Para-amino-benzyl linkers, methods for their preparation and their use in conjugates

Abstract

The present invention relates to p-amino-benzyl linker compounds that can be used to link a drug moiety to an antibody, to linker-drug compounds in which the p-amino-benzyl linker compound is covalently linked to a drug moiety, and to antibody-drug conjugates in which the p-amino-benzyl linker compound is covalently linked to a drug, wherein the drug is enzymatically cleaved from the conjugate at a specific cell or tissue type targeted by the antibody.

Description

[0001] This invention relates to para-amino-benzyl linker compounds that can be used to link a drug moiety to an antibody, to linker-drug compounds wherein the para-amino-benzyl linker compound is covalently linked to a drug moiety, and to antibody-drug conjugates comprising the para-amino-benzyl linker compound covalently linked to a drug, wherein the drug is enzymatically cleaved from the conjugate at a specific cell or tissue type targeted by the antibody. Background of the Invention

[0003] Targeted therapies are designed to reduce nonspecific toxicity and increase efficacy compared to conventional therapies. This approach manifests as the powerful targeting ability of monoclonal antibodies or antigen-binding fragments to specifically deliver highly potent conjugated small-molecule therapeutics to antigen-positive cells. To address toxicity concerns, chemotherapeutic agents (drugs) have been conjugated with targeting molecules (e.g., antibodies or ligands of protein receptors) that can bind highly specifically to tumor cells to form compounds called antibody-drug conjugates (ADCs) or immunoconjugates (Chau et al., Lancet 2019, 394, 793-804). ADCs or immunoconjugates should theoretically have lower toxicity because they guide cytotoxic drugs to diseased cells that overexpress specific cell surface antigens or receptors, compared to normal cells. Promising advances in ADCs or immunoconjugates have been seen in the use of cytotoxic drugs linked to antibodies via linkers that cleave at the tumor site or within tumor cells. Such advances can be applied to any disease requiring targeted delivery (Tumey, Innovations for Next-Generation Antibody-Drug Conjugates. Humana Press 2018, 187-214). Recent examples have also demonstrated their value in immune-mediated inflammatory diseases (Wang et al., J Am Chem Soc. 2015, 137, 3229-32), muscle-related diseases (Sugo et al., J. Control. Release 2016, 237, 1-13), and diseases caused by bacterial infections (Mariathasan et al., Trends Mol. Med. 2017, 23, 135-149). Furthermore, ADCs are not limited to cytotoxic drugs and can be used to specifically deliver imaging agents (Dammes et al., Theranostics 2020, 10, 938-955) or oligonucleotides (Dovgan et al., Bioconjug. Chem. 2019, 30, 2483-2501) to antigen-positive cells.

[0004] Linkers can influence the physicochemical properties of ADCs. Since many cytotoxic agents are inherently hydrophobic, linking them to antibodies with additional hydrophobic moieties can lead to aggregation. ADC aggregates are insoluble and often limit the achievable drug loading onto antibodies, which can adversely affect the potency of the ADC. Typically, protein aggregates in biologics are also associated with increased immunogenicity.

[0005] The chemical solution for targeted delivery of drugs conjugated to cell-specific ligands (e.g., cytotoxic or cell-inhibiting drugs) is the “self-degradable linker” PABC or PAB (p-aminobenzyloxycarbonyl or p-aminobenzyl), which links the drug portion to the ligand in the conjugate (Alouane et al. Angew. Chem. Int. Ed. 2015, 54, 7492-7509; Bargh et al. Chem. Soc. Rev. 2019, 48, 4361-4374). The PAB linker unit is also called an electron cascade spacer. The amide bond connecting the carboxyl terminus of the peptide unit and the p-aminobenzyl group of the PABC or PAB can be a substrate and can be cleaved by certain proteases. Aromatic amines become the electron donor and initiate an electron cascade effect that leads to the expulsion of the leaving group, releasing the free drug upon the elimination of carbon dioxide (de Groot et al. J. Org. Chem. 2001, 66, 8815-8830). Once the peptide bond adjacent to PABC or PAB is cleaved (i.e., cleaved by an intracellular enzyme), the drug is released from the ligand and thus does not bind to the remaining portion of the linker (de Groot et al., Molecular Cancer Therapeutics, 2002, 1, 901-911; de Groot et al., J. Med. Chem., 1999, 42, 5277-5283).

[0006] Linkers comprising p-aminobenzyl or p-aminobenzyloxycarbonyl (PAB or PABC) units bound to peptide units have been developed, exhibiting a “self-degradable” or “self-degradable” mechanism of 1,6-elimination and fragmentation under enzymatic, hydrolytic, or other metabolic conditions, thereby releasing the drug moiety from a target ligand such as an antibody (EP 1 718667; WO 2016 / 040684). For information on the use of PAB units in prodrugs and conjugates, see also: Alouane et al. Angew. Chem. Int. Ed. 2015, 54, 7492-7509; Bargh et al. Chem. Soc. Rev. 2019, 48, 4361-4374; Dal Corso et al. Chem. Eur. J. 2019, 25, 14740-14757.

[0007] The limiting factor for PAB-type self-degrading linkers is the resulting poor solubility and tendency to aggregate in conjugates. For example, in WO 2017 / 214282, PAB-type self-degrading linkers containing several hydrophilic groups (such as glucuronic acid, polyhydroxylated side chains, etc.) lead to high aggregation (>15%) in conjugates. Additionally, some PAB-containing conjugates may not be suitable substrates for certain lysinases or may cleave too slowly to achieve efficacy. Therefore, it is desirable to improve the properties of antibody-drug conjugates by optimizing the structure of self-degrading linkers to reduce aggregation while maintaining pharmacological activity and stability. Surprisingly, the novel p-amino-benzyl linkers disclosed herein, comprising p-amino-benzyl units and peptide units, overcome the aforementioned drawbacks, and their use results in ADCs with low aggregation levels, good stability, desired drug loading levels, and expected pharmacological activity, as demonstrated in the following examples. Brief description of the attached diagram

[0009] Figure 1 This demonstrates the use of site-specific antibody conjugation with bacterial transglutaminase (LAR2).

[0010] Figure 2 The image shows the conjugation of four interchain disulfide bonds using the antibody.

[0011] Figure 3 This demonstrates the use of bridging techniques that produce fragmentation.

[0012] definition

[0013] The term "antibody" or "Ab" is used in its broadest sense to refer to an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., a protein, polypeptide, carbohydrate, polynucleotide, lipid, or a combination thereof) through at least one antigen recognition site within the variable region of an immunoglobulin molecule. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. An "intact" antibody is typically a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and scattered with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Antibodies can be monoclonal antibodies, human antibodies, humanized antibodies, camelified antibodies, or chimeric antibodies. Antibodies can be any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgG1, and IgA2), or subclass. Antibodies can be intact antibodies or their antigen-binding fragments.

[0014] As used herein, the terms "antibody fragment," "antigen-binding fragment," or "fragment" refer to at least a portion of an antibody that retains the ability to specifically interact with an antigen via an epitope (e.g., through binding, steric hindrance, stabilization / destabilization, spatial distribution). Antigen-binding fragments may also retain the ability to be internalized into cells expressing the antigen. In some embodiments, antigen-binding fragments also retain immune effector activity. The terms antibody, antibody fragment, antigen-binding fragment, etc., are intended to encompass the use of binding domains from antibodies in the context of larger macromolecules such as ADCs. Fragments of full-length antibodies have been shown to perform the antigen-binding function of full-length antibodies. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAb (VL or VH), Camelidae VHH domains, multispecific antibodies formed from antibody fragments, such as bivalent fragments containing two Fab fragments linked by disulfide bonds in the hinge region, and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, giant antibodies, small bodies, nanobodies, intracellular antibodies, dimers, triantibodies, tetraantibodies, bispecific or multispecific antibody constructs, ADCs, v-NARs, and bis-scFv (see, for example, Holliger and Hudson, Nat. Biotechnol. 2005, 23, 1126-1136). Antigen-binding fragments can also be transplanted into a backbone based on peptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes the fibronectin polypeptide body).

[0015] The term "scFv" refers to a fusion protein comprising at least one antigen-binding fragment containing a light chain variable region and at least one antigen-binding fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are sequentially linked, for example, via a synthetic linker (e.g., a short flexible peptide linker), and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it originates. Unless otherwise stated, the scFv may have VL and VH variable regions in any order; for example, the scFv may contain VL-linker-VH or VH-linker-VL relative to the N-terminus and C-terminus of the polypeptide. Antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the utility (e.g., binding affinity, uptake) of the binding fragments is screened in the same manner as for intact antibodies. For example, antigen-binding fragments can be prepared by cleaving intact proteins, for example, by protease or chemical cleavage.

[0016] As used herein, the terms "drug," "pharmaceutical," "therapeutic agent," or "therapeutic agent" refer to a chemical compound, mixture of chemical compounds, biological macromolecule, extract made from biological material, or a combination of two or more thereof that can regulate biological processes and / or have biological activity. In particular, the term "drug" can mean "chemotherapy agent" or "anticancer agent."

[0017] The terms "chemotherapeutic agent" or "anticancer agent" are used herein to refer to all agents that are effective in treating cancer (regardless of their mechanism of action). Inhibition of metastasis or angiogenesis is often a characteristic of chemotherapeutic agents. Chemotherapy agents include antibodies, biomolecules, and small molecules. Chemotherapy agents can be cytotoxic agents or cell inhibitors. Examples of chemotherapeutic agents include, but are not limited to, monomethylaurestatin E, aurestatin E, SN-38, doxorubicin, etc.

[0018] The term "cell growth inhibitor" refers to drugs that inhibit or suppress cell growth and / or cell proliferation.

[0019] The term "cytotoxic agent" refers to a substance that causes cell death primarily by interfering with the expression activity and / or function of cells.

[0020] As used herein, the term "halogenated (C1-C4)alkyl" refers to a (C1-C4)alkyl group substituted with one or more halogen atoms such as fluorine, chlorine, bromine, or iodine, including but not limited to fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-1,1-dimethylethyl, 2,2,2-trichloroethyl, 3-fluoropropyl, 4-fluorobutyl, chloromethyl, trichloromethyl, iodomethyl, and bromomethyl.

[0021] The term "protecting group" refers to a substituent that is typically used to block or protect a specific functional group while allowing other functional groups on a compound to react. For example, an "amino protecting group" is a substituent attached to an amino group that blocks or protects the amino functional group in a compound. Suitable amino protecting groups include acetyl, trifluoroacetyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and 9-fluorenylmethoxycarbonyl (Fmoc). Similarly, a "hydroxyl protecting group" is a substituent that blocks or protects the hydroxyl group of a hydroxyl functional group. Suitable hydroxyl protecting groups include acetyl, benzyl, benzoyl, tetrahydropyranyl, and trialkylsilyl. Furthermore, a "carbonyl protecting group" is a substituent that blocks or protects the carbonyl group of a carbonyl functional group. Suitable carbonyl protecting groups include acetals, acyl acetals, and dithianes. Similarly, a "carboxyl protecting group" is a substituent that blocks or protects the carboxyl group of a carboxyl functional group. Suitable carboxyl protecting groups include methyl esters, benzyl esters, tert-butyl esters, silyl esters, and orthoesters. For a general description of protecting groups and their uses, see T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

[0022] As used herein, the term "part" refers to a specific segment, fragment, or functional group of a molecule or compound. A chemical part is sometimes referred to as a chemical entity that is inserted into or attached (i.e., a substituent or variable group) to a molecule, compound, or chemical formula.

[0023] As used herein, the term "bridging spacer" refers to one or more linker components covalently linked together to form a divalent moiety linking divalent peptides A1-A2 to a reactive group. In some embodiments, the "bridging spacer" comprises a carbonyl group linked to the N-terminus of the A2 group via an amide bond. In some embodiments, the "bridging spacer" comprises an amino group linked to the carbonyl group of the A2 group via an amide bond. In some embodiments, the bridging spacer comprises a polyoxyethylene (PEG) group. In other embodiments, the bridging spacer comprises butyryl, valeryl, hexanoyl, heptanyl, or octanoyl. In some embodiments, the bridging spacer comprises a hexanoyl group (i.e., a -CO-(CH2)5- group). In a preferred embodiment, the bridging spacer may comprise: -CO-CH2-CH2-PEG1-, -CO-CH2-PEG3-, or -NH-CH2-CH2-PEG1-.

[0024] As used herein, the terms "polyoxyethylene," "polyethylene glycol," or "PEG" refer to a straight-chain, branched, or star-shaped configuration consisting of (OCH2CH2) groups. In some embodiments, the polyoxyethylene or PEG groups are *-(OCH2CH2). t**, where t is 1-20, and where "*" indicates the end of the A2-A1 group, and "**" indicates the connection point with the reactive group. In some embodiments, the bridging spacer group comprises a PEG group, which is *-(OCH2CH2). t -**, where t is between 1 and 20, and where "*" indicates -(CH2). n The -C(O)- group is the connection point, where the carbonyl group is connected to the N-terminus of the A2 group and n is 1-5, and "**" indicates the connection point with a reactive group. In some embodiments, the bridging spacer group comprises a PEG group, which is *-(OCH2CH2). t -**, where t is between 1 and 20, and where "*" indicates -(CH2). n The -NH- group is the connection point where the amino group is attached to the carbonyl group of the A2 group, and n is 1-5, and "**" indicates the connection point with a reactive group. For example, as used herein, the term "PEG3" refers to a t of 3.

[0025] As used herein, the term "reactive group" is a functional group capable of forming a covalent bond with a functional group of an antibody or antibody fragment. In some embodiments, the reactive group is selected from, but is not limited to, thiols, maleimides, dibromomaleimides, haloacetamides, azides, alkynes, cyclooctene, triarylphosphine, oxaborheadiene, cyclooctyne, diaryltetraazine, monoaryltetraazine, norbornene, aldehydes, hydroxylamine, hydrazine, NH2-NH-C(=O)-, ketones, vinyl sulfones, aziridine, amino groups, amino acid residues, etc. In preferred embodiments, the reactive group is selected from maleimide groups, dibromomaleimide groups, thiols, cyclooctyne groups, or azides, more preferably maleimide groups, dibromomaleimide groups, or azides. For example, maleimide or dibromomaleimide groups may each have the following structures:

[0026]

[0027] For example, an azide group can have the following structure: -N=N + =N - .

[0028] As used herein, the term "linking group" refers to the attachment of a bridging spacer group to a divalent or trivalent portion of an antibody or a fragment thereof. A linking group is a divalent or trivalent portion formed by a reaction between a reactive group and a functional group on the antibody or a fragment thereof. In some embodiments, the reactive group used to form the linking group is selected from, but is not limited to, thiols, maleimides, dibromomaleimides, haloacetamides, azides, alkynes, cyclooctene, triarylphosphine, oxaborheadiene, cyclooctyne, diaryltetraazine, monoaryltetraazine, norbornene, aldehydes, hydroxylamine, hydrazine, NH₂-NH-C(=O)-, ketones, vinyl sulfones, aziridine, amino groups, amino acid residues, etc. In a preferred embodiment, the reactive group used to form the linking group is selected from maleimide groups, dibromomaleimide groups, thiols, cyclooctyne groups, or azides, more preferably maleimide groups, dibromomaleimide groups, or azides.

[0029] As used herein, the terms “spacer precursor” or “spacer precursor Z’” or “Z’” refer to a component that contains a bridging spacer group and a reactive group.

[0030] As used herein, the term "spacer unit" or "spacer unit Z" or "Z" refers to a component containing a bridging spacer group and a linking group.

[0031] As used herein, the terms "conjugate," "antibody-drug conjugate," "ADC," or "immunoconjugate" refer to an antibody covalently linked to a drug moiety via a p-amino-benzyl linker of formula (I). In one embodiment, an antibody-drug conjugate of formula (III), wherein the spacer unit Z is coupled to an antibody Ab via an amide group, can be prepared by reacting a free amine functional group on the antibody Ab with an active ester containing a precursor of spacer unit Z. For example, the carboxyl group on the spacer unit precursor Z' can be activated by reacting with N-hydroxysuccinimide and then reacting with Ab-NH2 to form a conjugate wherein Ab and Z are coupled via an amide group. Useful functional groups on antibodies naturally or chemically linked to spacer unit Z include, but are not limited to, thiol (-SH), amino, hydroxyl, anomeric hydroxyl, and carboxyl groups of carbohydrates. In one embodiment, the reactive functional groups on the antibody are thiol and amino groups. Thiol groups can be generated by reducing the intramolecular cysteine ​​disulfide bonds of the antibody. Alternatively, a thiol group can be generated by reacting the amino group of the lysine moiety of the antibody with 2-iminotetrahydrothiophene (Traut's reagent) or another thiol-generating reagent. In another embodiment, spacer unit Z is linked to antibody Ab via a disulfide bond between the sulfur atom of the antibody and the sulfur atom of spacer unit Z. In another embodiment, the reactive group of spacer unit Z contains a reactive site that can form a bond with the primary or secondary amino group of the antibody. Examples of such reactive sites include, but are not limited to, activated esters such as succinimidyl ester, 4-nitrobenzene ester, pentafluorophenyl ester, tetrafluorophenyl ester, acid anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. In another embodiment, the reactive group of spacer unit Z reacts with an aldehyde, acetal, or ketal group on the sugar (carbohydrate) of the glycosylated antibody. For example, carbohydrates can be gently oxidized using reagents such as sodium periodate, and the resulting (-CHO) unit of the oxidized carbohydrate can be condensed with a spacer unit containing the following functional groups: acylhydrazide, oxime, primary or secondary amine, hydrazine, thiourea, hydrazide carboxylates, and aryl acylhydrazide, as described by Kaneko et al. in Bioconjugate Chem. 1991, 2, 133-141.

[0032] The generation of an ADC can be accomplished using techniques known to those skilled in the art. In one exemplary embodiment, the present invention provides an ADC of formula (III) prepared by contacting a linker-drug compound of formula (II) with an antibody (or a fragment thereof) having a reactive thiol, amino, or carboxyl moiety under suitable conditions to achieve condensation of the reactive moiety with the spacer unit precursor Z' portion of the linker-drug compound of formula (II), wherein Z' is converted into Z generated by said contact.

[0033] In some embodiments, the ADC of formula (III) is incorporated with spacer unit Z, which is a divalent portion of the A2-A1 group coupled to the antibody. In some embodiments, the ADC of formula (III) is incorporated with spacer unit Z, which is a carbonyl group of the A2-A1 group coupled to the divalent portion of the antibody.

[0034] The spacer unit Z is covalently bonded to a functional group derived from the antibody, such as an amine (e.g., -NH2 from a Lys residue), a carboxyl group (-COOH from an Asp or Glu residue), or a thiol group (e.g., -SH from a Cys residue), forming an amide, thioether, or disulfide group. The antibody-drug conjugate of the present invention, wherein the spacer unit precursor Z' reacts with the thiol functional group of the antibody (e.g., a peptide containing Cys or a reduced antibody) to form a thioether bond, comprises a structure represented by the following formula:

[0035]

[0036] Wherein Ab is an antibody or its antigen-binding fragment. In other embodiments, the antibody-drug conjugate of the present invention, wherein the spacer unit precursor Z' reacts with two thiol functional groups of the antibody to form two thioether bonds, comprises a structure represented by the following formula:

[0037]

[0038] Ab represents an antibody or its antigen-binding fragment.

[0039] In some embodiments, the antibody or antigen-binding fragment is functionalized to prepare a functional group for reaction with a linker-drug intermediate. In some embodiments, the antibody or antigen-binding fragment is prepared using bacterial transglutaminase (BTG), which is a reactive glutamine specifically functionalized with an amine comprising the cyclooctyne BCN (N-[(1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl]-1,8-diamino-3,6-dioxaoctane) moiety. In some embodiments, the ADC of the present invention comprises a compound represented by the following formula, wherein the spacer unit precursor Z' reacts with the glutamine functional group of the antibody to form an amide bond:

[0040]

[0041] Ab represents an antibody or its antigen-binding fragment.

[0042] The term "drug load" is denoted by p and is also referred to herein as the drug-to-antibody ratio (DAR or drug:antibody ratio). Drug load can range from 1 to 8 drug fractions per antibody or antigen-binding fragment. In some embodiments, higher drug loads (e.g., p > 8) can cause aggregation, insolubility, toxicity, or loss of cell permeability in certain antibody-drug conjugates. Higher drug loads may also adversely affect the pharmacokinetics (e.g., clearance) of certain ADCs. In some embodiments, lower drug loads (e.g., p < 2) can reduce the potency of certain ADCs against cells expressing a target. In some embodiments, p is an integer from 2 to 8. In some embodiments, p is 2, or p is 4, or p is 8.

[0043] The drug loading of an ADC can be controlled in different ways, such as, but not limited to, limiting the molar excess of the drug-adaptor intermediate or adaptor reagent relative to the antibody, or limiting the conjugation reaction time or temperature.

[0044] In some embodiments, the drug loading in the ADC mixture produced by the conjugation reaction ranges from 1 to 8 drug moieties linked to each antibody or antigen-binding fragment. The average number of drug moieties per antibody or antigen-binding fragment (i.e., average drug loading or average p) can be calculated using any conventional method known in the art, such as mass spectrometry (e.g., liquid chromatography-mass spectrometry (LC-MS)) and / or high-performance liquid chromatography (e.g., HIC-HPLC). In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 1.5 to about 2.5, or about 7.0 to about 8. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 2. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is 2. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 4. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is 4. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 8. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is 8. Invention Details

[0046] In a first aspect, the present invention provides a p-amino-benzyl linker compound of formula (I):

[0047]

[0048] in:

[0049] • R1 represents a hydroxyl group or a halogen atom;

[0050] ·R2 represents the -S(O)2(OH) group, -S(O)2(O - M + ) group, straight-chain or branched -(C1-C4)alkyl-S(O)2(OH) group, straight-chain or branched -(C1-C4)alkyl-S(O)2(O - M + ) group, straight-chain or branched -halo(C1-C4)alkyl-S(O)2(OH) group or straight-chain or branched -halo(C1-C4)alkyl-S(O)2(O - M + ) group;

[0051] • A1 represents the -C(O)-CH(R3)-NH- group;

[0052] A2 represents the -C(O)-CH(R4)-NH- group. Groups, Group or Group;

[0053] • R3 and R4 represent amino acid side chains independently of each other;

[0054] • X represents a hydrogen atom, a hydroxyl group, or a protecting group;

[0055] ·M + This indicates a pharmaceutically acceptable monovalent cation.

[0056] In a preferred embodiment, R1 represents a hydroxyl group, a bromine atom, a chlorine atom, or an iodine atom. More preferably, R1 represents a hydroxyl group, a chlorine atom, or an iodine atom. Even more preferably, R1 represents a hydroxyl group.

[0057] Preferably, R2 represents a -S(O)2(OH) group or a -S(O)2(O - M + ) group, -CH2-S(O)2(OH) group, -CH2-S(O)2(O - M + ) group, -CH2-CH2-S(O)2(OH) group, -CH2-CH2-S(O)2(O - M + -CH2-CH2-CH2-S(O)2(OH) group, -CH2-CH2-CH2-S(O)2(O) group - M + ) group. M + Examples include Li + Na + or K + In a preferred embodiment, M + Na represents+ .

[0058] In one specific embodiment, A1 represents a -C(O)-CH(R3)-NH- group, and A2 represents a -C(O)-CH(R4)-NH- group, wherein R3 and R4 independently represent amino acid side chains. The carbon with the side chain can be in D or L (R or S) configuration. Preferably, the carbon with the side chain is in L configuration. In one specific embodiment, R3 and R4 independently represent, but are not limited to, hydrogen, methyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, phenyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH2SH, -CH2SeH-CH2CH2OH, -CH2CH2SH, -CH(OH)CH3, -CH2SCH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(C H2)3NHCOCH3,-(CH2)3NHCHO,-(CH2)4NHC(=NH)NH2,-(CH2)4NH2,-(CH2)4NHCO-(3-methyl-3,4-dihydro-2H-pyrrolo-2-yl),-(CH2)4NHCO-(3,4-dihydro-2H-pyrrolo-2-yl),-(CH2)4NHCOCH3,-(CH2)4NHCHO,-(CH2)3NHCONH2,-(CH2)4NHCONH2,-CH2CH2CH(OH)CH2NH2,4-imidazolylmethyl,3-indolemethyl,…

[0059] In a preferred embodiment, the A1-A2 groups form a dipeptide comprising two amino acid units, wherein the amino acid units are selected from natural and non-natural amino acids, preferably natural amino acids. The amino acid units may be alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), valine (Nva), leucine (Nle), selenocysteine ​​(Sec), pyrrolidone (Pyl), homoserine, homocysteine, and demethylpyrrolidone.

[0060] In one implementation, when A1 represents a proline (Pro) amino acid unit, A1 is defined by the following formula:

[0061]

[0062] The carbonyl group is attached to the amino group of the p-aminobenzyl moiety, and the wavy line indicates the covalent attachment site with the carbonyl group of the A2 group.

[0063] In one implementation, when A2 represents a proline (Pro) amino acid unit, A2 is defined by the following formula:

[0064]

[0065] The carbonyl group is connected to the amino group of the A1 group, and the wavy line indicates the covalent connection site with the X group, Z' group or Z group as defined in this invention.

[0066] In a specific implementation, the A1-A2 groups can represent: Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Ala; Ala-Phe; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-Ala; Phe-Cit; Cit-Phe; Leu-Cit; Cit-Leu; Ile-Cit; Cit-Ile; Phe-Arg; Arg-Phe; Cit-Trp; Trp-Cit. In a preferred embodiment, the A1-A2 groups represent Cit-Val or Ala-Val.

[0067] Preferably, A1 represents a -C(O)-CH(R3)-NH- group, wherein R3 represents a -(CH2)3-NH-CO-NH2 group or a methyl group.

[0068] Advantageously, A2 represents: -C(O)-CH(R4)-NH- group, where R4 represents isopropyl; Groups, Group or Group.

[0069] In a preferred embodiment, A1 represents a -C(O)-CH(R3)-NH- group, wherein R3 represents a -(CH2)3-NH-CO-NH2 group or a methyl group, and A2 represents a -C(O)-CH(R4)-NH- group, wherein R4 represents an isopropyl group.

[0070] In another preferred embodiment, A1 represents a -C(O)-CH(R3)-NH- group, where R3 represents a -(CH2)3-NH-CO-NH2 group, and A2 represents: Group or Group.

[0071] In one embodiment, X represents a hydrogen atom or a protecting group. Preferably, X represents a hydrogen atom, or a protecting group with an amino, carbonyl, or carboxyl functional group. More preferably, X represents a hydrogen atom or a protecting group selected from Fmoc, Boc, or CBz. Even more preferably, X represents an Fmoc protecting group.

[0072] The preferred p-amino-benzyl linker compound of formula (I) is:

[0073] -5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-(hydroxymethyl)benzenesulfonate sodium;

[0074] -5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]-2-(hydroxymethyl)benzenesulfonate sodium;

[0075] -5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]-2-(hydroxymethyl)benzenesulfonic acid;

[0076] -[5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-(hydroxymethyl)phenyl]methanesulfonate;

[0077] -2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]benzenesulfonic acid;

[0078] -2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]benzenesulfonic acid;

[0079] -5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-(iodomethyl)benzenesulfonic acid;

[0080] -N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-valine-N 5 -Carbamoyl-N-[4-(hydroxymethyl)-3-(2-sulfonate ethyl)phenyl]-L-ornithine sodium;

[0081] -N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-valine-N 5 -Carbamoyl-N-[4-(hydroxymethyl)-3-(3-sulfonylpropyl)phenyl]-L-ornithine.

[0082] The present invention also relates to a method for preparing p-amino-benzyl linker compounds of formula (I), characterized in that a compound of formula (IV) is used as a starting material:

[0083]

[0084] R1 and R2 are defined as in equation (I).

[0085] It undergoes a reduction reaction to produce a compound of formula (V):

[0086]

[0087] R1 and R2 are defined above.

[0088] The compound of formula (V) is further coupled with the compound of formula (VI):

[0089] HO-A1-P(VI)

[0090] Where P is an amino-protecting group, and A1 is as defined in formula (I).

[0091] The compound of formula (VII) was obtained.

[0092]

[0093] Where A1, P, R1, and R2 are defined as above.

[0094] The compound of formula (VII) is further coupled with the compound of formula (VIII):

[0095] HO-A2-X(VIII)

[0096] Where X and A2 are defined as in equation (I),

[0097] The p-amino-benzyl linker compound of formula (I) was obtained.

[0098] An alternative method for preparing p-amino-benzyl linker compounds of formula (I) is characterized by using a compound of formula (V) obtained from a compound of formula (IV) as a starting material, and coupling it with a compound of formula (IX):

[0099] HO-A1-A2-X(IX)

[0100] Where A1, A2, and X are as defined in equation (I),

[0101] The p-amino-benzyl linker compound of formula (I) was obtained.

[0102] In one embodiment, the p-amino-benzyl linker compound of formula (I) obtained at the end of the above method can be further purified according to conventional separation techniques, and if necessary, converted to its addition salt with a pharmaceutically acceptable acid or base, and isolated as its isomer according to conventional separation techniques. It should be understood that, as needed for the synthesis, at any suitable point in the process of the above method, some groups (hydroxyl, amino, etc.) of the starting reagent or synthetic intermediate can be protected, followed by deprotection and functionalization. The compounds of formulas (IV), (VI), (VIII), and (IX) are commercially available or can be obtained by those skilled in the art using conventional chemical reactions described in the literature.

[0103] This invention also relates to the use of p-amino-benzyl linker compounds of formula (I) in the preparation of antibody-drug conjugates. More specifically, this invention relates to the use of p-amino-benzyl linker compounds of formula (I) in the preparation of antibody-drug conjugates, said compounds being selected from:

[0104] Sodium 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-(hydroxymethyl)benzenesulfonate,

[0105] -5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]-2-(hydroxymethyl)benzenesulfonate sodium salt,

[0106] -5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]-2-(hydroxymethyl)benzenesulfonic acid,

[0107] Sodium 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-(hydroxymethyl)phenyl]methanesulfonate,

[0108] -2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]benzenesulfonic acid, or

[0109] -2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]benzenesulfonic acid,

[0110] -5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-(iodomethyl)benzenesulfonic acid,

[0111] -N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-valine-N 5 -Carbamoyl-N-[4-(hydroxymethyl)-3-(2-sulfonate ethyl)phenyl]-L-ornithine sodium, or

[0112] -N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-valine-N 5 -Carbamoyl-N-[4-(hydroxymethyl)-3-(3-sulfonylpropyl)phenyl]-L-ornithine.

[0113] In a second aspect, the present invention provides a connector-drug compound of formula (II):

[0114]

[0115] in:

[0116] •D indicates the drug component;

[0117] • T represents a valence bond, -OC(O)-N(CH3)-CH2-CH2-N(CH3)-C(O)-*, -O-*, -NR5-*,

[0118] -NR5-C(O)-* or -OC(O)-*, where * indicates the connection point with D;

[0119] ·R2 represents the -S(O)2(OH) group, -S(O)2(O - M + ) group, straight-chain or branched -(C1-C4)alkyl-S(O)2(OH) group, straight-chain or branched -(C1-C4)alkyl-S(O)2(O - M + ) group, straight-chain or branched -halo(C1-C4)alkyl-S(O)2(OH) group or straight-chain or branched -halo(C1-C4)alkyl-S(O)2(O - M + ) group;

[0120] • A1 represents the -C(O)-CH(R3)-NH- group;

[0121] A2 represents: -C(O)-CH(R4)-NH- group, Groups, Group or Group.

[0122] • R3 and R4 represent amino acid side chains independently of each other;

[0123] • R5 represents a hydrogen atom or a (C1-C4) alkyl group;

[0124] • Z' represents the precursor of the spacer unit;

[0125] ·M + This indicates a pharmaceutically acceptable monovalent cation.

[0126] In one embodiment of the second aspect of the invention, D is a pharmaceutical portion comprising a nitrogen atom or an oxygen atom, wherein D is directly linked to T via the nitrogen atom or oxygen atom of the pharmaceutical portion. In some embodiments, D is a pharmaceutical portion comprising a quaternized tertiary amine. In a specific embodiment, D is monomethylauratestatin E (IUPAC name: (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohep-4-yl) -N,3-Dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyramide or MMAE), Auristatin E (IUPAC name: (2S)-2-[[(2S)-2-(dimethylamino)-3-methyl-butyryl]amino]-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy] [1S]-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-N,3-dimethyl-butyramide), SN-38 (IUPAC name: (4S)-4,11-diethyl-4,9-dihydroxy-1H-pyrano[3',4':6, 7] Indazano[1,2-b]quinoline-3,14(4H,12H)-dione) or doxorubicin (IUPAC name: (1S,3S)-3,5,12-trihydroxy-3-(hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydro-terephthalen-1-yl-3-amino-2,3,6-trideoxy-α-L-lythreono-hexopyranoside).

[0127] Preferably, T is a valence bond or -OC(O)-*, where * represents a connection point with D. In some embodiments, when T is a valence bond, D is a pharmaceutical moiety comprising a quaternized tertiary amine.

[0128] Preferably, in the second aspect of the invention, R2 represents a -S(O)2(OH) group or a -S(O)2(O - M + ) group, -CH2-S(O)2(OH) group, -CH2-S(O)2(O - M + ) group, -CH2-CH2-S(O)2(OH) group, -CH2-CH2-S(O)2(O - M +-CH2-CH2-CH2-S(O)2(OH) group, -CH2-CH2-CH2-S(O)2(O) group - M + ) group. In a preferred embodiment of the second aspect of the invention, M + Na represents + .

[0129] Advantageously, in a second aspect of the invention, A1 represents a -C(O)-CH(R3)-NH- group, wherein R3 represents a -(CH2)3-NH-CO-NH2 group or a methyl group.

[0130] In a preferred embodiment of the second aspect of the invention, A2 represents: -C(O)-CH(R4)-NH- group, wherein R4 represents isopropyl; Groups, Group or Group.

[0131] In another preferred embodiment of the second aspect of the invention, A1 represents a -C(O)-CH(R3)-NH- group, wherein R3 represents a -(CH2)3-NH-CO-NH2 group, and A2 represents: Group or Group.

[0132] In another preferred embodiment of the second aspect of the invention, A1 represents a -C(O)-CH(R3)-NH- group, and A2 represents a -C(O)-CH(R4)-NH- group, wherein R3 and R4 independently represent amino acid side chains. Specifically, when R3 and R4 represent amino acid side chains, the carbon atom with the side chain has an L configuration. In a preferred embodiment, A1 represents a -C(O)-CH(R3)-NH- group, wherein R3 represents a -(CH2)3-NH-CO-NH2 group or a methyl group, and A2 represents a -C(O)-CH(R4)-NH- group, wherein R4 represents an isopropyl group. Preferably, in the second aspect of the invention, the A1-A2 groups represent Cit-Val or Ala-Val.

[0133] Preferably, the spacer unit precursor Z' is selected from:

[0134]

[0135] The wavy line represents the covalent connection point with the N-terminus or carbonyl group of the A2 group.

[0136] The preferred linker-drug compound of formula (II) is selected from:

[0137]

[0138]

[0139] R2 and D are defined as above.

[0140] A more preferred linker-drug compound of formula (II) is selected from:

[0141]

[0142]

[0143] R2 and D are defined as above.

[0144] In a preferred embodiment, the linker-drug compound of formula (II) is selected from:

[0145]

[0146]

[0147]

[0148]

[0149] Where D is as defined above.

[0150] In a specific implementation, the preferred linker-drug compound of formula (II) is:

[0151] -5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolo-1-yl)ethoxy]propionylamino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R, Sodium 2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidone-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonate;

[0152] -5-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrolo-1-yl)hexanoylamino]-3-methyl-butyryl]amino]-5-ureo-valeryl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonate sodium;

[0153] -5-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1 [R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidone-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonate sodium;

[0154] -5-[[(2S)-2-[[1-[2-[2-(2,5-dioxopyrrolo-1-yl)ethoxy]ethylcarbamoyl]cyclobutanecarbonyl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid;

[0155] -5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolo-1-yl)ethoxy]propionylamino]-3-methyl-butyryl]amino]propionyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonate sodium;

[0156] -5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolo-1-yl)ethoxy]propionylamino]-3-methyl-butyryl]amino]propionyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolid-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid;

[0157] -5-[[(2S)-2-[[3-[2-[2-(2,5-dioxopyrrolo-1-yl)ethoxy]ethylcarbamoyl]oxetane-3-carbonyl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R, 2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidone-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid;

[0158] -[5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolo-1-yl)ethoxy]propionylamino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R, 2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidone-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]phenyl]methanesulfonic acid;

[0159] -[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolo-1-yl)ethoxy]propionylamino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-sulfo-phenyl]methyl-[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[ (1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolid-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-dimethyl-ammonium; 2,2,2-trifluoroacetate;

[0160] -N-({[4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl]-L-valinel-L-alanyl}amino)-2-sulfophenyl]methoxy}carbonyl)-N-methyl-L-valinel-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylprop-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohep-4-yl]-N-methyl-L-valineamide;

[0161] -N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl]-L-valine-N 5-Carbamoyl-N-(4-{[({[(4S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzino[1,2-b]quinoline-4-yl]oxy}carbonyl)oxy]methyl}-3-sulfophenyl)-L-ornithine;

[0162] -(1S,3S)-3,5,12-trihydroxy-3-(hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydro-tetraphenyl-1-yl2,3,6-trideoxy-3-[({[4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl]-L-valine-N 5 [-carbamoyl-L-guanosyl}amino]-2-sulfophenyl]methoxy}carbonyl]amino]-α-L-lythreo-hexanoside

[0163] -N-{3-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy]propionyl}-L-valine-N-{4-[(5S,8S,11S,12R)-11-[(2S)-but-2-yl]-12-(2-{(2S)-2-[(1R,2R)-3-{(1S,2R)-1-hydroxy-1-phenylprop-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolo-1-yl}-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-5,8-di(prop-2-yl)-2,13-dioxa-4,7,10-triazatetradecane-1-yl]-3-(2-sulfoethyl)phenyl}-N 5 -Carbamoyl-L-ornithine;

[0164] -5-[[(2S)-2-[[(2S)-2-[6-(3,4-dibromo-2,5-dioxo-pyrrolo-1-yl)hexanoylamino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R ,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid.

[0165] The present invention also relates to a method for preparing a linker-drug compound of formula (II), the method being characterized by using a compound of formula (I) as a starting material:

[0166]

[0167] R1, R2, A1, A2, and X are defined as above.

[0168] Then, by modification, we obtain the compound of formula (X):

[0169]

[0170] Where A1, A2, D, R2, T and X are as defined in equation (II),

[0171] After removing the protecting group X, the compound of formula (X) is further coupled with the compound of formula (XI):

[0172] HO-Z'(XI)

[0173] Where Z' is defined in equation (II),

[0174] The connector-drug compound of formula (II) is obtained.

[0175] An alternative method for preparing linker-drug compounds of formula (II) is characterized by using a compound of formula (IV) obtained in two steps, as a starting material, and coupling it with the DT group to obtain a compound of formula (XII):

[0176]

[0177] Where A1, D, P, R2, and T are as defined above,

[0178] Further coupling of the compound of formula (XII) with the compound of formula (XIII):

[0179] HO-A2-Z'(XIII)

[0180] Z and A2 are defined as in equation (I).

[0181] The connector-drug compound of formula (II) is obtained.

[0182] In one embodiment, the linker-pharmaceutical compound of formula (II) obtained at the end of the above method can be further purified according to conventional separation techniques, and if necessary, converted to their addition salts with pharmaceutically acceptable acids or bases, and can be isolated into their isomers according to conventional separation techniques. It should be understood that, as needed for the synthesis, at any point in the process of the above method deemed appropriate, some groups (hydroxyl, amino, etc.) of the starting reagent or synthetic intermediate can be protected, followed by deprotection and functionalization. The compounds of formulas (IV), (XI), and (XIII) are commercially available or can be obtained by those skilled in the art using conventional chemical reactions described in the literature.

[0183] The present invention also relates to the use of the linker-drug compound of formula (II) in the preparation of antibody-drug conjugates.

[0184] In a third aspect of the invention, an antibody-drug conjugate (ADC) of formula (III) is provided:

[0185]

[0186] in

[0187] •Ab represents an antibody or its antigen-binding fragment;

[0188] • D represents the drug component;

[0189] • T represents a valence bond, -OC(O)-N(CH3)-CH2-CH2-N(CH3)-C(O)-*, -O-*, -NR5-*,

[0190] -NR5-C(O)-* or -OC(O)-*, where * indicates the connection point with D;

[0191] • Z represents the spacing unit;

[0192] • A1 represents the -C(O)-CH(R3)-NH- group;

[0193] A2 represents: -C(O)-CH(R4)-NH- group, Groups, Group or Group;

[0194] ·R2 represents the -S(O)2(OH) group, -S(O)2(O - M + ) group, straight-chain or branched -(C1-C4)alkyl-S(O)2(OH) group, straight-chain or branched -(C1-C4)alkyl-S(O)2(O - M +) group, straight-chain or branched -halo(C1-C4)alkyl-S(O)2(OH) group or straight-chain or branched -halo(C1-C4)alkyl-S(O)2(O - M + ) group;

[0195] • R3 and R4 represent amino acid side chains independently of each other;

[0196] R5 indicates (C1-C4) alkyl;

[0197] ·M + This indicates a pharmaceutically acceptable monovalent cation; and

[0198] ·p is an integer from 1 to 8.

[0199] In one embodiment of the third aspect of the invention, D is a pharmaceutical portion comprising a nitrogen atom or an oxygen atom, wherein D is directly linked to T via the nitrogen atom or oxygen atom of the pharmaceutical portion. In some embodiments of the third aspect of the invention, D is a pharmaceutical portion comprising a quaternized tertiary amine. In a specific embodiment of the third aspect of the invention, D is monomethylaurestatin E (IUPAC name: (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1- (Oxyhept-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyramide or MMAE), Auristatin E (IUPAC name: (2S)-2-[[(2S)-2-(dimethylamino)-3-methyl-butyryl]amino]-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S) -2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidone-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-N,3-dimethyl-butyramide), SN-38 (IUPAC name: (4S)-4,11-diethyl-4,9-dihydroxy-1H-pyrano[3',4': [6,7]Indazano[1,2-b]quinoline-3,14(4H,12H)-dione) or doxorubicin (IUPAC name: (1S,3S)-3,5,12-trihydroxy-3-(hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydro-terephthalen-1-yl-3-amino-2,3,6-trideoxy-α-L-lythreo-pyranoside).

[0200] Preferably, in the third aspect of the invention, T is a valence bond or -OC(O)-*, where * represents a connection point with D. In some embodiments of the third aspect of the invention, when T is a valence bond, D is a pharmaceutical moiety comprising a quaternized tertiary amine.

[0201] Preferably, in a third aspect of the invention, R2 represents a -S(O)2(OH) group or a -S(O)2(O - M + ) group, -CH2-S(O)2(OH) group, -CH2-S(O)2(O - M + ) group, -CH2-CH2-S(O)2(OH) group, -CH2-CH2-S(O)2(O - M + -CH2-CH2-CH2-S(O)2(OH) group, -CH2-CH2-CH2-S(O)2(O) group - M + ) group. In a preferred embodiment of the third aspect of the invention, M + Na represents + .

[0202] Advantageously, in a third aspect of the invention, A1 represents a -C(O)-CH(R3)-NH- group, wherein R3 represents a -(CH2)3-NH-CO-NH2 group or a methyl group.

[0203] In a preferred embodiment of the third aspect of the invention, A2 represents: -C(O)-CH(R4)-NH- group, wherein R4 represents isopropyl; Group; Group; or Group.

[0204] In another preferred embodiment of the third aspect of the invention, A1 represents a -C(O)-CH(R3)-NH- group, wherein R3 represents a -(CH2)3-NH-CO-NH2 group, and A2 represents: Group or Group.

[0205] In another preferred embodiment of the third aspect of the invention, A1 represents a -C(O)-CH(R3)-NH- group, and A2 represents a -C(O)-CH(R4)-NH- group, wherein R3 and R4 independently represent amino acid side chains. Specifically, when R3 and R4 represent amino acid side chains, the carbon with the side chain has an L configuration. In a preferred embodiment, A1 represents a -C(O)-CH(R3)-NH- group, wherein R3 represents a -(CH2)3-NH-CO-NH2 group or a methyl group, and A2 represents a -C(O)-CH(R4)-NH- group, wherein R4 represents an isopropyl group. Preferably, in the third aspect of the invention, the A1-A2 groups represent Cit-Val or Ala-Val.

[0206] In a preferred embodiment, the ADC of formula (III) is formed from a linker-drug compound selected from formula (II) below:

[0207]

[0208]

[0209] R2 and D are defined as above.

[0210] More preferably, the ADC of formula (III) is formed from a linker-drug compound selected from formula (II):

[0211]

[0212]

[0213] R2 and D are defined as above.

[0214] In a preferred embodiment, the ADC of formula (III) is formed from a linker-drug compound selected from formula (II) below:

[0215]

[0216]

[0217]

[0218]

[0219] Where D is as defined above.

[0220] In one specific embodiment, the present invention provides an antibody-drug conjugate of formula (III), wherein...

[0221]

[0222] Includes formulas selected from the following:

[0223]

[0224]

[0225] The wavy line represents the covalent connection point with the antibody, and R2 and D are as defined above.

[0226] In a preferred embodiment, the present invention provides an antibody-drug conjugate of formula (III), wherein

[0227]

[0228] Includes formulas selected from the following:

[0229]

[0230]

[0231] The wavy line represents the covalent connection point with the antibody, and R2 and D are as defined above.

[0232] In a preferred embodiment, the present invention provides an antibody-drug conjugate of formula (III), wherein

[0233]

[0234] Includes formulas selected from the following:

[0235]

[0236]

[0237]

[0238]

[0239] The wavy line represents the covalent connection point with the antibody, and D is as defined above.

[0240] The ADCs of the present invention can be used to treat diseases in mammals (e.g., patients) in need. Therefore, ADCs can be used in a variety of settings to treat conditions associated with the expression of antigens bound to the antibodies of the ADC, such as cancer. ADCs can be used to deliver drugs to target cells. Not bound by theory, in one embodiment, the antibody of the ADC binds or associates with a cell surface antigen or receptor, and after binding, the ADC can be taken up (ingested) within the cell via antigen- or receptor-mediated endocytosis or other internalization mechanisms. The antigen may be attached to the cell or may be an extracellular matrix protein associated with the cell. Once inside the cell, the drug is released intracellularly via an enzymatic or non-enzymatic cleavage mechanism, depending on the components of the adaptor system. In another embodiment, the drug is cleaved from the ADC near the cell, and subsequently, the drug permeates into the cell.

[0241] This invention provides pharmaceutical compositions comprising the ADC composition described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition can be any form that allows the ADC to be administered to a patient to treat a condition associated with the expression of an antigen bound to an antibody of the ADC. In particular, this invention relates to pharmaceutical compositions comprising at least one antibody-drug conjugate of formula (III) combined with one or more pharmaceutically acceptable excipients. For example, the pharmaceutical composition can be in liquid or lyophilized solid form. More particularly, those suitable for oral, parenteral, nasal, transdermal or transskin, rectal, lingual, ocular, or inhalation administration may be mentioned in the pharmaceutical compositions of this invention. A preferred route of administration is parenteral administration. Parenteral administration includes subcutaneous injection, intravenous, intramuscular, and intrasternal injection or infusion techniques. In a preferred embodiment, the pharmaceutical composition comprising the ADC is administered intravenously as a liquid solution. Liquids can be used for delivery by injection. Compositions intended for injection may also include one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, diluents, lubricants, binders, disintegrants, absorbents, and isotonic agents.

[0242] In another aspect, the present invention relates to the use of any antibody-drug conjugate as defined above in the preparation of a pharmaceutical formulation for treating mammals in need. In another aspect, the present invention relates to any antibody-drug conjugate as defined above for treating mammals in need. The invention also relates to a method of treating mammals in need, wherein the method comprises administering a pharmaceutical composition to the mammal at a therapeutically effective dose.

[0243] The invention is further illustrated by the following examples. These examples are for illustrative purposes and are not intended to limit the scope of the invention.

[0244] abbreviation

[0245] ACN acetonitrile (or CH3CN)

[0246] BBS (Borate Buffered Salt)

[0247] DBCO Dibenzocyclooctylene

[0248] DEA N,N-diethylamine

[0249] DCM dichloromethane

[0250] DIEA (Diisopropylethylamine)

[0251] DIPEA N,N-Diisopropylethylamine

[0252] DMA dimethylacetamide

[0253] DMAP 4-Dimethylaminopyridine

[0254] DMF (dimethylformamide or N,N-dimethylformamide)

[0255] DMSO (dimethyl sulfoxide)

[0256] EDC N-ethyl-N',N'-dimethylaminopropylcarbodiimide

[0257] EDTA (ethylenediaminetetraacetic acid)

[0258] EEDQ N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

[0259] EtOAc (ethyl acetate)

[0260] EtOH (ethanol)

[0261] FA Formic acid

[0262] HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0263] HBTU [benzotriazol-1-yloxy(dimethylamino)methylene]-dimethyl-ammonium; hexafluorophosphate

[0264] HOAt 1-hydroxy-7-azabenzotriazole

[0265] HOBt 1-hydroxy-benzotriazole

[0266] MeOH (methanol)

[0267] MMAE (2S)-N-[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]-3-methyl-2-(methylamino)butyramide

[0268] NMP (N-methyl-2-pyrrolidone)

[0269] PBS (Phosphate-Buffered Saline)

[0270] Pd / C Palladium / Carbon

[0271] rmp protein A multi-point connection

[0272] rt room temperature

[0273] TEA Triethylamine

[0274] TFA (trifluoroacetic acid)

[0275] THF Tetrahydrofuran

[0276] TSTU [dimethylamino-(2,5-dioxopyrrolidone-1-yl)oxy-methylene]-dimethyl-ammonium; tetrafluoroborate Materials and Methods / General Methods

[0277] All reagents obtained from commercial sources were used without further purification. Anhydrous solvents were obtained from commercial sources and were ready for use without further drying. Rapid chromatography was performed on a CombiFlash Rf (Teledyne ISCO) column pre-packed with silica gel (Macherey-Nagel Chromabond Flash). Thin-layer chromatography was performed on 5x10 cm plates coated with Merck Type 60F254 silica gel. (CEM) Microwave heating is performed in the instrument.

[0278] Using DMSO-d6 (also written as dmso-d6 or DMSO) or CDCl3 as solvents, the spectrometers were tested on a 400 MHz Bruker Avance or a 500 MHz Avance Neo spectrometer. 1 H-NMR measurements. 1¹H NMR data are presented as delta values ​​in parts per million (ppm), using residual solvent peaks (2.50 ppm for DMSO-d6, 7.26 ppm for CDCl3) as internal standards. Splitting modes are represented as: s (singleton), d (doublet), t (triplet), q (quartet), m (multiplex), brs (broad singlet), dd (doublet), brm (broad multiplet), td (triplet), dt (doublet), br dd (broad doublet).

[0279] IR measurements were performed on a Bruker Tensor 27 equipped with an ATR Golden Gate device (SPECAC). HRMS measurements were performed on an LTQ Orbitrap Velos Pro mass spectrometer (ThermoFisher Scientific). The sample was dissolved in CH3CN / H2O (2 / 1: v / v) at a concentration ranging from approximately 0.01 to 0.05 mg / ml, and 2 μl was injected into the source at a flow rate of 0.1 ml / min.

[0280] ESI ionization parameters are as follows: 3.5 kV and 350 °C for the transfer ion capillary. Full spectra were obtained in positive ion mode at a resolution of 30,000 or 60,000 using locked mass.

[0281] UPLC-MS:

[0282] A-grade Waters Aquity with a quality device featuring a diode array UV detector "PDA" and "ZQ detector 2" and MassLinks software.

[0283] ZQ detector 2: MS scan 0.15–6 min and 100–2372 Da

[0284] PDA detector: 190-400nm

[0285] Column: Acquity BEH column C 18 1.7μm 2.1x50mm

[0286] The column was used at 40°C with a flow rate of 0.6 mL / min.

[0287] Solvent A: Water + 0.02% TFA; Solvent B: Acetonitrile + 0.02% TFA

[0288] Gradient 2% B-100% B, 5 minutes, then wash with 100% B for 0.3 minutes and 0.5 minutes, equilibrate in 2% B for the next injection (total gradient 6 minutes).

[0289] Preparative HPLC:

[0290] Interchim Puriflash The maximum value is 100 bar, and the maximum flow rate is 250 mL / min, or Interchim Puriflash. The maximum value is 250 bar, and the maximum flow rate is 250 mL / min.

[0291] A quaternary solvent pump capable of using four solvents simultaneously; gradient UV: two wavelengths for acquisition between 200 and 400 nm.

[0292] Collection: 8 mL or 32 mL tubes

[0293] ColumnWaters 10μm

[0294] Three preparative HPLC methods were used:

[0295] 1) TFA method: Solvents: A = water + 0.05% TFA, B = acetonitrile + 0.05% TFA, gradient 5-100% B, 15-30 CV (column volume)

[0296] 2) NH4HCO3 method: Solvents: A = water + 0.02M NH4HCO3, B = acetonitrile / water 80 / 20 + 0.02M NH4HCO3, gradient 5-100% B, 15-30 CV

[0297] 3) Neutral method: Solvent: A = water, B = acetonitrile, gradient 5-100% B, 15-30 CV

[0298] All fractions containing the pure compound were combined and directly freeze-dried to obtain the compound as an amorphous powder.

[0299] Preparative SFC purification:

[0300] Preparative chiral SFC was performed on a PIC Solution Prep200 system. The sample was dissolved in ethanol at a concentration of 150 mg / mL. Mobile phase isotropy was maintained at 40% ethanol / CO2. The instrument was equipped with a Chiralpak IA column and a 3 mL loop. The ABPR (automatic back pressure regulator) was set to 100 bar.

[0301] IUPAC chemical names were generated using Biovia Draw version 18.1 .NET software or using ACD / Name2018.2.2 (file version N50E41, Build 103230, July 21, 2018) software.

[0302] Preparation Example 1 1-[2-[2-(2,5-dioxopyrrolidin-1-yl)ethoxy]ethylcarbamoyl]cyclobutanecarboxylic acid (2,5-dioxopyrrolidin-1-yl) ester

[0303]

[0304] Step 1: 1-[2-[2-(2,5-dioxopyrrole-1-yl)ethoxy]ethylcarbamoyl] tert-butyl cyclobutanecarboxylate

[0305] To a solution of 1-tert-butoxycarbonylcyclobutanecarboxylic acid (58.6 mg; 0.293 mmol) in DCM (5.85 mL), 1-[2-(2-aminoethoxy)ethyl]pyrrole-2,5-dione (53.9 mg; 0.293 mmol), EDC (84.2 mg; 0.439 mmol), HOBt (59.3 mg; 0.439 mmol), and DIPEA (204 μL; 1.17 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 18 hours, then concentrated to dryness and dissolved in DMF (1 mL). After dissolving the residue in DMF (1 mL), the crude product was passed through a C18 reversed-phase preparative HPLC system, allowing the reaction mixture to deposit directly onto the surface. The sample was purified on a column using the TFA method to give the title compound (57.3 mg; 0.156 mmol). IR (cm⁻¹) -1 ):3390,1697 / 1666. 1 H NMR(400MHz,dmso-d6)δppm7.5(t,1H),7.02(s,2H),3.55 / 3.5(2t,4H),3.38(t,2H),3.17(q,2H),2.33(m,4H),1.77(m,2H),1.38(s,9H). UPLC-MS: MS(ESI): m / z[M+Na]+=389.26, [M+H-tBu]+=311.22

[0306] Step 2: 1-[2-[2-(2,5-dioxopyrrole-1-yl)ethoxy]ethylcarbamoyl]cyclobutanecarboxylic acid

[0307] TFA (51.2 μL; 0.668 mmol) was added to a solution of tert-butyl cyclobutanecarboxylate (7 mg; 0.0191 mmol) in DCM (0.175 mL). The reaction mixture was stirred at room temperature for 3.5 h and then concentrated to dryness to give the title compound (5.8 mg; 0.0187 mmol) as a colorless oil. The crude product was used for the next step. UPLC-MS: MS (ESI): m / z [M+H]+ = 311.35, [M+Na]+ = 333.37

[0308] Step 3: Preparation of Example 1

[0309] TSTU (9.04 mg; 0.030 mmol) and DIPEA (9.5 μL; 0.0540 mmol) were added sequentially to a solution of 1-[2-[2-(2,5-dioxopyrrole-1-yl)ethoxy]ethylcarbamoyl]cyclobutanecarboxylic acid (8.47 mg; 0.0273 mmol) in DMF (0.560 mL). The reaction mixture was stirred at room temperature for 2 hours to obtain a solution of Preparation Example 1 in DMF. The crude product was used directly in the next step. UPLC-MS: MS (ESI): m / z [M+H]+=408.43, [M+Na]+=430.38

[0310] Preparation Example 2 3-[3-[2-(2,5-dioxopyrrolidin-1-yl)ethoxy]propionylamino]oxetane-3-carboxylic acid (2,5-dioxopyrrolidin-1-yl) ester]

[0311]

[0312] Step 1: Synthesis of 3-[3-[2-(2,5-dioxopyrrole-1-yl)ethoxy]propionylamino]oxetane-3-carboxylic acid

[0313] To a solution of 3-aminooxetane-3-carboxylic acid (115 mg; 0.982 mmol) in DMF (3.45 mL), 3-[2-(2,5-dioxopyrrolidin-1-yl)ethoxy]propionic acid (2,5-dioxopyrrolidin-1-yl) ester (274.3 mg; 0.883 mmol) and DIPEA (855 μL; 4.91 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 18 hours, then concentrated to dryness and dissolved in DMF (1 mL). The solution was then analyzed by C... 18 Reversed-phase preparative HPLC allows the reaction mixture to be directly deposited on... The crude product was purified on a column using TFA to give the title compound (16 mg; 0.0512 mmol). UPLC-MS: MS (ESI): m / z [M+H]+ = 313.09, [M+Na]+ = 335.06. 1 H NMR(400MHz,dmso-d6)δppm 8.92(s,1H),7.02(s,2H),4.8 / 4.45(2d,4H),3.57 / 3.48(2m,6H),2.32(t,2H).IR(cm -1 ):3700-2300,1769 / 1740 / 1697,692

[0314] Step 2: Preparation of Example 2

[0315] TSTU (6.32 mg; 0.0210 mmol) and DIPEA (6.7 μL; 0.0382 mmol) were added sequentially to a solution of 3-[2-[2-(2,5-dioxopyrrolo-1-yl)ethoxy]ethylcarbamoyl]oxetane-3-carboxylic acid (5.8 mg; 0.0187 mmol) in DMF (0.380 mL). The reaction mixture was stirred at room temperature for 2 hours to obtain a solution of Preparation Example 2 in DMF. The crude product was used directly in the next step. UPLC-MS: MS (ESI): m / z [M+H]+ = 310.29, [M+Na]+ = 332.27

[0316] Example 1 : Sodium 5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-(hydroxymethyl)benzenesulfonate

[0317]

[0318] Step 1: Sodium 2-(hydroxymethyl)-5-nitrobenzenesulfonate

[0319] Sodium 5-nitro-2-[(E)-2-(4-nitro-2-sulfonyl-phenyl)vinyl]benzenesulfonate (25.0 g; 52.7 mmol; 1 eq.) in water (336 mL) was introduced into an ozone stream for 1.5 h. After the reaction was complete, the mixture was purged with argon for 30 min to remove excess ozone. Then sodium carbonate (39.1 g; 368 mmol) and sodium bicarbonate (3.99 g; 105 mmol) were added, and the orange solution was stirred at room temperature for 16 h. The reaction mixture was concentrated to dryness to give the title compound (39.9 g; 156 mmol) as a brown solid. 1H NMR (DMSO): δ4.99 (d, 2H, J = 3.6Hz), 5.36 (t, 1H, J = 5.6Hz), 7.83 (d, 1H, J = 8.4Hz), 8.21 (d, 1H, J = 8.4Hz), 8.45 (s, 1H).

[0320] Step 2: Sodium 5-amino-2-(hydroxymethyl)benzenesulfonate

[0321] Sodium 2-(hydroxymethyl)-5-nitrobenzenesulfonate (26.9 g; 105 mmol) was dissolved in water (403 mL). The reaction mixture was then purged with argon. 10% Pd / C (2.65 g) was added, and the black suspension was purged with argon, then with hydrogen. The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 3.5 days. After filtration and washing with water and methanol, the filtrate was concentrated to dryness and evaporated three times with toluene to remove residual trace water. Purification was performed by silica gel column chromatography using ethyl acetate / methanol (90 / 10-70 / 30) as eluent to give the title compound (14.29 g; 63.46 mmol) as a pale yellow solid. 1 H NMR (DMSO): δ4.52 (d, 2H, J = 5.2Hz), 4.95 (t, 1H, J = 5.2Hz), 5.04 (s, 2H), 6.42 (d, 1H, J = 7.6Hz), 6.93 (d, 1H, J = 7.6Hz), 7.03 (s, 1H).

[0322] Step 3: Sodium 5-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-5-ureo-pentanoyl]amino]-2-(hydroxymethyl)benzenesulfonate

[0323] Sodium 5-amino-2-(hydroxymethyl)benzenesulfonate (500 mg; 2.22 mmol), HBTU (1.01 g; 2.66 mmol), and DIPEA (917 mL; 5.55 mmol) were added to a solution of Fmoc-L-Cit-OH (CAS No. 133174-15-9; 882 mg; 2.22 mmol) in DMF (32.5 mL). The reaction mixture was stirred at room temperature for 16 hours, then concentrated to dryness and co-evaporated with water (2 x 100 mL). The solution was then analyzed by C... 18 The crude product was purified by column chromatography using a neutral method to give the title compound (1.0 g; 1.40 mmol) as a pale red oil. 1H NMR (DMSO): δ4.30-4.12 (m, 4H), 4.74 (d, 2H, J = 4.4Hz), 5.05 (t, 1H, J = 5.6Hz), 5.37 (s, 2H) ,5.97(t,1H,J=4.8Hz),7.34-7.42(m,4H),7.62-7.90(m,7H),8.15(s,1H),10.05(s,1H).

[0324] Step 4: Sodium 5-[[(2S)-2-amino-5-ureido-pentanoyl]amino]-2-(hydroxymethyl)benzenesulfonate

[0325] To a solution of sodium 5-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-5-ureido-valeryl]amino]-2-(hydroxymethyl)benzenesulfonate (11.2 g; 15.73 mmol) in DMF (224 mL), piperidine (3.1 mL; 31.47 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours, and then water (400 mL) was added. The aqueous layer was extracted with ethyl acetate (2 x 300 mL) and DCM (300 mL). Sodium carbonate (5.01 g; 47.1 mmol; 3 eq.) was added to the aqueous layer, and the mixture was stirred at room temperature for 3 hours. The mixture was lyophilized to give the title compound (6.01 g; 15.73 mmol) as a white solid. 1 H NMR(DMSO): δ1.55-1.64(m,4H),2.99-3.01(m,2H),3.58(m,1H),4.75(s,2H),5.06(s,1H),5.38(s,2H),5.98 (t,1H,J=5.6Hz),7.38(d,1H,J=8.4Hz),7.72(dd,1H,J=8.4and2.4Hz),7.86(d,1H,J=2.4Hz),10.17(s,1H).

[0326] Step 5: Example 1

[0327] Fmoc-L-Val-OSu (CAS No. 130878-68-1; 6.85 g; 15.69 mmol) was added to a solution of sodium 5-[[(2S)-2-amino-5-ureido-valanoyl]amino]-2-(hydroxymethyl)benzenesulfonate (6.01 g; 15.73 mmol) in DMF (150 mL). The light brown solution was stirred at room temperature for 3 hours, and then the reaction mixture was diluted with saturated sodium bicarbonate (100 mL) and water (100 mL) and concentrated to dryness. The residue was purified with silica gel using ethyl acetate / methanol 90 / 10-50 / 50 as eluent to give the title compound (4.44 g; 6.31 mmol) as a white solid.1 H NMR(DMSO): 0.85-0.90(m,6H),1.31-1.76(m,4H),1.95-2.06(m,1H),2.91-3.05(m,2H) ),3.95(t,1H,J=8.4Hz),4.24-4.35(m,3H),4.37-4.45(m,1H),4.76(d,2H,J=6Hz),5.0 7(t,1H,J=6.4Hz,),5.40(s,2H),6.03(t,1H,J=5.6Hz),7.32-7.46(m,6H),7.67(d,1H, J=8Hz),7.76(t,2H,J=7.2Hz),7.88-7.91(m,3H),8.12(d,1H,J=7.6Hz),10.08(s,1H). 13 C10 NMR (DMSO): 18.25, 19.24, 26.70, 29.56, 30.45, 39.50, 46.67, 53.17, 60.01, 60.96, 65.66, 117.85, 119.15, 120.05, 125.36, 127.06, 127.62, 128.09, 134.39, 136.79, 140.67, 143.89, 145.34, 156.08, 158.82, 170.37, 171.16. LCMS (2-100 acetonitrile / H2O + 0.1% formic acid): 93.85%, Tr = 8.4 min. Positive mode: Detection value 682.15 (MH). + Negative mode: Detection value 680.17 (MH) - )

[0328] Example 2 5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]-2-(hydroxymethyl)benzenesulfonic acid

[0329]

[0330] Step 1: Sodium 5-[[(2S)-2-(tert-butoxycarbonylamino)propionyl]amino]-2-(hydroxymethyl)benzenesulfonate

[0331] To a solution of Boc-L-Ala-OH (CAS No. 15761-38-3; 588 mg; 3.11 mmol) in DMF (38.6 mL), HATU (1.77 g; 4.67 mmol), sodium 5-amino-2-(hydroxymethyl)benzenesulfonate (771 mg; 3.42 mmol), and DIPEA (1.29 mL; 7.78 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 16 hours, then concentrated to dryness and co-evaporated with water to obtain a crude reaction mixture. The residue was purified by silica gel column chromatography using ethyl acetate / methanol 95:5–80:20 as eluent to give the title compound (1.17 g; 2.95 mmol) as a white solid. 1 H NMR(DMSO): δ1.24(s,9H),1.38(m,3H),4.05-1.44(m,1H),4.73(d,2H,J=4.8Hz),5.04(t,1H,J =5.6Hz),6.97-7.02(m,1H),7.33(d,1H,J=8Hz),7.65-7.70(m,1H),7.83(s,1H),9.91(s,1H).

[0332] Step 2: 5-[[(2S)-2-aminopropionyl]amino]-2-(hydroxymethyl)benzenesulfonate

[0333] Sodium 5-[[(2S)-2-(tert-butoxycarbonylamino)propionyl]amino]-2-(hydroxymethyl)benzenesulfonate (1.17 g; 2.95 mmol; 1 eq.) was suspended in a dioxane solution (10 mL) of HCl 4N. The mixture was stirred at room temperature for 2 hours and then concentrated to dryness to give a crude mixture (982 mg; 2.95 mmol) as a white solid. 1 H NMR (DMSO): δ1.45 (d, 3H, J = 5.6Hz), 3.91-4.0 (m, 1H), 4.76 (s, 2H), 7.41 (d, 1H,J=7.6Hz),7.66(d,1H,J=7.6Hz),7.85(s,1H),8.17(s,2H),10.44(s,1H)

[0334] Step 3: Example 2

[0335] Fmoc-L-Val-OSu (CAS No. 130878-68-1; 1.29 g; 2.95 mmol; 1 eq.) and DIPEA (975 μL; 5.9 mmol) were added to a solution of 5-[[(2S)-2-aminopropionyl]amino]-2-(hydroxymethyl)benzenesulfonate (981 mg; 2.95 mmol) in DMF (34.5 mL). The mixture was stirred overnight at room temperature, then concentrated to dryness and co-evaporated with water to obtain a crude mixture. The residue was purified by silica gel column chromatography using ethyl acetate / methanol 95:5-80:20 as eluent to give Example 2 (1.28 g; 2.072 mmol) as a colorless oil. 1 H NMR(DMSO): δ0.80-0.92(m,6H),1.30(d,3H,J=6.4Hz),2.02-2.10(m,1H),4.17 -4.31(m,3H),4.37-4.44(m,1H),4.73(d,2H,J=5.6Hz),5.04(t,1H,J=6.4Hz), 7.28-7.36(m,3H),7.37-7.47(m,3H),7.66(d,1H,J=8.4Hz),7.71-7.77(m,2H) ,7.83-7.85(m,1H),7.88(d,2H,J=7.6Hz),8.14(d,1H,J=6.4Hz),9.99(s,1H).

[0336] Example 3 Sodium 5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]-2-(hydroxymethyl)phenyl]methanesulfonate

[0337]

[0338] Step 1: (2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-valeric acid

[0339] Fmoc-L-Val-OSu (CAS No. 130878-68-1; 1.59 g; 3.64 mmol; 1.0 eq.) was added to L(+)-citrulline (0.67 g; 3.82 mmol; 1.05 eq.) and sodium bicarbonate (0.32 g; 3.82 mmol; 1.05 eq.) in a mixture of 1,2-dimethoxyethane (9.5 mL), water (9.5 mL), and THF (4.7 mL) at room temperature. The reaction mixture was stirred at room temperature for 16 hours, then the solvent was removed under vacuum, and the residue was acidified to pH 1 with 1 N hydrochloric acid solution. The white suspension was filtered, washed with water (3 x 40 mL), ether (3 x 40 mL), and co-evaporated with acetonitrile (2 x 150 mL). The white solid obtained by rapid silica gel chromatography was eluent using (DCM / acetic acid (99 / 1)) / methanol (10 / 0-7 / 3), and then ground in diethyl ether (2 x 30 mL). The resulting solid was dissolved in a mixture of methanol (50 mL) and water (50 mL), concentrated to dryness, co-evaporated with acetonitrile (100 mL), and dried to give the title compound (0.75 g; 1.5 mmol) as a white solid. 1 H NMR(DMSO): δ0.84-0.88(m,6H),1.34-1.41(m,2H),1.51-1.73(m,2H),1.95-2 .04(m,1H),2.91-2.96(m,2H),3.88-3.92(m,1H),4.06-4.11(m,1H),4.19-4.3 1(m,3H),5.36(s,2H),5.91-5.94(m,1H),7.30-7.34(m,2H),7.39-7.45(m,3H ),7.75(t,2H,J=7.3Hz),7.89(d,2H,J=7.4Hz),8.02(brs,1H),12.56(brs,1H)

[0340] Step 2: Sodium (2-methoxycarbonyl-5-nitro-phenyl)methanesulfonate

[0341] A suspension of methyl 2-(bromomethyl)-4-nitrobenzene (10.43 g; 38.06 mmol) in methanol (37 mL) was added to a solution of sodium sulfite (14.39 g; 114.17 mmol) in water (228 mL). The reaction mixture was stirred at room temperature for 20 hours and then concentrated to dryness. The crude product was purified by silica gel chromatography using ethyl acetate / methanol (10 / 0–6 / 4) as eluent to give the title compound (13.9 g; 31.07 mmol) as a white solid. 1H NMR (DMSO): δ3.82(s,3H),4.29(s,2H),7.87(d,1H,J=8.6Hz),8.16(dd,1H,J=2.5and8.6Hz),8.25(d,1H,J=2.5Hz)

[0342] Step 3: Sodium [2-(hydroxymethyl)-5-nitro-phenyl]methanesulfonate

[0343] Lithium borohydride (0.59 g; 24.3 mmol) was added to a suspension of sodium (2-methoxycarbonyl-5-nitro-phenyl)methanesulfonate (10.1 g; 22.09 mmol) in THF (368 mL). The reaction mixture was stirred at room temperature for 16 hours, and then lithium borohydride (0.27 g; 11.04 mmol) was added, and the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with methanol (50 mL) and concentrated to dryness. The crude product was purified by silica gel chromatography using ethyl acetate / methanol (10 / 0-5 / 5) as eluent to give the title compound (5.09 g; 6.52 mmol) as a yellow solid. 1 H NMR (DMSO): δ3.90 (s, 2H), 4.76 (d, 2H, J = 5.7Hz), 5.38 (t, 1H, J = 5.6Hz), 7.66 (d, 1H, J = 8.8Hz), 8.07-8.09 (m, 2H)

[0344] Step 4: Sodium [5-amino-2-(hydroxymethyl)phenyl]methanesulfonate

[0345] Pd / C 10% (0.56 g; 0.53 mmol) was added to a solution of sodium [2-(hydroxymethyl)-5-nitro-phenyl]methanesulfonate (2 g; 3.57 mmol) in methanol (102 mL) under an argon atmosphere. The reaction mixture was purged with hydrogen and stirred for 2 hours at room temperature under a hydrogen pressure atmosphere. Then... Filter the reaction mixture, wash with methanol (3 x 50 mL), and concentrate to dryness. Then, pass through C... 18 The crude product, purified by rapid chromatography using acetonitrile / water (2 / 98-50 / 50), yielded the title compound (1.98 g; 3.46 mmol) as a gray solid. 1 H NMR (DMSO): δ3.72 (s, 2H), 4.3 (d, 2H, J = 5.9Hz), 4.89 (s, 2H), 4.96 (t, 1H, J = 5.9 Hz),6.38(dd,1H,J=2.4and8.1Hz),6.49(d,1H,J=2.4Hz),6.9(d,1H,J=8.1Hz)

[0346] Step 5: Example 3

[0347] To a solution of (2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-valeric acid (543 mg; 1.09 mmol) in dry DMF (6 mL), a suspension of HATU (600 mg; 1.58 mmol), sodium bicarbonate (102 mg; 1.22 mmol), and sodium [5-amino-2-(hydroxymethyl)phenyl]methanesulfonate (650 mg; 1.41 mmol) in dry DMF (14 mL) was added. The reaction mixture was stirred at room temperature for 3 hours, then co-evaporated with dioxane (200 mL). The crude product was purified by silica gel rapid chromatography using DCM / methanol (10 / 0–5 / 5) as the eluent, followed by C24 chromatography. 18 Rapid chromatography was performed using acetonitrile / water + 0.1% formic acid (2 / 98-50 / 50) as the eluent. After lyophilization, Example 3 (233 mg; 0.32 mmol) was obtained as a white solid. 1 H NMR(DMSO): δ0.84-0.89(m,6H),1.32-1.49(m,2H),1.54-1.73(m,2H),1.94-2.04(m,1H),2.89-3.09(m,2H ),3.81(s,2H),3.91-3.95(m,1H),4.20-4.33(m,3H),4.41-4.46(m,1H),4.47(s,2H),5.07(brs,1H),5.40 (brs, 2H), 5.98 (t, 1H, J = 5.7Hz), 7.21 (d, 1H, J = 8.2Hz), 7.31-7.36 (m, 3H), 7.39-7.45 (m, 3H), 7.63 (dd, 1H, J = 2.2 and 8.3Hz), 7.75 (t, 2H, J = 7.5Hz), 7.89 (d, 2H, J = 7.4Hz), 8.08 (d, 1H, J = 7.6Hz), 10.01-10.8 (m, 1H). LCMS (2-100ACN / H2O + 0.1% TFA): 93.75% Tr = 8.1min. Negative mode detection value: 694.14 (MH).

[0348] Example 4 2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]benzenesulfonic acid

[0349]

[0350] 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-(hydroxymethyl)benzenesulfonic acid (the free acid of Example 1; 300 mg, 0.4263 mmol) was dissolved in anhydrous NMP (6 mL) at room temperature. In parallel, a solution of SOCl2 (206 μL) in NMP (6 mL) was prepared. 900 μL of the SOCl2 solution was added to the reaction system in six portions, each lasting 75 minutes. After the last addition, the reaction mixture was stirred at room temperature for 15 minutes. The crude product was purified by TFA by direct deposition of the reaction mixture onto an Oasis column to give the title compound (138 mg; 0.1971 mmol) as a white powder. 1 ¹H NMR (400MHz, dmso-d6) δppm: 10.15 + 8.1 + 7.42 + 6.0 (s + 2d + m, 4H), 7.9 (m, 3H), 7.75 (m, 3H), 7.42 + 7.31 (2m, 5H), 5.23 (s, 2H), 4.4 (m, 1H), 4.3 - 4.2 (m, 3H), 3.95 (dd, 1H), 3.0 (m, 2H), 2.0 (m, 1H), 1.7 + 1.6 (2m, 2H), 1.48 + 1.37 (2m, 2H), 0.88 (2d, 6H). HR-ESI+: m / z [M + H]+ = 700.2199 / 700.2202 [Measured value / Theoretical value]

[0351] Example 5 2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]benzenesulfonic acid

[0352]

[0353] Sodium 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]-2-(hydroxymethyl)benzenesulfonate (sulfonate obtained from Example 2; 504.1 mg, 0.816 mmol) in NMP (5 mL) was added in 6 portions of SOCl2 (60 μL, 0.816 mmol) in NMP (500 μL) over a period of 75 minutes. The reaction mixture was stirred at room temperature for 15 minutes. The crude product was purified by TFA method by direct deposition of the reaction mixture onto an Oasis column to give (337 mg) a white powder. IR wavelength (cm) -1):3600-2400,1688+1648,1599,1518,1022. UPLC-MS: MS(ESI)m / z[M+H]+=614.17+616.18(Cl)

[0354] Example 6 : 5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolo-1-yl)ethoxy]propionylamino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R [2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonate sodium salt

[0355]

[0356] Step 1: Sodium 5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]benzenesulfonate

[0357] To the solution of Example 1 (450 mg; 0.64 mmol) in DMF (6 mL), DIEA (1.34 mL; 7.67 mmol) and bis(4-nitrophenyl) carbonate (778 mg; 2.56 mmol) were added. The solution was stirred at room temperature for 2 hours, and bis(4-nitrophenyl) carbonate (390 mg; 1.28 mmol) was added. After 1 hour, the solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (a gradient of methanol and acetic acid in DCM) to give the title compound (523 mg; 0.45 mmol). 1H NMR(400MHz,dmso-d6)δppm 10.2 / 8.1 / 5.95(m,3H),8.3(d,2H),7.95(s,1H),7.9(d,2H),7.75(dd,1H),7.75(m,2H),7.65(d,2H),7.4(d,4H),7.35(d,1H) ,5.7(s,2H),5.35(brs,2H),4.4(m,1H),4.3(t,1H),4.2(d,2H),3.95(m,1H),3(m,2H),2(m,1H),1.8-1.3(m,4H),0.85(2d,6H)

[0358] Step 2: 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]methylcarbamoyl]oxymethyl]benzenesulfonic acid trifluoroacetate

[0359] To a solution of MMAE (200 mg; 0.28 mmol) in DMF (5.6 mL), DIEA (0.19 mL; 1.39 mmol) was added, followed by sodium 5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]benzenesulfonate (472 mg; 0.55 mmol) and HOBt (75 mg; 0.56 mmol). The solution was stirred at room temperature for 3.5 hours, and then DEA (0.19 mL; 1.39 mmol) was added. After 2 hours, the solution was concentrated under reduced pressure and passed through a reverse-phase reactor. Preparative HPLC was used to purify the residue using the TFA method, yielding the title compound (99 mg; 0.08 mmol). 1H NMR(400MHz,dmso-d6)δppm 10.2 / 10(2s,1H),8.56 / 8.35(2m,1H),8.1(d,1H),7.9(brs,1H),7.88 / 7.6(2d,1H),7.68(b rs,1H),7.35-7.13(m,5H),7.3(brs,1H),6 / 5.96(2t,1H),5.5(m,2H),5.4(brs,2H),4.8-4. 15(m,5H),4.05-3.85(m,2H),3.8-3.6(5s,9H),3.62-3.5(m,2H),3.1-2.8(m,5H),3(brs,3 H),2.4 / 2.3(d+dd,2H),2.2-1.9(m,3H),1.9-1.75(m,11H),1.05 / 1(2d,9H),1-0.75(m,21H)

[0360] Step 3: Example 6

[0361] 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3 [-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid TFA salt (22 mg; 0.018 mmol) was added to a solution of DMF (0.18 mL) containing DIEA (6.3 μL mL; 36 μmmol) and 1-[2-[3-(2,5-dioxopyrrolidine-1-yl)-3-oxopropoxy]ethyl]pyrrole-2,5-dione (6.2 mg; 20 μmol). The solution was stirred at room temperature for 4.5 hours. C 18 The reaction system was purified by reversed-phase preparative HPLC using the NH4HCO3 method to obtain Example 6 (9.6 mg; 6.8 μmol). HRMS (ESI) [M+H] + Measured value = 1420.7041 (δ = -0.3 ppm)

[0362] Example 7:5-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrolo-1-yl)hexanoylamino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonate sodium salt

[0363]

[0364] 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propane] [1S]-1-pyrrolidone-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid TFA salt (obtained from step 2 of Example 6; 30 mg; 0.025 mmol) was added to a solution of DIEA (8.7 μL mL; 36 μmmol) and 6-(2,5-dioxopyrrolidone-1-yl)hexanoic acid (2,5-dioxopyrrolidone-1-yl) ester (8.46 mg; 0.027 mmol) in DMF (0.25 mL). The solution was stirred at room temperature for 3.5 hours. C 18 The reaction system was purified by reversed-phase preparative HPLC using the NH4HCO3 method to obtain Example 7 (20 mg; 14.09 μmol). HRMS (ESI) [M+H] + Measured value = 1418.7253 (δ = 0.1 ppm)

[0365] Example 8:5-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butyryl]amino]-5-ureo-valeryl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1 R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolid-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]sodium benzenesulfonate

[0366]

[0367] To a solution of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetic acid (6.4 mg; 0.027 mmol) in DMF (125 μL), DIEA (21.7 μL; 124 μmmol) and TSTU (7.88 mg; 0.026 mmol) were added. After activation, 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1- A solution of TFA salt (30 mg; 0.025 mmol) of [methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid in DMF (0.15 mL). The solution was stirred at room temperature for 2 hours. C 18 The reaction system was purified by reversed-phase preparative HPLC using the NH4HCO3 method to obtain Example 8 (16 mg; 11.10 μmol). HRMS (ESI) [M+H] + Measured value = 1440.7382 (δ = -2.6 ppm)

[0368] Example 95-[[(2S)-2-[[1-[2-[2-(2,5-dioxopyrrolo-1-yl)ethoxy]ethylcarbamoyl]cyclobutanecarbonyl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]methylcarbamoyl]oxymethyl]benzenesulfonic acid

[0369]

[0370] Step 1: Sodium 5-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-5-ureo-pentanoyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]benzenesulfonate

[0371] Sodium 5-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-5-ureo-pentanoyl]amino]-2-(hydroxymethyl)benzenesulfonate (261 mg; 0.448 mmol) in DMF (5.75 mL) was added sequentially with bis(4-nitrophenyl) carbonate (1.09 g; 3.58 mmol) and DIPEA (1.87 mL; 10.75 mmol). The reaction mixture was stirred at room temperature for 16 hours and then concentrated to dryness to give a crude mixture. The crude product was purified by silica gel chromatography (methanol gradient of DCM solution containing 2% AcOH) to give the title compound (137 mg; 0.183 mmol) as a white solid. UPLC-MS: MS (ESI): m / z [M+H]+=748.48

[0372] Step 2: Sodium 5-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-5-ureo-valeryl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonate

[0373] To a solution of 5-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-5-ureo-pentanoyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]benzenesulfonic acid (64.5 mg; 0.0863 mmol) in DMF (1.42 mL), MMAE (61.9 mg; 0.0863 mmol), DIPEA (75.1 μL; 0.431 mmol), and HOBt (23.3 mg; 0.173 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was then analyzed by C... 18 Reversed-phase preparative HPLC, through which the reaction mixture is directly deposited on The crude product was purified on a column using TFA to give the title compound (33.5 mg; 0.0253 mmol). UPLC-MS: MS (ESI): m / z [M+H]+ = 1326.88, [M+Na]+ = 1348.09

[0374] Step 3: 5-[[(2S)-2-amino-5-ureido-valeryl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid, TFA salt

[0375] 5-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyridine Sodium [1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonate (36.8 mg; 0.0292 mmol) was added to a solution of piperidine (23.1 μL; 0.234 mmol) in DMF (750 μL). The reaction mixture was stirred at room temperature for 17 hours. The reaction mixture was then subjected to C... 18 Reversed-phase preparative HPLC, through which the reaction mixture is directly deposited on The crude product was purified on a column using TFA to give the title compound (17 mg; 0.0139 mmol). UPLC-MS: MS (ESI): m / z [M+H]+=1038.10, [M+Na]+=1059.85

[0376] Step 4: Example 9

[0377] 5-[[(2S)-2-amino-5-ureido-valeryl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1 [-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid TFA salt (24.5 mg; 0.0215 mmol) was added sequentially to a solution of Preparation Example 1 (10.5 mg; 0.0258 mmol) and DIPEA (19 μL; 0.108 mmol) in DMF (735 μL). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was then subjected to C... 18 Reversed-phase preparative HPLC, through which the reaction mixture is directly deposited on On a column, the crude product was purified using the TFA method to obtain Example 9 (19.3 mg; 0.0147 mmol), as a white powder. HR-ESI+: m / z [M+H]+ = 1396.7040 / 1396.7074 [Measured value / Theoretical value]

[0378] Example 10 5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolo-1-yl)ethoxy]propionylamino]-3-methyl-butyryl]amino]propionyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid

[0379]

[0380] Step 1: 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]benzenesulfonic acid

[0381] Pyridine (875 μL; 10.8 mmol) was added to the suspension of Example 2 (1.28 g; 2.07 mmol) in THF (65 mL), followed by 4-nitrobenzene chloroformate (1.09 g; 5.41 mmol). The mixture was stirred overnight at room temperature. Then 4-nitrobenzene chloroformate (1.09 g; 5.41 mmol; 2.5 eq.) was added. After stirring at room temperature for 5 hours, the mixture was concentrated to dryness and then analyzed by C. 18 Purification was performed by column chromatography using a 90 / 10-0 / 100 water / acetonitrile eluent for 30 minutes. Acetonitrile was removed from the combined tube, and the residue was lyophilized to give the title compound (650 mg; 0.83 mmol) as a white solid. 1 H NMR(DMSO): δ0.88(m,6H),1.31(d,3H,J=4.8Hz),1.97-2.03(m,1H),3.92(t,1 H,J=6.8Hz),4.23(s,2H),4.24-4.34(m,1H),4.42(t,1H,J=5.6Hz),5.69(s,2H ),7.30-7.48(m,6H),7.62(d,2H,J=8Hz),7.72-7.76(m,3H),7.89(d,2H,J=6. 4Hz), 7.94 (s, 1H), 8.18 (d, 1H, J = 5.6Hz), 8.33 (d, 2H, J = 7.6Hz), 10.11 (s, 1H). 13 C NMR(DMSO): δ18.01,18.26,19.21,30.4,46.66,49.05,59.91,65.67,67.82,117.7,119.1,120.06,122.66,125.37,12 6.33,127.05,127.62,128.0,138.06,140.67,143.77,143.86,145.1,146.23,151.96,155.47,156.12,171.0,171.15. LCMS (2-100ACN / H2O+0.05% TFA): 90.41%Tr=12.7min. Positive mode detection value: 578.41. Negative mode detection value: 759.17.

[0382] Step 2: 5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]methylcarbamoyl]oxymethyl]benzenesulfonic acid

[0383] To a solution of 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]benzenesulfonic acid (29.3 mg; 0.0374 mmol) in DMF (1.20 mL), MMAE (26.9 mg; 0.0374 mmol), DIPEA (32.6 μL; 0.187 mmol), and HOBt (10.1 mg; 0.075 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was then subjected to C... 18 Reversed-phase preparative HPLC, through which the reaction mixture is directly deposited on The crude product was purified on a column using TFA to give the title compound (35.4 mg; 0.0264 mmol). UPLC-MS: MS (ESI): m / z [M+H]+ = 1340.9

[0384] Step 3: 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]propionyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]methylcarbamoyl]oxymethyl]benzenesulfonic acid, TFA salt

[0385] 5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3 [-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid (50.1 mg; 0.0374 mmol) was added to a solution of piperidine (29.6 μL; 0.299 mmol) in DMF (1.5 mL). The reaction mixture was stirred at room temperature for 1 hour. (The last sentence appears to be incomplete and requires further context.) 18 Reversed-phase preparative HPLC, through which the reaction mixture is directly deposited on On a column, the crude product was purified using TFA to give the title compound (23 mg; 0.0187 mmol) as a white powder. HR-ESI+: m / z [M+H]+=1117.6235 / 1117.6218, [M+2H] / 2+=559.3146 / 559.3148 [Measured / Theoretical]

[0386] Step 4: Example 10

[0387] 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]propionyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl [-Methylcarbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid TFA salt (10.4 mg; 0.00845 mmol) was added sequentially to a solution of 3-[2-(2,5-dioxopyrrolidin-1-yl)ethoxy]propionic acid (2,5-dioxopyrrolidin-1-yl) ester (2.9 mg; 0.00929 mmol) and DIPEA (7.36 μL; 0.0422 mmol) in DMF (0.34 mL). The reaction mixture was stirred at room temperature for 16 hours. (The last sentence appears to be incomplete and requires further context.) 18 Reversed-phase preparative HPLC, through which the reaction mixture is directly deposited on On a column, the crude product was purified using the TFA method to obtain Example 10 (7.7 mg; 0.0059 mmol), as a white powder. HR-ESI+: m / z [M+H]+ = 1312.6701 / 1312.6750, [Measured value / Theoretical value]

[0388] Example 11 : 5-[[(2S)-2-[[3-[2-[2-(2,5-dioxopyrrole-1-yl)ethoxy]ethylcarbamoyl]oxetane-3-carbonyl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R, [2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidone-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid

[0389]

[0390] 5-[[(2S)-2-amino-5-ureido-valeryl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropane] [4-O-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid; 2,2,2-trifluoroacetic acid (16 mg; 0.0131 mmol) was added sequentially to a solution of [480 μL] in DMF, along with Preparation Example 2 (10.24 mg; 0.0250 mmol) and DIPEA (11.4 μL; 0.0657 mmol). The reaction mixture was stirred at room temperature for 16 hours. [The following text appears to be incomplete and requires further context: "by C..."] 18 Reversed-phase preparative HPLC, through which the reaction mixture is directly deposited on On a column, the crude product was purified using the TFA method to obtain Example 11 (4.4 mg; 0.0031 mmol), as a white powder. HR-ESI+: m / z [M+2H]2+ = 699.8477 / 699.8472 [Measured value / Theoretical value]

[0391] Example 12:[5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolo-1-yl)ethoxy]propionylamino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R, 2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidone-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]phenyl]methanesulfonic acid

[0392]

[0393] Step 1: Sodium [5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]phenyl]methanesulfonate

[0394] To the solution of Example 3 (30 mg; 0.0418 mmol) in DMF (0.4 mL), DIEA (87 μL; 70.501 mmol) and bis(4-nitrophenyl) carbonate (51 mg; 0.167 mmol) were added. The solution was stirred at room temperature for 2 hours, and then bis(4-nitrophenyl) carbonate (25 mg; 0.08 mmol) was added. After 1 hour at room temperature, the solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (methanol gradient in DCM) to give the title compound (25 mg; 0.030 mmol).

[0395] Step 2: [5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]phenyl]methanesulfonic acid

[0396] DIEA (12 μL; 0.0696 mmol) was added to a solution of MMAE (10 mg; 0.0139 mmol) in DMF (0.6 mL), followed by the addition of sodium [5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]phenyl]methanesulfonate (24 mg; 0.028 mmol) and HOBt (3.77 mg; 0.0279 mmol). The solution was stirred at room temperature for 3.5 hours, and the solution was analyzed by... C 18 Reversed-phase preparative HPLC was used to purify the solution using the TFA method, yielding the title compound (5.4 mg; 0.003 mmol). HRMS (ESI) [M+H]+ determination value = 1439.7558 (δ = 1.9 ppm).

[0397] Step 3: [5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]-5-ureo-valeryl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]phenyl]methanesulfonic acid, TFA salt

[0398] [5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy] [2-Methyl-3-oxo-propyl]pyrrolidone-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]phenyl]methanesulfonic acid (5 mg; 3.4 μmol) was dissolved in DMF (0.2 mL), and then DEA (0.7 μL; 6.8 mmol) was added. The reaction system was stirred at room temperature for 1 hour, and the reaction was carried out by... C 18The reverse-phase preparative solution was purified using the TFA method to give the title compound (3.5 mg; 2.6 μmol).

[0399] Step 4: Example 12

[0400] [5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]-5-ureo-valeryl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropane] [3,5-Dioxopyrrolidone-1-yl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]phenyl]methanesulfonic acid TFA salt (3.5 mg; 2.6 μmol) in DMF (15 μL) was mixed with DIEA (2.3 μL; 13 μmmol) and 1-[2-[3-(2,5-dioxopyrrolidone-1-yl)-3-oxo-propoxy]ethyl]pyrrole-2,5-dione (0.9 mg; 2.9 μmol). The solution was stirred at room temperature for 2.5 hours. [The text abruptly ends here, likely due to an incomplete translation or missing information.] C of the column 18 The reaction system was purified by reversed-phase preparative HPLC using the NH4HCO3 method to obtain Example 12 (2.8 mg; 1.8 μmol). HRMS (ESI) [M+H]+ determination value = 1412.7397 (δ = 1.1 ppm).

[0401] Example 13 5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-(iodomethyl)benzenesulfonic acid

[0402]

[0403] Sodium iodide (51 mg; 340 μmol) was added to the solution of Example 4 (100 mg; 123 μmol) in acetone (6 mL). The reaction mixture was stirred at room temperature for 20 hours. The solvent was evaporated, and the compound was used for the next step without post-treatment. UPLC-MS: [M+H] +792.65; [M+Na] +814.49

[0404] Example 14:[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolo-1-yl)ethoxy]propionylamino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-sulfonyl-phenyl]methyl-[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[ [(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolid-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-dimethyl-ammonium; 2,2,2-trifluoroacetate

[0405]

[0406] Step 1: [4-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-sulfono-phenyl]methyl-[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]dimethyl-ammonium; iodide

[0407] To a solution of (2S)-2-[[(2S)-2-(dimethylamino)-3-methyl-butyryl]amino]-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-N,3-dimethyl-butyramide (auratestatin E) (42.1 mg; 57.5 μmol) in DMF (10 mL), 152 mg (95.8 μmol) from Example 13 and DIPEA (83.4 μL; 365 μmol) were added sequentially. The reaction mixture was stirred at room temperature for 17 hours. The desired product was determined by UPLC-MS, and the solution was used for the next step without post-treatment. UPLC-MS: [M+H]+1396.31; [M+Na]+1418.37

[0408] Step 2: [4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-sulfono-phenyl]methyl-[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-dimethyl-ammonium; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid

[0409] [4-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-sulfonyl-phenyl]methyl-[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]] [amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-dimethyl-ammonium; piperidine (45.4 μL; 460 μmol) was added to a solution of iodide in DMF, and the reaction system was stirred at room temperature for 1 hour. The reaction mixture was directly deposited onto a C18 reversed-phase preparative HPLC system. The crude product was purified by TFA on a column to obtain the desired product (15 mg; 10.7 μmol), a white solid. UPLC-MS: [M+H]+1174.51; [M+Na]+1197.03

[0410] Step 3: Example 14

[0411] [4-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-valeryl]amino]-2-sulfonyl-phenyl]methyl-[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methyl A solution of oxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-dimethyl-ammonium; iodide (15 mg; 10.7 μmol) in DMF (450 μL) was successively mixed with (2,5-dioxopyrrolidone-1-yl)-3-[2-(2,5-dioxopyrrolidone-1-yl)ethoxy] ester (3.7 mg; 11.8 μmol) and DIPEA (9.3 μL; 53.5 μmol). The solution was stirred at room temperature for 1 hour. The reaction mixture was directly deposited onto a C18 reversed-phase preparative HPLC system. On a column, the crude product was purified using the TFA method to obtain Example 14 (15.7 mg; 12.1 μmol), as a white solid. HRMS (ESI) [M-CF3CO2]+ 1368.7460 (δ = -1.7 ppm)

[0412] Example 23 :N-({[4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl]-L-valinel-L-alanyl}amino)-2-sulfophenyl]methoxy}carbonyl)-N-methyl-L-valinel-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylprop-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohep-4-yl]-N-methyl-L-valine

[0413]

[0414] To 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]propionyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4- [Oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid TFA salt (12.7 mg; 0.0103 mmol; obtained from step 3 of Example 10) was added to a solution in DMF (0.4 mL) with DIEA (9 μL; 51 μmol) and 6-(2,5-dioxopyrrolidin-1-yl)hexanoate (2,5-dioxopyrrolidin-1-yl) ester (3.5 mg; 11.3 μmol). The solution was stirred at room temperature for 6 hours. By using C of the column 18 Reversed-phase preparative HPLC was used to purify the reaction system using the TFA method, yielding Example 23. IR (cm) -1 ): 3278,1768 / 1703,1631,1159,829 / 696. RMN 1H(500MHz,DMSO-d6)δppm 9.96(s,1H),8.1(s,1H),7.87(m,2H),7.68(m,1H),7.6(d,1H),7.31(m,2H),7.26(m,3H),7.17(m,1H),6.99(s,2H), 5.48(m,2H),4.74(m,1H),4.49(m,1H),4.46(m,1H),4.41(m,1H),4.38(m,1H),4.33(m,1H),4.19(m,1H),3.98(m,1H ),3.98(m,1H),3.77(m,1H),3.6(m,2H),3.56(m,1H),3.46(m,1H),3.36(m,2H),3.32(m,1H),3.26(m,2H),3.24(m,3 H),3.23(m,2H),3.19(m,3H),3.13(m,2H),3.04(m,1H),2.98(m,3H),2.94(m,1H),2.9(m,1H),2.86(m,1H),2.37(br dd,2H),2.12(m,2H),2.12(m,2H),2.06(m,2H),1.97(m,2H),1.49(m,9H),1.29(d,3H),0.89(m,27H). HRMS(ESI)[M+H] + Measured value = 1310.6893 (δ = -4.5 ppm)

[0415] Example 24 :N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl]-L-valine-N 5 -Carbamoyl-N-(4-{[({[(4S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzino[1,2-b]quinoline-4-yl]oxy}carbonyl)oxy]methyl}-3-sulfophenyl)-L-guanine

[0416]

[0417] Step 1: 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-(hydroxymethyl)benzenesulfonic acid

[0418] DEA (212 μL; 2.053 mmol) was added to the solution of Example 1 (200 mg; 0.284 mmol) in DMF (8 mL), and the reaction system was stirred at room temperature for 1 hour. Excess DEA was evaporated under vacuum, and the crude solution of the desired compound in DMF was used directly for the next step.

[0419] Step 2: 5-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrolo-1-yl)hexanoylamino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-(hydroxymethyl)benzenesulfonic acid

[0420] Add DIEA (102 μL; 0.5866 mmol) and 6-(2,5-dioxopyrrolo-1-yl)hexanoic acid (2,5-dioxopyrrolo-1-yl) ester (99.4 mg; 0.3226 mmol) to a solution (8 mL) of the compound from the above steps. Stir the reaction mixture at room temperature for 18 hours. By using... C of the column 18 Reversed-phase preparative HPLC was used, and the reaction system was purified using the TFA method. The resulting compound was then lyophilized to obtain the desired compound. IR (cm⁻¹) -1 ): 3674-2999,1768 (weak) +1699 +1641,1238 / 1141,825 and 694. RMN 1 H(400MHz,DMSO-d6)δppm 9.97(s,1H),8.04(d,1H),7.85(d,1H),7.77(d,1H),7.65(dd,1H),7.32(d,1H),6.99(s,2H),6.02(m,1H),5.18(m,1H),4.72(s ,2H),4.36(m,1H),4.21(dd,1H),3.37(t,2H),2.98(m,2H),2.16(m,2H),1.97(m,1H),1.65(m,2H),1.39(m,8H),0.83(dd,6H). HRMS(ESI)[M+H]+measured value=653.2572(δ=-4.2ppm)

[0421] Step 3: [(19S)-10,19-diethyl-19-hydroxy-14,18-dioxo-17-oxa-3,13-dioneazapentacyclo[11.8.0.02,11.04,9.015,20] ticos(21),2,4(9),5,7,10,15(20)-hept-7-yl] tert-butyl carbonate

[0422] To a suspension of (19S)-10,19-diethyl-7,19-dihydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]carbon-1(21),2,4(9),5,7,10,15(20)-hepta-14,18-dione (220 mg; 0.5607 mmol) in DCM (22 mL), tert-butyl carbonate (128.5 mg; 0.5887 mmol) and piperidine (91 μL; 1.121 mmol) as powder were added. The yellow suspension was stirred at room temperature for 18 hours. The solvent and excess pyridine were evaporated under vacuum, and the crude product (DCM / MeOH) was purified by silica gel chromatography to give the desired compound as a yellow powder. IR (cm -1 ): 3700-3000,1753,1659,1254 / 1142. RMN 1 H(400MHz,DMSO-d6)δppm8.21(d,1H),8.09(d,1H),7.74(dd,1H),7.33(s,1H),6.5( s,1H),5.38(2s,4H),3.2(q,2H),1.86(m,2H),1.54(s,9H),1.3(t,3H),0.88(t,3H)

[0423] Step 4: [(19S)-19-chlorocarbonyloxy-10,19-diethyl-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20] docosa-1(21),2,4(9),5,7,10,15(20)-hept-7-yl] tert-butyl carbonate

[0424] DMAP (22.3 mg; 0.1827 mmol) was added to a solution of [(19S)-10,19-diethyl-19-hydroxy-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]docosa-1(21),2,4(9),5,7,10,15(20)-hept-7-yl]tert-butyl carbonate (30 mg; 0.0609 mmol) in DCM (2 mL), followed by triphosgene (7.2 mg; 0.0244 mmol). The reaction mixture was stirred at room temperature for 30 minutes, and the solution was used in the next step.

[0425] Step 5: 2-[[(19S)-7-tert-butoxycarbonyloxy-10,19-diethyl-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20] icosico-1(21),2,4(9),5,7,10,15(20)-hept-19-yl]oxycarbonyloxymethyl]-5-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrolo-1-yl)hexanoylamino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]benzenesulfonic acid

[0426] Add D to a solution of [(19S)-19-chlorocarbonyloxy-10,19-diethyl-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]docosa-1(21),2,4(9),5,7,10,15(20)-hept-7-yl]tert-butyl carbonate in DCM (2 mL) MAP (22.3 mg; 0.1827 mmol) was added, followed by 5-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrolo-1-yl)hexanoylamino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-(hydroxymethyl)benzenesulfonic acid (31.80 mg; 0.04873 mmol; obtained according to step 2 above). The reaction system was stirred at room temperature for 14 hours. By using... C of the column 18 The reaction system was purified by reverse-phase preparative HPLC, and then purified by TFA method. After lyophilization, the expected compound was obtained.

[0427] Step 6: Example 24

[0428] TFA (0.3 mL) was added to a solution of 2-[[(19S)-7-tert-butoxycarbonyloxy-10,19-diethyl-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]ticos-1(21),2,4(9),5,7,10,15(20)-hept-19-yl]oxycarbonyloxymethyl]-5-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrolo-1-yl)hexanoylamino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]benzenesulfonic acid (12.5 mg; 1.66 μmol) in DCM (2.5 mL), and the solution was stirred at room temperature for 1.5 hours. By using C of the column 18The reaction system was purified by reversed-phase preparative HPLC using the TFA method and then lyophilized to obtain Example 24. HRMS (ESI) [M+H]+ measured value = 1071.3768 (δ = 0.4 ppm).

[0429] Example 25 (1S,3S)-3,5,12-trihydroxy-3-(hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydro-tert-1-yl2,3,6-trideoxy-3-[({[4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl]-L-valine-N 5 [-carbamoyl-L-guanosyl}amino]-2-sulfophenyl]methoxy}carbonyl]amino]-α-L-lythreo-hexanoside

[0430]

[0431] Step 1: 5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[(2S,3S,4S,6R)-3-hydroxy-2-methyl-6-[[(1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-2,4-dihydro-1H-tetraphenyl-1-yl]oxy]tetrahydropyran-4-yl]carbamoyloxymethyl]benzenesulfonic acid

[0432] Sodium benzenesulfonate (157 mg; 0.1854 mmol; obtained from step 1 of Example 6) in DMF (11 mL) was added to a solution of 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-valeryl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]benzenesulfonate (157 mg; 0.1854 mmol; obtained from step 1 of Example 6). Then (7S,9S)-7-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyl-tetrahydropyran-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tetraphenyl-5,12-dione (107.5 mg; 0.1853 mmol) was added, followed by DIEA (323 μL; 1.854 mmol). The red mixture turns dark purple, and the conversion is complete after 2 hours. Use this solution for the next step.

[0433] Step 2: 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]-2-[[(2S,3S,4S,6R)-3-hydroxy-2-methyl-6-[[(1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-2,4-dihydro-1H-tetraphenyl-1-yl]oxy]tetrahydropyran-4-yl]carbamoyloxymethyl]benzenesulfonic acid

[0434] DEA (96 μL; 0.9268 mmol) was added to the 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[(2S,3S,4S,6R)-3-hydroxy-2-methyl-6-[[(1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-2,4-dihydro-1H-tetraphenyl-1-yl]oxy]tetrahydropyran-4-yl]carbamoyloxymethyl]benzenesulfonic acid prepared as described above. The reaction mixture was stirred at room temperature for 1 hour. Partial evaporation of the solvent was carried out, and the solvent was then used... C of the column 18 Reversed-phase preparative HPLC was used to purify the crude product using the TFA method, followed by lyophilization to obtain the desired compound. IR (cm) -1 ): 3357, 2661, 1666 / 1608, 1585 / 1531, 1201, 1082, 1020, 763 / 709. RMN 1H(500MHz,DMSO-d6)δppm14.06(s,1H),13.29(s,1H),10.15(s,1H),8.62(d,1H),8.04(m,3H),7.93(d,1H),7.93(t,1H),7.89(d,1H ),7.66(dd,1H),7.61(dd,1H),7.26(d,1H),6.88(d,1H),5.99(t,1H),5.49(brs,1H),5.37(m,2H),5.25(brs,1H),4.97(t,1H),4.79 (brm,2H),4.58(s,2H),4.46(q,1H),4.18(q,1H),3.99(s,3H),3.75(m,1H),3.63(m,1H),3.51(brs,1H),2.99(m,2H),2.99(m,2H), 2.16(m,2H),2.07(m,1H),1.87(td,1H),1.72(m,1H),1.61(m,1H),1.5(dt,1H),1.45(m,1H),1.37(m,1H),1.14(d,3H),0.93(2d,6H)

[0435] Step 3: Example 25

[0436] Add DIEA (1.6 μL; 5.2 μmmol) and 6-(2,5-dioxopyrrolo-1-yl)hexanoic acid (2,5-dioxopyrrolo-1-yl) ester (1.6 mg; 5.2 μmol) to a solution of 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]-5-ureido-valanoic acid]-2-[[(2S,3S,4S,6R)-3-hydroxy-2-methyl-6-[[(1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-2,4-dihydro-1H-tetraphenyl-1-yl]oxy]tetrahydropyran-4-yl]carbamoyloxymethyl]benzenesulfonic acid (5 mg; 4.7 μmol) in DMF (1 mL). The solution was stirred at room temperature for 12 hours. (The sentence fragment "using..." appears to be incomplete and lacks context. It's unclear what "using..." is.) C of the column 18 The reaction system was purified by reversed-phase preparative HPLC using the TFA method, yielding the HRMS (ESI) [M+H]+ value of Example 25, which was 1222.4153 (δ = 1.7 ppm).

[0437] Example 26 :N-{[(9H-fluorene-9-yl)methoxy]carbonyl}-L-valine-N 5-Carbamoyl-N-[4-(hydroxymethyl)-3-(2-sulfonic acid ethyl)phenyl]-L-ornithine sodium

[0438]

[0439] Step 1: (2-Allyl-4-nitro-phenyl)methoxy-tert-butyl-dimethyl-silane

[0440] A solution of tert-butyl-[(2-iodo-4-nitro-phenyl)methoxy]-dimethyl-silane (6.56 g; 16.68 mmol) and allyltributyltin (7.76 mL; 25.02 mmol) in 1,4-dioxane (165 mL) was purged with argon three times. Tetra(triphenylphosphine)palladium(0) (1.93 g; 1.67 mmol) was added, and the mixture was purged again with argon three times. The mixture was stirred at 100 °C for 18 hours. The reaction mixture was filtered and concentrated to dryness. The residue was dissolved in DCM (150 mL) and washed with 1 M sodium hydroxide aqueous solution. The aqueous layer was extracted with DCM, the combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by silica gel chromatography using cyclohexane / EtOAc (10 / 0-95 / 5) as eluent to give the expected compound (4.91 g; 15.97 mmol) as an orange oil. 1 H NMR(DMSO): δ0.11(s,6H),0.92(s,9H),3.49(d,2H,J=6.5Hz),4.84(s,2H),5.04-5.15(m,2H) ,5.92-6.02(m,1H),7.69(d,1H,J=8.5Hz),8.02(d,1H,J=2.5Hz),8.13(dd,1H,J=2.5 and 8.5Hz)

[0441] Step 2: 3-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-nitro-phenyl]prop-1,2-diol

[0442] To a solution of (2-allyl-4-nitro-phenyl)methoxy-tert-butyl-dimethylsilane (4.91 g; 15.97 mmol) in a mixture of acetone (147 mL) and water (20 mL), 4-methylmorpholine N-oxide (3.74 g; 31.94 mmol) and potassium (VI) osmium tetroxide dihydrate (0.29 g; 0.8 mmol) were added, and the mixture was stirred at room temperature for 20 hours. The reaction mixture was quenched with a saturated aqueous solution of sodium thiosulfate (40 mL) and water (40 mL), and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by rapid silica gel chromatography using cyclohexane / EtOAc (10 / 0–5 / 5) as eluent to give the desired compound (4.65 g; 13.62 mmol) as a yellow oil. 1 ¹H NMR (DMSO): δ 0.11 (s, 6H), 0.93 (s, 9H), 2.58 (dd, 1H, J = 8.8 and 14.2 Hz), 2.9 (dd, 1H, J = 3.5 and 14.2 Hz), 3.26–3.30 (m, 1H), 3.36–3.41 (m, 1H), 3.61–3.68 (m, 1H), 4.7 (t, 1H, J = 5.7 Hz), 4.75 (d, 1H, J = 5.4 Hz), 4.84–4.93 (m, 2H), 7.65–7.67 (m, 1H), 8.08–8.11 (m, 2H)

[0443] Step 3: 2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-nitro-phenyl]acetaldehyde

[0444] Sodium periodate (5.27 g; 24.65 mmol) was gradually added to a solution of 3-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-nitro-phenyl]prop-1,2-diol (3.05 g; 8.22 mmol) in a mixture of water (18 mL) and THF (51 mL) at 0 °C. The reaction mixture was stirred from 0 °C to 10 °C for 2 hours, and then quenched with a saturated aqueous solution of sodium thiosulfate (50 mL) and water (50 mL). The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried with sodium sulfate, filtered, and concentrated to dryness at 30 °C to give the desired compound (2.61 g; 8.22 mmol) as a yellow oil, which was used in the next step. 1HNMR(DMSO): δ0.08(s,6H),0.90(s,9H),4.04(s,2H),4.72(s,2H),7.7(d,1 H, J=8.8Hz), 8.13 (d, 1H, J=2.5Hz), 8.18 (dd, 1H, J=2.4 and 8.5Hz), 9.69 (s, 1H)

[0445] Step 4: 2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-nitro-phenyl]ethanol

[0446] Sodium borohydride (0.47 g; 12.33 mmol) was added to a solution of 2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-nitro-phenyl]acetaldehyde (2.61 g;) in MeOH (40 mL) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1 hour, then quenched by adding brine (25 mL) and water (25 mL). The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by silica gel chromatography using cyclohexane / EtOAc (10 / 0-6 / 4) to give the desired compound (1.36 g; 4.36 mmol) as a yellow solid. 1 H NMR (DMSO): δ0.11 (s, 6H), 0.92 (s, 9H), 2.82 (t, 2H, J = 6.4Hz), 3.67 (q, 2H, J = 5.9 Hz),4.74(t,1H,J=5.1Hz),4.87(d,2H),7.66(d,1H,J=8.6Hz),8.08-8.11(m,2H)

[0447] Step 5: [2-(2-bromoethyl)-4-nitro-phenyl]methoxy-tert-butyl-dimethyl-silane

[0448] TEA (1.12 mL; 8.09 mmol) and methanesulfonyl chloride (0.47 mL; 6.07 mmol) were added to a solution of 2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-nitro-phenyl]ethanol (1.26 g; 4.05 mmol) in THF (25 mL) at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for 5 hours, then filtered and washed with THF. The filtrate was added to lithium bromide (1.76 g; 20.23 mmol) in THF (25 mL) at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for 4 days, then quenched with a saturated aqueous solution of ammonium chloride (50 mL). The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to dryness to give the desired compound (1.38 g; 4.03 mmol) as a yellow oil, which was used for the next step. 1 H NMR (DMSO): δ0.12 (s, 6H), 0.92 (s, 9H), 3.27 (t, 2H, J = 7.2Hz), 3.81 (t, 2H, J = 7. 2Hz),4.87(s,2H),7.68(d,1H,J=8.4Hz),8.13-8.16(m,1H),8.18-8.19(m,1H)

[0449] Step 6: [2-(2-bromoethyl)-4-nitro-phenyl]methanol

[0450] Acetic acid (42 mL; 737.28 mmol) was added to a solution of [2-(2-bromoethyl)-4-nitro-phenyl]methoxy-tert-butyl-dimethyl-silane (1.38 g) in THF (28 mL) and water (14 mL) at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for 4 days, then diluted with water (100 mL). The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by rapid silica gel chromatography using cyclohexane / EtOAc (10 / 0-6 / 4) as eluent to give the desired compound (0.73 g; 2.81 mmol) as a light brown solid. 1 HNMR(DMSO): δ3.27(t,2H,J=7.2Hz),3.8(t,2H,J=7.2Hz),4.67(s,2H),5.51(brs,1H),7.7(d,1H,J=8.4Hz),8.11-8.14(m,1H),8.15-8.16(m,1H)

[0451] Step 7: Sodium 2-[2-(hydroxymethyl)-5-nitro-phenyl]ethylsulfonyloxy

[0452] A solution of sodium sulfite (273 mg; 2.17 mmol) in water (1.1 mL) and tetrabutylammonium iodide (13 mg; 0.036 mmol) was added sequentially to a solution of [2-(2-bromoethyl)-4-nitro-phenyl]methanol (188 mg; 0.72 mmol) in EtOH (750 μL). The reaction mixture was heated at 70 °C under microwave irradiation for 35 min. The reaction mixture was diluted with water (20 mL) and washed with EtOAc. The aqueous layer was concentrated to dryness, and the crude product was purified by rapid silica gel chromatography using DCM / MeOH (10 / 0-6 / 4) as the eluent, followed by chromatography. 18 Rapid chromatographic purification was performed using ACN / water (2 / 98-34 / 66) as the eluent to give the desired compound (80 mg; 0.28 mmol) as a white solid. 1 H NMR(DMSO): δ2.66-2.70(m,2H),2.94-2.98(m,2H),4.64(d,2H,J=5.3Hz),5.47(t,1H) ,J=5.3Hz),7.68(d,1H,J=8.5Hz),7.99(d,1H,J=2.3Hz),8.06(dd,1H,J=2.4 and 8.4Hz)

[0453] Step 8: Sodium 2-[5-amino-2-(hydroxymethyl)phenyl]ethylsulfonyloxy

[0454] Pd / C 10% (55 mg; 0.052 mmol) was added to a solution of [2-(2-bromoethyl)-4-nitro-phenyl]methanol (293 mg; 1.03 mmol) in MeOH (11 mL) and water (2 mL) under an argon atmosphere. The reaction system was purged with hydrogen and stirred at room temperature under hydrogen pressure for 16 hours. The reaction mixture was filtered through a 40 μm PTFE filter, washed with a mixture of MeOH / water (1 / 1), and concentrated to dryness to give the desired compound (274 mg; 1.03 mmol) as a yellow solid, which was used in the next step. 1 H NMR(DMSO): δ2.55-2.59(m,2H),2.75-2.79(m,2H),4.32(d,2H,J=5.3Hz),5. 66(t,1H,J=5.2Hz),4.86(brs,2H),6.33-6.35(m,2H),6.93(d,1H,J=7.8Hz)

[0455] Step 9: Example 26

[0456] To a solution of sodium 2-[5-amino-2-(hydroxymethyl)phenyl]ethylsulfonyloxy sodium (304 mg; 1.15 mmol) in DMF (6 mL), add (2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-valeric acid (474 ​​mg; 0.96 mmol), HATU (472 mg; 1.24 mmol), and sodium bicarbonate (160 mg; 1.91 mmol). Stir the reaction mixture at room temperature for 20 hours, dilute with water (50 mL), and wash with EtOAc. Concentrate the aqueous layer to dryness and then use C... 18 The sample was purified by rapid chromatography using ACN / water + TFA (0.1%) (2 / 98-50 / 50) as the eluent. After lyophilization, it was obtained as Example 26 (83 mg; 0.11 mmol) as a pale yellow solid. 1 H NMR(DMSO): δ0.84-0.88(m,6H),1.32-1.49(m,2H),1.54-1.74(m,2H),1.94-2.04(m,1H),2.58-2. 62(m,2H),2.83-2.87(m,2H),2.90-3.05(m,2H),3.93(t,1H,J=8Hz),4.23-4.34(m,3H),4.37-4.4 3 (m, 1H), 4.45 (s, 2H), 5.34 (brs, 2H), 5.98 (brs, 1H), 7.25 (d, 1H, J = 8.1Hz), 7.30-7.34 (m, 2H), 7.39-7.44 (m, 4H), 7.74 (t, 2H, J = 7.2Hz), 7.89 (d, 2H, J = 7.5Hz), 8.08 (d, 1H, J = 7.5Hz), 9.90 (s, 1H). LCMS (2-100ACN / H2O + 0.1% TFA): 76.60% Tr = 8.1 min. Negative mode detection value: 708.45 (MH).

[0457] Example 27:N-{3-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy]propionyl}-L-valine-N-{4-[(5S,8S,11S,12R)-11-[(2S)-but-2-yl]-12-(2-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylprop-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolo-1-yl}-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-5,8-di(prop-2-yl)-2,13-dioxa-4,7,10-triazatetradecane-1-yl]-3-(2-sulfoethyl)phenyl}-N 5 -Carbamoyl-L-ornithine

[0458]

[0459] Step 1: 2-[5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]phenyl]ethanesulfonic acid

[0460] To the solution of Example 26 (32 mg, 0.0436 mmol) in DMF (0.5 mL), bis(4-nitrophenyl) carbonate (53.1 mg; 0.1747 mmol) and DIEA (91 μL; 0.5240 mmol) were added. The solution was stirred at room temperature for 4 hours. After evaporation to dryness under vacuum, the crude product was purified by silica gel chromatography to obtain the desired compound.

[0461] Step 2: 2-[5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]phenyl]ethanesulfonic acid

[0462] To a solution of MMAE (17 mg, 0.02368 mmol) in DMF (6 mL), add 2-[5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]phenyl]ethanesulfonic acid (41.4 mg; 0.0473 mmol), DIEA (20 μL; 0.1184 mmol), and HOBt (6.4 mg; 0.047 mmol). Stir the reaction mixture overnight at room temperature and use the solution for the next step.

[0463] Step 3: 2-[5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]phenyl]ethanesulfonic acid, TFA

[0464] 2-[5-[[(2S)-2-[[(2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1 DEA (24 μL; 0.2338 mmol) was added to a solution of [-methoxy-2-methyl-3-oxo-propyl]pyrrolidone-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]phenyl]ethanesulfonic acid, and the solution was stirred at room temperature for 1.5 hours. By using... C of the column 18 The reaction system was purified by reversed-phase preparative HPLC using the TFA method, and the desired compound was obtained after lyophilization.

[0465] Step 4: Example 27

[0466] 2-[5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]-5-ureo-valeryl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolidine-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl] [-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]phenyl]ethanesulfonic acid, TFA (9.5 mg; 7.1 mmol) was added to a solution of DMF (0.2 mL) with DIEA (2.5 μL; 0.014 mmol), followed by the addition of 3-[2-(2,5-dioxopyrrolidin-1-yl)ethoxy]propionic acid (2,5-dioxopyrrolidin-1-yl) ester (2.4 mg; 7.8 μmol). The reaction system was stirred overnight at room temperature. By using... C of the column 18 The reaction system was purified by reversed-phase preparative HPLC using the TFA method and then lyophilized to obtain Example 27. HRMS (ESI) [M+H] + Measured value = 1426.7443 (δ = -6.7 ppm)

[0467] Example 28 : 5-[[(2S)-2-[[(2S)-2-[6-(3,4-dibromo-2,5-dioxo-pyrrolo-1-yl)hexanoylamino]-3-methyl-butyryl]amino]-5-ureo-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1 R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3-oxo-propyl]pyrrolid-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid

[0468]

[0469] HATU powder (11.3 mg; 29.8 μmol) was added to a solution of 6-(3,4-dibromo-2,5-dioxo-pyrrolo-1-yl)hexanoic acid in DMF (0.2 mL). The reaction mixture was stirred at room temperature for 10 min, and then rutin (6.3 μL; 54.2 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. Then 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butyryl]amino]-5-ureido-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2 [-methyl-3-oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methylcarbamoyl]oxymethyl]benzenesulfonic acid (10 mg; 8.3 μmol; obtained from step 2 of Example 6), the reaction system was stirred at room temperature for 15 hours. By using... C of the column 18 Reversed-phase preparative HPLC was used, and the reaction system was purified using the TFA method. The resulting compound was then lyophilized to obtain the desired compound. HRMS (ESI) [M+H] + Measured value = 1552.5595 (δ = -2.5 ppm)

[0470] Example 34 :N-{[(9H-fluorene-9-yl)methoxy]carbonyl}-L-valine-N 5 -Carbamoyl-N-[4-(hydroxymethyl)-3-(3-sulfopropyl)phenyl]-L-ornithine

[0471]

[0472] Step 1: 2-Iodo-4-nitrobenzoic acid

[0473] To a solution of 2-amino-4-nitrobenzoic acid (10.0 g; 54.9 mmol) in ACN (280 mL), p-toluenesulfonic acid monohydrate (32.0 g; 168 mmol) was added. The mixture was stirred at room temperature for 15 minutes, followed by the dropwise addition of sodium nitrite (8.00 g; 115.9 mmol) and potassium iodide (24.0 g; 144.6 mmol) in water (140 mL) over 15 minutes. The reaction mixture was stirred for 19 hours. After the reaction was complete, the mixture was quenched with sodium thiosulfate (13.02 g; 82.36 mmol) and acidified with 3N (25 mL) aqueous hydrogen chloride solution. The aqueous layer was extracted with DCM, the combined organic layers were washed with 1N aqueous hydrogen chloride solution, dried over sodium sulfate, filtered, and concentrated to dryness to give the title compound (15.0 g; 51.2 mmol) as an orange powder. 1 H NMR (DMSO): 7.86 (d, 1H, J = 8.4Hz), 8.27 (d, 1H, J = 8.4Hz), 8.64 (s, 1H), 13.8 (brs, 1H)

[0474] Step 2: (2-iodo-4-nitrophenyl)methanol

[0475] A solution of 1N borane in THF (85 mL; 85.0 mmol) was added to a solution of 2-iodo-4-nitrobenzoic acid (5.00 g; 17.1 mmol) in THF (70 mL). The reaction mixture was stirred at 65 °C for 4 hours. After the reaction was complete, the reaction mixture was cooled to room temperature and quenched with MeOH (200 mL). The mixture was stirred at room temperature for 30 minutes and then concentrated to dryness. The residue was purified by silica gel column chromatography using cyclohexane / EtOAc (80 / 20-50 / 50) as eluent to give the title compound (3.38 g; 12.1 mmol) as a yellow solid. 1 H NMR (DMSO): δ4.47 (d, 2H, J = 5.2Hz), 5.82 (t, 1H, J = 5.2Hz), 7.70 (d, 1H, J = 8.8Hz), 8.29 (dd, 1H, J = 8.8 and 2.0Hz), 8.54 (d, 1H, J = 2.0Hz)

[0476] Step 3: (4-Amino-2-iodophenyl)methanol

[0477] Iron (3.70 g; 66.3 mmol) and ammonium chloride (800 mg; 15.0 mmol) were added sequentially to a solution of (2-iodo-4-nitrophenyl)methanol (3.70 g; 13.3 mmol) in EtOH (100 mL) and water (25 mL). The reaction mixture was stirred at 80 °C for 3 hours. After the reaction was complete, [the solution was used]. The reaction mixture was filtered, washed with EtOH, and concentrated to dryness. The resulting residue was dissolved in EtOAc and washed with a saturated sodium bicarbonate solution. The organic layer was dried over sodium sulfate, filtered, and concentrated to dryness to give the title compound (2.48 g; 9.95 mmol) as a yellow oil. 1 H NMR (DMSO): δ4.28 (d, 2H, J = 5.2Hz), 4.97 (t, 1H, J = 5.2Hz), 5.16 (s, 2H), 6.57 (d, 1H, J = 8.4Hz), 7.02-7.10 (m, 2H)

[0478] Step 4: 4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3-iodoaniline

[0479] Imidazole (0.95 g; 14.0 mmol) was added to a solution of (4-amino-2-iodophenyl)methanol (3.51 g; 13.4 mmol) in DCM (150 mL). The mixture was cooled to 0 °C, and then a solution of tert-butylchlorodimethylsilane (2.40 mL; 13.85 mmol) in DCM (150 mL) was added dropwise over 15 minutes. The ice bath was removed, and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was quenched with MeOH (20 mL) and concentrated to dryness. The residue was purified by silica gel column chromatography using cyclohexane / EtOAc (100 / 0-90 / 10) as eluent to give the title compound (3.64 g; 10.0 mmol) as a yellow oil. 1 H NMR (DMSO): δ0.06 (s, 6H), 0.88 (s, 9H), 4.46 (s, 2H), 5.24 (s, 2H), 6.55 (d, 1H, J = 8.4Hz), 7.03 (d, 1H, J = 8.4Hz), 7.05 (s, 1H)

[0480] Step 5: N-[4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3-iodophenyl]-N 5 -Carbamoyl-N 2 -{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-ornithine

[0481] Fmoc-Cit-OH (12.0 g; 30.28 mmol) and EEDQ (8.17 g; 33.0 mmol) were added sequentially to a solution of 4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3-iodoaniline (10.0 g; 27.5 mmol) in MeOH (70 mL) and DCM (140 mL). The reaction mixture was stirred at room temperature for 14 hours. After the reaction was complete, the residue was purified by silica gel column chromatography using DCM / MeOH (100 / 0-88 / 12) as the eluent to give the title compound (17.09 g; 22.0 mmol) as a white solid. 1 H NMR(DMSO): δ0.09(s,6H),0.91(s,9H),1.38-1.48(m,2H),1.59-1.68(m,2H),2.93-3 .05(m,2H),4.06-4.15(m,1H),4.20-4.29(m,3H),4.56(s,2H),5.41(s,2H),5.98(t,1 H, J=5.5Hz),7.30-7.43(m,5H),7.55(dd,1H,J=8.8 and 2.1Hz),7.69(d,1H,J=7.8Hz),7 .74(dd,2H,J=7.2 and 3.4Hz),7.89(d,2H,J=7.5Hz),8.25(d,1H,J=1.5Hz),10.12(s,1H)

[0482] Step 6: N-[4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3-iodophenyl]-N 5 -Carbamoyl-L-ornithine

[0483] To N-[4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3-iodophenyl]-N 5 -Carbamoyl-N 2 -{[(9H-fluorene-9-yl)methoxy]carbonyl}-L-ornithine (17.1 g; 23.0 mmol) was added to a solution of dimethylamine in 2M THF (44.5 mL; 89.0 mmol) in 120 mL of THF. The reaction mixture was stirred at room temperature for 15 hours. After concentrating to dryness, the solution was analyzed by C 18 The residue obtained by column chromatography was purified using water / ACN (98 / 02-0 / 100) as the eluent to give the title compound (5.47 g; 10.5 mmol) as a white solid. 1H NMR(DMSO): δ0.0(s,6H),0.81(s,9H),1.27-1.38(m,3H),1.47-1.53(m,1H),2.83-2.89(m,2H),3.16-3.19(m,1H),4.4 6(s,2H),5.26(s,2H),5.82(t,1H,J=5.6Hz),7.24(d,1H,J=8.5Hz),7.50(dd,1H,J=8.3 and 2.0Hz),8.17(d,1H,J=2.0Hz)

[0484] Step 7: N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-valine-N-[4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3-iodophenyl]-N 5 -Carbamoyl-L-ornithine

[0485] To N-[4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3-iodophenyl]-N 5 Fmoc-Val-OSu (8.65 g; 8.65 mmol) and DIPEA (1.90 mL; 11.5 mmol) were added sequentially to a solution of 3-carbamoyl-L-ornithine (3.00 g; 5.76 mmol) in 2-methyltetrahydrofuran (240 mL). The reaction mixture was stirred at room temperature for 15 hours. The reaction mixture was filtered through a sintered funnel, the recovered solid was washed with 2-methyltetrahydrofuran, and then dried under high vacuum to give the title compound (3.57 g; 4.24 mmol) as a white solid. 1 H NMR(DMSO): δ0.10(s,6H),0.83-0.95(m,15H),1.27-1.52(m,2H),1.52-1.75(m,2H),1.93-2 .07(m,1H),2.88-3.09(m,2H),3.93(t,1H,J=8.0Hz),4.17-4.49(m,4H),4.56(s,2H),5.40( s,2H),5.96(t,1H,J=5.6Hz),7.27-7.37(m,3H),7.37-7.48(m,3H),7.54(d,1H,J=8.0Hz),7 .74(t,2H,J=7.2Hz),7.88(d,2H,J=7.6Hz),8.13(d,1H,J=7.6Hz),8.22(s,1H),10.11(s,1H)

[0486] Step 8: N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-valine-N-[4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3-(3-hydroxyprop-1-yn-1-yl)phenyl]-N 5 -Carbamoyl-L-ornithine

[0487] To N-{[(9H-fluorene-9-yl)methoxy]carbonyl}-L-valine-N-[4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3-iodophenyl]-N 5 1,3-Carbamoyl-L-ornithine (4.0 g; 4.55 mmol) and 2-propyn-1-ol (0.54 mL; 9.10 mmol) in a solution of 1,3-carbamoyl-L-ornithine (4.0 g; 4.55 mmol) and 2-propyn-1-ol (0.54 mL; 9.10 mmol) in dry DMF (36 mL) were added to DIPEA (2.26 mL; 13.66 mmol; 3.0 eq.). The reaction mixture was purged with argon, and then bis(triphenylphosphine)palladium(II) dichloride (0.64 g; 0.91 mmol) and cuprous iodide(I) (0.17 g; 0.91 mmol) were added. The reaction mixture was purged with argon again and stirred at room temperature for 16 hours. A saturated ammonium chloride solution (80 mL) was added, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by silica gel rapid chromatography using DCM / MeOH (10 / 0-85 / 15) as the eluent to obtain the title product (2.7 g; 3.52 mmol) as a brown solid. 1 H NMR(DMSO-d6):0.08(s,6H),0.85-0.89(m,6H),0.90(s,9H),1.32-1.48(m,2H), 1.54-1.74(m,2H),1.94-2.03(m,1H),2.89-3.06(m,2H),3.93(dd,1H,J=7.0and 9.1Hz),4.22-4.33(m,5H),4.36-4.41(m,1H),4.74(s,2H),5.35(t,1H,J=5.9Hz),5.40(s,2H),5.96(t,1H,J=6.0Hz),7.32(t,2H,J=7.4Hz), 7.36-7.43(m,4H),7.48(dd,1H,J=2.3 and 8.8Hz),7.74(t,2H,J=7.7Hz),7.78(s,1H),7.89(d,2H,J=7.8Hz),8.14(d,1H,J=7.1Hz),10.09(s,1H)

[0488] Step 9: N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-valine-N-[4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3-{3-[(methanesulfonyl)oxy]prop-1-yn-1-yl}phenyl]-N 5 -Carbamoyl-L-ornithine

[0489] At 0°C, N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-valine-N-[4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3-(3-hydroxyprop-1-yn-1-yl)phenyl]-N 5 1.0 g; 1.3 mmol of α-carbamoyl-L-ornithine (DIPEA) and 0.15 mL; 1.95 mmol of methanesulfonyl chloride were added sequentially to a solution of α-carbamoyl-L-ornithine (DMF) in 9 mL of dry DMF. The reaction mixture was slowly warmed to room temperature and stirred for 1 hour. A saturated solution of ammonium chloride (25 mL) was added, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to dryness to give the title product (1.1 g; 1.3 mmol) as an orange resin, which was used in the next step. 1 H NMR(DMSO-d6): 0.08(s,6H),0.84-0.89(m,6H),0.90(s,9H),1.32-1.49(m,2H),1.54-1.74(m,2H),1.94-2.03(m ,1H),2.91-3.05(m,2H),3.29(s,3H),3.93(dd,1H,J=7.1 and 9.0Hz),4.20-4.33(m,3H),4.37-4.42(m,1H),4.75(s, 2H),5.23(s,2H),5.4(s,2H),5.96(t,1H,J=5.8Hz),7.32(t,2H,J=7.2Hz),7.39-7.43(m,4H),7.56(dd,1H,J=2.3 and 8.8Hz), 7.74 (t, 2H, J = 7.4Hz), 7.83 (d, 1H, J = 2.3Hz), 7.89 (d, 2H, J = 7.3Hz), 8.14 (d, 1H, J = 7.3Hz), 10.15 (s, 1H)

[0490] Step 10: N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-valine-N-{3-[3-(acetylthioalkyl)prop-1-yn-1-yl]-4-({[tert-butyl(dimethyl)silyl]oxy}methyl)phenyl}-N 5 -Carbamoyl-L-ornithine

[0491] To N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-valine-N-[4-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3-{3-[(methanesulfonyl)oxy]prop-1-yn-1-yl}phenyl]-N 5 1-Carbamoyl-L-ornithine (1.1 g; 1.3 mmol) was added to a solution of potassium thioacetate (0.3 g; 2.6 mmol) in dry DMF (10 mL). The reaction mixture was stirred at room temperature for 16 hours. A saturated sodium bicarbonate solution (35 mL) was added, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by silica gel chromatography using DCM / MeOH (10 / 0–9 / 1) as eluent to give the title product (0.52 g; 0.62 mmol) as a pale yellow solid. 1 H NMR(DMSO-d6): 0.08(s,6H),0.84-0.89(m,6H),0.90(s,9H),1.31-1.49(m,2H),1.54-1.73(m,2H),1.95-2 .02(m,1H),2.4(s,3H),2.89-3.06(m,2H),3.93(dd,1H,J=6.9 and 9.1Hz),3.97(s,2H),4.20-4.33(m,3H),4. 36-4.41(m,1H),4.69(s,2H),5.39(s,2H),5.96(t,1H,J=5.8Hz),7.32(t,2H,J=7.2Hz),7.35-7.43(m,4H) ,7.49(dd,1H,J=2.2 and 8.5Hz),7.72-7.76(m,3H),7.89(d,2H,J=7.5Hz),8.12(d,1H,J=7.4Hz),10.08(s,1H)

[0492] Step 11: N-{[(9H-fluorene-9-yl)methoxy]carbonyl}-L-valine-N 5 -Carbamoyl-N-[4-(hydroxymethyl)-3-(3-sulfopropyl-1-yn-1-yl)phenyl]-L-ornithine

[0493] Formic acid (9.68 mL; 256.5 mmol) was added to a 35% hydrogen peroxide solution (1.08 mL; 12.57 mmol) at 0 °C. The solution was stirred for 1 hour, and then N-{[(9H-fluorene-9-yl)methoxy]carbonyl}-L-valine-N-{3-[3-(acetylthioalkyl)prop-1-yn-1-yl]-4-({[tert-butyl(dimethyl)silyl]oxy}methyl)phenyl}-N5 3-Carbamoyl-L-ornithine (367 mg; 0.44 mmol). The reaction mixture was stirred for 2 hours, diluted with water (20 mL), and concentrated to dryness at 35 °C. The residue was suspended in 1 N hydrochloric acid (25 mL) and washed with DCM / MeOH (7 / 3). The aqueous layer was concentrated to dryness at 35 °C. The solution was then passed through C... 18 The crude product was purified by rapid chromatography using ACN / water + TFA (0.1%) (2 / 98-50 / 50) as the eluent. After lyophilization, the title product (117 mg; 0.13 mmol) was obtained as a white solid. 1 H NMR(DMSO-d6): δ0.84-0.88(m,6H),1.33-1.50(m,2H),1.55-1.75(m,2H),1.9 5-2.02(m,1H),2.89-3.05(m,2H),3.58(s,2H),3.91-3.95(m,1H),4.20-4.33 (m,3H),4.37-4.42(m,1H),4.55(s,2H),5.22(brs,1H),5.38(s,2H),5.96(t, 1H, J=5.6Hz),7.32(t,2H,J=7.2Hz),7.36-7.43(m,4H),7.48(dd,1H,J=2.4and 9.2Hz), 7.65(d, 1H, J = 1.6Hz), 7.74(t, 2H, J = 6.8Hz), 7.89(d, 2H, J = 7.4Hz), 8.12(d, 1H, J = 7.6Hz), 10.03(s, 1H). LCMS (2-100ACN / H2O + 0.05% TFA): 77.27%, Rt = 8.3min. Positive mode detection value: 720.26(M+H) + )

[0494] Step 12: Example 34

[0495] In an argon atmosphere, N-{[(9H-fluorene-9-yl)methoxy]carbonyl}-L-valine-N 510 mg; 0.014 mmol of 1-carbamoyl-N-[4-(hydroxymethyl)-3-(3-sulfoprop-1-yn-1-yl)phenyl]-L-ornithine (10 mg; 0.014 mmol) was added to a suspension of 5% Pt / C (7 mg; 0.0018 mmol) in THF (600 μL) and acetic acid (60 μL). The reaction mixture was purged three times with hydrogen and then stirred at room temperature for 64 h. The reaction mixture was filtered through a 45 μm PTFE filter, washed with MeOH and MeOH / water (1 / 1), and then concentrated to dryness to give Example 34 (7 mg) as an orange oil. LCMS (2-100 ACN / H2O + 0.05% FA): 6.93%, Rt = 8.8 min. Negative mode detection value 722.19 (MH).

[0496] As performed in Examples 6-12, 14-25, 27 or 28, 34, the p-amino-benzyl linker compound of formula (I) can be used to prepare the linker-drug compound of formula (II) and to prepare the antibody-drug conjugate of formula (III) of the present invention.

[0497] Example A ADC conjugation and analytical characterization

[0498] As used throughout this application, antibody-drug conjugates can be identified using the general form of “target antigen / antibody-connector-drug” according to naming conventions. By way of example only, if an antibody-drug conjugate is referred to as “target X-example Y”, then such a conjugate comprises an antibody that binds to target X and a connector-drug exemplified in example Y.

[0499] 1. Adhesion

[0500] The exemplary ADC was synthesized using one of the methods described below. The antibodies used in the synthesis of the exemplary ADC are defined by the abbreviations Ab T TG and Ab T (Table 1).

[0501] Table 1. Antibodies used for the purposes of this invention

[0502]

[0503] Antibody T TG confers bacterial transglutaminase (BTG)-reactive glutamine with a cyclooctylene-containing amine BCN (or N-[(1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxane). This is performed using site-specific antibody conjugation of bacterial transglutaminase to the BCN moiety as described in InnatePharma 2013 (presented at ADC Summit, San Francisco, California, October 15, 2013), WO2017 / 059160A1, and WO 2016 / 144608A1. These modifications allow for the conjugation of the azide-containing precursor using method A. Figure 1 ).

[0504] By completely reducing the four interchain disulfide bonds and then conjugating with an excess of drug, a conjugate of Ab T was obtained. Figure 2 For the purposes of this invention, a water-soluble tertiary phosphine such as tri-(2-carboxyethyl)phosphine (TCEP) was used as a reducing agent. Complete reduction and conjugation primarily yielded a full load of 8 drugs / antibodies, and ultimately produced illustrative average DAR values ​​of 7.1 and 7.7 for 6 drug-loaded mAbs (Table 2).

[0505] Some Ab T conjugates were generated using the rebridging technique described in Method D below. The four interchain disulfide bonds of Ab T were completely reduced with TCEP, and the linker-load was reformed using dibromomaleimide-functionalized linkers. Figure 3 Due to the proximity of cysteine ​​residues in the interchain disulfide bridges on each heavy chain involved in the antibody, the conjugation produces two conformations.

[0506] Method A (DAR2)

[0507] Dimethyl sulfoxide (DMSO, 619 μl, 20% coupling volume) was added to AB solution (11.8 mg / mL; 2.5 mL, 4.5 mg). The mixture was vortexed for 30 seconds, and then a 4-molar excess of the linker-projectile effective load (20 mM, 6 μL, in DMSO) was added. The reaction system was stirred overnight at 64 rpm at room temperature. To remove unconjugated linker-effective load, a 10-molar excess of DBCO-containing TentaGel resin (0.1–0.2 mmol / g, Iris Biotech, CS-0477.0500) was added, and the mixture was stirred at room temperature for 6 hours. Using Vivaspin 20, 50KD, PES (Sartorius Stedim, VS2031), the buffer was exchanged with PBS 1X (Sigma Life Science, P3813, 10PAK) through 3 filtration cycles, then aseptically filtered through a 0.2μm sterile PES filter, 25mm (Whatmann, G896-2502), and stored at 4°C.

[0508] Method B (DAR8)

[0509] The Ab solution was diluted (10.3 mg / mL; 0.5 mL) in EDTA containing PBS buffer at pH 7.4 (10 mM; 0.5 mL) to fix the final EDTA concentration of 5 mM for the coupling reaction. Then, 849 μL of TCEP (1 mM, in PBS buffer at pH 7.4) was added to the antibody, followed by incubation at 37 °C for 2 h. After reduction, the antibody solution was cooled to 2–8 °C, and a 20-fold molar excess of adapter-effective loading (5 mM, 141 μL, in DMSO) was added. The reaction mixture was incubated at 4 °C for 1.5 h. The solution was centrifuged (14000 g, 4 °C) for 20 min and loaded onto a HiLoad 26 / 600 Superdex 200 pg (GE Healthcare, 28989336) SEC column. The ADC was eluted with 20% DMA in PBS (Sigma Life Science, P3813, 10PAK), followed by two cycles of dialysis (16 and 4 hours) in PBS 1X pH 7.4 (Sigma Life Science, P3813, 10PAK). The conjugate was concentrated using Vivaspin 20, 50 kDa, PES (Sartorius Stedim, VS2031), filtered through a 0.2 μm sterile PES filter, sterilely filtered through a 25 mm (Whatmann, G896-2502) filter, and stored at 4°C.

[0510] Method C (DAR8)

[0511] The Ab solution (10.3 mg / mL; 0.5 mL) was diluted in EDTA containing PBS buffer at pH 7.4 (10 mM; 0.5 mL) to fix a final EDTA concentration of 5 mM for the coupling reaction. Then, TCEP (1 mM, 849 μL in PBS buffer at pH 7.4) was added to the antibody, followed by incubation at 37 °C for 2 h. After reduction, the antibody solution was cooled to 2–8 °C, and a 20-fold molar excess of the adapter-effective load (5 mM, 141 μL in DMSO) was added. The reaction mixture was incubated at 4 °C for 1.5 h. The conjugate was then purified using rmp protein A resin (GE Healthcare, 17-5138-01) followed by two cycles of dialysis (16 and 4 h) in PBS 1X pH 7.4 (SigmaLife Science, P3813, 10 PAK). The ADC was concentrated using Vivaspin 20, 50KD, PES (Sartorius Stedim, VS2031), passed through a 0.2μm sterile PES filter, aseptically filtered through a 25mm (Whatmann, G896-2502) filter, and stored at 4°C.

[0512] Method D (DAR4)

[0513] Add 8 molar excess of TCEP (1 mM in pH 8 BBS, 137 μl) to an Ab solution (5 mg / ml, 0.5 ml) in pH 8 BBS buffer (prepared as described below), and incubate the reaction system at 37 °C for 2 h. Then, add 7.5 molar excess of adapter-effective loading (1 mM, 129 μL, in DMF), and mix the resulting solution at 600 rpm for 1 h at room temperature. Dialyze the resulting ADC in PBS 1X pH 7.4 (Sigma Life Science, P3813, 10 PAK) at 4 °C for 16 h. The conjugate was then purified using RMP protein A resin (GE Healthcare, 17-5138-01), followed by dialyzing in PBS 1X pH 7.4 (Sigma Life Science, P3813, 10 PAK) at room temperature for 2 hours. The ADC was concentrated using Vivaspin 20, 50 kDa, PES (Sartorius Stedim, VS2031), filtered through a 0.2 μm sterile PES filter, sterilely filtered through a 25 mm (Whatmann, G896-2502) filter, and stored at 4°C.

[0514] Preparation of BBS buffer at pH 8 ：Dissolve 528 mg of Na₂B₄O₇·10H₂O in 27.7 mL of deionized water. Dissolve 63 mg (25 mM) of sodium chloride and 16 mg (1 mM) of EDTA in 22.3 mL of 0.1 M hydrochloric acid. Then, mix the two solutions together and use the resulting solution directly in the conjugation step.

[0515] 2. LC-MS General Method

[0516] The drug-to-antibody ratio (DAR) of exemplary ADCs was determined by LC-I or LC-II using liquid chromatography-mass spectrometry (LC-MS) as follows. For the LC-I method, mobile phase A was purified by MS-grade water (Biosolve, Dieuze, France, 00232141B1BS), mobile phase B by MS-grade acetonitrile (Biosolve, Dieuze, France, 0001204101BS), and mobile phase D by MS-grade water supplemented with 1% formic acid (FA) (Honeywell / Fluka, Bucharest, Romania, 56302). Mobile phase D was kept constant at 10% to maintain a 0.1% FA mobile phase composition, and the column temperature was set at 80°C. General MS methods were optimized for all synthesized ADCs (Table 2). For the LC-II method, mobile phase A was purified by MS-grade water (Biosolve, Dieuze, France, 00232141B1BS), and mobile phase B was purified by MS-grade acetonitrile (Biosolve, Dieuze, France, 0001204101BS), both containing 0.1% formic acid (FA) (Honeywell / Fluka, Bucharest, Romania, 56302). The column temperature was set at 80 °C. The general MS method was optimized for all synthesized ADCs (Table 2).

[0517] Method LC-IThe ADC was loaded onto a Bioresolve RP mAb Polyphenyl, 450A, 2.7 μm, 2.1 x 150 mm (Waters, Saint-Quentin-en-Yvelines, France, 186008946). For analysis under both intact and reducing conditions, a desalting step was performed for 1.5 min at 0.6 mL / min in 20% B. Elution was performed in gradients from 1.5 min in 20% B to 16.5 min in 70% B at a flow rate of 0.3 mL / min. Washing was performed at 90% B for 16.8 min to 18.8 min at a flow rate of 0.6 mL / min. Finally, an adaptation step was performed at 19.1 min in 20% B for 1.9 min at a flow rate of 0.6 mL / min (total run time = 21 min).

[0518] Method LC-II The ADC was loaded onto a Bioresolve RP mAb Polyphenyl, 450A, 2.7 μm, 2.1 x 150 mm (Waters, Saint-Quentin-en-Yvelines, France, 186008946). For analysis under both intact and reducing conditions, a desalting step was performed for 1.5 min at 0.6 mL / min in 20% B. An elution step was then performed at the same flow rate, with a gradient from 20% B for 1.5 min to 50% B for 16.5 min. The subsequent washing step was set at 100% B for 16.8 min to 18.8 min at a flow rate of 0.6 mL / min. Finally, the column was stabilized using an adaptation step, transitioning from 100% to 20% B at 18.8 min to 19.2 min, followed by an additional 1.9 min at 20% B using the aforementioned flow rate (total run time = 21 min).

[0519] LC-MS analysis was performed using a Waters UPLC H-Class Bio chromatography system connected to a Xevo G2 XSQ-TOF ESI mass spectrometer (Waters, Manchester, UK). ADCs were analyzed under intact conditions (no pretreatment) or after reduction with 5 mM (final concentration) dithiothreitol DTT (Thermo Scientific, Rockford, IL, 20291). Subsequently, the ADCs were analyzed using the LC-I and LC-II methods described above (Table 2). MassLynx was used. TM Electrospray ionization time-of-flight mass spectra of the analytes were acquired using acquisition software (Waters, Manchester, UK). Then, MassLynx was used. TMThe software's maximum entropy method deconvolvees the extracted intensity-to-m / z spectra to determine the quality of each intact antibody species or each reduced antibody fragment, depending on the processing used. Finally, the DAR is determined from the deconvolution spectrum or UV chromatogram by summing the integrated MS (total ion current) or UV (280 nm) peak areas of unconjugated and conjugated species (MAH or related fragments). For DAR determination via UV chromatogram, the relative area percentage of each species is multiplied by the number of drug molecules linked. The summed weighted area of ​​each species is divided by the sum of the total relative area percentages, and the result yields an estimate of the final average DAR value for the intact ADC. For DAR determination via deconvolution spectrum, the percentage of each species identified is calculated from the intensity peaks from the deconvolution spectrum. The obtained percentage is multiplied by the number of drug molecules linked. The summation yields an estimate of the final average DAR value for the intact ADC.

[0520] 3. Size exclusion chromatography

[0521] Size exclusion chromatography (SEC) was performed to determine the quality of the ADC and its percentage of aggregates after purification. Analysis was conducted on an analytical column, Superdex 200 Increase 5 / 150 GL (GE Healthcare, 28990945), under isocratic conditions with 100% PBS pH 7.4 (Sigma Life Science, P3813, 10PAK), at a flow rate of 0.45 mL / min for 12 minutes. The percentage of aggregates in the conjugate sample was quantified based on the peak area absorbance at 280 nm. This was calculated by dividing the ratio between the high molecular weight eluents at 280 nm by the sum of the peak area absorbances of the high molecular weight and monomer eluents at the same wavelength, multiplied by 100.

[0522] 4. Hydrophobic interaction chromatography

[0523] Hydrophobic interaction chromatography (HIC) was performed to determine the effects of linker-load and bioconjugation techniques on the hydrophobicity of the antibody. Analysis was performed using a TSKgel Butyl-NPR column (Tosoh Bioscience, 0014947) with mobile phases A (1.5 M ammonium sulfate (NH4)2SO4, 25 mM dipotassium hydrogen phosphate (K2HPO4), adjusted to pH 7) and B (25 mM dipotassium hydrogen phosphate (K2HPO4), 20% isopropanol, adjusted to pH 7). Elution was initiated at 0.6 mL / min in 5% B. A gradient was applied from 5% to 100% B over 17 min, followed by washing at 100% B for 5 min. The final adaptation step was performed at 100%–5% B for 2 min, followed by 2 min in 5% B.

[0524] For the purposes of this invention, the relative retention time (RRT) of each ADC is calculated by dividing the ADC retention time (RT) by the antibody RT (Table 3).

[0525] 5. result

[0526] Table 2 summarizes the characterization of the exemplary ADCs (named Examples 15-22 and 29-33; coupling and LC-MS methods, aggregation status, and DAR). The average DAR value was determined using the LC-MS method LC-I or LC-II described above (Section 2), and the percentage of aggregates was measured by size exclusion chromatography (SEC) described above (Section 3).

[0527] Table 2. ADC Analysis, Characterization, and Coupling Methods

[0528]

[0529] The hydrophobicity of the exemplary ADCs was compared by calculating the relative retention times using the HIC chromatography method described above (Section 4). Table 3 summarizes the RRT values ​​of the conjugates.

[0530] Table 3. Comparison of ADC hydrophobicity using RRT via HIC chromatography

[0531]

[0532]

[0533] Stability studies were performed on all exemplary ADCs, with the conjugates incubated in PBS buffer at +37°C for one week under accelerated degradation conditions. Aggregation of the conjugates was measured using SEC chromatography. Examples of the results are shown in Table 4.

[0534] Table 4. Stability study of ADC in PBS buffer under accelerated degradation conditions

[0535]

[0536]

[0537] Example B: Detection of the effect of an exemplary ADC on cell viability using CTG assay

[0538] HCC1954 (Her+) and MOLT4 (Her-) cell lines were cultured in RPMI supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 IU / ml), streptomycin (100 μg / ml), and L-glutamine (2 mM). Cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO2. Cells were seeded in 96-well microplates and exposed to ADC for 120 hours (5-fold serial dilutions; 9 concentrations each, triplicate). After 5 days of incubation at 37°C / 5% CO2, the effect of ADC on cell viability was assessed by quantifying cellular ATP levels using 75 μL of CellTiterGlo reagent per well. All conditions were tested in triplicate. Quantitative luminescence was performed on a multi-purpose plate reader. IC50 was calculated using standard four-parameter curve fitting. 50 IC 50 The concentration of the compound is defined as the point at which the CTG signal decreases to 50% of the control CTG signal. Each experiment is performed at least twice to ensure reproducibility of results.

[0539] As shown in Table 5, in the CTG assay, the ADCs of all examples were active in antigen-positive cells.

[0540] Table 5. Exemplary ADC effects on cell viability using CTG assay

[0541]

Claims

1. A p-amino-benzyl linker compound of formula (I): ###0001### (I) wherein: • R1 represents a hydroxyl group or a halogen atom; • A1 represents a -C(O)-CH(R3)-NH- group; • R3 and R4 represent, independently from each other, an amino acid side chain; • X represents a hydrogen atom, a hydroxyl group or a protecting group; 2. The p-amino-benzyl linker compound of claim 1, wherein R1 represents a hydroxyl group, a bromine atom, a chlorine atom or an iodine atom.

4. The p-amino-benzyl linker compound of claim 1, wherein A1 represents a -C(O)-CH(R3)-NH- group, wherein R3 represents a -(CH2)3-NH-CO-NH2 group or a methyl group.

6. The p-amino-benzyl linker compound of claim 1, wherein A1 represents a -C(O)-CH(R3)-NH- group and A2 represents a -C(O)-CH(R4)-NH- group, wherein R3 and R4 represent, independently from each other, an amino acid side chain. • R2 represents a -S(O)2(OH) group, a -S(O)2(O - M + ) group, a linear or branched -(Ci-C4)alkyl-S(O)2(OH) group, a linear or branched -(Ci-C4)alkyl-S(O)2(O - M + ) group, a linear or branched -halo(Ci-C4)alkyl-S(O)2(OH) group or a linear or branched -halo(Ci-C4)alkyl-S(O)2(O - M + ) group; 7. The p-amino-benzyl linker compound of claim 1, which is: - sodium 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl- butyryl]amino]-5-ureido-pentanoyl]amino]-2-(hydroxymethyl)benzenesulfonate; - sodium 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl- butyryl]amino]propanoyl]amino]-2-(hydroxymethyl)benzenesulfonate; - 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl- butyryl]amino]propanoyl]amino]-2-(hydroxymethyl)benzenesulfonic acid; - sodium [5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl- butyryl]amino]-5-ureido-pentanoyl]amino]-2-(hydroxymethyl)phenyl]methanesulfonate; - 2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3- methyl-butyryl]amino]-5-ureido-pentanoyl]amino]benzenesulfonic acid; - 2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3- methyl-butyryl]amino]propanoyl]amino]benzenesulfonic acid; - 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl- butyryl]amino]-5-ureido-pentanoyl]amino]-2-(iodomethyl)benzenesulfonic acid. • A2 represents a -C(O)-CH(R4)-NH- group, group, group or group; 8. The p-amino-benzyl linker compound of formula (I) according to any one of claims 1 to 7 for use in the manufacture of an antibody-drug conjugate.

9. A linker-drug compound of formula (II): ###0002### (II) wherein: • D represents a drug moiety; • M + represents a pharmaceutically acceptable monvalent cation. ​ 3. The p-amino-benzyl linker compound of claim 1, wherein R2 represents a -S(O)2(OH) group, a -S(O)2(O - M + ) group, a -CH2-S(O)2(OH) group, a -CH2-S(O)2(O - M + ) group, a -CH2-CH2-S(O)2(OH) group, a -CH2-CH2-S(O)2(O - M + ) group, a -CH2-CH2-CH2-S(O)2(OH) group, or a -CH2-CH2-CH2-S(O)2(O - M + ) group. ​ 5. The p-amino-benzyl linker compound of claim 1, wherein A2 represents a -C(O)-CH(R4)-NH- group, wherein R4 represents isopropyl; group, group or group. ​ ​ ​ ​ ​ ​ ​ ​ ​ - N-{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-valyl-N 5 - Carbamoyl-N-[4-(hydroxymethyl)-3-(2-sulfonatoethyl)phenyl]-L- ornithinamide sodium; - N- { [(9H-fluoren-9-yl)methoxy] carbonyl} -L-valyl-N 5 - carbamoyl-N-[4-(hydroxymethyl)-3-(3-sulfonatopropyl)phenyl]-L- ornithinamide. ​ ​ ​ ​ • T represents a valence bond, -0-C(0)-N(CH3)-CH2-CH2-N(CH3)-C(0)-*, -0-*, -NR5-*, -NR5-C(0)-* or -0-C(0)-*, wherein * denotes the point of attachment to D; • R2 represents a -S(O)2(OH) group, a -S(O)2(O - M + ) group, a linear or branched -(Ci-C4)alkyl-S(O)2(OH) group, a linear or branched -(Ci-C4)alkyl-S(O)2(O - M + ) group, a linear or branched -halo(Ci-C4)alkyl-S(O)2(OH) group or a linear or branched -halo(Ci-C4)alkyl-S(O)2(O - M + ) group; • A1represents a -C(0)-CH(R3)-NH- group; • A2 represents: -C(O)-CH(R4)-NH- group; group, group or group; • R3and R4independently of one another represent an amino acid side chain; • R5represents a hydrogen atom or a (Ci-C4)alkyl group; • Z' represents a spacer unit precursor; • M + represents a pharmaceutically acceptable monvalent cation.

10. The linker-drug compound of claim 9, wherein R2 represents a -S(O)2(OH) group, a -S(O)2(O - M + ) group, a -CH2-S(O)2(OH) group, a -CH2-S(O)2(O - M + ) group, a -CH2-CH2-S(O)2(OH) group, a -CH2-CH2-S(O)2(O - M + ) group, a -CH2-CH2-CH2-S(O)2(OH) group, or a -CH2-CH2-CH2-S(O)2(O - M + ) group.

11. The linker-drug compound of claim 9, wherein A1represents a -C(0)-CH(R3)-NH- group, wherein R3represents a -(CH2)3-NH-CO-NH2group or a methyl group.

12. The linker-drug compound of claim 9, wherein A2 represents a -C(O)-CH(R4)-NH- group, wherein R4 represents isopropyl; group, group or group.

13. The linker-drug compound of claim 9, wherein T represents a valence bond or -0-C(0)-*, wherein * denotes the point of attachment to D.

14. The linker-drug compound of claim 9, wherein Z' represents a group selected from: wherein the wavy line denotes the point of covalent attachment to the N-terminus of the A2group or to the carbonyl group.

15. The linker-drug compound of claim 9, which is: wherein R2and D are as defined in claim 9.

16. The linker-drug compound of claim 9, which is: - 5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolidin-1-yl)ethoxy]propionylamino]-3- methyl-butyryl]amino]-5-carbamoyl-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4- [(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2- methyl-3-oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo- butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methyl- carbamoyl]oxymethyl]benzenesulfonic acid sodium salt; - 5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolidin-1-yl)ethoxy]propionylamino]-3- methyl-butyryl]amino]-5-carbamoyl-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4- [(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2- methyl-3-oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxo- butyl]-methyl-carbamoyl]-2-methyl-propyl]carbamoyl]-2-methyl-propyl]-methyl- carbamoyl]oxymethyl]benzenesulfonic acid sodium salt; ​ - 5 - [ [ (2S) -2 - [ [ (2S) -2 - [ [2 - [2 - (2-azidoethoxy) ethoxy] ethoxy] acetyl] amino] -3 - methyl-butyryl] amino] -5 -ureidovale nyl] amino] -2 - [ [ [ (1S) -1 - [ [ (1S) -1 - [ [ (1S, 2R) -4 - [ (2S) -2 - [ (1R, 2R) -3 - [ [ (1R, 2S) -2 -hydroxy-1 -methyl-2-phenyl-ethyl] amino] -1 - methoxy-2-methyl-3-oxo-propyl] pyrrolidin-1-yl] -2-methoxy-1 - [ (1S) -1 -methylpropyl] -4- oxo-butyl] -methyl-carbamoyl] -2-methyl-propyl] carbamoyl] -2-methyl-propyl] -methyl- carbamoyl] oxy-methyl] benzenesulfonic acid sodium salt; - 5 - [ [ (2S) -2 - [ [ (2S) -2 - [ [2 - [2 - (2, 5-dioxopyrrolidin-1-yl) ethoxy] ethoxy] ethoxy] acetyl] amino] -3 -methyl-butyryl] amino] -5 -ureidovale nyl] amino] -2 - [ [ [ (1S) -1 - [ [ (1S) -1 - [ [ (1S, 2R) -4 - [ (2S) -2 - [ (1R, 2R) -3 - [ [ (1R, 2S) -2 -hydroxy-1 -methyl-2-phenyl-ethyl] amino] -1 - methoxy-2-methyl-3-oxo-propyl] pyrrolidin-1-yl] -2-methoxy-1 - [ (1S) -1 -methylpropyl] -4- oxo-butyl] -methyl-carbamoyl] -2-methyl-propyl] carbamoyl] -2-methyl-propyl] -methyl- carbamoyl] oxy-methyl] benzenesulfonic acid; - 5 - [ [ (2S) -2 - [ [ (2S) -2 - [ [2 - [2 - (2, 5-dioxopyrrolidin-1-yl) ethoxy] ethoxy] ethoxy] acetyl] amino] -3 -methyl-butyryl] amino] -5 -ureidovale nyl] amino] -2 - [ [ [ (1S) -1 - [ [ (1S) -1 - [ [ (1S, 2R) -4 - [ (2S) -2 - [ (1R, 2R) -3 - [ [ (1R, 2S) -2 -hydroxy-1 -methyl-2-phenyl-ethyl] amino] -1 - methoxy-2-methyl-3-oxo-propyl] pyrrolidin-1-yl] -2-methoxy-1 - [ (1S) -1 -methylpropyl] -4- oxo-butyl] -methyl-carbamoyl] -2-methyl-propyl] carbamoyl] -2-methyl-propyl] -methyl- carbamoyl] oxy-methyl] benzenesulfonic acid; - 5 - [ [ (2S) -2 - [ [ (2S) -2 - [ [2 - [2 - (2, 5-dioxopyrrolidin-1-yl) ethoxy] ethoxy] ethoxy] acetyl] amino] -3 -methyl-butyryl] amino] -5 -ureidovale nyl] amino] -2 - [ [ [ (1S) -1 - [ [ (1S) -1 - [ [ (1S, 2R) -4 - [ (2S) -2 - [ (1R, 2R) -3 - [ [ (1R, 2S) -2 -hydroxy-1 -methyl-2-phenyl-ethyl] amino] -1 - methoxy-2-methyl-3-oxo-propyl] pyrrolidin-1-yl] -2-methoxy-1 - [ (1S) -1 -methylpropyl] -4- oxo-butyl] -methyl-carbamoyl] -2-methyl-propyl] carbamoyl] -2-methyl-propyl] -methyl- carbamoyl] oxy-methyl] benzenesulfonic acid; - 5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolidin-1-yl)ethoxy]ethylcarbamoyl]oxetan-3- yl]amino]-5-carbamimidoyl-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2- [(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3- oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methyl-propyl]-4-oxo-butyl]- methyl-carbamoyl]-2-methyl-propyl]aminoformyl]-2-methyl-propyl]-methyl- carbamoyl]oxymethyl]benzenesulfonic acid; - 5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolidin-1-yl)ethoxy]ethylcarbamoyl]oxetan-3- yl]amino]-5-carbamimidoyl-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2- [(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3- oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methyl-propyl]-4-oxo-butyl]- methyl-carbamoyl]-2-methyl-propyl]aminoformyl]-2-methyl-propyl]-methyl- carbamoyl]oxymethyl]benzenesulfonic acid; - 5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolidin-1-yl)ethoxy]ethylcarbamoyl]oxetan-3- yl]amino]-5-carbamimidoyl-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2- [(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3- oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methyl-propyl]-4-oxo-butyl]- methyl-carbamoyl]-2-methyl-propyl]aminoformyl]-2-methyl-propyl]-methyl- carbamoyl]oxymethyl]benzenesulfonic acid; - 5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolidin-1-yl)ethoxy]ethylcarbamoyl]oxetan-3- yl]amino]-5-carbamimidoyl-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2- [(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3- oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methyl-propyl]-4-oxo-butyl]- methyl-carbamoyl]-2-methyl-propyl]aminoformyl]-2-methyl-propyl]-methyl- carbamoyl]oxymethyl]benzenesulfonic acid; - 5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolidin-1-yl)ethoxy]ethylcarbamoyl]oxetan-3- yl]amino]-5-carbamimidoyl-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2- [(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3- oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methyl-propyl]-4-oxo-butyl]- methyl-carbamoyl]-2-methyl-propyl]aminoformyl]-2-methyl-propyl]-methyl- carbamoyl]oxymethyl]benzenesulfonic acid; - 5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrolidin-1-yl)ethoxy]ethylcarbamoyl]oxetan-3- yl]amino]-5-carbamimidoyl-pentanoyl]amino]-2-[[[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2- [(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenyl-ethyl]amino]-1-methoxy-2-methyl-3- oxo-propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methyl-propyl]-4-oxo-butyl]- methyl-carbamoyl]-2-methyl-propyl]aminoformyl]-2-methyl-propyl]-methyl- carbamoyl]oxymethyl]benzenesulfonic acid; - [(1 S)-1 -[[[(1 S)-1 -[[[(1 S,2R)-4-[(2S)-2-[(1 R,2R)-3-[[(1 R,2S)-2-hydroxy-1 - methyl-2-phenyl-ethyl]amino]-1 -methoxy-2-methyl-3-oxo-propyl]pyrrolidin-1 -yl]-2- methoxy-1 -[(1 S)-1 -methylpropyl]-4-oxo-butyl]-methyl-carbamoyl]-2-methyl-propyl] amino] carbamoyl]-2-methyl-propyl]-dimethyl-ammonium; 2,2,2-trifluoroacetate; - N-({[4-({N-[6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)hexanoyl]-L-valyl-L-alanyl}amino)-2-sulfooxyphenyl]methyloxy}carbonyl)-N- methyl-L-valyl - N-[(3R,4S,5S)-1 -{(2S)-2-[(1 R,2R)-3-{[(1 S,2R)-1 -hydroxy-1 -phenylpropan-2-yl]amino}- 1 -methoxy-2-methyl-3-oxopropyl]pyrrolidin-1 -yl}-3-methoxy-5-methyl-1 -oxoheptan-4- yl]-N-methyl-L-valinamide; - N-[6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanoyl]-L-valyl-N 5 - carbamoyl - N-(4-{[({[(4S)-4,11 -diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1 H- pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}carbonyl)oxy]methyl}-3-sulfooxyphenyl)- L-ornithinamide; - (1 S,3S)-3,5,12-trihydroxy-3-(hydroxyacetyl)-10-methoxy-6,11 -dioxo-1,2,3,4,6,11 - hexahydrobenztetramine -1-yl 2,3,6-trideoxy-3-[({[4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N 5 - carbamoyl-L-ornithyl}amino)-2-sulfooxyphenyl]methyloxy}carbonyl)amino]-α-L- lyxopyranoside - N-{3-[2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy]propionyl}-L-valinyl - N-{4-[(5S,8S,11 S,12R)-11 -[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1 R,2R)-3-{[(1 S,2R)-1 - hydroxy-1 -phenylpropan-2-yl]amino}-1 -methoxy-2-methyl-3-oxopropyl]pyrrolidin-1 -yl}- 2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo - 5,8-bis(prop-2-yl)-2,13-dioxa-4,7,10-triazatetradodecan-1 -yl]-3-(2- sulfoethyl)phenyl}-N 5 - carbamoyl - L-ornithinamide; - 5-[[(2S)-2-[[(2S)-2-[6-(3,4-dibromo-2,5-dioxo-pyrrol-1 -yl)hexanoylamino]-3-methyl- butanoyl]amino]-5-ureido-pentanoyl]amino]-2-[[[(1 S)-1 -[[[(1 S)-1 -[[[(1 S,2R)-4-[(2S)-2- [(1 R,2R)-3-[[(1 R,2S)-2-hydroxy-1 -methyl-2-phenyl-ethyl]amino]-1 -methoxy-2-methyl-3- oxopropyl]pyrrolidin-1 -yl]-2-methoxy-1 -[(1 S)-1 -methylpropyl]-4-oxo-butyl]-methyl- carbamoyl]-2-methyl-propyl]amino]carbamoyl]-2-methyl-propyl]amino]ethoxy]benzoic acid - 1 -methyl-2-phenyl-ethyl]amino]- 1 -methoxy-2-methyl-3-oxo- propyl]pyrrolidin- 1 -yl]-2-methoxy- 1 -[( 1 S)- 1 -methylpropyl]-4-oxo- butyl]-methyl-aminocarbonyl]-2-methyl-propyl]-methylaminocarbonyl] oxymethyl] benzenesulfonic acid.

17. An antibody-drug conjugate of formula (III): ###0003### (III) wherein • Ab represents an antibody or an antigen binding fragment thereof; • D is a drug moiety; • T is a bond, -0-C(0)-N(CH3)-CH2-CH2-N(CH3)-C(0)-*, -0-*, -NR5-*, -NR5-C(0)-* or -0-C(0)-*, wherein * indicates the point of attachment to D; • Z represents a spacer unit; • A1 represents a -C(0)-CH(R3)-NH- group; • R3 and R4 independently of each other represent an amino acid side chain; • A2 represents a -C(O)-CH(R4)-NH- group, group, group, or group; • R2 represents a -S(0)2(0H) group, a -S(0)2(0 - M + ) group, a linear or branched -(Ci-C4)alkyl-S(0)2(0H) group, a linear or branched -halo(Ci-C4)alkyl-S(0)2(0H) group or a linear or branched -halo(Ci-C4)alkyl-S(0)2(0 - M + ) group, a linear or branched -(Ci-C4)alkyl-S(0)2(0H) group, a linear or branched -halo(Ci-C4)alkyl-S(0)2(0H) group or a linear or branched -halo(Ci-C4)alkyl-S(0)2(0 - M + ) group; • R5 represents a (Ci-C4)alkyl group; • p is an integer from 1 to 8. • M + represents a pharmaceutically acceptable monvalent cation; and 18. The antibody-drug conjugate of claim 17, wherein T is a bond or -0-C(0)-*, wherein * indicates the point of attachment to D.

20. The antibody-drug conjugate of claim 17, wherein A1 represents a -C(0)-CH(R3)-NH- group, wherein R3 represents a -(CH2)3-NH-CO-NH2 group or a methyl group.

19. The antibody-drug conjugate of claim 17, wherein R2 represents a -S(O)2(OH) group, a -S(O)2(O - M + ) group, a -CH2-S(O)2(OH) group, a -CH2-S(O)2(O - M + ) group, a -CH2-CH2-S(O)2(OH) group, a -CH2-CH2-S(O)2(O - M + ) group, a -CH2-CH2-CH2-S(O)2(OH) group, or a -CH2-CH2-CH2-S(O)2(O - M + ) group.

22. The antibody-drug conjugate of claim 17, wherein the antibody-drug conjugate is formed from a linker-drug compound of formula (II) selected from: ###0004### (II) wherein R2 and D are as defined in claim 17.

21. The antibody-drug conjugate of claim 17, wherein A2 represents: a -C(O)-CH(R4)-NH- group, wherein R4 represents isopropyl; group, group or group.

23. The antibody-drug conjugate of claim 17, wherein the antibody-drug conjugate comprises a structure of formula selected from: ###0005### wherein the wavy line indicates the point of covalent attachment to the antibody or antigen binding fragment thereof, and D and R2 are as defined in claim 17.

24. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 17 to 23 and a pharmaceutically acceptable carrier.

25. Use of the antibody-drug conjugate of any one of claims 17 to 23 for the manufacture of a medicament for the treatment of cancer in a mammal in need thereof. ​ ​ ​

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