shows mean tumor volume as a function of time after start of antibody drug conjugate 3111 administration compared to vehicle control.
  • Figure 11B shows mean tumor volume as a function of time after start of antibody drug conjugate 3112 administration compared to vehicle control.
  • FIG.11C shows mean tumor volume as a function of time after start of antibody drug conjugate 3110 administration compared to vehicle control.
  • FIG.11D shows mean tumor volume as a function of time after start of antibody drug conjugate 3109 administration compared to vehicle control.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Disclosed herein, in part, are novel exatecan derivatives with novel chemical linkers that include cathepsin B cleavable moieties and conjugated to targeting antibodies. In a preferred embodiment the antibody is an anti-TROP2, an anti-EGFR or an anti-HER2 antibody.

Description

EXATECAN DERIVATIVES AND ANTIBODY-DRUG CONJUGATES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of, and priority to, U.S.S.N.63/422,517 filed on November 4, 2022; the content of which is incorporated herein by reference in its entirety. BACKGROUND [0002] Antibody-drug conjugates (ADC’s) provide a mechanism for selective delivery of small molecule therapeutic payloads to antigen-positive cancer cells, thereby attenuating systemic toxicity of cytotoxic drugs to antigen-negative normal cells. Three components of an ADC—the antibody, the cytotoxic payload, and the linker that joins them—are important in designing an effective therapeutic. Despite active development, challenges still exist, for example, toxicity due to the antibody binding to its target in normal tissue, and dispersion of the cytotoxic payload in normal tissue due to instability of the ADC linker. Thus, many ADC’s have not succeeded in clinical trials due to lack of safety and/or efficacy at tolerated doses. [0003] Topoisomerase I plays a critical role in DNA replication in both normal and diseased conditions (e.g., cancer). As inhibition of topoisomerase I leads to cell death, compounds that bind to and inhibit topoisomerase I may be useful as therapeutic agents. [0004] Camptothecin is a natural product with cytotoxic activity in a variety of cell lines. The binding of its active lactone ring to topoisomerase I inhibits DNA replication, thus causing cell apoptosis. However, its limitations for drug development include, for example, poor water solubility and an equilibrium between its active, lactone form and its inactive, ring-opened form. [0005] Exatecan is a water-soluble camptothecin derivative. As a chemotherapeutic agent, exatecan mesylate did not gain drug approval after several clinical trials due to lack of efficacy or high toxicity at tested doses. Efforts to enable the clinical utility of exatecan have been made by converting exatecan into a prodrug form, where exatecan is covalently linked to a carboxymethyldextran polyalcohol polymer via a peptidyl spacer (a substrate for intracellular cathepsin proteases). However, this prodrug did not succeed in clinical trials. [0006] Thus, a need exists for compounds more amenable to clinical development and success in treating human tumors. Moreover, preferential delivery of topoisomerase I inhibitors to diseased tissues through antibody-drug conjugates could lead to improved safety and efficacy, thereby providing therapeutic options for a larger number of patients and types of cancers. SUMMARY [0007] The present disclosure relates to compounds useful for the treatment of cancer. The present disclosure is directed, in part, to linker-payload constructs useful for attaching payloads to antibodies and exatecan-based drug conjugates. For example, provided herein are compounds representing a therapeutic payload, a linker-payload construct, or a drug conjugate. [0008] For example, the present disclosure provides linker-payload constructs and drug conjugates, each comprising a disclosed therapeutic payload. Further provided herein is the use of disclosed compounds as medicinal agents, processes for their preparation, and pharmaceutical compositions containing them as an active ingredient both alone or in combination with other agents, as well as provides for their use as medicaments and/or in the manufacture of medicaments for the treatment of cancer. [0009] For example, disclosed herein is a drug conjugate represented by Formula IA or Formula IB:
Figure imgf000003_0001
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: Lig is a targeting moiety; L is a linker moiety; R is selected from the group consisting of -R1, -C(O)-R1-, -C(O)-O-R1-, -C(O)-NH-R1-, - C(O)-C0-3alkyl-C(O)-NH-R1-, -C(O)-(5-6 membered heteroaryl)-R1-, -CH2-(5-6 membered heteroaryl)-R1-, -C(O)-(4-6 membered heterocyclyl)-, -C(O)-(4-6 membered heterocyclyl)-C(O)- R1-, -C(O)-(4-6 membered heterocyclyl)-NH-R1-, -C(O)-C3-4cycloalkyl-R1-, -C(S)-C3- 4cycloalkyl-R1-, and -C(O)-O-phenyl-R1-; wherein any aforementioned heteroaryl, heterocyclyl, alkyl, and cycloalkyl may optionally be substituted by one, two or three substituents each independently selected from the group consisting of halogen, hydroxyl, and oxo; R1 is selected from the group consisting of CH2-CH2O-, -CH2-O-, -NH-, -CH2ONH-, and -CH(CH3)-NH-; and s is 0, 1, 2, 3, 4, 5, 6, 7, or 8; and t is 0, 1, 2, 3, 4, 5, 6, 7, or 8. [0010] Also disclosed herein is a therapeutic payload represented by Formula II:
Figure imgf000004_0001
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: Y is hydrogen or -C1-3alkyl; R is selected from the group consisting of -C(O)-C3alkyl, -C(O)-C3cycloalkyl, -C(O)- C4cycloalkyl, -C1alkyl, -C(O)-(pyrrolidinyl), and -C(O)-CH2ONH-C(O)-(pyrrolidinyl); wherein: -C(O)-C3alkyl, -C(O)-C3cycloalkyl, -C(O)-C4cycloalkyl or -C1alkyl is substituted by one or two substituents each independently selected from the group consisting of hydroxyl, -NH2, -CHO, and -COOH; -C(O)-(pyrrolidinyl) and -C(O)-CH2ONH-C(O)-(pyrrolidinyl) may optionally be substituted on an available pyrrolidinyl carbon atom by one or two substituents each independently selected from the group consisting of halogen and hydroxyl, or two R1 join together to form oxo; and -C(O)-(pyrrolidinyl) may optionally be substituted on an available pyrrolidinyl nitrogen atom by -CHO, -C(O)CH3, or -C(O)CH2NH2. [0011] Methods of treating cancer are contemplated herein, comprising administering to a patient in need thereof an effective amount of a disclosed compound. For example, provided herein is a method of treating cancer in patient in need thereof, comprising administering to the patient an effective amount of a disclosed therapeutic payload, a disclosed linker-payload construct, or a disclosed drug conjugate. [0012] Pharmaceutical compositions comprising at least one disclosed compound and a pharmaceutically acceptable carrier are additionally described herein. For example, provided herein is a pharmaceutically acceptable composition comprising a disclosed compound, e.g., a disclosed therapeutic payload, a disclosed linker-payload construct, or a disclosed drug conjugate and a pharmaceutically acceptable excipient. BRIEF DESCRIPTION OF THE DRAWINGS [0013] FIG.1A, FIG.1B, and FIG.1C show tumor suppression effect of antibody drug conjugates 3031, 3036, and 3038 on lung carcinoma cell line NCI-H292 derived xenograft in athymic nude mice. Compounds exemplified here exhibit tumor suppression greater than control. [0014] FIG.2A and FIG.2B show tumor suppression effect of antibody drug conjugates 3058A and 3053B on human throat cancer cell line FaDu derived xenograft in athymic nude mice. Compounds exemplified here exhibit tumor suppression greater than control. [0015] FIG.3A, FIG.3B, FIG.3C, and FIG.3D show tumor suppression effect of antibody drug conjugates 3058A, 3058B, 3102, and 3053B on human lung cancer cell line H1975 derived xenograft in athymic nude mice. Compounds exemplified here exhibit tumor suppression greater than control. [0016] FIG.4A, FIG.4B, and FIG.4C show tumor suppression effect of antibody drug conjugates 3053B, 3058A, and 3102 on human breast cancer cell line MDA-MB-468 derived xenograft in NOD-SCID mice. Compounds exemplified here exhibit tumor suppression greater than control. [0017] FIG.5A, FIG.5B, FIG.5C, FIG 5D, FIG.5E, and FIG.5F show tumor suppression effect of antibody drug conjugates 3058A ,3102, 3059B, 3108, 3110, and 3053B on human throat cancer cell line FaDu derived xenograft in NOD-SCID mice. Compounds exemplified here exhibit tumor suppression greater than control. [0018] FIG.6A, FIG.6B, FIG.6C, and FIG.6D show tumor suppression effect of antibody drug conjugates 3058A, 3053B, 3102, and 3059B on human breast cancer cell line MDA-MB-468 and colon cancer cell line SW620-luc co-inoculated xenograft in NOD-SCID mice. Compounds exemplified here exhibit tumor suppression greater than control. [0019] FIG.7A, FIG.7B, FIG.7C, and FIG.7D show tumor suppression effect of antibody drug conjugates 3058A, 3053B, 3102, and 3059B on human breast cancer cell line MDA-MB-468 and colon cancer cell line SW620-luc co-inoculated xenograft in NOD-SCID mice. All four figures show luciferase signal expressed as total flux (photons per second) as a function of time after start of antibody drug conjugate administration compared to vehicle control group. Compounds exemplified here exhibit tumor suppression greater than control.  [0020] FIG.8A, FIG.8B, FIG.8C, and FIG.8D show tumor suppression effect of antibody drug conjugates 3111, 3112, 3110, and 3109 on human throat cancer cell line FaDu derived xenograft in NOD-SCID mice (10 mg per kg, 1 dose). Compounds exemplified here exhibit tumor suppression greater than control. [0021] FIG.9A, FIG.9B, FIG.9C, and FIG.9D show tumor suppression effect of antibody drug conjugates 3111, 3112, 3110, and 3109 on human throat cancer cell line FaDu derived xenograft in NOD-SCID mice (3 mg per kg, 1 dose). Compounds exemplified here exhibit tumor suppression greater than control. [0022] FIG.10A, FIG.10B, FIG.10C, and FIG.10D show tumor suppression effect of antibody drug conjugates 3111, 3112, 3110, and 3109 on human throat cancer cell line FaDu derived xenograft in NOD-SCID mice (1 mg per kg, 3 doses). Compounds exemplified here exhibit tumor suppression greater than control. [0023] FIG.11A, FIG.11B, FIG.11C, and FIG.11D shows tumor suppression effect of antibody drug conjugates 3111, 3112, 3110, and 3109 on human breast cancer cell line MDA- MB-468 derived xenograft in NOD-SCID mice (3 mg per kg, 1 dose). Compounds exemplified here exhibit tumor suppression greater than control. DETAILED DESCRIPTION [0024] The features and other details of the disclosure will now be more particularly described. Before further description of the present disclosure, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and as understood by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Definitions [0025] As used herein, the words “a” and “an” are meant to include one or more unless otherwise specified. For example, the term “an agent” encompasses both a single agent and a combination of two or more agents. [0026] The term “alkenyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Exemplary alkenyl groups include, but are not limited to, a straight or branched group of 2-6 or 3-4 carbon atoms, referred to herein as C2-6alkenyl, and C3-4alkenyl, respectively. Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, etc. [0027] The term “alkoxy” as used herein refers to a straight or branched alkyl group attached to oxygen (alkyl-O-). Exemplary alkoxy groups include, but are not limited to, alkoxy groups of 1-6 or 2-6 carbon atoms, referred to herein as C1-6alkoxy, and C2-6alkoxy, respectively. Exemplary alkoxy groups include, but are not limited to methoxy, ethoxy, isopropoxy, etc. [0028] The term “alkoxyalkyl” as used herein refers to a straight or branched alkyl group attached to oxygen, attached to a second straight or branched alkyl group (alkyl-O-alkyl-). Exemplary alkoxyalkyl groups include, but are not limited to, alkoxyalkyl groups in which each of the alkyl groups independently contains 1-6 carbon atoms, referred to herein as C1-6alkoxy-C1- 6alkyl. Exemplary alkoxyalkyl groups include, but are not limited to methoxymethyl, 2- methoxyethyl, 1-methoxyethyl, 2-methoxypropyl, ethoxymethyl, 2-isopropoxyethyl etc. [0029] The term “alkyoxycarbonyl” as used herein refers to a straight or branched alkyl group attached to oxygen, attached to a carbonyl group (alkyl-O-C(O)-). Exemplary alkoxycarbonyl groups include, but are not limited to, alkoxycarbonyl groups of 1-6 carbon atoms, referred to herein as C1-6alkoxycarbonyl. Exemplary alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, etc. [0030] The term “alkenyloxy” used herein refers to a straight or branched alkenyl group attached to oxygen (alkenyl-O-). Exemplary alkenyloxy groups include, but are not limited to, groups with an alkenyl group of 3-6 carbon atoms, referred to herein as C3-6alkenyloxy. Exemplary “alkenyloxy” groups include, but are not limited to allyloxy, butenyloxy, etc. [0031] The term “alkynyloxy” used herein refers to a straight or branched alkynyl group attached to oxygen (alkynyl-O). Exemplary alkynyloxy groups include, but are not limited to, groups with an alkynyl group of 3-6 carbon atoms, referred to herein as C3-6alkynyloxy. Exemplary alkynyloxy groups include, but are not limited to, propynyloxy, butynyloxy, etc. [0032] The term “alkyl” as used herein refers to a saturated straight or branched hydrocarbon. Exemplary alkyl groups include, but are not limited to, straight or branched hydrocarbons of 1-6, 1-4, or 1-3 carbon atoms, referred to herein as C1-6alkyl, C1-4alkyl, and C1- 3alkyl, respectively. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-butyl, 3-methyl-2-butyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4- methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, etc. [0033] The term “alkylcarbonyl” as used herein refers to a straight or branched alkyl group attached to a carbonyl group (alkyl-C(O)-). Exemplary alkylcarbonyl groups include, but are not limited to, alkylcarbonyl groups of 1-6 atoms, referred to herein as C1-6alkylcarbonyl groups. Exemplary alkylcarbonyl groups include, but are not limited to, acetyl, propanoyl, isopropanoyl, butanoyl, etc. [0034] “Alkylene” means a straight or branched, saturated aliphatic divalent radical having the number of carbons indicated. “Cycloalkylene” refers to a divalent radical of carbocyclic saturated hydrocarbon group having the number of carbons indicated. [0035] The term “alkynyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond. Exemplary alkynyl groups include, but are not limited to, straight or branched groups of 2-6, or 3-6 carbon atoms, referred to herein as C2-6alkynyl, and C3-6alkynyl, respectively. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, etc. [0036] The term “carbonyl” as used herein refers to the radical -C(O)-. [0037] The term “cyano” as used herein refers to the radical -CN. [0038] The term “cycloalkoxy” as used herein refers to a cycloalkyl group attached to oxygen (cycloalkyl-O-). Exemplary cycloalkoxy groups include, but are not limited to, cycloalkoxy groups of 3-6 carbon atoms, referred to herein as C3-6cycloalkoxy groups. Exemplary cycloalkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclohexyloxy, etc. [0039] The terms “cycloalkyl” or a “carbocyclic group” as used herein refers to a saturated or partially unsaturated hydrocarbon group of, for example, 3-6, or 4-6 carbons, referred to herein as C3-6cycloalkyl or C4-6cycloalkyl, respectively. Exemplary cycloalkyl groups include, but are not limited to, cyclohexyl, cyclopentyl, cyclopentenyl, cyclobutyl or cyclopropyl. [0040] The terms “halo” or “halogen” as used herein refer to F, Cl, Br, or I. [0041] The terms “heteroaryl” or “heteroaromatic group” as used herein refers to a monocyclic aromatic 5-6 membered ring system containing one or more heteroatoms, for example one to three heteroatoms, such as nitrogen, oxygen, and sulfur. Where possible, said heteroaryl ring may be linked to the adjacent radical though carbon or nitrogen. Examples of heteroaryl rings include but are not limited to furan, thiophene, pyrrole, thiazole, oxazole, isothiazole, isoxazole, imidazole, pyrazole, triazole, pyridine or pyrimidine etc. [0042] The terms “heterocyclyl” or “heterocyclic group” are art-recognized and refer to e.g., saturated or partially unsaturated, 4-10 membered monocyclic or bicyclic ring structures, or e.g., 4-9 or 4-6 membered saturated ring structures, including bridged, fused or spirocyclic rings, and whose ring structures include one to three heteroatoms, such as nitrogen, oxygen, and sulfur. Where possible, heterocyclyl rings may be linked to the adjacent radical through carbon or nitrogen. Examples of heterocyclyl groups include, but are not limited to, pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, oxetane, azetidine, tetrahydrofuran or dihydrofuran etc. [0043] The term “heterocyclyloxy” as used herein refers to a heterocyclyl group attached to oxygen (heterocyclyl-O-). [0044] The term “heteroaryloxy” as used herein refers to a heteroaryl group attached to oxygen (heteroaryl-O-). [0045] The terms “hydroxy” and “hydroxyl” as used herein refer to the radical -OH. [0046] The term “oxo” as used herein refers to the radical =O. [0047] “Pharmaceutically or pharmacologically acceptable” include molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologics standards. [0048] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” as used herein refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions. [0049] The term “pharmaceutical composition” as used herein refers to a composition comprising at least one compound as disclosed herein formulated together with one or more pharmaceutically acceptable carriers. [0050] “Individual,” “patient,” or “subject” are used interchangeably and include any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans. The compounds of the present disclosure can be administered to a mammal, such as a human, but can also be administered to other mammals such as an animal in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like). “Modulation” includes antagonism (e.g., inhibition), agonism, partial antagonism and/or partial agonism. [0051] “Treating” includes any effect, e.g., lessening, reducing, modulating, or eliminating, that results in the improvement of the condition, disease, disorder and the like. [0052] In the present specification, the term “therapeutically effective amount” or “effective amount” means the amount of the subject compound that will elicit the biological or medical response of a tissue, system or animal, (e.g., mammal or human) that is being sought by the researcher, veterinarian, medical doctor or other clinician. The compounds of the present disclosure are administered in therapeutically effective amounts to treat a disease. Alternatively, a therapeutically effective amount of a compound is the quantity required to achieve a desired therapeutic and/or prophylactic effect, such as an amount which results in weight loss. [0053] The term “pharmaceutically acceptable salt(s)” as used herein refers to salts of acidic or basic groups that may be present in compounds used in the compositions. Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including, but not limited to, malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1,1’-methylene-bis-(2-hydroxy-3- naphthoate)) salts. Compounds included in the present compositions that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts. Compounds included in the present compositions that include a basic or acidic moiety may also form pharmaceutically acceptable salts with various amino acids. The compounds of the disclosure may contain both acidic and basic groups; for example, one amino and one carboxylic acid group. In such a case, the compound can exist as an acid addition salt, a zwitterion, or a base salt. [0054] As will be understood by the skilled artisan, “H” is the symbol for hydrogen, “N” is the symbol for nitrogen, “S” is the symbol for sulfur, “O” is the symbol for oxygen. “Me” is an abbreviation for methyl. It will be appreciated that the present disclosure should be construed in congruity with the laws and principles of chemical bonding. [0055] The compounds of the disclosure may contain one or more chiral centers and, therefore, exist as stereoisomers. The term “stereoisomers” when used herein consist of all enantiomers or diastereomers. These compounds may be designated by the symbols “(+),” “(
Figure imgf000012_0001
” “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. The present disclosure encompasses various stereoisomers of these compounds and mixtures thereof. Mixtures of enantiomers or diastereomers may be designated “(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. [0056] The compounds of the disclosure may contain one or more double bonds and, therefore, exist as geometric isomers resulting from the arrangement of substituents around a carbon-carbon double bond. The symbol denotes a bond that may be a single, double or triple bond as described herein. Substituents around a carbon-carbon double bond are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the “E” and “Z” isomers. Substituents around a carbon-carbon double bond alternatively can be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond. [0057] Compounds of the disclosure may contain a carbocyclic or heterocyclic ring and therefore, exist as geometric isomers resulting from the arrangement of substituents around the ring. The arrangement of substituents around a carbocyclic or heterocyclic ring are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting carbocyclic or heterocyclic rings encompass both “Z” and “E” isomers. Substituents around a carbocyclic or heterocyclic rings may also be referred to as “cis” or “trans”, where the term “cis” represents substituents on the same side of the plane of the ring and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis/trans.” [0058] Individual enantiomers and diastereomers of compounds of the present disclosure can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, (3) direct separation of the mixture of optical enantiomers on chiral liquid chromatographic columns or (4) kinetic resolution using stereoselective chemical or enzymatic reagents. Racemic mixtures can also be resolved into their component enantiomers by well- known methods, such as chiral-phase liquid chromatography or crystallizing the compound in a chiral solvent. Stereoselective syntheses, a chemical or enzymatic reaction in which a single reactant forms an unequal mixture of stereoisomers during the creation of a new stereocenter or during the transformation of a pre-existing one, are well known in the art. Stereoselective syntheses encompass both enantio- and diastereoselective transformations and may involve the use of chiral auxiliaries. For examples, see Carreira and Kvaerno, Classics in Stereoselective Synthesis, Wiley-VCH: Weinheim, 2009. [0059] The compounds disclosed herein can exist in solvated as well as unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the present disclosure embrace both solvated and unsolvated forms. In one embodiment, the compound is amorphous. In one embodiment, the compound is a single polymorph. In another embodiment, the compound is a mixture of polymorphs. In another embodiment, the compound is in a crystalline form. [0060] The present disclosure also embraces isotopically labeled compounds of the disclosure which are identical to those recited herein, except that 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. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F, and 36Cl, respectively. For example, a compound of the disclosure may have one or more H atom replaced with deuterium. [0061] Certain isotopically labeled disclosed compounds (e.g., those labeled with 3H and 14C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., 3H) and carbon-14 (i.e., 14C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., 2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Isotopically labeled compounds of the present disclosure can generally be prepared by following procedures analogous to those disclosed in the examples herein by substituting an isotopically labeled reagent for a non-isotopically labeled reagent. [0062] The term “prodrug” refers to compounds that are transformed in vivo to yield a disclosed compound or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (such as by esterase, amidase, phosphatase, oxidative and or reductive metabolism) in various locations (such as in the intestinal lumen or upon transit of the intestine, blood or liver). Prodrugs are well known in the art (for example, see Rautio, Kumpulainen, et al, Nature Reviews Drug Discovery 2008, 7, 255). For example, if a compound of the present disclosure or a pharmaceutically acceptable salt, hydrate or solvate of the compound contains a carboxylic acid functional group, a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as (C1-8)alkyl, (C2-12)alkylcarbonyloxymethyl, 1-(alkylcarbonyloxy)ethyl having from 4 to 9 carbon atoms, 1-methyl-1-(alkylcarbonyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N-(C1-2)alkylamino(C2-3)alkyl (such as β- dimethylaminoethyl), carbamoyl-(C1-2)alkyl, N,N-di(C1-2)alkylcarbamoyl-(C1-2)alkyl and piperidino-, pyrrolidino- or morpholino(C2-3)alkyl. [0063] Similarly, if a disclosed compound contains an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as (C1-6)alkylcarbonyloxymethyl, 1-((C1-6)alkylcarbonyloxy)ethyl, 1-methyl-1-((C1- 6)alkylcarbonyloxy)ethyl (C1-6)alkoxycarbonyloxymethyl, N-(C1-6)alkoxycarbonylaminomethyl, succinoyl, (C1-6)alkylcarbonyl, α-amino(C1-4)alkylcarbonyl, arylalkylcarbonyl and α- aminoalkylcarbonyl, or α-aminoalkylcarbonyl-α-aminoalkylcarbonyl, where each ^- aminoalkylcarbonyl group is independently selected from the naturally occurring L-amino acids, P(O)(OH)2, -P(O)(O(C1-6)alkyl)2 or glycosyl (the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate). [0064] If a compound of the present disclosure incorporates an amine functional group, a prodrug can be formed, for example, by creation of an amide or carbamate, an N- alkylcarbonyloxyalkyl derivative, an (oxodioxolenyl)methyl derivative, an N-Mannich base, imine or enamine. In addition, a secondary amine can be metabolically cleaved to generate a bioactive primary amine, or a tertiary amine can be metabolically cleaved to generate a bioactive primary or secondary amine. For examples, see Simplício, et al., Molecules 2008, 13, 519 and references therein. [0065] Procedures for making compounds described herein are provided below in the working examples and may be supplemented or substituted by procedures known to those of skill in the art. Starting materials used in the working examples can be purchased or prepared by methods described in the chemical literature, or by adaptations thereof, using methods known by those skilled in the art. The order in which the steps are performed can vary depending on the groups introduced and the reagents used, but would be apparent to those skilled in the art. Disclosed compounds, or any of the intermediates described herein, can be further derivatized by using one or more standard synthetic methods known to those skilled in the art. [0066] Salts of compounds disclosed herein can be prepared by the reaction of a compound disclosed herein with an appropriate acid or base in a suitable solvent, or mixture of solvents (such as an ether, for example, diethyl ether, or an alcohol, for example ethanol, or an aqueous solvent) using conventional procedures. Salts of a compound disclosed herein can be exchanged for other salts by treatment using conventional ion-exchange chromatography procedures. Compounds [0067] Disclosed herein, for example, is a drug conjugate represented by Formula IA or Formula IB:
Figure imgf000016_0001
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: Lig is a targeting moiety; L is a linker moiety; R is selected from the group consisting of -R1, -C(O)-R1-, -C(O)-O-R1-, -C(O)-NH-R1-, - C(O)-C0-3alkyl-C(O)-NH-R1-, -C(O)-(5-6 membered heteroaryl)-R1-, -CH2-(5-6 membered heteroaryl)-R1-, -C(O)-(4-6 membered heterocyclyl)-, -C(O)-(4-6 membered heterocyclyl)-C(O)- R1-, -C(O)-(4-6 membered heterocyclyl)-NH-R1-, -C(O)-C3-4cycloalkyl-R1-, -C(S)-C3- 4cycloalkyl-R1-, and -C(O)-O-phenyl-R1-; wherein any aforementioned heteroaryl, heterocyclyl, alkyl, and cycloalkyl may optionally be substituted by one, two or three substituents each independently selected from the group consisting of halogen, hydroxyl, and oxo; R1 is selected from the group consisting of CH2-CH2O-, -CH2-O-, -NH-, -CH2ONH-, and -CH(CH3)-NH-; and s is 0, 1, 2, 3, 4, 5, 6, 7, or 8; and t is 0, 1, 2, 3, 4, 5, 6, 7, or 8. [0068] In some embodiments, a disclosed drug conjugate of Formula I may be represented by Formula IA:
Figure imgf000017_0001
or a pharmaceutically acceptable salt or stereoisomer thereof; wherein s is 1, 2, 3, 4, 5, 6, 7, or 8. [0069] In some embodiments, Lig is a monoclonal antibody. For example, in some embodiments Lig is selected from the group consisting of an anti-TROP2 antibody, an anti- EGRF antibody, an anti-HER2 antibody, an anti-B7-H3 antibody, an anti-CD30 antibody, an anti-CD33 antibody, and an anti-CD70 antibody. In other embodiments, Lig is selected from the group consisting of an anti-TROP2 antibody, an anti-EGRF antibody, and an anti-HER2 antibody. In certain embodiments, Lig is an anti-TROP2 antibody. In certain other embodiments, Lig is an anti-EGRF antibody. In still other embodiments, Lig is an anti-HER2 antibody. For example, in certain embodiments Lig may be selected from an antibody disclosed in Table 4. In further embodiments, s is 1 or 8. For example, is some embodiments s is 1. [0070] In some embodiments, L is selected, for example, from the group consisting of:
Figure imgf000017_0002
Figure imgf000018_0001
wherein * denotes the point of attachment to R. [0071] In other embodiments, R is selected from the group consisting of -CH2CH2O-, - C(O)-CH2ONH-, -C(O)-O-CH2CH2O-, -C(O)-NH-CH2CH2O-, -C(O)-C1alkyl-C(O)-NH- CH2CH2O-, -C(O)-C3alkyl-C(O)-NH-CH2CH2O-, and -C(O)-CH(CH3)-NH-. [0072] In still other embodiments, R is selected from the group consisting of -C(O)- triazolyl-CH2CH2O-, -CH2-triazolyl-CH2CH2O-, and -C(O)-furanyl-CH2O-. [0073] In further embodiments, R is selected from the group consisting of -C(O)- C3cycloalkyl-CH2CH2O-, -C(S)-C3cycloalkyl-CH2CH2O-,-C(O)-C3cycloalkyl-NH-, and -C(O)- O-phenyl-NH-. [0074] In certain embodiments, R is selected from the group consisting of -C(O)- pyrrolidinyl- and -C(O)-pyrrolidinyl-C(O)-CH(CH3)-NH-, wherein pyrrolidinyl may optionally be substituted by one or two fluoro atoms. [0075] For example, in some embodiments R is selected from the group consisting of
Figure imgf000018_0002
Figure imgf000019_0001
wherein ** denotes the point of attachment to L. [0076] For example, in some embodiments a disclosed drug conjugate may be selected from the group consisting of
Figure imgf000019_0002
, ,
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
, ,
Figure imgf000023_0001
,
Figure imgf000024_0001
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Lig is selected from the group consisting of an anti-TROP2 antibody, an anti-EGRF antibody, and an anti-HER2 antibody. [0077] In certain embodiments, Lig is an anti-TROP2 antibody. In certain other embodiments, Lig is an anti-EGRF antibody, for example, Panitumumab, Nimotuzumab, Matuzumab, or Cetuximab. In still other embodiments, Lig is an anti-HER2 antibody. In further embodiments, s is 1 or 8. For example, in some embodiments s is 1. [0078] For example, contemplated drug conjugates of the present disclosure are provided in Tables 1-3. Table 1. HER 2 Antibody Drug Conjugates
Figure imgf000025_0001
Figure imgf000026_0001
Table 2. TROP2 Antibody Drug Conjugates
Figure imgf000026_0002
Figure imgf000027_0001
Table 3. EGFR Antibody Drug Conjugates [0079] For compounds 3049-3107, Anti-EGFR IgG11 was used; for compound 3108, Anti-EGFR IgG12 was used; for compound 3109-3110, Anti-EGFR IgG13 was used, for compounds 3111-3112, Anti-EGFR IgG14 was used; and for compound 3113, Anti-EGFR IgG1 5 was used.
Figure imgf000027_0002
Figure imgf000028_0001
[0080] Contemplated targets and corresponding example antibodies of the present disclosure are provided in Table 4. Table 4
Figure imgf000028_0002
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0002
[0081] Also disclosed herein is a therapeutic payload represented by Formula II:
Figure imgf000031_0001
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: Y is hydrogen or -C1-3alkyl; R is selected from the group consisting of -C(O)-C3alkyl, -C(O)O-C3alkyl, -C(O)- C3cycloalkyl, -C(O)-C4cycloalkyl, -C1alkyl, -C(O)-(pyrrolidinyl), and -C(O)-CH2ONH-C(O)- (pyrrolidinyl); wherein: -C(O)-C3alkyl, -C(O)-C3cycloalkyl, -C(O)O-C3alkyl, -C(O)-C4cycloalkyl or - C1alkyl is substituted by one or two substituents each independently selected from the group consisting of hydroxyl, -NH2, -CHO, and -COOH; -C(O)-(pyrrolidinyl) and -C(O)-CH2ONH-C(O)-(pyrrolidinyl) may optionally be substituted on an available pyrrolidinyl carbon atom by one or two substituents each independently selected from the group consisting of halogen and hydroxyl, or two R1 join together to form oxo; and -C(O)-(pyrrolidinyl) may optionally be substituted on an available pyrrolidinyl nitrogen atom by -CHO, -C(O)CH3, or -C(O)CH2NH2. [0082] In some embodiments, a disclosed therapeutic payload of Formula II may be represented by Formula IIA:
Figure imgf000032_0001
; or a pharmaceutically acceptable salt or stereoisomer thereof. [0083] In some embodiments, Y is hydrogen. In other embodiments, Y is -C1-3alkyl. For example, in certain embodiments Y is -CH3. In some embodiments, R is selected, for example, from the group consisting of: ,
Figure imgf000032_0002
[0084] In some embodiments, a disclosed therapeutic payload may be selected, for example, from any one of the compounds disclosed in Table 5, or a pharmaceutically acceptable salt or stereoisomer thereof. Table 5.
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
[0085] For example, in certain embodiments a therapeutic payload disclosed herein may be selected from the group consisting of:
Figure imgf000040_0001
Figure imgf000041_0001
or a pharmaceutically acceptable salt thereof. Further disclosed herein is a linker-payload construct represented by Formula IIIA or Formula I
Figure imgf000041_0002
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L is selected from the group consisting of:
Figure imgf000042_0001
wherein * denotes the point of attachment to R; R is selected from the group consisting of:
Figure imgf000042_0002
a
Figure imgf000043_0001
wherein ** denotes the point of attachment to L; and s is 0, 1, 2, 3, 4, 5, 6, 7, or 8; and t is 0, 1, 2, 3, 4, 5, 6, 7, or 8. [0086] In some embodiments, a disclosed linker-payload construct of Formula III may be represented by Formula IIIC:
Figure imgf000043_0002
or a pharmaceutically acceptable salt or stereoisomer thereof wherein s is 1, 2, 3, 4, 5, 6, 7, or 8. [0087] In some embodiments, R is selected, for example, from the group consisting of:
Figure imgf000043_0003
Figure imgf000044_0001
wherein ** denotes the point of attachment to L. [0088] In certain embodiments, s is 1 or 8. In other embodiments, s is 1. [0089] In some embodiments, a disclosed linker-payload construct may be selected, for example, from any one of the compounds disclosed in Table 6, or a pharmaceutically acceptable salt or stereoisomer thereof. Table 6.
Figure imgf000044_0002
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
[0090] For example, in some embodiments a linker-payload construct contemplated herein may be selected from the group consisting of:
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
,
Figure imgf000055_0001
Figure imgf000056_0001
Methods [0091] Disclosed herein, for example, is a method of treating cancer in patient in need thereof, comprising administering to the patient an effective amount of a therapeutic payload disclosed herein, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, and esophageal cancer. [0092] Also disclosed herein is a method of treating cancer in patient in need thereof, comprising administering to the patient an effective amount of a linker-payload construct disclosed herein, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, and esophageal cancer. [0093] Further disclosed herein is method of treating cancer in patient in need thereof, comprising administering to the patient an effective amount of a drug conjugate comprising any of the payloads as disclosed herein, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, and esophageal cancer. [0094] Also disclosed herein is a method of delivering a therapeutically effective amount of a therapeutic payload moiety to a patient in need thereof, comprising administering to the patient any one of the drug conjugates disclosed herein. [0095] In certain embodiments, the patient is a human. [0096] In certain embodiments, administering a disclosed compound may comprise subcutaneous administration. In certain embodiments, administering a disclosed compound may comprise intravenous administration. In certain embodiments, administering a disclosed compound may comprise oral administration. [0097] Provided methods of treatment may include administering a disclosed compound once, twice, or three times daily; about every other day (e.g., every 2 days); twice weekly (e.g., every 3 days, every 4 days, every 5 days, every 6 days, or e.g., administered with an interval of about 2 to about 3 days between doses); once weekly; three times weekly; every other week; twice monthly; once a month; every other month; or even less often. [0098] In particular, in certain embodiments, the present disclosure provides a method of treating one or more of the above medical indications comprising administering to a subject in need thereof a therapeutically effective amount of a compound described herein. [0099] In certain embodiments, the compound utilized by one or more of the methods disclosed herein is one of the generic, subgeneric, or specific compounds described herein. [00100] The compounds of the present disclosure may be administered to patients (animals and humans) in need of such treatment in dosages that will provide optimal pharmaceutical efficacy. It will be appreciated that the dose required for use in any particular application will vary from patient to patient, not only with the particular compound or composition selected, but also with the route of administration, the nature of the condition being treated, the age and condition of the patient, concurrent medication or special diets then being followed by the patient, and other factors which those skilled in the art will recognize, with the appropriate dosage ultimately being at the discretion of the attendant physician. For treating clinical conditions and diseases noted herein, a compound of the present disclosure may be administered orally, subcutaneously, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. Parenteral administration may include subcutaneous injections, intravenous or intramuscular injections or infusion techniques. [00101] Treatment can be continued for as long or as short a period as desired. A suitable treatment period can be, for example, at least about one week, at least about two weeks, at least about one month, at least about six months, at least about 1 year, or indefinitely. A treatment period can terminate when a desired result is achieved. Pharmaceutical Compositions and Kits [00102] Another aspect of the present disclosure provides pharmaceutical compositions comprising compounds as disclosed herein formulated together with a pharmaceutically acceptable carrier. In particular, the present disclosure provides pharmaceutical compositions comprising compounds as disclosed herein formulated together with one or more pharmaceutically acceptable carriers. These formulations include those suitable for oral, rectal, topical, buccal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), vaginal, or aerosol administration, although the most suitable form of administration in any given case will depend on the degree and severity of the condition being treated and on the nature of the particular compound being used. For example, disclosed compositions may be formulated as a unit dose, and/or may be formulated for oral or subcutaneous administration. [00103] For example, disclosed herein is a pharmaceutical composition comprising a therapeutic payload disclosed herein, and a pharmaceutically acceptable excipient. Also disclosed herein is a pharmaceutical composition comprising a linker-payload construct disclosed herein, and a pharmaceutically acceptable excipient. Further disclosed herein is a pharmaceutical composition comprising a drug conjugate disclosed herein, and a pharmaceutically acceptable excipient. [00104] Exemplary pharmaceutical compositions of this disclosure may be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains one or more disclosed compounds, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. The active object compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of the disease. [00105] For preparing solid compositions such as tablets, the principal active ingredient may be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a disclosed compound, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. [00106] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the subject composition is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. [00107] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. [00108] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the subject composition, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, cyclodextrins and mixtures thereof. [00109] Suspensions, in addition to the subject composition, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. [00110] Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a subject composition with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent. [00111] Dosage forms for transdermal administration of a subject composition include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. [00112] The ointments, pastes, creams and gels may contain, in addition to a subject composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. [00113] Powders and sprays may contain, in addition to a subject composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. [00114] Compositions and compounds of the present disclosure may alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A non-aqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers may be used because they minimize exposing the agent to shear, which may result in degradation of the compounds contained in the subject compositions. Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of a subject composition together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular subject composition, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions. [00115] Pharmaceutical compositions of this disclosure suitable for parenteral administration comprise a subject composition in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. [00116] Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate and cyclodextrins. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. [00117] In another aspect, the present disclosure provides enteral pharmaceutical formulations including a disclosed compound and an enteric material; and a pharmaceutically acceptable carrier or excipient thereof. Enteric materials refer to polymers that are substantially insoluble in the acidic environment of the stomach, and that are predominantly soluble in intestinal fluids at specific pHs. The small intestine is the part of the gastrointestinal tract (gut) between the stomach and the large intestine, and includes the duodenum, jejunum, and ileum. The pH of the duodenum is about 5.5, the pH of the jejunum is about 6.5 and the pH of the distal ileum is about 7.5. Accordingly, enteric materials are not soluble, for example, until a pH of about 5.0, of about 5.2, of about 5.4, of about 5.6, of about 5.8, of about 6.0, of about 6.2, of about 6.4, of about 6.6, of about 6.8, of about 7.0, of about 7.2, of about 7.4, of about 7.6, of about 7.8, of about 8.0, of about 8.2, of about 8.4, of about 8.6, of about 8.8, of about 9.0, of about 9.2, of about 9.4, of about 9.6, of about 9.8, or of about 10.0. Exemplary enteric materials include cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP), polyvinyl acetate phthalate (PVAP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), cellulose acetate trimellitate, hydroxypropyl methylcellulose succinate, cellulose acetate succinate, cellulose acetate hexahydrophthalate, cellulose propionate phthalate, cellulose acetate maleate, cellulose acetate butyrate, cellulose acetate propionate, copolymer of methylmethacrylic acid and methyl methacrylate, copolymer of methyl acrylate, methylmethacrylate and methacrylic acid, copolymer of methylvinyl ether and maleic anhydride (Gantrez ES series), ethyl methyacrylate-methylmethacrylate-chlorotrimethylammonium ethyl acrylate copolymer, natural resins such as zein, shellac and copal collophorium, and several commercially available enteric dispersion systems (e. g. , Eudragit L30D55, Eudragit FS30D, Eudragit L100, Eudragit S100, Kollicoat EMM30D, Estacryl 30D, Coateric, and Aquateric). The solubility of each of the above materials is either known or is readily determinable in vitro. The foregoing is a list of possible materials, but one of skill in the art with the benefit of the disclosure would recognize that it is not comprehensive and that there are other enteric materials that would meet the objectives of the present invention. [00118] Advantageously, the present disclosure also provides kits for use by e.g., a consumer in need of treatment of cancer. Such kits include a suitable dosage form such as those described herein and instructions describing the method of using such dosage form to mediate, reduce or prevent inflammation. The instructions would direct the consumer or medical personnel to administer the dosage form according to administration modes known to those skilled in the art. Such kits could advantageously be packaged and sold in single or multiple kit units. An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process recesses are formed in the plastic foil. The recesses have the size and shape of the tablets or capsules to be packed. Next, the tablets or capsules are placed in the recesses and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are sealed in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening. [00119] It may be desirable to provide a memory aid on the kit, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the tablets or capsules so specified should be ingested. Another example of such a memory aid is a calendar printed on the card, e.g., as follows “First Week, Monday, Tuesday, . .. etc.... Second Week, Monday, Tuesday, ...” etc. Other variations of memory aids will be readily apparent. A “daily dose” can be a single tablet or capsule or several pills or capsules to be taken on a given day. Also, a daily dose of a first compound can consist of one tablet or capsule while a daily dose of the second compound can consist of several tablets or capsules and vice versa. The memory aid should reflect this. EXAMPLES [00120] The compounds described herein can be prepared in a number of ways based on the teachings contained herein and synthetic procedures known in the art. In the description of the synthetic methods described below, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be chosen to be the conditions standard for that reaction, unless otherwise indicated. It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule should be compatible with the reagents and reactions proposed. Substituents not compatible with the reaction conditions will be apparent to one skilled in the art, and alternate methods are therefore indicated. The starting materials for the examples are either commercially available or are readily prepared by standard methods from known materials. At least some of the compounds identified as “Intermediates” herein are contemplated as compounds of the present disclosure. [00121] Unless stated otherwise, all reactions were performed in a heat gun dried glassware under argon atmosphere, using standard septa techniques. All commercially available starting building blocks were purchased from commercial vendors. Example 1: General Methods [00122] Reactions were monitored by HPLC-MS analyses using a Shimadzu UFLC- MS-2020 system with ESI, and/or by thin-layer chromatography (TLC) using silica gel 60 F254 plates (Merck) and visualized by UV at 254 nm. Purifications were performed using automated flash chromatography system (ECOM), using prepacked column containing C18 or modified C18 silica gel (Interchim, PT-15C18AQ, 15 µm Puriflash 200, 5g, 12g, or 25g). Semipreparative HPLC were performed on ECOM HPLC system, using a modified C18 semipreparative column (YMC-Actus, Triart Prep C18, 250x20 mm, S-10 µm, 12nm). HPLC-MS analyses were performed on Shimadzu UFLC-MS-2020 system with ESI. Column: Acquity UPLC BEH C18 1.7 µm, 2.1 x 50 mm. Solvent A: H2O 0.1 % HCOOH; Solvent B: MeCN + 0.1 % HCOOH. Total flow 0.6 ml/min. Total time of the method 10 min. Mass spectrum was recorded in range 100-3000 m/z both in positive and negative mode with event time 0.2 s. UV-Vis spectra were recorded with a Shimadzu SPD-M2OA Prominence diode array detector, in the range 200-800 nm. NMR spectra were recorded using > 99% deuterated solvents, on a 400 MHz Bruker AVANCE III spectrometer (1H at 400 MHz) and/or on a Bruker AVANCE 500 (1H at 500.0 MHz). Chemical shifts (in ppm, δ scale) were solvent signal in 1H spectra. Intermediates and final products were freeze-dried using a Gregory instruments lyophilizer (model L4-110), from water or water mixtures of dioxane or acetonitrile. Abbreviations:
Figure imgf000065_0001
Figure imgf000066_0001
Monoclonal antibody gene synthesis, antibody expression and purification [00123] Anti-HER2 antibody amino acid sequence was based on heavy and light chain variable domain trastuzumab amino acid sequence KEGG database entry D03257, anti-EGFR antibody was based on heavy and light chain variable domain panitumumab amino acid sequence KEGG database entry D05350, cetuximab amino acid sequence KEGG database entry D03255, nimotuzumab amino acid sequence DrugBank accession number DB06192, or matuzumab amino acid sequence DrugBank accession number DB06192. Anti-HER2 light chain, SEQ ID NO: 1 [00124] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-HER2 IgG1 heavy chain, SEQ ID NO: 2 [00125] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYA MDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Anti-EGFR IgG11 light chain, SEQ ID NO: 3 [00126] DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-EGFR IgG11 heavy chain, SEQ ID NO: 4 [00127] QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEW IGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWG QGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Anti-EGFR IgG12 heavy chain, SEQ ID NO: 5 [00128] QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWI GGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGR GFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Anti-EGFR IgG12 light chain, SEQ ID NO: 6 [00129] DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAP KLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQ ITREVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-EGFR IgG13 heavy chain, SEQ ID NO: 7 [00130] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLE WIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYA GRYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Anti-EGFR IgG13 light chain, SEQ ID NO: 8 [00131] DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYD TSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-EGFR IgG14 heavy chain, SEQ ID NO: 9 [00132] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLE WIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYD GRYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Anti-EGFR IgG14 light chain, SEQ ID NO: 10 [00133] DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYD TSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-EGFR IgG15 heavy chain, SEQ ID NO: 11 [00134] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLG VIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAY WGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Anti-EGFR IgG15 light chain, SEQ ID NO: 12 [00135] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-EGFR IgG16 heavy chain (variant of anti-EGFR IgG15 heavy chain), SEQ ID NO: 13 [00136] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLG VIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSEDTAIYYCARALTYYDYEFAY WGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Anti-EGFR IgG16 light chain, variant, SEQ ID NO: 14 DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFS GSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Isotype antibody light chain, SEQ ID NO: 15 [00137] DIVLTQSPATLSVTPGNSVSLSCRASQSIGNDLHWYQQKSHESPRLLIKY ASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Isotype antibody human IgG1 heavy chain, SEQ ID NO: 16 [00138] DVQLQESGPSLVKPSQTLSLTCSVTGDSITSDYWSWIRKFPGNRLEYMG YVSYSGSTYYNPSLKSRISITRDTSKNQYYLDLNSVTTEDTATYYCANWDGDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Anti-TROP2 light chain, SEQ ID NO: 17 [00139] DIQMTQSPSSLSASVGDRVTITCQASQDVSIAVAWYQQKPGKAPKLLIYS ASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYITPLTFGGGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-TROP2 IgG1 heavy chain, SEQ ID NO: 18 [00140] QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEW MGWINTYTGEPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGGYGSSY WYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG [00141] Anti-HER2, anti-TROP2, anti-EGFR and IgG1 isotype control light chain and heavy chain protein sequences were generated by gene synthesis and codon optimized for expression in mammalian cells (GeneUniversal). The heavy and light chain genes were cloned into separate mammalian expression vectors and then transiently co-transfected in EXPI293F cells (ThermoFisher). Antibody expression was carried out in EXPI293F expression medium (ThermoFisher) and supernatant harvested 5 days post transfection. Respective antibodies were purified using MABSELECT Sure Resin (Cytiva) and buffer exchanged into 1x phosphate buffer saline pH 7.4. Analytical size-exclusion chromatography (Cytiva, Superdex 20010/300) data indicated that respective antibodies were approximately 99% monomer. Generation of antibody drug conjugate [00142] To generate an antibody drug conjugate where linker-drug is conjugated to heavy-heavy and heavy-light interchain cysteines of the antibody, the anti-HER2, anti-TROP2 and anti-EGFR antibody-drug conjugates were produced by reacting a linker-drug compound as described herein with an anti-HER2, anti-TROP2 or anti-EGFR antibody and forming a thioether bond at cysteine residues forming disulfide bond sites present between light and heavy chain antibody subunits and heavy and heavy chain antibody subunits respectively. Deruxtecan (CAS No: 1599440-13-7) was obtained from ChemScene, Inc. Procedure A: [00143] 3.3 mg/ml of anti-HER2 IgG1 monoclonal antibody (extinction coefficient at 280 nm, 215380 M-1cm-1) in 1x PBS pH 7.4, 10% sucrose, 5 mM EDTA, was partially reduced by adding 40 molar equivalents of TCEP solution (BondBreaker, ThermoFisher) relative to monoclonal antibody for 60 min at 20°C. Respective linker-drug lyophilized powder was dissolved in 100% DMSO (weight per volume) and added to the reduced antibody reaction mixture at 2-37 molar equivalents relative to monoclonal antibody as 5-8 % v/v solution of DMSO, and the reaction solution was nutated for 2 hours at 20°C. Afterwards, 20 mM water solution of N-acetylcysteine (SigmaAldrich) was added at 9 molar equivalent relative to antibody and reaction mixture incubated for 20 min at 20°C. Procedure B: [00144] 3.8 mg/ml of anti-TROP2 IgG1 monoclonal antibody (extinction coefficient at 280 nm, 203460 M-1cm-1) in 1x PBS pH 7.4, 10% sucrose, 5 mM EDTA, was partially reduced by adding 40 molar equivalents of TCEP (ThermoFisher) relative to monoclonal antibody for 60 min at 20°C. Respective linker-drug lyophilized powder was dissolved in 100% DMSO (weight per volume) and added to the reduced antibody reaction mixture at 6-37 molar equivalents relative to monoclonal antibody as 5-8 % v/v solution of DMSO, and the reaction solution was nutated for 2 hr at 20°C. Afterwards, 20 mM water solution of N-acetylcysteine (SigmaAldrich) was added at 9 molar equivalent relative to antibody and reaction mixture incubated for 20 min at 20°C. Procedure C: [00145] 2.4 to 10 mg/ml of respective anti-EGFR IgG1 monoclonal antibody 1, 2, 3, 4, or 5 in 1x PBS pH 7.4, 10% sucrose, 5 mM EDTA, was partially reduced by adding 40 molar equivalents of TCEP (ThermoFisher) relative to monoclonal antibody for 60 min at 20°C. Respective linker-drug lyophilized powder was dissolved in 100% DMSO (weight per volume) and added to the reduced antibody reaction mixture at 2-37 molar equivalents relative to monoclonal antibody as 5-8 % v/v solution of DMSO, and the reaction solution was nutated for 2 hr at 20°C. Afterwards, N-acetylcysteine (SigmaAldrich) was added at 1 molar equivalent relative to linker-payload and reaction mixture incubated for 20 min at 20°C. Anti-EGFR IgG1 monoclonal antibody 1 extinction coefficient at 280 nm, 232340 M-1cm-1. Anti-EGFR IgG1 monoclonal antibody 2 extinction coefficient at 280 nm, 217440 M-1cm-1. Anti-EGFR IgG1 monoclonal antibody 3 extinction coefficient at 280 nm, 227360 M-1cm-1. Anti-EGFR IgG1 monoclonal antibody 4 extinction coefficient at 280 nm, 227360 M-1cm-1. Anti-EGFR IgG1 monoclonal antibody 5 extinction coefficient at 280 nm, 217440 M-1cm-1. Removal of excess linker-drug from antibody-drug conjugate [00146] Excess quenched linker-drug was separated from respective antibody-drug conjugates by cation-exchange chromatography (Cytiva, HiTrap SP HP resin), composition of buffer A was 10% sucrose, 12.5 mM sodium acetate pH 5.0, 20 mM NaCl, and buffer B was 10% sucrose, 12.5 mM sodium acetate pH 5.0, 1M NaCl. Antibody drug conjugate and unconjugated linker-drug mixture in 1x PBS pH 7.4 were diluted in 10% sucrose, 12.5 mM Na- acetate pH 5.0, 0 mM NaCl buffer and loaded onto HiTrap SP HP resin column (Cytiva). Respective non-conjugated linker-drugs did not bind to the column, while respective antibody- drug conjugates bound to the column and were eluted in 10% sucrose, 100-150 mM NaCl, 12.5 mM Na-acetate pH 5.0 buffer. Analytical size-exclusion chromatography (Cytiva, Superdex 200 10/300 in 1x PBS, pH 7.4 buffer or Agilent AdvanceBio SEC 300Å, 4.6 x 150mm, 2.7 micron particle-size column, in 1x PBS pH 7.4, 10% isopropanol buffer) showed that antibody drug conjugates were >90 % monomeric. Determination of drug-linker to antibody ratio [00147] Drug-linker to antibody ratio of antibody drug conjugates was determined using reverse phase liquid chromatography mass spectrometry (RPLC-MS) of antibody’s heavy and light chain subunit species using the approach known in the art (Zhu X. et al.2020. J Pharm Anal.10(3): 209–220; Firth D. et al.2015 Analytical Biochemistry, 485, 34-42). Briefly, antibody drug conjugates were deglycosylated using PNGase F treatment (New England Biolabs Rapid PNGAse) and cysteine disulfide bridges reduced to separate heavy and light chain subunits and sample analyzed by reverse phase liquid chromatography coupled to mass spectrometry (RPLC-MS). Following reduction of cysteine disulfide bridges and removal of N- linked glycans, the heavy and light chains eluted in distinct peaks. Electrospray ionization baseline subtracted spectra and deconvoluted mass spectra of RPLC eluted peaks were used to determine respective heavy and light chain masses and abundance (mass peak intensity) of antibody’s heavy and light chain subunit species conjugated to 0, 1, 2 or 3 linker-drug for heavy chain subunit (here referred to as H0, H1, H2, H3) and 0 or 1 linker-drug for light chain subunit (here referred to as L0, L1). Data was used to determine the overall average linker-drug to antibody conjugate ratio (DAR) of each antibody drug conjugate. Reverse phase liquid chromatography condition was mobile phase A was 0.05% TFA in water, mobile phase B was 0.05% trifluoro acetic acid in acetonitrile. Gradient program was 10-20% of mobile phase B in 1 min, 20-50% mobile phase B in 9 min, flow rate 0.5 mL/min, post-column split, 2.1x50 mm Halo Diphenyl 2.7 µm, 80 ^C. Mass spectrometer was Waters Xevo G2-XS QTof, with Acquity i-Class UPLC. Mass spectra data processing was performed by ProMass HR for MassLyxn software (Waters Limited). [00148] Average number of conjugated drug molecules to antibody was determined for each antibody drug conjugate using following formulas: Average linker payload conjugated to light chain = (mass peak intensity of L0 x 0/ (1 x mass peak intensity of L1 + 0 x mass peak intensity of L0)) + (mass peak intensity of L1 x 1/ (1 x mass peak intensity of L1 + 0 x mass peak intensity of L0)). Average linker payload conjugated to heavy chain = (mass peak intensity of H0 x 0/ (0 x mass peak intensity of H0 + 1 x mass peak intensity of H1 + 2 x mass peak intensity of H2 + 3 x mass peak intensity of H3)) + (mass peak intensity of H1 x 1/ (0 x mass peak intensity of H0 + 1 x mass peak intensity of H1 + 2 x mass peak intensity of H2 + 3 x mass peak intensity of H3)) + (mass peak intensity of H2 x 2/ (0 x mass peak intensity of H0 + 1 x mass peak intensity of H1 + 2 x mass peak intensity of H2 + 3 x mass peak intensity of H3)) + (mass peak intensity of H3 x 3/ (0 x mass peak intensity of H0 + 1 x mass peak intensity of H1 + 2 x mass peak intensity of H2 + 3 x mass peak intensity of H3)). Average number of conjugated drug molecules to antibody composed of two light chain and two heavy chain subunits = (2x average linker payload conjugated to light chain + 2x average linker payload conjugated to heavy chain). [00149] Observed mass of anti-TROP2 antibody heavy chain without linker-drug (H0) was 49208.8 Da (daltons) and observed mass of light chain without linker-drug (L0) was 23343.8 Da. Observed mass of anti-HER2 antibody heavy chain without linker-drug (H0) was 49123.9 Da and observed light chain without linker-drug (L0) was 23456.2 (Da). Observed mass of anti- EGFR antibody 1 heavy chain without linker-drug (H0) was 48897.5 (Da) and observed light chain without linker-drug (L0) was 23357.3 (Da). Observed mass of anti-EGFR antibody 3 heavy chain without linker-drug (H0) was 49508 (Da) and observed light chain without linker- drug (L0) was 23317 (Da). Observed mass of anti-EGFR antibody 4 heavy chain without linker- drug (H0) was 49552 (Da) and observed light chain without linker-drug (L0) was 23317 (Da).Observed respective masses of heavy chain with one linker-drug (H1) was (H0 + 1x molecular weight of linker-drug), heavy chain with two linker-drug (H2) was (H0 + 2x molecular weight of linker-drug), heavy chain with three linker-drug (H3) was (H0 + 3x molecular weight of linker-drug). Observed respective masses of light chain with one linker-drug (L1) was (L0 + 1x molecular weight of linker-drug), where respective molecular weights of linker-drugs are provided in the synthetic procedures. [00150] In addition, DAR was determined by comparison of ADC conjugated to the same linker-drug using reduced reverse phase high performance liquid chromatography (RP- HPLC). Parent (unconjugated) antibodies in PBS (pH 7.4) were prepared with sample concentration of 1 mg/mL. ADC samples (in original sample buffer of 10 mM sodium acetate (pH 5.0), 130 mM NaCl, and 10% (wt./vol.) sucrose) were prepared with 1 mg/mL concentration (w.r.t. protein) and a volume of 0.1 M Tris (pH 8.5) was added to a final concentration of 20 mM Tris to help raise the pH. Dithiothreitol (DTT, No-Weigh™ Format ThermoScientific) was added to antibody and ADC samples to a concentration of 50 mM, and samples were incubated at 40 °C for 5 minutes. An equal volume of 0.1% (vol./vol.) trifluoroacetic acid (TFA, FisherChemical) in water was immediately added to each antibody and ADC sample following reduction with DTT. Denaturing reversed phase chromatography was performed on an Agilent 1260 HPLC system with thermostatted (10 °C) autosampler. Samples (5 ^g of total protein loaded) were analyzed with an Agilent AdvanceBio RP-mAb Diphenyl 2.1 ൈ 50 mm column (3.5 ^m particles with 450 Å superficial pores) using 0.5 mL/min. flow rate. Gradient elution was performed at 80 ˚C with 30–70% mobile phase ‘B’ (0.1% (vol./vol.) TFA in Acetonitrile) over 10 minutes, and mobile phase ‘A’ composed of 0.1% (vol./vol.) TFA in water. UV detection was monitored at 220, 280, and 370 nm, with baseline subtraction of the signal at 405 nm (for 220 and 280 nm) or 600 nm (for the 370 nm detection signal). As is known in the art, retention times depend on the hydrophobicity of the compound. Compared to heavy chain without linker-drug (H0) heavy chain conjugated to one linker-drug (H1), two linker-drugs (H2) and three linker-drugs (H3) were progressively more hydrophobic. Compared to light chain without linker-drug (L0), light chain conjugated to one linker-drug (L1) was more hydrophobic. For each ADC, detected peaks in RP-HPLC chromatograms were assigned to L0, L1, H0, H1, H2, and H3 by comparison of retention times with L0 and H0 of parent monoclonal antibody, comparison of 280 nm and 370 nm detection signal (here described linker-payloads absorb at 370 nm) and comparison with known LC/MS DAR values of heavy and light chains. The heavy and light chains were eluted in following order: L0, L1, H0, H1, H2, and H3. For ADCs for which DAR was determined based on RP-HPLC procedure respective peak areas of L0, L1, H0, H1, H2, and H3 detected at 280 nm were normalized by respective extinction coefficients of each DAR species (L0, L1, H0, H1, H2, and H3) at 280 nm. As is known in the art, L0 and H0 molar extinction coefficients were estimated based on respective amino acid sequence for each monoclonal antibody (M-1cm-1). Molar extinction coefficient of linker-payloads at 280 nm were estimated using Beer’s law, 5000-6100 M-1cm-1. L1 molar extinction coefficient = (molar extinction coefficient L0 + 1x molar extinction coefficient linker-payload). H1 molar extinction coefficient = (molar extinction coefficient H0 + 1x molar extinction coefficient linker-payload), H2 molar extinction coefficient = (molar extinction coefficient H0 + 2x molar extinction coefficient linker- payload), H3 molar extinction coefficient = (molar extinction coefficient H0 + 3x molar extinction coefficient linker-payload). Average number of conjugated drug molecules to antibody was determined for each antibody drug conjugate using following formulas and peak areas normalized by respective extinction coefficients: Average linker payload conjugated to light chain = (peak area of L0 x 0/ (1 x peak area of L1 + 0 x peak area of L0)) + (peak area of L1 x 1/ (1 x peak area of L1 + 0 x peak area of L0)). Average linker payload conjugated to heavy chain = peak area of H0 x 0/ (0 x peak area of H0 + 1 x peak area of H1 + 2 x peak area of H2 + 3 x peak area of H3)) + (peak area of H1 x 1/ (0 x peak area of H0 + 1 x peak area of H1 + 2 x peak area of H2 + 3 x area intensity of H3)) + (peak area of H2 x 2/ (0 x peak area of H0 + 1 x peak area of H1 + 2 x peak area of H2 + 3 x peak area of H3)) + (peak area of H3 x 3/ (0 x peak area of H0 + 1 x peak area of H1 + 2 x peak area of H2 + 3 x peak area of H3)). Average number of conjugated drug molecules to antibody composed of two light chain and two heavy chain subunits = (2x average linker drug conjugated to light chain + 2x average linker drug conjugated to heavy chain). Example 2: Synthesis of Compound 2
Figure imgf000076_0001
[00151] Intermediate 1. A mixture of ethanolamine (23 mg, 0.4142 mmol) and dimethoxysquarate (3 equiv., 177 mg, 1.242 mmol) were suspended in 10 mL of 1M borate buffer (pH = 9), and the mixture was stirred at 55 °C for 16 hours. Two mL of DMF were added, and solvents were evaporated under reduced pressure to a final volume of approx.3 mL. The crude reaction mixture was purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in water (0 ^ 50% ACN in H2O). The desired product was recovered as a white powder, after lyophilization from water (46 mg, 65 %). MS calc. for C7H10NO4: 172.06, found: 172.25, [M+H]+. [00152] Compound 2. Exatecan mesylate (20 mg, 0.0377 mmol) and the previously synthesized intermediate 1 (1,5 equiv., 9.7 mg, 0.0564) were suspended in 5 mL of 1M borate buffer (pH = 9), and the mixture was stirred at 55 °C for 16 hours.2 mL of DMF were added, and solvents were evaporated under reduced pressure to a final volume of approx.3 mL. The crude reaction mixture was purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in water (0 ^ 50% ACN in H2O). The desired product was recovered as a white powder, after lyophilization from water (12 mg, 57 %). MS calc. for C30H28FN4O7: 575.19, found: 575.45, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 8.45 (s, 1H), 7.83 (d, J = 10.9 Hz, 1H), 7.78 (s, 1H), 7.32 (s, 1H), 5.79 (s, 1H), 5.42 (s, 2H), 5.29 (d, J = 7.5 Hz, 2H), 3.68 – 3.43 (m, 4H), 3.23 (d, J = 7.7 Hz, 2H), 2.42 (d, J = 1.9 Hz, 3H), 2.33 (td, J = 5.7, 4.8, 2.9 Hz, 1H), 1.96 – 1.78 (m, 2H), 1.76 (s, 1H), 1.26 – 1.15 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).
Example 3: Synthesis of Compound 1001
Figure imgf000078_0001
[00153] Intermediate 1. Ethanolamine (100 mg, 1.637 mmol) and dimethoxysquarate (1.2 equiv., 1.964 mmol, 279 mg) were dissolved in 10 mL of 1 M borate buffer (pH 9). The reaction mixture was stirred at room temperature for 16 h. The solvent was evaporated under reduced pressure, the resulting solid was re-dissolved in DMF and directly loaded on column. The product was purified by reverse-phase flash chromatography, using a column containing 40 g of C18, and using a gradient of ACN in water (0 ^
Figure imgf000078_0002
20% ACN in water). The desired product was recovered as a white solid, after lyophilization from water (205 mg, 73 %). MS calc. for C7H10NO4: 172.06, found: 172.17, [M + H]+. [00154] Intermediate 2. Intermediate 1 (10 mg, 0.058 mmol) and the starting peptide FmocGGFG-OAc (1 equiv., 0.058 mmol, 37 mg) were dissolved in 2 mL of anhydrous DMF under an argon atmosphere, and 100 µL of HCl (2M in Et2O) were added. The reaction mixture was stirred for 1 h at room temperature, to be then directly loaded on column. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing 25 g of diol- modified C18, and using a gradient of ACN in water (0
Figure imgf000079_0001
80% ACN in water). The desired product was recovered as a white solid, after lyophilization from water (25 mg, 58 %). MS calc. for C38H40N6NaO10: 763.27, found: 763.80, [M + Na]+. [00155] Intermediate 3. Intermediate 2 (25 mg, 0.0338 mmol) and exatecan mesylate (1.5 equiv., 0.508 mmol, 27 mg) were suspended in 4 mL of 1M borate buffer (pH 9), and the reaction mixture was stirred at 55 °C for 4 hours. DMF (2 mL) was added and the solvents were evaporated under reduced pressure until a final volume of approx.2 mL. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing diol-modified C18, and using a gradient of ACN in water (0 ^
Figure imgf000079_0002
100% ACN in water). The desired product was recovered as a white solid, after lyophilization from water - dioxane (11 mg, 28 %). MS calc. for C61H59FN9O13: 1144.42: 1144.42, found: 1144.01, [M + H]+. [00156] Intermediate 4. Intermediate 3 (11 mg, 0.0096 mmol) was dissolved in 1 mL of DMF, and morpholine (20 µL) was added. The reaction mixture was stirred at room temperature for 30 min. The mixture was filtered through a 0.2 µm syringe filter and directly loaded on column. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing diol-modified C18, and using a gradient of ACN in water (0 ^ 100% ACN in water). The desired product was recovered as a yellowish solid, after lyophilization from water - dioxane (7.5 mg, 88 %). MS calc. for C46H50FN9O11: 923.36, found: 923.75, [M + H]+. [00157] Compound 1001. Intermediate 4 (7.5 mg, 0.0081 mmol) was dissolved in 1 mL of DMF.2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethoxy)propanoate (2 equiv., 0.0163 mmol, 5 mg) and DIPEA (20 µL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The mixture was then filtered through a 0.2 µm syringe filter and directly loaded on column. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing diol-modified C18, and using a gradient of ACN in water (0 ^ 80% ACN in water). The desired product was recovered as a white solid, after lyophilization from water (7 mg, 77 %). MS calc. for C55H58FN10O15: 1117.41, found: 1117.44, [M + H]+.1H NMR (500 MHz, DMSO-d6) δ 8.80 (s, 1H), 8.62 (d, J = 10.9 Hz, 1H), 8.52 (s, 1H), 8.27 (s, 1H), 8.15 (d, J = 33.5 Hz, 1H), 8.02 (d, J = 16.0 Hz, 1H), 7.82 (d, J = 11.5 Hz, 1H), 7.67 (s, 1H), 7.36 – 7.20 (m, 4H), 7.19 – 7.12 (m, 2H), 6.68 (s, 1H), 6.05 (t, J = 20.7 Hz, 1H), 5.86 (s, 1H), 5.75 (s, 1H), 5.30 (d, J = 23.9 Hz, 1H), 5.18 (m, 2H), 4.85 (d, J = 11.1 Hz, 2H), 4.66 – 4.54 (m, 1H), 4.54 (s, 2H), 4.44 (m, 1H), 3.85 – 3.79 (m, 1H), 3.70 – 3.65 (m, 3H), 3.56 – 3.47 (m, 2H), 3.47 – 3.42 (m, 1H), 3.41 – 3.36 (m, 1H), 3.17 (s, 1H), 3.07 – 2.97 (m, 2H), 2.85 – 2.69 (m, 4H), 2.67 – 2.52 (m, 2H), 2.44 – 2.36 (m, 3H), 2.33 – 2.23 (m, 1H), 1.97 (m, 2H), 1.82 (d, J = 7.9 Hz, 2H), 1.23 (s, 2H), 0.87 – 0.81 (m, 3H). Example 4: Synthesis of Compound 3002 [00158] Compound 3002 was prepared according to Procedure A of the General Methods section in Example 1 using 13 molar equivalents of linker payload compound 1001 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 3.7 based on a molecular weight of linker-drug 1117 of Da. Example 5: Synthesis of Compounds 3028A and 3028B [00159] Compound 3028A was prepared according to Procedure B of the General Methods section in Example 1 using 21 molar equivalents of linker payload compound 1001 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 4.21 based on a molecular weight of linker-drug of 1117 Da. When prepared according to Procedure B using 39 molar equivalents of linker payload compound 1001 to anti-TROP2 IgG1 monoclonal antibody, a DAR of 7.5 was achieved to afford compound 3028B. Example 6: Synthesis of Compound 3050 [00160] Compound 3050 was prepared according to Procedure C of the General Methods section in Example 1 using 13 molar equivalents of linker payload compound 1001 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 4.5 based on a molecular weight of linker-drug of 1117 Da. Example 7: Synthesis of Compound 12
Figure imgf000081_0001
[00161] Intermediate 1. Exatecan mesylate (39 mg, 0.0737 mmol), malonic acid (5 equiv., 0.3687 mmol, 38 mg) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM, 5 equiv., 0.3687 mmol, 102 mg) were dissolved in a 5:1 mixture of DMF and water (6 mL). Triethylamine (50 equiv., 3.6873 mmol, 514 µL) was added and the reaction mixture was stirred for 3 hours at room temperature. Solvents were evaporated under reduced pressure, and the crude reaction mixture was purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in 1% TFA (0 ^ 40% ACN in 1% TFA). The desired product was recovered as a white powder, after lyophilization from water (34 mg, 88 %). MS calc. for C27H23FN3O7: 520.15, found: 520.49 [M- H]-. [00162] Intermediate 2. The previously synthesized intermediate 1 (24 mg, 0.0461 mmol), 2-((tert-butyldimethylsilyl)oxy)ethan-1-amine (5 equiv., 0.2303 mmol, 48 µL) and 4- (4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM, 5 equiv., 0.2303 mmol, 64 mg) were dissolved in a 5:1 mixture of DMF and water (6 mL). Triethylamine (50 equiv., 2.303 mmol, 321 µL) was added and the reaction mixture was stirred for 3 hours at room temperature. Solvents were evaporated under reduced pressure, and the crude reaction mixture was purified by reverse-phase flash chromatography, using a column containing 25 g of diol- modified C18, and using a gradient of ACN in water (0 ^ 70% ACN in H2O). The desired product was recovered as a yellowish foam, after lyophilization from water-DMF (7 mg, 22 %). MS calc. for C35H44FN4O7Si: 679.30, found: 679.00, [M+H]+. [00163] Compound 12. The previously synthesized intermediate 2 (7 mg, 0.0103 mmol) was suspended in 1 % TFA (2 mL) and the mixture was stirred at room temperature for 1 h. The crude reaction mixture was directly loaded on column and purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in 1% TFA (0 ^ 40% ACN in 1% TFA). The desired product was recovered as a yellowish powder, after lyophilization from water (3 mg, 53 %). MS calc. for C29H30FN4O7: 565.21, found: 565.70, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 8.6 Hz, 1H), 8.05 (t, J = 5.6 Hz, 1H), 7.80 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 5.58 – 5.54 (m, 1H), 5.43 (s, 2H), 5.26 (d, J = 5.2 Hz, 2H), 3.38 (t, J = 6.3 Hz, 2H), 3.18 (s, 2H), 3.15 – 3.04 (m, 2H), 2.58 – 2.52 (m, 2H), 2.46 (m, 3H), 2.25 – 2.16 (m, 1H), 2.11 (s, 1H), 1.87 (m, 2H), 1.76 (s, 2H), 0.88 (t, J = 7.4 Hz, 3H). Example 8: Synthesis of Compound 1005
Figure imgf000082_0001
Figure imgf000083_0001
[00164] Intermediate 1. FmocGGFG-N3 (23 mg, 0.0350 mmol) was dissolved in 2 mL of dioxane. Pd/C (10% w/w, 5 mg) was suspended in the mixture, and H2 was bubbled using a balloon into the suspension while stirring at room temperature for 2 h. The suspension was taken with a syringe and filtrate through 0.2 µm syringe filter directly into a flask containing a previously prepared solution of malonic acid (5 equiv., 0.1750 mmol, 18 mg), DMTMM (5 equiv., 0,1750 mmol, 48 mg) and DIPEA (100 µL) in ACN (2 mL) and water (0.5 mL). The reaction mixture was stirred at room temperature for 2 h. The solvents were evaporated under reduced pressure, the resulting solid was re-dissolved in DMF and directly loaded on column. The product was purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in water (0 ^ 40% ACN in water). The desired product was recovered as a white solid, after lyophilization from water (15 mg, 60 %). MS calc. for C36H41N6O10: 715.27, found: 715.55 [M - H]-. [00165] Intermediate 2. Intermediate 1 (14 mg, 0.0195 mmol) was dissolved in a mixture of DMF (2 mL) and water (0.5 mL). To the solution were added exatecan mesylate (1.5 equiv., 0.0293 mmol, 16 mg), DMTMM (3 equiv., 0.0587 mmol, 16 mg) and DIPEA (20 µL), and the reaction mixture was stirred for 4 h at room temperature. The solvents were evaporated under reduced pressure, the resulting solid was re-dissolved in DMF and directly loaded on column. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing diol-modified C18, and using a gradient of ACN in water (0 ^
Figure imgf000083_0002
100% ACN in water). The desired product was recovered as a yellow solid, after lyophilization from water (15 mg, 66 %). MS calc. for C60H61FN9O13: 1134.44, found: 1134.40 [M + H]+. [00166] Intermediate 3. Intermediate 2 (15 mg, 0.0132 mmol) was dissolved in 1 mL of DMF, and morpholine (20 µL) was added. The reaction mixture was stirred at room temperature for 30 min. The mixture was filtered through a 0.2 µm syringe filter and directly loaded on column. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing diol-modified C18, and using a gradient of ACN in water (0 ^ 100% ACN in water). The desired product was recovered as a yellow solid, after lyophilization from water - dioxane (10 mg, 83 %). MS calc. for C45H51FN9O11: 912.37, found: 912.91 [M + H]+. [00167] Compound 1005. Intermediate 3 (10 mg, 0.0110 mmol) was dissolved in 1 mL of DMF.2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethoxy)propanoate (2 equiv., 0.0219 mmol, 7 mg) and DIPEA (20 µL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The mixture was then filtered through a 0.2 µm syringe filter and directly loaded on column. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing diol-modified C18, and using a gradient of ACN in water (0 ^
Figure imgf000084_0001
80% ACN in water). The desired product was recovered as a yellow solid, after lyophilization from water - dioxane (11 mg, 90 %). MS calc. for C54H60FN10O15: 1107.42, found: 1107.50 [M + H]+.1H NMR (500 MHz, DMSO-d6) δ 8.67 (m, 1H), 8.61 (m,1H), 8.52 (m, 1H), 8.38 (s, 1H), 8.24 (s, 1H), 8.12 (t, J = 5.6 Hz, 1H), 7.81 (d, J = 11.0 Hz, 1H), 7.67 (s, 1H), 7.28 (m, 4H), 7.25 – 7.24 (m, 2H), 7.212 (s, 1H), 7.15 (m, 1H), 6.68 (s, 1H), 6.09 – 6.00 (m, 1H), 5.58 – 5.47 (m, 1H), 5.19 – 5.08 (m, 2H), 4.88 (d, J = 11.8 Hz, 1H), 4.65 (d, J = 11.7 Hz, 1H), 4.50 (dd, J = 11.7, 6.6 Hz, 1H), 4.47 (m, 2H), 3.78 – 3.63 (m, 3H), 3.63 – 3.52 (m, 4H), 3.19 (m, 3H), 3.11 – 3.01 (m, 4H), 2.63 (p, J = 1.9 Hz, 5H), 2.43 – 2.36 (m, 6H), 2.24 – 2.14 (m, 1H), 1.75 (m, 2H), 1.23 (s, 2H), 0.85 (dd, J = 7.9, 6.3 Hz, 3H). Example 9: Synthesis of compound 3003 [00168] Compound 3003 was prepared according to Procedure A of the General Methods section in Example 1 using 2 molar equivalents of linker payload compound 1005 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 2.1 based on a molecular weight of linker-drug 1107 of Da. Example 10: Synthesis of Compounds 3029A and 3029B [00169] Compound 3029A was prepared according to Procedure B of the General Methods section in Example 1 using 2 molar equivalents of linker payload compound 1005 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 1.2 based on a molecular weight of linker-drug of 1107 Da. When 27 molar equivalents of linker payload compound 1005 to anti-TROP2 IgG1 monoclonal antibody was used, a DAR of 4.4 was achieved to provide compound 3029B. Example 11: Synthesis of Compounds 3051A and 3051B [00170] Compound 3051A was prepared according to Procedure C of the General Methods section in Example 1 using 2 molar equivalents of linker payload compound 1005 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 1.9 based on a molecular weight of linker-drug of 1107 Da. When 27 molar equivalents of linker payload compound 1005 to anti-EGFR IgG1 monoclonal antibody 1 was used, a DAR of 3.14 was achieved to provide compound 3051B. Example 12: Synthesis of Compound 16
Figure imgf000085_0001
[00171] Intermediate 1. Exatecan mesylate (20 mg, 0.0376 mmol), succinic acid (5 equiv., 0.1881 mmol, 22 mg) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM, 5 equiv., 0.1881 mmol, 53 mg) were dissolved in a 5:1 mixture of DMF and water (4 mL). Triethylamine (200 µL) was added and the reaction mixture was stirred for 3 hours at room temperature. Solvents were evaporated under reduced pressure, and the crude reaction mixture was purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in 1% TFA (0
Figure imgf000086_0001
40% ACN in 1% TFA). The desired product was recovered as a brown powder, after lyophilization from water-dioxane (18 mg, 89 %). MS calc. for C28H25FN3O7: 534.17, found: 534.80 [M-H]-. [00172] Intermediate 2. The previously synthesized intermediate 1 (15 mg, 0.0424 mmol), 2-((tert-butyldimethylsilyl)oxy)ethan-1-amine (3 equiv., 0.1272 mmol, 22 mg) and hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU, 3 equiv., 0.1272 mmol, 48 mg) were dissolved in DMF (1 mL). Diisopropylethylamine (50 µL) was added and the reaction mixture was stirred for 1 hour at room temperature. Solvents were evaporated under reduced pressure, and the crude reaction mixture was purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in water (0 ^ 80% ACN in H2O). The desired product was recovered as a yellow foam, after lyophilization from water-dioxane (22 mg, 74 %). MS calc. for C36H46FN4O7Si: 693.31, found: 693.55, [M+H]+. [00173] Compound 16. The previously synthesized intermediate 2 (22 mg, 0.0317 mmol) was suspended in 1% TFA (2 mL) and the mixture was stirred at room temperature for 1 h. The crude reaction mixture was directly loaded on column and purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in 1% TFA (0 ^ 40% ACN in 1% TFA). The desired product was recovered as a white powder, after lyophilization from water (11 mg, 60 %). MS calc. for C30H32FN4O7: 579.23, found: 579.25, [M+H]+.1
Figure imgf000086_0002
NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 8.7 Hz, 1H), 7.82 (m, 1H), 7.79 (d, J = 11.0 Hz, 1H), 7.31 (s, 1H), 5.56 (t, J = 4.8 Hz, 1H), 5.42 (s, 2H), 5.30 – 5.10 (m, 2H), 3.47 (m, 2H), 3.35 (t, J = 6.2 Hz, 2H), 3.18 (m, 2H), 3.07 (td, J = 6.1, 4.4 Hz, 2H), 2.56 – 2.52 (m, 2H), 2.42 – 2.32 (m, 5H), 2.12 (q, J = 5.3 Hz, 2H), 1.95 – 1.79 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). Example 13: Synthesis of Compound 1023
Figure imgf000087_0001
[00174] Intermediate 1. To the solution of FmocGGFG-N3 (1.0 equiv., 26 mg, 0.040 mmol) in dioxane (1.5 ml) was added Pd/C (10% w/w, 5 mg) and the resulting suspension was hydrogenated reaction mixture was hydrogenated (balloon) for 1.5 hour at room temperature. LC-MS analysis confirmed the full consumption of the starting material. The suspension was filtered and intermediate 1 was obtained as a solution in dioxane, which was used directly into next step. MS calc. for C33H39N6O7: 631.29, found: 631.30, [M+H]+. [00175] Intermediate 2. To the mixture of exatecan mesylate (1.0 equiv., 20 mg, 0.037 mmol) and succinic anhydride (1.1 equiv., 4.1 mg, 0.041 mmol) was added DMF (0.5 mL) and diisopropylethylamine (2.2 equiv., 14 µL, 0.082 mmol) and the mixture was stirred for 30 minutes at room temperature. Then, the solution of intermediate 1 in dioxane (obtained in the previous step) was added to the reaction mixture, followed by the addition of DMTMM (1.0 equiv., 11 mg, 0.037 mmol), diisopropylethylamine (1.0 equiv., 7 µL, 0.037 mmol) and water (0.25 mL), and the resulting solution was stirred 1 hour at room temperature. The mixture was concentrated on rotary evaporator and the residue was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 60% ACN/0.1% aq. TFA) to obtain the product intermediate 2 (15 mg, 35 %) as a pale-yellow solid after lyophilization. MS calc. for C61H63FN9O13: 1148.45, found: 1148.45, [M+H]+. [00176] Compound 1023. Morpholine (35 µL) was added to the solution of intermediate 2 (1.0 equiv., 15 mg, 0.013 mmol) in DMF (1 mL) and the reaction mixture was stirred for 1.5 hour at room temperature. Then, DMF and excess morpholine were evaporated using rotary evaporator. The residue was re-dissolved in DMF (0.5 mL), followed by the addition of Mal-PEG-NHS ester (1.1 equiv., 4.4 mg, 0.014 mmol) and diisopropylethylamine (1.1 equiv., 2.5 µL, 0.014 mmol), and the resulting solution was stirred for 1 hour at room temperature. Purification by reverse-phase flash chromatography using a semipreparative column (diol-modified C18, 0 ^ 70% ACN/0.1% aq. TFA) offered the desired product compound 1023 (7 mg, 48 %) as a pale-yellow solid after lyophilization. MS calc. for C52H62FN10O15: 1121.44, found: 1121.45, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ: 8.53 – 8.42 (m, 2H), 8.29 (t, J = 5.9 Hz, 1H), 8.14 – 8.07 (m, 2H), 7.99 (t, J = 5.8 Hz, 1H), 7.89 (t, J = 5.2 Hz, 1H), 7.79 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 7.27 – 7.20 (m, 4H), 7.20 – 7.12 (m, 1H), 6.99 (s, 2H), 5.55 (dt, J = 9.2, 4.8 Hz, 1H), 5.47 – 5.37 (m, 2H), 5.28 – 5.08 (m, 2H), 4.57 – 4.44 (m, 4H), 3.80 – 3.62 (m, 6H), 3.53 – 3.40 (m, 4H), 3.35 (t, J = 5.9 Hz, 2H), 3.16 (dq, J = 17.4, 5.8 Hz, 4H), 3.04 (dd, J = 13.9, 4.7 Hz, 1H), 2.87 – 2.73 (m, 2H), 2.43 – 2.35 (m, 7H), 2.32 (t, J = 6.5 Hz, 2H), 2.21 – 2.02 (m, 2H), 1.93 – 1.78 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). Example 14: Synthesis of Compound 3004 [00177] Compound 3004 was prepared according to Procedure A of the General Methods section in Example 1 using 17 molar equivalents of linker payload compound 1023 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 4.4 based on a molecular weight of linker-drug 1121 of Da. Example 15: Synthesis of Compound 3030 [00178] Compound 3030 was prepared according to Procedure B of the General Methods section in Example 1 using 17 molar equivalents of linker payload compound 1023 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 6.6 based on a molecular weight of linker-drug of 1121 Da. Example 16: Synthesis of Compound 3052 [00179] Compound 3052 was prepared according to Procedure C of the General Methods section in Example 1 using 17 molar equivalents of linker payload compound 1023 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 6.6 based on a molecular weight of linker-drug of 1121 Da. Example 17: Synthesis of Compound 22
Figure imgf000089_0001
[00180] Intermediate 1. 2-[[Tert-Butyl(dimethyl)silyl]oxy]ethanol (1.14 mmol, 200 mg) was dissolved in 4 mL of anhydrous dichloromethane under an argon atmosphere. The reaction mixture was cooled at 0 °C, and diisopropylethylamine (1.1 equiv., 1.25 mmol, 218 µL) was added, followed by triphosgene (1.2 equiv., 0.45 mmol, 135 mg). The reaction mixture was stirred at 0 °C for 2 h. Further diisopropylethylamine (1.2 equiv., 1.25 mmol, 218 µL) was added, followed by 2-mercaptopyridine (1.1 equiv., 1.25 mmol, 155 mg). After stirring for 2 more hours at 0 °C, the reaction mixture was diluted with 20 mL of dichloromethane and saturated NH4Cl (10 mL) was added. The organic phase was washed with saturated NH4Cl (2 x 100 mL) and brine (100 mL). Silica gel flash chromatography afforded, using a gradient of EtOAc in cyclohexane (0 ^
Figure imgf000089_0002
50% EtOAc in cyclohexane) afforded the desired product (100 mg, 28%). MS calc. for C14H24NO3SSi: 314.12, found: 314.10, [M+H]+. [00181] Compound 22. Exatecan mesylate (0.0376 mmol, 20 mg) and triethylamine (2 equiv., 0.0752 mmol, 11 µL) were dissolved in 2 mL of anhydrous DMF under an argon atmosphere. The intermediate 1 (1.5 equiv., 0.0752 mmol, 18 mg) was added, and the reaction mixture was stirred at room temperature for 48 h. Solvents were evaporated under reduced pressure, and the crude reaction mixture was purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in water (0 ^ 40% ACN in H2O). Fractions containing the desired reaction product (intermediate 2) were evaporated, the solid was re-suspended in 1% TFA and stirred at room temperature for 1 h. The desired product Sc-122 was re-purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in 1% TFA (0 ^ 40% ACN in 1% TFA), and recovered as a white powder, after lyophilization from water (16 mg, 81 % over 2 steps, calculated on Exatecan). MS calc. for C27H27FN3O7: 524.18, found: 524.20, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 7.97 (d, J = 9.0 Hz, 1H), 7.77 (d, J = 11.0 Hz, 1H), 7.32 (s, 1H), 5.43 (s, 2H), 5.32 – 5.16 (m, 2H), 4.11 (m, 2H), 3.33 (s, 1H), 3.25 (d, J = 6.0 Hz, 1H), 3.11 (d, J = 18.7 Hz, 1H), 2.68 (m, 1H), 2.55 (m, 2H), 2.39 – 2.29 (m, 4H), 2.24 – 2.12 (m, 2H), 1.87 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).
Example 18: Synthesis of Compound 1007
Figure imgf000091_0001
[00182] Intermediate 1. Compound 22 (28 mg, 0.0535 mmol) and FmocGGFG-OAc (2 equiv., 0.107 mmol, 67 mg) were dissolved in 1 mL of anhydrous DMF. HCl (100 µL, 2 M in Et2O) was added and the reaction mixture was stirred at room temperature for 2 h. The mixture was directly loaded on column. The product was purified by reverse-phase flash chromatography, using a column containing 25 g of C18, and using a gradient of ACN in water ^ 70% ACN in water). The desired product was recovered as a white solid, after lyophilization from water (23 mg, 39 %). MS calc. for C58H58FN8O13: 1093.41, found: 1093.63, [M + H]+. [00183] Intermediate 2. Intermediate 1 (23 mg, 0.0211 mmol) was dissolved in 1 mL of anhydrous DMF, and morpholine (100 µL) was added. The reaction mixture was stirred for 1 h at room temperature, to be then directly loaded on column. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing 25 g of diol-modified C18, and using a gradient of ACN in water (0 ^
Figure imgf000092_0001
100% ACN in water). The desired product was recovered as a white solid, after lyophilization from water (16 mg, 85 %). MS calc. for C43H48FN8O11: 871.34, found: 871.44, [M + H]+. [00184] Compound 1007. Intermediate 2 (16 mg, 0.0179 mmol) was dissolved in 1 mL of DMF.2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethoxy)propanoate (2 equiv., 0.0358 mmol, 11 mg) and DIPEA (20 µL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The mixture was then filtered through a 0.2 µm syringe filter and directly loaded on column. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing diol-modified C18, and using a gradient of ACN in water (0 ^ 100% ACN in water). The desired product was recovered as a white solid, after lyophilization from water (7 mg, 37 %). MS calc. for C52H57FN9O15: 1066.40, found: 1065.98, [M + H]+. 1H NMR (500 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.65 (d, J = 6.9 Hz, 1H), 8.58 – 8.49 (m, 1H), 8.45 (m, 1H), 8.37 (s, 1H), 8.31 – 8.22 (m, 1H), 8.15 – 8.08 (m, 2H), 8.07 – 7.96 (m, 1H), 7.91 (s, 1H), 7.79 (t, J = 11.5 Hz, 2H), 7.34 – 7.28 (m, 1H), 7.25 – 7.17 (m, 3H), 7.01 (d, J = 10.9 Hz, 2H), 6.67 (s, 1H), 6.61 – 6.46 (m, 1H), 5.42 (s, 1H), 5.39 – 5.28 (m, 1H), 5.26 – 5.23 (m, 2H), 5.15 (d, J = 7.3 Hz, 1H), 4.57 (m, 2H), 4.49 (d, J = 11.1 Hz, 2H), 4.18 (d, J = 4.8 Hz, 2H), 3.77 – 3.63 (m, 3H), 3.61 (m, 2H), 3.58 – 3.42 (m, 3H), 3.25 – 3.17 (m, 1H), 3.04 (s, 2H), 2.77 (dd, J = 8.5, 5.5 Hz, 1H), 2.47 – 2.29 (m, 4H), 2.20 – 2.11 (m, 1H), 2.00 (m, 1H), 1.86 (m, 2H), 1.23 (s, 2H), 0.97 – 0.82 (m, 3H). Example 19: Synthesis of Compound 3005 [00185] Compound 3005 was prepared according to Procedure A of the General Methods section in Example 1 using 12 molar equivalents of linker payload compound 1007 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 4.7 based on a molecular weight of linker-drug 1066 of Da. Example 20: Synthesis of Compound 3031 [00186] Compound 3031 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1007 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 6.2 based on a molecular weight of linker-drug of 1066 Da. Example 21: Synthesis of Compounds 3053A and 3053B [00187] Compound 3053A was prepared according to Procedure C of the General Methods section in Example 1 using 12 molar equivalents of linker payload compound 1007 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 3.5 based on a molecular weight of linker-drug of 1066 Da. Molar ratios of mAb and linker-payload were adjusted to achieve DAR 7.7 (compound 3053B). Example 22: Synthesis of Compound 42
Figure imgf000093_0001
[00188] Intermediate 1. 2-((Tert-butyldimethylsilyl)oxy)ethan-1-amine (21 mg, 0.121 mmol), triphosgene (0.95 equiv., 0.0383 mmol, 11 mg) and diisopropylethylamine (5 equiv., 0.605 mmol, 105 µL) were dissolved in dichloromethane (2 mL) under an argon atmosphere. The reaction mixture was stirred at room temperature for 1 h. The full reaction conversion to afford the intermediate 1 was confirmed by LCMS analysis. The reaction product was used in the next step without any further purification. [00189] Compound 42. Exatecan mesylate (0.0602 mmol, 32 mg) and diisopropylethylamine (2 equiv., 0.120 mmol, 21 µL) were dissolved in 1 mL of anhydrous DMF, and the solution was cooled at 0 °C. A dichloromethane solution of the previously prepared isocyanate intermediate 1 (2 mL, 0.1150 mmol) was added at 0 °C, and the reaction mixture was allowed to reach room temperature and stirred for 1 h. Solvents were evaporated under reduced pressure, and the crude reaction mixture was purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in water (0 ^
Figure imgf000094_0001
60% ACN in H2O). Fractions containing the desired reaction product (intermediate 2) were evaporated, the solid was re-suspended in 1% TFA and stirred at room temperature for 1 h. The desired product was re-purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in 1% TFA (0 ^ 40% ACN in 1% TFA), and recovered as a white powder, after lyophilization from water (15 mg, 48% over 3 steps, calculated from exatecan). MS calc. for C27H28FN4O6: 523.20, found: 523.25, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 7.76 (dd, J = 11.0, 2.0 Hz, 1H), 7.31 (s, 1H), 6.82 (d, J = 8.9 Hz, 1H), 6.61 (m, 1H), 5.43 (d, J = 2.6 Hz, 2H), 5.40 – 5.30 (m, 2H), 5.22 (s, 1H), 5.17 (s, 1H), 3.48 – 3.40 (m, 2H), 3.21 – 3.01 (m, 3H), 2.38 (d, J = 1.9 Hz, 3H), 2.23 – 2.06 (m, 2H), 1.96 – 1.80 (m, 2H), 1.76 (s, 2H), 0.90 (t, J = 7.3 Hz, 3H). Example 23: Synthesis of Compound 1008
Figure imgf000094_0002
Figure imgf000095_0001
[00190] Intermediate 1. Compound 48 (17 mg, 0.0325 mmol) and FmocGGFG-OAc (3 equiv., 0.0976 mmol, 61 mg) were dissolved in 1 mL of anhydrous DMF. HCl (100 µL, 2 M in Et2O) was added and the reaction mixture was stirred at room temperature for 2 h. The mixture was directly loaded on column. The product was purified by reverse-phase flash chromatography, using a column containing 25 g of C18, and using a gradient of ACN in water ^ 80% ACN in water). The desired product was recovered as a white solid, after lyophilization from water (15 mg, 44 %). MS calc. for C58H59FN9O12: 1092.43, found: 1093.03, [M + H]+. [00191] Intermediate 2. Intermediate 1 (15 mg, 0.0135 mmol) was dissolved in 1 mL of anhydrous DMF, and morpholine (100 µL) was added. The reaction mixture was stirred for 1 h at room temperature, to be then directly loaded on column. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing 25 g of diol-modified C18, and using a gradient of ACN in water (0 ^
Figure imgf000095_0002
100% ACN in water). The desired product was recovered as a white solid, after lyophilization from water (10 mg, 87 %). MS calc. for C43H49FN9O10: 870.36, found: 870.88, [M + H]+. [00192] Compound 1008. Intermediate 2 (10 mg, 0.0118 mmol) was dissolved in 1 mL of DMF.2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethoxy)propanoate (2 equiv., 0.0237 mmol, 7 mg) and DIPEA (20 µL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The mixture was then filtered through a 0.2 µm syringe filter and directly loaded on column. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing diol-modified C18, and using a gradient of ACN in water (0
Figure imgf000096_0001
100% ACN in water). The desired product was recovered as a white solid, after lyophilization from water (4 mg, 32 %). MS calc. for C52H58FN10O14: 1065.41, found: 1065.79, [M + H]+. Example 24: Synthesis of Compound 3007 [00193] Compound 3007 was prepared according to Procedure A of the General Methods section in Example 1 using 9 molar equivalents of linker payload compound 1008 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 4.9 based on a molecular weight of linker-drug 1065 of Da. Example 25: Synthesis of Compound 3032 [00194] Compound 3032 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1008 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 6.6 based on a molecular weight of linker-drug of 1065 Da. Example 26: Synthesis of Compound 3054 [00195] Compound 3054 was prepared according to Procedure C of the General Methods section in Example 1 using 9 molar equivalents of linker payload compound 1008 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 4.4 based on a molecular weight of linker-drug of 1065 Da. Example 27: Synthesis of Compound 52
Figure imgf000097_0001
[00196] Intermediate 1. To a solution of methyl propiolate (1.0 equiv., 55 mg, 0.65 mmol), (2-azidoethoxy)(tert-butyl)dimethylsilane (1.15 equiv., 150 mg, 0.74 mmol) and tris[(1- benzyltriazol-4-yl)methyl]amine (0.15 equiv., 50 mg, 0.094 mmol) in DMF (3 ml) was added 1M aqueous CuSO4.5H2O (0.1 equiv., 0.06 mmol, 60 µL) and 2M aqueous sodium ascorbate (0.2 equiv., 0.12 mmol, 60 µL) and the resulting mixture was stirred at room temperature for 2 hours. DMF was evaporated and the residue was taken into EtOAc and the organic phase was washed with water, 0.2M aqueous HCl, saturated aqueous NH4Cl and brine, and dried by Na2SO4. Purification by flash chromatography (silica, 0% to 30% EtOAc/cyclohexane) offered the triazole intermediate 1 (158 mg, 85%) as a white solid. [00197] Intermediate 2. To a solution of triazole intermediate 1 (1.0 equiv., 158 mg, 0.55 mmol) in MeOH (2 mL) was added 2M aqueous NaOH (1.0 equiv., 0.55 mL) and the resulting mixture was stirred at room temperature overnight. Solvents were then evaporated, and the residue was re-evaporated two times with toluene, suspended in EtOAc and filtered. Solid material was washed with Et2O and dried on vacuum, giving the triazole intermediate 2 (135 mg, 84%) as a white solid. [00198] Intermediate 3. To a suspension of exatecan mesylate (15 mg, 0.028 mmol), triazole intermediate 2 (2.1 equiv., 0.06 mmol, 17 mg), N-ethyl-N′-(3- dimethylaminopropyl)carbodiimide hydrochloride (2.0 equiv., 0.056 mmol, 11 mg) and 1- hydroxybenzotriazole (2.0 equiv., 0.056 mmol, 8 mg) in DMF (1 mL) was added diisopropylethylamine (5.0 equiv., 0.14 mmol, 18 mg, 25 µL) under an argon atmosphere and the mixture was stirred at room temperature for 5 hours. LC-MS indicated the full consumption of the starting material. Purification of the mixture by reverse-phase flash chromatography (25 g, diol-modified C18, 0% to 75% ACN/H2O) offered triazole intermediate 3 (14 mg, 73%) as a white powder after lyophilisation. [00199] Compound 52. The triazole intermediate 3 (14 mg, 0.02 mmol) was dissolved in ACN/0.1% aq. TFA mixture (1:1, 2 ml), followed by the addition of 2 drops of TFA and the resulting mixture was stirred at room temperature for 2 hours. LC-MS indicated the full consumption of the intermediate 1 and solvents were evaporated under reduced pressure. The residue was purified by reverse-phase flash chromatography using semipreparative column (diol- modified C18, 0 ^ 50% ACN/H2O), giving compound 52 (10 mg, 85 %) as a white powder after lyophilisation. MS calc. for C29H28FN6O6: 575.20, found: 575.25, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ: 8.63 (s, 1H), 7.77 (d, J = 10.9 Hz, 1H), 7.29 (s, 1H), 5.73 (dd, J = 7.7, 5.3 Hz, 1H), 5.36 (s, 2H), 5.14 (d, J = 3.7 Hz, 2H), 4.48 (t, J = 5.3 Hz, 2H), 3.81 (t, J = 5.4 Hz, 2H), 3.33 (s, 2H), 3.32 – 3.21 (m, 1H), 3.19 – 3.07 (m, 1H), 2.38 (d, J = 1.9 Hz, 3H), 2.34 – 2.20 (m, 2H), 1.93 – 1.76 (m, 2H), 0.85 (t, J = 7.3 Hz, 3H). Example 28: Synthesis of Compound 1010
Figure imgf000098_0001
Figure imgf000099_0001
[00200] Intermediate 1. The mixture of compound 52 (1.0 equiv., 30 mg, 0.052 mmol) and FmocGGFG-OAc (2.0 equiv., 0.104 mmol, 66 mg) was dissolved in anhydrous DMF (1.5 mL), followed by the addition of 2M HCl/Et2O (150 µL). The reaction mixture was stirred at room temperature for 4 h and during that time, several portions (ca.0.5 equiv. each) of FmocGGFG-OAc were added to the reaction mixture. Then the reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0% to 75% ACN/H2O). Fractions containing the product were lyophilized and the residue (product + co-eluting impurities) was used directly into next step. MS calc. for C60H59FN11O12: 1144.43, found: 1144.40, [M+H]+. [00201] Intermediate 2. Morpholine (140 µL) was added to the solution of intermediate 1 (as obtained in previous step) in anhydrous DMF (2 ml) and the reaction mixture was stirred for 1 h at room temperature. LC-MS indicated the full consumption of starting material. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0% to 60% ACN/H2O), giving the product as a white solid after lyophilization (20 mg, 42%, 2 steps). MS calc. for C45H49FN11O10: 922.36, found: 922.35, [M+H]+. [00202] Compound 1010. Mal-PEG-NHS ester (1.0 equiv., 0.022 mmol, 6.7 mg) and DIPEA (2.4 equiv., 0.053 mmol, 7 mg, 9 µL) were added to the solution of intermediate 2 (1.0 equiv., 20 mg, 0.022 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 40 minutes, as LC-MS indicated the full consumption of starting material. Purification by reverse-phase flash HPLC using a semipreparative column (diol-modified C18, 0% to 60% ACN/H2O) offered the desired product as a white solid after lyophilization (7.5 mg, 31%). MS calc. for C54H58FN12O14: 1117.42, found: 1117.35, [M + H]+.1H NMR (500 MHz, DMSO-d6) δ: 9.28 (t, J = 9.8 Hz, 1H), 8.65 (d, J = 2.1 Hz, 1H), 8.56 (t, J = 6.7 Hz, 1H), 8.32 (t, J = 5.9 Hz, 1H), 8.15 – 8.08 (m, 2H), 8.00 (t, J = 5.8 Hz, 1H), 7.80 (d, J = 10.8 Hz, 1H), 7.31 (d, J = 3.4 Hz, 1H), 7.28 – 7.21 (m, 5H), 7.20 – 7.15 (m, 1H), 7.00 (s, 1H), 6.51 (s, 1H), 5.78 – 5.71 (m, 1H), 5.41 – 5.33 (m, 2H), 5.25 – 5.11 (m, 2H), 4.63 – 4.60 (m, 2H), 4.58 (d, J = 6.7 Hz, 2H), 4.53 – 4.47 (m, 1H), 3.86 – 3.83 (m, 2H), 3.74 (td, J = 17.3, 5.8 Hz, 2H), 3.67 (d, J = 5.7 Hz, 2H), 3.64 – 3.50 (m, 5H), 3.46 (t, J = 5.7 Hz, 2H), 3.18 – 3.11 (m, 1H), 3.05 (dd, J = 14.0, 4.6 Hz, 1H), 2.84 – 2.75 (m, 1H), 2.40 (s, 3H), 2.33 (t, J = 6.5 Hz, 2H), 2.30 – 2.23 (m, 3H), 1.93 – 1.78 (m, 2H), 0.90 – 0.83 (m, 3H). Example 29: Synthesis of Compound 3006 [00203] Compound 3006 was prepared according to Procedure A of the General Methods section in Example 1 using 40 molar equivalents of linker payload compound 1010 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.9 based on a molecular weight of linker-drug 1117 of Da. Example 30: Synthesis of Compound 103
Figure imgf000100_0001
[00204] Compound 103. To the suspension of exatecan mesylate (10 mg, 0.019 mmol), 5-(hydroxymethyl)furan-2-carboxlic acid (3 equiv., 8 mg, 0.057 mmol), N-ethyl-N′-(3- dimethylaminopropyl)carbodiimide hydrochloride (2.5 equiv., 10 mg, 0.048 mmol) and 1- hydroxybenzotriazole (2.5 equiv., 7 mg, 0.048 mmol) in DMF (1 mL) was added diisopropylethylamine (5 equiv., 17 µL, 0.095 mmol) under an argon atmosphere and the mixture was stirred at room temperature for 40 minutes. LC-MS indicated the full consumption of the starting material. Purification by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 70 % ACN/H2O) followed by another purification on semipreparative column (diol- modified C18, 0 ^ 70 % ACN/H2O) offered the product (7 mg, 66 %) as a yellowish powder after lyophilization. MS calc. for C30H27FN3O7: 560.18, found: 560.15, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ: 8.90 (d, J = 8.6 Hz, 1H), 7.79 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 7.17 (d, J = 3.4 Hz, 1H), 6.50 (s, 1H), 6.45 (d, J = 3.4 Hz, 1H), 5.77–5.67 (m, 1H), 5.40 – 5.33 (m, 3H), 5.18 (d, J = 18.9 Hz, 1H), 5.10 (d, J = 19.0 Hz, 1H), 4.44 (d, J = 5.7 Hz, 2H), 3.29 – 3.09 (m, 1H), 2.40 (d, J = 1.9 Hz, 3H), 2.24 (q, J = 6.2 Hz, 2H), 0.94–1.75 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H). Example 31: Synthesis of Compound 1012
Figure imgf000101_0001
[00205] Intermediate 1. To the solution of FmocGGFGG-OAc (1.0 equiv., 50 mg, 0.079 mmol) and 5-(hydroxymethyl)furan-2-carboxylic acid (1.2 equiv., 14 mg, 0.095 mmol) in anhydrous DMF (0.5 ml) was added 2M HCl/Et2O (70 µL) and the resulting mixture was stirred at room temperature for 2 hours. Volatiles were removed and the residue was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 60% ACN/H2O), giving intermediate 1 as a white solid after lyophilization (23 mg, 41 %). MS calc. for C37H36FN5O10: 710.25, found: 710.25, [M-H]-. [00206] Intermediate 2. The mixture of intermediate 1 (1.05 equiv., 23 mg, 0.032 mmol), exatecan mesylate (1.0 equiv., 16.3 mg, 0.031 mmol) and DMTMM (1.05 equiv., 8.9 mg, 0.032 mmol) was added DMF/water (5:1, 1.2 ml) and diisopropylethylamine (2.1 equiv., 12 µL, 0.065 mmol) and the resulting mixture was stirred at room temperature for 40 minutes, as LC- MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 75% ACN/H2O), offering intermediate 2 as a white solid after lyophilization (27 mg, 77 %). MS calc. for C61H58FN8O13: 1129.41, found: 1129.45, [M+H]+. [00207] Intermediate 3. Morpholine (50 µL) was added to the solution of intermediate 2 (1.0 equiv., 27 mg, 0.024 mmol) in anhydrous DMF (1 ml) and the reaction mixture was stirred for 1.5 h at room temperature. LC-MS indicated the full consumption of starting material. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 60% ACN/H2O), giving the product as a white solid after lyophilization (15 mg, 69 %). MS calc. for C46H48FN8O11: 907.34, found: 907.35, [M+H]+. [00208] Compound 1012. Mal-PEG-NHS ester (1.0 equiv., 0.017 mmol, 5.2 mg) and DIPEA (1.05 equiv., 0.0173 mmol, 3.05 µL) were added to the solution of intermediate 3 (1.0 equiv., 15 mg, 0.017 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 1.5 h, as LC-MS indicated the full consumption of starting material. Purification by reverse-phase flash HPLC using a semipreparative column (diol-modified C18, 0 ^ 60% ACN/H2O) offered the desired product as a white solid after lyophilization (11 mg, 60 %). MS calc. for C55H57FN9O15: 1102.40, found: 1102.45, [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ: 9.06 – 8.93 (m, 1H), 8.59 (t, J = 6.4 Hz, 1H), 8.31 (t, J = 5.9 Hz, 1H), 8.16 – 8.05 (m, 2H), 8.04 – 7.94 (m, 1H), 7.80 (d, J = 10.8 Hz, 1H), 7.30 (s, 1H), 7.27 – 7.11 (m, 7H), 6.99 (s, 1H), 6.59 (d, J = 3.4 Hz, 1H), 6.51 (s, 1H), 5.78 – 5.63 (m, 1H), 5.38 (s, 2H), 5.28 – 4.97 (m, 2H), 4.59 (d, J = 7.0 Hz, 2H), 4.53 – 4.37 (m, 3H), 3.84 – 3.63 (m, 5H), 3.63 – 3.48 (m, 5H), 3.45 (t, J = 5.8 Hz, 2H), 3.29 – 3.22 (m, 1H), 3.20 – 3.08 (m, 1H), 3.04 (dd, J = 13.9, 4.6 Hz, 1H), 2.79 (dd, J = 13.9, 9.6 Hz, 1H), 2.40 (s, 3H), 2.32 (t, J = 6.6 Hz, 2H), 2.27 – 2.19 (m, 2H), 1.96 – 1.77 (m, 2H), 0.86 (t, J = 7.5 Hz, 3H). Example 32: Synthesis of Compound 3008 [00209] Compound 3008 was prepared according to Procedure A of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1012 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.7 based on a molecular weight of linker-drug 1102 of Da. Example 33: Synthesis of Compound 3033 [00210] Compound 3033 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1012 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 4.5 based on a molecular weight of linker-drug of 1102 Da. Example 34: Synthesis of Compound 3055 [00211] Compound 3055 was prepared according to Procedure C of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1012 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 7.3 based on a molecular weight of linker-drug of 1102 Da. Example 35: Synthesis of Compound 105
Figure imgf000103_0001
[00212] Compound 105. To the suspension of exatecan mesylate (30 mg, 0.056 mmol) in DMF (1 mL) were added diisopropylethylamine (3.5 equiv., 0.196 mmol, 34 µL) and 2- bromoethanol (2 equiv., 0.112 mmol, 14 mg, 8 µL) under an argon atmosphere and the mixture was heated to 80 °C for 2 days. LC-MS indicated the full consumption of the starting material. Purification by reverse-phase flash chromatography on semipreparative column (diol-modified C18, 0 ^ 50% ACN/1% TFA in H2O) offered the product (16 mg, 48 %) as a white powder after lyophilization. MS calc. for C26H27FN3O5: 480.19, found: 480.25, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ: 9.03 (s br, 1H), 8.80 (s br, 1H), 7.88 (d, J = 10.8 Hz, 1H), 7.34 (s, 1H), 6.57 (s, 1H), 5.57 – 5.36 (m, 4H), 5.30 (s, 1H), 5.16 – 5.02 (m, 1H), 3.69 (t, J = 5.5 Hz, 2H), 3.31 – 3.06 (m, 3H), 2.84 – 2.71 (m, 1H), 2.41 (s, 3H), 2.26 – 2.10 (m, 1H), 1.99 – 1.76 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).
Figure imgf000104_0001
Figure imgf000105_0001
Example 36: Synthesis of Compound 1013
Figure imgf000105_0002
[00213] Intermediate 1. The mixture of compound 105 (1.0 equiv., 20 mg, 0.034 mmol) and FmocGGFG-OAc (2.0 equiv., 0.068 mmol, 43 mg) was dissolved in anhydrous DMF (1 mL), followed by the addition of 2M HCl/Et2O (100 µL). The reaction mixture was stirred at room temperature for 4 h and during that time, several portions (ca.0.5 equiv. each) of FmocGGFG-OAc were added to the reaction mixture. Then the reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 75% ACN/H2O). Fractions containing the product were lyophilized and the residue (product + co-eluting impurities) was used directly into next step. MS calc. for C57H58FN8O11: 1049.42, found: 1049.40, [M+H]+. [00214] Intermediate 2. Morpholine (80 µL) was added to the solution of intermediate 1 (as obtained in previous step) in anhydrous DMF (2 ml) and the reaction mixture was stirred for 1,5 h at room temperature. LC-MS indicated the full consumption of starting material. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 60% ACN/H2O), giving the product as a white solid after lyophilization (12 mg, 43 %). MS calc. for C42H48FN8O9: 827.35, found: 827.30, [M+H]+. [00215] Compound 1013. Mal-PEG-NHS ester (1.0 equiv., 0.015 mmol, 4.3 mg) and DIPEA (2.2 equiv., 0.032 mmol, 4.2 mg, 5.7 µL) were added to the solution of intermediate 2 (12 mg, 0.015 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 40 minutes, as LC-MS indicated the full consumption of starting material. Purification by reverse-phase flash HPLC using a semipreparative column (diol-modified C18, 0 ^ 60% ACN/H2O) offered the desired product as a white solid after lyophilization (6.3 mg, 41 %). MS calc. for C51H57FN9O13: 1022.41, found: 1022.40, [M + H]+. 1H NMR (500 MHz, DMSO-d6) δ: 8.65 (t, J = 6.7 Hz, 1H), 8.33 – 8.26 (m, 1H), 8.15 – 8.07 (m, 2H), 8.03 – 7.96 (m, 1H), 7.76 – 7.70 (m, 1H), 7.60 (s, 1H), 7.26 – 7.18 (m, 5H), 7.18 – 7.12 (m, 1H), 6.99 (s, 2H), 5.49 – 5.24 (m, 3H), 4.81 (d, J = 11.8 Hz, 1H), 4.71 – 4.55 (m, 3H), 4.48 (ddd, J = 9.6, 8.1, 4.5 Hz, 1H), 4.28 (t, J = 4.2 Hz, 1H), 3.81 – 3.67 (m, 3H), 3.65 (d, J = 5.7 Hz, 2H), 3.62 – 3.47 (m, 6H), 3.47 – 3.41 (m, 2H), 3.22 – 3.12 (m, 1H), 3.06 – 2.90 (m, 2H), 2.87 – 2.71 (m, 2H), 2.38 – 2.29 (m, 5H), 2.26 – 1.98 (m, 4H), 1.26 – 1.16 (m, 1H), 0.90 – 0.82 (m, 3H). Example 37: Synthesis of Compound 3009 [00216] Compound 3009 was prepared according to Procedure A of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1013 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.5 based on a molecular weight of linker-drug 1022 of Da. Example 38: Synthesis of Compound 3034 [00217] Compound 3034 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1013 to anti- TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 6.2 based on a molecular weight of linker-drug of 1022 Da. Example 39: Synthesis of Compound 3056 [00218] Compound 3056 was prepared according to Procedure C of the General Methods section in Example 1 using 4 molar equivalents of linker payload compound 1013 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 3.9 based on a molecular weight of linker-drug of 1022 Da. Example 40: Synthesis of Compound 108
Figure imgf000107_0001
[00219] Diisopropylethylamine (2.5 equiv., 0.07 mmol, 9 mg, 13 µL) and propargyl bromide (2.5 equiv., 0.07 mmol, 8.5 mg, 9 µL 80% solution in toluene) were added to the suspension of exatecan mesylate (1.0 equiv., 15 mg, 0.028 mmol) in DMF (0.2 ml) and the resulting solution was stirred for 48 hours. Then, 2-azidoethanol (5.0 equiv., 0.14 mmol, 12 mg, 11 µL), tris(benzyltriazolylmethyl)amine (1.5 equiv., 0.042 mmol, 22 mg), CuSO4.5H2O (1.0 equiv., 0.028 mmol, 140 µL 2M aqueous solution) and sodium ascorbate (2.0 equiv., 0.056 mmol, 56 µL 1M aqueous solution) were successively added to the reaction mixture and the solution was stirred overnight. The crude reaction mixture was directly loaded on column and purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0% to 50% ACN/H2O), offering compound 108 (12 mg, 77%) as a white powder after lyophilisation. MS calc. for C29H30FN6O5: 561.23, found: 561.30, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ: 7.97 (s, 1H), 7.72 (d, J = 11.0 Hz, 1H), 7.29 (s, 1H), 5.43 (s, 2H), 5.28 (d, J = 19.0 Hz, 1H), 5.19 (d, J = 19.0 Hz, 1H), 4.39 (t, J = 5.5 Hz, 2H), 4.25 (t, J = 4.1 Hz, 1H), 3.97 (q, J = 13.9 Hz, 2H), 3.80 – 3.75 (m, 2H), 3.33 (s, 2H), 3.24 (ddd, J = 15.8, 10.4, 4.3 Hz, 1H), 3.01 (dt, J = 16.8, 4.8 Hz, 1H), 2.39 – 2.27 (m, 6H), 2.09 – 1.98 (m, 1H), 1.95 – 1.78 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). Example 41: Synthesis of Compound 1015 F
Figure imgf000108_0001
[00220] Intermediate 1. Compound 108 (1.0 equiv., 30 mg, 0.0536 mmol) and FmocGGFG-OAc (2.0 equiv., 0.107 mmol, 67 mg) were dissolved in anhydrous DMF (1.5 mL), followed by the addition of 2M HCl/Et2O (150 µL). The reaction mixture was stirred at room temperature for 4 h and during that time, several portions (ca.0.5 equiv. each) of FmocGGFG- OAc were added to the reaction mixture. Then the reaction mixture was purified by reverse- phase flash chromatography (25 g, diol-modified C18, 0 ^ 75% ACN/H2O). Fractions containing the product were lyophilised and the residue (product + co-eluting impurities) was used directly into next step. MS calc. for C60H61FN11O11: 1130.45, found: 1130.40, [M+H]+. [00221] Intermediate 2. Morpholine (100 µL) was added to the solution of Sc-260-F1 intermediate 1 (as obtained in previous step) in anhydrous DMF (2 ml) and the reaction mixture was stirred for 1 h at room temperature. LC-MS indicated the full consumption of starting material. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol- modified C18, 0 ^ 50% ACN/H2O), giving the product as a white solid after lyophilisation (14 mg, 29 % over 2 steps). MS calc. for C45H51FN11O9: 908.39, found: 908.55, [M+H]+. [00222] Compound 1015. Mal-PEG-NHS ester (1 equiv., 0.0154 mmol, 5 mg) and DIPEA (2.5 equiv., 0.039 mmol, 5 mg, 6.6 µL) were added to the solution of Sc-260-F1 intermediate 2 (1 equiv., 14 mg, 0.0154 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 30 minutes, as LC-MS indicated the full consumption of starting material. Purification by reverse-phase flash HPLC using a semipreparative column (diol-modified C18, 0 ^ 100% CAN in H2O) offered the desired product as a white solid after lyophilisation from water-acetonitrile (7 mg, 41 %). MS calc. for C54H60FN12O13: 1103.44, found: 1103.61, [M + H]+. 1H NMR (500 MHz, DMSO-d6) δ 8.54 (m, 1H), 8.30 (m, 1H), 8.12 (m, 2H), 8.01 (m, 1H), 7.97 (s, 1H), 7.73 (m, 1H), 7.65 (s, 1H), 7.29 (s, 1H), 7.25 – 7.17 (m, 4H), 7.17 – 7.12 (m, 1H), 7.01 (d, J = 5.9 Hz, 1H), 6.99 (s, 2H), 6.51 (s, 1H), 5.43 (s, 1H), 5.34 – 5.06 (m, 2H), 4.55 (m, 2H), 4.51 (m, 1H), 4.49 – 4.43 (m, 1H), 4.25 (d, J = 17.1 Hz, 1H), 4.10 – 3.88 (m, 2H), 3.80 (q, J = 4.9 Hz, 2H), 3.76 – 3.63 (m, 5H), 3.45 (t, J = 5.9 Hz, 2H), 3.22 (d, J = 10.9 Hz, 1H), 3.04 – 2.98 (m, 2H), 2.87 (t, J = 6.0 Hz, 1H), 2.82 – 2.73 (m, 2H), 2.59 (s, 1H), 2.36 (m, 4H), 2.32 (m, 2H), 2.06 – 1.97 (m, 2H), 1.86 (m 1H), 1.29 – 1.21 (m, 2H), 0.86 (m, 3H). Example 42: Synthesis of Compound 3010 [00223] Compound 3010 was prepared according to Procedure A of the General Methods section in Example 1 using 15 molar equivalents of linker payload compound 1015 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.6 based on a molecular weight of linker-drug 1103 of Da. Example 43: Synthesis of Compound 3035 [00224] Compound 3035 was prepared according to Procedure B of the General Methods section in Example 1 using 15 molar equivalents of linker payload compound 1015 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 6.3 based on a molecular weight of linker-drug of 1103 Da. Example 44: Synthesis of Compound 3057 [00225] Compound 3057 was prepared according to Procedure C of the General Methods section in Example 1 using 15 molar equivalents of linker payload compound 1015 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 6.0 based on a molecular weight of linker-drug of 1103 Da. Example 45: Synthesis of Compound 111
Figure imgf000110_0001
[00226] Compound 111. Methyl 2-(hydroxymethyl)cyclopropane-1-carboxylate (25 mg, 0.175 mmol) was dissolved in 1 mL of methanol, and 870 µL of 1M NaOH (1 equiv.) were added. The mixture was stirred at room temperature for 5h, thus, the solvents were evaporated and the crude product was lyophilized from water. To the obtained solid were added exatecan mesylate (46 mg, 0.5 equiv., 0,874 mmol), DMTMM (48 mg, 1 equiv., 0.175 mmol), and 10 mL of a 4:1 DMF/water mixture. The mixture was stirred at room temperature for 30 min. Solvents were evaporated under reduced pressure to a final volume of approx.2 mL. The crude reaction mixture was purified by reverse-phase flash chromatography, using a column containing 25 g of diol-modified C18, and using a gradient of ACN in water (0 ^
Figure imgf000110_0002
60% ACN in H2O). A second purification was performed using a semipreparative column loaded with diol-modified C18 and using a gradient of ACN in water (0 ^
Figure imgf000110_0003
80% ACN in H2O). Two isomers were separated during the semipreparative purification. The product was recovered separately as white powders, after lyophilization from water/dioxane (42 mg total, 91 % calculated from exatecan). MS calc. for C29H29FN3O6: 534.20, found: 534.10, [M+H]+. [00227] For isomer A: 1H NMR (400 MHz, DMSO-d6) δ 8.67 (d, J = 8.8 Hz, 1H), 7.78 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 6.52 (s, 1H), 5.56 (q, J = 6.6 Hz, 1H), 5.42 (s, 2H), 5.16 (d, J = 2.9 Hz, 2H), 4.63 (t, J = 5.5 Hz, 1H), 3.48 – 3.38 (m, 1H), 3.31 – 3.25 (m, 2H), 3.21 – 3.08 (m, 1H), 2,30 (m, 2H), 2.24 – 2.07 (m, 2H), 1.96 – 1.77 (m, 2H), 1.58 (d, J = 4.4 Hz, 1H), 1.49 (m, 1H), 0.99 (dt, J = 8.4, 4.3 Hz, 1H), 0.88 (t, J = 7.3 Hz, 3H), 0.71 (m, 1H). [00228] For isomer B (contains 7% of isomer A, based on NMR integrations): 1H NMR (400 MHz, DMSO-d6) δ 8.72 (d, J = 8.9 Hz, 1H), 7.77 (d, J = 10.9 Hz, 1H), 7.32 (s, 1H), 6.53 (s, 1H), 5.56 (m, 2H), 5.44 (s, 2H), 5.31 – 5.05 (m, 2H), 4.52 (dd, J = 6.1, 5.0 Hz, 1H), 3.45 (m 1H), 3.26 (m, 1H), 3.20 – 3.08 (m, 1H), 2.38 (m, 2H), 2.24 – 2.00 (m, 2H), 1.87 (m, 2H), 1.76 (s, 1H), 1.60 – 1.48 (m, 1H), 1.01 (m, 1H), 0.88 (t, J = 7.3 Hz, 3H), 0.75 (m, 1H). Example 46: Synthesis of Compound 1016 H
Figure imgf000111_0001
[00229] Intermediate 1.2-((Benzyloxy)methyl)cyclopropane-1-carboxylic acid (27.6 mg, 0.1338 mmol) was dissolved in 3 mL of anhydrous dioxane. Pd/C (10%) was added and hydrogen was bubbled into the solution for 5 h, while stirring at room temperature. The solution was filtered with 0.2 µm syringe filters, and the flask washed with acetonitrile. The filtrate was evaporated, re-dissolved in dioxane and lyophilized overnight. The crude filtrate was re- dissolved in 2 mL of anhydrous DMF, and FmocGGFG-OAc (1 equiv., 0.1338 mmol, 90 mg) was added, followed by 200 µL of a 2M HCl solution in ethyl ether. The reaction mixture was stirred at room temperature for 1 h, to be then directly loaded on column for purification. Purification was performed by reverse-phase flash chromatography (25 g, diol-modified C18, 0 à 75% ACN/H2O). Fractions containing the product were lyophilized from water (35 mg, 51%). MS calc. for C36H40N5O9: 686.28, found: 686.66, [M+H]+. [00230] Intermediate 2. To a solution of the previously prepared intermediate 1 (35 mg, 0.0505 mmol) in DMF (2 mL) were added exatecan mesylate (1 equiv., 0.0505 mmol, 27 mg), DMTMM (1.2 equiv., 0.0607 mmol, 17 mg), DIPEA (10 µL) and water (200 µL). The reaction mixture was stirred at room temperature for 30 min, to be then directly loaded on column for purification. Purification was performed by reverse-phase flash chromatography (25 g, diol- modified C18, 0 à 60% ACN/H2O). Fractions containing the product were lyophilized from water (28 mg, 50%). MS calc. for C60H60FN8O12: 1103.43, found: 1103.88, [M+H]+. [00231] Intermediate 3. The previously prepared intermediate 2 (28 mg, 0.0254 mmol) was dissolved in DMF (2mL) and morpholine (100 µL) was added. The reaction mixture was stirred at room temperature for 1 h. Purification was performed by reverse-phase HPLC chromatography (semipreparative, diol-modified C18, 0 à 100% ACN/H2O). Fractions containing the product were lyophilized from water (11 mg, 51%). MS calc. for C45H50FN8O10: 881.36, found: 881.12, [M+H]+. [00232] Compound 1016. The previously prepared intermediate 3 (11 mg,0.0130 mmol) was dissolved in 2 mL of anhydrous DMF.2,5-dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.1 equiv., 0.0143 mmol, 4 mg) and DIPEA (1.1 equiv., 0.0143 mmol, 2.5 µL) were added and the reaction mixture was stirred at room temperature for 1 h. Purification was performed by reverse-phase HPLC chromatography (semipreparative, diol-modified C18, 0 à 100% ACN/H2O). Fractions containing the product were lyophilized from water (7 mg, 50%). MS calc. for C54H59FN9O14: 1076.42, found: 1076.56, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ 8.80 – 8.70 (m, 2H), 8.53 (m, 1H), 8.45 (m, 1H), 8.27 (m, 1H), 8.10 (m, 2H), 7.99 (m, 2H), 7.93 (d, J = 6.1 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.80 (m, 2H), 7.34 – 7.20 (m, 2H), 7.18 – 7.08 (m, 2H), 6.67 (s, 2H), 6.51 (s, 1H), 5.62 (d, J = 10.0 Hz, 1H), 5.55 (s, 2H), 5.43 (t, J = 5.7 Hz, 2H), 5.19 – 5.07 (m, 3H), 5.06 – 5.02 (m, 1H), 4.74 (d, J = 6.0 Hz, 1H), 4.61 – 4.44 (m, 3H), 3.72 (m, 2H), 3.57 (m, 2H), 3.16 (d, J = 8.7 Hz, 1H), 3.08 – 3.00 (m, 1H), 2.79 (m, 2H), 2.39 (m, 3H), 2.34 (d, J = 1.8 Hz, 3H), 2.24 – 2.07 (m, 2H), 1.96 – 1.77 (m, 2H), 1.55 (m, 1H), 1.51 – 1.44 (m, 1H), 0.99 (m, 1H), 0.88 (t, J = 7.3 Hz, 3H), 0.83 – 0.71 (m, 1H). Example 47: Synthesis of Compound 3012 [00233] Compound 3012 was prepared according to Procedure A of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1016 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC was 2 based on a molecular weight of linker-drug 1076 of Da. Example 48: Synthesis of Compound 3037 [00234] Compound 3037 was prepared according to Procedure B of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1016 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC was 1.45 based on a molecular weight of linker-drug of 1076 Da. Example 49: Synthesis of Compound 1017
Figure imgf000114_0001
[00235] Intermediate 1. To the solution of (1S,2S)-2-(hydroxymethyl)cyclopropane-1- carboxylic acid (1.0 equiv., 5.7 mg, 0.049 mmol), Fmoc-GE(OBn)VCit-NH-CH2-OAc (1.5 equiv., 62 mg, 0.074 mmol) in anhydrous DMF (0.7 ml) was added 2M HCl/Et2O (100 µl) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using a semipreparative column (diol-modified C18, 0 ^ 75% ACN/0.1% HCl). Fractions containing the product (co-eluting with impurity) were lyophilised to obtain 31 mg of impure intermediate 1, which was used directly into the next step. MS calc. for C46H56FN7O12: 898.40, found: 898.40, [M-H]-. [00236] Intermediate 2. To the mixture of intermediate 1 (1.0 equiv., 31 mg, 0.034 mmol), exatecan mesylate (0.9 equiv., 17 mg, 0.031 mmol) and DMTMM (1.0 equiv., 10 mg, 0.034 mmol) was added DMF/water (5:1, 1.2 ml) and diisopropylethylamine (2.0 equiv., 12 µl, 0.068 mmol) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^
Figure imgf000115_0001
75% ACN/H2O), offering intermediate 2 as a white solid after lyophilisation (25 mg, 39 % (2 steps)). MS calc. for C70H78FN10O15: 1317.56, found: 1317.55, [M+H]+. [00237] Intermediate 3. To the solution of intermediate 2 (1.0 equiv., 25 mg, 0.019 mmol) in the mixture of dioxane (1.0 ml) and DMF (0.5 ml) was added 10% Pd/C (5 mg) and the reaction mixture was hydrogenated (balloon) for 2 hours at room temperature. LC-MS indicated the full consumption of starting material. The mixture was filtered through the layer of celite, and the filtrate was concentrated on rotary evaporator to remove dioxane. Morpholine (40 µl) was then added to the obtained solution and the reaction mixture was stirred at room temperature for 1 hour. Purification by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 60% ACN/H2O) offered the product intermediate 3 as a white solid after lyophilisation (8 mg, 42 %). MS calc. for C48H62FN10O13: 1005.43, found: 1005.40, [M+H]+. [00238] Compound 1017. Mal-PEG-NHS ester (1.0 equiv., 0.008 mmol, 2.5 mg) and DIPEA (1.05 equiv., 0.008 mmol, 1.5 µL) were added to the solution of intermediate 3 (1.0 equiv., 8 mg, 0.008 mmol) in anhydrous DMF (0.5 ml). The reaction mixture was stirred at room temperature for 1.5 h, as LC-MS indicated the full consumption of starting material. Purification by reverse-phase flash chromatography using a semipreparative column (diol-modified C18, 0 ^ 70% ACN/0.1% TFA) offered the desired product as a pale-yellow solid after lyophilisation (5 mg, 52 %). MS calc. for C57H71FN11O17: 1200.50, found: 1200.50, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ: 12.09 (s br, 1H), 8.71 (d, J = 8.7 Hz, 1H), 8.58 – 8.49 (m, 1H), 8.05 (t, J = 5.2 Hz, 1H), 8.01 – 7.92 (m, 2H), 7.83 – 7.70 (m, 2H), 7.31 (s, 1H), 7.00 (s, 2H), 6.58 (s br, 1H), 5.92 (s, 1H), 5.62 – 5.49 (m, 1H), 5.50 – 5.33 (m, 2H), 5.28 – 5.12 (m, 2H), 4.60 – 4.40 (m, 2H), 4.39 – 4.25 (m, 1H), 4.21 – 4.02 (m, 2H), 3.77 – 3.62 (m, 3H), 3.31 – 3.19 (m, 2H), 3.20 – 3.09 (m, 1H), 3.04 – 2.82 (m, 2H), 2.40 (s, 3H), 2.35 – 2.26 (m, 2H), 2.27 – 2.10 (m, 4H), 1.99 – 1.79 (m, 4H), 1.76 – 1.64 (m, 1H), 1.63 – 1.41 (m, 4H), 1.40 – 1.23 (m, 3H), 1.05 – 0.93 (m, 1H), 0.92 – 0.84 (m, 3H), 0.84 – 0.75 (m, 6H), 0.75 – 0.69 (m, 2H). Example 50: Synthesis of Compound 3013 [00239] Compound 3013 was prepared according to Procedure A of the General Methods section in Example 1 using 37 molar equivalents of linker payload compound 1017 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.6 based on a molecular weight of linker-drug 1200 of Da. Example 51: Synthesis of Compound 3038 [00240] Compound 3038 was prepared according to Procedure B of the General Methods section in Example 1 using 37 molar equivalents of linker payload compound 1017 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 6.6 based on a molecular weight of linker-drug of 1200 Da. Example 52: Synthesis of Compound 3059 [00241] Compound 3059A was prepared according to Procedure C of the General Methods section in Example 1 using 37 molar equivalents of linker payload compound 1017 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 6.4 based on a molecular weight of linker-drug of 1200 Da. Molar ratios of mAb and linker-payload were adjusted to achieve DAR 7.7 (compound 3059B). Example 53: Synthesis of Compound 1025
Figure imgf000117_0001
[00242] Intermediate 1. FmocGGFG-OAc (1 equiv., 0.0892 mmol, 60 mg) was dissolved into 2 mL of DMF and (1S,2S)-2-(hydroxymethyl)cyclopropane-1-carboxylic acid (2 equiv., 0.1784 mmol, 21 mg) was added, followed by 10 µL of a 4M HCl solution in dioxane. The reaction mixture was stirred at room temperature for 1 h, to be then directly loaded on column for purification. Purification was performed by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 100% ACN/H2O). Fractions containing the product were lyophilised from water (43 mg, 70 %). MS calc. for C36H40N5O9: 686.28, found: 686.34, [M+H]+. [00243] Intermediate 2. To a solution of the previously prepared intermediate 1 (43 mg, 0.0628 mmol) in DMF (2 mL) were added exatecan mesylate (1.1 equiv., 0.0691 mmol, 37 mg), DMTMM (1.2 equiv., 0.0754 mmol, 21 mg), DIPEA (20 µL) and water (400 µL). The reaction mixture was stirred at room temperature for 1 h, to be then directly loaded on column for purification. Purification was performed by reverse-phase flash chromatography (25 g, diol- modified C18, 0 ^ 100% ACN/0/1% TFA). Fractions containing the product were lyophilised from water (41 mg, 59 %). MS calc. for C60H60FN8O12: 1103.43, found: 1103.40, [M+H]+. [00244] Intermediate 3. The previously prepared intermediate 2 (41 mg, 0.0372 mmol) was dissolved in DMF (2mL) and morpholine (100 µL) was added. The reaction mixture was stirred at room temperature for 1 h. Purification was performed by reverse-phase HPLC chromatography (semipreparative, diol-modified C18, 0 ^ 100% ACN/ 0.1% TFA). Fractions containing the product were lyophilised from water (14 mg, 44%). MS calc. for C45H50FN8O10: 881.36, found: 881.65, [M+H]+. [00245] Compound 1025. The previously prepared intermediate 3 (14 mg, 0.0160 mmol) was dissolved in 2 mL of anhydrous DMF.2,5-dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.1 equiv., 0.0175 mmol, 5.4 mg) and DIPEA (2.5 µL) were added and the reaction mixture was stirred at room temperature for 1 h. Purification was performed by reverse-phase HPLC chromatography (semipreparative, diol-modified C18, 0 ^ 100% ACN/ 0.1% TFA). Fractions containing the product were lyophilised from water (7 mg, 41 %). MS calc. for C54H59FN9O14: 1076.42, found: 1076.42, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ 8.73 (t, J = 9.3 Hz, 1H), 8.42 (t, J = 6.6 Hz, 1H), 8.25 (t, J = 5.8 Hz, 1H), 8.10 (q, J = 7.5, 6.4 Hz, 2H), 7.99 (q, J = 6.7, 5.8 Hz, 1H), 7.79 (d, J = 10.8 Hz, 1H), 7.31 (s, 1H), 7.29 – 7.19 (m, 4H), 7.19 – 7.12 (m, 1H), 7.00 (s, 2H), 6.51 (s, 1H), 5.56 (m, 1H), 5.49 – 5.38 (m, 2H), 5.19 (s, 2H), 5.16 (d, J = 4.7 Hz, 1H), 4.56 (d, J = 6.7 Hz, 1H), 4.52 (d, J = 6.7 Hz, 1H), 4.48 (m, 1H), 3.78 – 3.69 (m, 2H), 3.67 (d, J = 5.7 Hz, 2H), 3.57 (d, J = 5.8 Hz, 3H), 3.53 (t, J = 6.6 Hz, 2H), 3.46 (t, J = 5.8 Hz, 3H), 3.26 (m, 1H), 3.14 (m, 1H), 3.02 (m, 1H), 2.80 – 2.75 (m, 1H), 2.39 (d, J = 3.9 Hz, 3H), 2.32 (t, J = 6.5 Hz, 2H), 2.14 (m, 2H), 1.86 (m, 2H), 1.61 – 1.49 (m, 2H), 1.03 (m, 1H), 0.88 (t, J = 7.3 Hz, 3H), 0.77 (p, J = 4.1 Hz, 1H). Example 54: Synthesis of Compound 3011 [00246] Compound 3011 was prepared according to Procedure A of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1025 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 5.4 based on a molecular weight of linker-drug 1076 of Da. Example 55: Synthesis of Compound 3036 [00247] Compound 3036 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1025 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 6.4 based on a molecular weight of linker-drug of 1076 Da. Example 56: Synthesis of Compounds 3058A and 3058B [00248] Compound 3058A was prepared according to Procedure C of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1025 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 7.1 based on a molecular weight of linker-drug of 1076 Da. Molar ratios of mAb and linker-payload were adjusted to achieve DAR 3.1 (compound 3058B). Example 57: Synthesis of Compound 130
Figure imgf000119_0001
[00249] Intermediate 1. To the mixture of exatecan mesylate (1 equiv., 40 mg, 0.075 mmol), 2-(((tert-butoxycarbonyl)amino)oxy)acetic acid (1.2 equiv., 18 mg, 0.090 mmol) and DMTMM (1.2 equiv., 25 mg, 0.090 mmol) was added DMF/water (5:1, 2 ml) and diisopropylethylamine (2.2 equiv., 0.165 mmol, 29 µL) and the resulting mixture was stirred at room temperature for 0.5 h. LC-MS indicated the full consumption of starting material. Purification of the mixture by reverse-phase flash chromatography (diol-modified C18, 0 ^ 100 % ACN/H2O) offered the intermediate 1 (43 mg, 94 %) as a white powder after lyophilization. [00250] Compound 130. Intermediate 1 (43 mg, 0.070 mmol) was dissolved in 4M HCl/dioxane (2 ml) and the mixture was stirred for 1.5 hour at room temperature. The resulting suspension was filtered and the solids were washed with dioxane and Et2O to give the hydrochloride of compound 130 (35 mg, 90 %) as a yellow powder. MS calc. for C26H26FN4O6: 509.18, found: 509.20, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ: 10.98 (s br, 3H), 8.89 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 11.0 Hz, 1H), 7.32 (s, 1H), 5.62 (dt, J = 8.5, 4.2 Hz, 1H), 5.43 (s, 2H), 5.37 – 5.24 (m, 2H), 4.63 – 4.52 (m, 2H), 3.20 (dd, J = 7.9, 4.7 Hz, 2H), 2.41 (d, J = 1.9 Hz, 3H), 2.32 – 2.21 (m, 1H), 2.21 – 2.09 (m, 1H), 1.96 – 1.77 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). Example 58: Synthesis of Compound 1018
Figure imgf000120_0001
[00251] Intermediate 1. The mixture of compound 130 (1.0 equiv., 30 mg, 0.055 mmol), FmocGGFGGG-OH (1.25 equiv., 47 mg, 0.069 mmol) and DMTMM (1.25 equiv., 19 mg, 0.069 mmol) was added DMF/water (5:1, 2.4 ml) and diisopropylethylamine (2.25 equiv., 22 µL, 0.124 mmol) and the resulting mixture was stirred at room temperature for 1 h, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 75% ACN/H2O), offering intermediate 1 as a white solid after lyophilization (48 mg, 75 %). MS calc. for C60H60FN10O14: 1163.43, found: 1163.40, [M+H]+. [00252] Intermediate 2. Morpholine (100 µL) was added to the solution of intermediate 1 (1.0 equiv., 48 mg, 0.041 mmol) in anhydrous DMF (1.5 ml) and the reaction mixture was stirred for 1 h at room temperature. LC-MS indicated the full consumption of starting material. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 60% ACN/H2O), giving the product as a white solid after lyophilization (26 mg, 67 %). MS calc. for C45H50FN10O12: 941.36, found: 941.40, [M+H]+. [00253] Compound 1018. Mal-PEG-NHS ester (1.0 equiv., 0.027 mmol, 8.4 mg) and DIPEA (1.1 equiv., 0.030 mmol, 5.2 µL) were added to the solution of intermediate 2 (1.0 equiv., 26 mg, 0.027 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 40 minutes, as LC-MS indicated the full consumption of starting material. DMF was removed and the residue was concentrated from the mixture of 0.1% aq. TFA and ACN. Purification by reverse-phase flash HPLC using a semipreparative column (diol-modified C18, 0 ^ 60% ACN/H2O) offered the desired product as a white solid after lyophilization (17 mg, 55 %). MS calc. for C54H59FN11O16: 1136.41, found: 1136.45, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ: 11.45 (s, 1H), 8.96 – 8.68 (m, 1H), 8.26 (t, J = 5.8 Hz, 1H), 8.22 – 8.04 (m, 3H), 8.04 – 7.93 (m, 2H), 7.87 – 7.75 (m, 1H), 7.31 (s, 1H), 7.23 (d, J = 6.9 Hz, 4H), 7.19 – 7.12 (m, 1H), 6.99 (s, 1H), 6.59 – 6.44 (m, 1H), 5.64 – 5.54 (m, 1H), 5.42 (s, 2H), 5.35 – 5.10 (m, 2H), 4.50 (td, J = 9.8, 9.1, 4.6 Hz, 1H), 4.45 – 4.28 (m, 2H), 3.79 – 3.39 (m, 17H), 3.25 – 3.10 (m, 2H), 3.09 – 2.99 (m, 1H), 2.88 – 2.72 (m, 1H), 2.39 (s, 3H), 2.32 (t, J = 6.5 Hz, 2H), 2.27 – 2.02 (m, 2H), 1.96 – 1.78 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H). Example 59: Synthesis of Compound 3015 [00254] Compound 3015 was prepared according to Procedure A of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1018 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 5 based on a molecular weight of linker-drug 1136 of Da. Example 60: Synthesis of Compound 3040 [00255] Compound 3040 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1018 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 4.8 based on a molecular weight of linker-drug of 1136 Da. E
Figure imgf000122_0001
3061 [00256] Compound 3061 was prepared according to Procedure C of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1018 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 6.3 based on a molecular weight of linker-drug of 1136 Da. Example 62: Synthesis of Compound 1019
Figure imgf000122_0002
[00257] Intermediate 1. The mixture of compound 130 (1.0 equiv., 35 mg, 0.064 mmol), FmocGGFGGP-OH (1.2 equiv., 55 mg, 0.077 mmol) and DMTMM (1.2 equiv., 22 mg, 0.077 mmol) was added DMF/water (5:1, 2.4 ml) and diisopropylethylamine (2.2 equiv., 25 µL, 0.141 mmol) and the resulting mixture was stirred at room temperature for 1 h, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 75% ACN/H2O), offering intermediate 1 as a white solid after lyophilization (65 mg, 84 %). MS calc. for C63H64FN10O14: 1203.46, found: 1203.50, [M+H]+. [00258] Intermediate 2. Morpholine (130 µL) was added to the solution of intermediate 1 (1.0 equiv., 65 mg, 0.054 mmol) in anhydrous DMF (1.7 ml) and the reaction mixture was stirred for 1 h at room temperature. LC-MS indicated the full consumption of starting material. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 60% ACN/H2O), giving the product as a white solid after lyophilization (34 mg, 64 %). MS calc. for C48H54FN10O12: 981.39, found: 981.40, [M+H]+. [00259] Compound 1019. Mal-PEG-NHS ester (1.0 equiv., 0.035 mmol, 10.8 mg) and DIPEA (1.05 equiv., 0.037 mmol, 6.4 µL) were added to the solution of intermediate 2 (1.0 equiv., 34 mg, 0.035 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 1 h, as LC-MS indicated the full consumption of starting material. DMF was removed and the residue was concentrated from the mixture of 0.1% aq. TFA and ACN. Purification by reverse-phase flash HPLC using a semipreparative column (diol-modified C18, 0 ^ 60% ACN/H2O) offered the desired product as a white solid after lyophilization (18 mg, 44 %). MS calc. for C57H63FN11O16: 1176.44, found: 1176.45, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ: 11.50 (s, 1H), 8.85 (d, J = 8.7 Hz, 1H), 8.31 – 8.25 (m, 1H), 8.16 – 8.02 (m, 3H), 7.96 (t, J = 5.7 Hz, 1H), 7.88 – 7.77 (m, 2H), 7.74 (t, J = 5.2 Hz, 1H), 7.35 – 7.28 (m, 2H), 7.28 – 7.18 (m, 5H), 7.18 – 7.11 (m, 1H), 6.99 (s, 1H), 6.57 – 6.46 (m, 1H), 5.70 – 5.58 (m, 1H), 5.43 (s, 2H), 5.35 – 5.11 (m, 3H), 4.54 – 4.46 (m, 1H), 4.32 (s, 2H), 4.08 – 4.03 (m, 1H), 3.90 – 3.39 (m, 14H), 3.24 – 3.10 (m, 2H), 3.03 (dd, J = 13.9, 4.4 Hz, 1H), 2.77 (dd, J = 13.9, 9.8 Hz, 1H), 2.40 (s, 3H), 2.32 (t, J = 6.5 Hz, 2H), 2.26 – 2.11 (m, 2H), 1.97 – 1.77 (m, 3H), 1.69 – 1.51 (m, 1H), 0.86 (t, J = 7.4 Hz, 3H). Example 63: Synthesis of Compound 3014 [00260] Compound 3014 was prepared according to Procedure A of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1019 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 5.2 based on a molecular weight of linker-drug 1176 of Da. Example 64: Synthesis of Compound 3039 [00261] Compound 3039 was prepared according to Procedure B of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1019 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 7.5 based on a molecular weight of linker-drug of 1176 Da. Example 65: Synthesis of Compound 3060 [00262] Compound 3060 was prepared according to Procedure C of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1019 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC-MS was 7.8 based on a molecular weight of linker-drug of 1176 Da. Example 66: Synthesis of Compound 177
Figure imgf000124_0001
[00263] Intermediate 1. To the solution of compound 111-B (1.0 equiv., 20 mg, 0.036 mmol) and imidazole (2.5 equiv., 0.094 mmol, 6.4 mg) in DMF (2 mL) was added tert- butyl(chloro)diphenylsilane (2 equiv., 0.075 mmol, 19.5 µL) under an argon atmosphere and the mixture was stirred over night at room temperature. Purification by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 80% ACN/H2O) offered the product intermediate 1 (22 mg, 79%) as an off-white powder after lyophilization. [00264] Intermediate 2. To the solution of intermediate 1 (22 mg, 0.029 mmol) in dry pyridine (2 mL) was added tert-butyldimethylsilyl trifluoromethanesulfonate (0.436 mmol, 100 µL) under an argon atmosphere and the mixture was heated to 80 °C for 4 hours. Then, it was concentrated on rotary evaporator and the residue was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 100% ACN/H2O), giving the product intermediate 2 (23 mg, 89 %) as a white powder after lyophilization. [00265] Compound 177. To the mixture of intermediate 2 (23 mg, 0.026 mmol) and Lawesson reagent (1.0 equiv., 0.026 mmol, 10.5 mg) was added anhydrous toluene (8 ml) and the resulting mixture was heated to 100 °C for 1 hours. The progress of the reaction was monitored by LC-MS. Then, volatiles were removed on rotary evaporator and the residue was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 100% ACN/H2O). The fractions containing the intermediate 3 were combined and concentrated on rotary evaporator. The residue was dissolved in DCM (2 ml) and trifluoroacetic acid (1 ml). The resulting mixture was stirred at room temperature for 48 hours and the progress of the reaction was monitored by LC-MS. Then, solvents were removed on rotary evaporator and the residue was purified by reverse-phase flash chromatography using semipreparative column (diol- modified C18, 0 ^ 70% ACN/H2O), giving the product compound 177 (3.3 mg, 23 %) as a yellow powder after lyophilization. MS calc. for C29H29FN3O5S: 550.17, found: 550.15, [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ: 10.63 (dd, J = 30.4, 8.6 Hz, 1H), 7.82 (d, J = 10.8 Hz, 1H), 7.32 (d, J = 9.4 Hz, 1H), 6.66 (s, 1H), 6.40 (dq, J = 14.0, 7.1 Hz, 1H), 5.42 (d, J = 5.7 Hz, 2H), 5.20 – 4.98 (m, 2H), 3.45 (dd, J = 11.5, 5.8 Hz, 1H), 3.35 (dd, J = 11.5, 6.2 Hz, 1H), 3.28 – 3.09 (m, 2H), 2.40 (s, 3H), 2.33 – 2.18 (m, 2H), 2.16 – 2.03 (m, 1H), 1.96 – 1.78 (m, 3H), 1.45 – 1.32 (m, 1H), 1.04 – 0.91 (m, 1H), 0.87 (t, J = 7.4 Hz, 3H). Example 67: Synthesis of Compound 1034
Figure imgf000126_0001
[00266] Intermediate 1. To the solution of compound 177 (1.0 equiv., 12 mg, 0.021 mmol) and FmocGGFG-NH-CH2-OAc (1.5 equiv., 21 mg, 0.032 mmol) in anhydrous DMF (1.0 ml) was added 2M HCl/Et2O (30 µl) and the resulting mixture was stirred at room temperature for 5 hours, with three more additions of FmocGGFG-NH-CH2-OAc (0.5 equiv., 6 mg, 0.011 mmol) during that time. The reaction mixture was then purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 80 % ACN/0.1% aq. TFA), offering intermediate 1 as an off-white solid after lyophilization (11 mg, 47 %). MS calc. for C60H60FN8O11S: 1119.41, found: 1119.40, [M+H]+. [00267] Intermediate 2. To the solution of intermediate 1 (1.0 equiv., 11 mg, 0.010 mmol) in DMF (0.75 ml) was added morpholine (40 µl) and the mixture was stirred 45 min at room temperature. The reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 60 % ACN/0.1% aq. TFA), giving the intermediate 2 (4.5 mg, 45 %) as a pale-yellow solid. MS calc. for C45H50FN8O9S: 897.34, found: 897.35, [M+H]+. [00268] Compound 1034. Mal-PEG-NHS ester (1.05 equiv., 1.45 mg, 0.0047 mmol) and DIPEA (2.1 equiv., 2.0 µl, 0.0095 mmol) were added to the solution of intermediate 2 (1.0 equiv., 4.5 mg, 0.0045 mmol) in anhydrous DMF (0.5 ml) and the reaction mixture was stirred at room temperature for 1 hour. Purification by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 70% ACN/H2O) gave the product compound 1034 (4.2 mg, 85%) as a white solid after lyophilization. MS calc. for C54H59FN9O13S: 1092.39, found: 1092.35, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ: 10.66 (d, J = 8.7 Hz, 1H), 8.48 (t, J = 6.7 Hz, 1H), 8.28 (t, J = 5.8 Hz, 1H), 8.14 – 8.06 (m, 2H), 8.00 (t, J = 5.9 Hz, 1H), 7.83 (d, J = 10.9 Hz, 1H), 7.35 – 7.30 (m, 1H), 7.28 – 7.12 (m, 5H), 7.00 (s, 2H), 6.52 (s, 1H), 6.45 – 6.34 (m, 1H), 5.42 (s, 1H), 5.21 – 5.02 (m, 3H), 4.58 – 4.52 (m, 2H), 4.52 – 4.44 (m, 1H), 3.78 – 3.63 (m, 5H), 3.62 – 3.50 (m, 6H), 3.46 (t, J = 5.8 Hz, 2H), 3.22 – 3.17 (m, 2H), 3.09 – 2.99 (m, 1H), 2.84 – 2.74 (m, 1H), 2.41 (s, 3H), 2.36 – 2.30 (m, 2H), 2.30 – 2.22 (m, 2H), 2.13 – 2.06 (m, 1H), 1.95 – 1.79 (m, 3H), 1.47 – 1.37 (m, 1H), 1.05 – 0.94 (m, 1H), 0.87 (t, J = 7.4 Hz, 3H). Example 68: Synthesis of Compound 3100 [00269] Compound 3100 was prepared according to Procedure A of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1034 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC was 3.6 based on a molecular weight of linker-drug 1092 of Da. Example 69: Synthesis of Compound 3101 [00270] Compound 3101 was prepared according to Procedure B of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1034 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC-MS was 3.7 based on a molecular weight of linker-drug of 1092 Da. Example 70: Synthesis of Compound 180
Figure imgf000127_0001
[00271] Intermediate 1. Exatecan mesylate (90 mg, 0.17 mmol), N-1-Fmoc-(2S,3S)-3-hydroxypyrrolidine-2-carboxylic acid (90 mg, 0.26 mmol) and DIPEA (100 µL) were dissolved in DMF (3 mL) and water (1 mL). DMTMM (70 mg, 0.26 mmol) was added and the reaction mixture was stirred for 30 min at room temperature, to be then directly loaded on column for purification. Purification was performed by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^
Figure imgf000128_0001
50% ACN/H2O(TFA)). Fractions containing the product were lyophilized from water (107 mg, 82%). MS calc. for C44H40FN4O8 +: 771.28, found: 771.35 [M+H]+. [00272] Compound 180. To a solution of intermediate 1 (33 mg, 0.0428 mmol) in DMF (2 ml), was added morpholine (100 µl) and the mixture was stirred 1 hour at room temperature. The reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^
Figure imgf000128_0002
60% ACN/0.1% TFA), giving compound 180 (15 mg, 57 %) as a white powder after lyophilization. MS calc. for C29H30FN4O6: 549.21, found: 549.48 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 1H), 9.26 (d, J = 8.6 Hz, 1H), 8.81 (s, 1H), 7.83 (d, J = 11.0 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.67 (s, 1H), 5.61 (m, 1H), 5.43 (s, 2H), 5.30 (d, J = 18.7 Hz, 1H), 5.08 (d, J = 18.7 Hz, 1H), 4.42 (d, J = 4.7 Hz, 1H), 4.05 (s, 1H), 3.45 (s, 2H), 3.22 (d, J = 7.6 Hz, 2H), 2.42 (d, J = 1.8 Hz, 3H), 2.29 – 2.15 (m, 1H), 2.10 – 1.97 (m, 1H), 1.87 (m, 3H), 0.89 (t, J = 7.3 Hz, 3H). Example 71: Synthesis of Compound 193
Figure imgf000128_0003
[00273] Compound 193. Compound 180 (10 mg, 0.018 mmol) and DIPEA (5 µL) were dissolved in DCM (2mL). The Ac2O (2.6 µL, 0.027) was added, and the reaction was stirred over night at room temperature. The mixture was evaporated, and residue was purified by reverse- phase HPLC chromatography (semipreparative, diol-modified C18, 0 ^ 50% ACN/H2O). Fractions containing the product were lyophilized from water (4.9 mg, 46%). MS calc. for C31H32FN4O7+: 591.22, found: 591.25 [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 8.5 Hz, 1H), 7.81 (t, J = 10.4 Hz, 1H), 7.32 (d, J = 3.6 Hz, 1H), 6.52 (d, J = 5.4 Hz, 1H), 5.51 (m, 2H), 5.44 (m, 3H), 5.30 (d, J = 18.7 Hz, 1H), 5.20 (s, 2H), 4.25 (s, 1H), 4.17 (s, 1H), 3.58 (m, 2H), 3.17 (d, J = 8.1 Hz, 3H), 2.41 (m, 3H), 2.17 (s, 1H), 2.09 (d, J = 12.9 Hz, 1H), 1.88 (m, 3H), 1.76 (s, 1H), 0.90 (q, J = 7.3, 6.7 Hz, 3H). Example 72: Synthesis of Compound 194
Figure imgf000129_0001
[00274] Compound 194. Formic acetic anhydride (2.2 equiv., 0.048, 4.2 µL) and diisopropylethylamine (2.5 equiv., 0.055 mmol, 10 µL) were added to the solution of compound 180 (1.0 equiv., 15 mg, 0.027 mmol) in DCM (2 ml) and the reaction was stirred over night at room temperature. Then, mixture was evaporated, and the residue was purified by reverse-phase HPLC chromatography (semipreparative, diol-modified C18, 0 ^ 50% ACN/H2O). Fractions containing the product were lyophilized from water (5.5 mg, 46%). MS calc. for C30H30FN4O7+: 577.20, found: 577.25 [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 8.85 (d, J = 8.6 Hz, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.20 – 8.12 (m, 1H), 7.80 (m, 1H), 7.32 (d, J = 0.9 Hz, 2H), 6.67 (s, 1H), 5.62 – 5.50 (m, 1H), 5.22 (d, J = 7.9 Hz, 2H), 4.39 – 4.26 (m, 2H), 3.66 – 3.61 (m, 1H), 3.17 (d, J = 5.7 Hz, 4H), 2.41 (d, J = 1.8 Hz, 3H), 2.21 – 2.12 (m, 1H), 2.06 – 1.96 (m, 1H), 1.93 – 1.84 (m, 2H), 1.76 (s, 2H), 0.93 – 0.85 (m, 3H). Example 73: Synthesis of Compound 195
Figure imgf000130_0001
[00275] Intermediate 1. To the mixture of exatecan mesylate (1.0 equiv., 40 mg, 0.075 mmol), (1S,2S)-2-((tert-butoxycarbonyl)amino)cyclopropane-1-carboxylic acid (1.2 equiv., 18 mg, 0.090 mmol) and DMTMM (1.2 equiv., 25 mg, 0.090 mmol) was added DMF/water (5:1, 1.8 ml) and diisopropylethylamine (2.2 equiv., 29 µl, 0.165 mmol) and the resulting mixture was stirred at room temperature for 40 minutes, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 75% ACN/H2O), offering intermediate 1 as a white solid after lyophilization (38 mg, 82 %). MS calc. for C33H36FN4O7: 619.26, found: 619.25, [M+H]+. [00276] Compound 195. The obtained intermediate 1 (38 mg, 0.061 mmol) was dissolved in 4M HCl/dioxane (3 ml) and the mixture was stirred 30 min at room temperature. The resulting suspension was filtered, the solid material was washed with dioxane and Et2O and dried under vacuum, giving compound 195 hydrochloride (25 mg, 74 %) as a yellow solid. MS calc. for C28H28FN4O5: 519.20, found: 519.20, [M+H]+. The product is unstable and undergoes decomposition during the preparation and further analyses. Example 74: Synthesis of Compounds 1035 and 1036
Figure imgf000131_0001
[00277] Intermediate 1. To the mixture of compound 195.HCl (1.0 equiv., 24 mg, 0.039 mmol), Fmoc-GGFGGP-OH (1.0 equiv., 31 mg, 0.039 mmol) and DMTMM (1.1 equiv., 14 mg, 0.043 mmol) was added DMF/water (5:1, 1.5 ml) and diisopropylethylamine (2.1 equiv., 16 µl, 0.082 mmol) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 80 % ACN/0.1% aq. TFA), offering intermediate 1 (two separated isomers A and B), both as white solids after lyophilization (isomer A: 19 mg, isomer B: 21 mg, combined yield: 87 %). MS calc. for C65H66FN10O13: 1213.48, found: 1213.45, [M+H]+. [00278] Compound 1035. To the solution of intermediate 1 isomer A (1.0 equiv., 19 mg, 0.016 mmol) in DMF (1.5 ml) was added morpholine (60 µl) and the mixture was stirred for 1 hour at room temperature. The reaction mixture was evaporated to dryness and the residue was redissolved in dry DMF (1 ml). Then, Mal-PEG-NHS ester (1.1 equiv., 5.4 mg, 0.017 mmol) and DIPEA (1.1 equiv., 3.0 µl, 0.017 mmol) were added and the reaction mixture was stirred at room temperature for 1 hour. Purification by reverse-phase flash chromatography using semipreparative column (diol-modified C18,
Figure imgf000131_0002
^ 65 % ACN/H2O) gave the product 1035 as a white solid after lyophilization (10 mg, 54 %). MS calc. for C59H65FN11O15: 1186.46, found: 1186.40, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ: 8.30 – 8.18 (m, 1H), 8.14 – 8.02 (m, 3H), 7.96 (t, J = 5.7 Hz, 1H), 7.91 – 7.78 (m, 1H), 7.74 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 7.27 – 7.18 (m, 5H), 7.18 – 7.12 (m, 1H), 6.99 (s, 2H), 6.51 (s, 1H), 5.72 – 5.56 (m, 1H), 5.47 – 5.36 (m, 2H), 5.27 – 5.03 (m, 2H), 4.49 (tt, J = 9.2, 5.4 Hz, 1H), 4.06 – 3.92 (m, 1H), 3.83 – 3.68 (m, 4H), 3.66 (d, J = 5.7 Hz, 2H), 3.62 – 3.50 (m, 7H), 3.45 (t, J = 5.8 Hz, 2H), 3.12 – 2.94 (m, 1H), 2.82 – 2.71 (m, 1H), 2.65 – 2.52 (m, 2H), 2.44 – 2.10 (m, 11H), 1.98 – 1.77 (m, 5H), 1.72 – 1.57 (m, 1H), 1.56 – 1.37 (m, 1H), 0.86 (t, J = 7.2 Hz, 3H). [00279] Compound 1036. The product was synthesized from intermediate 1 isomer B (1.0 equiv., 21 mg, 0.017 mmol) following the same procedure as for isomer A. Compound 1036 was obtained as a white solid after lyophilization (11 mg, 55 %). MS calc. for C59H65FN11O15: 1186.46, found: 1186.45, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ: 8.69 (d, J = 8.8 Hz, 1H), 8.26 (t, J = 6.0 Hz, 1H), 8.11 – 8.06 (m, 2H), 8.02 – 7.92 (m, 1H), 7.84 (t, J = 5.6 Hz, 1H), 7.76 (d, J = 11.0 Hz, 1H), 7.31 (s, 1H), 7.27 – 7.19 (m, 5H), 7.19 – 7.12 (m, 1H), 6.99 (s, 2H), 6.51 (s, 1H), 5.47 – 5.38 (m, 2H), 5.37 – 5.31 (m, 1H), 5.29 – 5.22 (m, 1H), 5.10 (d, J = 18.6 Hz, 1H), 5.00 (d, J = 18.6 Hz, 1H), 4.54 – 4.43 (m, 1H), 4.22 – 4.15 (m, 1H), 4.04 – 3.93 (m, 1H), 3.92 – 3.80 (m, 1H), 3.79 – 3.69 (m, 4H), 3.68 – 3.63 (m, 2H), 3.62 – 3.50 (m, 7H), 3.50 – 3.42 (m, 3H), 3.19 – 2.97 (m, 2H), 2.82 – 2.70 (m, 1H), 2.43 – 2.22 (m, 8H), 2.21 – 1.97 (m, 2H), 1.95 – 1.72 (m, 4H), 1.72 – 1.59 (m, 1H), 0.87 (t, J = 7.4 Hz, 3H). Example 75: Synthesis of Compound 3016 [00280] Compound 3016 was prepared according to Procedure A of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1035 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.5 based on a molecular weight of linker-drug 1186 of Da. Example 76: Synthesis of Compound 3041 [00281] Compound 3041 was prepared according to Procedure B of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1035 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC was 7.5 based on a molecular weight of linker-drug of 1186 Da. Example 77: Synthesis of Compounds 3062A and 3062B [00282] Compound 3062A was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1035 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC was 7.5 based on a molecular weight of linker-drug of 1186 Da. When 25 molar equivalents of linker payload compound 1035 to anti-EGFR IgG1 monoclonal antibody 1 was used, a DAR of 4.7 was achieved to provide compound 3062B. Example 78: Synthesis of Compound 3017 [00283] Compound 3017 was prepared according to Procedure A of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1036 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.5 based on a molecular weight of linker-drug 1186 of Da. Example 79: Synthesis of Compound 3042 [00284] Compound 3042 was prepared according to Procedure B of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1036 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC was 7.5 based on a molecular weight of linker-drug of 1186 Da. Example 80: Synthesis of Compound 3063 [00285] Compound 3063 was prepared according to Procedure C of the General Methods section in Example 1 using 6 molar equivalents of linker payload compound 1036 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC was 7.5 based on a molecular weight of linker-drug of 1186 Da. Example 81: Synthesis of Compound 1037
Figure imgf000134_0001
[00286] Intermediate 1. To the mixture of 195.HCl (1.0 equiv., 15 mg, 0.024 mmol), Fmoc-GGFG-OH (1.05 equiv., 14 mg, 0.025 mmol) and DMTMM (1.05 equiv., 6.9 mg, 0.025 mmol) was added DMF/water (5:1, 1.5 ml) and diisopropylethylamine (2.05 equiv., 8.6 µl, 0.049 mmol) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 80 % ACN/0.1% aq. TFA), offering intermediate 1 as a pale-yellow solid after lyophilization (12 mg, 47 %). MS calc. for C58H56FN8O11: 1059.40, found: 1059.35, [M+H]+. [00287] Compound 1037. To the solution of intermediate 1 (1.0 equiv., 12 mg, 0.011 mmol) in DMF (1.25 ml) was added morpholine (45 µl) and the mixture was stirred for 1 hour at room temperature. The reaction mixture was evaporated to dryness and the residue was redissolved in dry DMF (0.75 ml). Then, Mal-PEG-NHS ester (1.0 equiv., 3.4 mg, 0.011 mmol) and DIPEA (1.05 equiv., 2.0 µl, 0.012 mmol) were added and the reaction mixture was stirred at room temperature for 1 hour. Purification by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 65% ACN/H2O) gave the product 1037 (mixture of isomers) as a white solid after lyophilization (6.6 mg, 58%). MS calc. for C52H55FN9O13: 1032.39, found: 1032.35, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ: 8.58 (d, J = 9.1 Hz, 0.4H), 8.43 (t, J = 6.0 Hz, 0.4H), 8.32 – 8.13 (m, 2.2H), 8.13 – 8.03 (m, 2H), 8.03 – 7.92 (m, 1H), 7.79 – 7.66 (m, 1H), 7.34 (s, 0.4H), 7.31 (s, 0.6H), 7.27 – 7.17 (m, 3H), 7.17 – 7.09 (m, 2H), 6.99 (s, 2H), 6.53 (s, 0.4H), 6.50 (s, 0.6H), 5.85 – 5.75 (m, 0.6H), 5.64 – 5.52 (m, 0.6H), 5.49 – 5.35 (m, 2H), 5.39 – 4.93 (m, 2.8H), 4.52 – 4.42 (m, 0.4H), 4.32 – 4.20 (m, 0.6H), 3.83 – 3.59 (m, 4H), 3.59 – 3.47 (m, 5H), 3.44 (t, J = 5.8 Hz, 2H), 3.18 – 2.92 (m, 2H), 2.90 – 2.57 (m, 2H), 2.45 – 2.26 (m, 7H), 2.26 – 2.04 (m, 1H), 1.92 – 1.75 (m, 3H), 1.30 – 1.20 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H). Example 82: Synthesis of Compound 3018 [00288] Compound 3018 was prepared according to Procedure A of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1037 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.5 based on a molecular weight of linker-drug 1032 of Da. Example 83: Synthesis of Compound 3043 [00289] Compound 3043 was prepared according to Procedure B of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1037 to anti-TROP2 IgG1 monoclonal antibody. DAR based on RPLC was 7.5 based on a molecular weight of linker-drug of 1032 Da. Example 84: Synthesis of Compound 3064 [00290] Compound 3064 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1037 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RPLC was 7.5 based on a molecular weight of linker-drug of 1032 Da. Example 85: Synthesis of Compound 196
Figure imgf000136_0001
[00291] Intermediate 1. To a solution of exatecan mesylate (1.0 equiv., 30 mg, 0.0564 mmol) and diisopropylethyl amine (30 µL) in DMF (2 mL) was added bromoacetic acid (1.2 equiv., 0.0677 mmol, 9.4 mg) and the reaction mixture was stirred overnight at room temperature. The residue was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18,
Figure imgf000136_0002
^ 60% ACN/0.1% TFA), giving intermediate 1 (23 mg, 83 %) as a white powder after lyophilization. MS calc. for C26H23FN3O6: 492.16, found: 492.11 [M-H]-. [00292] Compound 196. To the solution of intermediate 1 (1.0 equiv., 23 mg, 0.0466 mmol) in formic acid (1 ml) was added 37% aqueous formaldehyde (0.25 ml) and the resulting mixture was stirred at 50 °C for 6 hours. Then, water was added, and the mixture was concentrated on rotary evaporator. The residue was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 60% ACN/0.1% TFA), giving 196 (14 mg, 61 %) as a white powder after lyophilization. MS calc. for C27H25FN3O6: 506.17, found: 506.08 [M-H]-.1H NMR (401 MHz, DMSO-d6) δ 7.73 (dd, J = 11.0, 3.8 Hz, 1H), 7.35 (d, J = 4.1 Hz, 1H), 7.31 (s, 1H), 5.54 – 5.43 (m, 2H), 5.43 (d, J = 2.1 Hz, 2H), 4.53 – 4.43 (m, 1H), 3.68 (s, 4H), 3.58 (s, 1H), 3.37 – 3.29 (m, 1H), 2.98 – 2.88 (m, 2H), 2.42 – 2.31 (m, 4H), 1.87 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H). Example 86: Synthesis of Compound 197
Figure imgf000136_0003
[00293] Intermediate 1. To the mixture of exatecan mesylate (1.0 equiv., 40 mg, 0.0753 mmol), Fmoc-L-proline (1.1 equiv., 28 mg, 0.0818 mmol) and DMTMM (1.1 equiv., 23 mg, 0.0818 mmol) was added DMF/water (4:1, 2 ml) and diisopropylethylamine (30 µl) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0
Figure imgf000137_0001
80 % ACN/H2O), offering intermediate 1 as a white solid after lyophilization (45 mg, 79 %). MS calc. for C44H39FN4O7: 755.29, found: 755.56, [M+H]+. [00294] Compound 197. To the solution of intermediate 1 (45 mg, 0.0595 mmol) in DMF (2 ml), was added morpholine (100 µl) and the mixture was stirred for 1 hour at room temperature. The reaction mixture was purified by reverse-phase flash chromatography (semipreparative column, diol-modified C18, 0 ^ 60 % ACN/ 0.1%TFA), giving 197 (26 mg, 82 %) as a white solid. MS calc. for C29H29FN4O5: 533.22, found: 533.08, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ: 8.65 (d, J = 8.7 Hz, 1H), 7.79 (d, J = 10.8 Hz, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.53 (m, 1H), 5.43 (s, 2H), 5.17 (s, 1H), 5.06 (d, J = 18.8 Hz, 1H), 3.73 (m, 1H), 3.73 (m, 2H), 2.93 (m, 2H), 2.39 (s, 3H), 2.18 (m, 3H), 1.88 (m, 3H), 1.72 (m, 1H), 0.89 (t, J = 7.35Hz, 3H). Example 87: Synthesis of Compound 198
Figure imgf000137_0002
[00295] Intermediate 1. To the mixture of exatecan mesylate (1.0 equiv., 45 mg, 0.084 mmol), Fmoc-D-proline (1.1 equiv., 31 mg, 0.092 mmol) and DMTMM (1.1 equiv., 26 mg, 0.092 mmol) was added DMF/water (5:1, 3 ml) and diisopropylethylamine (2.1 equiv., 30 µl, 0.176 mmol) and the resulting mixture was stirred at room temperature for 40 minutes, as LC- MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 80 % ACN/H2O), offering intermediate 1 as a white solid after lyophilization (59 mg, 93 %). MS calc. for C44H39FN4O7: 755.29, found: 755.25, [M+H]+. [00296] Compound 198. To the solution of intermediate 1 (59 mg, 0.078 mmol) in DMF (3 ml), was added morpholine (270 µl) and the mixture was stirred 45 min at room temperature. The reaction mixture was concentrated on rotavap followed by the addition of EtOAc. The precipitated product was isolated by filtration, washed with EtOAc and dried on high vacuum, giving 198 (31 mg, 75 %) as a white solid. MS calc. for C29H29FN4O5: 533.22, found: 533.20, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ: 8.56 (d, J = 9.2 Hz, 1H), 7.75 (d, J = 10.9 Hz, 1H), 7.29 (s, 1H), 6.50 (s, 1H), 5.58 – 5.46 (m, 1H), 5.42 (s, 2H), 5.20 (d, J = 19.0 Hz, 1H), 5.06 (d, J = 18.8 Hz, 1H), 3.67 (dd, J = 8.9, 6.0 Hz, 1H), 3.24 – 3.04 (m, 2H), 2.85 (td, J = 6.5, 2.6 Hz, 2H), 2.36 (s, 3H), 2.20 – 1.99 (m, 3H), 1.84 (m, 3H), 1.63 (m, 1H), 0.87 (t, J = 7.3 Hz, 3H). Example 88: Synthesis of Compound 199
Figure imgf000138_0001
[00297] Intermediate 1. To the mixture of exatecan mesylate (1.1 equiv., 38 mg, 0.0711 mmol), Fmoc-3-fluoro-D-proline (1 equiv., 23 mg, 0.0647 mmol) and DMTMM (1.5 equiv., 27 mg, 0.0971 mmol) was added DMF/water (4:1, 2 ml) and diisopropylethylamine (30 µl) and the resulting mixture was stirred at room temperature for 30 minutes, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^
Figure imgf000138_0002
80 % ACN/ 0.1% TFA), giving intermediate 1 as a white solid after lyophilization (40 mg, 73 %). MS calc. for C44H39F2N4O7: 773.28, found: 773.25, [M+H]+. [00298] Compound 199. To the solution of intermediate 1 (40 mg, 0.0518 mmol) in DMF (2 ml), was added morpholine (100 µl) and the mixture was stirred for 1 h at room temperature. The residue was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 60% ACN/0.1% TFA), giving 199 (20 mg, 69 %) as a white powder after lyophilization. MS calc. for C29H29F2N4O5: 551.21, found: 551.18 [M+H]+.1H NMR (401 MHz, DMSO-d6) δ 9.22 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 11.0 Hz, 1H), 7.33 (s, 1H), 6.56 (s, 1H), 5.71 – 5.62 (m, 2H), 5.53 – 5.48 (m, 1H), 5.41 (s, 1H), 5.39 (d, J = 23.3 Hz, 2H), 5.24 (d, J = 19.0 Hz, 1H), 4.50 (d, J = 3.8 Hz, 1H), 4.43 (d, J = 3.8 Hz, 1H), 3.26 (m, 1H), 3.18 – 3.05 (m, 1H), 2.42 (d, J = 1.9 Hz, 3H), 2.37 – 2.28 (m, 1H), 2.24 (m, 2H), 2.19 – 2.14 (m, 1H), 1.97 – 1.78 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). Example 89: Synthesis of Compounds 200 and 201
Figure imgf000139_0001
[00299] Intermediate 1. To a mixture of exatecan mesylate (1 equiv., 40 mg, 0.0752 mmol), Fmoc-3,3-difluoro-L-proline (1.2 equiv., 18 mg, 0.0903 mmol) and DMTMM (1.2 equiv., 25 mg, 0.0903 mmol) was added DMF/water (4:1, 2 ml) and diisopropylethylamine (30 µl) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^
Figure imgf000139_0002
100 % ACN in 0.1% TFA), giving intermediate 1 as a yellow solid after lyophilization (45 mg, 76 %). MS calc. for C44H38F3N4O7: 791.27, found: 791.35, [M+H]+. [00300] Compounds 200 and 201. To the solution of intermediate 1 (45 mg, 0.0570 mmol) in DMF (2 ml), was added morpholine (100 µl) and the mixture was stirred for 1 h at room temperature. The residue was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18,
Figure imgf000139_0003
^ 60% ACN in 0.1% TFA). The two isomers 200 (16 mg) and 201 (15 mg) were perfectly separated and lyophilized (total yield 95%). [00301] Compound 200. MS calc. for C29H28F3N4O5: 569.20, found: 569.19 [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 8.73 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 11.0 Hz, 1H), 7.32 (s, 1H), 6.53 (s, 1H), 5.58 (dt, J = 8.9, 4.5 Hz, 1H), 5.43 (s, 2H), 5.29 (d, J = 19.1 Hz, 1H), 5.10 (d, J = 19.1 Hz, 1H), 3.81 (m, 1H), 3.58 (s, 1H), 3.24 – 3.09 (m, 4H), 2.94 (m, 1H), 2.41 (d, J = 1.9 Hz, 3H), 2.29 – 2.19 (m, 2H), 1.87 (hept, J = 7.1 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H). [00302] Compound 201. MS calc. for C29H28F3N4O5: 569.20, found: 569.44 [M+H]+.1H NMR (401 MHz, DMSO-d6) δ 9.38 (d, J = 8.2 Hz, 1H), 7.86 (d, J = 10.9 Hz, 1H), 7.34 (s, 1H), 6.57 (s, 1H), 5.68 (dt, J = 7.8, 3.5 Hz, 1H), 5.44 (s, 1H), 5.44 – 5.22 (m, 2H), 4.44 (m, 1H), 3.60 – 3.52 (m, 2H), 3.38 – 3.22 (m, 3H), 3.15 – 3.02 (m, 1H), 2.68 – 2.52 (m, 1H), 2.43 (d, J = 1.9 Hz, 3H), 2.25 (m, 1H), 2.20 – 2.06 (m, 1H), 1.95 – 1.83 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). Example 90: Synthesis of Compound 202
Figure imgf000140_0001
[00303] Compound 202. To a mixture of compound 197 (1 equiv., 10 mg, 0.0188 mmol), Boc-Gly-OH (1.1 equiv., 4 mg, 0.0207 mmol) and DMTMM (1.1 equiv., 6 mg, 0.0207 mmol) was added DMF/water (4:1, 2 ml) and diisopropylethylamine (10 µl) and the resulting mixture was stirred at room temperature for 30 minutes, as LC-MS analysis indicated the full consumption of the starting material. Solvents were evaporated and the crude reaction mixture was re-dissolved in 4 mL of 2 M HCl in dioxane and stirred at room temperature for 4 hours. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 80 % ACN/ 0.1% TFA), giving 202 as a yellow solid after lyophilization (10 mg, 90 %). MS calc. for C31H33FN5O6: 590.24, found: 590.25, [M+H]+.1H NMR (500 MHz, Deuterium Oxide) δ 7.23 (t, J = 2.7 Hz, 1H), 7.15 (d, J = 9.2 Hz, 1H), 5.41 (d, J = 16.0 Hz, 2H), 5.28 (d, J = 16.2 Hz, 1H), 4.89 – 4.81 (m, 1H), 4.45 (dd, J = 8.1, 5.3 Hz, 1H), 4.41 (s, 1H), 4.16 – 4.06 (m, 1H), 3.97 (s, 2H), 3.73 (m, 1H), 3.70 – 3.66 (m, 1H), 3.66 – 3.56 (m, 2H), 3.11 – 3.04 (m, 1H), 2.87 (s, 1H), 2.34 (m, 2H), 2.14 – 2.02 (m, 6H), 1.87 – 1.83 (m, 2H), 0.85 (t, J = 7.3 Hz, 3H). Example 91: Synthesis of Compound 203
Figure imgf000141_0001
[00304] Compound 203. To the mixture of exatecan mesylate (1 equiv., 19 mg, 0.0354 mmol), Boc-Glu(OtBu)-OH (1.5 equiv., 16 mg, 0.0531 mmol) and DMTMM (1.5 equiv., 15 mg, 0.0531 mmol) were added DMF/water (4:1, 2 ml) and diisopropylethylamine (10 µl) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The solvent was removed under reduced pressure and the solid was re-dissolved in 2 mL of dioxane. HCl (4M in dioxane, 2mL) was added and the solution was stirred at room temperature for 2 hours. After the reaction was complete, as indicated by LC-MS analysis, the crude reaction mixture was re-dissolved in DMF and purified by reverse-phase flash chromatography using a semipreparative column (diol-modified C18, 0 ^ 50% ACN/0.1% TFA), giving 203 (12 mg, 62 %) as a yellowish powder after lyophilization. The final product consists of two isomers in equilibrium between each other. MS calc. for C29H30FN4O7: 565.21, found: 565.88 [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 9.01 (m, 1H), 8.26 (m, 2H), 7.84 (m, 1H), 7.34 (d, J = 7.6 Hz, 1H), 5.62 (m, 1H), 5.44 (d, J = 7.3 Hz, 2H), 5.43 – 5.26 (m, 2H), 5.06 (d, J = 18.8 Hz, 1H), 3.82 – 3.73 (m, 1H), 3.51 – 3.38 (m, 2H), 3.19 (d, J = 11.6 Hz, 1H), 2.42 (d, J = 2.0 Hz, 3H), 2.35 (m, 1H), 2.31 (s, 1H), 2.21 – 2.08 (m, 1H), 1.91 (m, 4H), 0.89 (t, J = 7.3 Hz, 3H). Example 92: Synthesis of Compound 204
Figure imgf000141_0002
[00305] Compound 204. To a mixture of exatecan mesylate (20 mg, 0.038 mmol), L-pyroglutamic acid (7.3 mg, 0.056 mmol) and DMTMM (21 mg, 0.076 mmol) was added DMF/water (4:1, 2.5 ml) and diisopropylethylamine (50 µl) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol- modified C18, 0 ^ 100 % ACN in 0.1% TFA), giving 204 as a yellow solid after lyophilization (18.5 mg, 89 %). MS calc. for C29H28FN4O6: 547.19, found: 547.10, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 8.6 Hz, 1H), 7.83 (s, 1H), 7.80 (d, J = 11.0 Hz, 1H), 7.32 (s, 1H), 5.57 (dt, J = 9.0, 4.8 Hz, 1H), 5.43 (d, J = 1.4 Hz, 2H), 5.20 (q, J = 18.8 Hz, 2H), 4.08 (dd, J = 8.4, 4.8 Hz, 1H), 3.58 (s, 3H), 3.19 (t, J = 6.3 Hz, 2H), 2.41 (d, J = 1.9 Hz, 3H), 2.29 – 2.22 (m, 1H), 2.21 – 2.12 (m, 2H), 2.00 (m, 1H), 1.86 (h, J = 7.0 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H). Example 93: Synthesis Compound 1038
Figure imgf000142_0001
Figure imgf000143_0001
[00306] Intermediate 1. To the mixture of exatecan mesylate (1 equiv., 40 mg, 0.0753 mmol), Boc-Glu(OBn)-OH (1.5 equiv., 38 mg, 0.113 mmol) and DMTMM (1.5 equiv., 31 mg, 0.113 mmol) were added DMF/water (3:1, 4 ml) and diisopropylethylamine (25 µl) and the resulting mixture was stirred at room temperature for 3 hours, as LC-MS analysis indicated the full consumption of the starting material. The solvent was removed under reduced pressure and the solid was re-dissolved in 3 mL of dioxane. HCl (4M in dioxane, 3mL) was added and the solution was stirred at room temperature for 2 hours. After the reaction was complete, as indicated by LC-MS analysis, the crude reaction mixture was re-dissolved in DMF and purified by reverse-phase flash chromatography (diol-modified C18, 0 ^
Figure imgf000143_0002
100% ACN/0.1% TFA), giving the desired intermediate 1 (43 mg, 87 %) as a yellowish powder after lyophilization. MS calc. for C36H36FN4O7: 655.26, found: 655.33 [M+H]+. [00307] Intermediate 2. A solution of intermediate 1 (40 mg, 0.0611 mmol), Fmoc- GGFGG-OH (1.1 equiv., 41 mg, 0.0673 mmol), DMTMM (1.1 equiv., 19 mg, 0.0673 mmol) and diisopropylethylamine (25 µl) in DMF/water (3:1, 4 ml) was stirred for 2 hours at room temperature. After the reaction was complete, as indicated by LC-MS analysis, the crude reaction mixture was purified by reverse-phase flash chromatography (diol-modified C18, 0 ^ 100% ACN/0.1% TFA), giving the desired intermediate 2 (52 mg, 71%) as a yellow powder after lyophilization. MS calc. for C66H64FN8O13: 1195.46, found: 1195.77 [M+H]+. [00308] Intermediate 3. The previously prepared intermediate 2 (52 mg, 0.0435 mmol) was dissolved in dioxane (10 mL) and Pd/C (10%, 10mg) was suspended in the solution. Hydrogen was bubbled into the reaction mixture for 3 hours, as LC-MS analysis indicated the full benzoyl deprotection. The mixture was filtered through a syringe filter (0.2 µm) and dioxane was evaporated under reduced pressure. The crude reaction product was re-dissolved in DMF (2 mL) and, after that morpholine (100 µl) was added, the mixture was stirred for one hour at room temperature. After the reaction was complete, as indicated by LC-MS analysis, the crude reaction mixture was purified by reverse-phase flash chromatography using a semipreparative column (diol-modified C18, 0 ^ 100% ACN/0.1% TFA), giving the desired intermediate 3 (17 mg, 44%) as a yellowish solid after lyophilization. MS calc. for C44H48FN8O11: 883.34, found: 883.01 [M+H]+. [00309] Compound 1038. A solution of the previously prepared intermediate 2 (17 mg, 0.0193 mmol), Mal-PEG NHS ester (1.2 equiv., 7 mg, 0.0217 mmol) and diisopropylethylamine (2 µl) in DMF (2 mL) was stirred at room temperature for 2 hours, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 100 % ACN/ 0.1% TFA). Compound 1038 was recovered as a white powder after lyophilization (11 mg, 53%). MS calc. for C53H57FN9O15: 1078.40, found: 1078.67 [M+H]+.1H NMR (500 MHz, DMSO-d6) δ: 8.63 (d, J = 12.7 Hz, 1H), 8.47 (m, 1H), 8.10 (m, 3H), 8.03 (m, 2H), 7.90 (m, 1H), 7.82 – 7.72 (m, 1H), 7.31 (s, 1H), 7.14 – 7.06 (m, 4H), 7.01 (s, 2H), 6.50 (m, 2H), 5.75 (m, 2H), 5.55 (m, 1H), 5.47 – 5.30 (m, 3H), 5.11 – 4.93 (m, 2H), 4.52 – 4.22 (m, 1H), 4.20 (s, 1H), 3.83 – 3.59 (m, 4H), 3.56 (m, 3H), 3.23 (m, 2H), 3.15 (tt, J = 11.7, 4.3 Hz, 2H), 3.10 – 2.92 (m, 3H), 2.90 – 2.57 (m, 2H), 2.42 – 2.38 (m, 4H), 2.06 (m, 1H), 1.94 (m, 1H), 1.87 (m, 1H), 1.22 – 1.06 (m, 1H), 0.92 (m, 3H). Example 94: Synthesis of Compound 205
Figure imgf000144_0001
[00310] N-Boc-GlyProOH. To the solution of N-Boc-Gly NHS ester (277 mg, 1.02 mmol) and L-Pro (117 mg, 1.02 mmol) in DMF (3 ml) was added DIPEA (300 µl), and the mixture was stirred for 1 hour at room temperature. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 100 % ACN), giving N-Boc- GlyProOH as a white solid after lyophilization (270 mg, 97 %). MS calc. for C12H21N2O5: 273.14, found: 273.20, [M+H]+. [00311] Compound 205. To the mixture of 130.HCl (15 mg, 0.028 mmol), N-Boc- GlyProOH (22 mg, 0.081 mmol) and DMTMM (25 mg, 0.089 mmol) were added DMF/water (4:1, 2.5 ml), and DIPEA (30 µl) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 100% ACN, 0.1 % TFA) and fractions containing the product were evaporated. The residue was dissolved in dioxane (1 ml), and HCl in dioxane was added (4M; 2ml). The resulting mixture was stirred at room temperature for 3 hours. Then, the product was isolated by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^
Figure imgf000145_0001
100% ACN, 0.1 % TFA), giving 205 as a yellow solid after lyophilization (7.0 mg, 38%). MS calc. for C33H36FN6O8: 663.25, found: 663.25, [M+H]+.1H NMR (600 MHz, DMSO-d6) δ 11.61 (s, 1H), 8.83 (d, J = 8.6 Hz, 1H), 8.04 (m, 2H), 7.81 (d, J = 10.9 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.62 (m, 1H), 5.44 (m, 2H), 5.37 – 5.19 (m, 2H), 4.45 – 4.30 (m, 2H), 4.13 (dd, J = 8.7, 3.7 Hz, 1H), 3.36 (t, J = 6.8 Hz, 2H), 3.19 (d, J = 6.3 Hz, 3H), 2.53 – 2.52 (m, 2H), 2.41 (d, J = 1.8 Hz, 2H), 2.28 – 2.17 (m, 2H), 2.01 – 1.92 (m, 1H), 1.87 (m, 2H), 1.72 (dd, J = 10.8, 5.4 Hz, 2H), 1.58 (m, 1H), 0.88 (td, J = 7.4, 3.8 Hz, 3H). Example 95: Synthesis of Compound 206
Figure imgf000145_0002
[00312] To a mixture of exatecan mesylate (1.0 equiv., 15 mg, 0.028 mmol), (R)-3- hydroxybutanoic acid (1.1 equiv., 3.2 mg, 0.030 mmol) and DMTMM (1.1 equiv., 8.5 mg, 0.030 mmol) was added DMF/water (5:1, 1.2 ml) and diisopropylethylamine (2.1 equiv., 10 µl, 0.059 mmol) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 60 % ACN/H2O), giving 206 as a white solid after lyophilization (10 mg, 69 %). MS calc. for C28H29FN3O6: 522.20, found: 522.20, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ: 8.41 (d, J = 8.6 Hz, 1H), 7.77 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.54 (dt, J = 9.3, 5.0 Hz, 1H), 5.42 (s, 2H), 5.28 – 5.10 (m, 2H), 4.66 (d, J = 4.6 Hz, 1H), 4.03 (dddd, J = 14.8, 7.3, 5.5, 4.4 Hz, 1H), 3.23 – 3.09 (m, 2H), 2.39 (d, J = 1.9 Hz, 3H), 2.30 (dd, J = 14.0, 7.5 Hz, 1H), 2.21 (dd, J = 13.9, 5.7 Hz, 1H), 2.17 – 2.05 (m, 2H), 1.93 – 1.79 (m, 2H), 1.09 (d, J = 6.1 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H). Example 96: Synthesis of Compound 207
Figure imgf000146_0001
[00313] To a 4:1 DMF/water mixture (4 mL) were added exatecan mesylate (20 mg, 0.038 mmol), cis-3-hydroxycyclobutane-1-carboxylic acid (1.25 equiv., 6 mg, 0.048 mmol), DMTMM (2.0 equiv., 21 mg, 0.076 mmol) and diisopropylethylamine (20 µL). The resulting solution was stirred for 1 hour at room temperature, as LC-MS indicated the full consumption of the starting material. The mixture was directly purified by reverse-phase HPLC chromatography using a semipreparative column (diol-modified C18, 0 ^ 100% ACN/H2O). The desired product 207 was obtained as a white powder after lyophilization (16 mg, 79%). MS calc. for C29H29FN3O6: 534.20, found: 534.45, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 6.49 (s, 1H), 5.60 – 5.51 (m, 1H), 5.42 (s, 2H), 5.11 (m, 2H), ), 5.07 (d, J = 6.2 Hz, 1H), 4.37 (m, 1H), 3.63 – 3.55 (m, 2H), 3.21 – 3.10 (m, 2H), 2.38 (d, J = 1.8 Hz, 3H), 2.29 (t, J = 5.2 Hz, 1H), 2.19 – 2.01 (m, 4H), 1.88 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). Example 97: Synthesis of Compound 1021
Figure imgf000147_0001
[00314] Intermediate 1. The mixture of exatecan mesylate (1.0 equiv., 50 mg, 0.094 mmol), FmocGE(OBn)VCitAP-OH (1.25 equiv., 111 mg, 0.118 mmol) and DMTMM (1.25 equiv., 33 mg, 0.118 mmol) was added DMF/water (5:1, 3.0 ml) and diisopropylethylamine (2.25 equiv., 37 µl, 0.212 mmol) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 75% ACN/H2O), offering intermediate 1 as a white solid after lyophilization (126 mg, 98 %). MS calc. for C72H81FN11O15: 1358.59, found: 1358.60, [M+H]+. [00315] Intermediate 2. To the solution of intermediate 1 (1.0 equiv., 126 mg, 0.092 mmol) in the mixture of DMF (1.0 ml) and dioxane (1.5 ml) was added 10% Pd/C (15 mg) and the reaction mixture was hydrogenated (balloon) for 3 hours at room temperature. LC-MS indicated the full consumption of starting material. The mixture was filtered through the layer of celite and the filtrate was concentrated on rotary evaporator. Morpholine (175 µl) was then added to the obtained solution and the reaction mixture was stirred at room temperature for 1 hour. Purification by reverse-phase flash chromatography (25 g, diol-modified C18,
Figure imgf000147_0002
^ 60% ACN/H2O) offered the product intermediate 2 as a white solid after lyophilization (58 mg, 60 %). MS calc. for C50H65FN11O13: 1046.47, found: 1046.50, [M+H]+. [00316] Compound 1021. Mal-PEG-NHS ester (1.0 equiv., 0.055 mmol, 17 mg) and DIPEA (1.05 equiv., 0.058 mmol, 10 µL) were added to the solution of intermediate 2 (1.0 equiv., 58 mg, 0.055 mmol) in anhydrous DMF (1.5 ml). The reaction mixture was stirred at room temperature for 1.5 h, as LC-MS indicated the full consumption of starting material. Purification by reverse-phase flash chromatography using a semipreparative column (diol- modified C18, 0 ^ 70% ACN/0.1% TFA) offered the desired product as a pale yellow solid after lyophilization (38 mg, 56 %). MS calc. for C59H74FN12O17: 1241.53, found: 1241.50, [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ: 8.45 (d, J = 8.5 Hz, 1H), 8.12 – 7.87 (m, 5H), 7.86 – 7.70 (m, 2H), 7.30 (s, 1H), 7.00 (s, 2H), 5.94 (s, 1H), 5.55 – 5.46 (m, 1H), 5.46 – 5.35 (m, 2H), 5.33 – 5.07 (m, 2H), 4.55 – 4.08 (m, 6H), 3.77 – 3.59 (m, 3H), 3.59 – 3.51 (m, 6H), 3.47 (t, J = 4.9 Hz, 2H), 3.22 – 3.12 (m, 2H), 2.99 – 2.88 (m, 2H), 2.39 (s, 4H), 2.35 – 2.28 (m, 2H), 2.28 – 2.16 (m, 2H), 2.16 – 2.04 (m, 2H), 2.05 – 1.93 (m, 1H), 1.93 – 1.78 (m, 6H), 1.79 – 1.66 (m, 1H), 1.67 – 1.52 (m, 1H), 1.47 – 1.26 (m, 3H), 1.10 (d, J = 6.6 Hz, 3H), 0.94 – 0.74 (m, 9H). Example 98: Synthesis of Compound 3024 [00317] Compound 3024 was prepared according to Procedure A of the General Methods section in Example 1 using 24 molar equivalents of linker payload compound 1021 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.3 based on a molecular weight of linker-drug 1241 of Da. Example 99: Synthesis of Compound 3048 [00318] Compound 3048 was prepared according to Procedure B of the General Methods section in Example 1 using 24 molar equivalents of linker payload compound 1021 to anti-TROP2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-TROP2 antibody drug conjugate based on RPLC-MS was 7.2 based on a molecular weight of linker-drug 1241 of Da.
Example 100: Synthesis of Compound 1022
Figure imgf000149_0001
[00319] Intermediate 1. (S,S)-3-fluoropyrrolidine-2-carboxylic acid (62.5 mg, 0.47 mmol) was dissolved in 1,4-dioxane (1 mL) and H2O (3 mL) and cooled to 0°C. K2CO3 (162 mg, 1.18 mmol) was added, and then Fmoc-Cl (115 mg, 0.45 mmol) was added. The mixture was stirred at RT overnight and H2O (10 mL) was added. The mixture acidified with aqueous HCl (1 M) to pH 2–3 and extracted with DCM (2 × 10 mL). Combined organic layers were dried Na2SO4, concentrated to dryness to give the product as a white solid (122 mg, 76% yield). MS calc. for C20H19FNO4: 356.12, found: 356.22, [M+H]+. [00320] Intermediate 2. Exatecan mesylate (30 mg, 0.056 mmol), previously prepared intermediate 1 (5 equiv., 100 mg) and DIPEA (200 µL) were dissolved in a 5:1 mixture of DMF and water (3 mL). DMTMM (5 equiv., 78 mg) was added, and the reaction mixture was stirred for 30 min at room temperature, to be then directly loaded on column for purification. Purification was performed by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 70% ACN/H2O). Fractions containing the product were lyophilised from water (35 mg, 81%). MS calc. for C44H39F2N4O8 +: 789.27, found: 789.43, [M+H]+. [00321] Intermediate 3. The previously prepared intermediate 2 (35 mg, 0.044 mmol) was dissolved in DMF (2mL) and morpholine (100 µL) was added. The reaction mixture was stirred at room temperature for 1 h. Purification was performed by reverse-phase HPLC chromatography (25 g, diol-modified C18, 0 ^ 50% ACN/H2O). Fractions containing the product were lyophilised from water (19 mg, 76%). MS calc. for C29H29F2N4O6 +: 567.20, found: 567.57, [M+H]+. [00322] Intermediate 4. The previously prepared intermediate 3 (19 mg, 0.035 mmol), Fmoc-GGFGG-COOH (2 equiv., 47 mg) and DIPEA (150 µL) were dissolved in a 5:1 mixture of DMF and water (3 mL). DMTMM (2 equiv., 19.4 mg) was added, and the reaction mixture was stirred for 30 min at room temperature, to be then directly loaded on column for purification. Purification was performed by reverse-phase flash chromatography (semipreparative, diol- modified C18, 0 ^ 50% ACN/H2O). Fractions containing the product were lyophilised from water (32 mg, 80%). MS calc. for C61H60F2N9O12+: 1149.43, found: 1149.25, [M+H]+. [00323] Intermediate 5. The previously prepared intermediate 4 (70 mg, 0.061 mmol) was dissolved in DMF (2mL) and morpholine (100 µL) was added. The reaction mixture was stirred at room temperature for 1 h. Purification was performed by reverse-phase HPLC chromatography (semipreparative, diol-modified C18, 0 ^
Figure imgf000150_0001
50% ACN/H2O). Fractions containing the product were lyophilised from water (21 mg, 38%). MS calc. for C46H50F2N9O10 +: 926.36, found: 926.78, [M+H]+. [00324] Compound 1022. The previously prepared intermediate 5 (21 mg, 0.023 mmol) was dissolved in 1.5 mL of anhydrous DMF.2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.1 equiv., 9.6 mg) and DIPEA (10 µL) were added and the reaction mixture was stirred at room temperature for 1 h. Purification was performed by reverse-phase HPLC chromatography (semipreparative, diol-modified C18, 0 ^ 50% ACN/H2O). Fractions containing the product were lyophilised from water (10 mg, 39%). MS calc. for C54H59FN9O14: 1122.42, found: 1122.45, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ 8.50 (d, J = 8.5 Hz, 1H), 8.29 (t, J = 5.9 Hz, 1H), 8.13 – 8.07 (m, 2H), 7.97 (t, J = 5.7 Hz, 1H), 7.84 – 7.76 (m, 2H), 7.34 (s, 1H), 7.24 (m, 5H), 7.17 (m, 1H), 7.00 (s, 2H), 6.52 (d, J = 4.1 Hz, 1H), 5.55 (dt, J = 8.5, 4.2 Hz, 1H), 5.51 – 5.39 (m, 2H), 5.38 – 5.32 (m, 1H), 5.26 – 5.23 (m, 2H), 4.57 – 4.45 (m, 2H), 4.07 (m, 1H), 3.86 (m, 1H), 3.75 (m, 3H), 3.67 (d, J = 5.6 Hz, 2H), 3.61 (d, J = 5.7 Hz, 1H), 3.55 (m, 4H), 3.46 (t, J = 5.8 Hz, 2H), 3.21 – 3.14 (m, 1H), 3.04 (m, 2H), 2.79 (m, 1H), 2.45 – 2.40 (m, 4H), 2.33 (t, J = 6.6 Hz, 3H), 2.21 (m, 1H), 2.15 (d, J = 5.0 Hz, 1H), 2.11 – 2.04 (m, 1H), 1.87 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H). Example 101: Synthesis of Compound 3021 [00325] Compound 3021 was prepared according to Procedure A of the General Methods section in Example 1 using 24 molar equivalents of linker payload compound 1022 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.9 based on a molecular weight of linker-drug 1121 of Da. Example 102: Synthesis of Compound 3045 [00326] Compound 3045 was prepared according to Procedure B of the General Methods section in Example 1 using 24 molar equivalents of linker payload compound 1022 to anti-TROP2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-TROP2 antibody drug conjugate based on RPLC-MS was 4.5 based on a molecular weight of linker-drug 1121 of Da. Example 103: Synthesis of Compound 3066 [00327] Compound 3066 was prepared according to Procedure C of the General Methods section in Example 1 using 24 molar equivalents of linker payload compound 1022 to anti-EGFR IgG1 monoclonal antibody 1. Drug to antibody ratios (DAR) for anti-EGFR antibody drug conjugate based on RPLC-MS was 7.4 based on a molecular weight of linker-drug 1121 of Da. Example 104: Synthesis of Compound 1028
Figure imgf000152_0001
[00328] Intermediate 1. The mixture of exatecan mesylate (1.0 equiv., 30 mg, 0.056 mmol), FmocGGFGGP-OH (1.5 equiv., 60 mg, 0.084 mmol) and DMTMM (1.5 equiv., 24 mg, 0.084 mmol) was added DMF/water (5:1, 2.4 ml) and diisopropylethylamine (2.5 equiv., 25 µL, 0.141 mmol) and the resulting mixture was stirred at room temperature for 40 minutes, as LC- MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 75% ACN/H2O), offering intermediate 1 as a white solid after lyophilization (68 mg, 98 %). MS calc. for C61H61FN9O12: 1130.44, found: 1130.40, [M+H]+. [00329] Intermediate 2. Morpholine (140 µL) was added to the solution of intermediate 1 (1.0 equiv., 62 mg, 0.055 mmol) in anhydrous DMF (2.0 ml) and the reaction mixture was stirred for 1 h at room temperature. LC-MS indicated the full consumption of starting material. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 ^ 60% ACN/H2O), giving the product as a white solid after lyophilization (45 mg, 90 %). MS calc. for C46H51FN9O10: 908.37, found: 908.40, [M+H]+. [00330] Compound 1028. Mal-PEG-NHS ester (1.0 equiv., 0.05 mmol, 15.5 mg) and DIPEA (1.5 equiv., 0.075 mmol, 13 µL) were added to the solution of intermediate 2 (1.0 equiv., 45 mg, 0.05 mmol) in anhydrous DMF (2 ml). The reaction mixture was stirred at room temperature for 1 h, as LC-MS indicated the full consumption of starting material. DMF was removed and the residue was concentrated from the mixture of 0.1% aq. TFA and ACN. Purification by reverse-phase flash HPLC using a semipreparative column (diol-modified C18, 0 ^ 60% ACN/H2O) offered the desired product as a white solid after lyophilization (20 mg, 37 %). MS calc. for C55H60FN10O14: 1103.43, found: 1103.45, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ: 8.51 (d, J = 8.7 Hz, 1H), 8.29 (t, J = 5.9 Hz, 1H), 8.18 – 8.03 (m, 2H), 7.98 (t, J = 5.7 Hz, 1H), 7.91 – 7.67 (m, 3H), 7.33 – 7.28 (m, 1H), 7.24 (d, J = 4.3 Hz, 5H), 7.21 – 7.10 (m, 2H), 6.99 (s, 2H), 6.57 – 6.47 (m, 1H), 5.56 – 5.46 (m, 1H), 5.46 – 5.37 (m, 2H), 5.28 – 5.00 (m, 2H), 4.59 – 4.44 (m, 1H), 4.37 – 4.26 (m, 1H), 4.00 (dd, J = 17.1, 5.7 Hz, 1H), 3.84 (dd, J = 17.1, 4.9 Hz, 1H), 3.79 – 3.64 (m, 5H), 3.65 – 3.49 (m, 6H), 3.45 (t, J = 5.8 Hz, 2H), 3.26 – 2.99 (m, 2H), 2.86 – 2.74 (m, 1H), 2.38 (s, 3H), 2.32 (t, J = 6.6 Hz, 2H), 2.26 – 1.98 (m, 3H), 1.97 – 1.77 (m, 3H), 0.87 (t, J = 7.3 Hz, 3H). Example 105: Synthesis of Compound 3019 [00331] Compound 3019 was prepared according to Procedure A of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1028 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 6.9 based on a molecular weight of linker-drug 1103 of Da. Example 106: Synthesis of Compound 1029
Figure imgf000153_0001
Figure imgf000154_0001
[00332] Intermediate 1. Exatecan mesylate (50 mg, 0.0941 mmol), Fmoc-Val-Cit-PAB- PNP (50 mg, 0.0652 mmol, 0.7 equiv.) and diisopropylethylamine (50 L) were dissolved in 1.3 mL of anhydrous DMF. The reaction mixture was stirred at room temperature for 12 hours, to be then directly loaded on column and purified by reverse-phase flash chromatography (25 g, diol- modified C18, 0 ^ 75% ACN/H2O). giving the product as a white solid after lyophilization from water-acetonitrile (47 mg, 69 %). MS calc. for C58H60FN8O11: 1063.44, found: 1063.40, [M+H]+. [00333] Intermediate 2. Morpholine (100 µL) was added to the solution of the previously prepared intermediate 1 (53 mg, 0.0443 mmol) in anhydrous DMF (1 ml) and the reaction mixture was stirred for 1 h at room temperature. LC-MS indicated the full consumption of starting material. The mixture was directly purified by reverse-phase HPLC (semipreparative HPLC, diol-modified C18, 0 ^ 100% ACN/H2O), giving the product as a white solid after lyophilization (20.5 mg, 55 %). MS calc. for C43H50FN8O9: 841.37, found: 841.30, [M+H]+. [00334] Compound 1029. Mal-PEG-NHS ester (1.0 equiv., 0.0244 mmol, 7.6 mg) and DIPEA (2 equiv., 0.0488 mmol, 6.4 mg, 8.7 µL) were added to a solution of the previously prepared intermediate 2 (20 mg, 0.0244 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 40 minutes, as LC-MS indicated the full consumption of starting material. Purification by reverse-phase flash HPLC using a semipreparative column (diol-modified C18, 0 ^ 100% ACN/H2O) offered the desired product as a white solid after lyophilization (8 mg, 32 %). MS calc. for C52H59FN9O13: 1036.42, found: 1036.60, [M + H]+.1H NMR (500 MHz, DMSO-d6) δ 9.99 (d, J = 5.7 Hz, 2H), 8.10 (d, J = 7.5 Hz, 1H), 8.06 (d, J = 8.6 Hz, 1H), 7.84 (d, J = 8.6 Hz, 1H), 7.78 (d, J = 10.9 Hz, 1H), 7.62 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 8.7 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.32 (s, 1H), 7.29 – 7.23 (m, 2H), 7.02 (s, 2H), 6.67 (s, 1H), 6.52 (s, 1H), 5.98 (t, J = 6.0 Hz, 1H), 5.45 (s, 2H), 5.44 – 5.40 (m, 3H), 5.29 (d, J = 7.1 Hz, 2H), 5.08 (s, 2H), 4.38 (q, J = 7.4 Hz, 2H), 4.21 (t, J = 7.4 Hz, 2H), 3.57 (m, 3H), 3.48 (m, 2H), 3.02 (m, 1H), 2.95 (m, 1H), 2.81 (s, 2H), 2.60 (s, 1H), 2.43 (m, 1H), 2.39 – 2.32 (m, 3H), 2.19 (m, 2H), 1.97 (m, 1H), 1.87 (m, 1H), 1.76 (s, 1H), 1.70 (m, 1H), 1.60 (m, 1H), 1.49 – 1.35 (m, 1H), 1.17 – 1.06 (m, 1H), 0.91 – 0.79 (m, 3H). Example 107: Synthesis of Compound 3025 [00335] Compound 3025 was prepared according to Procedure A of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1029 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC was 7 based on a molecular weight of linker-drug 1036 of Da. Example 108: Synthesis of Compound 3069 [00336] Compound 3069 was prepared according to Procedure C of the General Methods section in Example 1 using 20 molar equivalents of linker payload compound 1029 to anti-EGFR IgG1 monoclonal antibody 1. Drug to antibody ratios (DAR) for anti-EGFR antibody drug conjugate based on RPLC-MS was 7 based on a molecular weight of linker-drug 1036 of Da. Example 109: Synthesis of Compound 1030 and 1032
Figure imgf000156_0001
[00337] Compounds 1030 and 1032. To a solution of Mal-PEG-GGFGG-OH (1.2 equiv., 37 mg, 0.0633 mmol), compounds 200 and 201 (non-resolved mixture of L and D isomers, 1.0 equiv., 30 mg, 0.0528 mmol) and DMTMM (1.2 equiv., 18 mg, 0.0633 mmol) was added DMF/water (4:1, 2.0 ml) and diisopropylethylamine (1. equiv., 9.3 µl, 0.0528 mmol) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 100% ACN/ 0.1% TFA). The two L and D isomers 1030 and 1032 were separated and re-purified individually by a second flash chromatography using semipreparative column (diol-modified C18, 0 ^ 100 % ACN/ 0.1% TFA). The two isomers were obtained as yellowish solids after lyophilization (16 and 15 mg, 52% total yield). [00338] Compound 1030. MS calc. for C55H58F3N10O14: 1139.41, found: 1139.30, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ 9.02 (d, J = 8.2 Hz, 1H), 8.36 – 8.28 (m, 1H), 8.11 (m, 4H), 7.99 (t, J = 5.7 Hz, 2H), 7.92 (t, J = 5.5 Hz, 1H), 7.83 (m, 1H), 7.34 (d, J = 17.8 Hz, 1H), 7.27 – 7.18 (m, 2H), 7.00 (s, 2H), 5.64 – 5.48 (m, 2H), 5.43 (s, 2H), 5.39 – 5.30 (m, 2H), 4.99 (d, J = 19.1 Hz, 1H), 4.59 – 4.45 (m, 4H), 4.07 (m, 2H), 3.86 – 3.72 (m, 5H), 3.65 – 3.53 (m, 2H), 3.47 (d, J = 5.8 Hz, 2H), 3.24 – 3.01 (m, 2H), 2.80 (m, 2H), 2.42 (s, 3H), 2.34 (m, 3H), 2.20 (m, 1H), 2.08 (m, 1H), 1.86 (m, 3H), 1.24 (d, J = 5.4 Hz, 2H), 0.89 (q, J = 6.9 Hz, 3H). [00339] Compound 1032. MS calc. for C55H58F3N10O14: 1139.41, found: 1139.67, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ 9.09 (d, J = 8.3 Hz, 1H), 8.27 (t, J = 5.8 Hz, 1H), 8.09 (t, J = 7.8 Hz, 3H), 7.96 (t, J = 5.8 Hz, 1H), 7.84 (d, J = 10.9 Hz, 1H), 7.77 (t, J = 5.3 Hz, 1H), 7.36 (s, 1H), 7.24 (m, 5H), 7.18 (m, 1H), 7.00 (s, 2H), 6.54 (s, 2H), 5.56 (m, 1H), 5.49 – 5.39 (m, 2H), 5.26 (d, J = 8.4 Hz, 1H), 4.59 – 4.49 (m, 3H), 4.11 (m, 1H), 4.05 – 3.91 (m, 2H), 3.80 – 3.67 (m, 4H), 3.61 – 3.54 (m, 4H), 3.46 (m, 2H), 3.14 – 3.03 (m, 1H), 2.79 (m, 2H), 2.66 – 2.56 (m, 2H), 2.42 (s, 3H), 2.33 (m, 1H), 2.13 (m, 1H), 1.95 – 1.82 (m, 3H), 1.24 (d, J = 5.2 Hz, 2H), 0.89 (q, J = 7.4 Hz, 3H). Example 110: Synthesis of Compound 3022 [00340] Compound 3022 was prepared according to Procedure A of the General Methods section in Example 1 using 23 molar equivalents of linker payload compound 1030 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.4 based on a molecular weight of linker-drug 1139 of Da. Example 111: Synthesis of Compound 3046 [00341] Compound 3046 was prepared according to Procedure B of the General Methods section in Example 1 using 23 molar equivalents of linker payload compound 1030 to anti-TROP2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-TROP2 antibody drug conjugate based on RPLC was 6.5 based on a molecular weight of linker-drug 1139 of Da. Example 112: Synthesis of Compound 3067 [00342] Compound 3067 was prepared according to Procedure C of the General Methods section in Example 1 using 31 molar equivalents of linker payload compound 1030 to anti-EGFR IgG1 monoclonal antibody 1. Drug to antibody ratios (DAR) for anti-EGFR antibody drug conjugate based on RPLC was 7.4 based on a molecular weight of linker-drug 1139 of Da. Example 113: Synthesis of Compound 3023 [00343] Compound 3023 was prepared according to Procedure A of the General Methods section in Example 1 using 27 molar equivalents of linker payload compound 1032 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.4 based on a molecular weight of linker-drug 1139 of Da. Example 114: Synthesis of Compound 3047 [00344] Compound 3047 was prepared according to Procedure B of the General Methods section in Example 1 using 27 molar equivalents of linker payload compound 1032 to anti-TROP2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-TROP2 antibody drug conjugate based on RPLC was 7.4 based on a molecular weight of linker-drug 1139 of Da. Example 115: Synthesis of Compound 3068 [00345] Compound 3068 was prepared according to Procedure C of the General Methods section in Example 1 using 27 molar equivalents of linker payload compound 1032 to anti-EGFR IgG1 monoclonal antibody 1. Drug to antibody ratios (DAR) for anti-EGFR antibody drug conjugate based on RPLC was 7.4 based on a molecular weight of linker-drug 1139 of Da. Example 116: Synthesis of Compound 1031
Figure imgf000158_0001
[00346] Compound 1031. To the solution of Mal-PEG-GGFGG-OH (1.0 equiv., 22 mg, 0.037 mmol), Sc-598 (1.0 equiv., 20 mg, 0.037 mmol) and DMTMM (1.1 equiv., 12 mg, 0.041 mmol) was added DMF/water (5:1, 2.0 ml) and diisopropylethylamine (1.1 equiv., 7.2 µl, 0.041 mmol) and the resulting mixture was stirred at room temperature for 50 minutes, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 70% ACN/H2O), offering 1031 as a white solid after lyophilization (29 mg, 71%). MS calc. for C55H60FN10O14: 1103.43, found: 1103.30, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ: 8.53 (d, J = 8.6 Hz, 1H), 8.26 (t, J = 5.8 Hz, 1H), 8.15 – 8.02 (m, 2H), 7.96 (t, J = 5.7 Hz, 1H), 7.80 (d, J = 11.0 Hz, 1H), 7.67 (t, J = 5.1 Hz, 1H), 7.34 (s, 1H), 7.27 – 7.20 (m, 4H), 7.20 – 7.13 (m, 1H), 6.99 (s, 2H), 6.52 (s, 1H), 5.60 – 5.36 (m, 4H), 5.14 (d, J = 19.2 Hz, 1H), 4.58 – 4.46 (m, 1H), 4.26 (dd, J = 8.1, 4.2 Hz, 1H), 4.04 (dd, J = 17.0, 5.7 Hz, 1H), 3.87 (dd, J = 17.1, 4.4 Hz, 1H), 3.78 (t, J = 5.8 Hz, 1H), 3.73 (dd, J = 16.9, 5.8 Hz, 1H), 3.66 (d, J = 5.7 Hz, 2H), 3.60 (d, J = 5.1 Hz, 1H), 3.58 – 3.49 (m, 6H), 3.45 (t, J = 5.8 Hz, 2H), 3.22 – 3.06 (m, 2H), 3.03 (dd, J = 13.8, 4.5 Hz, 1H), 2.77 (dd, J = 13.8, 9.7 Hz, 1H), 2.40 (s, 3H), 2.32 (t, J = 6.5 Hz, 2H), 2.18 – 2.01 (m, 4H), 1.99 – 1.79 (m, 4H), 0.89 (t, J = 7.1 Hz, 3H). Example 117: Synthesis of Compound 3020 [00347] Compound 3020 was prepared according to Procedure A of the General Methods section in Example 1 using 31 molar equivalents of linker payload compound 1031 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.4 based on a molecular weight of linker-drug 1103 of Da. Example 118: Synthesis of Compound 3044 [00348] Compound 3044 was prepared according to Procedure B the General Methods section in Example 1 using 31 molar equivalents of linker payload compound 1031 to anti- TROP2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-TROP2 antibody drug conjugate based on RPLC was 7.4 based on a molecular weight of linker-drug 1103 of Da. Example 119: Synthesis of Compound 3065 [00349] Compound 3065 was prepared according to Procedure C of the General Methods section in Example 1 using 31 molar equivalents of linker payload compound 1031 to anti-EGFR IgG1 monoclonal antibody 1. Drug to antibody ratios (DAR) for anti-EGFR antibody drug conjugate based on RPLC was 7.4 based on a molecular weight of linker-drug 1103 of Da. Example 120: Synthesis of Compound 1033
Figure imgf000160_0001
[00350] Intermediate 1. To the mixture of exatecan mesylate (1.0 equiv., 25 mg, 0.0470 mmol), Fmoc-Val-Ala-OH (1.2 equiv., 23 mg, 0.0564 mmol) and DMTMM (1.2 equiv., 16 mg, 0.0564 mmol) was added DMF/water (4:1, 1.8 ml) and diisopropylethylamine (25 µl) and the resulting mixture was stirred at room temperature for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (25 g diol-modified C18, 0 ^
Figure imgf000160_0002
100% ACN/0.1% aq. TFA), offering intermediate 1 as a yellow solid after lyophilization (37 mg, 95%). MS calc. for C47H47FN5O8: 828.34, found: 828.45, [M+H]+. [00351] Intermediate 2. The previously prepared intermediate 1 (37 mg, 0.0447 mmol) was dissolved in 2 mL of DMF and morpholine (100 µL) was added. The reaction mixture was stirred for one hour at room temperature and the crude reaction mixture was purified by reverse- phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 70% ACN/ 0.1% TFA). The product was obtained as a yellow solid after lyophilization (25 mg, 92%). MS calc. for C32H37FN5O6: 606.27, found: 606.24, [M+H]+. [00352] Compound 1033. The previously prepared intermediate 2 (1.05 equiv.15 mg, 0.0248 mmol) was dissolved in 2 mL of DMF and MI-amido-PEG8-NHS ester (1 equiv., 0.0236 mmol, 16 mg) and diisopropylethylamine (1.1 equiv., 0.0260 mmol, 4.54 µL) were added, and the mixture was stirred at room temperature for 1 hour. The crude reaction mixture was purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18,
Figure imgf000161_0001
70% ACN/H2O). The product 1033 was obtained as a white solid after lyophilization (4.2 mg, 85%). MS calc. for C58H79FN7O18: 1180.55, found: 1180.10, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ: 8.40 (d, J = 8.6 Hz, 1H), 8.09 (d, J = 7.0 Hz, 1H), 8.00 (t, J = 5.7 Hz, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.79 (d, J = 10.9 Hz, 1H), 7.30 (s, 1H), 6.99 (s, 2H), 6.51 (s, 1H), 5.53 (dt, J = 9.2, 4.9 Hz, 1H), 5.42 (s, 2H), 5.23 (d, J = 18.7 Hz, 1H), 5.11 (d, J = 18.9 Hz, 1H), 4.27 (p, J = 7.0 Hz, 1H), 4.12 (dd, J = 8.6, 6.6 Hz, 1H), 3.58 (t, J = 7.3 Hz, 2H), 3.56 – 3.41 (m, 32H), 3.36 (t, J = 5.9 Hz, 2H), 3.23 – 3.10 (m, 4H), 2.47 – 2.38 (m, 4H), 2.32 (t, J = 7.2 Hz, 2H), 2.19 – 2.05 (m, 2H), 1.96 – 1.79 (m, 2H), 1.27 (d, J = 7.0 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H), 0.79 (dd, J = 6.9, 3.6 Hz, 6H). Example 121: Synthesis of Compound 3026 [00353] Compound 3026 was prepared according to Procedure A of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1033 to anti-HER2 IgG1 monoclonal antibody. Drug to antibody ratios (DAR) for anti-HER2 antibody drug conjugate based on RPLC-MS was 7.5 based on a molecular weight of linker-drug 1180 of Da. Example 122: Synthesis of Compound 3070 [00354] Compound 3070 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload compound 1033 to anti-EGFR IgG1 monoclonal antibody 1. Drug to antibody ratios (DAR) for anti-EGFR antibody drug conjugate based on RPLC was 7.5 based on a molecular weight of linker-drug 1180 of Da. Example 123: Synthesis of Compound 71
Figure imgf000162_0001
[00355] Intermediate 1. Bromoacetic acid (0.715 g, 10 mmol) was dissolved in 5 mL of water, then sodium azide (0.696 g, 5 mmol) was added and the solution was stirred at room temperature overnight. The solution was acidified with HCl until pH = 1, then the desired product was extracted with diethyl ether. The solvent was dried over Na2SO4, filtered, and then evaporated, affording the reaction product which was used into the next step without any further purification. [00356] Intermediate 2. Exatecan mesylate (100 mg, 0.188 mmol), 2-azidoacetic acid (1.1 equiv., 0.207 mmol, 21 mg), DMTMM (1.3 equiv., 0.244 mmol, 68mg) and DIPEA (50 µL) were dissolved into 5 mL of a 4:1 DMF/water mixture. The reaction mixture was stirred at room temperature for 1 hour. The mixture was directly purified by reverse-phase flash chromatography (diol-modified C18, 25g, 0 ^
Figure imgf000162_0002
100% ACN in water). The desired product was obtained as a white powder after lyophilization (79 mg, 74%). MS calc. for C26H24FN6O5: 519.18, found: 519.41, [M+H]+. [00357] Compound 71. The previously prepared intermediate 2 (10 mg, 0.0193 mmol), propargyl alcohol (1.2 equiv., 0.0232 mmol, 1.35 µL), and the catalyst CpRu(COD)Cl (10%, 0,7 mg) were suspended in anhydrous DCM (2 mL) under an argon atmosphere. The mixture was stirred at 40 C for 16 hours. The crude reaction product was directly purified by reverse-phase HPLC chromatography (semipreparative diol-modified C18, 0 ^ 100% ACN in water). The desired product was obtained as a yellowish powder after lyophilization from water-ACN (9 mg, 90%). MS calc. for C29H28FN6O6: 575.21, found: 575.14, [M+H]+. Example 124: Synthesis of Compound 72
Figure imgf000163_0001
[00358] Compound 72. The previously prepared intermediate 2 of Example 63 (10 mg, 0.0193 mmol), propargyl alcohol (1.2 equiv., 0.0232 mmol, 1.35 µL), sodium ascorbate (0.2 equiv., 0.00386 mmol, 2 M in water, 1.93 µL), copper sulphate pentahydrate (0.1 equiv., 0.00193 mmol, 1 M in water, 1.93 µL) and TBTA (0.15 equiv., 0.0029 mmol, 1.5 mg) were dissolved in 2 mL of a 4:1 mixture of DMF /water. The reaction mixture was stirred at room temperature for 2 hours. The crude reaction product was directly purified by reverse-phase HPLC chromatography (semipreparative diol-modified C18, 0 ^ 100% ACN in water). The desired product was obtained as a yellowish powder after lyophilization from water-ACN (10 mg, 95%). MS calc. for C29H28FN6O6: 575.21, found: 575.55, [M+H]+. Example 125: Synthesis of Compound 73
Figure imgf000163_0002
[00359] Compound 73. The previously prepared intermediate 2 of Example 63 (10 mg, 0.0193 mmol), 3-butyn-1-ol (1.2 equiv., 0.0232 mmol, 1.50 µL), and the catalyst CpRu(COD)Cl (10%, 0,7 mg) were suspended in anhydrous DCM (2 mL) under an argon atmosphere. The mixture was stirred at 40 C for 16 hours. The crude reaction product was directly purified by reverse-phase HPLC chromatography (semipreparative diol-modified C18, 0 ^ 100% ACN in water). The desired product was obtained as a yellowish powder after lyophilization from water- ACN (6 mg, 54%). MS calc. for C30H30FN6O6: 589.22, found: 589.23, [M+H]+. Example 126: Synthesis of Compound 74
Figure imgf000164_0001
[00360] Compound 74. The previously prepared intermediate 2 of Example 63 (10 mg, 0.0193 mmol), 3-butyn-1-ol (1.2 equiv., 0.0232 mmol, 1.50 µL), sodium ascorbate (0.2 equiv., 0.00386 mmol, 2 M in water, 1.93 µL), copper sulphate pentahydrate (0.1 equiv., 0.00193 mmol, 1 M in water, 1.93 µL) and TBTA (0.15 equiv., 0.0029 mmol, 1.5 mg) were dissolved in 2 mL of a 4:1 mixture of DMF /water. The reaction mixture was stirred at room temperature for 2 hours. The crude reaction product was directly purified by reverse-phase HPLC chromatography (semipreparative diol-modified C18, 0 ^ 100% ACN in water). The desired product was obtained as a white powder after lyophilization from water-ACN (7 mg, 62%). MS calc. for C30H30FN6O6: 589.22, found: 589.05, [M+H]+. Example 127: Synthesis of Compound 208
Figure imgf000164_0002
[00361] Intermediate 1. To a 4:1 DMF/water mixture (4 mL) were added exatecan mesylate (20 mg, 0.0376 mmol), 3-(1,3-dioxolan-2-yl)propanoic acid (2 equiv., 0.0753 mmol, 11 mg), DMTMM (1.5 equiv., 0.0564 mmol, 16 mg) and diisopropylethylamine (20 µL). The resulting solution was stirred for 1 hour at room temperature, as LC-MS indicated the full consumption of the starting material. The mixture was directly purified by reverse-phase HPLC chromatography using a semipreparative column (diol-modified C18, 0 ^ 100% ACN/H2O). The desired product was obtained as a white powder after lyophilisation (19 mg, 89%). MS calc. for C30H31FN3O7: 564.21, found: 564.34, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 8.7 Hz, 1H), 7.78 (d, J = 10.9 Hz, 1H), 7.30 (s, 1H), 6.52 (s, 1H), 5.56 (dt, J = 9.3, 5.0 Hz, 1H), 5.43 (s, 2H), 5.31 – 5.11 (m, 2H), 4.83 (t, J = 4.5 Hz, 1H), 3.89 – 3.80 (m, 2H), 3.79 – 3.69 (m, 2H), 3.24 – 3.10 (m, 2H), 2.40 (d, J = 1.9 Hz, 3H), 2.26 (t, J = 7.6 Hz, 2H), 2.14 (q, J = 7.3, 6.6 Hz, 2H), 1.98 – 1.77 (m, 4H), 0.88 (t, J = 7.3 Hz, 3H). [00362] Compound 208. Intermediate 1 (20 mg) was dissolved into 1 mL of 0.1% TFA aqueous solution and the resulting mixture was stirred for 5 hours at room temperature, as LC- MS indicated the full consumption of the starting material. The mixture was directly purified by reverse-phase HPLC chromatography using a semipreparative column (diol-modified C18, 0 ^100% ACN/1% TFA). The desired product was obtained as a white powder after lyophilization (17 mg, 94%). MS calc. for C28H27FN3O6: 520.19, found: 520.31, [M+H]+. NMR spectra were not obtained due to the instability of the material. Example 128: Synthesis of Compound 209
Figure imgf000165_0001
Figure imgf000165_0002
[00363] Intermediate 1. To the solution of (S)-1-(benzyloxy)propan-2-ol (1.0 equiv., 100 mg, 0.6 mmol) and bis(4-nitrophenyl) carbonate (2.0 equiv., 366 mg, 1.2 mmol) in DMF (4 mL) was added diisopropylethylamine (1.1 equiv., 115 µL, 0.66 mmol) and the resulting mixture was stirred at room temperature for 2 hours. DMF was removed on rotary evaporator and the residue was purified by flash chromatography (SiO2, 0 – 20% EtOAc/cyclohexane), giving the product Intermediate 1 (135 mg, 68 %) as a clear thick oil. [00364] Intermediate 2. To the mixture of Intermediate 1 (1.0 equiv., 40 mg, 0.12 mmol) and exatecan mesylate (1.0 equiv., 65 mg, 0.12 mmol) was added DMF (1.5 mL) and diisopropylethylamine (3.0 equiv., 0.36 mmol, 62 µL) and the resulting solution was stirred at room temperature overnight. Purification by reverse-phase flash chromatography (diol-modified C18, 0 ^ 60% ACN/10mM aq. NH4OAc) offered the product Intermediate 2 (55 mg, 73 %) as a white solid after lyophilization. [00365] Compound 209. To the solution of Intermediate 2 (1.0 equiv., 20 mg, 0.032 mmol) in dioxane/water (2:1, 1 mL) was added 10% Pd/C and the resulting mixture was hydrogenated (H2 in balloon) for 2 hours. Then, the suspension was filtered and the filtrate was evaporated on rotary evaporator. Purification of the residue by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 60% ACN/10mM aq. NH4OAc) offered compound 209 (9 mg, 53 %) as a white solid after lyophilization. MS calc. for C28H29FN3O7: 538.20, found: 538.10, [M+H]+.1H NMR (400 MHz, DMSO-d6) δ: 7.90 (d, J = 9.0 Hz, 1H), 7.71 (d, J = 10.9 Hz, 1H), 7.28 (s, 1H), 6.50 (s, 1H), 5.41 (s, 2H), 5.30 – 5.20 (m, 2H), 5.06 (d, J = 19.0 Hz, 1H), 4.86 – 4.76 (m, 2H), 3.53 – 3.40 (m, 2H), 3.30 – 3.19 (m, 1H), 3.17 – 3.00 (m, 1H), 2.34 (d, J = 1.8 Hz, 3H), 2.29 – 2.18 (m, 1H), 2.15 – 2.00 (m, 1H), 1.93 – 1.77 (m, 2H), 1.26 (d, J = 6.4 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H). Example 129: Synthesis of Compound 1039
Figure imgf000167_0001
[00366] Intermediate 1. Compound 209 (80 mg, 0.15 mmol) and Fmoc-GGFG-OAc (70 mg, 0.11 mmol) were co-evaporated 3 times from anhydrous DMF, then re-dissolved in anhydrous DMF (4 mL). HCl (4M in dioxane, 30 µL) was added and the reaction mixture was stirred for 60 min at 50 °C. Then DMF was removed on rotary evaporator and the residue was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0 ^ 50% ACN/10mM aq. NH4OAc), giving the product Intermediate 1 (30 mg, 25 %) as a white solid. MS calc. for C59H60FN8O13: 1107.42, found: 1107.45, [M + H]+. [00367] Intermediate 2. To a solution of Intermediate 1 (30 mg, 0.027 mmol) in 2 mL of anhydrous DMF was added morpholine (100 µL). The reaction mixture was stirred at room temperature for one hour and then DMF was removed on rotary evaporator. The reaction product was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0 ^ 100% ACN/0.1% TFA), giving the product Intermediate 2 (15 mg, 63 %) as a white solid. MS calc. for C44H49FN8O11: 885.35, found: 885.25, [M + H]+. [00368] Compound 1039. Intermediate 2 (15 mg, 0.017 mmol) was dissolved in 1 mL of DMF.2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.5 equiv., 0.025 mmol, 7.9 mg) and DIPEA (5 µL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing diol-modified C18 (0 ^ 100% ACN/10mM aq. NH4OAc). The desired product was recovered as a white solid, after lyophilization from water (11 mg , 60 %). MS calc. for C53H59FN9O15: 1080.40, found: 1080.35, [M + H]+.1H NMR (500 MHz, DMSO-d6) δ 8.54 (t, J = 6.7 Hz, 1H), 8.31 (t, J = 5.9 Hz, 1H), 8.14 – 8.08 (m, 2H), 8.00 (dd, J = 7.3, 4.1 Hz, 2H), 7.76 (d, J = 10.8 Hz, 1H), 7.31 (s, 1H), 7.25 (t, J = 4.4 Hz, 4H), 7.19 (m, 1H), 7.00 (s, 2H), 6.51 (s, 1H), 5.42 (d, J = 2.8 Hz, 2H), 5.30 – 5.24 (m, 2H), 5.13 (d, J = 19.0 Hz, 1H), 4.95 (m, 1H), 4.61 (m, 2H), 4.54 – 4.46 (m, 1H), 3.75 (m, 3H), 3.68 (s, 1H), 3.58 – 3.53 (m, 5H), 3.49 – 3.45 (m, 4H), 3.29 – 3.22 (m, 1H), 3.13 – 3.03 (m, 2H), 2.81 (dd, J = 13.9, 9.6 Hz, 1H), 2.39 – 2.36 (m, 3H), 2.33 (t, J = 6.6 Hz, 2H), 2.29 – 2.23 (m, 1H), 2.13 – 2.06 (m, 1H), 1.87 (m, 2H), 1.28 (d, J = 6.4 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H). Example 130: Synthesis of Compound 210
Figure imgf000168_0001
[00369] Intermediate 1. To the solution of (R)-1-(benzyloxy)propan-2-ol ( 100 mg, 0.6 mmol) and bis(4-nitrophenyl) carbonate (2.0 equiv., 366 mg, 1.2 mmol) in DMF (2 mL) was added diisopropylethylamine (1.1 equiv., 115 µL, 0.66 mmol) and the resulting mixture was stirred at room temperature for 2 hours. DMF was removed on rotary evaporator and the residue was purified by flash chromatography (SiO2, 0 – 20% EtOAc/cyclohexane), giving the product Intermediate 1 (161 mg, 82 %) as a yellowish oil. [00370] Intermediate 2. To the mixture of Intermediate 1 (1.0 equiv., 50 mg, 0.15 mmol) and exatecan mesylate (1.0 equiv., 80 mg, 0.15 mmol) was added DMF (3 mL) and diisopropylethylamine (200 µL) and the resulting solution was stirred at room temperature overnight. Purification by reverse-phase flash chromatography (diol-modified C18, 0 ^ 100% ACN/10mM aq. NH4OAc) offered the product Intermediate 2 (67 mg, 72 %) as a white solid after lyophilization. [00371] Compound 210. To a solution of Intermediate 2 (67 mg, 0.11 mmol)) in dioxane (5 mL) was added 10% Pd/C and the resulting mixture was hydrogenated (H2 in balloon) for 4 hours. The suspension was filtered and the filtrate was evaporated on rotary evaporator. Purification of the residue by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 60% ACN/10mM aq. NH4OAc) offered compound 210 (40 mg, 70 %) as a white solid after lyophilization. MS calc. for C28H29FN3O7: 538.20, found: 538.25, [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 7.92 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 6.53 (s, 1H), 5.43 (s, 2H), 5.31 – 5.20 (m, 2H), 5.15 (d, J = 19.1 Hz, 1H), 4.87 – 4.74 (m, 2H), 3.54 – 3.45 (m, 1H), 3.32 – 3.20 (m, 1H), 3.15 – 3.03 (m, 1H), 2.36 (d, J = 1.8 Hz, 3H), 2.27 – 2.18 (m, 1H), 2.17 – 2.08 (m, 1H), 1.88 (m, 2H), 1.21 (d, J = 6.4 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). Example 131: Synthesis of Compound 1040
Figure imgf000170_0001
[00372] Intermediate 1. Compound 210 (80 mg, 0.15 mmol) and Fmoc-GGFG-OAc (70 mg, 0.11 mmol) were co-evaporated 3 times from anhydrous DMF, then re-dissolved in anhydrous DMF (2 mL). HCl (4M in dioxane, 80 µL) was added and the reaction mixture was stirred for 60 min at 50 °C. Then DMF was removed on rotary evaporator and the residue was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0 ^ 50% ACN/10mM aq. NH4OAc), giving the product Intermediate 1 (64 mg, 52 %) as a white solid. MS calc. for C59H60FN8O13: 1107.42, found: 1107.35, [M + H]+. [00373] Intermediate 2. To a solution of Intermediate 1 (52 mg, 0.047 mmol) in 2 mL of anhydrous DMF was added morpholine (100 µL). The reaction mixture was stirred at room temperature for one hour and then DMF was removed on rotary evaporator. The reaction product was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0 ^ 100% ACN/0.1% TFA), giving the product Intermediate 2 (31 mg, 76 %) as a white solid. MS calc. for C44H49FN8O11: 885.35, found: 885.24, [M + H]+. [00374] Compound 1040. Intermediate 2 (31 mg, 0.017 mmol) was dissolved in 1 mL of DMF.2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.5 equiv., 0.025 mmol, 7.9 mg) and DIPEA (5 µL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing diol-modified C18 (0 ^ 100% ACN/10mM aq. NH4OAc). The product was re-purified by reverse-phase flash HPLC, using 0 ^
Figure imgf000171_0001
100% ACN/0.1% TFA and subsequently using 0 ^ 100% ACN/10mM aq. NH4OAc. The desired product 1040 was recovered as a white solid, after lyophilization from water (12 mg, 31 %), it contains 5% of compound 209. MS calc. for C53H59FN9O15: 1080.40, found: 1080.66, [M + H]+. 1H NMR (500 MHz, DMSO-d6) δ 8.50 (t, J = 6.2 Hz, 1H), 8.25 (m, 1H), 8.12 – 8.04 (m, 2H), 8.01 – 7.91 (m, 2H), 7.86 (s, 1H), 7.25 – 7.11 (m, 6H), 7.00 (s, 2H), 6.56 (s, 1H), 5.44 (m, 2H), 5.35 – 5.25 (m, 2H), 5.11 (m, 1H), 4.95 (m, 1H), 4.60 (m, 2H), 4.50 (m, 1H), 3.75 – 3.65 (m, 4H), 3.56 – 3.43 (m, 8H), 3.29 – 3.19 (m, 2H), 3.06 (m, 2H), 2.71 (m, 1H), 2.40 (m, 3H), 2.29 (m, 2H), 2.23 (m, 1H), 2.12 – 2.00 (m, 1H), 1.80 (m, 2H), 1.27 (d, J = 6.2 Hz, 3H), 0.89 (t, J = 7.7 Hz, 3H). Example 132: Synthesis of Compound 211
Figure imgf000171_0002
[00375] Intermediate 1. To the solution of (S)-2-(benzyloxy)propan-1-ol (1.0 equiv., 100 mg, 0.6 mmol) and bis(4-nitrophenyl) carbonate (2.0 equiv., 366 mg, 1.2 mmol) in DMF (2 mL) was added diisopropylethylamine (1.5 equiv., 157 µL, 0.9 mmol) and the resulting mixture was stirred at room temperature for 2 hours. DMF was removed on rotary evaporator and the residue was purified by flash chromatography (SiO2, 0 – 20% EtOAc/cyclohexane), giving the product Intermediate 1 (186 mg, 93 %) as a clear thick oil. [00376] Intermediate 2. To the mixture of Intermediate 1 (1.2 equiv., 60 mg, 0.18 mmol) and exatecan mesylate (1.0 equiv., 80 mg, 0.15 mmol) was added DMF (5 mL) and diisopropylethylamine (100 µL) and the resulting solution was stirred at room temperature overnight. DMF was removed on rotary evaporator and purification by reverse-phase flash chromatography (diol-modified C18, 0 ^ 50% ACN/10mM aq. NH4OAc) offered the product Intermediate 2 (90 mg, 96 %) as a white solid after lyophilization. MS calc. for C35H35FN3O7: 628.24, found: 628.20 [M+H]+. [00377] Compound 211. To the solution of Intermediate 2 (1.0 equiv., 90 mg, 0.14 mmol) in dioxane (5 mL) was added 10% Pd/C and the resulting mixture was hydrogenated (H2 in balloon) for 5 hours. Then, the suspension was filtered and the filtrate was evaporated on rotary evaporator. Purification of the residue by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 50% ACN/10mM aq. NH4OAc) offered compound 211 (71 mg, 92 %) as a white solid after lyophilization. MS calc. for C28H29FN3O7: 538.19, found: 508.15, [M+H]+. Example 133: Synthesis of Compound 1041
Figure imgf000173_0001
[00378] Intermediate 1. Compound 211 (70 mg, 0.13 mmol) and Fmoc-GGFG-OAc (65 mg, 0.10 mmol) were co-evaporated 3 times from anhydrous DMF, then re-dissolved in anhydrous DMF (4 mL). HCl (4M in dioxane, 30 µL) was added and the reaction mixture was stirred for 2 h at 50 °C. Then DMF was removed on rotary evaporator and the residue was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0 ^ 50% ACN/10mM aq. NH4OAc), giving the product Intermediate 1 (32 mg, 29 %) as a white solid. MS calc. for C59H60FN8O13: 1107.42, found: 1107.30, [M + H]+. [00379] Intermediate 2. To a solution of Intermediate 1 (32 mg, 0.029 mmol) in 2 mL of anhydrous DMF was added morpholine (100 µL). The reaction mixture was stirred at room temperature for one hour and then DMF was removed on rotary evaporator. The reaction product was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0 ^ 100% ACN/0.1% TFA), giving the product Intermediate 2 (15 mg, 59 %) as a white solid. MS calc. for C44H49FN8O11: 885.35, found: 885.20, [M + H]+. [00380] Compound 1041. Intermediate 2 (15 mg, 0.017 mmol) was dissolved in 1 mL of DMF.2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.5 equiv., 0.025 mmol, 7.9 mg) and DIPEA (5 µL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The product was purified by reverse-phase flash HPLC, using a semipreparative column containing diol-modified C18 (0 ^ 100% ACN/10mM aq. NH4OAc). The desired product was recovered as a white solid, after lyophilization from water (11 mg , 60 %). MS calc. for C53H59FN9O15: 1080.40, found: 1080.35, [M + H]+.
Figure imgf000174_0001
NMR (500 MHz, DMSO-d6) δ 8.49 (t, J = 6.6 Hz, 1H), 8.28 (q, J = 5.1 Hz, 1H), 8.10 (q, J = 4.7, 3.7 Hz, 2H), 7.99 (q, J = 7.0, 5.8 Hz, 2H), 7.78 (d, J = 10.8 Hz, 1H), 7.34 – 7.31 (m, 1H), 7.26 – 7.15 (m, 5H), 7.00 (s, 1H), 6.52 (s, 1H), 5.43 (d, J = 2.8 Hz, 2H), 5.25 (q, J = 6.9, 6.3 Hz, 3H), 4.66 – 4.61 (m, 2H), 4.48 (q, J = 8.8, 8.3 Hz, 1H), 4.12 – 4.00 (m, 1H), 3.98 (m, 1H), 3.82 (m, 1H), 3.77 – 3.63 (m, 5H), 3.63 – 3.48 (m, 5H), 3.46 (t, J = 5.8 Hz, 2H), 3.29 – 3.04 (m, 1H), 3.03 (m, 1H), 2.78 (dt, J = 13.8, 8.8 Hz, 1H), 2.38 (s, 3H), 2.33 (t, J = 6.5 Hz, 2H), 2.23 – 2.12 (m, 2H), 1.95 – 1.81 (m, 2H), 1.13 (t, J = 6.6 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). Example 134: Synthesis of Compound 1042
Figure imgf000174_0002
[00381] Intermediate 1. Fmoc-GGFG-OAc (60 mg, 0.095 mmol) and 2,2-difluoro-3- hydroxypropanoic acid (36 mg, 0.29 mmol) were co-evaporated 3 times from anhydrous DMF, then re-dissolved in anhydrous DMF (2 mL). HCl (4M in dioxane, 30 µL) was added and the reaction mixture was stirred for 60 min at 50 °C. Then DMF was removed on rotary evaporator and the residue was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0 ^ 50% ACN/0.1% TFA), giving the product Intermediate 1 (45 mg, 68 %) as a white solid. MS calc. for C34H36F2N5O9: 694.24, found: 694.25, [M - H]-. [00382] Intermediate 2. The mixture of exatecan mesylate (30 mg, 0.056 mmol), Intermediate 1 (45 mg, 0.065 mmol) and DMTMM (31 mg, 0.113 mmol) was added DMF/water (5:1, 2.5 ml) and diisopropylethylamine (30 µL) and the resulting mixture was stirred at room temperature for 1 h, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0 ^ 100% ACN/0.1% TFA), offering Intermediate 2 as a white solid after lyophilization (30 mg, 48 %). MS calc. for C58H56F3N8O12: 1113.39, found: 1113.40, [M+H]+. [00383] Intermediate 3. To a solution of Intermediate 2 (30 mg, 0.027 mmol) in 2 mL of anhydrous DMF was added morpholine (150 µL). The reaction mixture was stirred at room temperature for one hour and then DMF was removed on rotary evaporator. The reaction product was purified by reverse-phase flash chromatography (diol-modified C18, 25 g, 0 ^ 50% ACN/0.1% TFA), giving the product Intermediate 3 (10 mg, 42 %) as a white solid. MS calc. for C43H46F3N8O10: 891.32, found: 891.30, [M + H]+. [00384] Compound 1042. Intermediate 3 (10 mg, 0.011 mmol) was dissolved in 1.5 mL of DMF.2,5-Dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethoxy)propanoate (0.011 mmol, 3.5 mg) and DIPEA (4 µL) were added. The reaction mixture was stirred at room temperature for 30 minutes. The product was purified by reverse- phase flash HPLC, using a semipreparative column containing diol-modified C18 (0 ^ 100% ACN/10mM aq. NH4OAc). The desired product was recovered as a white solid, after lyophilization from water (6 mg, 50 %). MS calc. for C52H55F3N9O14: 1086.37, found: 1086.40, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ 9.50 (d, J = 8.4 Hz, 1H), 8.66 (t, J = 6.8 Hz, 1H), 8.32 (t, J = 5.8 Hz, 1H), 8.10 (dd, J = 6.9, 4.5 Hz, 2H), 7.98 (t, J = 5.8 Hz, 1H), 7.81 (d, J = 10.9 Hz, 1H), 7.33 (s, 1H), 7.27 – 7.20 (m, 4H), 7.20 – 7.11 (m, 1H), 7.00 (s, 2H), 6.53 (s, 1H), 5.60 (dt, J = 8.5, 5.5 Hz, 1H), 5.49 – 5.38 (m, 2H), 5.29 – 5.05 (m, 2H), 4.72 (m, 2H), 4.51 (m, 1H), 4.04 – 3.91 (m, 2H), 3.76 – 3.72 (m, 2H), 3.66 (s, 1H), 3.61 – 3.57 (m, 3H), 3.52 (s, 1H), 3.46 (t, J = 5.5 Hz, 2H), 3.24 – 3.12 (m, 2H), 3.05 (m, 1H), 2.79 (m, 1H), 2.40 (d, J = 1.9 Hz, 3H), 2.33 (t, J = 6.5 Hz, 2H), 2.26 – 2.15 (m, J = 6.8 Hz, 2H), 1.87 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). Example 135: Synthesis of Compound 1044
Figure imgf000176_0001
[00385] Intermediate 1. The mixture of compound 130 (60 mg, 0.11 mmol), (2S,3S)1(Fmoc)3fluoropyrrolidine-2-carboxylic acid (40 mg, 0.11 mmol) and DMTMM (30 mg, 0.11 mmol) was added DMF/water (3:1, 2 ml) and diisopropylethylamine (60 µL) and the resulting mixture was stirred at room temperature for 90 min. The reaction mixture was purified by reverse phase flash chromatography (25 g, diol-modified C18, 0 50% ACN/H2O), offering Intermediate 1 as a white solid after lyophilization (47 mg, 51 %). MS calc. for C46H42F2N5O9: 846.86, found: 846.30, [M+H]+. [00386] Intermediate 2. Morpholine (300 µL) was added to the solution of Intermediate 1 (83 mg, 0.098 mmol) in anhydrous DMF (2 ml) and the reaction mixture was stirred for 1 h at room temperature. LC-MS indicated the full consumption of starting material. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 50% ACN/H2O), giving the product as a white solid after lyophilization (34 mg, 56 %). MS calc. for C31H32F2N5O7: 624.61, found: 624.25, [M+H]+. [00387] Intermediate 3. The mixture of Intermediate 2 (34 mg, 0.055 mmol), FmocGGFGG-OH (37 mg, 0.06 mmol) and DMTMM (16.6 mg, 0.06 mmol) was added DMF/water (3:1, 2 ml) and diisopropylethylamine (60 µL) and the resulting mixture was stirred at room temperature for 60 min. The reaction mixture was purified by reverse phase flash chromatography (25 g, diol modified C18, 0 50% ACN/H2O), offering Intermediate 3 as a white solid after lyophilization (43 mg, 64 %). MS calc. for C63H62F2N10O14: 1221.94, found: 1221.40, [M+H]+. [00388] Intermediate 4. Morpholine (40 µL) was added to the solution of Intermediate 3 (25 mg, 0.020 mmol) in anhydrous DMF (1.5 ml) and the reaction mixture was stirred for 1 h at room temperature. LC-MS indicated the full consumption of starting material. The mixture was directly purified by reverse-phase flash chromatography (25 g, diol-modified C18, 0 50% ACN/H2O), giving the product as a white solid after lyophilization (15 mg, 75 %). MS calc. for C48H53F2N10O12: 999.00, found: 999.35, [M+H]+. [00389] Compound 1044. Mal-PEG-NHS ester (15 mg, 0.014 mmol) and DIPEA (20 µL) were added to the solution of Intermediate 4 (4.2 mg, 0.014 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 1 h, as LC-MS indicated the full consumption of starting material. The mixture was directly purified by reverse-phase flash HPLC using a semipreparative column (diol-modified C18, 0 50% ACN/H2O), offering compound 1044 as a white solid after lyophilization (2.5 mg, 15 %). MS calc. for C57H62F2N11O16: 1194.17, found: 1194.45, 15-54 [M+H]+. Example 136: Synthesis of Compound 1045
Figure imgf000178_0001
[00390] Intermediate 1. FmocGGFG-OAc (1 equiv., 0.0878 mmol, 54 mg) was dissolved into 2 mL of DMF and 5-(hydroxymethyl)-1H-pyrazole-3-carboxylic acid (1.1 equiv., 0.0966 mmol, 14 mg) was added, followed by 10 µL of a 4M HCl solution in dioxane. The reaction mixture was stirred at room temperature for 2 h, to be then directly loaded on column for purification. Purification was performed by reverse-phase flash chromatography (25 g, diol- modified C18, 0 ^ 100% ACN/H2O). Fractions containing the product were lyophilized from water. The product contained several impurities from peptide decomposition (72 mg). MS calc. for C36H38N7O9: 712.27, found: 712.24, [M+H]+. [00391] Intermediate 2. To a solution of the previously prepared intermediate 1 (72 mg, 0.101 mmol) in DMF (2 mL) were added exatecan mesylate (1 equiv., 0.101 mmol, 54 mg), DMTMM (1.3 equiv., 0.132 mmol, 36 mg), DIPEA (50 µL) and water (1 mL). The reaction mixture was stirred at room temperature for 1 h, to be then directly loaded on column for purification. Purification was performed by reverse-phase flash chromatography (25 g, diol- modified C18, 0 ^
Figure imgf000178_0002
100% ACN/0/1% TFA). Fractions containing the product were lyophilized from water (52 mg, 46 %). MS calc. for C60H58FN10O12: 1129.42, found: 1129.51, [M+H]+. [00392] Intermediate 3. The previously prepared intermediate 2 (52 mg, 0.0461 mmol) was dissolved in DMF (2mL) and morpholine (100 µL) was added. The reaction mixture was stirred at room temperature for 1 h. Purification was performed by reverse-phase HPLC chromatography (semipreparative, diol-modified C18, 0
Figure imgf000179_0001
100% ACN/ 0.1% TFA). Fractions containing the product were lyophilized from water (20 mg, 48 %). MS calc. for C45H48FN10O10: 907.35, found: 907.69, [M+H]+. [00393] Compound 1045. The previously prepared intermediate 3 (20 mg, 0.0221 mmol) was dissolved in 2 mL of anhydrous DMF.2,5-dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (1.2 equiv., 0.0265 mmol, 8.2 mg) and DIPEA (5 µL) were added and the reaction mixture was stirred at room temperature for 1 h. Purification was performed by reverse-phase HPLC chromatography (semipreparative, diol-modified C18, 0 ^ 100% ACN/ 0.1% TFA). Fractions containing the product were lyophilized from water (13 mg, 53 %). MS calc. for C54H57FN11O14: 1102.41, found: 1102.43, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ 9.13 (d, J = 8.5 Hz, 1H), 8.90 – 8.81 (m, 2H), 8.61 (t, J = 6.3 Hz, 1H), 8.31 (m, 3H), 8.09 (m, 2H), 7.97 (m, 2H), 7.82 (q, J = 10.7, 9.8 Hz, 1H), 7.32 (s, 1H), 7.21 (m, 3H), 7.00 (s, 2H), 6.52 (s, 2H), 5.87 (dd, J = 13.3, 6.6 Hz, 1H), 5.78 – 5.68 (m, 2H), 5.48 (m, 2H), 5.39 (d, J = 4.0 Hz, 1H), 5.29 – 5.13 (m, 2H), 4.68 (s, 1H), 4.54 – 4.47 (m, 1H), 4.40 (s, 2H), 3.75 (m, 4H), 3.66 (d, J = 5.7 Hz, 3H), 3.62 – 3.48 (m, 2H), 3.16 – 2.97 (m, 2H), 2.78 (td, J = 13.9, 9.6 Hz, 1H), 2.40 (d, J = 6.4 Hz, 3H), 2.32 (t, J = 6.5 Hz, 2H), 2.28 – 2.24 (m, 1H), 1.85 (dq, J = 14.1, 7.1 Hz, 2H), 1.24 (s, 1H), 0.87 (t, J = 7.5 Hz, 3H). Example 137: Synthesis of Compound 1046
Figure imgf000180_0001
[00394] Intermediate 1 (18 mg, 0.0204 mmol) was dissolved in 2 mL of anhydrous DMF. 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (1.1 equiv., 0.0225 mmol, 7 mg) and DIPEA (2.5 µL) were added and the reaction mixture was stirred at room temperature for 1 h. Purification was performed by reverse-phase HPLC chromatography (semipreparative, diol-modified C18, 0 ^ 100% ACN/ 0.1% TFA). A second purification was performed by reverse-phase HPLC chromatography (semipreparative, diol- modified C18, 0 ^ 100% ACN/ water). Fractions containing the product were lyophilized from water (9 mg, 41 %). MS calc. for C55H61FN9O13: 1074.44, found: 1076.42, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ 8.62 (t, J = 6.7 Hz, 1H), 8.50 (d, J = 8.9 Hz, 1H), 8.29 (t, J = 5.9 Hz, 1H), 8.05 (t, J = 5.8 Hz, 1H), 7.99 (t, J = 5.8 Hz, 1H), 7.78 (d, J = 10.9 Hz, 1H), 7.32 (s, 1H), 7.28 – 7.14 (m, 5H), 6.99 (s, 2H), 6.52 (s, 1H), 5.64 – 5.56 (m, 1H), 5.42 (d, J = 3.1 Hz, 3H), 5.21 (s, 2H), 4.64 (d, J = 6.7 Hz, 2H), 4.53 – 4.43 (m, 1H), 4.02 (d, J = 1.7 Hz, 2H), 3.74 (dd, J = 16.8, 6.0 Hz, 2H), 3.68 (d, J = 16.3 Hz, 2H), 3.59 – 3.44 (m, 4H), 3.27 – 3.06 (m, 2H), 3.02 (dd, J = 13.9, 4.6 Hz, 1H), 2.85 – 2.73 (m, 1H), 2.39 (d, J = 1.8 Hz, 2H), 2.32 (t, J = 6.5 Hz, 2H), 2.18 – 2.13 (m, 2H), 2.10 (t, J = 7.5 Hz, 2H), 1.86 (m, 2H), 1.56 (m, 2H), 1.47 (m, 2H), 1.24 (d, J = 4.3 Hz, 2H), 1.19 (t, J = 3.5 Hz, 1H), 1.03 (dt, J = 9.1, 4.9 Hz, 1H), 0.88 (t, J = 7.4 Hz, 3H), 0.79 – 0.73 (m, 1H). Example 138: Synthesis of Compound 1047
Figure imgf000181_0001
[00395] Intermediate 2. Morpholine (120 µL) was added to the solution of Intermediate 1 (1.0 equiv., 51 mg, 0.046 mmol) in DMF (1.8 ml) and the mixture was stirred for 1 hour at room temperature. The reaction mixture was concentrated on rotavap and re-evaporated from DMF to dryness. DMTMM (1.5 equiv., 19 mg, 0.069 mmol) and 3-(2-((((9H-Fluoren-9- yl)methoxy)carbonyl)amino)ethoxy)propanoic acid (1.5 equiv., 25 mg, 0.069 mmol) were added to the obtained residue, followed by the addition of DMF/water (5:1, 1.75 ml) and diisopropylethylamine (1.6 equiv., 13 µl, 0.074 mmol) and the resulting solution was stirred at room temperature for 1 hour. The reaction mixture was purified by reverse-phase flash chromatography (diol-modified C18, 0 ^
Figure imgf000181_0002
60% ACN/10mM aq. NH4OAc), offering Intermediate 2 as a white solid after lyophilization (47 mg, 84 %). MS calc. for C65H69FN9O14: 1218.49, found: 1218.35, [M+H]+. [00396] Intermediate 3. Morpholine (125 µL) was added to the solution of Intermediate 2 (1.0 equiv., 47 mg, 0.038 mmol) in DMF (1.75 ml) and the mixture was stirred for 2 hours at room temperature. The reaction mixture was concentrated on rotavap and purified by reverse- phase flash chromatography (diol-modified C18, 0 ^ 50% ACN/0.1% aq. TFA), offering Intermediate 3 as a pale-yellow solid after lyophilization (27 mg, 64 %). MS calc. for C50H59FN9O12: 996.43, found: 996.30, [M+H]+. [00397] Intermediate 4. The solution of Intermediate 3 (1.0 equiv., 27 mg, 0.024 mmol), Intermediate 5 (1.0 equiv., 7 mg, 0.024 mmol) and diisopropylethylamine (2.1 equiv., 9.5 µl, 0.050 mmol) in anhydrous DMF (0.75 ml) was stirred at room temperature for 40 minutes, as LC-MS indicated the full consumption of starting material. Purification by reverse-phase flash chromatography (diol-modified C18, 0 ^ 60% ACN/10mM aq. NH4OAc) gave the product Intermediate 4 as a white solid after lyophilization (20 mg, 73 %). MS calc. for C58H69FN9O14: 1134.49, found: 1134.35, [M+H]+. [00398] Compound 1047. The solution of Dess-Martin periodinane (1.3 equiv., 9.7 mg, 0.023 mmol) in anhydrous acetonitrile (2.5 ml) was added to the solution of Intermediate 4 (1.0 equiv., 20 mg, 0.018 mmol) in anhydrous DMF (1 ml) and the resulting mixture was stirred at room temperature for 3 hours, as LC-MS indicated the full consumption of starting material. The reaction mixture was concentrated on rotavap and purified by reverse-phase flash chromatography using semipreparative column (diol-modified C18, 0 ^ 60% ACN/10mM aq. NH4OAc), offering compound 1047 as a white solid after lyophilization (15 mg, 74 %). MS calc. for C58H67FN9O14: 1132.48, found: 1132.35, [M+H]+.1H NMR (500 MHz, DMSO-d6) δ: 8.71 (d, J = 8.8 Hz, 1H), 8.42 (t, J = 6.8 Hz, 1H), 8.25 (t, J = 5.9 Hz, 1H), 8.14 (t, J = 5.8 Hz, 1H), 8.09 (d, J = 8.1 Hz, 1H), 8.01 (d, J = 5.9 Hz, 1H), 7.95 (t, J = 5.7 Hz, 1H), 7.78 (d, J = 10.8 Hz, 1H), 7.30 (s, 1H), 7.25 – 7.17 (m, 4H), 7.15 (t, J = 6.9 Hz, 1H), 6.51 (s, 1H), 5.55 (q, J = 6.1 Hz, 1H), 5.49 – 5.37 (m, 2H), 5.18 (s, 2H), 4.59 – 4.41 (m, 3H), 3.77 – 3.65 (m, 5H), 3.64 – 3.53 (m, 4H), 3.46 (dd, J = 10.7, 5.9 Hz, 1H), 3.36 (t, J = 6.0 Hz, 2H), 3.29 – 3.08 (m, 4H), 3.02 (dd, J = 14.0, 4.5 Hz, 1H), 2.81 – 2.66 (m, 2H), 2.43 – 2.35 (m, 5H), 2.31 and 2.28 (s, 3H), 2.21 – 2.07 (m, 3H), 1.94 – 1.78 (m, 2H), 1.61 – 1.46 (m, 2H), 1.03 (t, J = 7.2 Hz, 3H), 0.87 (m, 4H), 0.81 – 0.72 (m, 1H). Example 139: Synthesis of Compound 3113: [00399] Compound 3113 was prepared according to Procedure C of the General Methods section in Example 1 using 34 molar equivalents of linker payload compound 1025 to anti-EGFR IgG1 monoclonal antibody 5. DAR based on RP-HPLC was 7.6. Example 140: Synthesis of compound 3053B [00400] Compound 3053B was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1007 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC was 7.6. Example 141: Synthesis of compound 3102 [00401] Compound 3102 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1046 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC was 7.7. Example 142: Synthesis of compound 3103 [00402] Compound 3103 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1047 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC was 3.6. Example 143: Synthesis of compound 3104 [00403] Compound 3104 was prepared according to Procedure C of the General Methods section in Example 1 using 25 molar equivalents of linker payload of compound 1045 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC was 7.8. Example 144: Synthesis of compound 3105 [00404] Compound 3105 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1044 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC-MS was 7.6 was based on molecular weight of the drug of 1194.17. Example 145: Synthesis of compound 3107 [00405] Compound 3107 was prepared according to Procedure C of the General Methods section in Example 1 using 25 molar equivalents of linker payload of compound 1042 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC was 7.7. Example 146: Synthesis of compound 3059B [00406] Compound 3059B was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1017 to anti-EGFR IgG1 monoclonal antibody 1. DAR based on RP-HPLC was 7.7. Example 147: Synthesis of compound 3108 [00407] Compound 3108 was prepared according to Procedure C of the General Methods section in Example 1 using 22 molar equivalents of linker payload of compound 1025 to anti-EGFR IgG1 monoclonal antibody 2. DAR based on RP-HPLC was 7.5. Example 148: Synthesis of compound 3110 [00408] Compound 3110 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1025 to anti-EGFR IgG1 monoclonal antibody 3. DAR based on RP-HPLC was 7.8. Example 149: Synthesis of compound 3111 [00409] Compound 3111 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1025 to anti-EGFR IgG1 monoclonal antibody 4. DAR based on RP-HPLC was 7.6, while DAR based on RPHPLC-MS was 7.8. Example 150: Synthesis of compound 3109 [00410] Compound 3109 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1007 to anti-EGFR IgG1 monoclonal antibody 3. DAR based on RP-HPLC was 7.7. Example 151: Synthesis of compound 3112 [00411] Compound 3112 was prepared according to Procedure C of the General Methods section in Example 1 using 30 molar equivalents of linker payload of compound 1007 to anti-EGFR IgG1 monoclonal antibody 4. DAR based on RP-HPLC was 7.6, while DAR based on RPHPLC-MS was 7.8. Example 152: Synthesis of Compound 1043: F
Figure imgf000185_0001
[00412] Intermediate 1. To the solution of Fmoc-GE(OBn)VCit-NH-CH2-OAc (1.0 equiv., 220 mg, 0.260 mmol) and ethylene glycol (3.0 equiv., 45 µl, 0.782 mmol) in anhydrous DMF (6 ml) was added 2M HCl/Et2O (450 µl) and the resulting mixture was stirred at room temperature for 2 hours, as LC-MS analysis indicated the full consumption of the starting material. The reaction mixture was purified by reverse-phase flash chromatography (diol- modified C18, 0 ^ 60% ACN/0.1% HCl), offering intermediate 1 as a white solid after lyophilisation (120 mg, 55%). MS calc. for C43H56N7O11: 846.40, found: 846.35, [M+H]+. [00413] Intermediate 2. The solution of 4-nitrophenyl chloroformate (3.0 equiv., 86 mg, 0.426 mmol) in dry THF (1.5 ml) was added dropwise to the ice-cold solution of intermediate 1 (1.0 equiv., 120 mg, 0.142 mmol) in dry pyridine (4 ml) and the resulting mixture was stirred at 0 °C for 1 hour, as LC-MS analysis indicated the full consumption of the starting material. Solvents were removed on rotavap and the residue was purified by reverse-phase flash chromatography (diol-modified C18, 0 ^ 75% ACN/10mM aq. NH4OAc) to give intermediate 2 as a pale-yellow solid after lyophilisation (50 mg, 35 %). MS calc. for C50H59N8O15: 1011.41, found: 1011.35, [M+H]+. [00414] Intermediate 3. Exatecan mesylate (1.0 equiv., 0.049 mmol, 26 mg) and DIPEA (4.0 equiv., 0.049 mmol, 34 µL) were added to the solution of intermediate 2 (1.0 equiv., 50 mg, 0.049 mmol) in anhydrous DMF (3 ml). The reaction mixture was stirred at room temperature for 40 hours, as LC-MS indicated the full consumption of starting material. Purification by reverse-phase flash chromatography (diol-modified C18, 0 ^ 75% ACN/10mM aq. NH4OAc) offered intermediate 3 as a white solid after lyophilisation (48 mg, 75 %). MS calc. for C68H76FN10O16: 1307.54, found: 1307.55, [M+H]+. [00415] Intermediate 4. To the solution of intermediate 3 (1.0 equiv., 48 mg, 0.036 mmol) in the mixture of dioxane/water (2:1, 2.5 ml) was added 10% Pd/C (5 mg) and the reaction mixture was hydrogenated (balloon) for 1 hour at room temperature. Then the mixture was filtered through the layer of celite, solids were washed with DMF and the filtrate was evaporated to dryness on rotavap. The residue was dissolved in DMF (1.5 ml), followed by the addition of morpholine (150 µl). The obtained solution was stirred at room temperature for 40 minutes. Purification by reverse-phase flash chromatography (diol-modified C18, 0 ^ 50% ACN/10mM aq. NH4OAc) gave intermediate 4 as a white solid after lyophilisation (15 mg, 42 %). MS calc. for C46H60FN10O14: 995.43, found: 995.35, [M+H]+. [00416] Compound 1043. Mal-PEG-NHS ester (1.05 equiv., 0.016 mmol, 4.9 mg) and DIPEA (2.1 equiv., 0.032 mmol, 5.6 µL) were added to the solution of intermediate 4 (1.0 equiv., 15 mg, 0.015 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 1 hour, as LC-MS indicated the full consumption of starting material. Purification by reverse-phase flash chromatography using a semipreparative column (diol- modified C18, 0 ^ 50% ACN/10mM aq. NH4OAc) offered 1043 as a white solid after lyophilisation (12 mg, 67 %). MS calc. for C55H69FN11O18: 1190.48, found: 1190.45, [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ: 12.05 (s, 1H), 8.63 (t, J = 6.7 Hz, 1H), 8.08 – 7.96 (m, 4H), 7.77 (t, J = 10.1 Hz, 2H), 7.31 (s, 1H), 7.00 (s, 2H), 6.51 (s, 1H), 5.96 – 5.92 (m, 1H), 5.47 – 5.33 (m, 4H), 5.29 – 5.16 (m, 2H), 4.57 (ddd, J = 34.3, 10.5, 6.7 Hz, 2H), 4.33 (td, J = 8.1, 5.2 Hz, 1H), 4.24 – 4.07 (m, 4H), 3.78 – 3.63 (m, 2H), 3.62 – 3.51 (m, 6H), 3.46 (t, J = 5.8 Hz, 2H), 3.30 – 3.18 (m, 1H), 3.18 – 3.03 (m, 1H), 3.01 – 2.86 (m, 2H), 2.37 (s, 3H), 2.32 (t, J = 6.5 Hz, 2H), 2.28 – 2.10 (m, 4H), 2.03 – 1.79 (m, 4H), 1.78 – 1.66 (m, 1H), 1.66 – 1.55 (m, 1H), 1.56 – 1.44 (m, 1H), 1.45 – 1.23 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H), 0.82 (dd, J = 11.6, 6.7 Hz, 6H). Example 153: Antibody-Drug Conjugates Effectively Kill Cognate Cells expressing HER2, TROP2 and EGFR [00417] To determine the relative cell-killing potency of ADCs of the instant disclosure compared to a similar ADC (DXD), cell-killing assays were run on multiple cells lines expressing HER2, TROP2 and EGFR. For testing anti-HER-2 ADCs, NCI-N87 and SK-BR-3 cell lines were used. For testing anti-TROP2 ADCs, MDA-MB-468 and FaDu cell lines were used. For testing anti-EGFR ADCs, MDA-MB-468, and HCC827, NCI-H292, FaDu, OVCAR3 cell lines were used. [00418] NCI-N87, MDA-MB-468, HCC827, NCI-H292 and OVCAR3 were cultured in RPMI-1640 media (Gibco, Life Technologies) supplemented with 10% v/v heat inactivated FBS (Corning), SK-BR-3 cells were maintained in McCoys 5A media (Gibco, Life Technologies) supplemented with 10% v/v heat inactivated FBS (Corning) at 37 ^C in a humidified incubator containing 5% CO2. FaDu cells were maintained in EMEM media (Gibco, Life Technologies) supplemented with 10% v/v heat inactivated FBS (Corning) at 37 ^C in a humidified incubator containing 5% CO2. [00419] The viability of cancer cells in the presence of ADCs was measured in a series of in vitro assays. For HER2 and TROP2-expressing cells, cells were plated in 384-well white flat-bottomed plates (Corning) at 0.5 × 103 per well in 30 µL culture medium. For EGFR- expressing cells, cells were plated in 96-well white flat-bottomed plates (Corning) at 2 × 103 per well in 100 µL culture medium. ADCs were added at a range of concentrations as eight-point serial dilution as quadruplicates. Following further incubation for 6 days in 37 ^C, 5% CO2, cell viability was assessed with the use of a CellTiter-Glo Luminescent Cell Viability Assay (Promega). Luminescence was measured using the SpectraMax iD3 plate reader (Molecular Devices). Luminescence values were plotted against log concentration of test compounds, and cell viability was calculated by dividing luminescence values at different antibody drug conjugate concentrations by luminescence values at antibody concentration of zero. ADC dose- response IC50 values were calculated by GraphPad Prism as best-fit values using four parameter dose-response curve fit, with R squared ranging from 0.97-0.999. Respective IC50 values from two to nine independent experiments were averaged and respective means and respective standard deviation between independent experiments calculated. Respective minimal cell viability from two to nine independent experiments were averaged and respective mean and respective standard deviation between independent experiments calculated. Mean IC50 values and mean percent cell killing calculated along with respective standard deviations are shown in Tables 7-9. [00420] Table 7A shows the percent cell viability of NCI-N87 cells after exposure to the different anti-HER2 ADCs of the instant disclosure. Table 7B shows the percent viability of SK- BR-3 cells after exposure to the different anti-HER2 ADCs of the instant disclosure. Table 7C shows the percent cell viability of NCI-N87 cells after exposure to the various exatecan release anti-HER2 ADCs described herein. Table 7D shows the percent cell viability of SK-BR-3 cells after exposure to the various exatecan release anti-HER2 ADCs described herein. IC50 ADC (nM) was calculated based on the molecular weights of monoclonal antibody (mAb) and linker payload conjugated to mAb, where MW ADC (g/mole) = MW mAb (g/mole) + DAR x MW linker-payload (g/mole). IC50 Linker-Payload (nM) was calculated from concentration of linker-payload delivered by the ADC (calculated as DAR multiplied by ADC (nM)). Table 7A
Figure imgf000189_0001
Table 7B
Figure imgf000189_0002
Figure imgf000190_0001
Table 7C
Figure imgf000190_0002
Table 7D
Figure imgf000191_0001
[00421] Table 8A shows the percent cell viability of FaDu cells after exposure to the different anti-TROP2 ADCs of the instant disclosure. Table 8B shows the percent viability of MDA-MB-468 cells after exposure to the different anti-TROP2 ADCs of the instant disclosure. Table 8C shows the percent cell viability of FaDu cells after exposure to the various exatecan release anti-TROP2 ADCs described herein. Table 8D shows the percent cell viability of MDA- MB-468 cells after exposure to the various exatecan release anti-TROP2 ADCs described herein. [00422] IC50 ADC (nM) was calculated based on the molecular weights of monoclonal antibody (mAb) and linker payload conjugated to mAb, where MW ADC (g/mole) = MW mAb (g/mole) + DAR x MW linker-payload (g/mole). IC50 Linker-Payload (nM) was calculated from concentration of linker-payload delivered by the ADC (calculated as DAR multiplied by ADC (nM)). Table 8A
Figure imgf000191_0002
Figure imgf000192_0001
Table 8B
Figure imgf000192_0002
Figure imgf000193_0001
Table 8C
Figure imgf000193_0002
Table 8D
Figure imgf000193_0003
Figure imgf000194_0002
[00423] Table 9A shows the percent cell viability of MDA-MB-468 cells after exposure to the different anti-EGFR ADCs of the instant disclosure. Table 9B shows the percent viability of HCC827 cells after exposure to the different anti-EGFR ADCs of the instant disclosure. Table 9C shows the percent cell viability of MDA-MB-468 cells after exposure to the various exatecan release anti-EGFR ADCs described herein. Table 9D shows the percent cell viability of HCC827 cells after exposure to the various exatecan release anti-EGFR ADCs described herein. Table 9E shows the percent cell viability of cell lines NCI-H292, OVCAR3 and FaDu cells after exposure to the different anti-EGFR ADCs of the present disclosure. IC50 (nM) of ADC was calculated based on the molecular weights (MW) of monoclonal antibody (mAb) and linker payload, where MW ADC (g/mole) = MW mAb (g/mole) + DAR x MW linker-payload). Table 9A
Figure imgf000194_0001
Figure imgf000195_0001
Table 9B
Figure imgf000195_0002
Figure imgf000196_0001
Table 9C
Figure imgf000196_0002
Table 9D
Figure imgf000196_0003
Table 9E
Figure imgf000197_0001
Example 154: Cytotoxicity (nmol/L) of Therapeutic Payloads Cell lines [00424] Human tumor cell lines, SK-BR-3, NCI-H292, HT-29, MCF-7, NCI-N87, and FaDu were obtained from ATCC. NCI-H292, HT-29, MCF-7, NCI-N87 cells MDA-MB-468, and HCC827 were cultured in RPMI-1640 media (Gibco, Life Technologies) supplemented with 10% v/v heat inactivated FBS (Corning), FaDu cells were maintained in EMEM media (Gibco, Life Technologies) supplemented with 10% v/v heat inactivated FBS (Corning) at 37 ^C in a humidified incubator containing 5% CO2. SK-BR-3 cells were maintained in McCoys 5A medium (Gibco, Life Technologies) supplemented with 10% v/w heat inactivated FBS (Corning) at 37 °C in a humidified incubator containing 5% CO2. Compound preparation [00425] For compounds 71, 72, 73, 74, 180, 193, 194, 195, 208, and 130, lyophilized compounds were dissolved in 100% dry DMSO, aliquots frozen and stored at -80 °C. Concentration of compound stock solutions in DMSO were determined by RP- HPLC Agilent 1100 platform with 1200 DAD and SofTA ELSD detectors. Shimadzu 3.0mm x 30mm XR ODS 2.2µm column was run at 50 °C, 1.5 mL/min. Solvent A: 0.1 % Formic acid in water, Solvent B: 0.08% Formic acid in methanol – Gradient: 5% - 100% B in 3.0min, 100% solvent B for 0.3min. Concentration of compounds in 100% DMSO determined by ELS ranged from 1-6 mM. For compounds 196, 197, 198, 199, 200, 201, 202, 203, 204, and 205, lyophilized compounds were dissolved in 100% dry DMSO, aliquots frozen and stored at -80oC. Concentration was calculated as W/V. Cytotoxicity Assay [00426] Cells were plated in 96-well white flat-bottomed plates (Corning) at 2.0 × 103 cells per well in 100 µL culture medium. Alternatively, cells were plated in 384-well white flat- bottomed plates (Corning) at 0.5 × 103 per well in 30 µL culture medium. After incubation for 24 hours, test compounds were added at a range of concentrations as ten-point serial dilution as duplicates or triplicates. Following further incubation for 6 days 37oC, 5% CO2, cell viability was assessed with the use of a CellTiter-Glo Luminescent Cell Viability Assay (Promega). Luminescence was measured using the GloMax instrument (Promega). Luminescence values were plotted against log concentration of test compounds, and the IC50 values were calculated by GraphPad Prism 9 as best-fit values using four parameter dose-response curve fit, with R squared values ranging from 0.97-0.999. For a subset of payloads, each treatment was independently repeated two to eight times, and IC50 values were averaged. [00427] Table 10 shows IC50 cytotoxicity values (nmol/L) of therapeutic payloads described herein for multiple tumor cell lines. Repeated treatments as two to eight independent experiments were used to determine the means of IC50 values and standard deviations (stdev). Standard deviation is shown as (N/A) for treatment performed once. Table 10 
Figure imgf000199_0001
Figure imgf000200_0001
Example 155: Tumor growth suppression effect on human lung carcinoma NCI-H292 of anti-TROP2 antibody drug conjugates 3038, 3036, and 3031 [00428] NCI-H292 human lung carcinoma cell line was purchased from ATCC. Athymic nude mice (Jackson Labs NU/J # 2019), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x106 NCI-H292 cells suspended 1:1 with Matrigel in serum-free medium. After tumor size reached ~100 to 200 mm3 mice were randomized into groups (5 animals per group, day 8). Respective anti-TRO2 antibody-drug conjugate was intravenously administered as a single dose of 3 mg/kg to the tail of each mouse (Start of dosing is referred to as Day 0 in the figures. Arrows indicate timing and frequency of dose administration). Phosphate buffer saline (PBS) was administered to the control group. Mean tumor volume was plotted until either the first death per group or end of the study. Error bars represent the standard error of the mean. FIG.1A shows mean tumor volume as a function of time after start of antibody drug conjugate 3031 administration compared to vehicle control group. FIG.1B shows mean tumor volume as a function of time after start of antibody drug conjugate 3036 administration. FIG. 1C shows mean tumor volume as a function of time after start of antibody drug conjugate 3038 administration. Example 156: Tumor growth suppression effect on human throat cancer cell line FaDu of anti-EGFR antibody drug conjugates 3058A and 3053B [00429] FaDu human throat carcinoma cell line was purchased from ATCC. Athymic nude mice (Jackson Labs NU/J # 2019), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x106 NCI-H292 cells suspended 1:1 with Matrigel in serum-free medium. After tumor size reached ~100 to 200 mm3 mice were randomized into groups (5 animals per group, day 10). Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 3 mg/kg to the tail of each mouse for total of 3 doses, 1 week apart (start of dosing is referred to as Day 0 in the figures. Arrows indicate timing and frequency of dose administration). Phosphate buffer saline (PBS) was administered to the control group. Mean tumor volume was plotted until either the first death per group or end of the study. Error bars represent the standard error of the mean. FIG.2A shows mean tumor volume as a function of time after start of antibody drug conjugate 3058A administration compared to vehicle control group. 5/5 mice were tumor free in antibody drug conjugate 3058A group. FIG.2B shows mean tumor volume as a function of time after start of antibody drug conjugate 3053B administration. 5/5 mice were tumor free in antibody drug conjugate 3053B group. Example 157: Tumor growth suppression effect on human lung cancer cell line NCI- H1975 of anti-EGFR antibody drug conjugates 3058A, 3058B, 3102, and 3053B [00430] NCI-H1975 human lung carcinoma cell line was purchased from ATCC. Athymic nude mice (Jackson Labs NU/J # 2019), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x106 NCI-H1975 cells suspended 1:1 with Matrigel in serum-free medium. After tumor size reached ~100 to 200 mm3 mice were randomized into groups (5 animals per group, day 15). Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 10 mg/kg to the tail of each mouse for total of 3 doses, 1 week apart (start of dosing is referred to as Day 0 in the figures. Arrows indicate timing and frequency of dose administration). Phosphate buffer saline (PBS) was administered to the control group. Mean tumor volume was plotted until either the first death per group or end of the study. Error bars represent the standard error of the mean. FIG.3A shows mean tumor volume as a function of time after start of antibody drug conjugate 3058A administration compared to vehicle control.3/5 mice were tumor free in antibody drug conjugate 3058A group. FIG.3B shows mean tumor volume as a function of time after start of antibody drug conjugate 3058B administration compared to vehicle control.1/5 mice were tumor free in antibody drug conjugate 3058B group. FIG.3C shows mean tumor volume as a function of time after start of antibody drug conjugate 3102 administration compared to vehicle control.3/5 mice were tumor free in antibody drug conjugate 3102 group. FIG.3D shows mean tumor volume as a function of time after start of antibody drug conjugate 3053B administration compared to vehicle control.2/5 mice were tumor free in antibody drug conjugate 3053B group. Example 158: Tumor growth suppression effect on human breast cancer cell line MDA- MB-468 of anti-EGFR antibody drug conjugates 3053B, 3058A, and 3102 [00431] MDA-MB-468 human breast cancer cell line was purchased from Accegen. NOD-SCID mice (Charles River Labs, strain code #394), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x106 MDA-MB-468 cells suspended in 10% Matrigel in serum-free medium. After tumor size reached ~100 to 200 mm3 mice were randomized into groups (5 animals per group, day 12). Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 3 mg/kg to the tail of each mouse for 1 dose, start of dosing is referred to as Day 0 in the figures as indicated by the arrow. Phosphate buffer saline (PBS) was administered to the control group. Mean tumor volume was plotted until either the first death per group or end of the study. Error bars represent the standard error of the mean. FIG. 4A shows mean tumor volume as a function of time after start of antibody drug conjugate 3053B, administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3053B group. FIG.4B shows mean tumor volume as a function of time after start of antibody drug conjugate 3058A administration compared to vehicle control.5/5 mice were tumor free in antibody drug conjugate 3058A group. FIG.4C shows mean tumor volume as a function of time after start of antibody drug conjugate 3102 administration compared to vehicle control. 3/5 mice were tumor free in antibody drug conjugate 3102 group. Example 159: Tumor growth suppression effect on human throat cancer cell line FaDu of anti-EGFR antibody drug conjugates 3058A, 3102, 3059B, 3108, 3110, and 3053B [00432] FaDu human throat carcinoma cell line was purchased from ATCC. NOD-SCID mice (Charles River Labs, strain code #394), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x106 FaDu cells suspended in 10% Matrigel in serum- free medium. After tumor size reached ~100 to 200 mm3 mice were randomized into groups (5 animals per group, day 9). Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 1 mg/kg to the tail of each mouse for total of 3 doses, 1 week apart (start of dosing is referred to as Day 0 in the figures. Arrows indicate timing and frequency of dose administration). Phosphate buffer saline (PBS) was administered to the control group. Mean tumor volume was plotted until either the first death per group or end of the study. Error bars represent the standard error of the mean. FIG.5A shows mean tumor volume as a function of time after start of antibody drug conjugate 3058A administration compared to vehicle control. FIG.5B shows mean tumor volume as a function of time after start of antibody drug conjugate 3102 administration compared to vehicle control. FIG.5C shows mean tumor volume as a function of time after start of antibody drug conjugate 3059B administration compared to vehicle control. FIG.5D shows mean tumor volume as a function of time after start of antibody drug conjugate 3108 administration compared to vehicle control. FIG.5E shows mean tumor volume as a function of time after start of antibody drug conjugate 3110 administration compared to vehicle control. FIG.5F shows mean tumor volume as a function of time after start of antibody drug conjugate 3053B administration compared to vehicle control. Compounds exemplified here exhibit tumor suppression greater than control. Example 160: Tumor growth suppression effect on in vivo bystander model using human breast cancer cell line MDA-MB-468 co-inoculated with human colon adenocarcinoma cell line SW620-luc of anti-EGFR antibody drug conjugates 3058A, 3053B, 3102, and 3059B [00433] MDA-MB-468 human breast cancer cell line was purchased from Accegen. SW620-luc (luciferase) cells was purchased from FenicsBIO. NOD-SCID mice (Charles River Labs, strain code #394), female, 6-8 weeks old were inoculated subcutaneously in the right hind flank with 5 x106 MDA-MB-468 cells and 5 x106 SW620-luc cells suspended in 10% Matrigel in serum-free medium.5 x106 SW620-luc cells suspended in 10% Matrigel in serum-free medium was inoculated subcutaneously in the left hind flank. After the right hind tumor size reached ~100 to 200 mm3 mice were randomized into groups (5 animals per group, day 9). Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 3 mg/kg to the tail of each mouse for 1 dose, start of dosing is referred to as Day 0 in the figures as indicated by the arrow. Phosphate buffer saline (PBS) was administered to the control group. Mean tumor volume was measured by caliper and plotted until either the first death per group or end of the study. Error bars represent the standard error of the mean. Bioluminescent imaging was performed once a week from start of dosing. Each mouse received 15 mg of VivoGlo luciferin (Promega) by intraperitoneal injection and imaged by the IVIS Lumina S5 System (Perkin Elmer). Bioluminescence signal was analyzed by Living Image Software (Perkin Elmer) and expressed in total flux as number of photons per second. FIG.6A shows mean tumor volume as a function of time after start of antibody drug conjugate 3058A administration compared to vehicle control.5/5 mice were tumor free in antibody drug conjugate 3058A group. FIG.6B shows mean tumor volume as a function of time after start of antibody drug conjugate 3053B administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3053B group. FIG.6C shows mean tumor volume as a function of time after start of antibody drug conjugate 3102 administration compared to vehicle control.5/5 mice were tumor free in antibody drug conjugate 3102 group. FIG.6D shows mean tumor volume as a function of time after start of antibody drug conjugate 3059B administration compared to vehicle control.3/5 mice were tumor free in antibody drug conjugate 3059B group. [00434] FIG.7A shows change in luciferase activity as a function of time after start of antibody drug conjugate 3058A administration compared to vehicle control. FIG.7B shows change in luciferase activity as a function of time after start of antibody drug conjugate 3053B administration compared to vehicle control. FIG.7C shows change in luciferase activity as a function of time after start of antibody drug conjugate 3102 administration compared to vehicle control. FIG.7D shows change in luciferase activity as a function of time after start of antibody drug conjugate 3059B administration compared to vehicle control. Example 161: Tumor growth suppression effect on human throat cancer cell line FaDu of anti-EGFR antibody drug conjugates 3111, 3112, 3110, and 3109 [00435] FaDu human throat carcinoma cell line was purchased from ATCC. NOD-SCID mice (Charles River Labs, strain code #394), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x106 FaDu cells suspended in 10% Matrigel in serum- free medium. After tumor size reached ~100 to 200 mm3 mice were randomized into groups (5 animals per group, day 9). Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 10, 3, or 1 mg/kg to the tail of each mouse for total of 1 or 3 doses, 1 week apart (start of dosing is referred to as Day 0 in the figures. Arrows indicate timing and frequency of dose administration). Phosphate buffer saline (PBS) was administered to the control group. Mean tumor volume was plotted until either the first death per group or end of the study. Error bars represent the standard error of the mean. [00436] FIG, 8A shows mean tumor volume as a function of time after start of antibody drug conjugate 3111 administration compared to vehicle control.5/5 mice were tumor free in antibody drug conjugate 3111 group. FIG.8B shows mean tumor volume as a function of time after start of antibody drug conjugate 3112 administration compared to vehicle control.5/5 mice were tumor free in antibody drug conjugate 3112 group. FIG.8C shows mean tumor volume as a function of time after start of antibody drug conjugate 3110 administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3110 group. FIG.8D shows mean tumor volume as a function of time after start of antibody drug conjugate 3109 administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3109 group. [00437] FIG.9A shows mean tumor volume as a function of time after start of antibody drug conjugate 3111 administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3111 group. FIG.9B shows mean tumor volume as a function of time after start of antibody drug conjugate 3112 administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3112 group. FIG.9C shows mean tumor volume as a function of time after start of antibody drug conjugate 3110 administration compared to vehicle control.2/5 mice were tumor free in antibody drug conjugate 3110 group. FIG.9D shows mean tumor volume as a function of time after start of antibody drug conjugate 3109 administration compared to vehicle control.5/5 mice were tumor free in antibody drug conjugate 3109 group. [00438] FIG.10A shows mean tumor volume as a function of time after start of antibody drug conjugate 3111 administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3111 group. FIG.10B shows mean tumor volume as a function of time after start of antibody drug conjugate 3112 administration compared to vehicle control.3/5 mice were tumor free in antibody drug conjugate 3112 group. FIG.10C shows mean tumor volume as a function of time after start of antibody drug conjugate 3110 administration compared to vehicle control.4/5 mice were tumor free in antibody drug conjugate 3110 group. FIG.10D shows mean tumor volume as a function of time after start of antibody drug conjugate 3109 administration compared to vehicle control.3/5 mice were tumor free in antibody drug conjugate 3109 group. Example 162. Tumor growth suppression effect on human breast cancer cell line MDA- MB-468 of anti-EGFR antibody drug conjugates 3111, 3112, 3110, and 3109 [00439] MDA-MB-468 human breast cancer cell line was purchased from Accegen. NOD-SCID mice (Charles River Labs, strain code #394), female, 6-8 weeks old were inoculated subcutaneously in the hind flank with 5 x106 MDA-MB-468 cells suspended in 10% Matrigel in serum-free medium. After tumor size reached ~100 to 200 mm3 mice were randomized into groups (5 animals per group, day 20). Respective anti-EGFR antibody-drug conjugate was intravenously administered as a dose of 3 mg/kg to the tail of each mouse for 1 dose, start of dosing is referred to as Day 0 in the figures as indicated by the arrow. Phosphate buffer saline (PBS) was administered to the control group. Mean tumor volume was plotted until either the first death per group or end of the study. Error bars represent the standard error of the mean. [00440] FIG.11A shows mean tumor volume as a function of time after start of antibody drug conjugate 3111 administration compared to vehicle control. Figure 11B shows mean tumor volume as a function of time after start of antibody drug conjugate 3112 administration compared to vehicle control. FIG.11C shows mean tumor volume as a function of time after start of antibody drug conjugate 3110 administration compared to vehicle control. FIG.11D shows mean tumor volume as a function of time after start of antibody drug conjugate 3109 administration compared to vehicle control. INCORPORATION BY REFERENCE [00441] All publications and patents mentioned herein are hereby incorporated by reference in their entirety for all purposes as if each individual publication or patent was specifically and individually incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. EQUIVALENTS [00442] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the present disclosure will become apparent to those skilled in the art upon review of this specification. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. [00443] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure.

Claims

CLAIMS What is claimed is: 1. A drug conjugate represented by Formula IA or Formula IB:
Figure imgf000208_0001
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: Lig is a targeting moiety; L is a linker moiety; R is selected from the group consisting of -R1, -C(O)-R1-, -C(O)-O-R1-, -C(O)-NH-R1-, - C(O)-C0-3alkyl-C(O)-NH-R1-, -C(O)-(5-6 membered heteroaryl)-R1-, -CH2-(5-6 membered heteroaryl)-R1-, -C(O)-(4-6 membered heterocyclyl)-, -C(O)-(4-6 membered heterocyclyl)-C(O)- R1-, -C(O)-(4-6 membered heterocyclyl)-NH-R1-, -C(O)-C3-4cycloalkyl-R1-, -C(S)-C3- 4cycloalkyl-R1-, and -C(O)-O-phenyl-R1-; wherein any aforementioned heteroaryl, heterocyclyl, alkyl, and cycloalkyl may optionally be substituted by one, two or three substituents each independently selected from the group consisting of halogen, hydroxyl, and oxo; R1 is selected from the group consisting of CH2-CH2O-, -CH2-O-, -NH-, -CH2ONH-, and -CH(CH3)-NH-; and s is 0, 1, 2, 3, 4, 5, 6, 7, or 8; and t is 0, 1, 2, 3, 4, 5, 6, 7, or 8.
2. The drug conjugate of claim 1, wherein Lig is a monoclonal antibody.
3. The drug conjugate of claim 1 or 2, wherein Lig is selected from the group consisting of an anti-TROP2 antibody, an anti-EGRF antibody, an anti-HER2 antibody, an anti-B7-H3 antibody, an anti-CD30 antibody, an anti-CD33 antibody, and an anti-CD70 antibody.
4. The drug conjugate of any one of claims 1-3, wherein Lig is selected from the group consisting of an anti-TROP2 antibody, an anti-EGRF antibody, and an anti-HER2 antibody.
5. The drug conjugate of any one of claims 1-4 wherein s is 1 or 8.
6. The drug conjugate of any one of claims 1-5, wherein L is selected from the group consisting of:
Figure imgf000209_0001
wherein * denotes the point of attachment to R.
7. The drug conjugate of any one of claims 1-6, wherein R is selected from the group consisting of -CH2CH2O-, -C(O)-CH2ONH-, -C(O)-O-CH2CH2O-, -C(O)-NH-CH2CH2O-, -C(O)-C1alkyl- C(O)-NH-CH2CH2O-, -C(O)-C3alkyl-C(O)-NH-CH2CH2O-, and -C(O)-CH(CH3)-NH-.
8. The drug conjugate of any one of claims 1-6, wherein R is selected from the group consisting of -C(O)-triazolyl-CH2CH2O-, -CH2-triazolyl-CH2CH2O-, and -C(O)-furanyl-CH2O-.
9. The drug conjugate of any one of claims 1-6, wherein R is selected from the group consisting of -C(O)-C3cycloalkyl-CH2CH2O-, -C(S)-C3cycloalkyl-CH2CH2O-,-C(O)-C3cycloalkyl-NH-, and -C(O)-O-phenyl-NH-.
10. The drug conjugate of any one of claims 1-6, wherein R is selected from the group consisting of -C(O)-pyrrolidinyl- and -C(O)-pyrrolidinyl-C(O)-CH(CH3)-NH-, wherein pyrrolidinyl may optionally be substituted by one or two fluoro atoms.
11. The drug conjugate of any one of claims 1-10, wherein R is selected from the group consisting of
Figure imgf000210_0001
wherein ** denotes the point of attachment to L.
12. The drug conjugate of any one of claims 1-11, wherein the drug conjugate is selected from the group consisting of:
Figure imgf000211_0001
Figure imgf000212_0001
Figure imgf000213_0001
Figure imgf000214_0001
,
Figure imgf000215_0001
Figure imgf000216_0001
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Lig is selected from the group consisting of an anti-TROP2 antibody, an anti-EGRF antibody, and an anti-HER2 antibody.
13. The drug conjugate of any one of claims 1-12, wherein Lig is an anti-TROP2 antibody.
14. The drug conjugate of any one of claims 1-12, wherein Lig is an anti-EGRF antibody.
15. The drug conjugate of any one of claims 1-12, wherein Lig is an anti-HER2 antibody.
16. A therapeutic payload represented by Formula II:
Figure imgf000216_0002
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: Y is hydrogen or -C1-3alkyl; R is selected from the group consisting of -C(O)-C3alkyl, -C(O)O-C3alkyl, -C(O)- C3cycloalkyl, -C(O)-C4cycloalkyl, -C1alkyl, -C(O)-(pyrrolidinyl), and -C(O)-CH2ONH-C(O)- (pyrrolidinyl); wherein: -C(O)-C3alkyl, -C(O)O-C3alkyl, -C(O)-C3cycloalkyl, -C(O)-C4cycloalkyl or - C1alkyl is substituted by one or two substituents each independently selected from the group consisting of hydroxyl, -NH2, -CHO and -COOH; -C(O)-(pyrrolidinyl) and -C(O)-CH2ONH-C(O)-(pyrrolidinyl) may optionally be substituted on an available pyrrolidinyl carbon atom by one or two substituents each independently selected from the group consisting of halogen and hydroxyl, or two R1 join together to form oxo; and -C(O)-(pyrrolidinyl) may optionally be substituted on an available pyrrolidinyl nitrogen atom by -CHO, -C(O)CH3, or -C(O)CH2NH2.
17. The therapeutic payload of claim 16, wherein R is selected from the group consisting of: ,
Figure imgf000217_0001
18. The therapeutic payload of claim 16 or 17, wherein the therapeutic payload is selected from the group consisting of:
Figure imgf000217_0002
Figure imgf000218_0001
Figure imgf000219_0001
or a pharmaceutically acceptable salt thereof.
19. A linker-payload construct represented by Formula IIIA or Formula IIIB:
Figure imgf000219_0002
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: L is selected from the group consisting of:
Figure imgf000219_0003
wherein * denotes the point of attachment to R; R is selected from the group consisting of:
a
Figure imgf000220_0001
wherein ** denotes the point of attachment to L; and s is 0, 1, 2, 3, 4, 5, 6, 7, or 8; and t is 0, 1, 2, 3, 4, 5, 6, 7, or 8.
20. The linker-payload construct of claim 19, wherein R is selected from the group consisting o
Figure imgf000220_0002
Figure imgf000221_0001
wherein ** denotes the point of attachment to L.
21. The linker-payload construct of claim 19 or 20, wherein s is 1 or 8.
22. The linker-payload construct of any one of claims 19-21, wherein the linker-payload construct is selected from the group consisting of:
Figure imgf000221_0002
,
Figure imgf000222_0001
,
,
Figure imgf000223_0001
Figure imgf000224_0001
Figure imgf000225_0001
23. A pharmaceutical composition comprising the drug conjugate of any one of claims 1-15, and a pharmaceutically acceptable excipient.
24. A pharmaceutical composition comprising the therapeutic payload of any one of claims 16- 18, and a pharmaceutically acceptable excipient.
25. A pharmaceutical composition comprising the linker-payload construct of any one of claims 16-18, and a pharmaceutically acceptable excipient.
26. A method of delivering a therapeutically effective amount of a therapeutic payload moiety to a patient in need thereof, comprising administering to the patient the drug conjugate of any one of claims 1-15.
27. A method of treating cancer in patient in need thereof, comprising administering to the patient an effective amount of a drug conjugate of any one of claims 1-15.
28. A method of treating cancer in patient in need thereof, comprising administering to the patient an effective amount of a therapeutic payload of any one of claims 16-18.
29. The method of claim 27 or 28, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, and esophageal cancer.
PCT/US2023/078842 2022-11-04 2023-11-06 Exatecan derivatives and antibody-drug conjugates thereof WO2024098066A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263422517P 2022-11-04 2022-11-04
US63/422,517 2022-11-04

Publications (1)

Publication Number Publication Date
WO2024098066A1 true WO2024098066A1 (en) 2024-05-10

Family

ID=89164291

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/078842 WO2024098066A1 (en) 2022-11-04 2023-11-06 Exatecan derivatives and antibody-drug conjugates thereof

Country Status (1)

Country Link
WO (1) WO2024098066A1 (en)

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3130608A1 (en) * 2014-04-10 2017-02-15 Daiichi Sankyo Co., Ltd. (anti-her2 antibody)-drug conjugate
WO2021151984A1 (en) * 2020-01-31 2021-08-05 Innate Pharma Treatment of cancer
CA3195515A1 (en) * 2020-09-30 2022-04-07 Duality Biologics (Suzhou) Co., Ltd. Anti-tumor compound and preparation method and use thereof
WO2022236136A1 (en) * 2021-05-07 2022-11-10 ALX Oncology Inc. Exatecan derivatives and antibody-drug conjugates thereof
WO2023088235A1 (en) * 2021-11-16 2023-05-25 Genequantum Healthcare (Suzhou) Co., Ltd. Exatecan derivatives, linker-payloads, and conjugates and thereof
WO2023125530A1 (en) * 2021-12-28 2023-07-06 Beigene, Ltd. Antibody drug conjugates
WO2023138635A1 (en) * 2022-01-18 2023-07-27 甘李药业股份有限公司 Exatecan derivative-antibody conjugate and medical use thereof
WO2023143365A1 (en) * 2022-01-28 2023-08-03 映恩生物制药（苏州）有限公司 Her3 antibody-drug conjugate and use thereof
WO2023173026A1 (en) * 2022-03-10 2023-09-14 Sorrento Therapeutics, Inc. Antibody-drug conjugates and uses thereof
WO2023174401A1 (en) * 2022-03-18 2023-09-21 映恩生物制药（苏州）有限公司 Anti-gpc3 antibody drug conjugate and use thereof
WO2023186015A1 (en) * 2022-03-30 2023-10-05 映恩生物制药（苏州）有限公司 B7h4 antibody-drug conjugate and use thereof

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3130608A1 (en) * 2014-04-10 2017-02-15 Daiichi Sankyo Co., Ltd. (anti-her2 antibody)-drug conjugate
WO2021151984A1 (en) * 2020-01-31 2021-08-05 Innate Pharma Treatment of cancer
CA3195515A1 (en) * 2020-09-30 2022-04-07 Duality Biologics (Suzhou) Co., Ltd. Anti-tumor compound and preparation method and use thereof
WO2022236136A1 (en) * 2021-05-07 2022-11-10 ALX Oncology Inc. Exatecan derivatives and antibody-drug conjugates thereof
WO2023088235A1 (en) * 2021-11-16 2023-05-25 Genequantum Healthcare (Suzhou) Co., Ltd. Exatecan derivatives, linker-payloads, and conjugates and thereof
WO2023125530A1 (en) * 2021-12-28 2023-07-06 Beigene, Ltd. Antibody drug conjugates
WO2023138635A1 (en) * 2022-01-18 2023-07-27 甘李药业股份有限公司 Exatecan derivative-antibody conjugate and medical use thereof
WO2023143365A1 (en) * 2022-01-28 2023-08-03 映恩生物制药（苏州）有限公司 Her3 antibody-drug conjugate and use thereof
WO2023173026A1 (en) * 2022-03-10 2023-09-14 Sorrento Therapeutics, Inc. Antibody-drug conjugates and uses thereof
WO2023174401A1 (en) * 2022-03-18 2023-09-21 映恩生物制药（苏州）有限公司 Anti-gpc3 antibody drug conjugate and use thereof
WO2023186015A1 (en) * 2022-03-30 2023-10-05 映恩生物制药（苏州）有限公司 B7h4 antibody-drug conjugate and use thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CARREIRAKVAERNO: "Classics in Stereoselective Synthesis", 2009, WILEY-VCH
FIRTH D. ET AL., ANALYTICAL BIOCHEMISTRY, vol. 485, 2015, pages 34 - 42
RAUTIO, KUMPULAINEN ET AL., NATURE REVIEWS DRUG DISCOVERY, vol. 7, 2008, pages 255
SIMPLICIO ET AL., MOLECULES, vol. 13, 2008, pages 519
ZHU X. ET AL., J PHARM ANAL., vol. 10, no. 3, 2020, pages 209 - 220

Similar Documents

Publication Publication Date Title
EP4334322A1 (en) Exatecan derivatives and antibody-drug conjugates thereof
EP4316524A2 (en) Camptothecin derivatives
US10590165B2 (en) Antibody drug conjugates
US20230140047A1 (en) 1H-PYRAZOLO[4,3-d]PYRIMIDINE COMPOUNDS AS TOLL-LIKE RECEPTOR 7 (TLR7) AGONISTS AND METHODS AND USES THEREFOR
US20230270868A1 (en) Pyrrolobenzodiazepine dimer prodrug and ligand-linker conjugate compound of the same
JP7103953B2 (en) Antibody drug conjugate with a derivative of amatoxin as a drug
TW202016081A (en) Small molecule modulators of human sting, conjugates and therapeutic applications
JP2016516035A (en) Cell growth inhibitors and their conjugates
US11691982B2 (en) Thailanstatin analogs
EA035625B1 (en) Novel cryptophycin compounds and conjugates, their preparation and their therapeutic use
KR20210094568A (en) Bis-octahydrophenanthrene carboxamide derivatives and protein conjugates thereof for use as LXR agonists
KR20210106467A (en) Tubulisin and Protein-Tubulin Conjugates
JP2023520605A (en) Camptothecin derivatives and their conjugates
KR20230137295A (en) Conjugates and their uses
EP3538098A1 (en) Antibody-drug conjugates
AU2016226083A1 (en) Cyclic peptide analogs and conjugates thereof
WO2024098066A1 (en) Exatecan derivatives and antibody-drug conjugates thereof
CA3226897A1 (en) Tumor-associated calcium signal transducer 2 (tacstd2) antibody-maytansine conjugates and methods of use thereof
KR20230028325A (en) Tubulysins and protein-tubulin conjugates
WO2018045245A1 (en) Cyclic peptide analogs and conjugates thereof