Antibody-drug conjugate linkers

Abstract

Disclosed are a linker compound, a linker-drug conjugate formed by coupling the linker compound with a drug, and an antibody-drug conjugate formed by coupling a drug with an antibody or an antigen binding fragment thereof through the linker compound, and a pharmaceutical composition for treating cancer comprising the antibody-drug conjugate.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Patent Application No. 63 / 529,828, filed on July 31, 2023, Korean Patent Application No. 10-2023-0017886, filed on February 10, 2023, and Korean Patent Application No. 10-2023-0024745, filed on February 24, 2023, the entire disclosures of which are incorporated herein by reference. Technical Field

[0003] The present invention discloses a linker compound, a linker-drug conjugate formed by coupling the linker compound with a drug, an antibody-drug conjugate formed by coupling a drug to an antibody or an antigen-binding fragment thereof via the linker compound, and a method for treating cancer by administering the antibody-drug conjugate to a subject in need. Background Art

[0004] Several approved or clinically advanced antibody-drug conjugates (ADCs) have encountered safety challenges in their anti-tumor applications, characterized by adverse side effects and inherent toxicity. Despite their impressive anti-tumor efficacy, these ADCs continue to face significant safety challenges in clinical practice. This hurdle highlights the urgent need to advance ADC therapeutics that combine superior efficacy with an improved safety profile.

[0005] 80% of approved ADCs utilize cleavable linkers to deliver their payload to tumors. However, a limitation of cleavable linkers is the potential for premature payload shedding during circulation, before the ADC reaches its tumor target. This premature release of the payload into the systemic circulation reduces the potency of the remaining ADC in circulation and can lead to nonspecific uptake and off-target toxicities, which can be dose-limiting. These challenges have significantly impacted ADC drug development. This not only complicates the assessment of antitumor efficacy in preclinical studies involving mice but also limits the dose that can be administered to patients, hindering their full therapeutic potential. Summary of the Invention

[0006] To overcome the above limitations, the present invention provides a novel cleavable linker system with excellent therapeutic efficacy and outstanding safety.

[0007] (1) Specifically, the present disclosure provides a compound as shown in formula (1):

[0008] Formula (1)

[0009]

[0010] in,

[0011] P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, -(Y-CH2)-, or -(dibenzocyclooctyne-NC(=O))-, wherein Y is Br, Cl, or I, and -(dibenzocyclooctyne-NC(=O))- has the following structure:

[0012]

[0013] L n is optionally included, and if included is cycloalkyl, alkyl, -(OCH2CH2)-, or -(CH2-C(=O)-NH-CH2-CH2)-, and n is an integer from 1 to 6;

[0014] A 1 is a peptide residue containing 0-3 amino acids;

[0015] A 2 is a peptide residue containing 0-3 amino acids;

[0016] Z is optionally included, and if included is a self-immolative spacer, -(NHCH2)-, or p-aminocarbamate; and

[0017] X1 is H, monosaccharide, disaccharide, oligosaccharide, polyethylene glycol, sulfate, phosphate or pyrophosphate.

[0018] (2) In addition, the present disclosure provides a linker-drug conjugate comprising:

[0019] The compound as described in embodiment (1);

[0020] and a drug coupled to the compound via Z of formula (1).

[0021] (3) The present disclosure also provides a linker-drug conjugate as described in embodiment (2), wherein the drug is conjugated to the compound in the form of -(NH-drug)- or -(O-drug)-.

[0022] (4) The present disclosure also provides a linker-drug conjugate as described in embodiment (2), wherein the drug is a topoisomerase I inhibitor.

[0023] (5) The present disclosure provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, a drug, and a linker.

[0024] Wherein, the linker is the compound as described in embodiment (1),

[0025] Wherein, the antibody or antigen-binding fragment thereof is coupled to the compound via P of formula (1),

[0026] Wherein, the drug is coupled to the compound via Z of formula (1).

[0027] (6) The present disclosure provides a pharmaceutical composition for treating cancer, which comprises the antibody-drug conjugate as described in embodiment (5) above.

[0028] (7) The present disclosure provides a pharmaceutical composition as described in embodiment (6), wherein the cancer includes one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), colorectal cancer, colon cancer, kidney cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, renal cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer and urothelial carcinoma.

[0029] (8) The present disclosure also provides a compound as shown in formula (2):

[0030] Formula (2)

[0031]

[0032] in

[0033] P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, or -(dibenzocyclooctyne-NC(=O))-;

[0034] L n is optionally included, and if included is a cycloalkyl group, an alkyl group, or a direct bond, and n is an integer from 1 to 6;

[0035] X is –(C=O)-, a direct bond or 1 Direct bonds formed by side chains;

[0036] A 1 For amino acids;

[0037] R m is a hydrophilic side chain connected to A1, m is an integer from 1 to 10; and

[0038] Z is optionally included, and if included is a self-immolative spacer, -(NHCH2)-, or p-aminocarbamate.

[0039] (9) The present disclosure provides the compound as described in embodiment (8), wherein the amino acid is aspartic acid, glycine, glutamic acid or lysine.

[0040] (10) The present disclosure provides a linker-drug conjugate comprising:

[0041] The compound according to embodiment (8);

[0042] and a drug coupled to the compound via Z of formula (2).

[0043] (11) The present disclosure provides a linker-drug conjugate as described in embodiment (10), wherein the drug is conjugated to the compound in the form of -(NH-drug)- or -(O-drug)-.

[0044] (12) The present disclosure provides a linker-drug conjugate as described in embodiment (10), wherein the drug is a topoisomerase 1 inhibitor.

[0045] (13) The present disclosure provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, a drug, and a linker.

[0046] Wherein, the linker is the compound as described in embodiment (8),

[0047] Wherein, the antibody or antigen-binding fragment thereof is coupled to the compound via P of formula (2),

[0048] Wherein, the drug is coupled to the compound via Z of formula (2).

[0049] (14) The present disclosure provides a pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate as described in embodiment (13).

[0050] (15) The present disclosure provides a pharmaceutical composition as described in embodiment (14), wherein the cancer includes one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), colorectal cancer, colon cancer, kidney cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, renal cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer and urothelial carcinoma.

[0051] (16) The present disclosure also provides a compound as shown in formula (3):

[0052] Formula (3)

[0053]

[0054] in:

[0055] P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, or -(dibenzocyclooctyne-NC(=O))-;

[0056] L n is optionally included, and if included is a cycloalkyl group, an alkyl group, or a direct bond, and n is an integer from 1 to 6;

[0057] X is -(C=O)- or a direct bond;

[0058] A 1 For amino acids;

[0059] R m Optionally included, if included then connected to A 1 A hydrophilic side chain, wherein m is an integer from 1 to 10; and

[0060] Z is optionally included, and if included is a self-immolative spacer, -(NHCH2)-, or p-aminocarbamate.

[0061] (17) The present disclosure provides a compound according to embodiment (16), wherein the amino acid is aspartic acid, glycine, glutamic acid or lysine.

[0062] (18) The present disclosure provides a linker-drug conjugate comprising:

[0063] The compound as described in Example (16);

[0064] and a drug coupled to the compound via Z of formula (2).

[0065] (19) The present disclosure provides a linker-drug conjugate as described in embodiment (18), wherein the drug is conjugated to the compound in the form of -(NH-drug)- or -(O-drug)-.

[0066] (20) The present disclosure provides a linker-drug conjugate as described in embodiment (18), wherein the drug is a topoisomerase 1 inhibitor.

[0067] (21) The present disclosure provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, a drug, and a linker.

[0068] Wherein, the linker is a compound as described in embodiment (16),

[0069] Wherein, the antibody or antigen-binding fragment thereof is coupled to the compound via P of formula (3),

[0070] Wherein, the drug is coupled to the compound via Z of formula (3).

[0071] (22) The present disclosure provides a pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate as described in embodiment (21).

[0072] (23) The present disclosure provides a pharmaceutical composition as described in embodiment (22), wherein the cancer includes one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), colorectal cancer, colon cancer, kidney cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, renal cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer and urothelial carcinoma.

[0073] Other implementations are also described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 The peptide cleavage specificity of cathepsins B and L was shown (J. Proteome Res. 2011, 105363-5373).

[0075] Figure 2 SEC of trastuzumab-LP2 is shown.

[0076] Figure 3 HIC of trastuzumab-LP2 is shown.

[0077] Figure 4 In vitro data are shown for Payload-001, Trastuzumab-LP1, and Trastuzumab-LP2.

[0078] Figure 5 SEC of trastuzumab-LP3 is shown.

[0079] Figure 6 HIC of trastuzumab-LP3 is shown.

[0080] Figure 7 HIC comparison of trastuzumab / trastuzumab-LP1 / trastuzumab-LP3 is shown.

[0081] Figure 8In vitro data are shown for trastuzumab-LP1 and trastuzumab-LP3.

[0082] definition

[0083] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments described herein, some preferred methods, compositions, apparatus, and materials are described herein. However, before describing the present materials and methods, it should be understood that the present invention is not limited to the specific molecules, compositions, methods, or protocols described herein, as these can vary according to routine experimentation and optimization. It should also be understood that the terminology used in the specification is used only to describe specific versions or embodiments and is not intended to limit the scope of the embodiments described herein.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in the event of a conflict, the present specification (including definitions) will control. Therefore, in the context of the embodiments described herein, the following definitions apply.

[0085] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" refers to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

[0086] As used herein, the term "comprising" and its linguistic variants indicate the presence of the described features, elements, method steps, etc., but does not exclude the presence of other features, elements, method steps, etc. In contrast, "consisting of..." and its linguistic variants indicate the presence of the described features, elements, method steps, etc., and excludes any undescribed features, elements, method steps, etc., except for impurities that are typically accompanied. The phrase "consisting essentially of..." indicates the described features, elements, method steps, etc. and any additional features, elements, method steps, etc. that do not materially affect the basic properties of the composition, system or method. Many embodiments herein are described using open-ended "comprising" language. Such embodiments encompass multiple closed "consisting of..." and / or "consisting essentially of..." embodiments, which may also be claimed or described using such language.

[0087] As used herein, the term "antibody-drug conjugate" refers to an antibody or antigen-binding fragment thereof linked to another anti-tumor compound (eg, chemotherapeutic drug, toxin, immunotherapeutic agent, imaging probe, etc.). This linkage can be a covalent bond.

[0088] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in the present disclosure can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, single-chain antibodies, and humanized antibodies.

[0089] As used herein, the term "antigen-binding fragment thereof" refers to a portion of a full-length antibody, typically its antigen-binding region or variable region. Examples of such antibody fragments include: Fab fragments, Fab' fragments, Fab'-SH fragments, Fv fragments, scFv fragments, F(ab')2 fragments, VL fragments, VH fragments, ScFv-Fc fragments, (ScFv)2-Fc fragments, diabodies, linear antibodies, fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity-determining regions), and any of the above epitope-binding fragments that can immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0090] As used herein, the term "linker" refers to a moiety that connects an antibody portion to an anti-tumor compound portion by covalent bonding. In some cases, the term "linker" as used herein may refer to a moiety comprising a cleavable unit and other moieties, such as a linking group, a group comprising one or more solubilizing groups, etc. In other cases, the term "linker" as used herein may refer to a specifically defined element, such as a linker that is "capable of being cleaved by cathepsin B" (described further below).

[0091] As used herein, the term "cleavable" refers to a linker that covalently links the antibody portion to the anti-tumor compound portion, but the linker is capable of cleavage under physiologically relevant conditions, thereby severing the covalent bond between the two portions. Cleavage typically results in release of the anti-tumor compound from the antibody. The term "non-cleavable" as used herein refers to a linker that is not readily cleaved under physiological conditions. Such linkers possess sufficient stability against degradation to maintain the anti-tumor compound attached to the antibody or antigen-binding fragment thereof until the antibody or antigen-binding fragment thereof itself is at least partially degraded.

[0092] As used herein, the term "peptide" refers to a compound comprising a continuous sequence of at least two amino acids linked to each other by peptide bonds. The terms "dipeptide," "tripeptide," and "tetrapeptide" refer to compounds comprising a continuous sequence of two, three, and four amino acids linked to each other by peptide bonds, respectively. The term "peptide bond" herein is intended to encompass (backbone) amide bonds as well as modified linkages; such modified linkages may be obtained when non-natural amino acids are introduced into the peptide sequence. In this case, the modified linkage replaces the (backbone) amide bond formed by the reaction of the amino and carboxyl groups of two amino acid residues in the continuous peptide sequence. For example, the modified linkage may be an ester bond, a thioester bond, a urea bond, a thiourea bond, or a triazole bond. Preferably, the amino acids forming the continuous peptide sequence are linked to each other by backbone amide bonds. The peptide may be linear or branched. In a preferred aspect, the peptide is a linear dipeptide, tripeptide, or tetrapeptide, more preferably a linear tripeptide or tetrapeptide.

[0093] As used herein, the term "amino acid" refers to a compound containing at least one amino group and at least one acidic group (preferably a carboxyl group), or a compound derived from such a compound. The distance between the amino group and the acidic group is not particularly limited. α-, β-, and γ-amino acids are all suitable, but α-amino acids, particularly α-aminocarboxylic acids, are particularly preferred. The term "amino acid" includes both naturally occurring amino acids, such as natural protein amino acids, and synthetic amino acids not found in nature. Hereinafter, amino acids may be referred to by a 3-letter amino acid code (Arg, Phe, Ala, Cys, Gly, Gln, etc.) or a single-letter amino acid code (R, F, A, C, G, Q, etc.). Unless otherwise indicated, when an amino acid is referred to by a 3-letter amino acid code, it refers to the corresponding (L)- or (D)-amino acid. Herein, amino acid sequences are written from N-terminus to C-terminus (from left to right). Unless otherwise indicated or the context requires otherwise, all connections between adjacent amino acid groups are formed by peptide (amide) bonds.

[0094] As used herein, the term "cycloalkyl" refers to a substituted or unsubstituted cyclic hydrocarbon group having 3 to 20 carbon atoms, preferably 5 to 8 carbon atoms. A cycloalkyl group may be composed of a single ring, but may also be composed of two or more fused rings. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl. More preferably, the cycloalkyl group is cyclopentyl or cyclohexyl.

[0095] As used herein, the term "alkyl" refers to a linear chain (straight chain) or branched, saturated or unsaturated hydrocarbon group having 1 to 20 carbon atoms, preferably 1 to 5 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, vinyl, allyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl and -3-methyl-1-butynyl. More preferably, alkyl is methyl or ethyl.

[0096] As used herein, the term "self-immolative spacer" refers to a bifunctional chemical group that is capable of covalently linking two spacer chemical groups into a generally stable ternary molecule; upon enzymatic cleavage, the group can release one of the spacer chemical groups from the ternary molecule; and after enzymatic cleavage, the group can spontaneously cleave from the remainder of the molecule to release the other spacer chemical group.

[0097] As used herein, the term "monosaccharide" refers to any sugar that cannot be hydrolyzed into simpler sugars. Monosaccharides are typically C5 (such as xylose) and C6 sugars (such as glucose), but may also include monosaccharides with other numbers of carbon atoms, such as C3, C4, C7, C8, etc. In other words, monosaccharides are the simplest building blocks of oligosaccharides and polysaccharides.

[0098] As used herein, "disaccharide" refers to a carbohydrate composed of two monosaccharides linked by a glycosidic bond. As used herein, "oligosaccharide" refers to a carbohydrate composed of 2 to 9 monosaccharides (e.g., linked by glycosidic bonds). Oligosaccharides may also be referred to herein as "oligomers." The monosaccharides that make up a disaccharide or oligosaccharide may be referred to, for example, as "monosaccharide units" or "monomer units." Preferred monosaccharides herein are fructose and glucose.

[0099] The term "oligosaccharide" as used herein refers to a linear or branched carbohydrate molecule composed of identical or different monosaccharide units linked by glycosidic bonds, with the general formula C x (H2O) y Oligosaccharides can be considered as short-chain polysaccharides, i.e. polysaccharides with a small number of monomer residues in the polymer chain. When oligosaccharides contain C6 monosaccharide residues, their general formula can be expressed as (C6H 10 O5) n , wherein n is from about 2 to about 9 (i.e., the number of hexose monomers in the oligosaccharide). As used herein, oligomers such as cellobiose have a degree of polymerization (DP) of from 2 to about 9, while polymers such as cellulose have a DP of at least about 10.

[0100] The term "drug" as used herein refers to a compound (payload or ADC payload) with an anti-tumor effect, which has a substituent or partial structure that allows connection to a linker structure. When part or all of the linker is cleaved in tumor cells, the drug (i.e., the anti-tumor compound portion) is released, thereby exerting the anti-tumor effect of the anti-tumor compound. When the linker is cleaved at the connection position with the drug, the anti-tumor compound can be released in an unmodified structure to exert its inherent anti-tumor effect. For example, an ideal payload may have the following characteristics. First, they may have sufficiently high cytotoxicity. Tumor-specific antigens are very limited, especially in solid tumors. In addition, due to the low permeability and poor internalization activity of monoclonal antibodies, the number of ADC payloads that can be internalized by tumor cells through antibody-antigen binding is extremely low. Secondly, the ADC payload may have sufficiently low immunogenicity.

[0101] As used herein, the term "topoisomerase 1 inhibitor" refers to compounds that inhibit the activity of topoisomerase 1. Topoisomerase 1 is an important nuclear enzyme crucial for maintaining genomic stability and DNA structure and has become a popular target for ADCs. Topoisomerase 1 (TOPO-I) inhibitors are associated with both innate and adaptive immune responses, suggesting that ADCs targeting TOPO-I may also be beneficial for anti-tumor immunotherapy.

[0102] As used herein, the term "hydrophilic side chain" (hydrophilic group or hydrophilic unit) refers to a group or unit that can improve overall water solubility and conjugation efficiency, thereby limiting accumulation and aggregation of the ADC during the conjugation process and in circulation.

[0103] As used herein, the term "treat" refers to partially or completely alleviating, ameliorating, alleviating, delaying the onset, inhibiting the progression, reducing the severity, and / or diminishing one or more symptoms or features of a particular disease, disorder, and / or condition. For example, "treating" cancer may refer to inhibiting the growth and / or spread of cancer cells, killing cancer cells, or shrinking cancer cells. Treatment can be applied to subjects who do not show signs of a disease, disorder, and / or condition, and / or to subjects who show only early signs of a disease, disorder, and / or condition, to reduce the risk of developing pathology associated with the disease, disorder, and / or condition.

[0104] As used herein, the terms "approximately," "about," "substantially," and the like indicate that the quantity or value in question may be exact or may provide an equivalent result or effect to that stated or described herein. That is, it should be understood that quantities, dimensions, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller as necessary to account for tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art to achieve an equivalent result or effect. In some cases, it is not possible to reasonably determine a value that provides an equivalent result or effect. In such cases, as used herein, unless otherwise stated or inferred, "approximately" and "about" are generally understood to refer to a range of ±10% of the nominal value. Generally speaking, regardless of whether or not a quantity, dimension, formulation, parameter, or other quantity or characteristic is explicitly stated, it should be understood that when "approximately," "approximately," or "about," is used before a quantitative value, the specific quantitative value itself is also included in that parameter, unless otherwise specifically stated.

[0105] As used herein, the term "cancer" refers to a disease characterized by the rapid, uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the blood and lymphatic systems. Examples of various cancers include, but are not limited to, kidney cancer, spleen cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

[0106] The term "effective amount" refers to an amount of a therapeutic, prophylactic and / or diagnostic agent that, when administered to a subject suffering from or susceptible to a disease, disorder and / or condition, is sufficient to treat, alleviate, ameliorate, palliate, relieve symptoms, prevent, delay onset, inhibit progression, reduce severity and / or reduce the incidence of the disease, disorder and / or condition.

[0107] The terms "subject," "patient," "individual," and the like are used interchangeably herein to refer to any animal or cell thereof, whether in vitro or in vivo, suitable for use in the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0108] Detailed description

[0109] 1.GGYG connection subsystem

[0110] (1) The present disclosure provides a compound as shown in formula (1):

[0111] Formula (1)

[0112]

[0113] in,

[0114] P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, -(Y-CH2)- or -(dibenzocyclooctyne-NC(=O))-, Y is Br, Cl or I, and -(dibenzocyclooctyne-NC(=O))- has the following structure:

[0115]

[0116] L n is optionally included, and if included is cycloalkyl, alkyl, -(OCH2CH2)-, or -(CH2-C(=O)-NH-CH2-CH2)-, and n is an integer from 1 to 6;

[0117] A 1 is a peptide residue containing 0-3 amino acids;

[0118] A 2 is a peptide residue containing 0-3 amino acids;

[0119] Z is optionally included, and if included is a self-immolative spacer, -(NHCH2)-, or p-aminocarbamate; and

[0120] X1 is H, monosaccharide, disaccharide, oligosaccharide, polyethylene glycol, sulfate, phosphate or pyrophosphate.

[0121] (2) In addition, the present disclosure provides a linker-drug conjugate comprising:

[0122] The compound as described in embodiment (1);

[0123] and a drug coupled to the compound via Z of formula (1).

[0124] In one embodiment, the drug (payload or ADC payload) may comprise: (i) microtubule targeting payloads such as maytansines, auristatins, eribulin, tubulolysins, cryptophycins, and EG5 inhibitors; (ii) DNA targeting payloads such as enediynes, topoisomerase 1 inhibitors, pyrrolo[2,1-c][1,4]benzodiazepines (iii) RNA-targeted payloads, such as talantins and amanitins; (iv) immunomodulatory ADC payloads, such as toll-like receptor agonists, stimulator of interferon genes (STING), and glucocorticoid receptor modulators; and (v) any new potential ADC payloads, such as Bcl-xL inhibitors, nicotinamide phosphoribosyltransferase (NAMPT), carbamycins, protein degradation targeting chimeric (PROTAC) molecules, near-infrared photoimmunotherapy (NIR-PIT) drugs, and dual payload combinations (e.g., MMAE and MMAF).

[0125] (3) The present disclosure also provides a linker-drug conjugate as described in embodiment (2), wherein the drug is conjugated to the compound in the form of -(NH-drug)- or -(O-drug)-.

[0126] (4) The present disclosure also provides a linker-drug conjugate as described in embodiment (2), wherein the drug is a topoisomerase I inhibitor.

[0127] "Topoisomerase 1 inhibitors" refer to compounds that inhibit the activity of topoisomerase 1. As a key nuclear enzyme in maintaining genomic stability and DNA structure, this enzyme has become a popular target for ADCs. Topoisomerase 1 (TOPO-I) inhibitors are associated with both innate and adaptive immune responses, suggesting that ADCs targeting TOPO-I may also synergize and enhance anti-tumor immunotherapy.

[0128] For example, linker-drug conjugates may include the following compounds.

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] (5) The present disclosure provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, a drug, and a linker.

[0135] Wherein, the linker is the compound as described in embodiment (1),

[0136] Wherein, the antibody or antigen-binding fragment thereof is coupled to the compound via P of formula (1),

[0137] Wherein, the drug is coupled to the compound via Z of formula (1).

[0138] In a certain embodiment, the antigen-binding fragment may comprise one or more of a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a Fv fragment, a scFv (single-chain variable region) fragment, a F(ab')2 fragment, a VL (light chain variable region) fragment, a VH (heavy chain variable region) fragment, a ScFv-Fc fragment, a (ScFv)2-Fc fragment, a bispecific antibody (diabody), a linear antibody (linearantibody), a fragment produced by a Fab expression library, an anti-idiotypic (anti-Id) antibody, a complementarity determining region (CDR), and an epitope binding fragment.

[0139] (6) The present disclosure provides a pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate as described in embodiment (5).

[0140] In one embodiment, the antibody-drug conjugate (ADC) can be administered to a subject by any route of administration. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, vaginal administration, intraocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal mucosal administration, and parenteral administration (including injection, such as intravenous injection, arterial injection, intramuscular injection, and subcutaneous injection). The mode of administration may be continuous or intermittent. Pharmaceutical preparations can be used for therapeutic administration, i.e., administration to treat existing diseases or conditions; and can also be used for prophylactic administration, i.e., administration to prevent cancer (such as hematological tumors or solid tumors).

[0141] An effective amount can be administered in one or more administrations, applications or dosages and is not limited to a particular formulation or route of administration. In one embodiment, administration is via a course of treatment comprising multiple treatment cycles and multiple rest periods.

[0142] In another embodiment, the antibody-drug conjugate can be administered to a subject at a concentration effective to treat the subject's cancer. For example, the antibody-drug conjugate can be in the range of 1 mg / kg to 4-5 mg / kg body weight.

[0143] (7) The present disclosure provides a pharmaceutical composition as described in embodiment (6), wherein the cancer includes one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), colorectal cancer, colon cancer, kidney cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, renal cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer and urothelial carcinoma.

[0144] 2.DGGFG or GGGFG connection subsystem

[0145] (8) The present disclosure also provides a compound as shown in formula (2):

[0146] Formula (2)

[0147]

[0148] in

[0149] P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, or -(dibenzocyclooctyne-NC(=O))-;

[0150] L n is optionally included, and if included is a cycloalkyl group, an alkyl group, or a direct bond, and n is an integer from 1 to 6;

[0151] X is –(C=O)-, a direct bond or 1 Direct bonds formed by side chains;

[0152] A 1 For amino acids;

[0153] R m is a hydrophilic side chain connected to A1, m is an integer from 1 to 10; and

[0154] Z is optionally included, and if included is a self-immolative spacer, -(NHCH2)-, or p-aminocarbamate.

[0155] In one embodiment, "hydrophilic side chain" refers to a group or unit that can improve overall water solubility and conjugation efficiency, thereby limiting accumulation and aggregation of the ADC during the conjugation process and in circulation.

[0156] In a certain embodiment, the hydrophilic side chains may be selected from the following compounds:

[0157]

[0158] wherein n may be an integer from 3 to 24. In a certain embodiment, the lower limit of n may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. In a certain embodiment, the upper limit of n may be 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, or 4. In a certain embodiment, n may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0159] (9) The present disclosure provides the compound as described in embodiment (8), wherein the amino acid is aspartic acid, glycine, glutamic acid or lysine.

[0160] (10) The present disclosure provides a linker-drug conjugate comprising:

[0161] The compound as described in embodiment (8);

[0162] and a drug coupled to the compound via Z of formula (2).

[0163] In one embodiment, the drug (payload or ADC payload) may comprise: (i) microtubule targeting payloads such as maytansines, auristatins, eribulin, tubulolysins, cryptophycins, and EG5 inhibitors; (ii) DNA targeting payloads such as enediynes, topoisomerase 1 inhibitors, pyrrolo[2,1-c][1,4]benzodiazepines (iii) RNA-targeted payloads, such as talantins and amanitins; (iv) immunomodulatory ADC payloads, such as Toll-like receptor agonists, stimulator of interferon genes (STING), and glucocorticoid receptor modulators; (v) novel potential ADC payloads, such as Bcl-xL inhibitors, nicotinamide phosphoribosyltransferase (NAMPT), carbamycins, protein degradation targeting chimeric (PROTAC) molecules, near-infrared photoimmunotherapy (NIR-PIT) drugs, and dual payload combinations (e.g., MMAE and MMAF).

[0164] (11) The present disclosure also provides a linker-drug conjugate as described in embodiment (10), wherein the drug is conjugated to the compound in the form of -(NH-drug)- or -(O-drug)-.

[0165] (12) The present disclosure also provides a linker-drug conjugate as described in embodiment (10), wherein the drug is a topoisomerase I inhibitor.

[0166] "Topoisomerase 1 inhibitors" refer to compounds that inhibit the activity of topoisomerase 1. As a key nuclear enzyme in maintaining genomic stability and DNA structure, this enzyme has become a popular target for ADCs. Topoisomerase 1 (TOPO-I) inhibitors are associated with both innate and adaptive immune responses, suggesting that ADCs targeting TOPO-I may also synergize and enhance anti-tumor immunotherapy.

[0167] For example, linker-drug conjugates may include the following compounds.

[0168]

[0169]

[0170]

[0171] (13) The present disclosure provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, a drug, and a linker.

[0172] Wherein, the linker is a compound as described in embodiment (8),

[0173] Wherein, the antibody or antigen-binding fragment thereof is coupled to the compound via P of formula (2),

[0174] Wherein, the drug is coupled to the compound via Z of formula (2).

[0175] (14) The present disclosure provides a pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate as described in embodiment (13).

[0176] In one embodiment, the antibody-drug conjugate (ADC) can be administered to a subject by any route of administration. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, vaginal administration, intraocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal mucosal administration, and parenteral administration (including injection, such as intravenous injection, arterial injection, intramuscular injection, and subcutaneous injection). The mode of administration may be continuous or intermittent. The pharmaceutical preparation can be used for therapeutic administration, i.e., administration to treat an existing disease or condition; it can also be used for prophylactic administration, i.e., administration to prevent cancer (such as a blood tumor or a solid tumor).

[0177] An effective amount can be administered in one or more administrations, applications or dosages and is not limited to a particular formulation or route of administration. In one embodiment, administration is via a course of treatment comprising multiple treatment cycles and multiple rest periods.

[0178] In another embodiment, the antibody-drug conjugate can be administered to a subject at a concentration effective to treat the subject's cancer. For example, the antibody-drug conjugate can be in the range of 1 mg / kg to 4-5 mg / kg body weight.

[0179] (15) The present disclosure provides a pharmaceutical composition as described in embodiment (6), wherein the cancer includes one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), colorectal cancer, colon cancer, kidney cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, renal cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer and urothelial carcinoma.

[0180] 3. GGFG connection subsystem

[0181] (16) The present disclosure also provides a compound of formula (3):

[0182] Formula (3)

[0183]

[0184] in:

[0185] P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, or -(dibenzocyclooctyne-NC(=O))-;

[0186] L n is optionally included, and if included is a cycloalkyl group, an alkyl group, or a direct bond, and n is an integer from 1 to 6;

[0187] X is -(C=O)- or a direct bond;

[0188] A 1 For amino acids;

[0189] R m To connect to A 1 A hydrophilic side chain, wherein m is an integer from 1 to 10; and

[0190] Z is optionally included, and if included is a self-immolative spacer, -(NHCH2)-, or p-aminocarbamate.

[0191] In one embodiment, "hydrophilic side chain" refers to a group or unit that can improve overall water solubility and conjugation efficiency, thereby limiting accumulation and aggregation of the ADC during the conjugation process and in circulation.

[0192] In a certain embodiment, the hydrophilic side chain can be selected from the following compounds.

[0193]

[0194] wherein n may be an integer from 3 to 24. In a certain embodiment, the lower limit of n may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. In a certain embodiment, the upper limit of n may be 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, or 4. In a certain embodiment, n may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0195] (17) The present disclosure provides a compound as described in embodiment (16), wherein the amino acid is aspartic acid, glycine, glutamic acid or lysine.

[0196] (18) The present disclosure provides a linker-drug conjugate comprising:

[0197] The compound as described in embodiment (16);

[0198] and a drug coupled to the compound via Z of formula (2).

[0199] In one embodiment, the drug (payload or ADC payload) may comprise: (i) microtubule targeting payloads such as maytansines, auristatins, eribulin, tubulolysins, cryptophycins, and EG5 inhibitors; (ii) DNA targeting payloads such as enediynes, topoisomerase 1 inhibitors, pyrrolo[2,1-c][1,4]benzodiazepines (iii) RNA-targeted payloads, such as talantins and amanitins; (iv) immunomodulatory ADC payloads, such as toll-like receptor agonists, stimulator of interferon genes (STING), and glucocorticoid receptor modulators; and (v) any new potential ADC payloads, such as Bcl-xL inhibitors, nicotinamide phosphoribosyltransferase (NAMPT), carbamycins, protein degradation targeting chimeric (PROTAC) molecules, near-infrared photoimmunotherapy (NIR-PIT) drugs, and dual payload combinations (e.g., MMAE and MMAF).

[0200] (19) The present disclosure also provides a linker-drug conjugate as described in embodiment (18), wherein the drug is conjugated to the compound in the form of -(NH-drug)- or -(O-drug)-.

[0201] (20) The present disclosure also provides a linker-drug conjugate as described in embodiment (18), wherein the drug is a topoisomerase I inhibitor.

[0202] For example, the linker-drug conjugate may include the following compounds.

[0203]

[0204] (21) The present disclosure provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, a drug, and a linker.

[0205] Wherein, the linker is a compound as described in embodiment (16),

[0206] wherein the antibody or antigen-binding fragment thereof is coupled to the compound via P of formula (3),

[0207] The drug is coupled to the compound via Z of formula (3).

[0208] (22) The present disclosure provides a pharmaceutical composition for treating cancer, which comprises the antibody-drug conjugate as described in embodiment (21).

[0209] In one embodiment, the antibody-drug conjugate (ADC) can be administered to a subject by any route of administration. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, vaginal administration, intraocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal mucosal administration, and parenteral administration (including injection, such as intravenous injection, arterial injection, intramuscular injection, and subcutaneous injection). The administration route may be continuous or intermittent. The preparation can be used for therapeutic administration, i.e., administration to treat an existing disease or condition; it can also be used for prophylactic administration, i.e., administration to prevent cancer (such as a blood tumor or a solid tumor).

[0210] An effective amount can be administered in one or more administrations, applications or dosages and is not limited to a particular formulation or route of administration. In one embodiment, administration is via a course of treatment comprising multiple treatment cycles and multiple rest periods.

[0211] In another embodiment, the antibody-drug conjugate can be administered to a subject at a concentration effective to treat the subject's cancer. For example, the antibody-drug conjugate can be in the range of 1 mg / kg to 4-5 mg / kg body weight.

[0212] (23) The present disclosure provides a pharmaceutical composition as described in embodiment (22), wherein the cancer includes one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), colorectal cancer, colon cancer, kidney cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, renal cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer and urothelial carcinoma.

[0213] 4. Other connection subsystems

[0214] The present disclosure also provides the following linker systems. For example, the linker-drug conjugates may include the following compounds.

[0215]

[0216]

[0217] In the above compounds, the hydrophilic group may be as shown below.

[0218] hydrophilic group

[0219] In the embodiments described in the present disclosure, a specific drug (payload PBX-7016) has been conjugated to a variety of linker systems. However, as discussed in the present disclosure, any drug (payload) for ADC can be conjugated to these linkers. For example, the drug (payload or ADC payload) can include: (i) microtubule targeting payloads such as maytansines, auristatins, eribulin, tubulolysins, cryptophycins, and EG5 inhibitors; (ii) DNA targeting payloads such as enediynes, topoisomerase 1 inhibitors, pyrrolo[2,1-c][1,4]benzodiazepines, (iii) RNA-targeted payloads, such as talantins and amanitins; (iv) immunomodulatory ADC payloads, such as toll-like receptor agonists, stimulator of interferon genes (STING), and glucocorticoid receptor modulators; and (v) any new potential ADC payloads, such as Bcl-xL inhibitors, nicotinamide phosphoribosyltransferase (NAMPT), carbamycins, protein degradation targeting chimeric (PROTAC) molecules, near-infrared photoimmunotherapy (NIR-PIT) drugs, and dual payload combinations (e.g., MMAE and MMAF).

[0220] In addition, any antibody or antigen-binding fragment thereof, or any portion in the field of antibody-drug conjugates or related technologies that binds to a specific target can be conjugated to the linkers discussed in this disclosure.

[0221] 5. Pharmaceutical compositions comprising antibody-drug conjugates

[0222] The antibody-drug conjugates of the present invention can be prepared into pharmaceutical compositions comprising the antibody-drug conjugates of the present invention and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be selected based on the specific anti-tumor compound used and its concentration, stability, and expected bioavailability, as well as the disease, disorder, or condition to be treated with the composition, the subject and their age, body shape, and general condition, as well as the route of administration. The ADC of the present invention can be mixed with solvents (such as sterile liquids, including water, oils (derived from petroleum, animals, plants, or synthetic oils (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)), physiological saline, aqueous glucose solution, or aqueous glycerol solution) and additives such as humectants, emulsifiers, pH buffers, etc., to prepare pharmaceutical compositions of the present invention. Pharmaceutically acceptable carriers for solid dosage forms may include sugars, starches, and other conventional substances, including polysorbates, histidine, lactose, talc, sucrose, gelatin, carboxymethylcellulose, agar, mannitol, sorbitol, calcium phosphate, calcium carbonate, sodium carbonate, kaolin, alginic acid, gum arabic, corn starch, potato starch, sodium saccharin, magnesium carbonate, gum tragacanth, microcrystalline cellulose, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, and stearic acid. In addition, such solid dosage forms may be uncoated or coated by known techniques (e.g., to delay disintegration and absorption). In addition, pharmaceutically acceptable carriers for liquid dosage forms for oral or parenteral administration include, for example, non-aqueous, pharmaceutically acceptable polar solvents such as oils, alcohols, amides, esters, ethers, ketones, hydrocarbons, and mixtures thereof, as well as water, saline solutions, glucose solutions, electrolyte solutions, or any other aqueous, pharmaceutically acceptable liquids. Example

[0223] (1) Importance of hydrophilic groups

[0224] The introduction of hydrophilic groups into the linker structure of antibody-drug conjugates (ADCs) is crucial for mitigating potential side effects. This strategic addition plays several key roles in improving the safety and efficacy of ADCs:

[0225] i. Reduce non-selective cellular uptake: Hydrophilic groups help improve water solubility, making it difficult for ADC to penetrate the cell wall.

[0226] This minimizes unintended exposure of normal cells to cytotoxic drugs.

[0227] ii. Enhanced circulation stability: Hydrophilic linkers can enhance the stability of ADCs, which is crucial for maintaining the integrity of the ADC structure before it reaches the tumor site to prevent its systemic release.

[0228] iii. Minimizing off-target toxicity: By reducing nonspecific interactions with normal cells, hydrophilic linkers help minimize off-target toxicity and preserve the therapeutic window of ADCs.

[0229] iv. Improved pharmacokinetics: Hydrophilic modification can affect the pharmacokinetics of ADCs and optimize factors such as circulation time and distribution.

[0230] Design: From GGFG to GGYG

[0231] To improve tumor specificity and tolerability, a stable cleavable linker is required. The inventors designed a novel peptide sequence that can be cleaved by lysosomal enzymes (cathepsins) with an efficiency similar to that of the GGFG linker system, while also allowing the introduction of functional groups to modulate the physicochemical properties of the ADC.

[0232] In this regard, the inventors investigated the recognition of peptide substrates by cathepsin B and L proteases. Figure 1 These proteases have broad substrate specificity and tend to prefer structurally related amino acids at specific subsites. With this in mind, the inventors designed a novel tetrapeptide sequence by replacing the aromatic amino acid phenylalanine (F) in the GGFG linker subsystem (LP1) with the structurally similar amino acid tyrosine (Y).

[0233]

[0234] Coupling:

[0235] 4 mg / ml of trastuzumab was reacted with a 30-fold molar excess of TCEP (tris(2-carboxyethyl)phosphine hydrochloride) at 25°C for 2 hours to generate cysteine ​​via disulfide bonds. The reduced trastuzumab was purified from unreacted TCEP using a PD-10 desalting column. 2 mg / ml of reduced trastuzumab was reacted with a 12-fold molar excess of linker-payload 2 (LP2) in 10% (v / v0) dimethyl sulfoxide (DMSO) at 25°C for 1 hour. The trastuzumab ADC was purified from unreacted linker-payload and DMSO using a PD-10 desalting column. Figures 2 to 4 .

[0236] (2) Improve hydrophilicity and tandem cleavage

[0237] The hydroxyl group on tyrosine (Y) serves as a linker for hydrophilic side chain groups such as carbohydrates, sulfates, phosphates, and polyethylene glycol (PEG) linkers. Studies have shown that β-glucuronidase levels are elevated in tumor tissue. This enzyme circulates at very low concentrations but is found at high concentrations in solid tumors, including lung, breast, pancreatic, colorectal, and ovarian cancers.

[0238] Taking advantage of the high expression of the lysosomal enzyme β-glucuronidase in malignant cells, the inventors designed a tandem cleavage linker in which β-glucuronic acid is attached to the hydroxyl group of tyrosine in a GGYG linker system. This linker is designed to enable sequential enzymatic cleavage in a specific and controlled manner.

[0239]

[0240] The introduction of β-glucuronic acid may have the following advantages.

[0241] Temporary hydrophilic unit:

[0242] (1) Improve overall water solubility and conjugation efficiency, thereby limiting the accumulation and aggregation of ADC during the conjugation process and circulation.

[0243] (2) Achieve the coupling of highly challenging hydrophobic payloads, thereby expanding the range of ADC cytotoxic payloads.

[0244] Sequential enzyme digestion:

[0245] (1) Cathepsin-mediated peptide cleavage is blocked until lysosomal β-glucuronidase cleaves the side chain groups.

[0246] (2) Limiting the exposure of the cleavage site in the linker-payload protects it from the action of serum proteases and neutrophil elastase, thereby effectively reducing the premature loss of the drug in the circulation and improving the preferential release of the drug in the tumor.

[0247] Coupling:

[0248] 4 mg / ml of trastuzumab was reacted with a 30-fold molar excess of TCEP (tris(2-carboxyethyl)phosphine hydrochloride) at 25°C for 2 hours to generate cysteine ​​via disulfide bonds. The reduced trastuzumab was purified from unreacted TCEP using a PD-10 desalting column. 2 mg / ml of reduced trastuzumab was reacted with a 12-fold molar excess of linker-payload 3 (LP3) in 10% (v / v0) DMSO at 25°C for 1 hour. The trastuzumab ADC was purified from unreacted linker-payload and DMSO using a PD-10 desalting column. Figures 5 to 8 .

[0249] (3) Improve stability

[0250] Traditionally, maleimides have been widely used for cysteine ​​modification due to their rapid and selective reaction with sulfhydryl groups. However, recent studies have found that thioether bonds can undergo uncoupling reactions via a retro-Michael addition pathway, leading to premature drug release in the circulation and reducing therapeutic efficacy. In addition, maleimide-based conjugates can undergo sulfhydryl exchange with other plasma sulfhydryl groups, such as human serum albumin (HSA), leading to ectopic delivery of toxic payloads, further compromising efficacy.

[0251] To address these challenges and improve the stability of plasma linkers beyond that of maleimidocaproic acid-containing antibody-drug conjugates (ADCs), para-acetamide-linked conjugates were developed by replacing the maleimide moiety with bromoacetamide. These novel linker derivatives (LP4 and LP5), featuring bromoacetamide, react with the sulfhydryl groups generated after reduction of antibody disulfide bonds. This results in the formation of stable conjugates and significantly reduces premature drug release and off-target binding.

[0252]

[0253] In addition to acetamide-linked conjugates, we have also developed a self-stabilizing maleimide conjugate. To this end, we introduced an amide functional group into the hexanoic acid spacer (LP6), which facilitates the rapid hydrolysis of the maleimide to its open-ring structure, thereby enhancing the stability of the maleimide-drug conjugate. This modification effectively reduces the retro-Michael addition reaction, ensuring that the drug-antibody ratio (DAR) of the ADC remains consistent throughout circulation. Alternatively, the maleimide conjugation site can be replaced with a DBCO-based conjugate (LP7) to improve stability.

[0254]

[0255] The inventors hypothesize that this design will maintain the effectiveness of the GGFG linker system while improving its safety profile, which will in turn reduce off-target toxicities such as neutropenia and interstitial lung disease.

[0256] In this innovation, the goal of limiting the effects of anti-tumor compounds on normal cells is achieved by integrating hydrophilic side chain groups into the backbone of the linker. This enhances the overall hydrophilicity of the linker payload, making it difficult for the decoupled linker payload to penetrate the cell wall. As a result, the non-selective uptake of the anti-tumor compound is reduced, resulting in less unintended cellular absorption, thereby reducing the incidence of off-target effects and achieving a high safety profile, as reflected in the reduction of interstitial lung disease (ILD).

[0257] Synthesis route and experimental steps:

[0258] Synthesis of linker-payload 2 (LP2)

[0259]

[0260] Step 1:

[0261]

[0262] 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetic acid (Fmoc-Gly-Gly-OH, 5.0 g, 14.1 mmol) was partially dissolved in tetrahydrofuran (THF, 125 mL), toluene (Tol, 42.6 mL), and pyridine (Pyr, 2.15 mL). Lead (IV) acetate (Pb(OAc)4, 7.8 g, 17.6 mmol) was added, and the reaction mixture turned orange. The mixture was heated to reflux temperature, stirred for 3 hours, then cooled to room temperature, filtered through celite, washed with ethyl acetate, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 10-100% ethyl acetate in heptane gradient) to give methyl [2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)acetamido]acetate (3.0 g, 57% yield) as a white solid. 1 H NMR (400MHz, CDCl3) δ7.77(d,J=7.4Hz,2H),7.59(d,J=7.5Hz,2H),7.45–7.37(m,2H),7.36–7.29(m,2H),6.98(s,1H) ,5.34(s,1H),5.26(d,J=7.3Hz,2H),4.46(d,J=6.8Hz,2H),4.23(t,J=6.8Hz,1H),3.94–3.84(m,2H),2.06(s,3H).m / z 391.2[M+Na] +

[0263] Step 2:

[0264]

[0265] To a solution of methyl [2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)acetamido]acetate (1.27 g, 3.45 mmol) in dichloromethane (DCM, 20 mL) was added (R)-benzyl lactate (6.21 g, 34.5 mmol) and pyridinium p-toluenesulfonate (PPTS, 0.087 g, 0.345 mmol), and the mixture was stirred at reflux overnight. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (100 mL), washed with water (3 x 50 mL), dried over sodium sulfate, filtered, and concentrated to give 7.10 g of a colorless oil. The residue was purified by flash column chromatography (80 g silica gel; gradient of 10-70% ethyl acetate in heptane). The product-containing fractions were combined and concentrated to give 1.68 g of a colorless, turbid oil. 1 HNMR (400MHz, CDCl3) δ7.77(d,J=7.5Hz,2H),7.58(d,J=7.6Hz,2H),7.45–7.28(m,8H),6.83–6.71(m,1H),5.30–5.21(m,1H), 5.21–5.10(m,2H),4.91–4.73(m,2H),4.45(d,J=6.7Hz,2H),4.30–4.17(m,2H),3.86–3.69(m,2H),1.41(d,J=6.9Hz,3H).m / z 511.2[M+H] +

[0266] Step 3:

[0267]

[0268] To a solution of (R)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester (1.0 g, 1 eq, 2.0 mmol) was added diethylamine (7.5 g, 11 mL, 50 eq, 0.10 mol). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was evaporated to dryness and azeotropically distilled twice with DCM. Yield: 926 mg; m / z 267.1 [M+H] +

[0269] Step 4:

[0270]

[0271] Benzyl (R)-2-[(2-aminoacetamido)methoxy]propanoate (0.71 g, estimated mass content 58%, 1 eq, 1.55 mmol) was dissolved in N,N-dimethylformamide (DMF, 10.0 mL). (S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-(4-hydroxyphenyl)propanoic acid (Fmoc-Tyr-OH, 624 mg, 1 eq, 1.55 mmol) and N,N-diisopropylethylamine (DIPEA, 300 mg, 404 μL, 1.5 eq, 2.32 mmol) were added, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 588 mg, 1 eq, 1.55 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The mixture is diluted with 100mL water, and extracted with 2x100mL ethyl acetate. The organic layer is dried over sodium sulfate, filtered, and evaporated to dryness. Purify the crude product by flash column chromatography (24g silica gel, ethyl acetate gradient 0-100% in heptane). After evaporating the solvent, solid product (455mg, 45%) is obtained. 1 H NMR(400MHz, DMSO-d6)δ9.17(s,1H),8.60(t,J=6.8Hz,1H),8.29(t,J=5.8Hz,1H),7.88 (d,J=7.5Hz,2H),7.68–7.58(m,3H),7.45–7.25(m,10H),7.10–7.04(m,2H),6.66–6.60 (m,2H),5.18–5.07(m,2H),4.67–4.53(m,2H),4.26–4.09(m,5H),3.79–3.65(m,2H),2. 92(dd,J=13.8,4.1Hz,1H),2.71–2.62(m,1H),1.26(d,J=6.8Hz,3H); m / z:674.4[M+Na] +

[0272] Step 5:

[0273]

[0274] Benzyl (5S,13R)-1-(9H-fluoren-9-yl)-5-(4-hydroxybenzyl)-13-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradec-14-oate (550 mg, 1 eq, 844 μmol) was suspended in DCM (4.3 mL). Diethylamine (3.09 g, 4.37 mL, 50 eq, 42.2 mmol) was added, and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was evaporated to dryness and azeotroped twice with DCM to give a cloudy oil. Yield: 570 mg. m / z 430.2 [M+H] +

[0275] Step 6:

[0276]

[0277] Crude benzyl (R)-2-[(2-[(S)-2-amino-3-(4-hydroxyphenyl)propionamido]acetamido)methoxy]propanoate (570 mg, estimated mass content 63%, 1 eq, 836 μmol) was dissolved in DCM (10 mL). 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetic acid (296 mg, 1.0 eq, 836 μmol), HATU (318 mg, 1.0 eq, 836 μmol), and DIPEA (162 mg, 218 μL, 1.5 eq, 1.25 mmol) were added sequentially. The yellow reaction mixture was stirred at room temperature. After 2 hours, an additional 0.3 eq of HATU and 0.5 eq of DIPEA were added. The reaction mixture was stirred continuously for a total of 5 hours before being evaporated to dryness under reduced pressure and purified by flash column chromatography (24 g silica gel, 0-6% MeOH gradient in DCM). The fractions containing the product were evaporated to dryness to give (11S,19R)-1-(9H-fluoren-9-yl)-11-(4-hydroxybenzyl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosano-20-oic acid benzyl ester (190 mg, 248 μmol, 29%). 1HNMR(400MHz,DMSO-d6)δ9.16(s,1H),8.54(t,J=6.8Hz,1H),8.26(t,J=5.9Hz,1H),8.09–7.98(m,2H),7.8 9(d,J=7.5Hz,2H),7.70(d,J=7.4Hz,2H),7.59(t,J=6.1Hz,1H),7.45–7.28(m,9H),7.04–6.97(m,2H),6.6 6–6.60(m,2H),5.20–5.09(m,2H),4.66–4.55(m,2H),4.46–4.37(m,1H),4.32–4.17(m,4H),3.82–3.53(m, 6H),3.19–3.07(m,1H),2.93(dd,J=14.0,4.6Hz,1H),2.68(dd,J=14.0,9.3Hz,1H),1.31–1.21(m,6H); 788.4[M+Na] +

[0278] Step 7:

[0279]

[0280] (11S,19R)-1-(9H-fluoren-9-yl)-11-(4-hydroxybenzyl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosane-20-oic acid benzyl ester (175 mg, 1 eq, 229 μmol) was dissolved in ethanol (EtOH, 5.0 mL) / ethyl acetate (EtOAc, 5.0 mL). Palladium on carbon (Pd / C, 10%, 50% wet) (48 mg, 5% by weight, 0.1 eq, 22.9 μmol) was added, and the reaction mixture was stirred under a hydrogen atmosphere for 2.5 hours. The reaction mixture was filtered through celite and washed with 2 x 20 mL of methanol. The filtrate was evaporated to dryness to give a colorless solid (169 mg). m / z 674.4 [MH] -

[0281] Step 8:

[0282]

[0283] The crude product (11S,19R)-1-(9H-fluoren-9-yl)-11-(4-hydroxybenzyl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazaeicosane-20-oic acid (154 mg, 1 eq, 228 μmol) was suspended in 1 mL of DCM. Diethylamine (833 mg, 1.18 mL, 50 eq, 11.4 mmol) was added and the reaction mixture was stirred at room temperature for 2 hours. A solid precipitated at the bottom of the bottle. The DCM layer was removed. The solid was dried under reduced pressure to give 154 mg of a white solid, which was used directly in the next reaction without further purification. m / z 454.2 [M+H] +

[0284] Step 9:

[0285]

[0286] Crude (2R,10S)-16-amino-10-(4-hydroxybenzyl)-2-methyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecanoic acid (154 mg, 67% by weight, 1 eq, 228 μmol) was suspended in 1.5 mL of DMF. DIPEA (88.2 mg, 119 μL, 3 eq, 683 μmol) was added. (2,5-dioxopyrrolidin-1-yl) 6-(2,5-dioxopyrrol-1-yl) hexanoate (MC-OSu, 105 mg, 1.5 eq, 341 μmol) was added. The reaction was stirred at room temperature for 20 minutes. The reaction mixture was diluted with DCM and added to a short silica gel column (approximately 30 g). The column was initially eluted with 100 mL of 9 / 1 DCM / MeOH. The product was then eluted with 75 mL of 1 / 1 DCM / MeOH. The fraction containing the product was evaporated to dryness under reduced pressure. The crude product was ground from DCM to give a white solid. The batch of product was dissolved in DMSO and purified by acidic preparative MPLC (Luna 5-40). The product fractions were lyophilized to give a white solid product. Yield: 80 mg, 54% yield. 1H NMR (400MHz, DMSO-d6) δ12.56(s,1H),9.16(s,1H),8.51(t,J=6.7Hz,1H),8.23(t,J=5.9Hz,1 H),8.12–7.99(m,3H),7.04–6.97(m,4H),6.66–6.59(m,2H),4.65–4.52(m,2H),4.44–4.35(m ,1H),4.08–3.99(m,1H),3.78–3.56(m,6H),3.37(t,J=7.1Hz,2H),2.92(dd,J=13.9,4.8Hz,1 H),2.68(dd,J=14.0,9.5Hz,1H),2.15–2.06(m,2H),1.54–1.41(m,4H),1.27–1.13(m,5H); m / z 645.4[MH] - ,669.4[M+Na] +

[0287] Step 10:

[0288]

[0289] (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-11,14-dione (20 mg, 1 eq, 45 μmol) was suspended in DMF (2 mL) and DIPEA (35 mg, 47 μL, 6 eq, 0.27 mmol) was added. Subsequently, (2R,10S)-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10-(4-hydroxybenzyl)-2-methyl-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosanoic acid (26 mg, 0.9 eq, 40 μmol) and HATU (34 mg, 2 eq, 89 μmol) were added. The reaction mixture was stirred at room temperature for 1 hour and then directly purified by acidic preparative MPLC. Fractions containing the product were combined and lyophilized to obtain a yellow solid. Yield: 22 mg, 51% yield. 1H NMR (400MHz, DMSO-d6) δ9.14 (s, 1H), 8.57 (t, J = 6.7Hz, 1H), 8.49 (d, J = 9.2Hz ,1H),8.24(t,J=5.9Hz,1H),8.08–7.96(m,3H),7.40(s,1H),7.23(s,1H),7. 01–6.94(m,4H),6.64–6.58(m,2H),6.47(s,1H),6.27(d,J=5.8Hz,2H),5.60 –5.52(m,1H),5.44–5.34(m,2H),5.17–5.03(m,2H),4.67(dd,J=10.1,6.6Hz ,1H),4.53(dd,J=10.2,6.6Hz,1H),4.40–4.32(m,1H),4.15–4.06(m,1H),3. 77–3.54(m,6H),3.39–3.33(m,2H),3.15–2.97(m,2H),2.87(dd,J=13.9,4.7 Hz,1H),2.67–2.59(m,1H),2.17–2.05(m,4H),1.92–1.77(m,2H),1.51–1.41 (m,4H),1.39(d,J=6.8Hz,3H),1.23–1.13(m,2H),0.87(t,J=7.3Hz,3H).m / z 1076.4[M+H] +

[0290] Synthesis of linker-payload 3 (LP3)

[0291]

[0292] Step 1:

[0293]

[0294] To a suspension of (R)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester (211 mg, 1 eq, 432 μmol) in DCM (2.0 mL) was added diethylamine (0.71 g, 1.0 mL, 22 eq, 9.7 mmol). The reaction mixture was stirred at room temperature for 90 minutes. After 90 minutes, the reaction mixture was concentrated under reduced pressure and azeotroped with DCM (6x) to give the product as a thick, colorless oil. m / z 267.2 [M+H] +

[0295] Step 2:

[0296]

[0297] To a solution of benzyl (R)-2-((2-aminoacetamido)methoxy)propanoate (115 mg, 1 eq, 432 μmol) in DMF (5.0 mL) were added DIPEA (167 mg, 226 μL, 3 eq, 1.30 mmol), (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)propanoic acid (466 mg, 1.5 eq, 648 μmol), and HATU (246 mg, 1.5 eq, 648 μmol). The reaction mixture was stirred at room temperature for 1 hour, diluted with ethyl acetate, and washed with water (2x) and saturated brine (2x). The organic layer was dried over sodium sulfate, filtered, and concentrated to give a thick yellow oil (656 mg). The crude product was purified by flash column chromatography (24 g silica gel, 0-100% ethyl acetate in heptane gradient). The fractions containing the product were collected, concentrated, and azeotropically distilled to give the product as a white solid (317 mg, 71%). 1 H NMR(400MHz, CDCl3)δ7.76(d,J=7.5Hz,2H),7.56–7.48(m,2H),7.40(t,J=7.5Hz,2H),7.37–7.27(m,7H) ,7.14–7.05(m,2H),6.94(d,J=8.1Hz,2H),6.87(s,1H),6.29(s,1H),5.36–5.19(m,4H),5.18–5.05(m,3 H),4.86–4.78(m,1H),4.72–4.64(m,1H),4.53–4.44(m,1H),4.43–4.26(m,2H),4.24–4.08(m,2H),3.79 (m,2H),3.70(s,3H),3.15–2.91(m,2H),2.08–2.00(m,9H),1.61–1.53(m,2H),1.38(d,J=7.0Hz,3H).m / z 990.2[M+Na] + Step 3:

[0298]

[0299] To a solution of (2S,3R,4S,5S,6S)-2-(4-((4R,12S)-12-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methyl-3,8,11-trioxo-1-phenyl-2,5-dioxa-7,10-diazatridecan-13-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (317 mg, 1 eq, 327 μmol) in DMF (4.5 mL) was added piperidine (61.3 mg, 71.2 μL, 2.2 eq, 720 μmol) and the reaction mixture was stirred at room temperature for 30 min. The reaction was quenched by adding acetic acid (43.3 mg, 41.2 μL, 2.2 eq, 720 μmol) and purified by acidic preparative MPLC (Luna 10-50). The fractions containing the product were combined and lyophilized to give a white solid (200 mg, 82%). 1 H NMR(400MHz, CDCl3)δ7.81(t,J=5.8Hz,1H),7.38–7.30(m,5H),7.17–7.11(m,2H),6.99–6.92(m, 2H),6.83(t,J=6.8Hz,1H),5.39–5.25(m,3H),5.18(d,J=4.7Hz,2H),5.16–5.12(m,1H),4.88–4.7 3(m,2H),4.29–4.14(m,2H),3.89–3.85(m,2H),3.73(s,3H),3.63(dd,J=8.9,4.2Hz,1H),3.17(dd ,J=13.7,4.2Hz,1H),2.80–2.70(m,1H),2.10–2.01(m,9H),1.42(d,J=6.9Hz,3H).m / z746.4[M+H] +

[0300] Step 4:

[0301]

[0302] To a solution of (2S,3R,4S,5S,6S)-2-(4-((4R,12S)-12-amino-4-methyl-3,8,11-trioxo-1-phenyl-2,5-dioxa-7,10-diazatridecan-13-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (197 mg, 1 eq, 264 μmol) in DMF (3.0 mL) were added DIPEA (102 mg, 138 μL, 3 eq, 793 μmol), Fmoc-Gly-Gly-OH (140 mg, 1.5 eq, 396 μmol), and HATU (151 mg, 1.5 eq, 396 μmol) in sequence. The reaction mixture was stirred at room temperature for 45 minutes and directly purified by acidic preparative MPLC (Luna 30-70). The fractions containing product were combined and lyophilized to give a white solid (215 mg, 75%). 1 H NMR(400MHz, CDCl3)δ7.75(d,J=7.6Hz,2H),7.57(t,J=6.7Hz,2H),7.49–7.27(m,10H),7.20–7.04(m,5H) ,6.92–6.85(m,2H),5.97(t,J=5.6Hz,1H),5.39–5.27(m,2H),5.27–5.20(m,1H),5.18–5.06(m,3H),4.80– 4.72(m,1H),4.71–4.63(m,1H),4.56(q,J=7.1Hz,1H),4.44(d,J=6.7Hz,2H),4.27–4.15(m,3H),3.94–3.7 2(m,6H),3.68(s,3H),3.21–3.12(m,1H),2.99–2.89(m,1H),2.08–2.01(m,9H),1.36(d,J=6.9Hz,3H).m / z 1104.2[M+Na] +

[0303] Step 5:

[0304]

[0305] A suspension of (2S,3R,4S,5S,6S)-2-(4-((4R,12S)-12-(2-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetamido)-4-methyl-3,8,11-trioxo-1-phenyl-2,5-dioxa-7,10-diazatridecan-13-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (193 mg, 1 eq, 178 μmol) in methanol (8.0 mL) was purged with nitrogen for 10 minutes. Palladium on carbon (10%, 50% wet) (38.0 mg, 5% by weight, 0.1 eq, 17.8 μmol) was then added, and the mixture was stirred under a hydrogen atmosphere for 30 minutes. The reaction mixture was purged with nitrogen again for 10 minutes, filtered through celite, washed with methanol, and concentrated to give a white solid (155 mg). The crude product was purified by acidic preparative MPLC (Luna 20-60), and the fractions containing the product were combined and lyophilized to give a white solid product (117 mg, 66%). 1 HNMR (400MHz, DMSO) δ12.60(s,1H),8.64–8.57(m,1H),8.31(t,J=5.8Hz,1H),8.22–8.04(m,2H),7.89(d,J=7.5Hz,2H),7.71(d, J=7.4Hz,2H),7.61(t,J=6.1Hz,1H),7.41(t,J=7.4Hz,2H),7.32(t,J=7.4Hz,2H),7.22–7.15(m,2H),6.91–6.85(m,2H),5.61(d, J=8.0Hz,1H),5.45(t,J=9.6Hz,1H),5.11–5.01(m,2H),4.72–4.52(m,3H),4.52–4.42(m,1H),4.32–4.26(m,2H),4.26–4.15(m,1 H),4.03(q,J=6.9Hz,1H),3.83–3.55(m,9H),3.04–2.95(m,1H),2.81–2.71(m,1H),2.04–1.96(m,9H),1.23(d,J=6.9Hz,3H).m / z 1014.0[M+Na] +

[0306] Step 6:

[0307]

[0308] (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-11,14-dione methanesulfonate (55 mg, 1 eq, 0.10 mmol) was suspended in DMF (2.5 mL), and (11S,19R)-1-(9H-fluoren-9-yl)-19-methyl-3,6,9, 1,2,15-pentaoxo-11-(4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)-2,18-dioxa-4,7,10,13,16-pentaazaeicosane-20-oic acid (0.10 g, 1 eq, 0.10 mmol), DIPEA (39 mg, 53 μL, 3 eq, 0.30 mmol), and HATU (48 mg, 1.25 eq, 0.13 mmol). The reaction mixture was stirred at room temperature for 15 minutes and then directly purified by acidic preparative MPLC (Luna 20-60). Fractions containing the product were combined and lyophilized to give the product as a yellow solid (100 mg, 70%). 1 H NMR(400MHz, DMSO-d6)δ8.65(t,J=6.6Hz,1H),8.49(d,J=9.1Hz,1H),8.31(t, J=5.8Hz,1H),8.10(d,J=7.9Hz,1H),8.03(t,J=5.7Hz,1H),7.87(d,J=7.6Hz,2 H),7.69(d,J=7.4Hz,2H),7.57(t,J=6.0Hz,1H),7.43–7.26(m,5H),7.23(s,1 H),7.19–7.11(m,2H),6.91–6.84(m,2H),6.47(s,1H),6.26(d,J=7.2Hz,2H),5 .64–5.51(m,2H),5.45(t,J=9.7Hz,1H),5.42–5.32(m,2H),5.17–4.99(m,4H) ,4.72–4.63(m,2H),4.54(m,1H),4.48–4.38(m,1H),4.31–4.07(m,4H),3.81–3 .55(m,9H),3.15–2.88(m,3H),2.75–2.67(m,1H),2.16–2.05(m,2H),2.05–1. 95(m,9H),1.92–1.77(m,2H),1.39(d,J=6.8Hz,3H),0.86(t,J=7.3Hz,3H).m / z 1421.8[M+H]+

[0309] Step 7:

[0310]

[0311] (2S,3R,4S,5S,6S)-2-(4-((S)-11-((2-((((R)-1-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-1,2,3,10,13,16-hexahydro-1H,13H-benzo[de][1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)- A solution of 1-(9H-fluoren-9-yl)-3,6,9-trioxo-2-oxa-4,7,10-triazadodec-12-yl)-1-(1-oxopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamoyl)-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2-oxa-4,7,10-triazadodec-12-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (50 mg, 1 eq, 35 μmol) was dissolved in MeOH / THF (1:1, 6 mL) and cooled to 0°C. A solution of lithium hydroxide monohydrate (15 mg, 10 eq, 0.35 mmol) in water (600 μL) was added, and the reaction mixture was stirred at 0°C for 2 hours. Glacial acetic acid (0.11 g, 0.10 mL, 50 eq, 1.8 mmol) was added, and the organic solvent was removed under reduced pressure. DMSO (2 mL) was added to the residual aqueous phase and the solution was purified by acidic preparative MPLC (Luna 5-40). The fractions containing the product were combined and lyophilized to give the product as a white solid (27 mg, 72%). 11H NMR (400 MHz, DMSO-d6) δ 8.71 (t, J = 6.6 Hz, 1H), 8.54 (d, J = 9.2 Hz, 1H), 8.38 (t, J = 5.9 Hz, 1H), 8.29–8.13 (m, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.40 (s, 1H), 7.23 (s, 1H), 7.08 (d, J = 8.3 Hz, 2H), 6.86 (d, J = 8.2 Hz, 2H), 6.48 (s, 1H), 6.26 (d, J = 5.0 Hz, 2H), 5.61–5.51 (m, 1H), 5.45–5.34 (m, 2H), 5.29–4.98 (m, 4H), 4.84 (d, J = 7.5 Hz, 1H), 4.74–4.64 (m, 1H), 4.58–4.41 (m, 2H), 4.12 (q, J = 6.7 Hz, 1H), 3.82–3.48 (m, 9H, coincides with H2O peak), 3.24–2.92 (m, 13H, coincides with H2O peak), 2.64–2.53 (m, 1H), 2.17–2.08 (m, 2H), 1.92–1.78 (m, 2H), 1.39 (d, J = 6.8 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H). m / z 1059.6 [M+H] +

[0312] Step 8:

[0313]

[0314] (2S,3S,4S,5R,6S)-6-(4-((S)-2-(2-(2-aminoacetamido)acetamido)-3-((2-((((R)-1-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-1,2,3,10,13,16-hexahydro-1H,13H-benzo[de][1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-1- A mixture of 1,4-dihydro-2-[[(1,4-dihydro-3-[ ... 1 H NMR (400MHz, DMSO-d6) δ8.62(t,J=6.6Hz,1H),8.50(d,J=9.1Hz,1H),8.27(t ,J=5.9Hz,1H),8.11–7.98(m,3H),7.39(s,1H),7.23(s,1H),7.15–7.07(m,2 H),6.98(s,2H),6.92–6.84(m,2H),6.47(s,1H),6.26(d,J=4.3Hz,2H),5.61 –5.52(m,1H),5.46–5.32(m,3H),5.24–4.93(m,4H),4.67(dd,J=10.1,6.6Hz, 1H),4.53(dd,J=10.1,6.6Hz,1H),4.46–4.36(m,1H),4.11(q,J=6.7Hz,1H), 3.86(d,J=9.5Hz,1H),3.80–3.53(m,7H),3.43–3.36(m,2H),3.29–3.19(m,2H ),3.16–2.89(m,3H),2.69(dd,J=13.9,9.1Hz,1H),2.19–2.03(m,4H),1.94– 1.76(m,2H),1.52–1.37(m,7H),1.23–1.11(m,2H),0.87(t,J=7.3Hz,3H).m / z 1252.2[M+H] +

[0315] Synthesis of compound 11

[0316]

[0317] Step 1:

[0318]

[0319] To a suspension of methyl-1,2,3,4-tetra-O-acetyl-β-D-glucuronide (3.39 g, 1 eq, 9.01 mmol) in THF (40 mL) was added benzylamine (1.16 g, 1.18 mL, 1.2 eq, 10.8 mmol). The reaction mixture was stirred at room temperature overnight and then concentrated under reduced pressure to yield a red oil. The crude product was initially purified by silica gel flash column chromatography (80 g silica gel, 0-50% ethyl acetate in heptane gradient) to yield a red oil (3.12 g). This material was adsorbed on celite and then purified again by silica gel flash column chromatography (80 g silica gel, 0-50% ethyl acetate in heptane gradient) to yield the product as a light yellow oil (2.35 g, 76%). 1 H NMR (400 MHz, CDCl3) showed a mixture of anomers: δ 5.62–5.53 (m, 2H), 5.35–5.15 (m, 1.5H), 4.96–4.89 (m, 1.25H), 4.83–4.78 (m, 0.25H), 4.59 (d, J = 10.0 Hz, 1H), 4.17–4.07 (m, 1H), 3.79–3.72 (m, 4H), 3.72–3.53 (m, 1H), 2.13–2.07 (m, 4H), 2.07–1.99 (m, 8H). m / z 691.0 [2M+Na] +

[0320] Step 2:

[0321]

[0322] A solution of (3R,4S,5S,6S)-2-hydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2.35 g, 1 eq, 7.03 mmol) in DCM (40 mL) was cooled to 0°C in an ice bath. 2,2,2-trichloroacetonitrile (5.07 g, 3.52 mL, 5 eq, 35.2 mmol) and DBU (214 mg, 210 μL, 0.2 eq, 1.41 mmol) were added sequentially. The reaction mixture was warmed to room temperature and stirred for 3 hours. The mixture was concentrated under reduced pressure to afford a red oil. The crude product was adsorbed on celite and purified by flash column chromatography (80 g silica gel, gradient 0-35% ethyl acetate in heptane). Fractions containing the product were collected and concentrated under reduced pressure to afford the product as an off-white solid (1.86 g, 55%).1 H NMR (400MHz, CDCl3) δ8.74(s,1H),6.64(d,J=3.6Hz,1H),5.63(t,J=9.9Hz,1H),5.31–5.23(m,1H),5 .15(dd,J=10.2,3.6Hz,1H),4.50(d,J=10.2Hz,1H),3.75(s,3H),2.07–2.04(m,6H),2.02(s,3H).m / z 499.8[M+Na] +

[0323] Step 3:

[0324]

[0325] To a solution of H-Tr-OBzl (4.0 g, 1 eq, 15 mmol) in chloroform (100 mL) was added N-(9-Fluorenylmethyloxycarbonyloxy)succinimide (Fmoc-OSu, 5.0 g, 1 eq, 15 mmol). The reaction mixture was stirred at room temperature overnight. After dilution with DCM (100 mL), the mixture was washed with water (2 x 100 mL) and saturated brine (100 mL). The organic phase was concentrated under reduced pressure to afford an off-white solid (7.22 g). The solid was filtered, rinsed with DCM (3 x 10 mL), and then dried in a vacuum oven at 40°C for 2 days to afford the product as a white solid (5.77 g, 79%). 1 H NMR (400MHz, DMSO) δ9.25 (s, 1H), 7.95–7.81 (m, 3H), 7.66 (t, J = 7.4Hz, 1H), 7.53–7.20 (m, 9H), 7. 08–6.89(m,2H),6.70–6.60(m,2H),5.15–5.01(m,2H),4.48–3.97(m,4H),3.02–2.61(m,2H).m / z 494.4[M+H] + ,516.4[M+Na] +

[0326] Step 4:

[0327]

[0328] (2S,3S,4S,5R)-2-(methoxycarbonyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (51 mg, 1 eq, 0.11 mmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)-L-tyrosine benzyl ester (53 mg, 1 eq, 0.11 mmol) were mixed in a mixture containing A suspension of molecular sieves in anhydrous DCM (1.5 mL) was cooled to 0°C. Boron trifluoride etherate (BF3OEt2, approximately 48% BF3) (17 mg, 15 μL, 1.1 eq, 0.12 mmol) was added, and after removing the ice bath, the mixture was stirred at room temperature for 2 hours. The reaction was quenched with triethylamine (2M in THF) (7.0 mg, 59 μL, 1.1 eq, 0.12 mmol), and the reaction mixture was directly purified by flash column chromatography (12 g silica gel, 0-60% ethyl acetate in heptane gradient). Fractions containing the product were collected and concentrated under reduced pressure to yield the product as a white solid (32 mg, 37%). 1 H NMR (400MHz, CDCl3) δ7.78(d,J=7.6Hz,2H),7.59–7.51(m,2H),7.45–7.28(m,9H),6.93–6.75(m,4H),5.37–5.09(m,6H),5.02(d,J=7.1Hz,1H),4.7 3–4.65(m,1H),4.49–4.42(m,1H),4.37–4.29(m,1H),4.23–4.18(m,1H), 4.16–4.06(m,1H),3.71(s,3H),3.15–2.99(m,2H),2.09–2.02(m,9H).m / z 810.6[M+H] + ,832.6[M+Na] +

[0329] Step 5:

[0330]

[0331] A suspension of (2S,3R,4S,5S,6S)-2-(4-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(benzyloxy)-3-oxopropyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (527 mg, 1 eq, 651 μmol) in ethyl acetate (17 mL) was purged with nitrogen for 10 minutes. Palladium on carbon (10%, 50% wet) (139 mg, 5% by weight, 0.1 eq, 65.1 μmol) was added, and the mixture was stirred under a hydrogen atmosphere for 5 hours. The reaction mixture was purged with nitrogen and then filtered through celite, washed with ethyl acetate, and the filtrate was concentrated under reduced pressure and azeotroped with DCM (2x) to give the product as a white solid (468 mg, quantitative yield). 1H NMR(400MHz, CDCl3)δ7.77(d,J=7.6Hz,2H),7.59–7.49(m,2H),7.41(t,J=7.5Hz,2H),7.3 5–7.28(m,2H),7.07(d,J=8.1Hz,2H),6.91(d,J=8.0Hz,2H),5.36–5.29(m,2H),5.29–5.18 (m,2H),5.06(d,J=7.3Hz,1H),4.70–4.61(m,1H),4.52–4.43(m,1H),4.39–4.30(m,1H),4. 23–4.06(m,2H),3.68(s,3H),3.21–3.11(m,1H),3.11–3.02(m,1H),2.09–1.98(m,9H).m / z 742.0[M+Na] +

[0332] Synthesis of linker payload 4 (LP4)

[0333]

[0334] Step 1:

[0335]

[0336] To a solution of potassium hydroxide (0.43 g, 1 eq, 7.6 mmol) in water (2.1 mL) at 0°C was added 6-aminohexanoic acid (1.0 g, 1 eq, 7.6 mmol). Bromoacetyl bromide (1.8 g, 0.80 mL, 1.2 eq, 9.1 mmol) was added dropwise, while 2.8 M aqueous potassium carbonate was added dropwise to adjust the pH to >7.8. After the addition was complete, the reaction mixture was stirred at 0°C for 1 hour. The reaction solution was acidified to a pH of approximately 1 with 0.5 N HCl and extracted with ethyl acetate (3x). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to yield 1.47 g of a colorless oil. The product was purified by flash column chromatography (40 g silica gel; a 0-2.5% methanol in dichloromethane gradient). The product-containing fractions were combined and concentrated to yield 661 mg (34%) of a colorless oil that solidified upon standing at room temperature. 1 H NMR(400MHz, CDCl3)δ6.52(s,1H),3.89(s,2H),3.35–3.26(m,2H),2.38(t, J=7.3Hz,2H),1.73–1.63(m,2H),1.63–1.52(m,2H),1.46–1.34(m,2H).m / z 525.2[M+H] + ,Br isotope characteristic peak

[0337] Step 2:

[0338]

[0339] To a solution of 6-(2-bromoacetamido)hexanoic acid (631 mg, 1 eq, 2.50 mmol) in dichloromethane (30 mL) at 0°C were added pentafluorophenyl trifluoroacetate (1.02 g, 624 μL, 1.45 eq, 3.63 mmol) and pyridine (Py, 792 mg, 810 μL, 4 eq, 10.0 mmol). The reaction mixture was stirred at 0°C for 10 minutes and then washed with 0.5 M aqueous hydrochloric acid. The organic phase was dried over sodium sulfate, filtered, and concentrated to yield 1.2 g of a colorless oil. The product was purified by flash column chromatography (40 g silica gel; 0-50% ethyl acetate in n-heptane gradient). The product-containing fractions were combined and concentrated to yield 812 mg (77%) of a flocculent white solid. 1 H NMR (400MHz, CDCl3) δ6.53 (s, 1H), 3.89 (s, 2H), 3.39–3.26 (m, 2H), 2.69 (t, J = 7. 3Hz,2H),1.88–1.76(m,2H),1.69–1.57(m,2H),1.54–1.42(m,2H).SC_ACID:m / z 420.2[M+H] +

[0340] Step 3:

[0341]

[0342] (2S,3S,4S,5R,6S)-6-(4-((S)-2-(2-(2-aminoacetamido)acetamido)-3-((2-(((((R)-1-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxa[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl)amino)-1-oxoprop-2-yl)oxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (50 mg, 1 eq, 47 μmol) was dissolved in 3.5 mL of To DMF, perfluorophenyl 6-(2-bromoacetamido)hexanoate (39 mg, 2 eq, 94 μmol) and DIPEA (18 mg, 25 μL, 3 eq, 0.14 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 1 hour, then acidified with glacial acetic acid (10 mg, 10 μL, 3.7 eq, 0.17 mmol) and purified by acidic preparative MPLC (Luna 5-40). The product-containing fractions were combined and lyophilized to obtain 45 mg of a yellow solid. LCMS showed insufficient purity. Purification was performed again by acidic preparative MPLC (Luna 5-40) to obtain 19 mg of the desired product as a yellow solid. m / z 1292.2, 1294.2 [M+H] + ,Br isotope characteristic peak

[0343] Synthesis of linker payload 5 (LP5)

[0344]

[0345] To (2S,3S,4S,5R,6S)-6-(4-((S)-2-(2-(2-aminoacetamido)acetamido)-3-((2-(((((R)-1-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxa[4,5-g]pyrano[3',4':6,7]indole To a solution of (64 mg, 1 eq, 60.4 μmol) in DMF (2 mL) was added 2,5-dioxopyrrolidin-1-yl-2-bromoacetate (18.5 mg, 1.3 eq, 78.6 μmol). The resulting yellow solution was stirred at room temperature for 25 minutes. The reaction mixture was then syringe-purified by acidic preparative MPLC (Luna 5-40). Fractions containing the product were combined and lyophilized to yield 35 mg (49%) of a light yellow solid. NMR (400MHz, DMSO-d6) δ8.64(t,J=6.6Hz,1H),8.57–8.47(m,2H),8.29(t,J=5.9Hz,1H),8.19–8.03(m,2H),7.39(s,1H),7.23(s,1H),7. 11(d,J=8.7Hz,2H),6.97–6.84(m,2H),6.47(s,1H),6.26(d,J=4.3Hz,2H),5.61–5.48(m,1H),5.48–5.34(m,3H),5.23–5.03(m,3H),4.9 7 (d, J = 7.4 Hz, 1H), 4.74–4.63 (m, 1H), 4.58–4.37 (m, 2H), 4.16–4.05 (m, 1H), 3.92 (s, 2H), 3.88–3.54 (m, 7H), 3.42–3.35 (m, 1H), 3.29–3.19 (m, 2H), 3.15–2.88 (m, 3H), 2.76–2.58 (m, 1H), 2.18–2.07 (m, 2H), 1.92–1.76 (m, 2H), 1.39 (d, J = 6.6 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H); m / z 1179.0, 1181.0 [M+H]+, characteristic Br isotope peaks

[0346] Synthesis of linker payload 6 (LP6)

[0347]

[0348] To (2S,3S,4S,5R,6S)-6-(4-((S)-2-(2-(2-aminoacetamido)acetamido)-3-((2-(((((R)-1-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxa[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinolin-1-yl) To a solution of 2,5-dioxopyrrolidin-1-yl-3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanoate (23 mg, 1.5 eq, 71 μmol) in DMF (3.0 mL) was added 2,5-dioxopyrrolidin-1-yl-3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanoate (23 mg, 1.5 eq, 71 μmol). After reacting at room temperature for 45 minutes, the mixture was purified by acidic preparative MPLC (Luna 5-40). Fractions containing the product were combined and lyophilized to give 16 mg (27%) of the product as a light yellow solid. 1 H NMR (400MHz, DMSO-d6) δ8.63(t,J=6.6Hz,1H),8.51(d,J=9.1Hz,1H),8.29( t,J=5.9Hz,1H),8.26–8.15(m,2H),8.09–8.01(m,2H),7.40(s,1H),7.23(s ,1H),7.15–7.04(m,4H),6.92–6.84(m,2H),6.47(s,1H),6.27(d,J=4.0Hz, 2H),5.61–5.52(m,1H),5.43–5.34(m,3H),5.20–5.03(m,3H),4.95(d,J=7.4 Hz,1H),4.73–4.63(m,1H),4.57–4.50(m,1H),4.46–4.37(m,1H),4.15–4.0 8(m,1H),3.99(s,2H),3.84–3.54(m,8H),3.39–3.34(m,1H),3.29–3.19(m,4 H),3.15–2.89(m,3H),2.73–2.63(m,1H),2.30(t,J=7.1Hz,2H),2.17–2.07 (m,2H),1.92–1.78(m,2H),1.39(d,J=6.8Hz,3H),0.87(t,J=7.3Hz,3H); m / z 1267.4[M+H] +

[0349] Synthesis of linker payload 7 (LP7)

[0350]

[0351] (2S,3S,4S,5R,6S)-6-(4-((S)-2-(2-(2-aminoacetamido)acetamido)-3-((2-(((((R)-1-(((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxa[4,5-g]pyrano[3',4':6,7]indole (40 mg, 1 eq, 38 μmol) of 1,2-difluoro-1,2-difluoro-2-nitropropane-2-yl (1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (40 mg, 1 eq, 38 μmol) was dissolved in DMF (1.5 mL) and dibenzocyclooctyne-N-hydroxysuccinimide ester (DBCO-NHS, 15 mg, 1 eq, 38 μmol) was added. The yellow reaction solution was stirred at room temperature for 1 hour, and then DBCO-NHS (3.0 mg, 0.2 eq, 7.6 μmol) was added and the reaction continued for 45 minutes. The mixture was purified by acidic preparative MPLC (Luna 20-60). Fractions containing the product were combined and lyophilized to yield 39 mg (77%) of a light yellow solid. 1 H NMR (400MHz, DMSO-d6) δ8.69–8.57(m,1H),8.52(d,J=8.9Hz,1H),8.33–8.21(m,1H),8.19–8.07(m,1H),8.07–7.92(m,2H),7.70–7.53( m,2H),7.49–7.21(m,8H),7.10(d,J=8.3Hz,2H),6.91–6.84(m,2H),6.48(s,1H),6.29–6.22(m,2H),5.63–5.49(m,1H),5.39(s,3H),5.2 3–4.89(m,5H),4.72–4.60(m,1H),4.57–4.47(m,1H),4.44–4.34(m,1H),4.17–4.04(m,1H),3.85–3.46(m,8H),3.27–3.18(m,2H),3.14 –2.86(m,4H),2.74–2.57(m,2H),2.31–2.21(m,1H),2.18–1.99(m,3H),1.92–1.72(m,3H),1.42–1.35(m,3H),0.86(t,J=7.3Hz,3H); m / z 1346.2[M+H]+ 。

Claims

1. A compound as shown in formula (1): Formula (1) in, P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, -(Y-CH2)- or -(dibenzocyclooctyne-NC(=O))-, Y is Br, Cl or I, and -(dibenzocyclooctyne-NC(=O))- has the following structure: L n is optionally included, and if included is cycloalkyl, alkyl, -(OCH2CH2)-, or -(CH2-C(=O)-NH-CH2-CH2)-, and n is an integer from 1 to 6; A 1 is a peptide residue containing 0-3 amino acids; A 2 is a peptide residue containing 0-3 amino acids; Z is optionally included, and if included is a self-immolative spacer, -(NHCH2)-, or p-aminocarbamate; and X1 is H, monosaccharide, disaccharide, oligosaccharide, polyethylene glycol, sulfate, phosphate or pyrophosphate.

2. A linker-drug conjugate comprising: The compound according to claim 1; and a drug coupled to the compound via Z of formula (1).

3. The linker-drug conjugate according to claim 2, wherein The drug is coupled to the compound in the form of -(NH-drug)- or -(O-drug)-. The linker-drug conjugate of claim 2 , wherein the drug is a topoisomerase I inhibitor.

5. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, a drug and a linker, in, The linker is the compound according to claim 1, Wherein, the antibody or antigen-binding fragment thereof is coupled to the compound via P of formula (1), Wherein, the drug is coupled to the compound via Z of formula (1). 6 . A pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate according to claim 5 .

7. The pharmaceutical composition according to claim 6, wherein The cancer includes one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, colorectal cancer, colon cancer, kidney cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, kidney cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer and urothelial cancer.

8. A compound represented by formula (2): Formula (2) in P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, or -(dibenzocyclooctyne-NC(=O))-; L n is optionally included, and if included is a cycloalkyl group, an alkyl group, or a direct bond, and n is an integer from 1 to 6; X is –(C=O)-, a direct bond or 1 Direct bonds formed by side chains; A 1 For amino acids; R m is a hydrophilic side chain connected to A1, m is an integer from 1 to 10; and Z is optionally included, and if included is a self-immolative spacer, -(NHCH2)-, or p-aminocarbamate.

9. The compound according to claim 8, wherein The amino acid is aspartic acid, glycine, glutamic acid or lysine.

10. A linker-drug conjugate comprising: The compound according to claim 8; and a drug coupled to the compound via Z of formula (2).

11. The linker-drug conjugate according to claim 10, wherein The drug is coupled to the compound in the form of -(NH-drug)- or -(O-drug)-.

12. The linker-drug conjugate according to claim 10, wherein The drug is a topoisomerase 1 inhibitor.

13. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, a drug, and a linker. in, The linker is the compound according to claim 8, Wherein, the antibody or antigen-binding fragment thereof is coupled to the compound via P of formula (2), Wherein, the drug is coupled to the compound via Z of formula (2).

14. A pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate according to claim 13.

15. The pharmaceutical composition according to claim 14, wherein The cancer includes one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, colorectal cancer, colon cancer, kidney cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, kidney cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer and urothelial cancer.

16. A compound represented by formula (3): Formula (3) in: P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, or -(dibenzocyclooctyne-NC(=O))-; L n is optionally included, and if included is a cycloalkyl group, an alkyl group, or a direct bond, and n is an integer from 1 to 6; X is -(C=O)- or a direct bond; A 1 For amino acids; R m Optionally included, if included then connected to A 1 A hydrophilic side chain, wherein m is an integer from 1 to 10; and Z is optionally included, and if included is a self-immolative spacer, -(NHCH2)-, or p-aminocarbamate.

17. The compound according to claim 16, wherein The amino acid is aspartic acid, glycine, glutamic acid or lysine.

18. A linker-drug conjugate comprising: The compound according to claim 16; and a drug coupled to the compound via Z of formula (2).

19. The linker-drug conjugate of claim 18, wherein: The drug is coupled to the compound in the form of -(NH-drug)- or -(O-drug)-.

20. The linker-drug conjugate of claim 18, wherein The drug is a topoisomerase 1 inhibitor.

21. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, a drug, and a linker. in, The linker is the compound according to claim 16, Wherein, the antibody or antigen-binding fragment thereof is coupled to the compound via P of formula (3), Wherein, the drug is coupled to the compound via Z of formula (3).

22. A pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate according to claim 21.

23. The pharmaceutical composition according to claim 22, wherein The cancer includes one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, colorectal cancer, colon cancer, kidney cancer, esophageal cancer, leukemia, hepatocellular carcinoma, bone cancer, bladder cancer, sarcoma, kidney cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, endometrial cancer and urothelial cancer.

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