Linkers for antibody-drug conjugates

Abstract

Provided herein are a linker compound, a linker-drug conjugate in which the linker compound is conjugated to a drug, an antibody-drug conjugate in which a drug is conjugated to an antibody or antigen-binding fragment thereof via a linker compound, and a pharmaceutical composition for treating cancer comprising the antibody-drug conjugate.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Patent Application No. 63 / 529,828, filed July 31, 2023, Korean Patent Application No. 10-2023-0017886, filed February 10, 2023, and Korean Patent Application No. 10-2023-0024745, filed February 24, 2023, the entire disclosures of which are incorporated herein by reference. [Technical Field]

[0002] Provided herein are linker compounds, linker-drug conjugates in which the linker compounds are conjugated to a drug, antibody-drug conjugates in which a drug is conjugated to an antibody or antigen-binding fragment thereof via a linker compound, and methods for treating cancer by administering the antibody-drug conjugates to a subject in need thereof. [Background technology]

[0003] Several antibody-drug conjugates (ADCs) approved for antitumor use or in various clinical stages face safety challenges characterized by adverse side effects and inherent toxicities. Despite the excellent antitumor efficacy of these ADCs, safety concerns have hindered their clinical utility. This obstacle highlights the imperative to advance ADC therapeutics characterized by both excellent efficacy and an improved safety profile.

[0004] Eighty percent of approved ADCs use cleavable linkers for payload delivery to tumors. A limitation associated with cleavable linkers is the potential for payload shedding during circulation before the antibody-drug conjugate (ADC) reaches its tumor target. Premature systemic release of the payload can reduce the efficacy of the remaining circulating ADC, potentially leading to nonspecific uptake and potentially dose-limiting off-target toxicity. These challenges have a significant impact on ADC drug development. This not only complicates the evaluation of antitumor efficacy in preclinical mouse studies, but also limits the ADC's dosing in patients, preventing its full therapeutic potential from being realized. Summary of the Invention [Problem to be solved by the invention]

[0005] To overcome the above limitations, the present disclosure provides a novel cleavable linker system that is characterized by excellent therapeutic efficacy and an exemplary safety profile. [Means for solving the problem]

[0006] (1) In particular, the present disclosure: A compound of formula (1), Formula (1) [ka] In formula (1), P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, -(Y-CH)-, or -(dibenzocyclooctyne-NC(=O))-, Y is Br, Cl, or I, and -(dibenzocyclooctyne-NC(=O))- has the structure: [ka] L nis optionally included, and when included, is cycloalkyl, alkyl, or —(OCHCH)—, —(CH—C(═O)—NH—CH—CH)—, and n is an integer of 1 to 6; A 1 is a peptide residue containing 0 to 3 amino acids, A 2 is a peptide residue containing 0 to 3 amino acids, Z is optionally included and, if included, is a self-immolative spacer, —(NHCH)—, or p-aminocarbamate; X1 is H, a monosaccharide, a disaccharide, an oligosaccharide, a polyethylene glycol, a sulfate, a phosphate, or a pyrophosphate; A compound is provided.

[0007] (2) This disclosure also provides: A compound according to the embodiment of (1) above, a drug conjugated to said compound via Z of formula (1); The present invention provides a linker-drug conjugate comprising:

[0008] (3) The present disclosure also provides a linker-drug conjugate according to the embodiment (2) above, wherein the drug is conjugated to the compound in the form of -(NH-drug)- or -(O-drug)-.

[0009] (4) The present disclosure also provides a linker-drug conjugate according to the embodiment of (2) above, wherein the drug is a topoisomerase I inhibitor.

[0010] (5) This disclosure comprising an antibody or antigen-binding fragment thereof, a drug, and a linker; the linker is a compound according to embodiment (1) above, the antibody or antigen-binding fragment thereof is conjugated to the compound via P of formula (1), the drug is conjugated to the compound via Z of formula (1); Antibody-drug conjugates are provided.

[0011] (6) The present disclosure provides a pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate according to the embodiment of (5) above.

[0012] (7) The present disclosure provides a pharmaceutical composition according to the embodiment (6) above, wherein the cancer comprises one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain tumor, renal clear 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's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, renal 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.

[0013] (8) Furthermore, this disclosure A compound of formula (2): Formula (2) [ka] In formula (2), P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, or -(dibenzocyclooctyne-NC(=O))-; L n is optionally included and, when included, is cycloalkyl, alkyl, or a direct bond, and n is an integer from 1 to 6; X is -(C=O)-, a direct bond, or A 1 is a direct bond to the side chain of A 1 is an amino acid, R m is A 1 wherein m is an integer from 1 to 10; Z is optionally included and, if included, is a self-immolative spacer, —(NHCH)—, or p-aminocarbamate; A compound is provided.

[0014] (9) The present disclosure provides a compound according to the embodiment (8) above, wherein the amino acid is aspartic acid, glycine, glutamic acid, or lysine.

[0015] (10) The present disclosure also provides: A compound according to the embodiment of (8) above, a drug conjugated to said compound via Z of formula (2); The present invention provides a linker-drug conjugate comprising:

[0016] (11) The present disclosure also provides a linker-drug conjugate according to the embodiment (10) above, wherein the drug is conjugated to the compound in the form of -(NH-drug)- or -(O-drug)-.

[0017] (12) The present disclosure also provides a linker-drug conjugate according to the embodiment of (10) above, wherein the drug is a topoisomerase 1 inhibitor.

[0018] (13) This disclosure comprising an antibody or antigen-binding fragment thereof, a drug, and a linker; the linker is a compound according to embodiment (8) above, the antibody or antigen-binding fragment thereof is conjugated to the compound via P of formula (2), The drug is conjugated to the compound via Z in formula (2). Antibody-drug conjugates are provided.

[0019] (14) The present disclosure provides a pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate according to the embodiment of (13) above.

[0020] (15) The present disclosure provides a pharmaceutical composition according to the embodiment (14) above, wherein the cancer comprises one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, renal clear 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's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, renal 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.

[0021] (16) The present disclosure also provides: A compound of formula (3): Formula (3) [ka] In formula (3), P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, or -(dibenzocyclooctyne-NC(=O))-; L n is optionally included and, when included, is cycloalkyl, alkyl, or a direct bond, and n is an integer from 1 to 6; X is —(C═O)— or a direct bond; A 1 is an amino acid, R m is optionally included, and if included, A 1 wherein m is an integer from 1 to 10; Z is optionally included and, if included, is a self-immolative spacer, —(NHCH)—, or p-aminocarbamate; A compound is provided.

[0022] (17) The present disclosure provides a compound according to the embodiment (16) above, wherein the amino acid is aspartic acid, glycine, glutamic acid, or lysine.

[0023] (18) The present disclosure also provides: A compound according to the embodiment of (16) above, a drug conjugated to said compound via Z of formula (2); The present invention provides a linker-drug conjugate comprising:

[0024] (19) The present disclosure also provides a linker-drug conjugate according to the embodiment (18) above, wherein the drug is conjugated to the compound in the form of -(NH-drug)- or -(O-drug)-.

[0025] (20) The present disclosure also provides a linker-drug conjugate according to the embodiment of (18) above, wherein the drug is a topoisomerase 1 inhibitor.

[0026] (21) This disclosure provides: comprising an antibody or antigen-binding fragment thereof, a drug, and a linker; the linker is a compound according to embodiment (16) above, the antibody or antigen-binding fragment thereof is conjugated to the compound via P of formula (3), The drug is conjugated to the compound via Z of formula (3). Antibody-drug conjugates are provided.

[0027] (22) The present disclosure provides a pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate according to the embodiment of (21) above.

[0028] (23) The present disclosure provides a pharmaceutical composition according to the embodiment (22) above, wherein the cancer comprises one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, renal clear 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's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, renal 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.

[0029] Further embodiments are described herein. [Brief explanation of the drawings]

[0030] [Figure 1] Figure 1 shows the peptide cleavage specificity of cathepsins B and L. (J. Proteome Res. 2011, 10, 5363-5373) [Figure 2] FIG. 2 shows the SEC of trastuzumab-LP2. [Figure 3] Figure 3 shows the HIC of trastuzumab-LP2. [Figure 4] FIG. 4 shows the in vitro data for Payload-001, Trastuzumab-LP1, and Trastuzumab-LP2. [Figure 5] FIG. 5 shows the SEC of trastuzumab-LP3. [Figure 6] FIG. 6 shows the HIC of trastuzumab-LP3. [Figure 7] Figure 7 shows the HIC comparison of Tra / Tra-LP1 / Tra-LP3. [Figure 8] FIG. 8 shows in vitro data for trastuzumab-LP1 and trastuzumab-LP3. DETAILED DESCRIPTION OF THE INVENTION

[0031] 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, certain preferred methods, compositions, devices, and materials are now described. However, before describing these materials and methods, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies, or protocols described herein, as these may vary depending on routine experimentation and optimization. It is also to be understood that the terminology used herein is for the purpose of describing particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[0032] 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 case of conflict, the present specification, including definitions, will control. Therefore, in the context of the embodiments described herein, the following definitions apply:

[0033] As used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural forms 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.

[0034] As used herein, the term "comprise" and its linguistic variants indicate the presence of one or more stated features, elements, method steps, etc., but do not exclude the presence of additional features, elements, method steps, etc. Conversely, the term "consisting of" and its linguistic variants indicate the presence of one or more stated features, elements, method steps, etc., and excludes one or more unrecited features, elements, method steps, etc., except for impurities normally associated therewith. The phrase "consisting essentially of" refers to one or more stated features, elements, method steps, etc., as well as any additional features, elements, method steps, etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open-ended phrase "comprising." Such embodiments encompass multiple closed embodiments of "consisting of" and / or "consisting essentially of," which may also be alternatively claimed or described using such language.

[0035] As used herein, the term "antibody-drug conjugate" refers to a linkage between an antibody or antigen-binding fragment thereof and another anti-tumor compound, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, etc. This linkage can be a covalent bond.

[0036] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or an immunoreactive portion of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in this disclosure may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, single-chain antibodies, and humanized antibodies.

[0037] As used herein, the term "antigen-binding fragment thereof" refers to a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of 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 a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity-determining regions), epitope-binding fragments of any of the above that immunospecifically bind to a cancer cell antigen, a viral antigen, or a microbial antigen, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0038] As used herein, the term "linker" refers to a moiety that covalently links two moieties, an antibody and an anti-tumor compound. In some cases, the term "linker" as used herein may refer to a moiety that includes an additional element, such as a cleavable element, a linking group, or a group that includes one or more solubilizing groups. In other cases, the term "linker" as used herein may refer to a specifically defined element, such as a "cathepsin B-cleavable" linker (described further below).

[0039] The term "cleavable" as used herein refers to a linker that covalently links two moieties, an antibody and an anti-tumor compound, but that degrades under physiologically relevant conditions to cleave the covalent bond between the moieties. Cleavage generally releases the anti-tumor compound from the antibody. The term "cleavable" as used herein refers to a linker that is not particularly susceptible to degradation under physiological conditions. Such a linker is sufficiently resistant to degradation to keep the anti-tumor compound linked to the antibody or antigen-binding fragment until the antibody or antigen-binding fragment itself is at least partially degraded.

[0040] As used herein, the term "peptide" refers to a compound comprising a consecutive sequence of at least two amino acids linked together via a peptide linkage. The terms "dipeptide," "tripeptide," and "tetrapeptide" refer to compounds comprising a consecutive sequence of two, three, and four amino acids linked together via a peptide linkage, respectively. The term "peptide linkage" is intended to encompass (backbone) amide bonds and modified linkages that can be obtained when non-natural amino acids are introduced into a 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 consecutive peptide sequence. For example, the modified linkage can be an ester linkage, a thioester linkage, a carbamide linkage, a thiocarbamide linkage, or a triazole linkage. Preferably, the amino acids forming the consecutive peptide sequence are linked together via a backbone amide bond. Peptides can be linear or branched. In a preferred embodiment, the peptide is a linear dipeptide, tripeptide, or tetrapeptide, more preferably a linear tripeptide or tetrapeptide.

[0041] As used herein, the term "amino acid" refers to compounds containing at least one amino group and at least one acidic group, preferably a carboxyl group, or compounds derived from such compounds. The distance between the amino and acidic groups is not particularly limited. α-, β-, and γ-amino acids are preferred, with α-amino acids, especially α-aminocarboxylic acids, being particularly preferred. The term "amino acid" encompasses both natural amino acids, such as naturally occurring proteinogenic amino acids, and synthetic amino acids not found in nature. Hereinafter, references to amino acids may be made by their three-letter amino acid code (e.g., Arg, Phe, Ala, Cys, Gly, Gln, etc.) or by their one-letter amino acid code (e.g., R, F, A, C, G, Q, etc.). Unless otherwise specified, references to amino acids by their three-letter amino acid code refer to the corresponding (L)- or (D)-amino acid. Herein, amino acid sequences are written from N-terminus to C-terminus (left to right). Unless otherwise specified or dictated by context, all bonds between adjacent amino acid groups are formed by peptide (amide) bonds.

[0042] The term "cycloalkyl group" as used herein 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 consist of a single ring or may be formed of two or more condensed 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 a cyclopentyl or cyclohexyl group.

[0043] The term "alkyl group" as used herein refers to a linear (straight chain) or branched, saturated or unsaturated hydrocarbon group having from 1 to 20 carbon atoms, preferably from 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, the alkyl group is a methyl or ethyl group.

[0044] The term "self-immolative spacer," as used herein, refers to a bifunctional moiety that can covalently link two spaced apart moieties into a normally stable tripartite molecule, one of the spaced apart moieties can be released from the tripartite molecule by enzymatic cleavage, and can spontaneously cleave from the remainder of the molecule following enzymatic degradation to release the other of the spaced apart moieties.

[0045] As used herein, "monosaccharide" refers to any of the classes of sugars that cannot be hydrolyzed to produce simpler sugars. Monosaccharides are typically C5 sugars (e.g., xylose) and C6 sugars (e.g., glucose), but may also include monosaccharides with other carbon numbers, such as C3, C4, C7, and C8. In other words, monosaccharides are the simplest building blocks of oligosaccharides and polysaccharides.

[0046] The term "disaccharide" herein refers to a carbohydrate having two monosaccharides joined by a glycosidic linkage. The term "oligosaccharide" herein refers to a carbohydrate consisting of, for example, two to nine monosaccharides joined by glycosidic linkages. Oligosaccharides may also be referred to herein as "oligomers." The monosaccharides contained in a disaccharide or oligosaccharide may be referred to, for example, as "monosaccharide units" or "monomer units." Preferred monosaccharides herein are fructose and glucose.

[0047] As used herein, an "oligosaccharide" refers to a sugar having the general formula C x (H2O) y Oligosaccharides refer to linear or branched carbohydrate molecules of identical or different monosaccharide units joined by glycosidic bonds having the formula: 10 O5) n where n is from about 2 to about 9 (i.e., the number of hexose monomers in the oligosaccharide). As used herein, oligomers (e.g., cellooligosaccharides) have a DP of 2 to about 9, while polymers (e.g., cellulose) have a DP of at least about 10.

[0048] As used herein, the term "drug" refers to a compound (payload or ADC payload) that has an anti-tumor effect and has a substituent or moiety that allows it to be linked 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. As the linker is cleaved at the linking position to the drug, the anti-tumor compound is released without altering its structure, allowing it to exert its inherent anti-tumor effect. For example, an ideal payload may have the following properties: First, it may have sufficiently high cytotoxicity. Tumor-specific antigens are very limited, especially in solid tumors. In addition, due to the low permeability and internalization activity of monoclonal antibodies, the number of ADC payloads that can be taken up by tumor cells via antibody-antigen binding is very limited. Second, the ADC payload may have sufficiently low immunogenicity.

[0049] As used herein, "topoisomerase 1 inhibitors" refers to compounds that inhibit the activity of topoisomerase 1, a ribozyme important for genome stability and DNA structural maintenance, and are attracting attention as targets for ADCs. Topoisomerase 1 (TOPO-I) inhibitors are associated with innate and adaptive immune responses, suggesting that ADCs targeting TOPO-I may also contribute to antitumor immunotherapy.

[0050] As used herein, "hydrophilic adduct" (hydrophilic group or unit) refers to a group or unit that may increase overall water solubility and conjugation efficiency, thereby reducing accumulation and aggregation of the ADC upon conjugation and in the circulation.

[0051] As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, relieving, delaying the onset, inhibiting progression, reducing severity, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. For example, "treating" cancer may refer to inhibiting the growth and / or metastasis of cancer cells, killing cancer cells, or shrinking cancer cells. Treatment may be administered to subjects who do not exhibit symptoms associated with the disease, disorder, and / or condition and / or who exhibit only early signs of the disease, disorder, and / or condition, with the intent of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0052] As used herein, the terms "about," "approximate," "roughly (at) or about," and "substantially" mean that the amount or value in question may be an exact value or a value that will produce an equivalent result or effect as recited in the claims or taught herein. That is, it is understood that amounts, sizes, 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 produce an equivalent result or effect, reflecting tolerances, conversion factors, rounding, measurement errors, and the like, as well as other factors known to those skilled in the art. In some circumstances, a value that will produce an equivalent result or effect may not be reasonably determinable. In such cases, "about" and "approximately," as used herein, are generally understood to mean a ±10% variation from the stated nominal value, unless otherwise indicated or inferred. In general, a quantity, size, formulation, parameter, or other quantity or characteristic is "about," "approximately," or "approximately," whether or not expressly stated as such. When "about," "approximately," or "generally" is used before a quantitative value, it is understood that the parameter also includes the particular quantitative value itself, unless specifically stated otherwise.

[0053] The term "cancer," as used herein, is defined as a disease characterized by the rapid and uncontrollable growth of abnormal cells. Cancer cells metastasize locally or to other parts of the body via the bloodstream and lymphatic system. 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, colon cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0054] The term "effective amount" means the 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, relieve, alleviate the symptoms of, prevent, delay the onset of, inhibit the progression of, reduce the severity of, and / or reduce the incidence of, the disease, disorder, and / or condition.

[0055] The terms "subject," "patient," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof amenable to the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0056] Detailed Description 1. GGYG linker system (1) This disclosure A compound of formula (1), Formula (1) [ka] In formula (1), P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, -(Y-CH)-, or -(dibenzocyclooctyne-NC(=O))-, Y is Br, Cl, or I, and -(dibenzocyclooctyne-NC(=O))- has the structure: [ka] L n is optionally included, and when included, is cycloalkyl, alkyl, or —(OCHCH)—, —(CH—C(═O)—NH—CH—CH)—, and n is an integer of 1 to 6; A 1 is a peptide residue containing 0 to 3 amino acids, A 2 is a peptide residue containing 0 to 3 amino acids, Z is optionally included and, if included, is a self-immolative spacer, —(NHCH)—, or p-aminocarbamate; X1 is H, a monosaccharide, a disaccharide, an oligosaccharide, a polyethylene glycol, a sulfate, a phosphate, or a pyrophosphate; A compound is provided.

[0057] (2) This disclosure also provides: A compound according to the embodiment of (1) above, a drug conjugated to said compound via Z of formula (1); The present invention provides a linker-drug conjugate comprising:

[0058] In one embodiment, the drug (payload or ADC payload) may include (i) microtubule-targeting payloads such as maytansinoids, auristatins, eribulin, tubulysins, cryptophycins, and EG5 inhibitors; (ii) DNA-targeting payloads such as enediynes, topoisomerase 1 inhibitors, pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), and duocarmycins; (iii) RNA-targeting payloads such as tylanstatin and amatoxins; (iv) immuno-ADC payloads such as Toll-like receptor agonists, stimulator of interferon genes (STING), and glucocorticoid receptor modulators; and (v) any novel potential ADC payloads such as Bcl-xL inhibitors, niacinamidophosphate ribose transferase (NAMPT), carmaphycin, PROTAC molecules, near-infrared photoimmunotherapy (NIR-PIT) drugs, and dual payloads (e.g., MMAE and MMAF).

[0059] (3) The present disclosure also provides a linker-drug conjugate according to the embodiment (2) above, wherein the drug is conjugated to the compound in the form of -(NH-drug)- or -(O-drug)-.

[0060] (4) The present disclosure also provides a linker-drug conjugate according to the embodiment of (2) above, wherein the drug is a topoisomerase I inhibitor.

[0061] "Topoisomerase 1 inhibitors" refer to compounds that inhibit the activity of topoisomerase 1, a ribozyme important for genome stability and DNA structural maintenance, and are attracting attention as targets for ADCs. Topoisomerase 1 (TOPO-I) inhibitors are associated with innate and adaptive immune responses, suggesting that ADCs targeting TOPO-I may also contribute to antitumor immunotherapy.

[0062] For example, the linker-drug conjugate may include the following compound: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0063] (5) This disclosure comprising an antibody or antigen-binding fragment thereof, a drug, and a linker; the linker is a compound according to embodiment (1) above, the antibody or antigen-binding fragment thereof is conjugated to the compound via P of formula (1), the drug is conjugated to the compound via Z of formula (1); Antibody-drug conjugates are provided.

[0064] In one embodiment, the antigen-binding fragment may include one or more of a Fab fragment, a Fab' fragment, a Fab'-SH, an Fv fragment, an scFv fragment, a F(ab')2 fragment, a VL fragment, a VH fragment, an ScFv-Fc fragment, and an (ScFv)2-Fc fragment, a diabody, a linear antibody, a fragment produced by a Fab expression library, an anti-idiotypic (anti-Id) antibody, a complementarity-determining region (CDR), and an epitope-binding fragment.

[0065] (6) The present disclosure provides a pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate according to the embodiment of (5) above.

[0066] In one embodiment, the antibody-drug conjugate can be administered to a subject by any method. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, intranasal administration, topical administration, intravaginal administration, intraocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injections such as intravenous, intraarterial, intramuscular, and subcutaneous administration. Administration can be continuous or intermittent. The formulation can be administered therapeutically, i.e., to treat an existing disease or condition. The formulation can also be administered prophylactically, i.e., to prevent cancer, such as a blood cancer or solid tumor.

[0067] An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or route of administration. In one embodiment, administration occurs over a course of treatment that includes multiple treatment cycles and multiple drug-free periods.

[0068] In another embodiment, the antibody-drug conjugate may be administered to a subject at a concentration effective to treat cancer in the subject, for example, in the range of 1 mg to 4-5 mg per kg of body weight.

[0069] (7) The present disclosure provides a pharmaceutical composition according to the embodiment (6) above, wherein the cancer comprises one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain tumor, renal clear 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's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, renal 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.

[0070] 2. DGGFG or GGGFG linker system (8) Furthermore, this disclosure A compound of formula (2): Formula (2) [ka] In formula (2), P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, or -(dibenzocyclooctyne-NC(=O))-; L n is optionally included and, when included, is cycloalkyl, alkyl, or a direct bond, and n is an integer from 1 to 6; X is -(C=O)-, a direct bond, or A 1 is a direct bond to the side chain of A 1 is an amino acid, R m is A 1 wherein m is an integer from 1 to 10; Z is optionally included and, if included, is a self-immolative spacer, —(NHCH)—, or p-aminocarbamate; A compound is provided.

[0071] In one embodiment, "hydrophilic adduct" refers to a group or unit that may increase overall water solubility and conjugation efficiency, thereby reducing accumulation and aggregation of the ADC upon conjugation and in the circulation.

[0072] In one embodiment, the hydrophilic adduct may be selected from the following compounds: [ka]

[0073] Here, n may be an integer ranging from 3 to 24. In one 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, or 22. In one 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 one 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.

[0074] (9) The present disclosure provides a compound according to the embodiment (8) above, wherein the amino acid is aspartic acid, glycine, glutamic acid, or lysine.

[0075] (10) The present disclosure also provides: A compound according to the embodiment of (8) above, a drug conjugated to said compound via Z of formula (2); The present invention provides a linker-drug conjugate comprising:

[0076] In one embodiment, the drug (payload or ADC payload) may include (i) microtubule-targeting payloads such as maytansinoids, auristatins, eribulin, tubulysins, cryptophycins, and EG5 inhibitors; (ii) DNA-targeting payloads such as enediynes, topoisomerase 1 inhibitors, pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), and duocarmycins; (iii) RNA-targeting payloads such as tylanstatin and amatoxins; (iv) immuno-ADC payloads such as Toll-like receptor agonists, stimulator of interferon genes (STING), and glucocorticoid receptor modulators; and (v) any novel potential ADC payloads such as Bcl-xL inhibitors, niacinamidophosphate ribose transferase (NAMPT), carmaphycin, PROTAC molecules, near-infrared photoimmunotherapy (NIR-PIT) drugs, and dual payloads (e.g., MMAE and MMAF).

[0077] (11) The present disclosure also provides a linker-drug conjugate according to the embodiment (10) above, wherein the drug is conjugated to the compound in the form of -(NH-drug)- or -(O-drug)-.

[0078] (12) The present disclosure also provides a linker-drug conjugate according to the embodiment of (10) above, wherein the drug is a topoisomerase 1 inhibitor.

[0079] "Topoisomerase 1 inhibitors" refer to compounds that inhibit the activity of topoisomerase 1, a ribozyme important for genome stability and DNA structural maintenance, and are attracting attention as targets for ADCs. Topoisomerase 1 (TOPO-I) inhibitors are associated with innate and adaptive immune responses, suggesting that ADCs targeting TOPO-I may also contribute to antitumor immunotherapy.

[0080] For example, the linker-drug conjugate may include the following compound: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0081] (13) This disclosure comprising an antibody or antigen-binding fragment thereof, a drug, and a linker; the linker is a compound according to embodiment (8) above, the antibody or antigen-binding fragment thereof is conjugated to the compound via P of formula (2), The drug is conjugated to the compound via Z in formula (2). Antibody-drug conjugates are provided.

[0082] (14) The present disclosure provides a pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate according to the embodiment of (13) above.

[0083] In one embodiment, the antibody-drug conjugate can be administered to a subject by any method. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, intranasal administration, topical administration, intravaginal administration, intraocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injections such as intravenous, intraarterial, intramuscular, and subcutaneous administration. Administration can be continuous or intermittent. The formulation can be administered therapeutically, i.e., to treat an existing disease or condition. The formulation can also be administered prophylactically, i.e., to prevent cancer, such as a blood cancer or solid tumor.

[0084] An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or route of administration. In one embodiment, administration occurs over a course of treatment that includes multiple treatment cycles and multiple drug-free periods.

[0085] In another embodiment, the antibody-drug conjugate may be administered to a subject at a concentration effective to treat cancer in the subject, for example, in the range of 1 mg to 4-5 mg per kg of body weight.

[0086] (15) The present disclosure provides a pharmaceutical composition according to the embodiment (14) above, wherein the cancer comprises one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, renal clear 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's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, renal 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.

[0087] 3. GGFG Linker System (16) The present disclosure also provides: A compound of formula (3): Formula (3) [ka] In formula (3), P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, or -(dibenzocyclooctyne-NC(=O))-; L n is optionally included and, when included, is cycloalkyl, alkyl, or a direct bond, and n is an integer from 1 to 6; X is —(C═O)— or a direct bond; A 1 is an amino acid, R m is optionally included, and if included, A 1 wherein m is an integer from 1 to 10; Z is optionally included and, if included, is a self-immolative spacer, —(NHCH)—, or p-aminocarbamate; A compound is provided.

[0088] In one embodiment, "hydrophilic adduct" refers to a group or unit that may increase overall water solubility and conjugation efficiency, thereby reducing accumulation and aggregation of the ADC upon conjugation and in the circulation.

[0089] In one embodiment, the hydrophilic adduct may be selected from the following compounds: [ka]

[0090] Here, n may be an integer ranging from 3 to 24. In one 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, or 22. In one 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 one 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.

[0091] (17) The present disclosure provides a compound according to the embodiment (16) above, wherein the amino acid is aspartic acid, glycine, glutamic acid, or lysine.

[0092] (18) The present disclosure also provides: A compound according to the embodiment of (16) above, a drug conjugated to said compound via Z of formula (2); The present invention provides a linker-drug conjugate comprising:

[0093] In one embodiment, the drug (payload or ADC payload) may include (i) microtubule-targeting payloads such as maytansinoids, auristatins, eribulin, tubulysins, cryptophycins, and EG5 inhibitors; (ii) DNA-targeting payloads such as enediynes, topoisomerase 1 inhibitors, pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), and duocarmycins; (iii) RNA-targeting payloads such as tylanstatin and amatoxins; (iv) immuno-ADC payloads such as Toll-like receptor agonists, stimulator of interferon genes (STING), and glucocorticoid receptor modulators; and (v) any novel potential ADC payloads such as Bcl-xL inhibitors, niacinamidophosphate ribose transferase (NAMPT), carmaphycin, PROTAC molecules, near-infrared photoimmunotherapy (NIR-PIT) drugs, and dual payloads (e.g., MMAE and MMAF).

[0094] (19) The present disclosure also provides a linker-drug conjugate according to the embodiment (18) above, wherein the drug is conjugated to the compound in the form of -(NH-drug)- or -(O-drug)-.

[0095] (20) The present disclosure also provides a linker-drug conjugate according to the embodiment of (18) above, wherein the drug is a topoisomerase 1 inhibitor.

[0096] For example, the linker-drug conjugate may include the following compound: [ka] [ka]

[0097] (21) This disclosure provides: comprising an antibody or antigen-binding fragment thereof, a drug, and a linker; the linker is a compound according to embodiment (16) above, the antibody or antigen-binding fragment thereof is conjugated to the compound via P of formula (3), The drug is conjugated to the compound via Z of formula (3). Antibody-drug conjugates are provided.

[0098] (22) The present disclosure provides a pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate according to the embodiment of (21) above.

[0099] In one embodiment, the antibody-drug conjugate can be administered to a subject by any method. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, intranasal administration, topical administration, intravaginal administration, intraocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injections such as intravenous, intraarterial, intramuscular, and subcutaneous administration. Administration can be continuous or intermittent. The formulation can be administered therapeutically, i.e., to treat an existing disease or condition. The formulation can also be administered prophylactically, i.e., to prevent cancer, such as a blood cancer or solid tumor.

[0100] An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or route of administration. In one embodiment, administration occurs over a course of treatment that includes multiple treatment cycles and multiple drug-free periods.

[0101] In another embodiment, the antibody-drug conjugate may be administered to a subject at a concentration effective to treat cancer in the subject, for example, in the range of 1 mg to 4-5 mg per kg of body weight.

[0102] (23) The present disclosure provides a pharmaceutical composition according to the embodiment (22) above, wherein the cancer comprises one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, renal clear 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's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, renal 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.

[0103] 4. Other Linker Systems The present disclosure also provides the following linker systems: For example, the linker-drug conjugate may include the following compounds: [ka] [ka] [ka]

[0104] In the above compounds, the hydrophilic groups may be as follows: [ka]

[0105] In the embodiments described in this disclosure, a particular drug (payload (PBX-7016)) is conjugated to various linker systems, although, as discussed in this disclosure, any drug (payload) used in an ADC may be conjugated to such a linker. For example, the drug (payload or ADC payload) may include (i) microtubule-targeting payloads such as maytansinoids, auristatins, eribulin, tubulysins, cryptophycins, and EG5 inhibitors; (ii) DNA-targeting payloads such as enediynes, topoisomerase 1 inhibitors, pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), and duocarmycins; (iii) RNA-targeting payloads such as tylanstatin and amatoxins; (iv) immuno-ADC payloads such as Toll-like receptor agonists, stimulator of interferon genes (STING), and glucocorticoid receptor modulators; and (v) any novel potential ADC payloads such as Bcl-xL inhibitors, niacinamidophosphate ribose transferase (NAMPT), carmaphycin, PROTAC molecules, near-infrared photoimmunotherapy (NIR-PIT) drugs, and dual payloads (e.g., MMAE and MMAF).

[0106] Also, in the field of antibody-drug conjugates or related art, any antibody or antigen-binding fragment thereof or any moiety that binds to a specific target may be conjugated to a linker discussed in this disclosure.

[0107] 5. Pharmaceutical Compositions Comprising Antibody-Drug Conjugates The antibody-drug conjugate of the present disclosure may be prepared in the form of a pharmaceutical composition comprising the antibody-drug conjugate of the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be selected based on the specific anti-tumor compound used, its concentration, stability, and intended bioavailability, the disease, disorder, or condition being treated with the composition, the subject, their age, size, and general condition, and the route of administration. The antibody-drug conjugate of the present disclosure may be mixed with a solvent such as a sterile liquid (including water and oils (oils derived from petroleum, animal, or plant sources, or synthetic oils (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)), saline, aqueous glucose solution, or aqueous glycerol solution, and additives such as humectants, emulsifiers, or pH buffering agents) to prepare a pharmaceutical composition of the present disclosure. Pharmaceutically acceptable carriers for solid dosage forms include sugars, starches, and other conventional materials, including polysorbates, histidine, lactose, talc, sucrose, gelatin, carboxymethylcellulose, agar, mannitol, sorbitol, calcium phosphate, calcium carbonate, sodium carbonate, kaolin, alginic acid, acacia, corn starch, potato starch, sodium saccharin, magnesium carbonate, tragacanth, microcrystalline cellulose, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, and stearic acid. Additionally, such solid dosage forms may be uncoated or may be coated by known techniques (e.g., to delay disintegration and absorption). Additionally, pharmaceutically acceptable carriers used in preparing 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, glucose solutions, electrolyte solutions, or other aqueous pharmaceutically acceptable liquids.

[0108] Example (1) Importance of hydrophilic groups: The incorporation of hydrophilic groups into the linker structure of antibody-drug conjugates (ADCs) is crucial in vivo for mitigating potential side effects. This strategic introduction plays several important roles in improving the safety and efficacy profile of ADCs. i. Reduced non-selective cellular uptake: Hydrophilic groups contribute to increased water solubility, making it difficult for the ADC to penetrate cell walls, thereby minimizing unintended exposure of the cytotoxic payload in normal cells. ii. Improved stability in circulation: Hydrophilic linkers are essential to increase the stability of ADCs, maintain the structural integrity of the ADC until it reaches the tumor site, and prevent systemic release. iii. Minimizing off-target toxicity: By reducing non-specific interactions with normal cells, hydrophilic linkers help minimize off-target toxicity and maintain the therapeutic window of the ADC. iv. Improved pharmacokinetics: Hydrophilic modifications can affect the pharmacokinetics of ADCs, optimizing factors such as circulation time and distribution.

[0109] Design: GGFG to GGYG To enhance tumor specificity and tolerability, a stable and cleavable linker is necessary. We designed a novel peptide sequence that can be cleaved by lysosomal enzymes (cathepsins) with efficiency comparable to that of the GGFG linker system, enabling the introduction of functional moieties to adjust the physicochemical properties of ADCs.

[0110] In this regard, we investigated the peptide substrate recognition of cathepsin B and L proteases (Figure 1). These proteases are characterized by broad substrate specificity and tend to prefer structurally related amino acids at specific subsites. Taking this into consideration, we designed a novel tetrapeptide sequence by substituting the aromatic amino acid phenylalanine (F) in the GGFG linker system (LP1) with the structurally similar amino acid tyrosine (Y). [ka]

[0111] Conjugate: Trastuzumab (4 mg / ml) was reacted with a 30-fold molar excess of TCEP (tris(2-carboxyethyl)phosphine hydrochloride) at 25°C for 2 hours to generate cysteines from disulfide bonds. Reduced trastuzumab was purified from unreacted TCEP using a PD-10 desalting column. Reduced trastuzumab (2 mg / ml) was reacted with a 12-fold molar excess of linker-payload 2 (LP2) in 10% (v / v) DMSO at 25°C for 1 hour. Trastuzumab ADC was purified from unreacted linker-payload and DMSO using a PD-10 desalting column. See Figures 2-4.

[0112] (2) Enhanced hydrophilicity and tandem cleavage The hydroxyl group on tyrosine (Y) serves as the attachment point for hydrophilic pendant groups such as sugars, sulfates, phosphates, PEG linkers, etc. Studies have reported elevated levels of the enzyme β-glucuronidase in tumor tissue. This enzyme is found in very low concentrations in the circulation but is present in high concentrations in solid tumors, including lung, breast, pancreatic, colon, and ovarian cancers.

[0113] Taking advantage of the high expression of the lysosomal enzyme β-glucuronidase in malignant cells, we designed a tandem cleavage linker in which β-glucuronic acid is attached to the hydroxyl group of tyrosine in the GGYG linker system, which is designed to undergo sequential enzymatic cleavage in a specific and controlled manner. [ka] The introduction of a β-glucuronide moiety may offer the following advantages:

[0114] Temporary hydrophilic units: (1) Improved overall water solubility and conjugation efficiency reduce accumulation and aggregation of ADCs during conjugation and in circulation. (2) Expanding the range of cytotoxic payloads in ADCs by enabling the conjugation of difficult-to-conjugate hydrophobic payloads.

[0115] Sequential enzymatic cleavage: (1) Cathepsin-mediated peptide cleavage is inhibited until cleavage of the pendant group by lysosomal β-glucuronidase. (2) By reducing the exposure of the linker-payload cleavage site to the action of serum proteases and neutrophil elastase, it effectively reduces premature drug loss in the circulation and improves preferential drug release in tumors.

[0116] Conjugation: Trastuzumab (4 mg / ml) was reacted with a 30-fold molar excess of TCEP (tris(2-carboxyethyl)phosphine hydrochloride) at 25°C for 2 hours to generate cysteines from disulfide bonds. Reduced trastuzumab was purified from unreacted TCEP using a PD-10 desalting column. Reduced trastuzumab (2 mg / ml) was reacted with a 12-fold molar excess of linker-payload 3 (LP3) in 10% (v / v) DMSO at 25°C for 1 hour. Trastuzumab ADC was purified from unreacted linker-payload and DMSO using a PD-10 desalting column. See Figures 5-8.

[0117] (3) Improved stability Traditionally, maleimides have been widely used for cysteine ​​modification due to their rapid and selective reaction with thiols. However, recent findings have shown that thioether linkages undergo unconjugation via a retro-Michael pathway, resulting in premature drug release in the circulation and reduced therapeutic efficacy. Furthermore, maleimide-based conjugates undergo thiol exchange reactions with other plasma thiol groups, such as human serum albumin (HSA), leading to toxic off-site drug delivery and further reduced efficacy.

[0118] To address these challenges and enhance plasma linker stability compared to maleimide-caproic acid-containing antibody-drug conjugates (ADCs), acetamide-linked conjugates were developed, replacing the maleimide moiety with bromoacetamide. These new linker derivatives (LP4 and LP5), featuring bromoacetamide, react with thiols generated after disulfide bond reduction in antibodies. This results in the formation of stable conjugates and significantly reduces premature drug release and off-target binding. [ka] [ka]

[0119] In addition to acetamide-linked conjugates, we have also developed self-stabilizing maleimide conjugates. To achieve this, we introduced an amide functional group (LP6) into the caproic acid spacer, which allows the maleimide to rapidly hydrolyze to form an open ring structure, improving the stability of the maleimide-drug conjugate. This modification effectively suppresses retro-Michael unconjugation and ensures a consistent DAR throughout the ADC's lifetime in circulation. Alternatively, stability can be improved by replacing the maleimide conjugation site with a DBCO-based conjugate (LP7). [ka] [ka]

[0120] We believe that this design maintains the efficacy of the GGFG linker system while improving its safety profile, resulting in reduced off-target toxicities such as neutropenia and interstitial lung disease.

[0121] This innovation pursues the goal of suppressing the effects of antitumor compounds on normal cells by incorporating hydrophilic pendant groups into the linker backbone. This increases the overall hydrophilicity of the linker-payload, making the unconjugated linker-payload less likely to penetrate the cell wall. This results in reduced nonselective uptake of antitumor compounds and suppression of unintended cellular absorption, resulting in a lower incidence of off-target effects and a higher level of safety reflected in reduced ILD.

[0122] Synthetic scheme and procedure: Synthesis of Linker-Payload 2 (LP2) [ka]

[0123] Step 1: [ka] Fmoc-Gly-Gly-OH (5.0 g, 14.1 mmol) was partially dissolved in tetrahydrofuran (125 mL), toluene (42.6 mL), and pyridine (2.15 mL). Lead(IV) acetate (7.8 g, 17.6 mmol) was added, causing the reaction mixture to turn orange. The mixture was heated to reflux. After stirring for 3 h, the reaction mixture was cooled to room temperature, then filtered through a bed of Celite, rinsed with ethyl acetate, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica, 10–100% ethyl acetate in heptane) to give methyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-acetate (3.0 g, 57% yield) as a white solid. 1HNMR (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, 1 H), 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 / z391.2[M+Na] +

[0124] Step 2: [ka] To a solution of (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (1.27 g, 3.45 mmol) in dichloromethane (20 mL) was added (R)-benzyl lactate (6.21 g, 34.5 mmol), followed by pyridinium p-toluenesulfonate (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 (50 mL × 3), dried over sodium sulfate, filtered, and concentrated to give 7.10 g of a colorless oil. The residue was purified by flash chromatography (Si80 g; 10% to 70% ethyl acetate in heptane). The combined product fractions were concentrated to give 1.68 g of a colorless cloudy 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 / z511.2[M+H] +

[0125] Step 3: [ka] To a solution of (R)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-atobenzyl (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 coevaporated twice with DCM. Yield: 926 mg, m / z 267.1 [M+H] +

[0126] Step 4: [ka] (R)-benzyl 2-((2-aminoacetamido)methoxy)propanoate (0.71 g, estimated 58% wt, 1 eq, 1.55 mmol) was dissolved in DMF (10.0 mL). (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxyphenyl)propanoic acid (624 mg, 1 eq, 1.55 mmol) and DIPEA (300 mg, 404 μL, 1.5 eq, 2.32 mmol) were added, followed by HATU (588 mg, 1 eq, 1.55 mmol). The reaction mixture was stirred at room temperature for 30 min. The mixture was diluted with 100 mL of water and extracted twice with 100 mL of ethyl acetate. The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The crude product was purified by flash column chromatography (Si24 g, 0-100% EtOAc in heptane). Evaporation of the solvent gave the product (455 mg, 45%) as a solid. 1HNMR(400MHz,DMSO-d6)δ9.17(s,1H),8.60(t,J=6.8Hz,1H),8.29(t,J=5.8Hz,1H),7.8 8(d,J=7.5Hz,2H),7.68-7.58(m,3H),7.45-7.25(m,10H),7.10-7.04(m,2H),6.66-6.6 0(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] +

[0127] Step 5: [ka] (5S,13R)-1-(9H-Fluoren-9-yl)-5-(4-hydroxybenzyl)-13-methyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazatetradecane-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 coevaporated twice with DCM to give a cloudy oil. Yield: 570 mg. m / z 430.2 [M+H] +

[0128] Step 6: [ka] Crude benzyl (R)-2-((2-((S)-2-amino-3-(4-hydroxyphenyl)propanamido)acetamido)methoxy)propanoic acid (570 mg, estimated 63% wt, 1 eq, 836 μmol) was dissolved in dichloromethane (10 mL). 2-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetic acid (296 mg, 1.0 eq, 836 μmol) was added, followed by HATU (318 mg, 1.0 eq, 836 μmol) and DIPEA (162 mg, 218 μL, 1.5 eq, 1.25 mmol). The yellow reaction mixture was stirred at room temperature. After 2 h, 0.3 eq of HATU and 0.5 eq of DIPEA were obtained. DIPEA was added. The reaction mixture was stirred for a total of 5 h. The mixture was evaporated to dryness and purified by flash column chromatography (Si 24 g, 0–6% MeOH in DCM). The product fractions were evaporated to dryness to give benzyl (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-pentaazaicosan-20-oate (190 mg, 248 μmol, 29%). 1 HNMR(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. 89(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 .66-6.60(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). m / z788.4[M+Na] +

[0129] Step 7: [ka] Benzyl(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-pentaazaicosan-20-oate (175 mg, 1 eq, 229 μmol) was dissolved in ethanol (5.0 mL) / ethyl acetate (5.0 mL). Pd / C (10%, 50% wet) (48 mg, 5% Wt, 0.1 eq, 22.9 μmol) was added, and the reaction mixture was stirred under a hydrogen atmosphere for 2.5 h. The reaction mixture was filtered through Celite and washed twice with 20 mL of methanol. The filtrate was evaporated to dryness to give a colorless solid (169 mg). m / z 674.4 [M−H] -

[0130] Step 8: [ka] (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-pentaazaicosan-20-oic acid (154 mg, 1 eq, 228 μmol) (crude product) 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 formed at the bottom of the flask. The DCM layer was removed. The resulting solid was dried under reduced pressure to give 154 mg of a white solid. This solid was used directly in the next reaction without further purification. m / z 454.2 [M+H] +

[0131] Step 9: [ka] (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% wt, 1 eq, 228 μmol) (crude product) 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 (105 mg, 1.5 eq, 341 μmol) was added. The reaction was stirred at room temperature for 20 min. The reaction mixture was diluted with DCM and applied to a short column of silica gel (~30 g). The column was first eluted with 100 mL of 9 / 1 DCM / MeOH. The product was then eluted with 75 mL 1 / 1 DCM / MeOH. The product-containing fractions were evaporated to dryness under reduced pressure. The crude product was triturated from DCM to give a white solid. The product batch was dissolved in DMSO and purified by preparative MPLC (Luna 5-40). The product fractions were lyophilized to give the product as a white solid. Yield: 80 mg, 54%. 1 HNMR(400MHz,DMSO-d6)δ12.56(s,1H),9.16(s,1H),8.51(t,J=6.7Hz,1H),8.23(t,J=5.9Hz,1H),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,1H),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 / z645.4[MH]-,669.4[M+Na] +

[0132] Step 10: [ka] (1S,10S)-1-Amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[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. (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) was added, followed by HATU (34 mg, 2 eq, 89 μmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was directly purified by acidic preparative MPLC. The product fractions were combined and lyophilized to give a yellow solid. Yield: 22 mg, 51%. 1 HNMR(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),3.77-3.5 4(m,6H),3.39-3.33(m,2H),3.15-2.97(m,2H),2.87(dd,J=13.9,4.7Hz,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 / z1076.4[M+H] +

[0133] Synthesis of Linker-Payload 3 (LP3) [ka]

[0134] Step 1: [ka] To a suspension of benzyl (R)-1-(9H-fluoren-9-yl)-10-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oate (211 mg, 1 eq, 432 μmol) in dichloromethane (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 coevaporated with dichloromethane (6 times) to give the product as a colorless thick oil. m / z 267.2 [M+H] +

[0135] Step 2: [ka] To a solution of benzyl (R)-2-((2-aminoacetamido)methoxy)propanoate (115 mg, 1 eq, 432 μmol) in DMF (5.0 mL) was added DIPEA (167 mg, 226 μL, 3 eq, 1.30 mmol), followed by (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. The reaction mixture was diluted with ethyl acetate and washed with water (twice) and saturated brine (twice). The organic phase was dried over Na2SO4, filtered, and concentrated to give a thick yellow oil (656 mg). The crude product was purified by flash column chromatography (Si24 g, 0-100% EtOAc in heptane). The product fractions were collected, concentrated, and co-evaporated to give a white solid (317 mg, 71%). 1 HNMR(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,7 H),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 ,3H),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 / z990.2[M+Na] +

[0136] Step 3: [ka] 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 minutes. The reaction mixture was quenched with acetic acid (43.3 mg, 41.2 μL, 2.2 eq, 720 μmol) and purified by acidic preparative MPLC (Luna 10-50). The product fractions were combined and lyophilized to give a white solid (200 mg, 82%). 1 HNMR(400MHz,CDCl3)δ7.81(t,J=5.8Hz,1H),7.38-7.30(m,5H),7.17-7.11(m,2H),6.99-6.9 2(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 .88-4.73(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,1 H),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] +

[0137] Step 4: [ka] 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) was added DIPEA (102 mg, 138 μL, 3 eq, 793 μmol), followed by Fmoc-Gly-Gly-OH (140 mg, 1.5 eq, 396 μmol) and HATU (151 mg, 1.5 eq, 396 μmol). The reaction mixture was stirred at room temperature for 45 min. The reaction mixture was directly purified by acidic preparative MPLC (Luna 30-70). The product fractions were combined and lyophilized to give a white solid (215 mg, 75%). 1 HNMR(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),4.8 0-4.72(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~ 3.72(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 / z1104.2[M+Na] +

[0138] Step 5: [ka] 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. Pd / C (10%, 50% wet) (38.0 mg, 5% Wt, 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 flushed with nitrogen for 10 minutes, filtered through diatomaceous earth, eluted with methanol, and concentrated to give a white solid (155 mg). The crude product was purified by acidic preparative MPLC (Luna 20-60). The product fractions were combined and lyophilized to give a white solid (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 , 1H), 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 / z1014.0[M+Na] +

[0139] Step 6: [ka] (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione methanesulfonate (55 mg, 1 eq, 0.10 mmol) was suspended in DMF (2.5 mL). (11S,19R)-1-(9H-Fluoren-9-yl)-19-methyl-3,6,9,12,15-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-pentaazaicosan-20-oic acid (0.10 g, 1 eq, 0.10 mmol) and DIPEA (39 mg, 53 μL, 3 eq, 0.30 mmol) were added, followed by HATU (48 mg, 1.25 eq, 0.13 mmol). The reaction mixture was stirred at room temperature for 15 min. The reaction mixture was directly purified by acidic preparative MPLC (Luna 20-60). The product fractions were combined and lyophilized to give a yellow solid (100 mg, 70%). 1HNMR(400MHz, DMSO-d6) δ 8.65 (t, J = 6.6 Hz, 1H), 8.49 (d, J = 9.1 Hz, 1H), 8.31 (t, J = 5.8 Hz, 1H), 8.10 (d, J = 7.9 Hz, 1H), 8.03 (t, J = 5.7 Hz, 1H), 7.87 (d, J = 7.6 Hz, 2H), 7.69 (d, J = 7.4 Hz, 2H), 7.57 (t, J = 6.0 Hz, 1H), 7.43 - 7.26 (m, 5H), 7.23 (s, 1H), 7.19 - 7.11 (m, 2H), 6.91 - 6.84 (m, 2H), 6.47 (s, 1H), 6.26 (d, J = 7.2 Hz, 2H), 5.64 - 5.51 (m, 2H), 5.45 (t, J = 9.7 Hz, 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 = 6.8 Hz, 3H), 0.86 (t, J = 7.3 Hz, 3H). m / z 1421.8 [M + H] +

[0140] Step 7:

Chem.

[0141] ステップ8:

change

[0142] Synthesis of Compound 11

change

[0143] ステップ1:

change

[0144] Step 2: [ka] 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, and 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 allowed to warm to room temperature and stirred for 3 h. The reaction mixture was concentrated under reduced pressure to give a red oil. The crude product was coated onto isolute and purified by flash column chromatography (80 g Si, 0–35% EtOAc in heptane). The product fractions were collected and concentrated under reduced pressure to give the product as a beige solid (1.86 g, 55%). 1 HNMR(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 / z499.8[M+Na] +

[0145] Step 3: [ka] To a solution of H-Tyr-OBzl (4.0 g, 1 eq, 15 mmol) in chloroform (100 mL) was added Fmoc-OSu (5.0 g, 1 eq, 15 mmol), and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with DCM (100 mL) and washed with HO (2 x 100 mL) and saturated brine (100 mL). The organic layer was concentrated under reduced pressure to give an off-white solid (7.22 g). The solid was placed on a filter and washed with DCM (3 x 10 mL). The solid was removed and dried in a vacuum oven at 40 °C for 2 days to give the product (5.77 g, 79%) as a white solid. 1HNMR (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 / z494.4[M+H] + ,516.4[M+Na] +

[0146] Step 4: [ka] A suspension of (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 benzyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-tyrosinate (53 mg, 1 eq, 0.11 mmol) in 1.5 mL of dry DCM containing 4A molecular sieves was cooled to 0 °C. BF3.OEt2 (ca. 48% BF3) (17 mg, 15 μL, 1.1 eq, 0.12 mmol) was added. The ice bath was removed, and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with trimethylamine (2M THF solution) (7.0 mg, 59 μL, 2.0 mol, 1.1 eq, 0.12 mmol) and directly purified by flash column chromatography (12 g Si, 0-60% EtOAc in heptane). The product fractions were collected and concentrated under reduced pressure to give a white solid (32 mg, 37%). 1HNMR(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 .73-4.65(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 / z810.6[M+H] + ,832.6[M+Na] +

[0147] Step 5: [ka] 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 EtOAc (17 mL) was purged with nitrogen for 10 minutes. Pd / C (10%, 50% wet) (139 mg, 5% Wt, 0.1 eq, 65.1 μmol) was then added, and the mixture was stirred under a hydrogen atmosphere for 5 hours. The reaction mixture was flushed with nitrogen, then filtered through a bed of diatomaceous earth, washed with ethyl acetate, concentrated under reduced pressure, and co-evaporated with dichloromethane (twice) to give the product as a white solid (468 mg, quantitative yield). 1HNMR(400MHz,CDCl3)δ7.77(d,J=7.6Hz,2H),7.59-7.49(m,2H),7.41(t,J=7.5Hz,2H),7. 35-7.28(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 / z742.0[M+Na] +

[0148] Synthesis of Linker-Payload 4 (LP4) [ka]

[0149] Step 1: [ka] To a solution of potassium hydroxide (0.43 g, 1 eq, 7.6 mmol) in water (2.1 mL) was added 6-aminohexanoic acid (1.0 g, 1 eq, 7.6 mmol) at 0 °C. While adding bromoacetyl bromide (1.8 g, 0.80 mL, 1.2 eq, 9.1 mmol), the pH was adjusted to pH > 7.8 by the dropwise addition of 2.8 M aqueous potassium carbonate. After the addition, the reaction mixture was stirred at 0 °C for 1 h. The reaction mixture was acidified to pH ≈ 1 with 0.5 N HCl and extracted three times with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated to give 1.47 g of a colorless oil. Purification was performed by flash column chromatography (Si40 g, 0-2.5% methanol in dichloromethane). The combined product fractions were concentrated to give 661 mg (34%) of a colorless oil, which solidified upon standing at room temperature. 1HNMR (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 / z525.2[M+H] + , bromine isotope pattern

[0150] Step 2: [ka] To a solution of 6-(2-bromoacetamido)hexanoic acid (631 mg, 1 eq, 2.50 mmol) in dichloromethane (30 mL) was added pentafluorophenyltrifluoroacetic acid (1.02 g, 624 μL, 1.45 eq, 3.63 mmol) and pyridine (792 mg, 810 μL, 4 eq, 10.0 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 10 min. The reaction mixture was washed with 0.5 M aqueous HCl, and the organic layer was dried over sodium sulfate, filtered, and concentrated to give 1.2 g of a colorless oil. Purification was carried out by flash column chromatography (Si 40 g, 0 to 50% EtOAc in heptane). The combined product fractions were concentrated to give 812 mg (77%) of a fluffy white solid. 1 HNMR (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 / z420.2[M+H] +

[0151] Step 3: [ka] (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]dioxolo[4,5-g]pyrano [3',4':6,7]indolizino[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 (50 mg, 1 eq, 47 μmol) was dissolved in 3.5 mL of 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. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was acidified with acetic acid (10 mg, 10 μL, 3.7 eq, 0.17 mmol) and then purified by acidic preparative MPLC (Luna 5-40). The product fractions were combined and lyophilized to give 45 mg of a yellow solid. LCMS purity was insufficient. The product was purified again by acidic preparative MPLC (Luna 5-40) to give 19 mg of the desired product as a yellow solid. m / z 1292.2, 1294.2 [M+H] + , Br isotope pattern.

[0152] Synthesis of Linker-Payload 5 (LP5) [ka] (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]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1 To a solution of (1-(2-yl)amino)-1-oxopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)amino)-3-oxopropyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (64.0 mg, 1 eq, 60.4 μmol) in DMF (2.0 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 min. The reaction mixture was collected in a syringe and directly purified by acidic preparative MPLC (Luna 5-40). The product fraction was lyophilized to give 35 mg (49%) of a pale yellow solid. 1 HNMR(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),4.97(d,J=7. 4Hz, 1H), 4.74-4.63(m, 1H), 4.58-4.37(m, 2H), 4.16-4.05(m, 1H), 3.9 2(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=6.6Hz, 3H), 0.87(t, J=7.3Hz, 3H); m / z1179.0,1181.0[M+H] + , bromine isotope pattern

[0153] Synthesis of Linker-Payload 6 (LP6) [ka] (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]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopro To a solution of (2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (50 mg, 1 eq, 47 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 45 min at room temperature, the reaction mixture was purified by acidic preparative MPLC (Luna 5-40). The product fractions were combined and lyophilized to give 16 mg (27%) of the product as a pale yellow solid. 1HNMR(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,1 H),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=7.4Hz,1H ), 4.73-4.63(m, 1H), 4.57-4.50(m, 1H), 4.46-4.37(m, 1H), 4.15-4.08(m, 1H) ,3.99(s,2H),3.84-3.54(m,8H),3.39-3.34(m,1H),3.29-3.19(m,4H),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),1.9 2-1.78(m,2H),1.39(d,J=6.8Hz,3H),0.87(t,J=7.3Hz,3H);m / z1267.4[M+H] +

[0154] Synthesis of リンカー-ペイロード7(LP7)

change

Claims

1. A compound of formula (1), Formula (1) 【Chemistry 1】 In formula (1), P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, -(Y-CH ２ )-, or -(dibenzocyclooctyne-N-C(=O))-, where Y is Br, Cl, or I, and -(dibenzocyclooctyne-N-C(=O))- has the structure: 【Chemistry 2】 L ｎ is optionally included, and when included, is cycloalkyl, alkyl, or —(OCH ２ CH ２ ) -, -(CH ２ -C(=O)-NH-CH ２ -CH ２ )-, where n is an integer from 1 to 6; A １ is a peptide residue containing 0 to 3 amino acids, A ２ is a peptide residue containing 0 to 3 amino acids, Z is optionally included and, if included, is a self-immolative spacer, -(NHCH ２ )- or p-aminocarbamate, X １ is H, a monosaccharide, a disaccharide, an oligosaccharide, a polyethylene glycol, a sulfate, a phosphate, or a pyrophosphate; compound.

2. A compound according to claim 1; a drug conjugated to said compound via Z of formula (1); A linker-drug conjugate comprising:

3. 3. The linker-drug conjugate of claim 2, wherein the drug is conjugated to the compound in the form of -(NH-drug)- or -(O-drug)-.

4. 3. The linker-drug conjugate of claim 2, wherein the drug is a topoisomerase 1 inhibitor.

5. comprising an antibody or antigen-binding fragment thereof, a drug, and a linker; The linker is a compound according to claim 1, the antibody or antigen-binding fragment thereof is conjugated to the compound via P of formula (1); The drug is conjugated to the compound via Z of formula (1). Antibody-drug conjugates.

6. A pharmaceutical composition for treating cancer, comprising the antibody-drug conjugate of claim 5.

7. 7. The pharmaceutical composition of claim 6, wherein the cancer comprises one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, renal clear 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's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, renal 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 of formula (2): Formula (2) 【Transformation 3】 In formula (2), P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, or -(dibenzocyclooctyne-N—C(═O))-; L ｎ is optionally included and, when included, is cycloalkyl, alkyl, or a direct bond, and n is an integer from 1 to 6; X is —(C═O)—, a direct bond, or A １ is a direct bond to the side chain of A １ is an amino acid, R ｍ is A １ wherein m is an integer from 1 to 10; Z is optionally included and, if included, is a self-immolative spacer, -(NHCH ２ )- or p-aminocarbamate; compound.

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

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

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

12. 11. The linker-drug conjugate of claim 10, wherein the drug is a topoisomerase 1 inhibitor.

13. comprising an antibody or antigen-binding fragment thereof, a drug, and a linker; The linker is a compound according to claim 8, the antibody or antigen-binding fragment thereof is conjugated to the compound via P of formula (2), The drug is conjugated to the compound via Z of formula (2). Antibody-drug conjugates.

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

15. 15. The pharmaceutical composition of claim 14, wherein the cancer comprises one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, renal clear 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's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, renal 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 of formula (3): Formula (3) 【Chemistry 4】 In formula (3), P is -(maleimide-N)-, -(dibromomaleimide-N)-, -(bromoacetamide-N)-, or -(dibenzocyclooctyne-N—C(═O))-; L ｎ is optionally included and, when included, is cycloalkyl, alkyl, or a direct bond, and n is an integer from 1 to 6; X is —(C═O)— or a direct bond; A １ is an amino acid, R ｍ is optionally included, and if included, A １ wherein m is an integer from 1 to 10; Z is optionally included and, if included, is a self-immolative spacer, -(NHCH ２ )- or p-aminocarbamate; compound.

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

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

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

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

21. comprising an antibody or antigen-binding fragment thereof, a drug, and a linker; The linker is a compound according to claim 16, the antibody or antigen-binding fragment thereof is conjugated to the compound via P of formula (3), The drug is conjugated to the compound via Z of formula (3). Antibody-drug conjugates.

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

23. 23. The pharmaceutical composition of claim 22, wherein the cancer comprises one or more of breast cancer, liver cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, brain cancer, renal clear 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's lymphoma, non-Hodgkin's lymphoma (NHL), colorectal cancer, colon cancer, renal 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.

Images