Peptide-based linker

Abstract

The present invention relates to novel linkers comprising two or three basic, acidic, or hydrophobic natural or unnatural amino acids. The present invention also relates to drug conjugates comprising the linkers, pharmaceutical compositions comprising the drug conjugates, and the use of the drug conjugates in preventing, inhibiting, or treating cancer.

Description

[Technical Field]

[0001] (Field of Invention) The present invention relates to a novel linker comprising two or three basic, acidic, or hydrophobic natural or non-natural amino acids. The present invention also relates to a drug conjugate comprising the linker, a pharmaceutical composition comprising the drug conjugate, and the use of the drug conjugate in preventing, suppressing, or treating cancer. [Background technology]

[0002] (Background of the invention) Cancer therapeutic conjugates containing a conjugate of a binding agent (i.e., peptide, antibody, etc.) and a cytotoxic agent have been evaluated for many years. This concept involves the binding agent being configured to bind to a target, typically an epitope on cancer cells, and the presence of the cytotoxic agent intended to act as a payload for killing cancer cells. However, the synthesis of these drug conjugates usually involves the incorporation of a linker between the binding agent and the cytotoxic agent, and after administration to the target, this linker is often subjected to premature cleavage by other proteases that recognize the linker sequence. Such cleavage results in the release of the cytotoxic agent before it can bind to the cancer target, increasing the risk of undesirable side effects.

[0003] This issue has been attempted to be addressed by several research groups. For example, WO 98 / 19705 describes the existence of branched peptide linkers containing two or more amino acid parity that provide an enzymatic cleavage site. US 2017 / 360952 describes a linker having an azide-containing non-natural amino acid between a cell-binding agent and a cytotoxic agent. US 2016 / 046721 describes an antibody-drug conjugate containing a Val-Cit linker. US 2015 / 087810 describes an antibody-toxin conjugate having a linker containing 1 to 20 amino acids.

[0004] Therefore, there is a need to provide another linker that enables selective cleavage of cytotoxic agents at or near the target binding site, resulting in increased stability of the resulting conjugate. [Overview of the project]

[0005] (Summary of the invention) According to a first aspect of the present invention, a linker comprising a -P1-P2-P3- portion: P1 represents a basic non-natural amino acid or its derivative; P2 represents a hydrophobic amino acid or a hydrophobic unnatural amino acid; and P3 does not exist, or ,acid It represents either a sexual amino acid or an acidic non-natural amino acid. That is, If P1 represents Cit and P2 represents Val, then P3 must represent an acidic, unnatural amino acid. A linker is provided.

[0006] A further aspect of the present invention provides a drug conjugate comprising a target-binding binder and a cytotoxic agent, wherein the binder is linked to the cytotoxic agent via a linker as described herein.

[0007] A further aspect of the present invention provides a pharmaceutical composition comprising a drug conjugate described herein in combination with one or more excipients that are pharmaceutically acceptable.

[0008] A further aspect of the present invention provides a drug conjugate described herein for use in preventing, suppressing, or treating cancer. [Brief explanation of the drawing]

[0009] (Brief explanation of the drawing) [Figure 1] Figure 1: Pharmacokinetic analysis of BCY7761 in mouse plasma. [Figure 2] Figure 2: Pharmacokinetic analysis of BCY10980 in mouse plasma. [Figure 3] Figure 3: Pharmacokinetic analysis of BCY10981 in mouse plasma. [Figure 4] Figure 4: Pharmacokinetic analysis of BCY10989 in mouse plasma. [Figure 5] Figure 5: Pharmacokinetic analysis of BCY10984 in mouse plasma. [Figure 6] Figure 6: Pharmacokinetic analysis of BCY10985 in mouse plasma. [Figure 7] Figure 7: Pharmacokinetic analysis of BCY10984 in rat plasma. [Figure 8] Figure 8: Pharmacokinetic analysis of BCY7761 in rat plasma. [Figure 9] Figure 9: Tumor-reducing efficacy of BCY10984. [Figure 10] Figure 10: Tumor-reducing efficacy of BCY7761. [Figure 11] Figure 11: Analysis of toxin levels of BCY10984 and BCY7761. [Figure 12] Figure 12: Analysis of toxin levels of BCY10984 and BCY7761. [Figure 13] Figure 13: Analysis of toxin levels of BCY10984 and BCY7761. [Figure 14] Figure 14: Analysis of toxin levels of BCY10984 and BCY7761. [Figure 15] Figure 15: Analysis of toxin levels of BCY10984 and BCY7761. [Figure 16] Figure 16: Traces of tumor volume after administration of BCY10984 and BCY12951 to female BALB / c nude mice carrying HT1080 tumors. Error bars represent the standard error of the mean (SEM). [Figure 17] Figure 17: The results from Example 6, showing mice from Groups 5 and 6 (administered with 45 μM BCY10984), demonstrated potent inhibition of tumor growth. [Modes for carrying out the invention]

[0010] (Detailed description of the invention) (Linker) According to a first aspect of the present invention, a linker comprising a -P1-P2-P3- portion: P1 represents a basic non-natural amino acid or its derivative; P2 represents a hydrophobic amino acid or a hydrophobic unnatural amino acid; and P3 does not exist, or ,acid It represents either a sexual amino acid or an acidic non-natural amino acid. That is, If P1 represents Cit and P2 represents Val, then P3 must represent an acidic, unnatural amino acid. A linker is provided.

[0011] Therefore, the present invention relates to a linker molecule containing at least one non-natural amino acid and two or three amino acids, which require the presence of either a basic-hydrophobic-motif or a basic-hydrophobic-acidic-motif.

[0012] The linker molecule of the present invention offers the advantage of increased plasma stability, as demonstrated by the extended half-life shown in Example 1, compared to the Cit-Val control linker. Furthermore, the linker molecule of the present invention provides the ability to adjust the CatB cleavage rate to the required level as needed (see Example 2). Furthermore, the linker molecule of the present invention provides the ability to modulate the plasma protein binding capacity of the bicyclic peptide toxin conjugate, as demonstrated in Example 3. In addition, the linker molecule of the present invention showed an extended half-life and a decrease in the relative level of free toxin in plasma, as demonstrated by pharmacokinetic studies in mice and rats shown in Example 4. Furthermore, one example of the linker molecule of the present invention (BCY10984) showed a higher tumor volume reduction efficacy compared to the Cit-Val reference bicyclic peptide toxin conjugate (BTC) (see Figures 9 and 10 and Example 5). Furthermore, compared to the Cit-Val reference bicyclic peptide toxin conjugate (BTC), higher levels of toxin are observed in tumors using a linker molecule (BCY10984), which is one example of the present invention (see Figures 11-15 and 5).

[0013] In this specification, "basic non-natural amino acids or their derivatives" refers to any amino acid other than the standard 20 natural amino acids that have basic properties. Within the scope of the term "basic" are non-natural amino acids that contain a basic side chain at neutral pH. Such basic non-natural amino acids are typically polar, positively charged, and highly hydrophilic at pH values ​​below their pKa.

[0014] In one embodiment, P1 represents a basic non-natural amino acid selected from 2-amino-4-guanidinobutanoic acid (Agb); 2-amino-4-(3-methylguanidino)butanoic acid (Agb(Me)); 2,4-diaminobutyric acid (Dab); 2,3-diaminopropanoic acid (Dap); 2-amino-3-guanidinopropanoic acid (Dap(CNNH2)); and citrulline (Cit). In a further embodiment, P1 represents citrulline (Cit).

[0015] The term "hydrophobic amino acid or hydrophobic non-natural amino acid" as used herein includes any amino acid, including both the 20 standard natural amino acids and any non-natural amino acids that possess hydrophobic properties. The term "hydrophobic" encompasses both natural and non-natural amino acids that contain hydrophobic side chains, i.e., side chains that do not prefer to be present in an aqueous (i.e., water) environment.

[0016] In one embodiment, P2 represents a hydrophobic amino acid selected from Ala, Gly, Ile, Leu, Met, Phe, Pro, Trp, and Val, or a hydrophobic unnatural amino acid selected from cyclobutyl, diphenylalanine (Dpa), 1-naphthylalanine (1Nal), 2-naphthylalanine (2Nal), and methyltryptophan (Trp(Me)), for example, a hydrophobic amino acid selected from Val, or an unnatural amino acid selected from cyclobutyl, Dpa, 1Nal, and 2Nal. In a further embodiment, P2 represents 1-naphthylalanine (1Nal).

[0017] The term “acidic amino acid or acidic non-natural amino acid” as used herein includes any amino acid, including both the 20 standard natural amino acids and any non-natural amino acids that have acidic properties. Within the scope of the term “acidic” are both natural and non-natural amino acids that contain acidic side chains at an acidic pH. Typically, these side chains have a carboxylic acid group whose pKa is low enough to lose a proton and in the process acquire a negative charge.

[0018] In one embodiment, P3 is absent. In an alternative embodiment, P3 represents an acidic amino acid selected from Asp and Glu. In a further embodiment, P3 represents Glu.

[0019] In one embodiment, the -P1-P2-P3- portion represents the following: [Table 1] .

[0020] In a further embodiment, the -P1-P2-P3- portion represents: Cit-1Nal-Glu(BCY10984).

[0021] (Drug conjugates) A further aspect of the present invention provides a drug conjugate comprising a target-binding binder and a cytotoxic agent, wherein the binder is connected to the cytotoxic agent via a linker as defined herein.

[0022] In one embodiment, the binder is a peptide, for example, an antibody or a bicyclic peptide, particularly a bicyclic peptide.

[0023] (Bicyclic peptide) While peptides and antibodies are recognized terms in the art, it will be apparent to those skilled in the art that the use of bicyclic peptides (or bicycles) herein refers to peptide sequences having two loops via cyclization at three reactive amino acid groups (i.e., cysteine ​​residues). These bicyclic peptides were identified in 2009 by a phage display-based combinatorial method for constructing and screening large libraries of bicyclic peptides against target organisms (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). Preferably, bicyclic peptides are configured to bind to anti-cancer targets. Preferred examples of cancer cell-binding bicyclic peptides include WO 2016 / 067035 (MT1-MMP-binding bicyclic peptide), WO 2017 / 191460 (MT1-MMP-binding bicyclic peptide), and WO Examples include those described in PCT / GB2019 / 025811 (CD137-binding bicyclic peptide), PCT / GB2018 / 053675 (EphA2-binding bicyclic peptide), PCT / GB2018 / 053676 (EphA2-binding bicyclic peptide), PCT / GB2018 / 053678 (EphA2-binding bicyclic peptide), PCT / GB2019 / 050485 (CD137-binding bicyclic peptide), PCT / GB2019 / 051740 (Nectin-4-binding bicyclic peptide), and PCT / GB2019 / 051741 (Nectin-4-binding bicyclic peptide), the bicyclic peptides disclosed in said documents are incorporated herein by reference.

[0024] As used herein, bicyclic peptides refer to peptides covalently bonded to a molecular scaffold. Typically, such peptides comprise two or more reactive groups (i.e., cysteine ​​residues) capable of forming a covalent bond with the scaffold, and an inherent sequence between these reactive groups, which forms a loop when the peptide binds to the scaffold and is therefore called a loop sequence. In this case, the peptide comprises at least three cysteine ​​residues and forms at least two loops on the scaffold.

[0025] (Molecular scaffold) In one embodiment, a bicyclic peptide is covalently bonded to a non-aromatic molecular scaffold. The term “non-aromatic molecular scaffold” as used herein refers to any molecular scaffold as defined herein that does not contain aromatic (i.e., unsaturated) carbocyclic or heterocyclic ring systems.

[0026] A suitable example of a non-aromatic molecule scaffold is described in Heinis et al.'s (2014) Angewandte Chemie, International Edition 53(6) 1602-1606.

[0027] As described in the aforementioned document, the molecular scaffold may be made of low-molecular-weight molecules, such as low-organic molecules.

[0028] In one embodiment, the molecular scaffold may be a polymer. In one embodiment, the molecular scaffold is a polymer composed of amino acids, nucleotides, or carbohydrates.

[0029] In one embodiment, the molecular scaffold includes a reactive group that can react with the functional group of the polypeptide to form a covalent bond.

[0030] The molecular scaffold may contain chemical groups that form bonds with peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and acyl halides.

[0031] An example of an αβ-unsaturated carbonyl-containing compound is 1,1',1''-(1,3,5-triazinan-1,3,5-triyl)triporpa-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606).

[0032] In an alternative embodiment, the bicyclic peptide is covalently bonded to an aromatic molecular scaffold. The term “aromatic molecular scaffold” as used herein refers to any molecular scaffold as defined herein that contains an aromatic carbocyclic or heterocyclic ring system.

[0033] It will be understood that aromatic molecule scaffolds may contain aromatic moieties. Examples of suitable aromatic moieties within aromatic scaffolds include biphenylene, terphenylene, naphthalene, or anthracene.

[0034] It will also be understood that aromatic molecule scaffolds may contain heteroaromatic moieties. Examples of suitable heteroaromatic moieties within aromatic scaffolds include pyridine, pyrimidine, pyrrole, furan, and thiophene.

[0035] It will also be understood that aromatic molecule scaffolds may contain halomethylarene moieties, such as bis(bromomethyl)benzene, tris(bromomethyl)benzene, tetra(bromomethyl)benzene, or derivatives thereof.

[0036] Non-limiting examples of aromatic molecule scaffolds include: bis-, tris-, or tetra(halomethyl)benzene; bis-, tris-, or tetra(halomethyl)pyridine; bis-, tris-, or tetra(halomethyl)pyridazine; bis-, tris-, or tetra(halomethyl)pyrimidine; bis-, tris-, or tetra(halomethyl)pyrazine; bis-, tris-, or tetra(halomethyl)-1,2,3-triazine; bis-, tris-, or tetra(halomethyl)-1,2,4-triazine; bis-, tris-, or tetra(halomethyl)pyro Examples include ru, -furan, -thiophene; bis-, tris-, or tetra(halomethyl)imidazole, -oxazole, -thiazole; bis-, tris-, or tetra(halomethyl)-3H-pyrazole, -isoxazole, -isothiazole; bis-, tris-, or tetra(halomethyl)biphenylene; bis-, tris-, or tetra(halomethyl)terphenylene; 1,8-bis(halomethyl)naphthalene; bis-, tris-, or tetra(halomethyl)anthracene; and bis-, tris-, or tetra(2-halomethylphenyl)methane.

[0037] More specific examples of aromatic molecule scaffolds include: 1,2-bis(halomethyl)benzene; 3,4-bis(halomethyl)pyridine; 3,4-bis(halomethyl)pyridazine; 4,5-bis(halomethyl)pyrimidine; 4,5-bis(halomethyl)pyrazine; 4,5-bis(halomethyl)-1,2,3-triazine; 5,6-bis(halomethyl)-1,2,4-triazine; 3,4-bis(halomethyl)pyrrole, -furan, -thiophene, and other positional isomers; 4,5-bis(halomethyl)imidazole, -oxazole, -thiazole; 4,5-bis(halomethyl)-3H-pyrazole, -isoxazole, -isothiazole; 2,2'-bis(halomethyl)biphenylene; 2,2''-bis(halomethyl)terphenylene; 1,8-Bis(halomethyl)naphthalene; 1,10-Bis(halomethyl)anthracene; Bis(2-halomethylphenyl)methane; 1,2,3-Tris(halomethyl)benzene; 2,3,4-Tris(halomethyl)pyridine; 2,3,4-Tris(halomethyl)pyridazine; 3,4,5-Tris(halomethyl)pyrimidine; 4,5,6-Tris(halomethyl)-1,2,3-triazine; 2,3,4-Tris(halomethyl)pyrrole,-furan,-thiophene; 2,4,5-Bis(halomethyl)imidazole,-oxazole,-thiazole; 3,4,5-Bis(halomethyl)-1H-pyrazole,-isoxazole,-isothiazole; 2,4,2'-Tris(halomethyl)biphenylene; 2,3',2''-Tris(halomethyl)terphenylene; 1,3,8-Tris(halomethyl)naphthalene; 1,3,10-Tris(halomethyl)anthracene; Bis(2-halomethylphenyl)methane; 1,2,4,5-Tetra(halomethyl)benzene; 1,2,4,5-Tetra(halomethyl)pyridine; 2,4,5,6-Tetra(halomethyl)pyrimidine; 2,3,4,5-Tetra(halomethyl)pyrrole,-furan,-thiophene; 2,2',6,6'-Tetra(halomethyl)biphenylene; 2,2'',6,6''-Tetra(halomethyl)terphenylene; 2,3,5,6-Tetra(halomethyl)naphthalene and 2,3,7,8-Tetra(halomethyl)anthracene;Also mentioned is bis(2,4-bis(halomethyl)phenyl)methane.

[0038] In one embodiment, the molecular scaffold may contain or consist of tris(bromomethyl)benzene, in particular 1,3,5-tris(bromomethyl)benzene ("TBMB"), or derivatives thereof.

[0039] In one embodiment, the molecular scaffold is 2,4,6-tris(bromomethyl)mesitylene. This molecule is similar to 1,3,5-tris(bromomethyl)benzene but contains three additional methyl groups attached to the benzene ring. This has the advantage that these additional methyl groups can form further contact with the polypeptide and thus add additional structural constraints.

[0040] The molecular scaffold of the present invention contains a chemical group that enables the functional groups of polypeptides in the encoded library of the present invention to form covalent bonds with the molecular scaffold. The chemical group is selected from a broad range of functional groups, including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides, and acyl halides.

[0041] The scaffold reactants that can be used on a molecular scaffold to react with the thiol group of cysteine ​​are alkyl halides (also called halogenoalkanes or haloalkanes).

[0042] Examples include bromomethylbenzene (a scaffold reactant exemplified by TBMB) or iodoacetamide. Other scaffold reactants used to selectively couple compounds with cysteine ​​in proteins are maleimides, αβ-unsaturated carbonyl-containing compounds, and α-halomethylcarbonyl-containing compounds. Examples of maleimides that can be used as molecular scaffolds in the present invention include tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, and tris-(maleimido)benzene. An example of an α-halomethylcarbonyl-containing compound is N,N',N''-(benzene-1,3,5-triyl)tris(2-bromoacetamide). Selenocysteine ​​is also a natural amino acid that has similar reactivity to cysteine ​​and can be used in the same reaction. Therefore, whenever cysteine ​​is mentioned, it is generally acceptable to substitute selenocysteine ​​unless the context suggests otherwise.

[0043] (synthesis) Bicyclic peptides can be synthesized using standard techniques and then reacted in vitro with molecular scaffolds. Standard chemistry can be used to carry this out. This allows for the rapid, large-scale preparation of soluble materials for further downstream experiments or validation. Such methods can be achieved using conventional chemistry, for example, those disclosed in the literature of Timmerman et al. (above).

[0044] Accordingly, the present invention also relates to the production of polypeptides selected as described herein, wherein the production includes any further steps as described below. In one embodiment, these steps are carried out on a final product polypeptide produced by chemical synthesis.

[0045] By extending the peptide, for example, another loop can be incorporated, and therefore multiple specificities can be introduced.

[0046] To extend the peptide, it may simply be chemically extended at its N-terminus or C-terminus or within the loop using orthogonally protected lysine (and analogues) with standard solid-phase or liquid-phase chemistry. An activated or activatable N- or C-terminus may be introduced using standard (bio)conjugation techniques. Alternatively, the addition may be carried out by fragment condensation or native chemical ligation as described in (Dawson et al., 1994, Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779), or by enzymes, for example, using sub-tillary gauze as described in (Chang et al., Proc Natl Acad Sci US A. 1994 Dec 20; 91(26):12544-8 or Hikari et al., Bioorganic & Medicinal Chemistry Letters, Vol. 18, No. 22, November 15, 2008, pp. 6000-6003).

[0047] Alternatively, the peptide may be extended or modified by further conjugation via disulfide bonds. This has the additional advantage of allowing the first and second peptides to dissociate from each other in the reducing environment of the cell. In this case, a molecular scaffold (e.g., TATA) can be added during the chemosynthesis of the first peptide to react with three cysteine ​​groups; thereafter, additional cysteine ​​or thiol can be added to the N or C-terminus of the first peptide, so that this cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide to form a disulfide-bonded bicyclic peptide-peptide conjugate.

[0048] A similar technique is equally applicable to the synthesis / coupling of two bicyclic bispecific macrocyclic molecules, which potentially give rise to a quadruspecific molecule.

[0049] Furthermore, the addition of other functional groups or effector groups may be achieved in the same manner by coupling at the N- or C-terminus or via the side chain using appropriate chemistry. In one embodiment, the coupling is carried out in such a manner that it does not block the activity of any of the entities.

[0050] (Cytotoxic agents) Examples of suitable "cytotoxic agents" include: cisplatin and carboplatin, as well as alkylating agents such as oxaliplatin, mechloretamine, cyclophosphamide, chlorambucil, and ifosfamide; antimetabolites including the purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids including vinca alkaloids such as vincristine, vinblastine, vinorelbine, and vindesine; podophyllotoxin and its derivatives etoposide and teniposide; taxanes, including paclitaxel, originally known as taxol; topoisomerase inhibitors including camptothecin; irinotecan and topototecan, as well as type II inhibitors including amsacrin, etoposide, phosphate etoposide, and teniposide. Further medications include the immunosuppressant dactinomycin (used in kidney transplants), and antitumor antibiotics such as doxorubicin, epirubicin, bleomycin, and calicheamicin.

[0051] In one embodiment, the cytotoxic agent is selected from maytansinoids (e.g., DM1) or monomethyl auristatin (e.g., MMAE).

[0052] DM1 is a thiol-containing derivative of maytansine and has the following structure: [ka] It is a cytotoxic agent that possesses [certain properties].

[0053] Monomethyl auristatin E (MMAE) is a synthetic antineoplastic agent with the following structure: [ka] It has.

[0054] In a further embodiment, the cytotoxic agent is MMAE.

[0055] (Pharmaceutical composition) A further aspect of the present invention provides a pharmaceutical composition comprising a drug conjugate described herein in combination with one or more excipients that are pharmaceutically acceptable.

[0056] Typically, drug conjugates are used in a purified form with pharmacologically appropriate excipients or carriers. These excipients or carriers typically include aqueous or alcohol / aqueous solutions, emulsions, or suspensions, including physiological saline and / or buffering media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, as well as lactated Ringer's dextrose. Physiologically acceptable adjuvants may be selected from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates, if necessary to maintain the polypeptide complex in suspension.

[0057] Intravenous vehicles include fluids, nutritional supplements, and electrolyte supplements, such as those based on Ringer dextrose. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present (Mack (1982), Remington's Pharmaceutical Sciences, 16th edition).

[0058] The drug conjugates of the present invention may be administered separately as a composition or used in combination with other agents. These include antibodies, antibody fragments, and various immunotherapeutic agents, such as silcosporine, methotrexate, adriamycin, or cisplatin, and immunotoxins. The pharmaceutical composition may include a "cocktail" of various cytotoxic agents or other agents combined with the drug conjugates of the present invention, or even combinations of selected drug conjugates according to the present invention having different specificities, such as polypeptides selected using different target ligands, whether or not they are pooled before administration.

[0059] The route of administration of the pharmaceutical composition according to the present invention may be any that is generally known to those skilled in the art. For therapeutic purposes, the drug conjugate of the present invention can be administered to any patient according to standard techniques. Administration may be by any suitable mode, including parenteral, intravenous, intramuscular, intraperitoneal, percutaneous, pulmonary routes, or, as appropriate, direct infusion using a catheter. Preferably, the pharmaceutical composition according to the present invention is administered by inhalation. The dosage and frequency of administration will depend on the patient's age, sex, and condition, concurrent administration of other drugs, contraindications, and other parameters considered by the clinician.

[0060] The drug conjugate of the present invention can be lyophilized before storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective, and lyophilization and reconstitution techniques known in the art can be utilized. It will be understood by those skilled in the art that lyophilization and reconstitution may result in varying degrees of activity loss, and that it may be necessary to adjust the levels upward to compensate for this.

[0061] Compositions containing this drug conjugate or cocktail thereof may be administered for prophylactic and / or therapeutic purposes. In a particular therapeutic use, the amount sufficient to achieve at least partial inhibition, suppression, modulation, death, or any other measurable parameter of a selected population of cells is defined as the “therapeutic effective dose.” The amount required to achieve this dose depends on the severity of the disease and the overall state of the patient’s own immune system, but is generally in the range of 0.005 to 5.0 mg of the selected drug conjugate per kilogram of body weight, with doses of 0.05 to 2.0 mg / kg / dose being more commonly used. For prophylactic purposes, compositions containing this drug conjugate or cocktail thereof may also be administered in similar or slightly lower doses.

[0062] Compositions containing the drug conjugates according to the present invention can be used in prophylactic and therapeutic settings to assist in altering, inactivating, killing, or removing selective target cell populations in mammals. Furthermore, the drug conjugates described herein can be selectively used in vitro or in vitro to selectively kill, deplete, or otherwise effectively remove target cell populations from cellular heterogeneous aggregates. Mammalian blood can be combined in vitro with selected drug conjugates to kill or otherwise remove unwanted cells from the blood for return to the mammal according to standard techniques.

[0063] (therapeutic use) A further aspect of the present invention provides a drug conjugate described herein for use in preventing, suppressing, or treating cancer.

[0064] Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other carcinomas), e.g., bladder and urinary tract, breast, gastrointestinal tract (including esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidney, lung (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma, and mesothelioma), head and neck (e.g., tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands), Cancers of the nasal cavity and sinuses), ovaries, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (e.g., follicular thyroid carcinoma), adrenal gland, prostate, skin, and adnexa (melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthomatoma, dysplastic nevi); hematological malignancies (i.e., leukemia, lymphoma) and borderline malignancies including pre-malignant hematological disorders and lymphatic hematological malignancies and related diseases (e.g., acute lymphoblastic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphoma, e.g., diffuse large B-cell lymphoma [D]). (e.g., LBCL), follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammaglobulinemia of unknown significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), as well as myeloid hematological malignancies and related diseases (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], eosinophilia, myeloproliferative disorders, e.g., polycythemia vera, essential platelet disease) Bloodylasma, and primary myelofibrosis, myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia; tumors of mesenchymal origin, e.g., sarcomas of soft tissue, bone, or cartilage, e.g., osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma, and glioblastoma, meningioma, ependymoma, pineal tumor, and Schwann cell tumor);Endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary carcinoma of the thyroid); ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblast tumors (e.g., teratomas, seminomas, undifferentiated germ cell tumors, hydatidiform moles, and choriocarcinoma); and pediatric and embryonic tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and undifferentiated neuroectodermal tumors); or congenital or other syndromes that predispose a patient to malignant tumors (e.g., xeroderma pigmentosum).

[0065] In further embodiments, cancer is selected from hematopoietic malignancies, such as: non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), multiple myeloma (MM), chronic lymphocytic leukemia B (B-CLL), acute lymphocytic leukemia B and T (ALL), T-cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), and chronic myeloid leukemia (CML).

[0066] In this specification, the term “prevention” includes the administration of a protective composition before the induction of disease. “Suppression” refers to the administration of a composition after an inducible event but before the clinical manifestation of the disease. “Treatment” includes the administration of a protective composition after disease symptoms have become apparent.

[0067] Animal model systems are available that can be used to screen the efficacy of drug conjugates in the protection against or treatment of disease. The use of animal model systems is facilitated by the present invention, which enables the development of drug conjugates that can cross-react with human and animal targets.

[0068] The present invention will be further described below with reference to the following examples. [Examples]

[0069] (Examples) (Materials and Methods) (Peptide synthesis) Peptide synthesis was based on Fmoc chemistry using the Symphony peptide synthesizer manufactured by Peptide Instruments and the Syro II synthesizer manufactured by MultiSynTech. Standard Fmoc amino acids (Sigma, Merck) were used with appropriate side-chain protecting groups: where applicable, standard coupling conditions were used in each case, followed by deprotection using standard methodologies. Peptides were purified by HPLC, isolated, and then modified with 1,3,5-tris(bromomethyl)benzene (TBMB, Sigma). For this purpose, linear peptides were diluted with H2O to approximately 35 mL, approximately 500 μL of 100 mM TBMB in acetonitrile was added, and the reaction was initiated with 5 mL of 1 M NH4HCO3 in H2O. The reaction was allowed to proceed at RT for approximately 30 to 60 minutes, and once the reaction was complete (as determined by MALDI), it was lyophilized. After lyophilization, the modified peptides were purified as described above, while the Luna C8 column was replaced with a Gemini C18 column (Phenomenex) and the acid was changed to 0.1% trifluoroacetic acid. The pure fractions containing the correct TMB-modified material were pooled, lyophilized, and stored at -20°C.

[0070] Unless otherwise specified, all amino acids were used in their L-stereoconfiguration.

[0071] In some cases, the peptide is converted to an activated disulfide and then coupled with the free thiol group of the toxin using the following method: A solution of 4-methyl(succinimidyl 4-(2-pyridylthio)pentanoate) (100 mM) in anhydrous DMSO (1.25 mol equivalent) was added to a solution of peptide (20 mM) in anhydrous DMSO (1 mol equivalent). The reaction mixture was thoroughly mixed, and DIPEA (20 mol equivalent) was added. The reaction was monitored by LC / MS until completion.

[0072] (Biring conjugate synthesis) (General method) [ka] (Step (a): Solid-phase synthesis of peptide linker compounds) [ka] The peptide was synthesized on chlorotrityl resin (2 mmol) using standard Fmoc chemistry. The first amino acid was loaded onto the resin by incubation for 2 hours with a mixture of Fmoc-AA-OH (1 equivalent) and DIEA (4 equivalents) in DMF. The resin was drained, washed, and then treated with MeOH for 30 minutes. The remaining sequence was constructed using the standard SPPS method with Fmoc-AA-OH (or an acidic capping group, e.g., azidoacetic acid) (3 equivalents), HBTU (2.85 equivalents), and DIEA (6 equivalents). The coupling reaction was carried out for 1 hour. Fmoc deprotection was performed with 20% piperidine / DMF for 30 minutes. The peptide was cleaved from the resin by incubation with HFIP / DCM (20:80) for 30 minutes. The crude peptide was dried and used directly in the next step without purification.

[0073] (Step (b): Addition of (4-aminophenyl)methanol to the peptide linker compound) [ka] To a solution of compound 2 (1.0 equivalent) in MeOH (100 mg / mL), a DCM solution of EEDQ (2.0 equivalents) and (4-aminophenyl)methanol (2.0 equivalents) was added. The mixture was stirred at 35°C for 16 hours. After completion, the reaction mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC.

[0074] (Step (c): Reaction of bis(2,4-dinitrophenyl)carbonate with peptide linker-(4-aminophenyl)methanol) [ka] To a solution of compound 3 (1.0 equivalent) in DMF (50 mg / mL), DIEA (5.0 equivalents) and bis(4-nitrophenyl)carbonate (4.0 equivalents) were added, and the mixture was stirred at 25°C for 1 hour (or until compound 3 was consumed). The reaction mixture was purified directly by preparative HPLC.

[0075] (Step (d): MMAE conjugation with peptide linker compound) [ka] To a solution of compound 4 (1.5 equivalents) in DMF (10 mg / mL), HOBt (1.5 equivalents), DIEA (5.0 equivalents), and MMAE (1.0 equivalent) were added. The mixture was stirred at 40°C for 16 hours until compound 4 was completely consumed. The reaction mixture was purified directly by preparative HPLC.

[0076] (Step (e): Removal of Boc protecting group) [ka] (In the case of linkers synthesized using amino acids with Boc-protected side chains) A Boc-protecting amine-containing linker (1.0 equivalent) was added to a mixture of 10% TFA / DCM (30 mg / mL). The mixture was stirred at 0°C for 1 hour, and then concentrated under reduced pressure to remove the DCM. The crude product was used directly in the next step without purification.

[0077] (Step (f): Copper-catalyzed cycloaddition of an azide-functionalized toxin-linker to an alkyne-functionalized biring) [ka] To a solution of compound 5 (1.0 equivalent) in t-BuOH / H2O (1:1, 6.5 mg / mL), CuSO4 (0.4 M, 2.0 equivalents), THPTA (1.0 equivalent), BCY3900 (0.9 equivalents), and VcNa (2.0 equivalents) were added. The mixture was adjusted to pH ~7 and then stirred at 40°C for 2 hours (or until compound 5 was consumed). The reaction mixture was concentrated under reduced pressure to remove t-BuOH. If DMAB or methyl ester protecting groups were present in the compound, deprotection was performed on the crude material (general method F or G). Otherwise, the crude residue was purified by preparative HPLC to obtain the final conjugate.

[0078] (Step (g): Removal of DMAB protecting group) [ka] (In the case of linkers synthesized using amino acids with DMAB-protected side chains) To a solution of DMAB-protected linker compound (1.0 equivalent) in DMF (36 mg / mL), N2H4-H2O (75 equivalents) was added. The mixture was stirred at 25°C for 0.5 hours, and the reaction mixture was then purified by preparative HPLC to obtain the final conjugate.

[0079] (Step (h): Removal of methyl ester protecting group) [ka] (In the case of linkers synthesized using amino acids with methyl ester-protected side chains) NaOH (20.0 equivalents) was added to a solution of a methyl ester-protected linker compound (1.0 equivalent) in H2O (100 mg / mL). The mixture was stirred at 25°C for 2 hours. The reaction mixture was purified directly by preparative HPLC to obtain the final conjugate.

[0080] Using the general method described above, the following biring conjugate was prepared: [Table 2]

[0081] (BCY10989-(Dab-Val)) [ka] Following a standard method, BCY10989 was obtained. Predicted MW = 3882.4, observed m / z: 1294 [M+3H] 3+ , 971[M+4H] 4+ .

[0082] (BCY10988-(Dab-cBu-Glu)) [ka] Following a standard method, BCY10988 was obtained. Predicted MW = 4009.5, observed m / z: 1337 [M+3H] 3+ , 1003[M+4H] 4+ .

[0083] (BCY10986(Dab-2Nal-Glu)) [ka] Following a standard method, BCY10986 was obtained. Predicted MW = 4109.6, observed m / z: 1370 [M+3H] 3+ , 1028[M+4H] 4+ .

[0084] (BCY10987-(Dab-DPA-Glu)) [ka] Following a standard method, BCY10987 was obtained. Predicted MW = 4135.7, observed m / z: 1379 [M+3H] 3+ , 1034[M+4H] 4+ .

[0085] (BCY10983-(Agp-Val-Glu)) [ka] The MMAE-PAB-Dap-Val-Glu(OMe)-AcAz intermediate was prepared as described for the synthesis of BCY10982. Subsequently, the side-chain amine of Dap was converted to the corresponding guanidine. MMAE-PAB-Dap-Val-Glu(OMe)-AcAz (1 equivalent) was stirred in DMF, and DIEA (9 equivalents) and 1H-pyrazole-1-carboxamidine hydrochloride (24 equivalents) were added thereto. The mixture was stirred at 45 °C for 24 hours, and then the mixture was diluted and purified by preparative HPLC. When the remaining synthetic steps were carried out using a general method, BCY10983 was obtained. Predicted MW = 4039.5, observed m / z: 1346 [M+3H] 3+ , 1010 [M+4H] 4+ .

[0086] (BCY10980-(Cit-Val-Glu)) [Chemical formula] BCY10980 was obtained according to a general method. Predicted MW = 4068.6, observed m / z: 1356 [M+3H] 3+ , 1017 [M+4H] 4+ .

[0087] (BCY10981-(Dab-Val-Glu)) [Chemical formula] BCY10981 was obtained according to a general method. Predicted MW = 4011.5, observed m / z: 1338 [M+3H] 3+ , 1003 [M+4H] 4+ .

[0088] (BCY10982-(Dap-Val-Glu)) [Chemical formula] BCY10982 was obtained according to a general method. Predicted MW = 3997.5, observed m / z: 1333 [M+3H] 3+, 1000 [M+4H] 4+ .

[0089] (BCY10984-(Cit-1Nal-Glu)) [ka] Following a standard method, BCY10984 was obtained. Predicted MW = 4166.7, observed m / z: 1388 [M+3H] 3+ , 1042[M+4H] 4+ .

[0090] (BCY10985-(Dab-1Nal-Glu)) [ka] Following a standard method, BCY10985 was obtained. Predicted MW = 4109.6, observed m / z: 1370 [M+3H] 3+ , 1028[M+4H] 4+ .

[0091] (BCY10298-(Dap-Val)) (Preparation of Compound 1) [ka] DCM was added to a container containing CTC resin (5 mmol, 4.50 g, 1.10 mmol / g), and then Fmoc-Dap(Boc)-OH (2.13 g, 5 mmol, 1.0 equivalent) was added while bubbling with N2. DIEA (4.0 equivalents) was added dropwise, and the resin was mixed for 2 hours. MeOH (4.5 mL) was added, and the resin was mixed again for 30 minutes. After that, the resin was drained and washed 5 times with DMF.

[0092] The Fmoc groups were removed by adding 20% ​​piperidine / DMF and allowing the mixture to react for 30 minutes. The resin was then drained and washed five times with DMF.

[0093] To couple the following amino acids, a DMF solution of Fmoc-amino acids was added to the resin and mixed for 30 seconds, after which an activator and base were added. The coupling reaction was allowed to proceed for 1 hour while continuously bubbling with N2. The coupling and Fmoc deprotection rounds were repeated using the following amino acids. [Table 3]

[0094] After the final coupling reaction, the resin was washed three times with MeOH and then dried under vacuum. The side-chain protected peptide was cleaved from the resin by adding 20% ​​HFIP / 80% DCM at room temperature to a flask containing the peptide. This cleavage was then repeated with continuous N2 bubbling (for 1 hour each). The resin was filtered, the filtrate was collected, and then concentrated to remove the solvent. The crude peptide was freeze-dried to obtain compound 1 (1.64 g, 94.1% purity, 73.7% yield). Expected MW = 444.53, observed m / z: 445.12 [M+H] + .

[0095] (Preparation of Compound 2) [ka] To a solution of compound 1 (800 mg, 1.80 mmol, 1.0 equivalent) in DCM (16.0 mL) and MeOH (8.00 mL), (4-aminophenyl)methanol (266 mg, 2.16 mmol, 1.2 equivalents) and EEDQ (890 mg, 3.60 mmol, 2.0 equivalents) were added in the dark. The mixture was stirred at 25°C for 16 hours. TLC (DCM:MeOH = 10:1, R fCalculation by (0.46) showed that compound 1 was completely consumed. LC-MS showed that compound 1 was completely consumed and that one major peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40g SepaFlash® silica flash column, eluent of 0-20% MeOH / DCM @ 60 mL / min) to obtain compound 2 (550 mg, 1.00 mmol, 55.6% yield) as a light brown solid. Expected MW = 549.66, observed m / z: 450.04 [(M-Boc)+H] + and 550.08[M+H] + .

[0096] (Preparation of Compound 3) [ka] To a solution of compound 2 (550 mg, 1.00 mmol, 1.0 equivalent) in DMF (7.00 mL), bis(4-nitrophenyl)carbonate (913 mg, 3.00 mmol, 3.0 equivalents) and DIEA (517 mg, 4.00 mmol, 697 μL, 4.0 equivalents) were added under an N2 atmosphere. The mixture was stirred at 25°C for 2 hours. LC-MS showed that compound 2 was completely consumed and a single main peak with the desired m / z was detected. Purification of the reaction mixture by preparative HPLC (neutral conditions) yielded compound 3 (560 mg, 783 μmol, 78.30% yield) as a pale yellow solid. Expected MW = 714.76, observed m / z: 614.96 [(M-Boc)+H] + and 715.01[M+H] + .

[0097] (Preparation of Compound 4) [ka] To a solution of compound 3 (550 mg, 769 μmol, 1.0 equivalent) in DMF (6.00 mL), DIEA (398 mg, 3.08 mmol, 536 μL, 4.0 equivalents) was added and the mixture was stirred for 10 minutes under an N2 atmosphere. Subsequently, 1-ethyl-6-fluoro-4-oxo-7-piperazine-1-ylquinoline-3-carboxylic acid (491 mg, 1.54 mmol, 2.0 equivalents) and HOBt (208 mg, 1.54 mmol, 2.0 equivalents) were added to the mixture. The mixture was stirred at 25°C for 2 hours. LC-MS showed that compound 3 was completely consumed and a single main peak with the desired m / z was detected. The reaction mixture was washed with H2O (250 mL) at 25°C, filtered, and concentrated under reduced pressure to obtain crude compound 4 (580 mg, crude product) as a yellow solid. This was used in the next step without further purification. Expected MW = 894.98, observed m / z: 398.04 [(M-Boc) / 2+H] + , 895.06[M+H] + .

[0098] (Preparation of BCY10298) [ka] TFA (924 mg, 8.10 mmol, 0.60 mL, 29.0 equivalents) was added to a solution of compound 4 (250 mg, 279 μmol, 1.0 equivalent) in DCM (2.40 mL). The mixture was stirred at 25°C for 2 hours. LC-MS showed that compound 4 was completely consumed and a single main peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to obtain a residue. Purification of the residue by preparative HPLC (A: 0.075% TFA in H2O, B: ACN) yielded BCY10298 (160 mg, 194 μmol, 69.6% yield) as a white solid. Expected MW = 794.87, observed m / z: 398.06 [M / 2 + H] + , 795.02[M+H] + .

[0099] (BCY10300-(Dap(CNNH2)-Val)) [ka] To a solution of BCY10298 (85.0 mg, 107 μmol, 1.0 equivalent) in DMF (1.00 mL), chloro(pyrazole-1-carboxyimidoyl)ammonium (15.6 mg, 107 μmol, 1.0 equivalent) and DIEA (41.5 mg, 321 μmol, 60.0 μL, 3.0 equivalent) were added under an N2 atmosphere. The mixture was stirred at 25°C for 16 hours. LC-MS showed that compound 5 was completely consumed and a single main peak with the desired m / z was detected. Purification of the reaction mixture by preparative HPLC (A: 0.075% TFA in H2O, B: ACN) yielded BCY10300 (39.3 mg, 46.2 μmol, 43.1% yield, 98.3% purity) as a white solid. Predicted MW = 836.91, observed m / z: 419.11 [M / 2 + H] + and 836.95[M+H] + .

[0100] (BCY9474-(Dab-Val)) (Preparation of Compound 2) [ka] DCM was added to a container containing CTC resin (10 mmol, 9.10 g, 1.10 mmol / g), and then Fmoc-Dab(Boc)-OH (4.40 g, 10 mmol, 1.0 equivalent) was added while bubbling with N2. DIEA (4.0 equivalents) was added dropwise, and the resin was mixed for 2 hours. MeOH (9.1 mL) was added, and the resin was mixed again for 30 minutes. After that, the resin was drained and washed 5 times with DMF.

[0101] The Fmoc groups were removed by adding 20% ​​piperidine / DMF and allowing the mixture to react for 30 minutes. The resin was then drained and washed five times with DMF.

[0102] To couple the following amino acids, a DMF solution of Fmoc-amino acids was added to the resin and mixed for 30 seconds, after which an activator and base were added. The coupling reaction was allowed to proceed for 1 hour while continuously bubbling with N2. The coupling and Fmoc deprotection rounds were repeated using the following amino acids. [Table 4]

[0103] After the final coupling reaction, the resin was washed three times with MeOH and then dried under vacuum. The side-chain protected peptide was cleaved from the resin by adding 20% ​​HFIP / 80% DCM at room temperature to a flask containing the peptide. This cleavage was then repeated with continuous N2 bubbling (for 1 hour each). The resin was filtered, the filtrate was collected, and then concentrated to remove the solvent. The crude peptide was freeze-dried to obtain compound 2 (2.50 g, 96.6% purity, 54.40% yield). Expected MW = 458.56, observed m / z: 459.4 [M+H] + .

[0104] (Preparation of Compound 3) [ka] To a solution of compound 2 (2.50 g, 5.45 mmol, 1.0 equivalent) in DCM (50.0 mL) and MeOH (25.0 mL), (4-aminophenyl)methanol (806 mg, 6.54 mmol, 1.2 equivalents) and EEDQ (2.70 g, 10.9 mmol, 2.0 equivalents) were added in the dark. The mixture was stirred in the dark at 25°C for 16 hours. TLC (DCM:MeOH = 10:1, R) fA 0.35 MW test indicated that compound 2 was completely consumed. LC-MS showed that the majority of compound 2 was consumed, and a single main peak with the desired m / z for compound 3 was detected. Concentration of the reaction mixture under reduced pressure yielded a residue. Purification of this residue by flash silica gel chromatography (ISCO®; 80g SepaFlash® silica flash column, 0-20% MeOH / DCM eluent @ 60 mL / min) yielded compound 3 (1.65 g, 2.93 mmol, 53.7% yield) as a pale yellow solid. Expected MW = 563.69, observed m / z: 464.3[(M-Boc)+H] + and 564.3[M+H] + .

[0105] (Preparation of Compound 4) [ka] To a solution of compound 3 (1.65 g, 2.93 mmol, 1.0 equivalent) in DMF (10.0 mL), bis(4-nitrophenyl)carbonate (2.67 g, 8.78 mmol, 3.0 equivalents) and DIEA (1.51 g, 11.7 mmol, 2.04 mL, 4.0 equivalents) were added under an N2 atmosphere. The mixture was stirred at 25°C for 2 hours. LC-MS showed the detection of a single main peak with the desired m / z for compound 4. Purification of the reaction mixture by preparative HPLC (neutral conditions) yielded compound 4 (1.56 g, 1.33 mmol, 45.3% yield, 62.0% purity) as a pale yellow solid. Expected MW = 728.79, observed m / z: 729.3 [M+H] + .

[0106] (Preparation of Compound 5) [ka] To a solution of compound 4 (1.56 g, 2.14 mmol, 1.0 equivalent) in DMF (10.0 mL), DIEA (1.38 g, 10.7 mmol, 1.86 mL, 5.0 equivalents) was added and the mixture was stirred for 10 minutes. Subsequently, 1-ethyl-6-fluoro-4-oxo-7-piperazine-1-ylquinoline-3-carboxylic acid (1.37 g, 4.28 mmol, 2.0 equivalents) and HOBt (578 mg, 4.28 mmol, 2.0 equivalents) were added to the mixture under an N2 atmosphere. The mixture was stirred at 35°C for 2 hours. LC-MS showed the detection of one major peak with the desired m / z for compound 5. Purification of the reaction mixture by preparative HPLC (neutral conditions) yielded compound 5 (1.45 g, 1.60 mmol, 74.5% yield) as a pale yellow solid. Predicted MW = 909.01, observed m / z: 909.3 [M+H] + .

[0107] (Preparation of BCY9474) [ka] To a solution of compound 5 (1.45 g, 1.60 mmol, 1.0 equivalent) in DCM (9.00 mL), TFA (1.54 g, 13.5 mmol, 1.00 mL, 8.47 equivalents) was added. The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 5 was completely consumed and a single main peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to obtain a residue. Purification of the residue by preparative HPLC (neutral conditions) yielded BCY9474 (850 mg, 1.05 mmol, 65.9% yield, 98.74% purity) as a pale yellow solid. Expected MW = 808.90, observed m / z: 405.3 [M / 2 + H] + and 809.3[M+H] + .

[0108] (BCY9423-(Agb-Val)) [ka] To a solution of BCY9474 (150 mg, 185 μmol, 1.0 equivalent) in DMF (2.00 mL), tert-butyl(NZ)-N-[(tert-butoxycarbonylamino)-pyrazole-1-ylmethylene]carbamate (86.3 mg, 278 μmol, 1.5 equivalents) and DIEA (47.9 mg, 371 μmol, 64.6 μL, 2.0 equivalents) were added. The mixture was stirred at 25°C for 16 hours. LC-MS showed that most of the BCY9474 was consumed, and one main peak with the desired m / z for compound 2 was detected. Purification of the reaction mixture by preparative HPLC (neutral conditions) yielded compound 2 (110 mg, 105 μmol, 56.4% yield) as a white solid. Expected MW = 1050.52, observed m / z: 525.70 [M / 2 + H] + and 1050.82[M+H] + .

[0109] [ka] To a solution of compound 2 (110 mg, 105 μmol, 1.0 equivalent) in DCM (2.00 mL), TFA (770 mg, 6.75 mmol, 500 μL, 64.5 equivalents) was added. The mixture was stirred at 25°C for 0.5 hours. LC-MS showed that compound 2 was completely consumed and a single main peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to obtain a residue. Purification of the residue by preparative HPLC (TFA conditions) yielded BCY9423 (20.8 mg, 24.2 μmol, 23.1% yield, 98.8% purity) as a white solid. Expected MW = 850.94, observed m / z: 425.72 [M / 2 + H] + , 850.67[M+H] + .

[0110] (BCY9477-(Agb(Me)-Val)) [ka] To a solution of BCY9474 (100 mg, 124 μmol, 1.0 equivalent) in DMF (3 mL), N-methylpyrazole-1-carboxyamidine (59.6 mg, 371 μmol, 3.0 equivalents, HCl salt form) and DIEA (95.9 mg, 742 μmol, 129 μL, 6.0 equivalents) were added under an N2 atmosphere. The mixture was stirred at 60°C for 16 hours. LC-MS showed the detection of a single main peak with the desired m / z. Purification of the reaction mixture by preparative HPLC (TFA conditions) yielded BCY9477 (40.9 mg, 46.9 μmol, 37.9% yield, 99.2% purity) as a white solid. Expected MW = 864.96, observed m / z: 432.68 [M / 2 + H] + and 864.62[M+H] + .

[0111] (BCY9696-(Cit-Val-Glu)) (Preparation of Compound 2) [ka] DCM was added to a container containing CTC resin (5 mmol, 4.50 g, 1.10 mmol / g), and then Fmoc-Cit-OH (1.98 g, 5 mmol, 1.0 equivalent) was added while bubbling with N2. DIEA (4.0 equivalents) was added dropwise, and the resin was mixed for 2 hours. MeOH (4.5 mL) was added, and the resin was mixed again for 30 minutes. After that, the resin was drained and washed 5 times with DMF.

[0112] The Fmoc groups were removed by adding 20% ​​piperidine / DMF and allowing the mixture to react for 30 minutes. The resin was then drained and washed five times with DMF.

[0113] To couple the following amino acids, a DMF solution of Fmoc-amino acids was added to the resin and mixed for 30 seconds, after which an activator and base were added. The coupling reaction was allowed to proceed for 1 hour while continuously bubbling with N2. The coupling and Fmoc deprotection rounds were repeated using the following amino acids. [Table 5]

[0114] After the final coupling reaction, the resin was washed three times with MeOH and then dried under vacuum. The side-chain protected peptide was cleaved from the resin by adding 20% ​​HFIP / 80% DCM at room temperature to a flask containing the peptide. This cleavage was then repeated with continuous N2 bubbling (for 1 hour each). The resin was filtered, the filtrate was collected, and then concentrated to remove the solvent. The crude peptide was freeze-dried to obtain compound 2 (1.9 g, 100% purity, 63.3% yield). Expected MW = 600.71, observed m / z: 601.3 [M+H] + .

[0115] (Preparation of Compound 3) [ka] To a solution of compound 2 (500 mg, 832 μmol, 1.0 equivalent) in DCM (10 mL) and MeOH (5 mL), (4-aminophenyl)methanol (123 mg, 999 μmol, 1.2 equivalents) and EEDQ (412 mg, 1.66 mmol, 2.0 equivalents) were added in the dark. The mixture was stirred at 25°C for 12 hours. TLC (DCM:MeOH = 10:1, R) f Calculation by (0.23) showed that compound 2 was completely consumed, and many new spots were formed. LC-MS showed that compound 2 was completely consumed, and one major peak with the desired m / z for compound 3 was detected. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40g SepaFlash® silica flash column, eluent of 0-20% MeOH / DCM @ 40 mL / min) to obtain compound 3 (350 mg, 496 μmol, 59.6% yield) as a pale yellow solid. Expected MW = 705.84, observed m / z: 706.3[M+H] + .

[0116] (Preparation of Compound 4) [ka] To a solution of compound 3 (350 mg, 496 μmol, 1.0 equivalent) in DMF (4 mL), bis(4-nitrophenyl)carbonate (453 mg, 1.49 mmol, 3.0 equivalents) and DIEA (256 mg, 1.98 mmol, 345 μL, 4.0 equivalents) were added under an N2 atmosphere. The mixture was stirred at 25°C for 2 hours. LC-MS showed that compound 3 was completely consumed and a single main peak with the desired m / z was detected. Purification of the reaction mixture by preparative HPLC (neutral conditions) yielded compound 4 (370 mg, 425 μmol, 85.7% yield) as a white solid. Expected MW = 870.41, observed m / z: 870.66 [M+H] + .

[0117] (Preparation of Compound 5) [ka] To a solution of compound 4 (360 mg, 413 μmol, 1.0 equivalent) in DMF (4 mL), DIEA (267 mg, 2.07 mmol, 360 μL, 5.0 equivalents) was added and the mixture was stirred for 10 minutes under an N2 atmosphere. Subsequently, 1-ethyl-6-fluoro-4-oxo-7-piperazine-1-ylquinoline-3-carboxylic acid (264 mg, 827 μmol, 2.0 equivalents) and HOBt (112 mg, 827 μmol, 2.0 equivalents) were added to the mixture. The mixture was stirred at 35°C for 2 hours. LC-MS showed that compound 4 was completely consumed and that one major peak with the desired m / z was detected. Purification of the reaction mixture by preparative HPLC (neutral conditions) yielded compound 5 (390 mg, 371 μmol, 89.8% yield) as a white solid.

[0118] (Preparation of BCY9696) [ka] To a 5 mL solution of compound 5 (150 mg, 143 μmol, 1.0 equivalent) in DCM, TFA (1.93 g, 16.9 mmol, 1.25 mL, 118.0 equivalents) was added. The mixture was stirred at 25°C for 0.5 hours. LC-MS showed that compound 5 was completely consumed and a single main peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to obtain a residue. Purification of the residue by preparative HPLC (A: 0.075% TFA in H2O, B: ACN) yielded compound BCY9696 (66.5 mg, 65.8 μmol, 46.1% yield, 98.4% purity) as a white solid. Expected MW = 994.46, observed m / z: 497.68 [M / 2 + H] + and 994.64[M+H] + .

[0119] (BCY10299-(Dap(CNNH2)-Val-Glu)) (Preparation of Compound 5) [ka] To a 1.9 mL solution of compound 4 (which can be prepared as described in BCY10297; 150 mg, 139 μmol, 1.0 equivalent) in DCM, TFA (150 mg, 1.32 mmol, 0.10 mL, 9.5 equivalents) was added at 0°C and the mixture was stirred for 1 hour. LC-MS (ES10336-123-P1A3) showed that compound 4 was completely consumed, and two main peaks were formed (in this case, one being BCY10297 (a fully deprotected substance) and the other being the desired compound 5). The reaction mixture was concentrated under reduced pressure to obtain a residue. Purification of the residue by preparative HPLC (neutral conditions) yielded compound 5 (50.0 mg, 51.0 μmol, 36.7% yield) as a white solid. Expected MW = 980.09, observed m / z: 490.67 [M / 2 + H] + and 980.07[M+H] + .

[0120] (Preparation of Compound 6) [ka] To a solution of compound 5 (92.0 mg, 93.9 μmol, 1.0 equivalent) in DMF (2 mL), DIEA (48.5 mg, 375 μmol, 65.4 μL, 4.0 equivalents) and pyrazole-1-carboxyamidine (13.8 mg, 93.9 μmol, 1.0 equivalent) were added under an N2 atmosphere. The mixture was stirred at 25°C for 16 hours. LC-MS showed that most of compound 5 was consumed, and one main peak with the desired m / z for compound 6 was detected. Purification of the reaction mixture by preparative HPLC (neutral conditions) yielded compound 6 (72.0 mg, 70.4 μmol, 75.0% yield) as a white solid. Expected MW = 1222.36, observed m / z: 512.1[(M-2 * Boc) / 2+H] + , 1022.7[(M-2 * Boc)+H] + .

[0121] (Preparation of BCY10299) [ka] To a solution of compound 6 (72.0 mg, 70.4 μmol, 1.0 equivalent) in DCM (2.4 mL), TFA (900 mg, 8.00 mmol, 0.60 mL, 114.0 equivalents) was added and the mixture was stirred at 25°C for 2 hours. LC-MS showed that compound 6 was completely consumed and a single main peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (A: 0.075% TFA in H2O, B: ACN) to obtain BCY10299 (15.2 mg, 14.0 μmol, 96.0% purity and 1.1 mg, 1.09 μmol, 97.1% purity; overall yield 21.4%) as a white solid. Expected MW = 966.02, observed m / z: 483.65 [M / 2 + H] + and 966.12[M+H] + .

[0122] (BCY10297-(Dap-Val-Glu)) (Preparation of Compound 1) [ka] DCM was added to a container containing CTC resin (5 mmol, 4.50 g, 1.10 mmol / g), and then Fmoc-Dap(Boc)-OH (2.13 g, 5 mmol, 1.0 equivalent) was added while bubbling with N2. DIEA (4.0 equivalents) was added dropwise, and the resin was mixed for 2 hours. MeOH (4.5 mL) was added, and the resin was mixed again for 30 minutes. After that, the resin was drained and washed 5 times with DMF.

[0123] The Fmoc groups were removed by adding 20% ​​piperidine / DMF and allowing the mixture to react for 30 minutes. The resin was then drained and washed five times with DMF.

[0124] To couple the following amino acids, a DMF solution of Fmoc-amino acids was added to the resin and mixed for 30 seconds, after which an activator and base were added. The coupling reaction was allowed to proceed for 1 hour while continuously bubbling with N2. The coupling and Fmoc deprotection rounds were repeated using the following amino acids. [Table 6]

[0125] After the final coupling reaction, the resin was washed three times with MeOH and then dried under vacuum. The side-chain protected peptide was cleaved from the resin by adding 20% ​​HFIP / 80% DCM at room temperature to a flask containing the peptide. This cleavage was then repeated with continuous N2 bubbling (for 1 hour each). The resin was filtered, the filtrate was collected, and then concentrated to remove the solvent. The crude peptide was freeze-dried to obtain compound 2 (2.27 g, 93.4% purity, 69.1% yield). Expected MW = 629.75, observed m / z: 630.10 [(M-Boc)+H] + , 630.10[M+H] + .

[0126] (Preparation of Compound 2) [ka] To a solution of compound 1 (800 mg, 1.27 mmol, 1.0 equivalent) in DCM (16.0 mL) and MeOH (8.00 mL), (4-aminophenyl)methanol (188 mg, 1.52 mmol, 1.2 equivalents) and EEDQ (628 mg, 2.54 mmol, 2.0 equivalents) were added in the dark. The mixture was stirred at 25°C for 16 hours. TLC (DCM:MeOH = 10:1, R) f Compound 1 was shown to be completely consumed by 0.46. LC-MS showed that compound 1 was completely consumed and that one major peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was then purified by flash silica gel chromatography (ISCO®; 40g SepaFlash® silica flash column, 0-20% MeOH / DCM eluent @ 60 mL / min) to obtain compound 2 (600 mg, 816 μmol, 64.3% yield) as a light brown solid. Expected MW = 734.88, observed m / z: 635.09 [(M-Boc)+H] + and 735.10[M+H] + .

[0127] (Preparation of Compound 3) [ka] To a solution of compound 2 (600 mg, 816 μmol, 1.0 equivalent) in DMF (8.00 mL), bis(4-nitrophenyl)carbonate (745 mg, 2.45 mmol, 3.0 equivalents) and DIEA (422 mg, 3.27 mmol, 569 μL, 4.0 equivalents) were added under an N2 atmosphere. The mixture was stirred at 25°C for 2 hours. LC-MS showed that compound 2 was completely consumed, and a single main peak with the desired m / z was detected. Purification of the reaction mixture by preparative HPLC (neutral conditions) yielded compound 3 (630 mg, 700 μmol, 85.7% yield) as a pale yellow solid. Expected MW = 899.98, observed m / z: 799.99 [(M-Boc)+H] + and 900.02[M+H] + .

[0128] (Preparation of Compound 4) [ka] To a solution of compound 3 (630 mg, 700 μmol, 1.0 equivalent) in DMF (8.00 mL), DIEA (362 mg, 2.80 mmol, 488 μL, 4.0 equivalents) was added and the mixture was stirred for 10 minutes under an N2 atmosphere. Subsequently, 1-ethyl-6-fluoro-4-oxo-7-piperazine-1-ylquinoline-3-carboxylic acid (447 mg, 1.40 mmol, 2.0 equivalents) and HOBt (189 mg, 1.40 mmol, 2.0 equivalents) were added to the mixture. The mixture was stirred at 25°C for 2 hours. LC-MS showed that compound 3 was completely consumed and a single main peak with the desired m / z was detected. The reaction mixture was washed with 250 mL of H2O at 25°C, filtered, and concentrated under reduced pressure to obtain crude compound 4 (630 mg, crude product) as a yellow solid. This was used in the next step without further purification. Expected MW = 1080.20, observed m / z: 490.63 [(M-Boc) / 2+H] + and 1080.09[M+H] + .

[0129] (Preparation of BCY10297) [ka] To a solution of compound 4 (130 mg, 120 μmol, 1.0 equivalent) in DCM (1.60 mL), TFA (596 mg, 5.23 mmol, 400 μL, 43.6 equivalents) was added. The mixture was stirred at 25°C for 0.5 hours. LC-MS showed that compound 4 was completely consumed and a single main peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to obtain a residue. Purification of the residue by preparative HPLC (A: 0.075% TFA in H2O, B: ACN) yielded BCY10297 (45.3 mg, 46.6 μmol, 38.7% yield, 95.0% purity) as a white solid. Expected MW = 923.68, observed m / z: 462.58 [M / 2 + H] + and 924.07[M+H] + .

[0130] (BCY9695-(Agb-Val-Glu)) (Preparation of Compound 2) [ka] DCM was added to a container containing CTC resin (5 mmol, 4.50 g, 1.10 mmol / g), and then Fmoc-Dab(Boc)-OH (2.20 g, 5 mmol, 1.0 equivalent) was added while bubbling with N2. DIEA (4.0 equivalents) was added dropwise, and the resin was mixed for 2 hours. MeOH (4.5 mL) was added, and the resin was remixed for 30 minutes. After that, the resin was drained and washed 5 times with DMF.

[0131] The Fmoc groups were removed by adding 20% ​​piperidine / DMF and allowing the mixture to react for 30 minutes. The resin was then drained and washed five times with DMF.

[0132] To couple the following amino acids, a DMF solution of Fmoc-amino acids was added to the resin and mixed for 30 seconds, after which an activator and base were added. The coupling reaction was allowed to proceed for 1 hour while continuously bubbling with N2. The coupling and Fmoc deprotection rounds were repeated using the following amino acids. [Table 7]

[0133] After the final coupling reaction, the resin was washed three times with MeOH and then dried under vacuum. The side-chain protected peptide was cleaved from the resin by adding 20% ​​HFIP / 80% DCM at room temperature to a flask containing the peptide. This cleavage was then repeated with continuous N2 bubbling (for 1 hour each). The resin was filtered, the filtrate was collected, and then concentrated to remove the solvent. The crude peptide was freeze-dried to obtain compound 2 (2.60 g, 90.0% purity, 72.6% yield). Expected MW = 643.78, observed m / z: 644.4 [M+H] + .

[0134] (Preparation of Compound 3) [ka] To a solution of compound 2 (500 mg, 777 μmol, 1.0 equivalent) in DCM (10 mL) and MeOH (5 mL), (4-aminophenyl)methanol (115 mg, 932 μmol, 1.2 equivalents) and EEDQ (384 mg, 1.55 mmol, 2.0 equivalents) were added in the dark. The mixture was stirred at 25°C for 12 hours. TLC (DCM:MeOH = 10:1, R) f= 0.38), it was shown that Compound 2 was completely consumed and several new spots were formed. By LC-MS, it was shown that Compound 2 was completely consumed and one main peak with the desired m / z was detected. When the reaction mixture was concentrated under reduced pressure, a residue was obtained. Then, the residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica flash column, eluent 0 - 20% MeOH / DCM at 40 mL / min), and Compound 3 (390 mg, 521 μmol, 67.1% yield) was obtained as a pale yellow solid. Predicted MW = 748.44, observed m / z: 749.4 [M + H] + .

[0135] (Preparation of Compound 4) [Chemical formula] To a solution of Compound 3 (390 mg, 521 μmol, 1.0 equivalent) in DMF (4 mL), bis(4-nitrophenyl) carbonate (475 mg, 1.56 mmol, 3.0 equivalents) and DIEA (269 mg, 2.08 mmol, 363 μL, 4.0 equivalents) were added under N2 atmosphere. The mixture was stirred at 25 °C for 2 h. By LC-MS, it was shown that Compound 3 was completely consumed and one main peak with the desired m / z for Compound 4 was detected. The residue was purified by preparative HPLC (neutral conditions), and Compound 4 (400 mg, 438 μmol, 84.0% yield) was obtained as a white solid. Predicted MW = 913.44, observed m / z: 913.60 [M + H] + .

[0136] (Preparation of Compound 5) [Chemical formula] To a solution of compound 4 (400 mg, 438 μmol, 1.0 eq) in DMF (5 mL) was added DIEA (283 mg, 2.19 mmol, 381 μL, 5.0 eq), and the mixture was stirred for 10 minutes. Then, 1-ethyl-6-fluoro-4-oxo-7-piperazin-1-yl-quinoline-3-carboxylic acid (280 mg, 875 μmol, 2.0 eq) and HOBt (118 mg, 875 μmol, 2.0 eq) were added to the mixture under a N2 atmosphere. The mixture was stirred at 35 °C for 2 hours. LC-MS showed that compound 4 was completely consumed and one main peak with the desired m / z for compound 5 was detected. The residue was purified by preparative HPLC (neutral conditions) to give compound 5 (310 mg, 283 μmol, 64.7% yield) as a white solid. Predicted MW = 1093.55, observed m / z: 1093.77 [M+H] + and 547.18 [M / 2+H] + .

[0137] (Preparation of compound 6) [Chemical formula] To a solution of compound 5 (305 mg, 279 μmol, 1.0 eq) in DCM (5.70 mL) was added TFA (462 mg, 4.05 mmol, 0.30 mL, 14.5 eq). The mixture was stirred at 0 °C for 1 hour. LC-MS showed that compound 5 was completely consumed and two main peaks (in this case, one is the desired compound 6 and the other corresponds to the fully protected substance) were formed. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (neutral conditions) to give compound 6 (205 mg, 206 μmol, 74.0% yield) as a white solid. Predicted MW = 993.50, observed m / z: 993.72 [M+H] + and 497.26 [M / 2+H] + .

[0138] (Preparation of compound 7) [Chemical formula] To a solution of compound 6 (205 mg, 206 μmol, 1.0 equivalent) in DMF (3 mL), tert-butyl(NZ)-N-[(tert-butoxycarbonylamino)-pyrazole-1-ylmethylene]carbamate (96.0 mg, 309 μmol, 1.5 equivalents) and DIEA (53.3 mg, 412 μmol, 71.8 μL, 2.0 equivalents) were added under an N2 atmosphere. The mixture was stirred at 25°C for 16 hours. LC-MS showed that compound 6 was completely consumed and a single main peak with the desired m / z for compound 7 was detected. Purification of the residue by preparative HPLC (neutral conditions) yielded compound 7 (70.0 mg, 56.6 μmol, 27.5% yield) as a white solid. Expected MW = 1235.62, observed m / z: 1257.66 [M+Na] + and 618.21[M / 2+H] + .

[0139] (Preparation of BCY9695) [ka] To a solution of compound 7 (70.0 mg, 56.6 μmol, 1.0 equivalent) in DCM (0.90 mL), TFA (154 mg, 1.35 mmol, 100 μL, 23.8 equivalents) was added. The mixture was stirred at 25°C for 0.5 hours. LC-MS showed that compound 7 was completely consumed and a single main peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was then purified by preparative HPLC (A: 0.075% TFA in H2O, B: ACN) to obtain compound BCY9695 (21.9 mg, 22.1 μmol, 39.0% yield, 98.9% purity) as a white solid. Expected MW = 979.46, observed m / z: 979.60 [M+H] + and 490.18[M / 2+H] + .

[0140] (BCY10122-(Dab-Val-Glu)) (Preparation of BCY10122) [ka] To a solution of compound 5 (305 mg, 279 μmol, 1.0 equivalent) in DCM (5.70 mL), TFA (462 mg, 4.05 mmol, 0.30 mL, 14.5 equivalents) was added. The mixture was stirred at 0°C for 1 hour. LC-MS showed that compound 5 was completely consumed and a single main peak with the desired m / z (calculated MW:, observed m / z:) was detected. The reaction mixture was concentrated under reduced pressure to obtain a residue. Purification of the residue by preparative HPLC (neutral conditions) yielded BCY10122 (50.2 mg, 52.0 μmol, 18.6% yield, 97.1% purity) as a white solid. Expected MW = 937.43, observed m / z: 469.24 [M / 2+H] + , 937.65[M+H] + .

[0141] (BCY7761-(Cit-Val) (Preparation of Compound 2) [ka] To a solution of compound 1 (1.50 g, 3.24 mmol, 1.0 equivalent) in DMF (20 mL), bis(4-nitrophenyl)carbonate (2.96 g, 9.73 mmol, 3.0 equivalents) and DIEA (1.68 g, 13.0 mmol, 2.26 mL, 4.0 equivalents) were added under an N2 atmosphere. The mixture was stirred at 15°C for 2 hours. LC-MS showed that compound 1 was completely consumed and a single main peak with the desired m / z was detected. Purification of the reaction mixture by preparative HPLC (neutral conditions) yielded compound 2 (1.20 g, 1.91 mmol, 58.9% yield) as a pale yellow solid. Expected MW = 627.61, observed m / z: 627.94 [M+H] + .

[0142] (Preparation of Compound 3) [ka] To a 10 mL solution of compound 2 (905 mg, 1.44 mmol, 1.15 equivalents) in DMF, DIEA (486 mg, 3.76 mmol, 655 μL, 3.0 equivalents) was added and the mixture was stirred for 10 minutes. Then, HOBt (195 mg, 1.44 mmol, 1.15 equivalents) and MMAE (900 mg, 1.25 mmol, 1.0 equivalent) were added to the mixture. The mixture was stirred at 35°C for 16 hours. LC-MS showed that compound 2 was completely consumed and a single main peak with the desired m / z was detected. Purification of the reaction mixture by preparative HPLC (neutral conditions) yielded compound 3 (1.08 g, 895 μmol, 71.6% yield) as a pale yellow solid. Expected MW = 1206.48, observed m / z: 1206.25 [M+H] + .

[0143] (Preparation of BCY7761) [ka] To a solution of compound 3 (971 mg, 805 μmol, 1.1 equivalents) and BCY3900 (2.00 g, 732 μmol, 1.0 equivalent) in t-BuOH (10 mL) and H2O (10 mL), CuSO4 (0.4 M, 1.83 mL, 1.0 equivalent) and tris(3-hydroxypropyl-triazolylmethyl)amine (THPTA, 318 mg, 732 μmol, 1.0 equivalent) was added. Subsequently, VcNa (0.4 M, 3.66 mL, 2.0 equivalents) was added to the mixture under an N2 atmosphere. The mixture was stirred at 15°C for 2 hours. LC-MS showed that compound 3 was completely consumed and a single main peak with the desired m / z was detected. The reaction was quenched by adding EDTA (0.5 M, 1.5 mL) to the reaction mixture. Subsequently, the reaction mixture was purified by preparative HPLC (A: 0.075% TFA in H2O, B: ACN), yielding BCY7761 (2.20 g, 539 μmol, 73.4% yield, 96.5% purity) as a white solid. Expected MW = 3939.45, observed m / z: 985.47 [M / 4 + H] + and 1313.56[M / 3+H] + .

[0144] (BCY9422-(Cit-Val) (Preparation of Compound 2) [Chemical Structure] DCM was added to a container containing CTC resin (10 mmol, 9.10 g, 1.10 mmol / g), and then Fmoc-Cit-OH (3.98 g, 10 mmol, 1.0 equivalent) was added while bubbling with N2. DIEA (4.0 equivalents) was added dropwise, and the resin was mixed for 2 hours. MeOH (9.1 mL) was added, and the resin was remixed for 30 minutes. Then, the resin was drained and washed 5 times with DMF.

[0145] 20% piperidine / DMF was added and reacted for 30 minutes to remove the Fmoc group. Then, the resin was drained and washed 5 times with DMF.

[0146] To couple the next amino acid, a DMF solution of Fmoc-amino acid was added to the resin, mixed for 30 seconds, and then an activator and a base were added. The coupling was reacted for 1 hour while continuously bubbling with N2. The rounds of coupling and Fmoc deprotection were repeated using the following amino acids. [Table 8]

[0147] After the last amino acid coupling, the resin was washed 3 times with MeOH and then dried under vacuum. Cleavage from the resin was performed by adding 20% HFIP / 80% DCM to the flask containing the side-chain protected peptide at room temperature. Then, the cleavage was repeated while continuously bubbling with N2 (1 hour each). The resin was filtered, the filtrate was collected, and then concentrated to remove the solvent. Freeze-drying of the crude peptide gave Compound 2 (crude, 1.80 g, 91.82% purity, 39.7% yield). Predicted MW = 415.49, observed m / z: 416.2 [M+H] + .

[0148] (Preparation of Compound 3) [ka] To a solution of compound 2 (500 mg, 1.20 mmol, 1.0 equivalent) in DCM (10 mL) and MeOH (5 mL), (4-aminophenyl)methanol (178 mg, 1.44 mmol, 1.2 equivalents) and EEDQ (595 mg, 2.41 mmol, 2.0 equivalents) were added in the dark. The mixture was stirred at 25°C for 12 hours. TLC (DCM:MeOH = 10:1, R f A reaction (0.53) indicated that compound 2 was completely consumed. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40g SepaFlash® silica flash column, 0-25% MeOH / DCM eluent @ 40 mL / min) to obtain compound 3 (380 mg, 730 μmol, 60.7% yield) as a pale yellow solid. [ka]

[0149] (Preparation of Compound 4) [ka] To a solution of compound 3 (380 mg, 730 μmol, 1.0 equivalent) in DMF (8 mL), bis(4-nitrophenyl)carbonate (666 mg, 2.19 mmol, 3.0 equivalents) and DIEA (377 mg, 2.92 mmol, 509 μL, 4.0 equivalents) were added. The mixture was stirred at 25°C for 2 hours. LC-MS showed that compound 3 was completely consumed and a single main peak with the desired m / z for compound 4 was detected. Purification of the reaction mixture by preparative HPLC (neutral conditions) yielded compound 4 (368 mg, 537 μmol, 73.5% yield) as a white solid. Expected MW = 685.32, observed m / z: 686.1 [M+H] + .

[0150] (Preparation of BCY9422) [ka] To a solution of compound 4 (150 mg, 219 μmol, 1.0 equivalent) in DMF (3 mL), DIEA (141 mg, 1.09 mmol, 191 μL, 5.0 equivalents) was added and the mixture was stirred at 25°C for 10 minutes. Subsequently, 1-ethyl-6-fluoro-4-oxo-7-piperazine-1-ylquinoline-3-carboxylic acid (105 mg, 328 μmol, 1.5 equivalents) and HOBt (29.6 mg, 219 μmol, 1.0 equivalent) were added to the mixture. The mixture was stirred at 35°C for 16 hours. LC-MS showed the detection of a single main peak with the desired m / z. The reaction mixture was purified by preparative HPLC (A: 0.075% TFA in H2O, B: ACN) to obtain BCY9422 (107.8 mg, 119 μmol, 54.6% yield, 95.9% purity) as a white solid. Expected MW = 865.95, observed m / z: 433.7 [M / 2 + H] + and 866.2[M+H] + .

[0151] (Plasma stability analysis) Pooled frozen plasma was thawed in a 37°C water bath. The plasma was centrifuged at 4000 rpm for 5 minutes, and any blood clots were removed. The pH was adjusted to 7.4 ± 0.1 if necessary. A 1 mM stock solution was prepared using DMSO. Propantheline (positive control) was prepared by diluting 5 μL of stock solution (10 mM) with 495 μL of ultrapure water to prepare a 100 μM working solution. A 100 μM working solution of the test compound was prepared by diluting 10 μL of stock solution (1 mM) with 90 μL of DMSO. 2 μL of the administration solution (100 μM) was spiked into 98 μL of blank plasma to achieve a final concentration of 2 μM in two consecutive steps, and the samples were incubated in a water bath at 37°C. At each time point (0, 1, 2, 4, 6, and 24 hours), 400 μL of 200 ng / mL tolbutamide and labetalol in 100% MeOH were added and thoroughly mixed to precipitate the protein. The sample plate was centrifuged at 4,000 rpm for 15 minutes. Aliquots (150 μL) of the supernatant were transferred from each well and subjected to LC-MS / MS analysis.

[0152] The percentage of the test compound remaining after incubation in plasma was calculated using the following equation: % remaining = 100 × (PAR at specified incubation time / PAR at T0 time) (Here, PAR is the peak area ratio of the analyte to the internal standard (IS).)

[0153] The specified incubation times are T0 (0 hours) and Tn (n = 0, 1, 2, 4, 6, 24 hours). The half-life (T1 / 2) was calculated from a logarithmic linear plot of concentration versus time.

[0154] If the percentage survival rate at the maximum incubation time (which is 24 hours in this study) is higher than 75%, it is considered to be within the acceptable range of experimental variability. Therefore, we reported a corresponding t1 / 2 of >57.8 hours.

[0155] (Cathepsin B (CatB) assay) 15 μL of the test compound solution (2 mM in DMSO) was added in duplicate to the incubation plate. 30 μL of the cathepsin B stock solution (16 μM) was pre-activated at room temperature for 10 minutes using 1500 μL of the activation buffer. The cathepsin B solution was diluted in 13.17 mL of water, and then 735 μL of the activated enzyme mixture was added to the incubation plate. The mixture was incubated at 37 °C in a water bath. At various time points (e.g., 0 h, 1 h, 2 h, 4 h, 6 h, 24 h), 100 μL aliquots were taken and the reaction was terminated by quenching with 400 μL of cold IS-enhanced quenching solution. The samples were mixed and centrifuged at 4000 rpm for 20 minutes. 50 μL of the supernatant was taken into a new plate containing 150 μL of ultrapure water, and after thoroughly mixing the samples, they were subjected to LC-MS / MS analysis.

[0156] (Xenograft model) For the cell-derived xenograft (CDX) model, mice (balb / c nude, female, 18 - 23 g at the start of the test) were inoculated with HT1080 cells (5.0 × 10 6 cells / mouse) in 0.2 ml PBS in the right flank. When the mean tumor volume reached a pre-specified starting size, the animals were randomly assigned. The group size was n = 4. All tests included a vehicle-treated control.

[0157] Administration was by rapid intravenous injection. Tumor volume was measured two-dimensionally using calipers, and the volume was expressed in mm 2 using the formula: V = 0.5a × b 3 (where a and b are the long and short diameters of the tumor, respectively). All xenograft tests were conducted at Wuxi AppTec Co., Ltd. (Shanghai).

[0158] (Plasma pharmacokinetics of the bicyclic conjugate and free payload in CD-1 mice) Male CD-1 mice were administered each bicyclic conjugate, formulated in 25 mM histidine HCl, 10% sucrose, pH 7, via tail vein injection. Continuous blood collection (approximately 80 μL blood / time point) was performed at each time point via the submandibular or saphenous vein. All blood samples were immediately transferred to pre-cooled microcentrifuge tubes containing 2 μL of K2-EDTA (0.5 M) as an anticoagulant and placed on wet ice. Blood samples were immediately processed for plasma by centrifugation at approximately 4°C and 3000 g. A precipitate containing an internal standard (350 μL) was immediately added to 35 μL of plasma sample, mixed thoroughly, and centrifuged at 3220 g and 4°C for 15 minutes. The supernatant was transferred to pre-labeled polypropylene microcentrifuge tubes and then flash-frozen on dry ice. If necessary, samples were stored below 70°C until analysis. The supernatant sample was mixed with 50 μL of water, thoroughly vortexed, and centrifuged at 3220 g at 4°C for 15 minutes. The supernatant sample was injected into an Acquity UPLC in positive ion mode with an AB Sciex 6500+Triple Quad MS for LC-MS / MS analysis, and the concentrations of the bicyclic conjugate and free payload were determined. Plasma concentration-time data were analyzed using a non-compartmental method with the Phoenix WinNonlin 6.3 software program. C0, Cl, Vd ss , T 1 / 2 AUC (0-last) AUC (0-inf) , MRT (0-last) , MRT (0-inf) We also reported graphs of plasma concentration versus time profiles.

[0159] (Measurement of MMAE in plasma, muscle, and tumor samples) Tumor samples from in vivo xenograft studies were weighed and homogenized (diluted 10× in homogenization buffer containing a protease inhibitor). Tumor homogenates and plasma were then analyzed by LC-MS / MS according to standard procedures.

[0160] (Test compound) The compounds used in the following tests were constructed as described above using norfloxacin, used as a surrogate payload, conjugated to the di / tripeptide linker of the present invention via a PAB self-sacrificing group. The peptide linker was capped at the N-terminus with 5-(dimethylamino)-5-oxopentanoic acid, as schematically shown below: [ka]

[0161] (Bicyclic toxin conjugate (BTC)) BTCs incorporating the di / tripeptide linker of the present invention were synthesized by preparing azide-supported toxin / linker sequences. Here, MMAE cytotoxin was linked to a peptide-cleaving linker via a PAB self-sacrificing group, and this was conjugated to a bicyclic peptide MT1-MMP binder (BCY3900; described as SEQ ID NO: 5 in WO 2016 / 067035) using copper-catalyzed azide-alkyne cyclization. [ka]

[0162] (Example 1: Plasma stability analysis using the linker of the present invention) The exchange of a citrulline residue with a basic non-natural amino acid in the CatB-sensitive dipeptide linker Cit-Val was shown to increase the linker's stability against nonspecific cleavage when incubated with mouse plasma in vitro. This is demonstrated by the extension of the half-lives of the test compounds compared to the Cit-Val linker (BCY9422) shown in Table 1. Table 1: Exchange of P1 Cit with basic non-natural amino acids in dipeptide linkers [Table 9]

[0163] In some cases, for example, with Agb and Dab, an additive effect is observed when citrulline is exchanged within a linker containing the Cit-Val-Glu motif (which has been reported to have higher mouse plasma stability than Cit-Val). In this case, similar linkers incorporating basic residues show a further increase in mouse plasma stability over Cit-Val-Glu, as shown in Table 2. Table 2: Exchange of P1 Cit with basic non-natural amino acids in tripeptide linkers [Table 10]

[0164] BTCs incorporating a linker with a basic non-natural amino acid at the P1 position exhibit increased stability in mouse plasma. For example, a BTC with a Dab-Val cleavage linker (see BCY10989 in Table 3) has a half-life of 30.8 hours in mouse plasma (with EDTA anticoagulant) compared to 6.8 hours for a Cit-Val linker (see BCY7761 in Table 3).

[0165] When Glu is incorporated at the P3 position, linkers with Dab at the P1 position exhibit enhanced stability in plasma compared to their Cit counterpart (see Cit-Val-Glu compared to Dab-Val-Glu in human plasma, and Cit-1Nal-Glu compared to Dab-1Nal-Glu in rat and mouse plasma EDTA anticoagulant). Table 3: Plasma stability of BTC [Table 11]

[0166] (Example 2: CatB cleavage rate analysis using the linker of the present invention) The exchange of citrulline residues with basic non-natural amino acids in the CatB-sensitive dipeptide linker Cit-Val also modulates the linker's cathepsin B cleavage rate. For example, Dab, Agb, and Agb(Me) each increase the in vitro cathepsin B cleavage rate compared to Cit-Val (see Table 4). Dap and Dap(CNNH2) decrease the cleavage rate (see Table 4). Table 4: Exchange of P1 Cit with basic non-natural amino acids in dipeptide linkers [Table 12]

[0167] The exchange of citrulline residues in the CatB-sensitive tripeptide linker Cit-Val-Glu with basic non-natural amino acids modulates the linker's cathepsin B cleavage rate. For example, Dab and Agb exhibit similar in vitro cathepsin B cleavage rates to Cit-Val (see Table 5). Substitution with Dap and Dap(CNNH2) reduces the cleavage rate (see Table 5). Table 5: Exchange of P1 Cit with basic non-natural amino acids in tripeptide linkers [Table 13]

[0168] The rate of cathepsin B cleavage in linkers can be regulated by introducing various non-natural amino acids at the P1 and P2 positions. Results of CatB cleavage analysis using BTC are shown in Table 6, where the exchange of Cit and Dab in the Cit-Val linker results in a linker that cleaves more slowly. When Glu is introduced at the P3 position in these sequences, the cleavage rates between the two linkers are similar. Exchanging the P1 position with Dap significantly delays CatB cleavage of the linker. Exchanging Val at the P2 position with 1Nal significantly delays CatB cleavage, while its positional isomer 2Nal only slightly reduces the cleavage reaction rate. Incorporation of Dpa at P2 dramatically reduces the CatB cleavage rate, and cBu completely inhibits cleavage. Table 6: CatB Cut of BTC [Table 14]

[0169] (Example 3: Plasma protein binding analysis using the linker of the present invention) Altering the amino acids at the P1 and P2 positions can modulate the plasma protein binding of BTC. Table 7 shows that replacing Cit at the P1 position with Dab increases the percentage of unbound BTC. Replacing Val at the P2 position with 1Nal decreases the percentage of unbound BTC. Table 7: Plasma protein binding of BTC [Table 15]

[0170] (Example 4: Pharmacokinetic analysis using the linker of the present invention) The exchange of dipeptide linker amino acids can alter the pharmacokinetic (PK) profile of BTC.

[0171] (mouse) The results shown in Figures 1-6 and Table 8 demonstrate that the linker containing 1Nal at the P2 position exhibits an extended half-life in mouse PK tests. The linker with increased mouse plasma stability shows lower relative levels of free MMAE to Cit-Val in plasma (compared to the parent compound). Table 8: Summary of pharmacokinetic analysis in mice [Table 16]

[0172] (Rat) The results of rat PK experiments, shown in Figures 7 and 8 and Table 9, indicate that BCY10984 has an extended half-life compared to the Cit-Val analog BCY7761. Free MMAE toxins in plasma are also lower (compared to the intact parent). Table 9: Summary of pharmacokinetic analysis in rats [Table 17]

[0173] (Example 5: Tumor-reducing efficacy and toxin levels in tumors) The results shown in Figures 9 and 10 demonstrate that BCY10984 (Cit-1Nal-Glu linker) showed higher efficacy than BCY7761 (BT1769-Cit-Val linker) in a mouse CDX model (HT1080 cells), with complete tumor clearance observed after a single dose at 1 mg / kg and 3 mg / kg. Animal body weight was not affected by these doses.

[0174] The results shown in Figures 11-15 indicate that when BCY10984 was administered to mice carrying HT1080 tumors, higher levels of MMAE toxins were observed in the tumors compared to the same dose of BT1769. Similar MMAE levels were present in plasma and muscle tissue.

[0175] (Example 6: In vivo efficacy test using the linker of the present invention in an HT1080 xenograft model in BALB / c nude mice) ((a) Test Objectives) The purpose of this study was to evaluate the in vivo therapeutic efficacy of BCY10984 and BCY12951 (same as BCY10984 but containing a linker conjugated to an unbound bicyclic peptide ligand, i.e., a drug conjugate with the composition: MMAE-PAB-(Dab-Val-Glu)-unbound bicyclic peptide) in an HT1080 xenograft model in BALB / c nude mice.

[0176] ((b) Experimental design) [Table 18] Note: Mice were monitored for 2-3 dose cycles until day 39. Mice in groups 5 and 6 were administered 45 μM BCY10984 on days 14 and 28.

[0177] ((c) Material) (i) Animals and living conditions (animal) Species: Mouse (Mus Musculus) Category: BALB / c nude Age: 6-8 weeks Sex: Female Weight: 18-22g Number of animals: 35 mice + spares Animal supplier: Shanghai Lingchang Biotechnology Experimental Animal Co. LTD

[0178] (Breeding conditions) The mice were housed in individual ventilated cages with a constant temperature and humidity, with five animals in each cage. ·Temperature: 20~26℃ ·Humidity 40~70% Cage: Made of polycarbonate. Dimensions: 375mm x 215mm x 180mm. The bedding material for the animals is corn cobs, which are replaced twice a week. Diet: Throughout the entire study period, the animals had free access to irradiated dry granule food. Water: Animals had free access to sterile drinking water. Cage Identification: The identification markings for each cage included the following information: number of animals, sex, strain, arrival date, treatment, number of tests, group number, and start date of treatment. Animal identification: Animals were marked using ear coding.

[0179] (d) Experimental methods and procedures ((i) Cell culture) HT1080 cells were maintained in EMEM medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C under an atmosphere of 5% CO2 in air. Tumor cells were regularly subcultured twice a week. Cell proliferations in the exponential growth phase were collected and counted for tumor inoculation.

[0180] (ii) Tumor inoculation To induce tumor formation, HT1080 tumor cells (5 × 10) in 0.2 ml of PBS were placed in the right flank of each mouse. 6 (1) was administered subcutaneously. The average tumor volume was 320 mm². 3 When the target level was reached, animals were randomly assigned for efficacy testing. The number of animals administered the test substance and the number of animals in each group are shown in the experimental design table.

[0181] (iii) Preparation of test product formulation) [Table 19]

[0182] (iv) Observation All procedures for handling, managing, and treating animals in the study were carried out in accordance with the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec. During periodic monitoring, animals were checked for any effects of tumor growth and treatment on normal behaviors such as mobility, food and water consumption (by observation only), weight gain / loss, eye / hair matting, and any other abnormal effects described in the protocol. Deaths and observed clinical symptoms were recorded based on the number of animals in each subset.

[0183] (v) Tumor measurement and endpoints The primary endpoint was to determine whether tumor growth could be delayed or whether the mice could be cured. Tumor volume was measured three times a week in two dimensions using calipers, and the volume was given by the formula: V = 0.5a × b 2 (In the formula, a and b are the long and short diameters of the tumor, respectively) using mm 3 This was expressed as follows. Next, this tumor size was used to calculate the T / C ratio. The T / C ratio (in %) is an indicator of the antitumor effect; T and C are the average volumes of the treated group and the control group on a given day, respectively.

[0184] TGI is given by the formula: TGI(%)=[1-(T i -T0) / (V i Using -V0) × 100, calculate for each group; T i is the mean tumor volume of the treatment group on a given day, T0 is the mean tumor volume of the treatment group on the start of treatment, and Vi is T i V0 represents the mean tumor volume of the vehicle control group on the same day, while V0 represents the mean tumor volume of the vehicle group on the treatment initiation day.

[0185] ((vi) Statistical analysis) Simple statistics, including the mean and standard error of the mean (SEM), are provided for the tumor volume of each group at each time point.

[0186] Statistical analysis of the difference in tumor volume between groups was performed on data obtained at the best treatment time after the final dose.

[0187] One-way ANOVA was performed to compare tumor volumes between groups. If a significant F-statistic (ratio of treatment variance to error variance) was obtained, the group comparison was performed using the Games-Howell test. A two-sided t-test was performed to compare tumor volumes between the two groups. All data were analyzed using GraphPad 5.0. A p-value of <0.05 was considered statistically significant.

[0188] ((e) Results) (i) Tumor growth curve The tumor growth curves are shown in Figures 16 and 17.

[0189] (ii) Tumor volume tracing The mean tumor volume over time in female BALB / c nude mice carrying HT1080 tumors is shown in Table 10. Table 10: Tumor volume trace over time [Table 20]

[0190] (iii) Tumor growth inhibition analysis The tumor growth inhibition rates of BCY10984 and BCY12951 in the HT1080 xenograft model were calculated based on tumor volume measurements 11 days after the start of treatment. Table 11: Tumor growth inhibition analysis [Table 21] a. Mean ± SEM; b. Tumor growth inhibition is calculated by dividing the group-average tumor volume of the treatment group by the group-average tumor volume of the control group (T / C).

[0191] ((f) Summary and discussion of results) This study evaluated the therapeutic efficacy of BCY10984 and BCY12951 in an HT1080 xenograft model. Measured tumor volumes for all treatment groups at various time points are shown in Figures 16 and 17 and Tables 10 and 11.

[0192] The average tumor volume of vehicle-treated mice was 1731 mm³ on day 11 after the start of treatment. 3 It reached 5μM qw(TV=1257mm). 3 , TGI=33.6%, p>0.05), 15μM qw(TV=288mm 3 , TGI=102.3%, p<0.001), and 45μM qw(TV=15mm 3 BCY10984 at a dose-dependent level (TGI=121.6%, p<0.001) showed dose-dependent antitumor activity. Among these, 45 μM qw of BCY10984 completely eradicated tumors on day 16. 5 μM qw (TV=10¹¹ mm) 3 (TGI=51.1%, p<0.01) and 45μM qw(TV=901mm) 3 BCY12951 showed significant antitumor activity (TGI = 58.9%, p < 0.001).

[0193] When comparing the antitumor efficacy between the two test samples, 5 μM BCY10984 showed comparable antitumor efficacy to 5 μM BCY12951 (p>0.05), while 15 μM and 45 μM BCY10984 showed stronger efficacy than BCY12951 at the same molar concentration dose (15 μM BCY10984 vs. 15 μM BCY12951, p<0.001; 45 μM BCY10984 vs. 45 μM BCY12951, p<0.001).

[0194] The seven animals in groups 5 and 6 were measured at 1291 mm. 3 The average starting tumor size was 45 μM BCY10984, and the mice were treated with it. After the first dose, all mice showed rapid tumor regression, and after the second dose, all tumors were completely eradicated. This application provides the invention in the following embodiments. (Aspect 1) A linker that includes the -P1-P2-P3- section: P1 represents a basic non-natural amino acid or its derivative; P2 represents a hydrophobic amino acid or a hydrophobic unnatural amino acid; and The linker wherein P3 is either absent or represents an acidic amino acid or an acidic non-natural amino acid, and if P1 represents Cit and P2 represents Val, then P3 must represent an acidic non-natural amino acid. (Aspect 2) P1 is 2-amino-4-guanidinobutanoic acid (Agb); 2-amino-4-(3-methylguanidino)butanoic acid (Agb(Me)); 2,4-diaminobutanoic acid (Dab); 2,3-diaminopropanoic acid (Dap); 2-amino-3-guanidinopropanoic acid (Dap(CNNH) 2 A linker according to embodiment 1, representing a basic non-natural amino acid selected from )); and citrulline (Cit): for example, citrulline (Cit). (Aspect 3) A linker according to Embodiment 1 or Embodiment 2, wherein P2 represents a hydrophobic amino acid selected from Ala, Gly, Ile, Leu, Met, Phe, Pro, Trp, and Val, or a hydrophobic unnatural amino acid selected from cyclobutyl, diphenylalanine (Dpa), 1-naphthylalanine (1Nal), 2-naphthylalanine (2Nal), and methyltryptophan (Trp(Me)), for example, a hydrophobic amino acid selected from Val, or an unnatural amino acid selected from cyclobutyl, Dpa, 1Nal, and 2Nal, for example, 1-naphthylalanine (1Nal). (Aspect 4) A linker according to any one of embodiments 1 to 3, wherein P3 represents an acidic amino acid selected from Asp and Glu, for example, an acidic amino acid selected from Glu. (Appendix 5) The linker according to any one of the embodiments 1 to 4, wherein the aforementioned -P1-P2-P3- represents the following: (Table 1) TIFF0007879099000088.tif106170 。 (Aspect 6) A drug conjugate comprising a target-binding binder and a cytotoxic agent, wherein the binder is connected to the cytotoxic agent via a linker according to any one of embodiments 1 to 5. (Aspect 7) The drug conjugate according to embodiment 6, wherein the binder is a peptide, for example, an antibody or a bicyclic peptide, particularly a bicyclic peptide. (Pattern 8) The drug conjugate according to embodiment 6 or embodiment 7, wherein the cytotoxic agent is DM1 or MMAE, for example, MMAE. (Aspect 9) A drug conjugate according to any one of embodiments 6 to 8, which is protease resistant, for example, resistant to cathepsin B cleavage, compared to a conjugate in the absence of the linker. (Aspect 10) A drug conjugate according to any one of embodiments 6 to 9, which is plasma stable when compared to a conjugate in the absence of the linker. (Aspect 11) A drug conjugate according to any one of embodiments 6 to 10, selected from BCY10989, BCY10980, BCY10982, BCY10983, BCY10984, BCY10981, BCY10985, BCY10986, BCY10987, and BCY10988, for example, BCY10984. (Aspect 12) A pharmaceutical composition comprising a drug conjugate according to any one of embodiments 6 to 11 in combination with one or more excipients that are acceptable as pharmaceuticals. (Aspect 13) A drug conjugate according to any one of embodiments 6 to 11, for use in preventing, suppressing, or treating cancer.

Claims

1. A linker including a -P1-P2-P3- portion, wherein the -P1-P2-P3- portion is in the direction from C-terminus to N-terminus from P1 to P3, and the -P1-P2-P3- portion represents one of the -P1-P2-P3- portions shown in Table 1 below. (Table 1) Table 1 The linker wherein Cit is citrulline, 1Nal is 1-naphthylalanine, Glu is glutamic acid, Dab is 2,4-diaminobutyric acid, cBu is 1-aminocyclobutane-1-carboxylic acid, Dpa is diphenylalanine, and 2Nal is 2-naphthylalanine.

2. The linker according to claim 1, wherein the -P1-P2-P3- portion represents -Cit-1Nal-Glu.

3. A drug conjugate comprising a target-binding binder and a cytotoxic agent, wherein the binder is connected to the cytotoxic agent via a linker according to claim 1 or 2.

4. The drug conjugate according to claim 3, wherein the binder is a peptide, and optionally an antibody or a bicyclic peptide.

5. The drug conjugate according to claim 3, wherein the cytotoxic agent is DM1 or MMAE.

6. The drug conjugate according to claim 3, which is protease-resistant and optionally cathepsin B-resistant when compared to the conjugate in the absence of the linker.

7. The drug conjugate according to claim 3, which is plasma stable compared to the conjugate in the absence of the linker.

8. (i) The -P1-P2-P3- portion is -Cit-1Nal-Glu-, and the drug conjugate is 【Chemistry 1】 Is it, (ii) The -P1-P2-P3- portion is -Dab-1Nal-Glu- and the drug conjugate is 【Chemistry 2】 Is it, (iii) The -P1-P2-P3- portion is -Dab-2Nal-Glu- and the drug conjugate is 【Transformation 3】 Is it, (iv) The -P1-P2-P3- portion is -Dab-Dpa-Glu- and the drug conjugate is 【Chemistry 4】 is or (v) The -P1-P2-P3- portion is -Dab-cBu-Glu- and the drug conjugate is 【Transformation 5】 And, Here, the structure of BCY3900 is: 【Transformation 6】 The drug conjugate according to claim 3, which is a bicyclic peptide having [a specific characteristic].

9. A pharmaceutical composition comprising a drug conjugate according to any one of claims 3 to 8.

10. A pharmaceutical composition comprising a drug conjugate according to any one of claims 3 to 8 for the purpose of preventing, suppressing, or treating cancer.

11. Use of a composition comprising a drug conjugate according to any one of claims 3 to 8 in the manufacture of a pharmaceutical product for preventing, suppressing, or treating cancer in a patient.

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