Ligand-drug conjugates with improved pharmacokinetics and drug release properties

Abstract

The present invention relates to ligand-drug-conjugates for use in the treatment of disease. In particular, the present invention relates to ligand-drug-conjugates comprising a linker system that allow for improved delivery of drugs to target cells while retaining the advantageous pharmacokinetic properties of antibodies. The present invention also relates to a ligand-drug-conjugate which achieves a high drug-antibody ratio and exhibits excellent pharmacokinetic properties, resulting in significantly improved efficacy. In certain aspects, the invention also relates to ligand-drug-conjugates for intracellular delivery of a cytotoxic drug to a tumor or cancer cell.

Description

Technical Field

[0001] The present invention relates to ligand-drug-conjugates (LDCs) for treating diseases. Specifically, the present invention relates to ligand-drug-conjugates comprising a linker system that allows for improved delivery of drugs to target cells while retaining the favorable pharmacokinetic properties of antibodies. The present invention also relates to ligand-drug-conjugates that achieve a high drug-antibody ratio (DAR) and exhibit excellent pharmacokinetic properties, resulting in significantly improved efficacy. In certain aspects, the present invention also relates to ligand-drug-conjugates for intracellular delivery of cytotoxic drugs to tumors or cancer cells. Background of the Invention

[0003] Recently, a lot of interest has been focused on the use of enzyme-triggered drug release systems (such as antibody-drug-conjugates (ADCs)) for targeted delivery of cytotoxic agents to tumor cells. Antibody-drug-conjugates are usually composed of three components: antibodies targeting antigens highly expressed on tumor cells, cytotoxic agents (sometimes referred to as "toxins" or "payloads"), and a linker system that can release cytotoxic agents from antibodies when internalized into cancer cells. Ideally, antibody-drug-conjugates should retain the favorable pharmacokinetic and functional properties of antibodies, remain intact and non-toxic in the systemic circulation (blood), and become active at the target site, releasing sufficient amounts of drugs to kill target cells. Therefore, one of the biggest challenges in developing antibody-drug-conjugates is to design a linker system for conjugating antibodies to drugs, which is non-toxic and stable in the systemic circulation, but can still release drugs at high rates and in sufficient amounts in target cells while retaining the favorable pharmacokinetic properties of antibodies.

[0004] Many linker systems have been developed for specific intracellular release of cytotoxic drugs. There are two main families of linkers: cleavable and non-cleavable. Cleaved linkers typically utilize intrinsic properties of target cells, such as protease sensitivity, to selectively release drugs, such as cytotoxic agents, from the conjugate. Non-cleavable linkers typically rely on complete degradation of the antibody after the conjugate is internalized into the target cell. Examples of antibody-drug-conjugates using non-cleavable linkers are the humanized anti-HER2 (anti-ErbB2) antibody-maytansine conjugate emtansine trastuzumab (T-DM1, or LoRusso et al. Clin. Cancer Res. 2011, 17, 6437-6447), which comprises the monoclonal antibody trastuzumab conjugated to the tubulin polymerization inhibitor mertansine (DM1) via a non-cleavable maleimidomethylcyclohexane-1-carboxylate (MCC) linker.

[0005] Although non-cleavable antibody-drug-conjugates such as T-DM1 Because of its stability in the systemic circulation, it is considered to be advantageous, but they may not show the desired efficacy. In particular, for example, due to the slow degradation of the antibody portion, the drug may not be released in sufficient amounts to achieve the desired pharmacological action, and / or may be released in a less effective modified form (e.g., as a "linker-drug" construct). For example, the lysosomal degradation of the antibody component of T-DM1 releases lysine-MCC-DM1 moiety, which efficiently binds to tubulin, but has only a limited ability to induce cytotoxic bystander effects (Ogitani et al. Cancer Sci. 2016, 107, 1039-1046). The failure of treatment due to inherent and acquired resistance to T-DM1 also remains a major clinical challenge (Hunter et al. Br. J. Cancer 2020, 122 (5), 603-612).

[0006] Peptide linkers have also been proposed because they combine good stability in systemic circulation with rapid intracellular drug release by specific enzymes (e.g., proteases). In particular, peptide linkers comprising valine-citrulline (Val-Cit) dipeptides as substrates for intracellular cleavage by cathepsin B (Cat B) have been described (Lu et al. Int. J. Mol. Sci. 2016, 17, 561-582; Jain et al. Pharm. Res. 2015, 32 (11), 3526-3540; Dubowchik et al. Bioconj. Chem. 2002, 13, 855-859). Cat B is a lysosomal cysteine ​​protease involved in many physiological processes. It differs from other cysteine ​​proteases in that it has endopeptidase and exopeptidase (carboxydipeptidase) activities, which means that it can remove dipeptide units from the C-terminus of proteins and peptides (Turk et al. Biochim. Biophys. Acta 2012, 1824(1), 68-88).

[0007] Typically, enzymatic cleavage of the conjugate releases the antibody and linker-drug conjugate at the target site. The linker must in turn allow rapid release of the drug from the linker-drug conjugate. Therefore, "self-immolative" spacers between the linker and the drug have been proposed to increase the rate of drug release after enzymatic cleavage. Self-immolative spacers typically release the drug by a mechanism based on elimination or cyclization.

[0008] An example of a linker system comprising a self-immolative spacer is, for example, in the bremtuximab-vedotin conjugate The p-aminobenzyloxycarbonyl (PABC) linker used in (Younes et al. N. Engl. J. Med. 2010, 363, 1812-1821; Jain et al. Pharm. Res. 2015, 32 (11), 3526-3540). The PABC linker system used in antibody-drug-conjugates utilizes a protease-sensitive Val-Cit-PABC dipeptide linker that can be recognized and cleaved by cathepsin B. The maleimidocaproyl (MC) moiety is typically used to attach the linker unit to the antibody and serves as a spacer between the drug and the antibody to avoid steric conflicts when cathepsin B recognizes the substrate. After enzymatic cleavage of the citrulline-PABC amide bond, the resulting PABC-substituted drug, such as monomethyl auristatin E (MMAE), spontaneously undergoes 1,6-elimination, releasing free drug (e.g., MMAE) as a product. However, the efficacy of the PABC linker system may be limited due to slow intracellular drug release and the tendency of the Val-Cit-PABC moiety to break off in the systemic circulation (Dorywalska et al. Mol. Cancer Ther. 2016, 15(5), 958-970).

[0009] In addition, in vivo studies have shown that the pharmacokinetic properties of conjugates based on Val-Cit-PABC, such as distribution and liver clearance, depend on the number of drug molecules attached to the antibody portion (Strop et al. Chem. & Biol. 2013, 20, 161-167). Therefore, an important parameter of antibody-drug-conjugates is the drug-antibody ratio (DAR) (or drug loading), which refers to the average number of one or more drug molecules attached to an antibody portion. DAR not only affects efficacy, but also affects the pharmacokinetic properties and toxicity of the conjugate. Due to the increased tendency of conjugate molecules to aggregate and / or premature rupture, high DAR is associated with reduced pharmacokinetic properties and / or higher toxicity.

[0010] In order to overcome these problems, it has been proposed to use a hydrophilic linker system containing a negatively charged sulfonate group, a polyethylene glycol group or a pyrophosphate diester group to reduce conjugate aggregation. Similarly, WO 2015 / 057699 A2 discloses an antibody-drug-conjugate based on a combination of a drug (such as MMAE) and a linker system, wherein the linker system comprises a cleavable Val-Cit-PABC part and a hydrophilic untethered polyethylene glycol group. The conjugate disclosed in WO 2015 / 057699 A2 is said to show good pharmacokinetic properties even at high DAR (e.g., 8) in an in vivo model. However, the effect of the linker system disclosed in, for example, WO 2015 / 057699 A2 may be limited due to nonspecific enzymatic and / or premature cleavage, slow intracellular drug release, and possible increased lysosomal capture.

[0011] Therefore, there is a need for new compounds containing linker systems that are stable in the systemic circulation and can rapidly release and deliver sufficient amounts of drug to target cells to kill the target cells while allowing retention of favorable pharmacokinetic properties.

[0012] Therefore, an object of the present invention is to provide a compound comprising a linker system that is stable in the systemic circulation and allows rapid release and delivery of the drug to target cells in a traceless manner while retaining favorable pharmacokinetic properties. Another object of the present invention is to provide a pharmaceutical composition comprising such a compound. The present invention also relates to a ligand-drug-conjugate characterized by a high DAR and exhibiting excellent pharmacokinetic properties, resulting in significantly improved efficacy.

[0013] Yet another object of the present invention is to provide compounds comprising a linker system which can release multiple drug molecules while allowing to maintain favorable pharmacokinetic properties, wherein the individual drug molecules can be the same or different.

[0014] Another object of the present invention is to provide compounds or compositions that can be used in methods for treating or preventing cancer, autoimmune or inflammatory diseases and / or infectious diseases. SUMMARY OF THE INVENTION

[0016] The present invention provides a new hydrophilic cleavable joint system that can be used for ligand-drug-conjugates. The joint system is preferably characterized by the presence of a C-terminal peptide unit carrying a drug, which is covalently attached to its N-terminus via a specific spacer group and a solubilizing group on its side chain. The C-terminal peptide unit is a highly specific substrate for cathepsin B, preferably a highly specific substrate for the exopeptidase (i.e., carboxydipeptidase) activity of cathepsin B, resulting in increased intracellular cleavage and drug release. The joint system is stable in the systemic circulation and enables high DAR to be achieved while maintaining excellent pharmacokinetic properties, thereby resulting in significantly improved efficacy.

[0017] Therefore, the present invention relates to a compound represented by the following general formula (I):

[0018]

[0019] in,

[0020] D represents a moiety derived from a drug selected from a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug, and a hydroxyl-containing drug; if more than one (D) is present, each (D) is independently selected from a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug, and a hydroxyl-containing drug, and the moieties (D) are preferably identical to each other;

[0021] X represents a divalent group comprising 1 to 7 backbone atoms independently selected from C, N, O and S; X is covalently attached to (D) via an atom selected from C, S, N and O derived from a carboxyl, thiol, amino or hydroxyl functional group contained in (D);

[0022] Y is a divalent group comprising one or more atoms selected from C, N, O, P and S;

[0023] L represents a linker that can be cleaved by cathepsin B;

[0024] T represents a (2+n)-valent branched group;

[0025] S represents a moiety derived from a compound comprising one or more, for example two, three or four, solubilizing groups;

[0026] V represents a moiety derived from a carrier group capable of interacting with target cells;

[0027] n is an integer from 1 to 4; and

[0028] m is an integer of 1-12.

[0029] The present invention also relates to the compounds as described above or compositions thereof for use in methods for treating or preventing cancer, autoimmune diseases or inflammatory diseases and / or infectious diseases.

[0030] The present invention particularly comprises the following embodiments ("items"):

[0031] 1. A compound represented by the general formula (I):

[0032]

[0033] in,

[0034] D represents a moiety derived from a drug, the drug being selected from carboxyl-containing drugs such as auristatin F (AF), thiol-containing drugs such as maytansine (DM1) or ravtansine (DM4), amino-containing drugs such as monomethyl auristatin F (MMAF) or exatecan and hydroxyl-containing drugs such as Maaa-1181a, preferably selected from thiol-containing drugs, amino-containing drugs and hydroxyl-containing drugs; if more than one (D) is present, each (D) is independently selected from carboxyl-containing drugs, thiol-containing drugs, amino-containing drugs and hydroxyl-containing drugs, and the moieties (D) are preferably the same as each other;

[0035] X represents a divalent group comprising 1 to 7, preferably 2 to 6, more preferably 2 to 5 backbone atoms independently selected from C, N, O and S; X is covalently attached to (D) via an atom selected from C, S, N and O derived from a carboxyl, thiol, amino or hydroxyl functional group contained in (D);

[0036] Y is a divalent group containing one or more atoms selected from C, N, O, P and S, preferably a divalent group derived from a compound selected from maleimides, triazoles, hydrazones, carbonyl-containing compounds and derivatives thereof, more preferably a divalent group derived from maleimides and derivatives thereof such as ring-opening hydrolyzed maleimides, most preferably a group derived from ring-opening hydrolyzed maleimide;

[0037] L represents a linker that can be cleaved by cathepsin B;

[0038] T represents a (2+n)-valent branched group;

[0039] S represents a moiety derived from a compound comprising one or more, for example two, three or four, solubilizing groups;

[0040] V represents a moiety derived from a carrier group capable of interacting with target cells;

[0041] n is an integer from 1 to 4, preferably 1 or 2, more preferably 1; and

[0042] m is an integer of 1-12, preferably 2-10, more preferably 4-8.

[0043] 2. The compound of item 1, wherein (L) is represented by the general formula (II) or (II'):

[0044]

[0045] in,

[0046] Axx represents a moiety derived from a trifunctional amino acid, provided that Axx in formula (II) is not a moiety derived from an amino acid in the (D) configuration;

[0047] Ayy represents a moiety derived from an amino acid selected from the group consisting of Phe, Ala, Trp, Tyr, phenylglycine (Phg), Met, Val, His, Lys, Arg, citrulline (Cit), 2-amino-butyric acid (Abu), ornithine (Orn), Ser, Thr, Leu and Ile; or Ayy in formula (II) represents a moiety derived from an amino acid selected from the group consisting of homotyrosine (homo-Tyr), homophenylalanine (homo-Phe), β-phenylalanine (β-Phe) and β-homophenylalanine (β-homo-Phe), Tyr (OR1) and homo-Tyr (OR1), wherein R1 is -(CH2CH2O) n1 -R2, wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer from 2 to 24; provided that Ayy in formula (II') is not a moiety derived from an amino acid in configuration (D);

[0048] Dxx represents a single covalent bond or a moiety derived from an amino acid having a hydrophobic side chain, preferably a single covalent bond or a moiety derived from an amino acid selected from Phe, Val, Tyr, homo-Phe and Ala, more preferably a single covalent bond or a moiety derived from Phe or Val;

[0049] Dyy represents a single covalent bond, a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain, preferably a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), more preferably a moiety derived from Arg or Cit; provided that if Dxx is a moiety derived from an amino acid with a hydrophobic side chain, Dyy is a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain, and if Dxx is a single covalent bond, Dyy is a single covalent bond, a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain;

[0050] Z represents a group selected from -OH and -N(H)(R) covalently bonded to the C-terminus of Ayy or Axx, wherein R represents a hydrogen atom, an alkyl group or a cycloalkyl group, preferably represents -OH;

[0051] * indicates covalent attachment to (T); and

[0052] ** indicates covalent attachment to (X).

[0053] 3. The compound of item 2, wherein at least one of Axx and Ayy is defined as follows:

[0054] Axx represents a moiety derived from an amino acid selected from the group consisting of Glu, 2-amino-pimelic acid (Apa), 2-aminoadipic acid (Aaa), Dap, Dab, Lys, Orn, Ser, Ama, and homolysine (homo-Lys), preferably a moiety derived from an amino acid selected from the group consisting of Dap, Dab, Lys, Orn, and homo-Lys, more preferably a moiety derived from Orn or Lys, most preferably a moiety derived from Lys;

[0055] Ayy in formula (II) represents a moiety derived from an amino acid selected from the group consisting of Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, homo-Tyr, Tyr(OR1) and homo-Tyr(OR1) (wherein R1 is -(CH2CH2O) n1 -R2, wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer from 2 to 24), preferably a moiety derived from Phe, homo-Phe, Tyr, homo-Tyr, Tyr(OR1) and homo-Tyr(OR1), more preferably a moiety derived from Phe or Tyr, most preferably a moiety derived from Tyr;

[0056] Ayy in formula (II') represents a moiety derived from an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr and Ser, preferably a moiety derived from Phe, homo-Phe and Ser, more preferably a moiety derived from Phe or Ser, most preferably a moiety derived from Phe.

[0057] 4. The compound according to any one of items 1 to 3, wherein (X) represents a divalent carbonyl- or thiocarbonyl-containing group, preferably a group represented by one of the following formulae (IIIa) to (IIIf):

[0058] ***-(CH2)n2-(C=A)-**' (IIIa)

[0059] ***-(C=A)-(CH2)n3-**' (IIb)

[0060] ***-(CH2)n2-(C=A)-(CH2)n3-**' (IIIc)

[0061] ***-(CH2)n2-(C=A)-(C(CH3)2-(CH2)n3**' (IIId)

[0062] ***-(CH2)n2-(C(CH3)2-(C=A)-(CH2)n3-**' (IIIe)

[0063] ***-(C=A)-(NH)n4-(CH2)n3-(NH)n5-(C=A)-**' (IIIf)

[0064] in

[0065] n2 and n3 are each independently selected from 0 to 5, preferably 0, 1 or 2, more preferably 0 or 1;

[0066] n4 and n5 are each selected from 0 or 1;

[0067] Each A is independently selected from O and S, preferably O;

[0068] *** represents covalent attachment to (D); and

[0069] **' represents covalent attachment to (L).

[0070] 5. The compound according to any one of items 1 to 3, wherein (X) is represented by one of the following formulae (IVa) to (IVj'):

[0071]

[0072]

[0073] in,

[0074] *** represents covalent attachment to (D);

[0075] **' represents covalent attachment to (L);

[0076] Provided that if (X) is represented by Formula (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVp), (IVr), (IVt), (IVv), (IVw), (IVx), (IVy) or (IVz), then (D) in Formula (I) represents an amino-containing drug; if (X) is represented by Formula (IVj), (IVq), (IVs) or (IVu), then (D) in Formula (I) represents an amino-containing drug or a hydroxyl-containing drug; and if (X) is represented by Formula (IVa'), (IVb'), (IVc'), (IVd'), (IVe'), (IVf'), (IVg'), (IVh'), (IVi') or (IVj'), then (D) in Formula (I) represents a carboxyl-containing drug.

[0077] 6. The compound of item 5, wherein (X) is represented by formula (IVb'), (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), preferably represented by formula (IVc) or (IVm).

[0078] 7. The compound of any one of items 1 to 6, wherein each solubilizing group contained in (S) is independently selected from the group consisting of:

[0079] a moiety comprising one or more ionic or ionizable groups, such as an ammonium, guanidinium, sulfate or sulfonate group, preferably a moiety derived from Arg, (D)-Arg, Dap, (D)-Dap, Dab, (D)-Dab, Orn, (D)-Orn, Lys, D-Lys or carnitine;

[0080] - a sugar moiety selected from monosaccharides, disaccharides and linear or branched oligosaccharides, in particular linear or branched oligosaccharides having 3 to 10 monosaccharide units linked by glycosidic bonds, wherein each monosaccharide unit in the monosaccharide, disaccharide and oligosaccharide is independently selected from glucose, fructose, mannose, ribose and galactose; and

[0081] - a polyoxyalkylene group, preferably a C2-3 polyoxyalkylene group, more preferably a C2-3 polyoxyalkylene group, which independently comprises 6 to 200, preferably 10 to 150, more preferably 12 to 80 repeating units.

[0082] 8. The compound of any one of items 1 to 7, wherein (S) is a moiety derived from a compound comprising one or more polyethylene oxide groups, wherein preferably each polyethylene oxide group independently comprises 6 to 200, more preferably 10 to 150, most preferably 12 to 80 repeating units;

[0083] (S) is preferably a moiety represented by formula (V):

[0084] ＊＊＊＊ -X 1 -(CH2CH2O)n3-X 2 (V)

[0085] in,

[0086] n3 is an integer from 6 to 200, preferably from 10 to 150, more preferably from 12 to 80;

[0087] **** indicates covalent attachment to (T);

[0088] X 1 is selected from a single covalent bond, -(C=O)- and -N(R)-, wherein R represents a hydrogen atom, an alkyl group or a cycloalkyl group;

[0089] X 2represents an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group such as an acetyl group or a group of the formula -(CH2)n4-CO2H, a thiocarbonyl-containing group, a group of the formula -(CH2)n4OR, a group of the formula -(CH2)n4-SO3H or an amino-containing group such as a group of the formula -(CH2)n4-(C=A)-N(R)2 or -(CH2)n4-N(R)2, wherein A is O or S, each R is independently selected from a hydrogen atom, an alkyl group and a cycloalkyl group, and n4 is an integer from 1 to 6;

[0090] X 2 It is preferably -CH3, -CH2CH2OH or a group represented by the following formula (VI):

[0091] -(CH2)n5-(C=A)N(R)-(CH2)n6-(C=A)N(H)(R) (VI)

[0092] in,

[0093] Each A is independently selected from O and S, preferably O;

[0094] Each R is independently selected from a hydrogen atom, an alkyl group, and a cycloalkyl group; and

[0095] n5 and n6 are each independently an integer from 1 to 6, preferably 1 or 2;

[0096] X 2 Most preferably -CH3; and

[0097] If more than one (S) is present, each (S) is preferably a moiety of formula (V) above.

[0098] 9. The compound according to any one of items 1 to 8, wherein (T) is represented by the following formula (VII):

[0099]

[0100] in,

[0101] each AA independently represents a moiety derived from a trifunctional amino acid such as a diaminocarboxylic acid, an aminodicarboxylic acid, an azidoamino acid or an alkyne-containing amino acid, preferably a moiety derived from an amino acid selected from N-ε-propynyloxycarbonyl-L-lysine (Lys(Poc)), Asp, Glu, Orn, Lys, Dab and Dap, more preferably a moiety derived from Lys(Poc), Glu, Orn or Lys, most preferably a moiety derived from Lys;

[0102] α represents covalent attachment to (Y);

[0103] If n=1, the side chain derived from the trifunctional amino acid is covalently attached to (L) or (S), and the C-terminus is covalently attached to the further moiety (S) or (L), respectively;

[0104] If n = 2, 3, or 4:

[0105] *' indicates covalent attachment to (L);

[0106] ****' indicates covalent attachment to (S); and

[0107] n is as defined in item 1.

[0108] 10. The compound of any one of items 1 to 8, wherein (T) is represented by formula (VIII) or (IX):

[0109]

[0110] in,

[0111] Each AA 1 and AA 2 is independently a moiety derived from a trifunctional amino acid, such as a diaminocarboxylic acid, an aminodicarboxylic acid, an azido amino acid or an alkyne-containing amino acid, preferably a moiety derived from an amino acid selected from Lys(Poc), Asp, Glu, Orn, Lys, Dab and Dap, more preferably a moiety derived from Lys(Poc), Glu, Orn or Lys, most preferably a moiety derived from Lys;

[0112] α represents covalent attachment to (Y);

[0113] In formula (IX), the side chain derived from the trifunctional amino acid is covalently attached to (L) or (S), and the C-terminus is covalently attached to the other moiety (S) or (L), respectively;

[0114] In formula (VIII), *' indicates covalent attachment to (L), and ****' indicates covalent attachment to (S).

[0115] 11. The compound of any one of items 1 to 10, wherein (D) is a moiety derived from a drug selected from:

[0116] (i) Antineoplastic agents, such as

[0117] o DNA alkylating agents, such as duocarmycin,

[0118] o Topoisomerase inhibitors, such as doxorubicin,

[0119] o RNA-polymerase II inhibitors, such as α-amanitin,

[0120] o DNA cleaving agents, such as calicheamicin,

[0121] o Antimitotic or microtubule disrupting agents, such as taxanes, auristatins, or maytansines,

[0122] oAntimetabolites, such as gemcitabine derivatives,

[0123] o Spindle kinesin inhibitors, such as filaments,

[0124] o kinase inhibitors, such as patasertib or gefitinib,

[0125] o Nicotinamide phosphoribosyltransferase inhibitors,

[0126] o Matrix metallopeptidase 9 inhibitors,

[0127] o Phosphatase inhibitors such as Microcystis toxin-LR,

[0128] (ii) Immunomodulators such as fluticasone

[0129] (iii) anti-infective drugs, such as rifamycin, clindamycin or retapamulin, and

[0130] (iv) radioactive isotopes, metabolites, pharmaceutically acceptable salts and / or prodrugs of any of the foregoing;

[0131] Provided that the drug selected from (i) to (iv) is a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug or a hydroxyl-containing drug; and

[0132] If more than one (D) is present, each (D) is independently selected from the above-mentioned parts (i) to (iv), and the parts (D) are preferably identical to each other.

[0133] 12. The compound of any one of items 1 to 11, wherein (D) is a moiety derived from a drug selected from the group consisting of amanitin, duocarmycin, auristatin, auristatin F (AF), monomethyl auristatin F (MMAF), maytansine, maytansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, paclitaxel, daunorubicin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepine (PBD) and its dimer, indenobenzodiazepine (IBD) and its dimer, or its radioactive isotope and / or pharmaceutically acceptable salt; preferably a portion derived from a drug selected from auristatin, MMAF, exatecan, maytansinoid, DM1 and DM4; more preferably a portion derived from DM1 or DM4.

[0134] 13. The compound according to any one of items 1 to 12, which is represented by the general formula (X) or (X'):

[0135]

[0136] in,

[0137] Axx in formula (X) and formula (X') represents a moiety derived from an amino acid selected from Glu, Apa, Aaa, Dap, Dab, Lys, Orn, Ser, Ama and homo-Lys, preferably a moiety derived from an amino acid selected from Dap, Dab, Lys, Orn and homo-Lys, more preferably a moiety derived from a moiety derived from Lys;

[0138] Ayy in formula (X) represents a moiety derived from an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, homo-Tyr, Tyr(OR1) and homo-Tyr(OR1), wherein R1 is -(CH2CH2O) n1 -R2, wherein R2 is a hydrogen atom or a methyl group and n1 is an integer from 2 to 24, preferably a moiety derived from Phe, homo-Phe, Tyr, homo-Tyr, Tyr(OR1) or homo-Tyr(OR1), more preferably a moiety derived from Tyr;

[0139] Ayy in formula (X') represents a moiety derived from an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr and Ser, preferably a moiety derived from Phe, homo-Phe or Ser, more preferably a moiety derived from Phe or Ser;

[0140] D, Dxx, Dyy, X, Y, T, S, V, Z, m and n have the same meanings as specified in items 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12; and wherein preferably at least one, for example two, three, four, five, six, seven or eight of D, Dxx, Dyy, X, Y, T, S and Z are defined as follows:

[0141] (a) D is a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansinoids, DM1 and DM4, preferably a moiety derived from DM1 or DM4;

[0142] (b) Dxx is a moiety derived from an amino acid selected from Phe, Val, Tyr, homo-Phe and Ala, preferably a moiety derived from Phe or Val;

[0143] (c) Dyy is a covalent bond or a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap and Dab, preferably a covalent bond or a moiety derived from Arg or Cit;

[0144] (d) X is a group of formula (III) wherein n2 is 1 or 2, or a group represented by any one of formulae (IVa) to (IVz), preferably a group represented by any one of formulae (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm);

[0145] (e) Y is a group derived from a compound selected from maleimides, triazoles, hydrazones, carbonyl-containing compounds and derivatives thereof, preferably selected from maleimides and derivatives thereof such as ring-opening hydrolyzed maleimide, and more preferably a group derived from ring-opening hydrolyzed maleimide;

[0146] (f) T is a group of formula (VII), (VIII) or (IX);

[0147] (g) S is a moiety of formula (V); and

[0148] (h) Z is -OH.

[0149] 14. The compound of item 13, wherein in formula (X), each Dyy-Dxx-Axx-Ayy is independently selected from wherein Dyy is covalently bonded Arg-Lys-Phe, wherein Dyy is covalently bonded Arg-Lys-homo-Phe, wherein Dyy is covalently bonded Arg-Lys-Tyr, wherein Dyy is covalently bonded Cit-Lys-Phe, wherein Dyy is covalently bonded Cit-Lys-Tyr, wherein Dyy is covalently bonded Arg-Lys-homo-Tyr, wherein Dyy is covalently bonded Cit-Lys-homo-Tyr, Phe-Cit-Lys-Phe, Phe-Cit-Lys-Tyr, Phe-Arg-Lys-Tyr, Phe-Cit-Lys-homo-Tyr, Phe-Lys-Lys-Phe, homo-Phe-Arg-Lys-Phe, homo-Phe-Cit-Lys-Tyr; and

[0150] In formula (X'), each Dyy-Dxx--Ayy-Axx is independently selected from Arg-Phe-Lys where Dyy is covalently bonded, Arg-Ser-Lys where Dyy is covalently bonded, Cit-Phe-Lys where Dyy is covalently bonded, Cit-Ser-Lys where Dyy is covalently bonded, Cit-homo-Phe-Lys where Dyy is covalently bonded, Phe-Cit-Phe-Lys, homo-Phe-Cit-Phe-Lys and Phe-Arg-Phe-Lys.

[0151] 15. The compound of any one of items 1 to 14, which is represented by one of the following formulae:

[0152]

[0153] wherein D, X, Y, T, S, V, Z, m and n have the same meanings as specified in item 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12; and wherein preferably at least one, for example two, three, four, five or six of D, X, Y, T, S and Z are defined as follows:

[0154] (a) D is a moiety derived from a drug selected from auristatin, AF, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4;

[0155] (d) X is a group of formula (III), wherein n2 is 1 or 2, or is a group represented by any one of formulae (IVa) to (IVz), preferably a group represented by any one of formulae (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm);

[0156] (e) Y is a group derived from a compound selected from maleimides, triazoles, hydrazones, carbonyl-containing compounds and derivatives thereof, preferably selected from maleimides and derivatives thereof such as ring-opening hydrolyzed maleimide derivatives, and more preferably a group derived from ring-opening hydrolyzed maleimide;

[0157] (f) T is a group of formula (VII), (VIII) or (IX);

[0158] (g) S is a moiety of formula (V); and

[0159] (h) Z is -OH.

[0160] 16. The compound according to any one of items 1 to 15, which is represented by one of the following formulae:

[0161]

[0162]

[0163] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8;

[0164] wherein the number of oxyethylene repeating units (17) may be replaced by 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups; and / or wherein the maleimide attachment to (V) may be replaced by a maleimide attachment by ring-opening hydrolysis.

[0165] 17. The compound of any one of items 1 to 16, wherein (V) represents a moiety derived from a carrier group capable of interacting with a target cell, wherein the target cell is selected from tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, cells involved in autoimmune diseases, activated cells, myeloid cells, lymphoid cells, melanocytes, and infectious agents including bacteria, viruses, mycobacteria, and fungi;

[0166] Preferably, the target cell is selected from lymphoma cells, myeloma cells, myeloid cells, lymphoid cells, renal cancer cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell cancer cells, small cell lung cancer cells, testicular cancer cells, skin cancer cells, pancreatic cancer cells, liver cancer cells and any cell that grows and divides at an unregulated and accelerated rate, thereby causing cancer.

[0167] 18. The compound of any one of items 1 to 17, wherein (V) represents a moiety derived from a carrier group selected from antibodies, antibody fragments, proteins, peptides and non-peptide molecules;

[0168] Preferred are moieties derived from antibodies or antibody fragments such as single chain antibodies, monoclonal antibodies, single chain monoclonal antibodies, monoclonal antibody fragments, chimeric antibodies, chimeric antibody fragments, domain antibodies or fragments thereof, cytokines, hormones, growth factors, colony stimulating factors, neurotransmitters or nutrient transport molecules.

[0169] 19. The compound according to any one of items 1 to 18, wherein (V) represents a moiety derived from:

[0170] Monoclonal antibodies, preferably antibodies selected from the group consisting of adalimumab, aducanumab, alemtuzumab, atumomab triamine pentaacetic acid, ervantumab, atezolizumab, anetuzumab, avelumab, bapilumab, basiliximab, betumomab, belantamab, beramezumab, bevacizumab, belotuzumab, vebutin, vebutin, bodalumab, catumaxomab, cemiplimab, cetuximab, simpanumab, clituzumab, crenetuzumab, tetraxeta n, daclizumab, daratumumab, denosumab, dinutuximab, dotalimumab, durvalumab, edrecolomab, elotuzumab, epavacumab, enrofloxacin, enrofloxacin vedotin, icoretuzumab, epratuzumab, epratuzumab-SN-38, edamasumab, gemtuzumab, gemtuzumab ozogamicin), genmab, grofituzumab, gemtuzumab, gonelizumab, ibritumomab tiuxetan, inelizumab, infliximab, inotuzumab, ozogamicin inotuzumab, ipilimumab, isatuximab, ixetuzumab, J591 PSMA-antibodies, labetuzumab, lencanezumab, lentuximab tesirin, moglizumab, motuzumab, necituzumab, nimotuzumab, natalizumab, natalizumab, nasituximab, nivolumab, omezumab, ofatumumab, olaratumumab, ogovumab, panitumumab, pembrolizumab, pertuzumab, polotuzumab, vepotuzumab vedotin, putinumab, rituximab, ramucirumab, rituximab, certolizumab pegol Antibody, gosartan, ceritumomab, siltuximab, solanezumab, tetuzumab, tancitumomab, tetuximab, tipranezumab, tocilizumab, tositumomab, trastuzumab, detrastuzumab, emtansuzumab, TS23, ustekinumab, vedolizumab, futumomab, zagetelumab, zalumab, zalumumab, fragments and derivatives thereof; more preferably selected from atezolizumab, durvalumab, pembrolizumab, rituximab or trastuzumab; or

[0171] - Antibody fragments introduced into Fc-fusion proteins, which are preferably selected from the group consisting of belatacept, aflibercept, Ziv-aflibercept, dulaglutide, rilocept, romiplostim, abatacept and alefacept.

[0172] 20. The compound or salt of any one of items 1 to 19, wherein (V) represents a moiety derived from an anti-HER2, anti-CD37, anti-PDL1 or anti-EGFR antibody, preferably derived from an antibody selected from trastuzumab, pembrolizumab, natalizumab, atezolizumab, durvalumab, avelumab, panitumumab and cetuximab, more preferably derived from natalizumab, trastuzumab and cetuximab, most preferably derived from natalizumab and trastuzumab.

[0173] 21. The compound of claim 20, wherein (D) is a moiety derived from an antitumor agent, and is preferably a moiety derived from a drug selected from the group consisting of amanitin, duocarmycin, auristatin, auristatin F (AF), monomethyl auristatin F (MMAF), maytansine, maytansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, paclitaxel, daunorubicin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepine (PBD) and its dimer, indenobenzodiazepine (IBD) and its dimer, or its radioactive isotope and / or pharmaceutically acceptable salt.

[0174] 22. The compound of claim 21, wherein (D) represents a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansinoids, DM1 and DM4; more preferably a moiety derived from DM1 or DM4.

[0175] 23. The compound of item 22, wherein (D) represents a moiety derived from DM1, and wherein the compound is represented by one of the following formulae:

[0176]

[0177] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; and wherein the number of oxyethylene repeating units (17) can be replaced by 12 to 30, preferably from 14 to 25, more preferably from 15 to 19 oxyethylene groups, and / or wherein the maleimide attachment to (V) can be replaced by a maleimide attachment by ring-opening hydrolysis.

[0178] 24. The compound of item 23, which is represented by the following formula:

[0179]

[0180] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; and wherein the number of oxyethylene repeating units (17) can be replaced by 12 to 30, preferably from 14 to 25, more preferably from 15 to 19 oxyethylene groups, and / or wherein the maleimide attachment to (V) can be replaced by a maleimide attachment by ring-opening hydrolysis.

[0181] 25. A composition comprising a therapeutically effective amount of a compound according to any one of items 1 to 24 or a pharmaceutically acceptable salt thereof, and one or more components selected from carriers, diluents and other excipients.

[0182] 26. A compound or composition according to any one of items 1 to 25 for use in a method for treating or preventing cancer, autoimmune diseases and / or infectious diseases.

[0183] 27. The compound or composition for use according to item 26, wherein the method is a method for treating blood and bone marrow cancer, preferably acute myeloid leukemia (AML).

[0184] 28. The compound or composition for use according to item 26 or 27, wherein the compound is as defined in any one of items 20, 21, 22, 23 and 24, preferably as defined in item 23 or 24, most preferably as defined in item 24.

[0185] 29. A compound or composition for use according to any one of items 26 to 28, wherein the compound or composition is administered simultaneously with, before or after one or more other therapeutic agents or therapies such as chemotherapeutic agents, radiotherapy, immunotherapeutic agents, autoimmune disease agents, anti-infective agents or other compounds of formula (I) in a method for treating or preventing cancer, autoimmune diseases and / or infectious diseases.

[0186] 30. A compound represented by the general formula (XI):

[0187]

[0188] in,

[0189] D, X, L, T, S and n have the same meanings as specified in item 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0190] Y' represents a moiety comprising a conjugating group capable of forming a covalent attachment with a molecule capable of interacting with a target cell (V'), such as a monoclonal antibody or an antibody fragment introduced into an Fc-fusion protein; Y' is preferably a moiety comprising a conjugating group selected from:

[0191] - an optionally substituted maleimide, preferably capable of reacting with one or both thiol groups of (V');

[0192] - an optionally substituted haloacetamide, preferably capable of reacting with the thiol group of (V');

[0193] - an ester, preferably an ester capable of reacting with the side chain of the amino acid of (V'), such as an acyl halide, an N-hydroxysuccinimide ester or a phenolic ester;

[0194] - a carbonate, preferably capable of reacting with the side chain of the amino acid of (V'), such as a haloformate, or a carbonate comprising a leaving group, such as N-hydroxysuccinimide or phenol;

[0195] - an isocyanate or an isothiocyanate, preferably capable of reacting with the side chain of the amino acid of (V');

[0196] - an azide, preferably capable of reacting with the alkyne group contained in (V');

[0197] - an alkyne, preferably capable of reacting with the azide group contained in (V');

[0198] - an amino group, preferably capable of reacting with molecule (V') in the presence of an enzyme such as transglutaminase;

[0199] The compound preferably has the structure as shown in item 13 or 15, provided that (Y') replaces (Y) and m is 1.

[0200] 31. A kit for modifying a molecule capable of interacting with a target cell, comprising the compound of item 30 and optionally a buffer, the buffer preferably having a pH of 6.0 to 10, more preferably a pH of 6.5 to 8.0.

[0201] 32. A method for modifying a molecule capable of interacting with a target cell, comprising reacting a molecule capable of interacting with a target cell, such as a monoclonal antibody or an antibody fragment introduced into an Fc-fusion protein, with the compound according to item 30.

[0202] Attached photos

[0203] Figure 1 - Mechanism of exo-Cat B-induced drug release from compounds of formula (I), wherein L represents a moiety of formula (II). Intracellular exo-Cat B cleavage at the N-terminus of the dipeptide Axx-Ayy releases the drug, e.g., moiety DX-Dxx-Dyy, in target cells.

[0204] Figure 2- Mechanism of exo-Cat B-induced drug release from compounds of formula (I), wherein L represents a moiety of formula (II'). Intracellular exo-Cat B cleavage at the N-terminus of the dipeptide Ayy-Axx releases the drug, e.g., moiety DX-Dxx-Dyy, in target cells.

[0205] Figure 3 - Chromatograms of reverse phase liquid chromatography (RPLC) of trastuzumab (Tmab). A: Chromatogram with basic gradient; B: Chromatogram with gradient dev2; C: Chromatogram with gradient dev7; D: Chromatogram with gradient dev7 / short.

[0206] Figure 4 - RPLC chromatograms of natalizumab. A: Chromatogram with basic gradient; B: Chromatogram with gradient dev7.

[0207] Figure 5 - RPLC chromatograms of the prepared ADCs (DAR properties). A: Chromatogram of ADC-1 with basic gradient; B: Chromatogram of ADC-2 with gradient dev7; C: Chromatogram of ADC-3 with gradient dev7 / short; D: Chromatogram of ADC-4 with gradient dev7.

[0208] Figure 6 - RPLC chromatograms of the prepared ADCs (DAR properties). A: Chromatogram of ADC-5 with basic gradient; B: Chromatogram of ADC-6 with basic gradient; C: Chromatogram of ADC-7 with gradient dev7 / short; D: Chromatogram of ADC-8 with gradient dev7 / short; E: Chromatogram of ADC-9 with gradient dev2.

[0209] Figure 7 - Results of an in vitro cytotoxicity assay using trastuzumab and ADC-2 in JIMT-1 (HER2 positive) cells (Example 8.6.1).

[0210] Figure 8 - Results of an in vitro cytotoxicity assay using trastuzumab, ADC-2 and ADC-4 in JIMT-1 cells (Example 8.6.1).

[0211] Fig. 9 - Results of in vitro cytotoxicity assays in JIMT-1 cells using trastuzumab, ADC-2, ADC-1, ADC-3 and ADC-9 (Example 8.6.1).

[0212] Fig.10 - Results of in vitro cytotoxicity assays using trastuzumab, ADC-2 and ADC-7 in JIMT-1 cells (Example 8.6.1).

[0213] Fig.11 - Results of an in vitro cytotoxicity assay using natalizumab, ADC-5 and ADC-6 in SU-DHL-5 (CD37 positive) cells (Example 8.6.1).

[0214] Fig.12 - Results of in vivo efficacy assays (tumor volume) using ADC-2 and ADC-4 (Example 8.6.2).

[0215] Fig.13 - Results of in vivo efficacy assays (body weight) using ADC-2 and ADC-4 (Example 8.6.2).

[0216] Fig.14 - Results of in vivo efficacy assays (tumor volume) (Example 8.6.3) performed using ADC-1, ADC-2, ADC-3 and ADC-9.

[0217] Fig.15 - Results of in vivo efficacy assays (body weight) (Example 8.6.3) performed using ADC-1, ADC-2, ADC-3 and ADC-9.

[0218] Fig.16 - Results of in vivo efficacy assays (tumor volume) using natalizumab and ADC-4 (Example 8.6.4).

[0219] Fig.17 - Results of in vivo efficacy assays (survival) (Example 8.6.4) performed with natalizumab, ADC-2 and ADC-4.

[0220] Fig.18a and 18b - Results of in vivo efficacy assays (tumor growth and survival) using natalizumab, ADC-2 and ADC-4 (Example 8.6.4).

[0221] Fig.19a , Fig.19b , Fig.20a and Fig.20b - Results of in vivo efficacy assays (tumor growth and survival) (Example 8.6.4) using naratuximab emtansine (Debio 1562) or ADC-4 (0.3 mg / kg, 1 mg / kg or 3 mg / kg).

[0222] Fig.21a- Results of the cell binding assay - Natuximab antibody bound to cells of the AML cell lines MV-4-11, MOLM-13, HL-60 and THP-1 expressing CD37 (Example 8.6.5).

[0223] Figure 21b - Results of the internalization assay - Internalization of the natalizumab antibody in the CD37 expressing AML cell lines MV-4-11, MOLM-13, HL-60 and THP-1 (Example 8.6.5).

[0224] Fig.21c - Results of cytotoxicity assays - Cytotoxicity of ADC-4 on CD37-expressing AML cell lines MV-4-11, MOLM-13, HL-60 and THP-1 (Example 8.6.5).

[0225] Fig.22a - Results of cytotoxicity assays - Comparison of the cytotoxicity of ADC-4, natalizumab and Debio 1562 on the CD37-expressing AML cell line MV-4-11 (Example 8.6.6).

[0226] Figure 22b - Results of cytotoxicity assays - Comparison of the cytotoxicity of ADC-4 and Debio 1562 on the CD37 expressing AML cell line THP-1 (Example 8.6.6).

[0227] Fig.23 - Results of in vivo efficacy assays - In vivo tumor growth in NCG mice (inoculated with MV4; Luc AML cells) following treatment with ADC-4 (1 mg / kg), ADC-4 (5 mg / kg), or venetoclax and azacytidine (Example 8.6.7).

[0228] Fig.24a - Results of in vivo pharmacokinetic assay - In vivo pharmacokinetic profile (plasma concentration) of total antibody after administration of ADC-4 (5 mg / kg) and natalizumab (5 mg / kg) to male Swiss mice (Example 8.6.8).

[0229] Figure 24b - Results of in vivo pharmacokinetic assays - In vivo pharmacokinetic profiles (plasma concentrations) of total antibody and total ADC following administration of ADC-4 to female CD1 mice (Example 8.6.8).

[0230] Fig.25a - In vitro plasma stability results - Comparison of human and mouse plasma stability of ADC-4, Enhertu and Adcetris (DAR reduction) (Example 8.6.9).

[0231] Fig.25b - Results of in vivo pharmacokinetic assays - Comparison of the human and mouse plasma stability of ADC-2, Euhede and Adcetris (DAR reduction) (Example 8.6.9).

[0232] Fig.26 - Results of in vivo efficacy assay - In vivo efficacy (% survival) of ADC-4 (1 mg / kg), ADC-4 (1 mg / kg) + rituximab, Debio 1562 (10 mg / kg) and Debio 1562 (10 mg / kg) + rituximab in female SCID beige mice (inoculated with FarageDLBCL cells) (Example 8.6.11).

[0233] Fig. 27 - Results of Cat B cleavage assay - Cat B cleavage of DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH releases DM1-Ac-Cit (Example 8.7.3).

[0234] Fig.28 - Results of Cat B cleavage assay - Cat B cleavage of Exatecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH releases Exatecan-Suc-Phe-Cit (Example 8.7.3).

[0235] Fig.29 - Scheme of the retro-Michael reaction of LDC with (blocked) maleimide attachment.

[0236] Detailed description of the invention

[0237] 1. Definition

[0238] The term "drug" as used herein characterizes a substance (e.g., a naturally occurring or synthetic substance) that is capable of inhibiting or preventing cellular function and / or killing cells. In some embodiments, the term "drug" should be understood as synonymous with other terms commonly used in the art, such as "cytotoxic agent," "toxin," or "payload" used in the field of cancer treatment. The drug may include a group that can be derived from a functional group that allows the drug to be covalently attached to the rest of the compound, such as a divalent group (X) in formula (I), such as a carboxylic acid, a thiol, a primary amine, a secondary amine, a hydroxyl, and the like.

[0239] The expression "part derived from a drug" as used herein characterizes a part containing a group, which is identical to a natural drug except for the structural modification required for bonding the drug to the rest of the compound of the present invention. Depending on the functional groups available in the natural drug, bonding can be achieved using one of the functional groups already present in the natural drug, or bonding can be achieved by introducing a new functional group. Therefore, the (natural) drug can be used for bonding in an unmodified or modified form. That is, except that hydrogen atoms are replaced by covalent bonds, the drug can be unmodified (existing in its natural form), or it can be chemically modified to introduce a functional group (e.g., a group selected from hydroxyl, carboxyl, amino and sulfhydryl groups) to allow covalent attachment to adjacent groups or parts, such as the divalent group (X) in formula (I). However, after being cleaved by cathepsin B, the adjacent group or part will remain attached to the drug. Thus, the expression "drug-derived moiety" as used herein refers to a moiety that differs from the unmodified (native) or modified (introduction of a functional group) drug only in the covalent bond required for bonding to the rest of the molecule, and is not meant to encompass any other groups, for example, groups or moieties that remain attached thereto after cleavage by cathepsin B.

[0240] The term "carboxyl-containing drug" characterizes an unmodified (natural) or modified drug that includes a carboxylic acid group that allows covalent attachment to an adjacent group or moiety. The term "sulfhydryl-containing drug" characterizes an unmodified (natural) or modified drug that includes a sulfhydryl group that allows covalent attachment to an adjacent group or moiety. Non-limiting examples of sulfhydryl-containing drugs include maytansine (DM1) and ravtansine (DM4). The term "amino-containing drug" characterizes an unmodified (natural) or modified drug that includes a primary or secondary amino group that allows covalent attachment to an adjacent group or moiety. Non-limiting examples of amino-containing drugs include monomethyl auristatin F (MMAF), exatecan, and indenobenzodiazepine (indilinobenzodiazepine, IBD) derivative 2554618-79-8 (shown below). The term "hydroxyl-containing drug" characterizes unmodified (natural) or modified drugs including hydroxyl groups that allow covalent attachment to adjacent groups or moieties. An example of a hydroxyl-containing drug is the exatecan derivative Maaa-1181a (shown below).

[0241]

[0242] In a similar manner, the term "derivative" is used to characterize moieties bonded to adjacent moieties that differ from the molecule from which they are derived only in the structural element responsible for the bond to the adjacent moiety. This may include covalent bonds formed by existing functional groups or covalent bonds and adjacent functional groups newly introduced for this purpose.

[0243] Likewise, unless otherwise stated or the context dictates otherwise, the expression "derived from" when used in conjunction with other groups or moieties (such as in "derived from a compound") is intended to describe a group or moiety that is identical to the referenced compound, etc., except for the structural modifications necessary to bond the group or moiety to one or more adjacent groups or moieties, typically by replacing a hydrogen atom or group of atoms with a covalent bond (e.g., the OH in a carboxyl group is replaced by a covalent bond when forming an amide bond with an amino group; other examples are given at the end of the "Divalent Group (X)" section of the table below).

[0244] The term "natural medicine" refers to a compound for which efficacy has been determined by in vitro and / or in vivo testing. In a preferred embodiment, a natural medicine is a compound for which efficacy has been determined by clinical trials. Most preferably, a natural medicine is a commercially available medicine. The type of efficacy to be determined and the appropriate test to be applied will of course depend on the type of medical indication to be treated.

[0245] When referring to specific classes of drug molecules, such as anti-neoplastic agents, topoisomerase inhibitors, RNA-polymerase II inhibitors, DNA cleaving agents, anti-mitotic or microtubule disrupting agents, antimetabolites, kinase inhibitors, immunomodulators, or anti-infectious disease agents, these terms are intended to have the meaning generally accepted in the medical field, as reflected, for example, in Mosby's Medical Dictionary, Mosby, Elsevier 10th edition (2016) or Oxford Textbook of Oncology, David J. Kerr, OUP Oxford 3rd edition (2016).

[0246] Thus, the drug to be used in the ligand-drug-conjugate of the present invention may be a natural drug (e.g., a drug that naturally contains one or more functional groups that allow for covalent attachment to the conjugate), or may be a drug that has been chemically modified to incorporate a functional group that allows for covalent attachment to an adjacent group or moiety (e.g., a group selected from hydroxyl, carboxyl, amino, and thiol), as long as the modified drug has pharmacological activity. In this regard, pharmacological activity refers to at least 20%, preferably at least 50%, and more preferably at least 80% of the pharmacological activity of the natural drug.

[0247] In addition, after the drug is cleaved by cathepsin B, in the case where the group or part remains attached to it, the drug is released in a modified form, for example, in the case of release in the form of a part DX or DX-Dxx-Dyy, the modified drug can be referred to as an "intra-drug". In some cases, the remaining groups X, Dxx and Dyy can be subsequently removed by other mechanisms, such as by hydrolysis of an ester bond that may be present between D and X. For those linkages that are not cleaved by other mechanisms, it is advantageous if the remaining modified drug DX or DX-Dxx-Dyy is pharmacologically active. In this regard, pharmacological activity means that when released intracellularly by the ADC, the released modified drug, such as the DX or DX-Dxx-Dyy part, retains at least 20%, preferably at least 50%, and more preferably at least 80% of the pharmacological activity of the native drug. In order to ensure real conditions, such activity testing should not be performed by comparing the cytotoxicity of the released modified drug and the native drug, because these conditions require the modified drug to enter the cell, which may introduce cell permeability bias. Due to the intracellular release of the modified drug, the permeability difference between the two entities is not relevant here. It is possible to compare the activity of the modified drug with the native drug in a cell-free binding assay to determine the Ki value (binding affinity) of the appropriate target receptor of the drug. If it is not possible to determine the Ki value, the cytotoxicity IC50 of two trastuzumab ADCs (one designed to release the modified drug and the other designed to release the native drug) with exactly the same linker system (e.g., Val-Cit-PAB) in HER2+ cell lines can be compared.

[0248] The term "pharmaceutically acceptable salt" as used herein refers to derivatives (including reactive conjugates) of disclosed compounds in which the parent compound is modified by forming an acid salt or a base salt thereof. Pharmaceutically acceptable salts include, for example, non-toxic salts or quaternary ammonium salts of the parent compound formed by non-toxic inorganic or organic acids or bases. Lists of suitable salts can be found in the following literature: Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, PA, 1985, page 1418, SM Berge, LM Bighley and DC Monkhouse, "Pharmaceutical Salts" J. Pharm. Sci. 1977, 66 (1), 1-19; P. H. Stahl and C. G. Wermuth, eds., Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zürich, Wiley-VCH, 2008, and A. K. Bansal et al., Pharmaceutical Technology, 3 (32), 2008. Pharmaceutically acceptable salts can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. For reactive conjugates, this can be done before or after the drug moiety is incorporated into the compound of the invention. Unless the context indicates otherwise, all references to the compounds of the invention (conjugates, modified antibodies, etc.) should also be understood as references to pharmaceutically acceptable salts of the corresponding compounds.

[0249] The term "skeleton" refers to a chain of atoms across the length of a molecule. Therefore, the expression "a divalent group comprising X to Y skeleton atoms" defines a group in which atoms such as C, N, O, S are covalently linked to each other to form a chain of atoms, which is covalently attached to an adjacent group or part at its end. A divalent group may include a side atom or group attached to a skeleton portion. Hydrogen atoms of the valence of saturated skeleton atoms are not counted as skeleton atoms. If a cyclic group is present, the skeleton is considered to be the shortest connection between the ends.

[0250] The term "functional group" refers to a group that is capable of bonding to another functional group by forming at least one covalent bond without breaking any CC or CH covalent bonds.

[0251] The expression "capable of being cleaved by cathepsin B" characterizes any compound (or a moiety that can be incorporated into a compound) that is cleaved when in contact with cathepsin B (Cat B) under suitable conditions, for example, as described in WO2019096867. In some preferred embodiments, the cleavage is (a) rapid and / or (b) the cleavage is performed by the exopeptidase activity of Cat B. The embodiment (b) relates to a compound or moiety that is "capable of being cleaved by the exopeptidase activity of Cat B", which is defined in more detail in the next paragraph. The above-mentioned "rapid" cleavage of embodiment (a) generally means for the target compound that the corresponding unconjugated compound (i.e., a compound that does not have a carrier group V and is quenched at the conjugation group, for example, wherein cysteine ​​is covalently attached to a maleimide conjugation group) has a cleavage rate T of 25 minutes or less, preferably 20 minutes or less, more preferably 18 minutes or less, even more preferably 16 minutes or less, and most preferably 14 minutes or less. 1 / 2 There is no particular lower limit. However, since solubilizing groups (PEG, etc.) tend to reduce the cleavage rate, the cleavage rate T is expected to be 1 / 2 A period of 1 minute or more, typically 2 minutes or more, even more typically 5 minutes or more is realistic.

[0252] The expression "capable of being cleaved by the exopeptidase activity of Cat B" as used herein means that the corresponding part of the compound, in particular the linker, such as the C-terminal peptide unit, is capable of being specifically recognized and cleaved by the exopeptidase (i.e., carboxydipeptidase) of cathepsin B. The cleavage results in the rapid release of the drug (or a modified drug having a group or part that remains attached after cleavage by cathepsin B, an "internal drug") into the target cell. The cleavage of the linker (e.g., the C-terminal peptide unit) by the exopeptidase activity of Cat B can be evaluated by an in vitro enzymatic cleavage assay using recombinant human Cat B and UHPLC-MS / MS analysis further described below. Taking into account that the exopeptidase activity of Cat B is generally associated with a higher cleavage rate compared to the endopeptidase activity of Cat B, in some aspects, the expression "a compound capable of being cleaved by the exopeptidase activity of Cat B" can be confirmed by demonstrating a high Cat B cleavage rate. According to this aspect, a "compound capable of being cleaved by the exopeptidase activity of Cat B" refers to a compound that meets the following criteria: the cleavage rate T of the corresponding unconjugated compound (i.e., a compound without a carrier group V and quenched at the conjugation group, e.g., wherein cysteine ​​is covalently attached to a maleimide conjugation group) is 1 / 2 The cleavage rate T is 25 minutes or less, preferably 20 minutes or less, more preferably 18 minutes or less, even more preferably 16 minutes or less, and most preferably 14 minutes or less. There is no particular lower limit. However, since solubilizing groups (PEG, etc.) tend to reduce the cleavage rate, the expected cleavage rate T 1 / 2In some aspects, the expression "a compound capable of being cleaved by the exopeptidase activity of Cat B" may refer to a compound having a cleavage ratio compared to a reference compound A or a reference Cys-quenched compound B, the structure of which is shown below:

[0253]

[0254]

[0255] Wherein the ratio (T 1 / 2化合物 / T 1 / 2参照 ) is 0.6 or less, preferably 0.4 or less, more preferably 0.2 or less. Likewise, there is no particular lower limit to this range, but a typical cut ratio is 0.001 or greater, more typically 0.01 or greater, especially 0.05 or greater.

[0256] The term "solubilizing group" or "solubilizing part" used herein refers to a hydrophilic group or hydrophilic part that can enhance (improve) the water solubility of the part or compound to which it is attached. The solubilizing group can be, for example, a polyoxyalkylene group, such as a polyethylene oxide (PEO) or polypropylene oxide (PPO) group, a sugar group, or a part comprising one or more ionic or ionizable groups, i.e., a functional group of (anion or cation) charged at physiological pH (7.4), such as derived from amino acids, for example, derived from Lys, Glu, Asp, His, Arg, diaminopropionic acid (Dap), diaminobutyric acid (Dab), 2-aminoadipic acid (Aad), carnitine, Orn parts. Examples of ionic or ionizable groups include ammonium, guanidine, sulfate, phosphate, phosphonate, and sulfonate. The example of sugar group comprises monosaccharide, disaccharide and straight or branched oligosaccharide, particularly has 3 to 10 straight or branched oligosaccharide of monosaccharide units connected by glycosidic bond, wherein each monosaccharide unit in monosaccharide, disaccharide and oligosaccharide is independently selected from glucose, fructose, mannose, ribose and galactose.In the present invention, term " solubilizing part " refers to the part of the compound that is derived from comprising one or more, for example two, three or four solubilizing groups.In other embodiments, solubilizing part can be made up of one or more solubilizing groups for example amino acid, PEO group.

[0257] As used herein, the term "polyalkylene oxide" (or "polyalkylene glycol", "polyoxyalkylene") refers to a substance having the general structure HO-(XO)nH, wherein X represents an alkylene group having 2 or 3 carbon atoms, and n represents the number of repeating units, for example 6 to 200, 10 to 150 or 12 to 80 repeating units, for example 16 or 40 repeating units, such as 17, 18, 20 or 24 PEO repeating units. Thus, the term "polyoxyalkylene group" should be understood as a substance of the formula* -O-(XO)n- ** A divalent group wherein X and n are as defined above, and * and ** Indicates covalent attachment to an adjacent moiety. In some embodiments, the term "polyoxyalkylene" may refer to polyethylene oxide (or polyethylene glycol, C2-polyoxyalkylene) or polypropylene oxide (or polypropylene glycol, C3-polyoxyalkylene). Polyoxyalkylene groups may also be provided in which two or more different alkylene groups (as defined above) are arranged in a random or block manner.

[0258] The term "peptide" as used herein refers to a compound comprising a continuous sequence of at least two amino acids connected to each other by peptide bonds. The terms "dipeptide", "tripeptide" and "tetrapeptide" refer to compounds comprising a continuous sequence of two, three and four amino acids connected to each other by peptide bonds, respectively. In this regard, the term "peptide bond" refers to a (backbone) amide bond and a modified bond, which can be obtained if a non-natural amino acid is introduced into the peptide sequence. In this case, the modified bond replaces the (backbone) amide bond formed in the continuous peptide sequence by reacting the amino group of two amino acid residues with the carboxyl group. For example, the modified bond can be an ester, thioester, carbonamide (carbamide), thiourea (thiocarbamide) or triazole bond. Preferably, the amino acids forming the continuous peptide sequence are connected to each other by a backbone amide bond. The peptide can be straight or branched. In a preferred aspect, the peptide is a linear dipeptide, tripeptide, tetrapeptide, more preferably a linear tripeptide or tetrapeptide.

[0259] The term "amino acid" as used herein refers to a compound containing or derived from at least one amino group and at least one acidic group (preferably carboxyl). The distance between the amino group and the acidic group is not particularly limited. α-, β- and γ-amino acids are suitable, but α-amino acids, especially α-aminocarboxylic acids are particularly preferred. The term "amino acid" encompasses naturally occurring amino acids, such as naturally occurring proteinogenic amino acids (proteinogenic amino acid), and synthetic amino acids that do not exist in nature. In the following, amino acids can be represented by 3-letter amino acid codes (Arg, Phe, Ala, Cys, Gly, Gln, etc.), or by 1-letter amino acid codes (R, F, A, C, G, Q, etc.).

[0260] In the following, the amino acid sequences are written from N-terminus to C-terminus (from left to right). Unless otherwise stated or the context dictates otherwise, all linkages between adjacent amino acid groups are formed by peptide (amide) bonds.

[0261] As used herein, the expression "amino acid in the (D) configuration" refers to the (D)-isomer of any naturally occurring or synthetic amino acid. This applies to α-amino acids as well as β- and γ-amino acids. As used herein, the expression "amino acid in the (D) configuration" is not meant to encompass achiral amino acids such as glycine or other achiral amino acids such as aminoisobutyric acid.

[0262] As used herein, the expression "side chain of an amino acid" may refer to the moiety attached to the α-carbon of an amino acid. For example, the side chain of Ala is a methyl group, the side chain of Phe is a benzyl group, the side chain of Cys is a thiomethyl group, the side chain of Tyr is a 4-hydroxybenzyl group, and the like. Both naturally occurring side chains and non-naturally occurring side chains are included in this definition.

[0263] The term "trifunctional" as used herein refers to a compound or moiety having three functional groups that can form or have formed three covalent bonds with adjacent moieties. Therefore, the term "trifunctional amino acid" refers to a compound comprising or derived from a compound containing at least an amino group, an acidic group (e.g., a carboxyl group) and another functional group such as an amino group or a carboxyl group. Non-limiting examples of trifunctional amino acids include Ser, Cys, Tyr, N-ε-propynyloxycarbonyl-L-lysine (Lys(Poc)), Asp, Glu, Orn, Lys, Dab, and Dap.

[0264] The term "C-terminus" as used herein refers to the C-terminus of an amino acid (peptide) chain. Binding to the "C-terminus" means the formation of a covalent bond between an acidic group in the main chain (skeleton) of an amino acid residue and a binding partner. For example, binding of a group "X" to the C-terminus of an amino acid residue "Axx" produces an ester or amide type structural unit. * -C(O)-X, wherein the carbonyl group is derived from the acidic group of Axx, and ( * ) indicates attachment to the backbone. The term "C-terminal peptide unit" is used herein to characterize a peptide sequence of 2, 3 or 4 amino acids, wherein the C-terminal amino acid forms the C-terminus of the peptide sequence.

[0265] As used herein, the term "N-terminus" refers to the N-terminus of an amino acid (peptide) chain. Binding to the "N-terminus" means the formation of a covalent bond between an amino group in the main chain (skeleton) of an amino acid residue and a binding partner (which replaces one hydrogen atom). For example, binding of a group "X" to the N-terminus of an amino acid residue "Axx" produces the structural element X-NH- * , wherein the amino group is derived from Axx, and ( * ) indicates attachment to the main chain.

[0266] The term "hydrophobic" is used herein to characterize compounds, groups or parts that lack affinity for water. For example, the term "amino acid with a hydrophobic side chain" is used to characterize amino acids with hydrophobic or partially hydrophobic aliphatic side chains or amino acids with aromatic side chains, such as Phe, Leu, Ile, Val, Tyr, Trp, Ala. Of course, in the sense of the present invention, any other amino acid that exhibits the same or higher degree of hydrophobicity should also be considered hydrophobic. The comparison of the degree of hydrophobicity can be carried out by measuring the n-octanol / water partition coefficient (at 25°C and pH 7): if the concentration ratio of another amino acid in n-octanol / water is equal to or higher than the concentration ratio of one or more of the amino acids Phe, Leu, Ile, Val, Tyr, Trp, Ala, then this other amino acid will be considered a hydrophobic amino acid.

[0267] The term "amino acid with a basic side chain" is used herein to characterize a natural or non-natural amino acid, wherein the side chain contains one or more ionizable groups with a pKa value equal to or greater than 6. Examples of natural amino acids with basic side chains include Arg (guanidino, pKa = 12.5), Lys (amino, pKa = 10.5), His (imidazolyl, pKa = 6). Examples of non-natural amino acids with basic side chains include citrulline (Cit), ornithine (Orn), 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab).

[0268] The term "alkyl" as used herein refers to a straight or branched hydrocarbon group having 1 to 20 carbon atoms, preferably 1 to 5 carbon atoms, more preferably a methyl or ethyl group, or a cycloalkyl group having 3 to 20 carbon atoms, preferably 5 to 8 carbon atoms. The cycloalkyl group may consist of a single ring, but may also be formed by two or more condensed rings.

[0269] As used herein, the term "divalent maleimide derivative" (or, for example, "a divalent group derived from a compound selected from maleimide...") refers to a divalent moiety derived from maleimide (e.g., a succinimide moiety) in which the double bond is hydrogenated and the two hydrogen atoms are replaced by two covalent bonds, thereby allowing attachment to an adjacent moiety. For example, a divalent maleimide derivative can have the following structure (wherein R and R' represent the adjacent moiety to which the maleimide derivative is attached):

[0270]

[0271] Such moieties contain chiral carbon atoms (ie atoms carrying sulphur atoms). Unless otherwise stated, references to divalent maleimide derivatives are to be understood as references to pure stereoisomers and to mixtures thereof, especially racemic mixtures thereof.

[0272] The term "divalent maleimide derivative" or "divalent radical derived from a compound selected from maleimide" is further understood to cover any derivative of maleimide additionally substituted at positions other than 2 and 3 (as described above), as well as ring-opening hydrolyzed maleimide derivatives.

[0273] A divalent maleimide-type disulfide bridge (e.g., formula -SX 2 -S- / -SX 3 -S- divalent group, where X 2 / X 3 represents a divalent group derived from maleimide) can be obtained by side-chain-to-side-chain cyclization in the presence of, for example, 2,3-dibromomaleimide or another suitable reagent as described by Kuan et al. in Chem. Eur. J. 2016, 22, 17112-17129.

[0274] In the present specification, "ring-opening hydrolyzed maleimide derivative" refers to a divalent moiety derived from maleimide (as described above), wherein the maleimide ring has been opened by hydrolysis. For example, the hydrolysis of a divalent maleimide derivative RXS-R' (wherein X represents an unsubstituted divalent group derived from maleimide, R / R' represents an adjacent group or moiety; in X, the double bond of maleimide no longer exists) results in a ring-opening hydrolyzed maleimide derivative of the formula R-NH-C(=O)-CH(S-R')-CH2-COOH or R-NH-C(=O)-CH2-CH(S-R')-COOH, or a mixture thereof. The ring hydrolysis can be carried out, for example, under alkaline conditions. The following conditions are particularly suitable: at the end of the cysteine-maleimide conjugation reaction (e.g., after the maleimide moiety (Y') reacts with the side chain of a cysteine ​​residue contained in a molecule (V') capable of interacting with a target cell), the pH is adjusted to pH 8 by adding 10x pH 8 DPPS (0.2 to 0.5 reaction volumes), and excess reactive drug linker and reducing agent (TCEP) are removed by gel filtration using a suitable gel filtration column (e.g., a PF column, eluted with a pH 8 buffer), and the eluate is then stirred overnight for 16 hours to complete the opening, and the final buffer is then exchanged into an Amicon concentration unit with DPBS. Unless otherwise specified, any reference to "ring-opened hydrolyzed maleimide derivatives" should be understood as a reference to one of these structures alone or any mixture of these structures. In addition, the carbon carrying the sulfur atom is chiral. Unless otherwise stated, any reference to a "ring-opening hydrolysed maleimide derivative" is to be understood as a reference to the pure stereoisomers and any mixtures thereof, especially racemic mixtures thereof.

[0275] Furthermore, in the present specification, "maleimide attachment" refers to a divalent moiety derived from maleimide as described above, which contains two covalent bonds, thereby allowing the formation of an attachment to an adjacent group or moiety. For example, in a maleimide derivative of the formula RXS-R', where R / R' represents an adjacent group or moiety, X represents maleimide attachment (a divalent group derived from maleimide, where the double bond of maleimide no longer exists). Therefore, the term "maleimide attachment" is synonymous with "maleimide derivative attachment".

[0276] Similarly, in the present specification, "ring-opening hydrolytic attachment" refers to a divalent moiety derived from maleimide as described above, which contains two covalent bonds, thereby allowing the formation of an attachment to an adjacent group or part. For example, in a ring-opening hydrolyzed maleimide derivative of the formula RXS-R', where R / R' represents an adjacent group or part, X represents a ring-opening hydrolyzed maleimide attachment. Therefore, the term "ring-opening hydrolytic attachment" is synonymous with "ring-opening hydrolyzed derivative attachment".

[0277] Reference to a "divalent radical derived from a compound selected from ... triazoles" is intended to characterize a divalent radical resulting from a 3+2 cycloaddition of an alkyne and an azide. Such divalent radicals are generally characterized by the following structure:

[0278]

[0279] Wherein a single bond characterizes the attachment to the adjacent group, there is no particular restriction as to which adjacent group is bonded to the nitrogen atom and which adjacent group is bonded to the carbon atom. In the case of the group Y, a divalent group can be formed by reaction of an alkynyl group attached to V with an azide group attached to T, or vice versa.

[0280] Reference to "a divalent group derived from a compound selected from ... hydrazones" is intended to characterize a divalent group -CH=N-NH- resulting from the condensation of a carbonyl group with a hydrazine group. In the case of group Y, the divalent group can be formed by reacting a carbonyl group attached to V with a hydrazine group attached to T, or vice versa.

[0281] Reference to "a divalent group derived from a compound selected from ... a carbonyl-containing compound" is intended to characterize a divalent group -C(=O)-X- (wherein X represents O, S or NH), which results from the reaction of an (activated) carbonyl group with a nucleophilic group, such as the formation of an amide group, an ester group, a thioester group. In the case of the group Y, the divalent group can be formed by reacting a carbonyl-containing group attached to V (e.g. -C(=O)-Cl) with a nucleophilic group attached to T (e.g. -NH2), or vice versa.

[0282] In the above-mentioned divalent groups, the term "derivatives thereof" means that any hydrogen atom may be substituted with a substituent as defined below as long as the substitution does not interfere with the formation of the divalent group.

[0283] The term "leaving group" as used herein refers to an atom or group (which may be charged or uncharged) that is separated from an atom or molecule that is considered to be the remainder or major portion of a molecule that participates in a specific reaction, such as a nucleophilic substitution reaction (Pure Appl. Chem. 1994, 66, 1134). Examples of leaving groups include thiophenolates, phenolates, carboxylates, and sulfonates.

[0284] The expression "derived from a moiety of a carrier group" as used herein means that the carrier group may be in unmodified or modified form. That is, the carrier group may be unmodified (in its native form) except for the replacement of hydrogen atoms by covalent bonds, or may be chemically modified to introduce one or more functional groups (e.g., a group selected from hydroxyl, carboxyl, thiol and / or amino groups) to allow covalent attachment of the carrier group to T, provided that such modification does not significantly interfere with the interaction between the carrier group and the target cell.

[0285] As used herein, the expression "capable of interacting with a target cell" means that the carrier group can bind, complex or react with a moiety such as a protein or receptor exposed on the surface of a target cell. The interaction can produce a targeting effect (i.e., locally increase the concentration of the carrier-carrying compound near the target cell) and / or it can result in the internalization of the carrier-carrying compound of the present invention into the target cell.

[0286] The term "antibody" (also synonymously referred to as "immunoglobulin" (Ig)) as used herein encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), veneered antibodies, and small immunoproteins. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to specific antigens. Target antigens typically have many binding sites, also called epitopes, that are recognized by the complementary determining regions on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, there may be more than one corresponding antibody for an antigen. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to the antigen or portion thereof of the target of interest. The antibody may be IgG, such as IgG1, IgG2, IgG3, IgG4. Preferably, the antibody is an IgG protein, more preferably an IgG1, IgG2, or IgG4 protein. Most preferably, the antibody is an IgG1 protein. The antibody may be human or derived from other species. Preferably, the antibody is a human antibody.

[0287] The term "monoclonal antibody" as used herein characterizes antibodies that are identical in that they are produced by one type of immune cell and are clones of a single parent cell.

[0288] As used herein, the term "antibody fragment" refers to a molecule comprising at least one polypeptide chain derived from an antibody that is less than the full length.

[0289] As used herein, the term "Fc-fusion protein" refers to a protein comprising at least an Fc-containing antibody fragment (i.e., an immunoglobulin-derived portion comprising at least one Fc region) and a portion derived from a second non-immunoglobulin protein. The Fc-containing antibody fragment forms part of the Fc-fusion protein and is therefore incorporated into the Fc-fusion protein. The Fc-containing antibody fragment may be derived from an antibody as described above, in particular from IgG, such as IgG1, IgG2, IgG3, IgG4. Preferably, the Fc-containing portion is derived from an IgG1 protein, more preferably from a human IgG1 protein. The non-Ig protein may be a therapeutic protein, for example, derived from erythropoietin (EPO), thrombopoietin (THPO) such as a THPO binding peptide, growth hormone, interferon (IFN) such as IFNα, IFNβ or IFNγ, platelet-derived growth factor (PDGF), interleukin (IL) such as IL1α or IL1β, transforming growth factor (TGF) such as TGFα or TGFβ, or tumor necrosis factor (TNF) such as TNFα or TNFβ, or derived from a receptor, in particular a ligand binding fragment from the extracellular domain of a receptor, for example, derived from a ligand binding fragment of a receptor. Therapeutic proteins of Fc cluster 2 (CD2), CD4, CD8, CD11, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD58 (LFA3), CD80, CD86, CD147, CD164, IL2 receptor, IL4 receptor, IL6 receptor, IL12 receptor, epidermal growth factor (EGF) receptor, vascular endothelial growth factor (VEGF) receptor, epithelial cell adhesion molecule (EpCAM) or cytotoxic T lymphocyte-associated protein 4 (CTLA4). Examples of Fc-fusion proteins include Belatacept Aflibercept Lelocip Romiplostim Abatacept Alphaset Etanercept

[0290] The term "cancer" as used herein means a physiological condition of a mammal characterized by uncontrolled cell growth. Tumors include one or more cancer cells. Examples of cancer include carcinomas, lymphomas, blastomas, sarcomas, and lymphoid malignancies. Other examples of cancer include squamous cell carcinomas (e.g., epithelial squamous cell carcinomas), non-Hodgkin's lymphomas (NHL) such as diffuse large B-cell lymphomas, lung cancers including small cell lung cancer, non-small cell lung cancer, acute myeloid leukemia (AML), lung adenocarcinomas, and lung squamous cell carcinomas, peritoneal cancer, hepatocellular carcinoma, gastric cancer (gastric cancer) or stomach cancer (stomach cancer) including gastrointestinal cancer and gastrointestinal stromal tumors, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, thyroid cancer, and liver cancer.

[0291] The term "drug-antibody ratio" (or "DAR", drug-antibody-ratio) used herein refers to the average number of drug molecules attached to one (e.g., antibody) part (V). In the art, DAR is sometimes referred to as "drug loading" or "drug loading". The DAR in the compounds of the present invention can be calculated by multiplying n by m in formula (1). If n=1, then m in formula (1) represents DAR. However, it should also be understood that when used to describe a sample containing many molecules, due to a certain degree of heterogeneity (which is usually related to the conjugation step), DAR is usually an average value. For example, the average DAR can be in the range of about 1 to about 10, and can be about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10. In some aspects, the DAR can be about 3 to about 8, and typically can be about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, or 9. In some aspects, the DAR can be about 4. In some aspects, the DAR can be about 8. In some aspects, a DAR of "about n" means that the DAR is measured within ±20% of n (e.g., between 80% of n and 120% of n).

[0292] As used herein, the term "emtansine" refers to a complex formed by covalent attachment of the anti-mitotic agent maytansine (DM1) via the non-reducible linker N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC", named "MCC" after conjugation to, for example, an antibody). Emtansine is used in the known antibody-drug-conjugate emtansine trastuzumab

[0293] The term "patient" as used herein refers to a subject to whom a compound of the invention (ie, ligand-drug-conjugate) is administered. In the context of the present invention, a patient is a mammal, preferably a human (male or female).

[0294] Insofar as the present description mentions “preferred” embodiments / features, combinations of these “preferred” embodiments / features are also to be regarded as disclosed, insofar as this combination of “preferred” embodiments / features makes technical sense.

[0295] Hereinafter, in the description and claims of the present invention, the use of the terms "containing" and "comprising" should be understood so that in addition to the mentioned elements, there may be additional unmentioned elements. However, these terms should also be understood to also disclose the term "consisting of..." as a more limited embodiment, so that there may be no additional unmentioned elements, as long as this is technically meaningful. For example, the expression "a group containing a divalent carbonyl group" includes a divalent group consisting of a carbonyl group (-CO-) as a preferred embodiment. In addition, the expression "at least one of X and Y" should be broadly understood to disclose one or both of X and Y, that is, it is equivalent to the expression "at least one selected from the group of X and Y".

[0296] Unless otherwise stated or the context dictates otherwise, reference to a "substituted" or "optionally substituted" group is to be understood as a reference to the presence (or optional presence, as the case may be) of at least one substituent selected from the group consisting of F, Cl, Br, I, CN, NO2, NH-C 1-6 -alkyl, N(C 1-6 -alkyl)2, -XC 1-6 -alkyl, -XC 2-6 -Alkenyl, -XC 2-6 -Alkynyl, -XC 6-14 -aryl, -X-(5-14 membered heteroalkyl having 1-3 heteroatoms selected from N, O, S), wherein X represents a single bond, -(CH2)-, -O-, -S-, -S(O)-, -S(O)2-, -NH-, -CO- or any combination thereof, including, for example, -C(O)-NH-, -NH-C(O)-. The number of substituents is not particularly limited and can be from 1 to the maximum number of valences that can be saturated by substituents. It is usually 1, 2 or 3, usually 1 or 2, most usually 1.

[0297] Unless otherwise indicated, all valences of the individual atoms of the compounds or moieties described herein are saturated. In particular, they are saturated with the specified binding partners. If no binding partner is indicated or the number of binding partners indicated is too small, the remaining valences of the corresponding atoms are saturated with the corresponding number of hydrogen atoms.

[0298] Unless otherwise stated, chiral compounds and moieties may exist in the form of pure stereoisomers or in the form of stereoisomer mixtures (including 50:50 racemates). In the present description, references to specific stereoisomers are to be understood as references to compounds or moieties in which the specified stereoisomer is present in at least 90% enantiomeric excess (ee), more preferably at least 95% ee, and most preferably 100% ee, wherein % ee is defined as (|RS|) / (R+S)*100%

[0299] Where R and S represent the molar amounts of the corresponding enantiomers.

[0300] Unless the context dictates otherwise, and / or other meanings are expressly provided herein, all terms are intended to have the meanings generally accepted in the art, as given in the IUPAC Gold Book (status of 1 st Aug.2020) or as reflected in the Dictionary of Chemistry, Oxford, 6th edition.

[0301] 2. Overview

[0302] The present invention is based on the discovery of a new cleavable linker system comprising a linker, such as a C-terminal peptide unit, carrying (1) a drug covalently attached thereto via a specific spacer group and (2) a solubilizing group. The linker, such as a C-terminal peptide unit, acts as a highly specific substrate that is rapidly cleaved by Cat B and / or cleaved by the exopeptidase (i.e., carboxydipeptidase) activity of Cat B, resulting in improved (high rate) cleavage and release of the drug into target cells. The linker system is not prone to premature cleavage and remains stable in the systemic circulation (blood). Therefore, the linker system of the present invention can be used in ligand-drug-conjugates (LDCs).

[0303] Cat B is a lysosomal cysteine ​​protease of the papain superfamily that plays a role in intracellular protein turnover and in various physiological and pathological processes. Extensive structural and functional data are currently available, making this protease a versatile tool in intracellular drug delivery.

[0304] The papain fold consists of two domains called left (L-) and right (R-) domains. The L-domain contains three α-helices, while the R-domain forms a β-barrel as described by Turk et al. (Biochim.Biophys.Acta 2012, 1824 (1), 68-88). The interface of the two domains opens at the top, forming the active site cleft of the enzyme. At the center of the active site cleft are residues Cys25 (at the N-terminus of the central helix, L-domain) and His163 (inside the β-barrel residues, R-domain). These two catalytic residues form a thiol-imidazolium ion pair, which is necessary for the proteolytic activity of the enzyme. As described by Turk et al. (Biochem.Soc.Symp.2003, 70, 15-30), the substrate binds along the active site cleft in an extended conformation, thereby contacting alternately with the L- and R-domains. Most cysteine ​​cathepsins mainly show endopeptidase activity (F, L, K, S, V), while Cat X and C only show exopeptidase activity. In contrast, Cat B shows both endopeptidase and exopeptidase activity. X-ray analysis reveals that exopeptidases / carboxydipeptidases such as Cat B contain additional structural features, i.e., additional ("closed") rings, which modify the active site cleft and serve as the basic principle for substrate binding in endopeptidase and exopeptidase activity. Specifically, as shown in Renko et al. (FEBS 2010 January, 277, 4338-4345), the closed ring provides a structural basis for the significant exo-contrast endo-Cat B activity.

[0305] Cat B-cleavable linker systems described in the prior art (e.g., Val-Cit-PABC linker systems) are primarily based on the endopeptidase activity of Cat B. On the other hand, the linker system of the present invention is specifically designed to meet the structural requirements for being used as a specific substrate for rapid cleavage by Cat B and / or the exopeptidase activity of Cat B. Therefore, the linker system can be used in LDC as a highly specific substrate for rapid cleavage by Cat B and / or the exopeptidase activity of Cat B, i.e., in the compounds of formula (I) below, resulting in an improved cleavage pattern (e.g., rapid intracellular drug release). The linker system can also release multiple drug molecules in the cell, wherein each drug molecule can be the same or different. If the drug is a cytotoxic agent, the linker system can release multiple drug molecules in the cell, and the multiple drug molecules can be multiple molecules of the same drug or multiple molecules of different drugs (e.g., 2 or more different drugs).

[0306] Furthermore, it was surprisingly found that any effect on Cat B binding due to the presence of a sterically demanding solubilizing moiety was fully compensated by the rapidity of Cat B, and thus sterically demanding solubilizing groups can be introduced into ADCs to improve ADC PK and manufacturability without affecting cytotoxicity. For many substrates, cleavage by the exopeptidase activity of Cat B, especially Cat Bis, is very fast, and this high cleavage rate can compensate for a certain slowing of the cleavage rate due to the introduction of the solubilizing moiety, so that the resulting cleavage rate is still quite fast. Without wishing to be bound by any theory, it is believed that the solubilizing moiety is directed to the outside of the Cat B binding groove, thereby, for example, leading to excellent selectivity and cleavage rate of Cat B by the exopeptidase activity of Cat B, while allowing a high DAR to be achieved.

[0307] Still further, it was found that the attachment of the drug to the linker via a specific spacer group and the presence of a solubilizing moiety resulted in a surprising increase in efficacy and allowed for overcoming drug resistance issues that may occur with other conjugates. Without wishing to be bound by any theory, it is believed that the spacer group and the solubilizing moiety act synergistically in such a way that, after being cleaved by Cat B, the drug is not only able to bind to its molecular target or targets in the cytoplasm, but also to escape capture by lysosomes and diffuse to proximal cells to induce a bystander effect, such as a cytotoxic bystander effect.

[0308] 3. Compounds of formula (I)

[0309] The present invention relates to compounds represented by the general formula (I), namely ligand-drug-conjugates (LDCs):

[0310]

[0311] The compound of formula (I) comprises a linker (L), such as a C-terminal peptide unit, which serves as a substrate specifically recognized and cleaved by cathepsin B, especially rapidly cleaved and / or cleaved by the exopeptidase activity of Cat B. The linker is covalently attached to the branching group (T) and the divalent group (X).

[0312] In addition, in formula (I), D represents a part derived from a drug, wherein the drug is selected from a carboxyl-containing drug such as auristatin F (AF), a sulfhydryl-containing drug such as maytansine (DM1) or ravtansine (DM4), an amino-containing drug such as monomethyl auristatin F (MMAF), isatecan or 2554618-79-8 and a hydroxyl-containing drug such as Maaa-1181a. The drug is covalently attached to the divalent group (X) through the functional group (i.e., carboxyl, sulfhydryl, amino or hydroxyl functional group) contained therein. Preferably, the drug is selected from a sulfhydryl-containing drug, an amino-containing drug and a hydroxyl-containing drug. More preferably, the drug is a sulfhydryl-containing drug, such as DM1 or DM4.

[0313] If there is more than one (D) (n>1 and / or m>1) in the conjugate molecule, at least one (D), preferably each (D) is independently selected from carboxyl-containing drugs, thiol-containing drugs, amino-containing drugs and hydroxyl-containing drugs, preferably selected from thiol-containing drugs, amino-containing drugs and hydroxyl-containing drugs, and multiple parts (D) are preferably the same as each other.

[0314] X represents a divalent group comprising 1 to 7, preferably 2 to 6, more preferably 2 to 5, for example 2, 3, 4 or 5 backbone atoms independently selected from C, N, O and S; X is covalently attached to (D) via an atom selected from C, S, N and O, which atom is derived from a carboxyl, thiol, amino or hydroxyl functional group contained in (D).

[0315] Y represents a divalent group containing one or more atoms selected from C, N, O, P and S, preferably a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound and derivatives thereof, more preferably a divalent group derived from maleimide and its derivatives such as ring-opening hydrolyzed maleimide derivatives, most preferably a divalent group derived from ring-opening hydrolyzed maleimide.

[0316] T represents a (2+n)-valent branched group.

[0317] S represents a moiety derived from a compound comprising one or more, for example 2, 3, 4 or 5, solubilising groups.

[0318] V represents a moiety derived from a carrier group capable of interacting with a target cell.

[0319] n is an integer of 1 to 4, preferably 1 or 2, more preferably 1; m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8.

[0320] In one embodiment, n is 1 and m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8, such as from 5 to 8 or from 6 to 8.

[0321] 3.1 Cleavable linker (L)

[0322] The compound of formula (I) contains a linker (L), such as a C-terminal peptide unit, which acts as a substrate that is specifically recognized and cleaved by cathepsin B, especially rapidly cleaved and / or cleaved by the exopeptidase activity of Cat B. The linker enables rapid (high rate) release of the drug-containing moiety, such as the DX or DX-Dxx-Dyy moiety, from the compound into the target cell.

[0323] In some embodiments of the present invention, the linker (L) is represented by the general formula (II) or (II'):

[0324]

[0325] In formula (II) and (II'), Axx represents a portion derived from a trifunctional amino acid. Axx can be a portion derived from any natural or non-natural trifunctional amino acid, provided that Axx in formula (II) is not a portion derived from an amino acid in (D) configuration. Examples of trifunctional amino acids include amino-dicarboxylic acids and diamino-carboxylic acids, such as α-aminoadipic acid (Aaa), diaminopropionic acid (Dap), diaminobutyric acid (Dab) and aminomalonic acid (Ama). Other suitable trifunctional amino acids include Glu, 2-aminopimelic acid (Apa), Lys, Orn, Ser and homolysine (homo-Lys).

[0326] According to one embodiment, Axx represents a portion derived from an amino acid selected from Glu, Apa, Aaa, Dap, Dab, Lys, Orn, Ser, Ama and homolysine (high-Lys). According to a preferred embodiment, Axx represents a portion derived from an amino acid selected from Dap, Dab, Lys, Orn and high-Lys. More preferably, Axx represents a portion derived from Orn or Lys, most preferably a portion derived from Lys.

[0327] Ayy represents a portion derived from an amino acid selected from Phe, Ala, Trp, Tyr, phenylglycine (Phg), Met, Val, His, Lys, Arg, citrulline (Cit), 2-aminobutyric acid (Abu), Orn, Ser, Thr, Leu and Ile; or Ayy in formula (II) represents a portion derived from an amino acid selected from homotyrosine (homo-Tyr), homophenylalanine (homo-Phe), β-phenylalanine (β-Phe), β-homophenylalanine (β-homo-Phe), Tyr (OR1) and homo-Tyr (OR1), wherein R1 is a solubilizing group, preferably -(CH2CH2O)n1-R2, wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer from 2 to 24, for example an integer from 5 to 20 or from 8 to 16; provided that Ayy in formula (II') is not an amino acid in the (D) configuration. Without wishing to be bound by theory, it is believed that Ayy provides the compounds of the invention with structural features that are specifically recognized and cleaved by the exopeptidase activity of Cat B. As a result, the compounds can release the drug at a significantly higher rate compared to compounds cleaved by the endopeptidase activity of CatB, such as compounds comprising the Val-Cit-PABC linker system.

[0328] According to one embodiment, Ayy in formula (II) represents a moiety derived from an amino acid selected from the group consisting of Phe, homo-Phe, Ala, Trp, Leu, Tyr, Phg, Met, Abu, Val, Lys, Cit, Tyr(OR1) and homo-Tyr(OR1), preferably a moiety derived from an amino acid selected from the group consisting of Phe, homo-Phe, Ala, Trp, Leu, Val, Tyr, homo-Tyr, Tyr(OR1) and homo-Tyr(OR1), more preferably a moiety derived from Phe, homo-Phe, Tyr, homo-Tyr, Tyr(OR1) or homo-Tyr(OR1). wherein R1 is as described above, in particular a moiety derived from Phe or Tyr, most preferably a moiety derived from Tyr; and Ayy in formula (II') represents a moiety derived from an amino acid selected from Phe, homo-Phe, Ala, Ser, Thr, Leu, Val, Tyr, Phg, Trp, Ile and Arg, preferably a moiety derived from an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr and Ser, more preferably a moiety derived from Phe, homo-Phe or Ser, in particular a moiety derived from Phe or Ser, most preferably a moiety derived from Phe.

[0329] With respect to the above formulae (II) and (II′), Dxx represents a single covalent bond or a moiety derived from an amino acid having a hydrophobic side chain, preferably a single covalent bond or a moiety derived from an amino acid selected from Phe, Val, Tyr, homo-Phe and Ala. More preferably, Dxx represents a single covalent bond or a moiety derived from Phe or Val.

[0330] Dyy represents a single covalent bond, a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain, preferably a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap and Dab; ​​provided that if Dxx is a moiety derived from an amino acid with a hydrophobic side chain, then Dyy is a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain, and if Dxx is a single covalent bond, then Dyy is a single covalent bond, a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain. More preferably, Dyy represents a moiety derived from Arg or Cit.

[0331] Z in formula (II) and (II') represents a group covalently bonded to the C-terminus of Ayy or Axx and selected from -OH and -N(H)(R), wherein R represents a hydrogen atom, an alkyl group or a cycloalkyl group, preferably -OH.

[0332] In formula (II) and (II'), * represents covalent attachment to (T), ** represents covalent attachment to (X).

[0333] 3.2 Divalent group (X)

[0334] The compound of formula (I) comprises a divalent group (X) comprising 1-7, preferably 2-6, more preferably 2-5, for example 2, 3, 4 or 5 backbone atoms independently selected from C, N, O and S; X is covalently attached to (D) via an atom selected from C, S, N and O derived from a carboxyl, thiol, amino or hydroxyl functional group contained in (D).

[0335] After Cat B cleavage of the linker (L), the divalent group (X) remains covalently attached to (D), so that the modified drug (internal drug), such as moiety DX, is released into the target cell. In the present invention, it is surprisingly found that such modified drugs exhibit improved efficacy, such as cytotoxic efficacy, compared to the same drug not carrying the group (X) attached thereto. Without wishing to be bound by any theory, it is believed that the spacer group (X) has a suitable size, such as 1-7 backbone atoms, which does not adversely affect the pharmacokinetic properties of the conjugate even at high DAR (DAR>4), while contributing to the membrane permeability properties of the released drug moiety, such as due to an appropriate level of hydrophobicity. As a result, upon high-speed cleavage induced by Cat B, the drug can not only bind to one or more molecular targets in the cytoplasm, but can also diffuse to proximal cells to induce a bystander effect, such as a cytotoxic bystander effect.

[0336] In one embodiment, (X) represents a divalent carbonyl or thiocarbonyl-containing group, preferably a group represented by one of the following formulae (IIIa) to (IIIf):

[0337] ***-(CH2)n2-(C=A)-**' (IIIa)

[0338] ***-(C=A)-(CH2)n3-**' (IIIb)

[0339] ***-(CH2)n2-(C=A)-(CH2)n3-**' (IIIc)

[0340] ***-(CH2)n2-(C=A)-(C(CH3)2)-(CH2)n3-**' (IIId)

[0341] ***-(CH2)n2-(C(CH3)2)-(C=A)-(CH2)n3-**' (IIIe)

[0342] ***-(C=A)-(NH)n4-(CH2)n3-(NH)n5-(C=A)-**' (IIf)

[0343] in

[0344] n2 and n3 are each independently selected from 0 to 5, preferably 0, 1 or 2, more preferably 0 or 1;

[0345] n4, n5 are each selected from 0 or 1, wherein, in one embodiment, n4 is 0 and n5 is 1, in another specific embodiment, n4 is 1 and n5 is 0, in another specific embodiment, n4 and n5 are both 1, and in yet another specific embodiment, n4 and n5 are both 0;

[0346] Each A is independently selected from O and S, preferably O;

[0347] *** represents covalent attachment to (D); and

[0348] **' represents covalent attachment to (L).

[0349] In one embodiment, (X) is represented by one of the following general formulae (IVa) to (IVj'):

[0350]

[0351]

[0352] in,

[0353] *** represents covalent attachment to (D);

[0354] **' represents covalent attachment to (L);

[0355] Provided that if (X) is represented by Formula (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVp), (IVr), (IVt), (IVv), (IVw), (IVx), (IVy) or (IVz), then (D) in Formula (I) represents an amino-containing drug; if (X) is represented by Formula (IVj), (IVq), (IVs) or (IVu), then (D) in Formula (I) represents an amino-containing drug or a hydroxyl-containing drug; and if (X) is represented by Formula (IVa'), (IVb'), (IVc'), (IVd'), (IVe'), (IVf'), (IVg'), (IVh'), (IVi') or (IVj'), then (D) in Formula (I) represents a carboxyl-containing drug.

[0356] In a preferred embodiment, (X) is represented by formula (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt). More preferably, (X) is represented by formula (IVc) or (IVm).

[0357] It is advantageous to select a structure of X that can conveniently bond to the functional group of (D). According to one embodiment, if (D) has a carboxyl group, (X) preferably has an amino group to allow bonding to (D) by formation of an amide bond (linkage); if (D) has an amino group, (X) preferably has a carbonyl group (or a thiocarbonyl group) to allow bonding to (D) by formation of an amide bond; if (D) has a sulfhydryl group, (X) preferably has a methylene group to allow bonding to (D) by formation of a thioether bond; and if (D) has a hydroxyl group, (X) preferably has a methylene group or a carbonyl group (or a thiocarbonyl group) to allow bonding to (D) by formation of an ether bond or an ester (or thioester) bond, respectively. Further matching of structural groups and the resulting linkages are also conceivable. The following table provides an overview of the available options.

[0358]

[0359] In the above table, R1 characterizes the remainder of the group (X), while R2 represents the remainder of the drug (D).

[0360] 3.3 Solubilized fraction (S)

[0361] The compound of formula (I) comprises a solubilizing part (S) which is derived from a compound comprising one or more, for example 2, 3, 4 or 5, solubilizing groups. The presence of the solubilizing part makes it possible to reduce (or prevent) the tendency of the conjugate molecules to aggregate, thereby obtaining excellent pharmacokinetic properties, such as biodistribution, liver clearance, even at high DAR (DAR>4, for example DAR=8). In some cases, the aggregation of the conjugate molecules can be completely inhibited even at high DAR. The inventors surprisingly found that even in the presence of a sterically demanding solubilizing part, it is possible to be cleaved by Cat B. Preferably, the rapid cleavage of the linker by Cat B can be achieved preferably by the exopeptidase mechanism of Cat B. Without being bound by any theory, it is believed that the solubilizing part is directed to the outside of the Cat B binding groove, thus allowing, for example, excellent selectivity and cleavage rate to be obtained by the exopeptidase mechanism. In addition, it was also surprisingly found that the solubilizing part can compensate for the potential hydrophobicity of the drug part DX, so that even if multiple drug parts are attached to the linker (for example, n>1), excellent pharmacokinetic properties can be maintained.

[0362] In one embodiment, S represents a moiety derived from a compound comprising one or more, for example two, three or four, solubilizing groups; wherein each solubilizing group comprised in (S) is independently selected from:

[0363] - a moiety comprising one or more ionic or ionizable groups, such as an ammonium, guanidinium, sulfate or sulfonate group, preferably a moiety derived from Arg, (D)-Arg, Dap, (D)-Dap, Dab, (D)-Dab, Orn, (D)-Orn, Lys, D-Lys or carnitine;

[0364] - a sugar moiety selected from monosaccharides, disaccharides and linear or branched oligosaccharides, in particular linear or branched oligosaccharides having 3 to 10 monosaccharide units linked by glycosidic bonds, wherein each monosaccharide unit in the monosaccharide, disaccharide and oligosaccharide is independently selected from glucose, fructose, mannose, ribose and galactose; and

[0365] - a polyoxyalkylene group, preferably a C2-3 polyoxyalkylene group, more preferably a C2-3 polyoxyalkylene group, which independently comprises 6 to 200, preferably 10 to 150, more preferably 12 to 80 repeating units.

[0366] There are no particular restrictions on the general arrangement of the solubilizing groups in the solubilizing part. Thus, the solubilizing part may have a linear structure, for example, in which several solubilizing groups are arranged in a random or block manner, a cyclic structure or a branched structure, for example, in which several solubilizing groups are attached to a core molecule, such as pentaerythritol or glycerol, in a grafted or dendritic manner. The solubilizing part may also comprise several blocks, each block independently having a linear or branched structure.

[0367] In one aspect, the solubilizing portion comprises one or more solubilizing groups arranged in a linear block. For example, the solubilizing portion may comprise -(So) n’ - represents, as represented by the following formula: -(Sol)-(Sol)-[…]-(Son) structure represented in more detail, wherein each So1 to Son represents a solubilizing group, such as a polyoxyalkylene group, for example PEO having 6 to 200 repeating units, or a moiety containing one or more ionic or ionizable groups, such as Arg, and n' is an integer from 1 to 20, for example 1 to 10, provided that directly connected polyoxyalkylene groups of the same structure are regarded as multiple repeating units of the same solubilizing group (rather than adjacent So groups). That is, adjacent polyoxyalkylene groups must have different structures and / or be connected by functional groups such as -C(O)-O-, etc., in order to be treated as independent So groups.

[0368] In another aspect, the solubilizing moiety comprises one or more solubilizing groups attached to a core moiety such as pentaerythritol or glycerol in a non-tethered, grafted or dendritic manner. For example, the solubilizing moiety may have a moiety consisting of -((-Y'-X'(So m’ )) n’ -H represents the grafted structure:

[0369]

[0370] wherein X' is a (m'+2)-valent group, such as a trivalent or tetravalent group, Y' is a divalent group, each So is independently selected to be a solubilizing group, such as a polyoxyalkylene group, such as a PEO group having 4 to 600 repeating units, or a moiety comprising one or more ionic groups, m' is 1, 2, 3 or more, preferably 1 or 2, and n' is an integer from 1 to 20, such as 1 to 10;

[0371] or as described in more detail below -X'(So) n’ The tree-like and dendritic structures represented by:

[0372]

[0373] wherein X' is an n-valent (branched) group, each So1 to Son is independently selected to be a solubilizing group as described above, such as a polyoxyalkylene group, for example a PEO group having 4 to 600 repeating units, or a portion comprising one or more ionic groups, and n' is an integer from 1 to 20, for example 1 to 10.

[0374] In one embodiment, (S) is a moiety derived from a compound comprising one or more polyoxyalkylene groups, wherein preferably each polyoxyalkylene group independently comprises 6 to 200, more preferably 10 to 150, most preferably 12 to 80 repeating units.

[0375] In a preferred embodiment, (S) is a group represented by formula (V):

[0376] ****-X 1 -(CH2CH2O)n3-X 2 (V)

[0377] in,

[0378] n3 is an integer from 6 to 200, preferably from 10 to 150, more preferably from 12 to 80;

[0379] **** indicates covalent attachment to (T);

[0380] X 1 is selected from a single covalent bond, -(C=O)- and -N(R)-, wherein R represents a hydrogen atom, an alkyl group or a cycloalkyl group;

[0381] X 2 represents an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group such as acetyl or a group of the formula -(CH2) n4 -CO2H group, thiocarbonyl-containing group, formula -(CH2) n4 OR group, formula -(CH2) n4-SO3H group or amino group such as -(CH2) n4 -(C=A)-N(R)2 or -(CH2) n4 -N(R)2 group, wherein A is O or S, each R is independently selected from a hydrogen atom, an alkyl group and a cycloalkyl group, and n4 is an integer from 1 to 6;

[0382] X 2 It is preferably -CH3, -CH2CH2OH or a group represented by the following formula (VI):

[0383] -(CH2)n5-(C=A)N(R)-(CH2)n6-(C=A)N(H)(R) (VI)

[0384] in,

[0385] Each A is independently selected from O and S, preferably O;

[0386] Each R is independently selected from a hydrogen atom, an alkyl group, and a cycloalkyl group; and

[0387] n5 and n6 are each independently an integer of 1-6, preferably 1 or 2; and

[0388] X2 is most preferably -CH3.

[0389] If more than one group is present, each group is preferably moiety of formula (V) as described above.

[0390] 3.4 Branched Group (T)

[0391] T represents a (2+n)-valent, for example, a 3-valent, 4-valent, 5-valent, 6-valent branching group. The branching group connects the carrier group (V), the solubilizing part (S) and one or more (n) linker parts (L) to form a branched structure. Preferably, T is a 3-valent (n=1) or 4-valent (n=2) branching group. More preferably, T is a 3-valent branching group.

[0392] In one embodiment, branched group is to comprise at least one trifunctional amino acid, for example, a group derived from the part of Lys. In addition to the above-mentioned trifunctional amino acid, branched group may also comprise other (optional) joints and / or amino acid, provided that the other joints described do not contain solubilizing groups, such as polyoxyalkylene groups, and / or the other amino acids described are not trifunctional amino acids or comprise the part of one or more ions or ionizable groups. Such other amino acids may for example be selected from high-Phe (hPHe) and Phe. In some respects, branched group is made up of the part derived from trifunctional amino acid (that is, excluding any other joints and / or amino acid).

[0393] In one embodiment, (T) is a moiety represented by the following formula (VII):

[0394]

[0395] in,

[0396] each AA independently represents a moiety derived from a trifunctional amino acid such as a diaminocarboxylic acid, an aminodicarboxylic acid, an azidoamino acid or an alkyne-containing amino acid, preferably derived from an amino acid selected from N-ε-propynyloxycarbonyl-L-lysine (Lys(Poc)), Asp, Glu, Orn, Lys, Dab and Dap, more preferably derived from Lys(Poc), Glu, Orn or Lys, most preferably derived from Lys;

[0397] α represents covalent attachment to (Y);

[0398] If n=1, the side chain derived from the trifunctional amino acid is covalently attached to (L) or (S), and the C-terminus is covalently attached to the other moiety (S) or (L), respectively;

[0399] If n = 2, 3, or 4:

[0400] *’ represents covalent attachment to (L);

[0401] ****’ represents covalent attachment to (S); and

[0402] n is as defined in formula (I).

[0403] In one other embodiment, (T) is a moiety represented by formula (VIII) or (IX):

[0404]

[0405] in,

[0406] Each AA1 and AA2 is independently a moiety derived from a trifunctional amino acid, such as a diaminocarboxylic acid, an aminodicarboxylic acid, an azidoamino acid, or an alkyne-containing amino acid, preferably a moiety derived from an amino acid selected from Lys(Poc), Asp, Glu, Orn, Lys, Dab, and Dap, more preferably a moiety derived from Lys(Poc), Glu, Orn, or Lys, most preferably a moiety derived from Lys;

[0407] α represents covalent attachment to (Y);

[0408] In formula (IX), the side chain derived from the trifunctional amino acid is covalently attached to (L) or (S), and the C-terminus is covalently attached to another moiety (S or (L), respectively;

[0409] In formula (VIII), *’represents covalent attachment to (L), and ****’ represents covalent attachment to (S).

[0410] 3.5 Divalent group (Y)

[0411] The compound of formula (I) contains a divalent group (Y) comprising one or more atoms selected from C, N, O, P and S. The divalent group connects the carrier group (V) to the branching group (T). The divalent group is usually attached to the side chain of an amino acid such as Cys contained in the carrier. Preferably, Y is a divalent group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound and derivatives thereof. More preferably, Y is a divalent group derived from maleimide and derivatives thereof, such as a divalent group of maleimide derivatives of ring-opening hydrolysis. Most preferably, Y is a divalent group of maleimide derived from ring-opening hydrolysis.

[0412] The hydrolysis of the maleimide attachment to V is typically performed under alkaline conditions as the final step of the conjugation of the maleimide derivative to V, as described in the general procedures 1 to 3 in the examples herein. The following conditions are particularly suitable: At the end of the cysteine ​​maleimide conjugation reaction, the pH is adjusted to pH 8 by adding 110x pH 8 DPPS (0.2 to 0.5 reaction volumes), and excess reactive drug linker and reducing agent (TCEP) are removed by gel filtration using a suitable gel filtration column (PF column, eluted with pH 8 buffer). The eluate is then stirred overnight for 16 hours to complete the opening, and the final buffer is then exchanged into an Amicon concentration unit with DPBS.

[0413] In some cases, when m in the compound of formula (I) is at least 2, the compound may comprise a mixture of (closed) maleimide derivatives (Y) and ring-opening hydrolyzed maleimide derivatives (Y) attached to V. Thus, in compounds described herein in which group (R) is attached to a support (V) via maleimide (as shown below, left hand side), hydrolysis may be performed such that when m is at least 2, the compound of the invention may comprise a closed maleimide attachment to V (A) and a ring-opening hydrolyzed maleimide attachment (B) (as shown below, left hand side).

[0414]

[0415] In a preferred embodiment, in compounds of formula (I) where m is at least 2, at least 50% of the attachments of Y to V are ring-opening hydrolyzed maleimide attachments (B), and the remaining attachments are closed maleimide attachments (A). In some cases, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, preferably at least 98% of the attachments of Y to V are ring-opening hydrolyzed maleimide attachments (B).

[0416] The presence of one or more ring-opening hydrolyzed maleimide attachments (even if present together with one or more closed maleimide attachments) can contribute to the stability and therapeutic effect of the compounds of the invention. Without being bound by any theory, it is believed that the ring-opening hydrolyzed maleimide attachments, for example, prevent the retro-Michael reaction (e.g. Fig.29 As shown), this reaction leads to the release of active maleimide in the circulation and ultimately leads to the transfer of linker-payload to other sulfhydryl-containing molecules in the body, such as albumin. The ring-opened maleimide can also synergize with the divalent group X and the solubilizing part S to obtain improved stability and therapeutic effect.

[0417] In a preferred embodiment, Y is a divalent group derived from maleimide and its derivatives, such as ring-opening hydrolyzed maleimide, preferably a divalent group represented by any one of the following formulae (XIIIa) to (XIIIc):

[0418]

[0419] in,

[0420] R 3 Represents -(CH2) n7 -(C=A) n9 -or-(CH2CH2O) n8 -(C=A) n9 -, preferably -(CH2) n7 -(C=A) n9 -,in,

[0421] n7 is 1 to 6, preferably 1 or 2, more preferably 1,

[0422] n8 is 1 to 6, preferably 1,

[0423] n9 is 0 or 1, preferably 1, and

[0424] A is O or S, preferably O;

[0425] wherein the methylene carbon atom is covalently attached to the nitrogen atom of formula (XIIIa)-(XIIIc), and the carbonyl carbon or thiocarbonyl carbon is covalently attached to T;

[0426] β represents a covalent attachment to V; and

[0427] α' indicates covalent attachment to T.

[0428] In a more preferred embodiment, Y is represented by formula (XIIIb) or (XIIIc), wherein R 3 Preferably, it is of the formula -(CH2) n7 -(C=A) n9-represented group, wherein n7 is 1 or 2, n9 is 1, and A is O. Most preferably, R 3 It is -CH2-C=O-.

[0429] Section 3.6 (D)

[0430] The compound of formula (I) comprises a part derived from a drug, wherein the drug is selected from a carboxyl-containing drug, a sulfhydryl-containing drug, an amino-containing drug and a hydroxyl-containing drug. Preferably, the drug is selected from a sulfhydryl-containing drug, an amino-containing drug and a hydroxyl-containing drug. More preferably, (D) is a part derived from a sulfhydryl-containing drug such as DM1 or DM4.

[0431] If more than one (D) (n>1 and / or m>1) is present in the compound of formula (I), each (D) is independently selected from carboxyl-containing drugs, thiol-containing drugs, amino-containing drugs and hydroxyl-containing drugs. However, it is preferred that the multiple moieties (D) are identical to each other.

[0432] The drug may be unmodified (in its native form except for the replacement of hydrogen atoms by covalent bonds) or may be chemically modified so as to introduce one or more functional groups (e.g. one or more groups selected from hydroxyl, carboxyl, amino and thiol) allowing the formation of one or more covalent attachments to the divalent group (X), the drug moiety, e.g. moiety DX or moiety DX-Dxx-Dyy, preferably being pharmacologically active once it is released from the conjugate. According to one embodiment, the drug moiety, e.g. moiety DX or moiety DX-Dxx-Dyy released from the conjugate is pharmacologically active in the sense that it retains at least 20%, preferably at least 35%, more preferably at least 50%, even more preferably at least 70% of the pharmacological activity of the corresponding unmodified (native) drug.

[0433] In some aspects of the invention, each moiety derived from the drug independently represents a prodrug group that is pharmacologically inactive in conjugated form, for example, when present in a compound of formula (I), but becomes pharmacologically active once released from the conjugate.

[0434] According to one embodiment, each moiety derived from a drug is independently selected from:

[0435] (i) Antineoplastic agents, such as

[0436] o DNA alkylating agents, such as duocarmycin,

[0437] o Topoisomerase inhibitors, such as doxorubicin,

[0438] o RNA-polymerase II inhibitors, such as α-amanitin,

[0439] o DNA cleaving agents, such as calicheamicin,

[0440] o antimitotic or microtubule disrupting agents, such as taxanes, auristatins or maytansinoids,

[0441] o antimetabolites, such as derivatives of gemcitabine,

[0442] o Spindle kinesin inhibitors, such as filaments,

[0443] o kinase inhibitors, such as patasertib or gefitinib,

[0444] ο Nicotinamide phosphoribosyltransferase inhibitors,

[0445] ο matrix metallopeptidase 9 inhibitors,

[0446] o Phosphatase inhibitors such as Microcystis toxin-LR,

[0447] (ii) Immunomodulators such as fluticasone

[0448] (iii) anti-infective drugs, such as rifamycin, clindamycin or retapamulin, and

[0449] (iv) radioactive isotopes, metabolites, pharmaceutically acceptable salts and / or prodrugs of any of the foregoing;

[0450] Provided that the drug selected from the above (i) to (iv) is a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug or a hydroxyl-containing drug.

[0451] If there is more than one (D) in the compound of formula (I), each (D) can be independently selected from (i) to (iv) above, provided that each drug selected from (i) to (iv) is a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug or a hydroxyl-containing drug.

[0452] The following are exemplary drugs that can be used in the ligand-drug-conjugates of the present invention, provided that the drug is a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug, or a hydroxyl-containing drug.

[0453] (i) Anti-tumor agents include:

[0454] (a) Alkylating agents such as nitrogen mustard analogs (e.g., cyclophosphamide, chlorambucil, melphalan, mechlorethamine, ifosfamide, trofosfamide, prednimustine, bendamustine, naphthyl mustard, estramustine, mechlorethamine oxide hydrochloride, hydrochloride), mannomustine, dibromodulan, nembixin, phenamic acid, uracil mustard); alkyl sulfonates (e.g., busulfan, trooxosulfan, mannosulfan, improsulfan, and piposulfan); ethyleneimines (e.g., thiotepa, triazinequinone, carboquinone); nitrosoureas (e.g., carmustine, lomustine, semustine, streptozotocin, chlorozotocin, fotemustine, nimustine, ranimustine); epoxides (e.g., etoglucon); other alkylating agents (e.g., dibromodulan, pipobroman, temozolomide, dacarbazine);

[0455] (b) alkaloids, for example, vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine, navelbine, vinflunide, vintafolide); taxanes (e.g., paclitaxel, docetaxel, paclitaxel polyglumex, cabazitaxel) and their analogs, maytansinoids (e.g., DM1, DM2, DM3, DM4, maytansinoids and ansamitocin) and their analogs, cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); epothilones, chrysoprolol, bryostatin, dolostatins, auristatins (e.g., monomethyl auristatin E, monomethyl auristatin F), tubulysins (e.g., tubulysin); , cephalostatins; pancratistatin; spongistatin; spongistatin; demeclocycline; epipodophyllotoxins (e.g., 9-aminocamptothecin, camptothecin, crestofol, daunorubicin, etoposide, etoposide phosphate, irinotecan and its metabolites such as SN-38, mitoxantrone, novatrexone, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS 2000)); mitomycins (e.g., mitomycin C);

[0456] (c) antimetabolites, such as DHFR inhibitors (e.g. methotrexate, trimetrexate, dimethoate, pteropterin, aminopterin (4-aminopteroic acid) or other folic acid analogs such as raltitrexed, pemetrexed, pralatrexate); IMP dehydrogenase inhibitors (e.g. mycophenolic acid, thiazofurine, ribavirin, EICAR); ribonucleotide reductase inhibitors (e.g. hydroxyurea, deferoxamine); pyrimidine analogs (e.g. cytarabine, fluorouracil, 5-fluorouracil and its metabolites, tegafur); , carmofur, gemcitabine, capecitabine, azacitidine, decitabine, fluorouracil combination, tegafur combination, trifluridine combination, cytosine arabinoside, ancitabine, floxuridine, deoxyfluridine), uracil analogs (e.g., 6-azauridine, deoxyuridine); cytosine analogs (e.g., enocitabine); purine analogs (e.g., azathioprine, fludarabine, mercaptopurine, thioimidazole, thioguanine, cladribine, clofarabine, nelarabine); folic acid supplements such as folinic acid;

[0457] (d) endocrine therapies specifically used to treat neoplastic diseases, such as estrogens, progestins, gonadotropin-releasing hormone analogues, antiestrogens, antiandrogens, and aromatase inhibitors;

[0458] (e) kinase inhibitors such as BIBW 2992 (anti-EGFR / Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetinib, afatinib, vemurafenib, crizotinib, regorafenib, masitinib, dabrafenib, tramatinib, ibrutinib, ceritinib, lenvatinib, nintedanib, cediranib, indopalbociclib, osimertinib, icotinib, ectinib, abamivir, rucaparib, cobimetinib, midostaurin, omotinib, E7080 (anti-VEGFR2), muritinib, ponatinib (AP24534), bafatinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, tivozanib, ispinax, temsirolimus, everolimus, ridaforolimus;

[0459] (f) Other drugs, such as duocarboxamicin (including synthetic analogs: adoz, kazelesin, bicelesin, KW-2189 and CBI-TMI); benzodiazepines Dimer-like (pyrrolobenzodiazepine (pyrrolobenzodiazepine) or tomamycin, indole benzodiazepine indolinobenzodiazepines, imidazobenzothiadiazepines imidazobenzothiadiazepines or oxazolidinone benzodiazepines dimers of the type); platinum-containing compounds (e.g., carboplatin, cisplatin, oxaliplatin, satraplatin, polyplatin); aziridines such as benzodepa, metodepa and uredepa; methylmelamines, including hexamethylmelamine, triethylenemelamine, triacetylene phosphoramide, triethylenethiophosphoramide and trihydroxymethylmelamine; dynemicin, esperamycin, kedacetin, maprotiline, aclarubicin, actinomycin, anthramycin, azoserine, bleomycin, actinomycin C, carrubicin, carmorubicin, carmosin; chromomycin, dacarbamycin, daunorubicin, deoxydaunorubicin, 6-diazo- 5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marseromycin, nitomycins, mycophenolic acid, nogamycin, oliveromycin, peplomycin, porfiromycin, puromycin, triferric doxorubicin, rhodorubicin, streptozocin, streptozotocin, truffle, ubenimex, netastatin, daunorubicin; polyketides (e.g., annona); gemcitabine, epoxomicins (e.g., bortezomib).

[0460] (ii) Immunomodulators include immunostimulants, immunosuppressants, cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g., amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, flucodone danazol, dexamethasone, prednisone, triamcinolone acetonide, beclomethasone dipropionate), DHEA, hydroxychloroquine, meloxicam, methotrexate, mycophenolate mofetil, mycophenylate, sirolimus, tacrolimus, everolimus, fingolimod, ibrutinib.

[0461] (iii) Anti-infectious agents include antibacterial drugs, anti-mitotic drugs, anti-mycobacterial drugs and anti-viral drugs. A non-limiting example of an antibiotic used in an antibiotic-antibody drug conjugate is rifalogue (a rafamycin derivative).

[0462] As used herein, a drug also includes its radioactive isotopes. Examples of radioactive isotopes (radionuclides) are e.g. 3 H. U C. 14 C. 18 F. 32 P. 35 S. 64 Cu, 68 Ga, 86 Y.99 Tc, 111 In, 123 I. 124 I. 125 I. 131 I, 177 Lu, 186 Re, 188 Re, 211 At 212 Bi, 213 Bior 225 Radioactive isotope-labeled drugs can be used for targeted imaging experiments or targeted therapy (Wu et al. Nat. Biotech. 2005, 23, 1137-1146).

[0463] The drug used herein also includes its pharmaceutically acceptable salt, acid or derivative.

[0464] According to a preferred embodiment, each moiety derived from a drug is independently derived from a drug selected from the group consisting of amanitin, duocarmycin, auristatin, auristatin F (AF), monomethyl auristatin F (MMAF), maytansine, maytansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, paclitaxel, daunorubicin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepine (PBD) and its dimer, indenobenzodiazepine (IBD) and its dimer, or its radioisotope and / or pharmaceutically acceptable salt. More preferably, each part derived from the drug is independently derived from a drug selected from auristatin, MMAF, exatecan, maytansinoids, DM1 and DM4; and even more preferably, it is derived from a drug selected from DM1 and DM4.

[0465] 3.7 Compounds of formula (I) comprising a linker of formula (II) / (II')

[0466] In some cases, the compound of formula (I) may include a linker of formula (II) or (II') as described above. Therefore, the compound of the present invention may be represented by the following general formula (X) or (X'):

[0467]

[0468] Axx in formula (X) and formula (X') represents a moiety derived from an amino acid selected from Glu, Apa, Aaa, Dap, Dab, Lys, Orn, Ser, Ama and homo-Lys, preferably a moiety derived from an amino acid selected from Dap, Dab, Lys, Orn and homo-Lys, more preferably a moiety derived from a moiety derived from Lys;

[0469] Ayy in formula (X) represents a moiety derived from an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, homo-Tyr, Tyr(OR1) and homo-Tyr(OR1), wherein R1 is -(CH2CH2O) n1 -R2, wherein R2 is a hydrogen atom or a methyl group, n1 is an integer from 2 to 24, preferably a moiety derived from Phe, homo-Phe, Tyr, homo-Tyr, Tyr(OR1) or homo-Tyr, more preferably a moiety derived from Tyr;

[0470] Ayy in formula (X') represents a moiety derived from an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr and Ser, preferably a moiety derived from Phe, homo-Phe or Ser, more preferably a moiety derived from Phe or Ser;

[0471] D, Dxx, Dyy, X, T, S, V, Z, m and n in formulae (X) and (X′) have the same meanings as described above.

[0472] According to a preferred embodiment, at least one, for example two, three, four, five, six, seven or eight of D, Dxx, Dyy, X, Y, T, S and Z are defined as follows:

[0473] (a) D is a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansinoids, DM1 and DM4, preferably a moiety derived from DM1 or DM4;

[0474] (b) Dxx is a moiety derived from an amino acid selected from the group consisting of Phe, Val, Tyr, homo-Phe and Ala, preferably selected from Phe or Val;

[0475] (c) Dyy is a covalent bond or a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap and Dab, preferably a covalent bond or a moiety derived from Arg or Cit;

[0476] (d) X is a group of formula (III) wherein n2 is 1 or 2, or a group represented by any one of formulae (IVa) to (IVz), preferably a group represented by any one of formulae (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm);

[0477] (e) Y is a group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound and derivatives thereof, preferably a group derived from maleimide and derivatives thereof such as maleimide derivatives obtained by ring-opening hydrolysis, more preferably a group derived from maleimide obtained by ring-opening hydrolysis;

[0478] (f) T is a group of formula (VII), (VIII) or (IX);

[0479] (g) S is a moiety of formula (V); and

[0480] (h) Z is -OH.

[0481] According to a preferred embodiment, at least one, for example two, three, four, five, six, seven, eight or nine of D, Dxx, Dyy, X, Y, T, S, V and Z are defined as follows:

[0482] (a) D is a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansinoids, DM1 and DM4, preferably a moiety derived from DM1 or DM4;

[0483] (b) Dxx is a moiety derived from an amino acid selected from the group consisting of Phe, Val, Tyr, homo-Phe and Ala, preferably selected from Phe or Val;

[0484] (c) Dyy is a covalent bond or a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap and Dab, preferably a covalent bond or a moiety derived from Arg or Cit;

[0485] (d) X is a group of formula (III) wherein n2 is 1 or 2, or a group represented by any one of formulae (IVa) to (IVz), preferably a group represented by any one of formulae (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm);

[0486] (e) Y is a group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compound and derivatives thereof, preferably a group derived from maleimide and derivatives thereof such as maleimide derivatives obtained by ring-opening hydrolysis, more preferably a group derived from maleimide obtained by ring-opening hydrolysis;

[0487] (f) T is a group of formula (VII), (VIII) or (IX);

[0488] (g) S is a moiety of formula (V);

[0489] (h) V is a portion derived from natalizumab; and

[0490] (i) Z is -OH.

[0491] According to other preferred embodiments, each Dyy-Dxx-Axx-Ayy in formula (X) is independently selected from wherein Dyy is covalently bound Arg-Lys-Phe, wherein Dyy is covalently bound Arg-Lys-homo-Phe, wherein Dyy is covalently bound Arg-Lys-Tyr, wherein Dyy is covalently bound Cit-Lys-Phe, wherein Dyy is covalently bound Cit-Lys-Tyr, wherein Dyy is covalently bound Arg-Lys-homo-Tyr, wherein Dyy is covalently bound Arg-Lys-homo-Tyr, wherein Dyy is covalently bound Arg-Lys-Phe, Phe-Cit-Lys-Tyr, Phe-Arg-Lys-Tyr, Phe-Cit-Lys-homo-Tyr, -Tyr, Phe-Lys-Lys-Phe, homo-Phe-Arg-Lys-Phe, homo-Phe-Cit-Lys-Tyr, and each Dyy-Dxx-Ayy-Axx in formula (X') is independently selected from Arg-Phe-Lys wherein Dyy is covalently bonded, Arg-Ser-Lys wherein Dyy is covalently bonded, Cit-Ser-Lys wherein Dyy is covalently bonded, Cit-Ser-Lys wherein Dyy is covalently bonded, Cit-homo-Phe-Lys wherein Dyy is covalently bonded, Phe-Cit-Phe-Lys, homo-Phe-Cit-Phe-Lys and Phe-Arg-Phe-Lys.

[0492] In one embodiment, the compounds of the invention are represented by one of the following formulae:

[0493]

[0494]

[0495] wherein D, X, Y, T, S, V, Z, m and n have the same meanings as described above.

[0496] According to a preferred embodiment, at least one, for example two, three, four, five or six of D, X, Y, T, S and Z are defined as follows:

[0497] (a) D is a moiety derived from a drug selected from auristatin, AF, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4;

[0498] (d) X is a group of formula (III) wherein n2 is 1 or 2, or is a group represented by any one of formulae (IVa) to (IVz), preferably a group represented by any one of formulae (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm);

[0499] (e) Y is a group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compounds and derivatives thereof, preferably selected from maleimide and derivatives thereof such as ring-opening hydrolyzed maleimide derivatives and more preferably selected from ring-opening hydrolyzed maleimide;

[0500] (f) T is a group of formula (VII), (VIII) or (IX);

[0501] (g) S is a moiety of formula (V); and

[0502] (h) Z is -OH.

[0503] According to a preferred embodiment, at least one, for example two, three, four, five, six or seven of D, X, Y, T, S, V and Z are defined as follows:

[0504] (a) D is a moiety derived from a drug selected from auristatin, AF, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4;

[0505] (d) X is a group of formula (III) wherein n2 is 1 or 2, or is a group represented by any one of formulae (IVa) to (IVz), preferably a group represented by any one of formulae (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm);

[0506] (e) Y is a group derived from a compound selected from maleimide, triazole, hydrazone, carbonyl-containing compounds and derivatives thereof, preferably selected from maleimide and derivatives thereof such as ring-opening hydrolyzed maleimide derivatives and more preferably selected from ring-opening hydrolyzed maleimide;

[0507] (f) T is a group of formula (VII), (VIII) or (IX);

[0508] (g) S is a moiety of formula (V);

[0509] (h) V is a portion derived from narasimab; and

[0510] (h) Z is -OH.

[0511] In one embodiment, the compounds of the invention are represented by one of the following formulae (this is evident from formula (I) in which the carrier group (V) is understood to be outside the brackets, so that the designation m does not apply to (V), and all other described structural elements are understood to be within the brackets and thus present m times in the molecule):

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520] wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8.

[0521] According to one embodiment, in the above compounds wherein the solubilizing moiety (S) comprises a C2 polyoxyalkylene group, the number of oxyethylene repeating units (17) may be replaced by 12 to 22, preferably 15 to 19 oxyethylene groups.

[0522] According to one embodiment, in the above compounds, the maleimide attachment to (V) may be replaced by a ring-opening hydrolyzed maleimide attachment. In some cases, where m is at least 2, the compound may comprise a mixture of (closed) maleimide derivatives and ring-opening hydrolyzed maleimide derivatives attached to V, preferably at least 50% of the attachment to V is a ring-opening hydrolyzed maleimide attachment.

[0523] In a preferred embodiment, the compound (LDC) is represented by one of the following formulae:

[0524]

[0525]

[0526]

[0527] wherein m is an integer of 1 to 12, preferably 2 to 10, more preferably 4 to 8.

[0528] As is evident from formula (I), in the above compounds, the carrier group (V) is understood to be outside the brackets, so that the designation m does not apply to (V), whereas all other described structural elements are understood to be within the brackets, so that they are present m times in the molecule.

[0529] According to one embodiment, in the above compounds wherein the solubilizing moiety (S) comprises C2 polyoxyalkylene groups, the number of oxyethylene repeating units (17 or 24) may be replaced by 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups.

[0530] According to one embodiment, in the above compounds, the maleimide attachment to (V) may be replaced by a ring-opening hydrolyzed maleimide attachment. In some cases, where m is at least 2, the compound may comprise a mixture of (closed) maleimide derivatives and ring-opening hydrolyzed maleimide derivatives attached to V, preferably at least 50% of the attachment to V is a ring-opening hydrolyzed maleimide attachment.

[0531] In a preferred embodiment, the compound (LDC) is represented by one of the following formulae:

[0532]

[0533]

[0534] in,

[0535] V is as defined in formula (I);

[0536] Y is a group derived from maleimide and its derivatives such as maleimide derivatives derived from ring-opening hydrolysis, preferably maleimide derived from ring-opening hydrolysis; and

[0537] m is an integer of 1-12, preferably 2-10, more preferably 4-8.

[0538] As is evident from formula (I), in the above compounds, the carrier group (V) is understood to be outside the brackets, so that the designation m does not apply to (V), whereas all other described structural elements are understood to be within the brackets, so that they are present m times in the molecule.

[0539] According to one embodiment, in the above compounds wherein the solubilizing moiety (S) comprises C2 polyoxyalkylene groups, the number of oxyethylene repeating units (17 or 24) may be replaced by 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups.

[0540] According to a preferred embodiment, in the above compounds, Y is represented by any one of the following general formulae (XIIIa) to (XIIIc):

[0541]

[0542] in,

[0543] R 3 Represents -(CH2) n7 -(C=A) n9 -or-(CH2CH2O) n8 -(C=A) n9 -, priority represents priority -(CH2) n7 -(C=A) n9 -,in,

[0544] n7 is 1 to 6, preferably 1 or 2, more preferably 1,

[0545] n8 is 1 to 6, preferably 1,

[0546] n9 is 0 or 1, preferably 1, and

[0547] A is O or S, preferably O;

[0548] wherein the methylene carbon atom is covalently attached to the nitrogen atom of formula (XIIIa)-(XIIIc), and the carbonyl or thiocarbonyl carbon is covalently attached to the nitrogen atom of T (covalently attached to the amino group of the Lys residue originating from T);

[0549] β represents a covalent attachment to V; and

[0550] α' indicates covalent attachment to T (to the amino acid derived from the Lys residue of T).

[0551] In a more preferred embodiment, Y is represented by formula (XIIIb) or (XIIIc), wherein R 3 Preferably, it is of the formula -(CH2) n7 -(C=A) n9 -represented group, wherein n7 is 1 or 2, n9 is 1, and A is O. Most preferably, R 3 It is -CH2-C=O-.

[0552] 3.8 Vector group (V)

[0553] The carrier group (V) in formula (I), (X) and (X') represents a portion of the carrier group that is derived from the carrier group that can interact with the target cell. As used herein, the expression "can interact with the target cell" means that the carrier group can bind, compound or react with a portion (e.g., an antigen or receptor) on the surface of the target cell. This interaction with the target cell can be experimentally verified by methods known in the art, such as by providing a compound of formula (I) with a label (e.g., a fluorescent label), by contacting the compound with a tissue containing the target cell, and by detecting the distribution of the fluorescent label in the tissue (e.g., by fluorescence microscopy). According to the present invention, an increase in fluorescence intensity at the target cell indicates interaction with the target cell. In some preferred embodiments, the carrier group can also cause or contribute to the internalization of the targeted drug conjugate (i.e., the compound of formula (I)) into the target cell.

[0554] In one embodiment, (V) represents a moiety derived from a carrier group selected from the group consisting of an antibody, an antibody fragment, a protein, a peptide and a non-peptide molecule.

[0555] In one embodiment, (V) represents a portion derived from an antibody or antibody fragment (such as a single chain antibody, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment, a chimeric antibody, a chimeric antibody fragment, a domain antibody or a fragment thereof, a cytokine, a hormone, a growth factor, a colony stimulating factor, a neurotransmitter or a nutrient transport molecule).

[0556] In a preferred embodiment, (V) represents a portion derived from the following antibody:

[0557] a monoclonal antibody, preferably a portion derived from an antibody selected from the group consisting of adalimumab, aducanumab, alemtuzumab, atumomab triamine pentaacetic acid, ervantumab, atezolizumab, anetuzumab, avelumab, bapilumab, basiliximab, betumomab, belantamab, beramezumab, bevacizumab, belotuzumab, vebutuximab, vebutuximab, bodalumab, catumaxomab, cemiplimab, cetuximab, simpanumab, clituzumab, crenezumab, tetrazodone, Daclizumab, daratumumab, denosumab, dinutuximab, dotalimumab, durvalumab, edrecolomab, elotuzumab, epavacumab, enrofloxacin, enrofloxacin, icoretuzumab, epratuzumab, epratuzumab-SN-38, edamazumab, gemtuzumab, gemtuzumab ozogamicin, genmab, grofituzumab, gemtuzumab, gonelizumab, ibritumomab tiuxetan, inelizumab, infliximab, inotuzumab, ozogamicin inotuzumab, ipilimumab, isatuximab, ixetuzumab, J591 PSMA-antibodies, labetuzumab, lencanezumab, lantuzumab, moglizumab, motuzumab, necituzumab, nimotuzumab, natalizumab, natalizumab, nasituzumab, nivolumab, omezumab, ofatumumab, olaratumumab, ogovumab, panitumumab, pembrolizumab, pertuzumab, polotuzumab, vepotuzumab, putuzumab, rantumomab, ramucirumab, rituximab, certolizumab pegol, goxatuzumab Monoclonal antibody, ceritumomab, siltuximab, solanezumab, temuzumab, tancitumomab, tetumumab, tipranezumab, tocilizumab, tositumomab, trastuzumab, detrastuzumab, emtansuzumab, TS23, ustekinumab, vedolizumab, futumomab, zegtenemab, zalumab, zalumumab, fragments and derivatives thereof; more preferably selected from atezolizumab, durvalumab, pembrolizumab, rituximab or trastuzumab; or

[0558] - Antibody fragments introduced into Fc-fusion proteins, said antibody fragments are preferably selected from the group consisting of belatacept, aflibercept, Ziv-aflibercept, dulaglutide, lelocept, romiplostim, abatacept and alefacept.

[0559] In a preferred embodiment, (V) represents a portion derived from an anti-HER2, anti-CD37, anti-PDL1 or anti-EGFR antibody, preferably derived from an antibody selected from trastuzumab, pembrolizumab, natalizumab, atezolizumab, durvalumab, panitumumab, avelumab and cetuximab, more preferably derived from natalizumab, trastuzumab and cetuximab, most preferably derived from natalizumab or trastuzumab.

[0560] As used herein, the term "natuximab" (also referred to herein as K7153A) refers to the antibody huCD37-3 (version 1.0) described in WO 2019 / 229677 (incorporated herein by reference). On the other hand, natuximab is characterized as the monoclonal antibody huCD37-3v1.0 in WO2011 / 112978, which specifically describes its heavy chain (SEQ ID NO: 90) and light chain (SEQID 107). In a further aspect, the antibody comprises CDRs represented by SEQ ID NOs: 2-7 in Tables 1 and 2 of WO 2019 / 229677, VH of SEQ ID NO: 8 in Table 3, and VL of SEQ ID NO: 10 in Table 4.

[0561] In some specific aspects, natalizumab may comprise:

[0562] · The full-length light chain of Seq No 11 in Table 5 of WO 2019 / 229677,

[0563] · The full-length heavy chain of Seq No. 12 in Table 6 of WO 2019 / 229677,

[0564] · The full-length light chain of Seq No. 11 in Table 5 and the full-length heavy chain of Seq No. 12 in Table 6 of WO 2019 / 229677,

[0565] · A light chain or light chain variable region having the same amino acid sequence as the amino acid sequence encoded by the recombinant plasmid DNA phuCD37-3LC (ATCC deposit number PTA-10722, deposited with ATCC on March 18, 2010),

[0566] · A heavy chain or heavy chain variable region comprising an amino acid sequence identical to the amino acid sequence encoded by the recombinant plasmid DNA phuCD37-3HCv.1.0 (ATCC deposit number PTA-10723, deposited with ATCC on March 18, 2010),

[0567] · A light chain or light chain variable region comprising an amino acid sequence identical to the amino acid sequence encoded by the recombinant plasmid DNA phuCD37-3LC (PTA-10722), and a heavy chain or heavy chain variable region comprising an amino acid sequence identical to the amino acid sequence encoded by the recombinant plasmid DNA phuCD37-3HCv.1.0 (PTA-10723),

[0568] · It includes (i) a VL-CDR containing the same amino acid sequence as the VL-CDR encoded by the recombinant plasmid DNA phuCD37-3LC (PTA-10722) and (ii) a VH-CDR containing the same amino acid sequence as the VH-CDR encoded by the recombinant plasmid DNA phuCD37-3HCv.1.0 (PTA-10723).

[0569] In one embodiment, (V) represents a moiety derived from an anti-CD37 antibody, preferably natalizumab or an anti-HER2 antibody, preferably trastuzumab.

[0570] In another embodiment, (V) represents a moiety derived from an anti-CD37 antibody (preferably natalizumab) or an anti-HER2 antibody (preferably trastuzumab), and (D) represents a moiety derived from an anti-tumor agent, and preferably a moiety derived from a drug selected from the group consisting of amanitin, duocarmycin, auristatin, auristatin F (AF), monomethyl auristatin F (MMAF), maytansine, maytansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, paclitaxel, daunorubicin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepine (PBD) and its dimer, indenobenzodiazepine (IBD) and its dimer, or its radioactive isotope and / or pharmaceutically acceptable salt.

[0571] In a preferred embodiment, (V) represents a portion derived from an anti-CD37 antibody (preferably natalizumab) or an anti-HER2 antibody (preferably trastuzumab), and (D) represents a portion derived from a drug selected from auristatin, MMAF, exatecan, maytansinoids, DM1, and DM4. More preferably, (V) represents a portion derived from an anti-CD37 antibody (preferably natalizumab) or an anti-HER2 antibody (preferably trastuzumab), and (D) represents a portion derived from DM1 or M4.

[0572] In another preferred embodiment, (V) represents a moiety derived from an anti-CD37 antibody (preferably natalizumab) or an anti-HER2 antibody (preferably trastuzumab), (D) represents a moiety derived from DM1, and the compound is represented by one of the following formulae:

[0573]

[0574] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; wherein the number of oxyethylene repeating units (17) can be replaced by 12 to 30, preferably from 14 to 25, more preferably from 15 to 19 oxyethylenes; and / or wherein the maleimide attachment to (V) can be replaced by a maleimide that is ring-opening and hydrolyzed.

[0575] As is evident from formula (I), in the above compounds, the carrier group (V) is understood to be outside the brackets, so that the designation m does not apply to (V), whereas all other described structural elements are understood to be within the brackets, so that they are present m times in the molecule.

[0576] According to one embodiment, in the above compounds, the maleimide attachment to (V) may be replaced by a ring-opening hydrolyzed maleimide attachment. In some cases, where m is at least 2, the compound may comprise a mixture of (closed) maleimide derivatives and ring-opening hydrolyzed maleimide derivatives attached to V, preferably at least 50% of the attachment to V is a ring-opening hydrolyzed maleimide attachment.

[0577] In another preferred embodiment, (V) represents a moiety derived from an anti-CD37 antibody (preferably natalizumab) or an anti-HER2 antibody (preferably trastuzumab), (D) represents a moiety derived from DM1, and the compound is represented by one of the following formulae:

[0578]

[0579] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; wherein Y is represented by any one of formulae (XIIIa) to (XIIIc), preferably by formula (XIIIb) or (XIIIc); and / or wherein the number of oxyethylene repeating units (17) may be replaced by 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups.

[0580] In a more preferred embodiment, (V) represents a moiety derived from an anti-CD37 antibody, preferably natalizumab, or an anti-HER2 antibody, preferably trastuzumab, (D) represents a moiety derived from DM1, and the compound is represented by the following formula:

[0581]

[0582] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; wherein the number of oxyethylene repeating units (17) can be replaced by 12 to 30, preferably from 14 to 25, more preferably from 15 to 19 oxyethylenes; and / or wherein the maleimide attachment to (V) can be replaced by a maleimide that is ring-opening and hydrolyzed.

[0583] In an even more preferred embodiment, (V) represents a moiety derived from an anti-CD37 antibody (preferably natalizumab) or an anti-HER2 antibody (preferably trastuzumab), (D) represents a moiety derived from DM1, and the compound is represented by the following formula:

[0584]

[0585] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; wherein Y is represented by any one of formulae (XIIIa) to (XIIIc), preferably by formula (XIIIb) or (XIIIc); and / or wherein the number of oxyethylene repeating units (17) may be replaced by 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups.

[0586] As is evident from formula (I), in the above compounds, the carrier group (V) is understood to be outside the brackets, so that the designation m does not apply to (V), whereas all other described structural elements are understood to be within the brackets, so that they are present m times in the molecule.

[0587] According to one embodiment, in the above compounds, the maleimide attachment to (V) may be replaced by a ring-opening hydrolyzed maleimide attachment. In some cases, where m is at least 2, the compound may comprise a mixture of (closed) maleimide derivatives and ring-opening hydrolyzed maleimide derivatives attached to V, preferably at least 50% of the attachment to V is a ring-opening hydrolyzed maleimide attachment.

[0588] In another preferred embodiment, (V) represents a portion derived from a peptide capable of interacting with a target of interest. Non-limiting examples of peptides include somatostatin or its analogs, such as octreotide, Angiopep-2, gastrin-releasing peptide, transferrin-derived peptides, derivatives of neuropeptide Y, RGD peptides, α-melanocyte stimulating hormone peptide analogs, vasoactive intestinal peptide, neurotensin and luteinizing hormone-releasing hormone (LHRH) analogs.

[0589] According to another preferred embodiment, (V) represents a moiety derived from a non-peptide molecule such as folic acid, hyaluronic acid, neurotensin receptor 1 (NRT1) antagonists such as SR 142948A derivatives and ligands of prostate specific membrane antigen (PSMA) such as PSMA-617 and PSMA-11.

[0590] According to one embodiment, the target cell is selected from tumor cells, virus-infected cells, microbial-infected cells, parasite-infected cells, cells involved in autoimmune diseases, activated cells, myeloid cells, lymphoid cells, melanocytes and infectious agents including bacteria, viruses, mycobacteria, fungi.

[0591] According to a preferred embodiment, the target cell is any tumor cell from a solid or liquid tumor, including but not limited to lymphoma cells, myeloma cells, myeloid cells, lymphoid cells, renal cancer cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell cancer cells, small cell lung cancer cells, testicular cancer cells, or any cell that grows and divides at an uncontrolled and accelerated rate to cause cancer.

[0592] 4. Pharmaceutical Composition

[0593] The compounds of the present invention can be provided in the form of pharmaceutical compositions for human or animal use in human and veterinary medicine. Such compositions generally comprise a therapeutically effective amount of LDC according to the present invention or a pharmaceutically acceptable salt thereof, and one or more components selected from carriers, diluents and other excipients.

[0594] In one embodiment, the pharmaceutical composition comprises a mixture of LDCs according to the invention.

[0595] In a more specific embodiment, the pharmaceutical composition comprises a mixture of LDCs according to the invention, wherein said LDCs (compounds of formula I) comprise a (closed) maleimide and / or a ring-opening hydrolyzed maleimide attachment to V (as described above).

[0596] The ratio of the (closed) maleimide derivative (A) attached to V and the ring-opening hydrolyzed maleimide derivative (B) (based on the total attached amount of total maleimide in the composition) can be A:B / 0-50:50-100%, preferably A:B / 10-40:60-90%, more preferably A:B / 15-35:65-85%, and most preferably about A:B / 30:70%.

[0597] The respective ratios of the closed maleimide derivative and the ring-opened hydrolyzed maleimide derivative (and thus the closed (A) and the ring-opened (B) maleimide attachments) in the composition can be determined by MS techniques such as Tof or Orbitrap analysis of reduced LDC (compound of formula (I)). An example of a detailed scheme can be obtained in Chapter 6.d. of Chem. Eur. J. 2019, 25, 8208-8213.

[0598] Suitable carriers, diluents and other excipients for pharmaceutical compositions are well known in the art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (Gennaro AR, 1985). Carriers, diluents and / or other excipients can be selected according to the intended route of administration and pharmaceutical practice. The pharmaceutical composition may contain a diluent and / or other excipient as a carrier, or in addition thereto, any one or more suitable binders, one or more lubricants, one or more suspending agents, one or more coating agents, one or more solubilizing agents.

[0599] The therapeutically effective amount can be determined by a physician on a routine basis. The specific dosage level and dosing frequency of any particular subject / patient may vary and depend on a variety of factors, including the activity of the specific drug compound used, the metabolic stability and duration of action of the compound, the patient's age, weight, general health, sex, diet, mode and time of administration, excretion rate, drug combination, severity of the specific illness, and the individual being treated. The physician will take these factors into account when determining the therapeutically effective dose.

[0600] 5. Use of LDC or its composition in a method for preventing or treating a disease

[0601] The compounds of the present invention can be used to treat diseases. Treatment can be therapeutic and / or prophylactic treatment, the purpose of which is to prevent, reduce or stop undesirable physiological changes or symptoms. In some aspects, treatment can prolong the survival of the subject compared to the expected survival if not treated.

[0602] The disease treated by LDC can be any disease that benefits from the treatment, including chronic and acute conditions or diseases and those pathological conditions that cause the condition. In some aspects, the disease is a tumor disease, such as a cancer that can be treated by targeted destruction of tumor cells. Non-limiting examples of cancers that can be treated include benign and malignant tumors (solid or liquid); lymphatic system malignancies, such as non-Hodgkin's lymphoma (NHL), such as diffuse large B-cell lymphoma (DLBCL), and breast cancer, ovarian cancer, gastric cancer, endometrial cancer, salivary gland cancer, lung cancer, kidney cancer, colon cancer, thyroid cancer, pancreatic cancer, prostate cancer or bladder cancer, and bone cancer and bone marrow cancer, such as acute myeloid leukemia (AML). The disease can be a neuronal disease, a glial disease, an astrocyte disease, a hypothalamic disease or other glandular disease, a macrophage disease, an epithelial disease, an interstitial disease and a blastocyst cavity disease; or an inflammatory disease, angiogenic disease or an immune disease. An exemplary disease is a solid malignant tumor, and another exemplary disease is a liquid malignant tumor.

[0603] According to one embodiment, the compounds of the invention or compositions thereof are used in a method for treating or preventing cancer, autoimmune diseases or inflammatory diseases and / or infectious diseases, for example by administering a therapeutically effective amount of the compounds of the invention or compositions thereof to a patient in need thereof.

[0604] Non-limiting examples of autoimmune, inflammatory and / or infectious diseases include rheumatoid arthritis, multiple sclerosis, type I diabetes, idiopathic inflammatory myopathy, systemic lupus erythematosus (SLE), myasthenia gravis, Graves' disease, dermatomyositis, polymyositis, Crohn's disease, ulcerative colitis, gastritis, Hashimoto's thyroiditis, asthma, psoriasis, psoriatic arthritis, dermatitis, systemic sclerosis and sclerosis, inflammatory bowel disease (IBD), respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, atherosclerosis, leukocyte adhesion deficiency, Raynaud's syndrome, Sjogren's syndrome, Reiter's disease, Behcet's disease, immune complex nephropathy, and inflammatory bowel disease. complexnephritis), IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupus nephritis, atopic dermatitis, pemphigus vulgaris, opsoclonus-myoclonus syndrome, pure red cell aplasia, mixed cryoglobulinemia, ankylosing spondylitis, hepatitis C-associated cryoglobulinemic vasculitis, chronic focal encephalitis, bullous pemphigoid, hemophilia A, membranoproliferative glomerulonephritis, dermatomyositis in adults and juveniles, polymyositis in adults, chronic urticaria, primary biliary cirrhosis, neuromyelitis optica, Graves' thyroid disease, bullous pemphigoid, membranoproliferative glomerulonephritis, Churg-Strauss syndrome, juvenile-onset diabetes mellitus onset diabetes), hemolytic anemia, atopic dermatitis, systemic sclerosis, Sjögren's syndrome and glomerulonephritis, dermatomyositis, ANCA, aplastic anemia, autoimmune hemolytic anemia (AIHA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, Castleman syndrome, Goodpasture syndrome, solid organ transplant rejection, graft-versus-host disease (GVHD), autoimmune hepatitis, lymphoid interstitial pneumonitis, bronchiolitis obliterans (non-transplant), Guillain-Barré syndrome, large vessel vasculitis, giant cell (Takayasu's) arteritis, medium vessel vasculitis, Kawasaki disease, polyarteritis nodosa. Wegener's granulomatosis, microscopic polyangiitis (MPA), Omenn syndrome, chronic renal failure, acute infectious mononucleosis, HIV, hepatitis, herpes, and bacterial infections. Furthermore, reference is made to the medical indications mentioned in WO 2012 / 135740 A, for example in paragraphs

[0004] and

[0039] of this document.

[0605] In a specific embodiment, the compounds of the invention or compositions thereof are used in a method for treating or preventing cancer of the bone and bone marrow, preferably acute myeloid leukemia (AML). Preferably, the compound is a compound of the invention, wherein (V) represents a moiety derived from an anti-CD37 antibody, preferably natalizumab.

[0606] In another specific embodiment, the compound of the present invention or a composition thereof is used in a method for treating or preventing lymphoma (preferably NHL, more preferably DLBCL). Preferably, the compound is a compound of the present invention, wherein (V) represents a moiety derived from an anti-CD37 antibody (preferably natalizumab).

[0607] In a more specific embodiment, the compound of the present invention or a composition thereof is used in a method for treating or preventing cancer of the bone and bone marrow, preferably acute myeloid leukemia (AML), wherein the compound is a compound of the present invention, wherein (V) represents a moiety derived from an anti-CD37 antibody, preferably natalizumab, and wherein (D) is a moiety derived from an anti-tumor agent, and preferably a moiety derived from a drug selected from the group consisting of amanitin, duocarmycin, auristatin, auristatin F (AF), monomethyl auristatin F (MMAF), maytansine, maytansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, paclitaxel, daunorubicin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepine (PBD) and its dimer, indenobenzodiazepine (IBD) and its dimer, or its radioactive isotope and / or pharmaceutically acceptable salt.

[0608] In an even more specific embodiment, the compounds of the present invention or compositions thereof are used in a method for treating or preventing bone cancer and bone marrow cancer, preferably acute myeloid leukemia (AML), wherein the compound is a compound of the present invention, wherein (V) represents a moiety derived from an anti-CD37 antibody (preferably natalizumab), and wherein (D) is a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1 and DM4; more preferably a moiety derived from DM1 or DM4.

[0609] In a preferred embodiment, the compound of the present invention or a composition thereof is used in a method for treating or preventing cancer of the bone and bone marrow, preferably acute myeloid leukemia (AML), wherein the compound is a compound of the present invention, wherein (V) represents a moiety derived from an anti-CD37 antibody (preferably natalizumab), (D) represents a moiety derived from DM1, and the compound is represented by one of the following formulae:

[0610]

[0611] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; wherein the number of oxyethylene repeating units (17) can be replaced by 12 to 30, preferably from 14 to 25, more preferably from 15 to 19 oxyethylene groups; and / or wherein the maleimide attachment to (V) can be replaced by a ring-opening hydrolyzed maleimide,

[0612] And preferably the compound is represented by the following formula:

[0613]

[0614] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; wherein the number of oxyethylene repeating units (17) can be replaced by 12 to 30, preferably from 14 to 25, more preferably from 15 to 19 oxyethylenes; and / or wherein the maleimide attachment to (V) can be replaced by a maleimide that is ring-opening and hydrolyzed.

[0615] As is evident from formula (I), in the above compounds, the carrier group (V) is understood to be outside the brackets, so that the designation m does not apply to (V), whereas all other described structural elements are understood to be within the brackets, so that they are present m times in the molecule.

[0616] In a more preferred embodiment, the compound of the present invention or a composition thereof is used in a method for treating or preventing cancer of the bone and bone marrow (preferably acute myeloid leukemia (AML)), wherein the compound is a compound of the present invention, wherein (V) represents a moiety derived from an anti-CD37 antibody (preferably natalizumab), (D) represents a moiety derived from DM1, and the compound is represented by the following formula:

[0617]

[0618] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; and wherein the number of oxyethylene repeating units (17) may be replaced by 12 to 30, preferably from 14 to 25, more preferably from 15 to 19 oxyethylene groups.

[0619] As is evident from formula (I), in the above compounds, the carrier group (V) is understood to be outside the brackets, so that the designation m does not apply to (V), whereas all other described structural elements are understood to be within the brackets, so that they are present m times in the molecule.

[0620] According to one embodiment, in the above compounds, the maleimide attachment to (V) may be replaced by a ring-opening hydrolyzed maleimide attachment. In some cases, where m is at least 2, the compound may comprise a mixture of (closed) maleimide derivatives and ring-opening hydrolyzed maleimide derivatives attached to V, preferably at least 50% of the attachment to V is a ring-opening hydrolyzed maleimide.

[0621] In a preferred embodiment, the compound of the present invention or a composition thereof is used in a method for treating or preventing bone cancer and bone marrow cancer (preferably acute myeloid leukemia (AML)), wherein the compound is a compound of the present invention, wherein (V) represents a moiety derived from an anti-CD37 antibody (preferably natalizumab), (D) represents a moiety derived from DM1, and the compound is represented by one of the following formulae:

[0622]

[0623] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; wherein Y is represented by any one of formulae (XIIIa) to (XIIIc), preferably by formula (XIIIb) or (XIIIc); and / or wherein the number of oxyethylene repeating units (17) can be replaced by 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups.

[0624] In a more preferred embodiment, the compound of the present invention or a composition thereof is used in a method for treating or preventing cancer of the bone and bone marrow, preferably acute myeloid leukemia (AML), wherein the compound is a compound of the present invention, wherein (V) represents a moiety derived from an anti-CD37 antibody (preferably natalizumab), (D) represents a moiety derived from DM1, and the compound is represented by the following formula:

[0625]

[0626] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; wherein Y is represented by any one of formulae (XIIIa) to (XIIIc), preferably represented by formula (XIIIb) or (XIIIc); and / or wherein the number of oxyethylene repeating units (17) may be replaced by 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups.

[0627] As is evident from formula (I), in the above compounds, the carrier group (V) is understood to be outside the brackets, so that the designation m does not apply to (V), whereas all other described structural elements are understood to be within the brackets, so that they are present m times in the molecule.

[0628] The compounds / molecules of the present invention can be administered to a subject (e.g., a patient) at any therapeutically effective dose. The therapeutically effective dose may depend on the characteristics of the patient, such as body size and body weight. The molecule may be administered to a subject (e.g., a patient) at one time or by a series of treatments. Depending on the type and severity of the disease, a drug of about 0.1 μg / kg to 1 mg / kg may be used as an initial candidate dose for the first human trial, for example, by one or more separate administrations, or by continuous infusion. Typical dosage ranges for daily, weekly (QW), biweekly (Q2W), three-weekly (Q3W), or monthly administrations may be about 0.1 mg / kg to 50 mg / kg of patient weight or more, or about 0.5 to about 25 mg / kg of patient weight. The subject to whom the compound / molecule is administered may be a patient in need, i.e., a patient in need of treatment, such as a patient suffering from cancer (e.g., AML), an autoimmune disease, or an infectious disease.

[0629] When treating cancer, the observed therapeutic effect may be a decrease in the number of cancer cells; a decrease in tumor size; an inhibition or delay in the infiltration of cancer cells into peripheral organs; an inhibition of tumor growth; and / or amelioration of one or more symptoms associated with cancer.

[0630] Administration (delivery) routes include one or more of the following: oral (e.g., tablets, capsules, ingestible solutions), topical, mucosal (e.g., nasal sprays, aerosols for inhalation), nasal, parenteral (e.g., injectable forms), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, intraocular (including intravitreal or intracameral), transdermal, rectal, oral, vaginal, epidural, sublingual. According to a preferred embodiment, the compounds of the present invention are administered by injection, such as parenteral, intravenous, subcutaneous, intramuscular, transdermal administration.

[0631] According to another embodiment, the compounds of the present invention are used in methods for treating or preventing cancer, autoimmune diseases or inflammatory diseases and / or infectious diseases, and are administered simultaneously with one or more other therapeutic agents such as chemotherapeutic agents, radiotherapy, immunotherapeutic agents, autoimmune disease agents, anti-infective agents or one or more other compounds of formula (I). Additional therapeutic agents may also be administered before or after the compounds of the present invention.

[0632] In a more specific embodiment, the compound of the present invention is used in a method for treating or preventing cancer (preferably lymphoma or cancer of the blood and bone marrow), wherein the compound of the present invention is administered simultaneously with an anti-CD20 antibody (preferably rituximab), and wherein the compound is a compound of the present invention, wherein (V) represents a moiety derived from an anti-CD37 antibody (preferably rituximab), (D) represents a moiety derived from DM1, and the compound is represented by the following formula:

[0633]

[0634] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; and wherein the number of oxyethylene repeating units (17) may be replaced by 12 to 30, preferably from 14 to 25, more preferably from 15 to 19 oxyethylenes; and wherein the maleimide attachment to V may be replaced by a maleimide attachment by ring-opening hydrolysis; and

[0635] Preferably, the lymphoma is DLBCL and the cancer of the bone and bone marrow is AML.

[0636] In a preferred embodiment, the compound of the present invention is used in a method for treating or preventing cancer (preferably lymphoma or cancer of the blood and bone marrow), wherein the compound of the present invention is administered simultaneously with an anti-CD20 antibody (preferably rituximab), and wherein the compound is a compound of the present invention, wherein (V) represents a moiety derived from an anti-CD37 antibody (preferably narasimab), (D) represents a moiety derived from DM1, and the compound is represented by the following formula:

[0637]

[0638] wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; wherein Y is represented by any one of formulae (XIIIa) to (XIIIc), preferably by formula (XIIIb) or (XIIIc); and / or wherein the number of oxyethylene repeating units (17) may be replaced by 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylenes; and wherein preferably the lymphoma is DLBCL and the cancer of the bone and bone marrow is AML.

[0639] As is evident from formula (I), in the above compounds, the carrier group (V) is understood to be outside the brackets, so that the designation m does not apply to (V), whereas all other described structural elements are understood to be within the brackets, so that they are present m times in the molecule.

[0640] As used herein, the term "anti-CD20 antibody" or "antibody that binds to CD20" refers to an antibody that is capable of binding to CD20 with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent in targeting CD20. The extent of binding of the anti-CD20 antibody to unrelated, non-CD20 proteins can be less than about 10% of the extent of binding of the antibody to CD20, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to CD20 has a dissociation constant (Kd) of ≤1 μμ, ≤1100 nM, ≤110 nM, ≤11 nM, or ≤1.1 nM.

[0641] Similarly, as used herein, the terms "anti-CD37 antibody", "anti-HER2 antibody", "anti-PDL1 antibody" and "anti-EGFR antibody" (or "antibody that binds to CD37", "antibody that binds to HER2", "antibody that binds to PDL1" and "antibody that binds to EGFR") refer to antibodies that are capable of binding to CD37, HER2, PDL1 or EGFR, respectively, with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent in targeting the corresponding antigen CD37, HER2, PDL1 or EGFR. The degree of binding of the anti-CD37, anti-HER2, anti-PDL1 or anti-EGFR antibody to each unrelated non-CD37, non-HER2, non-PDL1 or non-EGFR protein may be less than about 10% of the binding of the antibody to CD37, HER2, PDL1 or EGFR, as measured by, for example, radioimmunoassay (RIA). In certain embodiments, an antibody that binds to any of CD37, HER2, PDL1, and EGFR has a dissociation constant (Kd) of ≤1 μM, ≤1100 nM, ≤110 nM, ≤11 nM, or ≤1.1 nM.

[0642] According to one embodiment, in the above compounds, the maleimide attachment to (V) may be replaced by a ring-opening hydrolyzed maleimide attachment. In some cases, where m is at least 2, the compound may comprise a mixture of (closed) maleimide derivatives and ring-opening hydrolyzed maleimide derivatives attached to V, preferably at least 50% of the attachment to V is a ring-opening hydrolyzed maleimide.

[0643] Rituximab may be administered prior to, subsequent to, or concurrently with the administration of the compound of the invention.

[0644] 6. Compounds of formula (XI), kits for modifying molecules capable of interacting with target cells, and methods for modifying molecules capable of interacting with target cells

[0645] In some aspects, the present invention relates to compounds that can be used to modify carrier molecules, such as antibodies, that are capable of interacting with target cells as described above. Therefore, the present invention relates to compounds represented by general formula (XI):

[0646]

[0647] in,

[0648] D, X, L, T, S and n have the same meanings as described above for the compounds of formula (I); and

[0649] Y' represents a moiety comprising a conjugating group capable of forming a covalent attachment with a molecule capable of interacting with a target cell (V'), such as a monoclonal antibody or an antibody fragment optionally incorporated into an Fc-fusion protein.

[0650] According to one embodiment, Y' in formula (XI) is a moiety comprising a conjugated group selected from:

[0651] - an optionally substituted maleimide, which is preferably capable of reacting with one or two thiol groups of (V'), wherein said thiol groups of (V') may each independently and optionally be substituted or protected, for example by monomethoxytrityl,

[0652] - an optionally substituted haloacetamide, preferably capable of reacting with the thiol group of (V'),

[0653] - an ester, which is preferably capable of reacting with the side chain of the amino acid (e.g., Lys) of (V′), such as an acyl halide, an N-hydroxysuccinimide ester (the structure is shown below, on the left hand side, σ represents the covalent attachment to the rest of the compound of formula (XI)) or a phenolic ester (the structure is shown below, on the right hand side, σ represents the covalent attachment to the rest of the compound of formula (XI), each R is independently selected from H, F, NO2 and CN),

[0654]

[0655] - a carbonate which is preferably capable of reacting with the side chain of the amino acid (eg Lys) of (V'), such as a haloformate or a carbonate comprising a leaving group, such as N-hydroxysuccinimide or a phenol derivative,

[0656] - an isocyanate or isothiocyanate, which is preferably capable of reacting with the side chain of an amino acid (eg Lys) of (V'),

[0657] - an azide, which is preferably capable of reacting with an alkyne group contained in the molecule (V');

[0658] - an alkyne, which is preferably capable of reacting with the azide group contained in the molecule (V'); and

[0659] - an amino group, such as a primary or secondary amino group, which is preferably capable of reacting with a molecule (V'), such as an antibody, the presence of an enzyme such as transglutaminase.

[0660] According to a preferred embodiment, the compound has a structure represented by the general formula (XII) or (XII'):

[0661]

[0662]

[0663] in,

[0664] Axx in formula (XII) and (XII') has the same meaning as in formula (X) and (X') above; Ayy in formula (XII) has the same meaning as in formula (X) above; Ayy in formula (XII') has the same meaning as in formula (X') above, and Y' has the same meaning as described above with respect to formula (XI); and

[0665] D, Dxx, Dyy, X, T, S, Z and n have the same meanings as described above for the Ligand-Drug-Conjugate.

[0666] According to a preferred embodiment, at least one, for example two, three, four, five, six or seven of D, Dxx, Dyy, X, T, S and Z in formula (XII) and (XII') are defined as follows:

[0667] (a) D is a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4;

[0668] (b) Dxx is a moiety derived from an amino acid selected from the group consisting of Phe, Val, Tyr, homo-Phe and Ala, preferably selected from Phe or Val;

[0669] (c) Dyy is a covalent bond or a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap and Dab, preferably selected from Arg or Cit;

[0670] (d) X is a group of formula (III) wherein n2 is 1 or 2, or a group represented by any one of formulae (IVa) to (IVz), preferably a group represented by any one of formulae (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm);

[0671] (e) T is a group of formula (VII), (VIII) or (IX);

[0672] (f) S is a moiety of formula (V); and

[0673] (g) Z is -OH.

[0674] According to other preferred embodiments, each Dyy-Dxx-Axx-Ayy in formula (XII) is independently selected from wherein Dyy is covalently bound Arg-Lys-Phe, wherein Dyy is covalently bound Arg-Lys-homo-Phe, wherein Dyy is covalently bound Arg-Lys-Tyr, wherein Dyy is covalently bound Cit-Lys-Phe, wherein Dyy is covalently bound Cit-Lys-Tyr, wherein Dyy is covalently bound Arg-Lys-homo-Tyr, wherein Dyy is covalently bound Cit-Lys-homo-Tyr, Phe-Cit-Lys-Phe, Phe-Cit-Lys-Tyr, Phe-Arg-Lys-Tyr, Phe-Cit -Lys-homo-Tyr, Phe-Lys-Lys-Phe, homo-Phe-Arg-Lys-Phe, homo-Phe-Cit-Lys-Ty; and each Dyy-Dxx-Ayy-Axx in formula (XII') is independently selected from Arg-Phe-Lys wherein Dyy is covalently bonded, Arg-Ser-Lys wherein Dyy is covalently bonded, Cit-Phe-Lys wherein Dyy is covalently bonded, Cit-Ser-Lys wherein Dyy is covalently bonded, Cit-homo-Phe-Lys wherein Dyy is covalently bonded, Phe-Cit-Phe-Lys, homo-Phe-Cit-Phe-Lys and Phe-Arg-Phe-Lys.

[0675] According to yet another preferred embodiment, the compound has a structure represented by one of the following formulae:

[0676]

[0677] wherein D, X, T, S, Y', Z and n have the same meanings as described above.

[0678] According to a preferred embodiment, at least one, for example two, three, four or five, of D, X, T, S and Z in the above formula are defined as follows:

[0679] (a) D is a moiety derived from a drug selected from auristatin, AF, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4;

[0680] (d) X is a group of formula (III) wherein n2 is 1 or 2, or is a group represented by any one of formulae (IVa) to (IVz), preferably a group represented by any one of formulae (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm);

[0681] (e) T is a group of formula (VII), (VIII) or (IX);

[0682] (f) S is a moiety of formula (V); and

[0683] (g) Z is -OH.

[0684] In one embodiment, the compound is represented by one of the following formulae:

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693] In some aspects, the present invention relates to a kit comprising a compound as described above (i.e., a compound according to formula (XI), (XII) or (XII')) and a buffer, which can be used to modify a carrier molecule capable of interacting with a target cell, such as an antibody or a fragment thereof, the antibody fragment being optionally incorporated into an Fc-fusion protein, in particular for modifying a therapeutic antibody.

[0694] Compound and buffer (together forming a test kit) can be present alone in, for example, a separate primary container (which can be transported to consumers in a single box), which can be stored for a long time without degradation. For a given amount of antibody to be modified or its fragment, compound and buffer can be prepared and proportioned. In some respects, the compound of the present invention can be in solid form (for example, as a lyophilized powder, or non-covalently adsorbed or covalently bound to a solid phase matrix, as further described below), or in a solution form in a suitable solvent (such as a polar aprotic solvent miscible with water (for example, DMF, DMSO)), which can be mixed with buffer shortly before the antibody or antibody fragment is modified.

[0695] The buffer used in the kit of the present invention is not particularly limited. Preferably, the pH value of the buffer is 6.0 to 10, more preferably 6.5 to 8.0. The buffer can be selected from, for example, 2-bis(2-hydroxyethyl)aminoacetic acid (Bicine), carbonate-bicarbonate, tris(hydroxymethyl)methylaminopropanesulfonic acid (TAPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

[0696] In some aspects, the compounds of the invention can be used in a method of modifying a carrier molecule (eg, an antibody) capable of interacting with a target cell. Thus, the method produces a ligand-drug-conjugate of formula (I) as described above.

[0697] In one embodiment, the method includes the step of reacting (contacting) a molecule capable of interacting with a target cell, such as a monoclonal antibody or an antibody fragment optionally introduced into an Fc fusion protein. The reaction mixture can be purified by techniques known in the art, such as diafiltration techniques or gel permeation chromatography using a suitable solvent. Examples of suitable stationary phases for separating clean conjugates include polyacrylamide gels, such as Bio- P-30 and cross-linked dextran, such as or

[0698] The method can be applied to any molecule that can interact with the target cell. Preferably, the method is applied to a molecule selected from antibodies, antibody fragments, proteins, peptides and non-peptide molecules. In one embodiment, the method is applied to a monoclonal antibody (mAb) or an antibody fragment introduced into an Fc fusion protein, preferably to an antibody selected from the group consisting of adalimumab, aducanumab, alemtuzumab, atumomab triamine pentaacetic acid, ervantumab, atezolizumab, anetomab, avelumab, bapilumab, basiliximab, betumomab, belantamab, beramezumab, bevacizumab, belotuzumab, vebutin, vebutin, bodalumab, catumaxomab, cimiprilimumab, cetuximab, simpanumab, cletoxema ... ezetimibe, crenetuzumab, tetrazodone, daclizumab, daratumumab, denosumab, dinutuximab, dotalimumab, durvalumab, edrecolomab, elotuzumab, epavacumab, enrofloxacin, enrofloxacin, icoretuzumab, epratuzumab, epratuzumab-SN-38, edatuzumab, gemtuzumab, gemtuzumab ozogamicin), genmab, gemtuzumab, grofituzumab, gonelizumab, ibritumomab tiuxetan, inelizumab, infliximab, inotuzumab, ozogamicin inotuzumab, ipilimumab, isatuximab, ixetuzumab, J591 PSMA-antibodies, labetuzumab, lencanezumab, lantuzumab, moglizumab, motuzumab, necituzumab, nimotuzumab, natalizumab, natalizumab, nasituximab, nivolumab, omezumab, ofatumumab, olaratumumab, ogavuzumab, panitumumab, pembrolizumab, pertuzumab, polotuzumab, vepotuzumab, putuzumab, rantumomab, ramucirumab, rituximab, certolizumab pegol, gosartumumab, ceruleinumab, siltuximab, solanezumab, tancitumomab, tuximab , titumumab, tipranezumab, tocilizumab, tositumomab, trastuzumab, detrastuzumab, emtansine trastuzumab, TS23, ustekinumab, vedolizumab, futumomab, zagetelumab, zalumab, zalumab, fragments and derivatives thereof; more preferably selected from atezolizumab, durvalumab, pembrolizumab, rituximab or trastuzumab; or an antibody fragment for introduction into an Fc fusion protein, preferably selected from belatacept, aflibercept, ziv-aflibercept, dulaglutide, lelocept, romiplostim, abatacept and afacept.

[0699] In one embodiment, the method is applied to an anti-HER2, anti-CD37, anti-PDL1 or anti-EGFR antibody, preferably to an antibody selected from trastuzumab, pembrolizumab, natalizumab, atezolizumab, durvalumab, avelumab, panitumumab and cetuximab, more preferably to an antibody selected from natalizumab, trastuzumab and cetuximab, most preferably to natalizumab and trastuzumab.

[0700] 7. Preparation of the compounds of the present invention

[0701] Hereinafter, methods for preparing linkers, drug-linkers and ligand-drug-conjugates are provided. The compounds of the present invention can be synthesized by standard organic chemical reactions or solid phase peptide synthesis (SPPS) based on Fmoc (including peptide coupling and convergence strategies in solution and on resin). The introduction of various maleimide derivatives and the subsequent chemical selective connection with the part derived from the carrier group are also illustrated below. The general strategies and methods that can be used to prepare the compounds of the present invention are well known to those skilled in the art. 8. Examples

[0702] 8.1 List of abbreviations used in the examples:

[0703] Ac:Acetyl

[0704] ADC: Antibody-drug conjugate

[0705] ACN: Acetonitrile

[0706] AMAS: N-α-Maleimidoacetoxysuccinimide ester

[0707] AML: Acute myeloid leukemia

[0708] Arg: Arginine

[0709] Bn: benzyl

[0710] Boc: tert-butyloxycarbonyl

[0711] Bu: Butyl

[0712] Citrulline

[0713] DAR: Drug-Antibody Ratio

[0714] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

[0715] DCM: dichloromethane

[0716] DIEA: Diisopropylethylamine

[0717] DLBCL: diffuse large B-cell lymphoma

[0718] DM1: Maytansine

[0719] DMF: dimethylformamide

[0720] DMSO: dimethyl sulfoxide

[0721] DOC: Degree of Conjugation

[0722] DPBS: Dulbecco's phosphate buffered saline

[0723] EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0724] Et: Ethyl

[0725] eq.: equivalent

[0726] FA: Formic acid

[0727] Fmoc: 9-fluorenylmethoxycarbonyl

[0728] g: grams

[0729] Gly: Glycine

[0730] Glu: Glutamic acid

[0731] h: hours

[0732] HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide

[0733] HPLC: High Performance Liquid Chromatography

[0734] HRMS: High Resolution Mass Spectrometry

[0735] IR: infrared

[0736] L: Lift

[0737] Lys: lysine

[0738] m: millimeter

[0739] ma: maleimidoacetic acid

[0740] mAb: monoclonal antibody

[0741] Me:Methyl

[0742] min: minutes

[0743] Mtt: 4-methyltrityl

[0744] mol: mole

[0745] MS: Mass spectrometry

[0746] m / z: mass-to-charge ratio

[0747] NHS: N-hydroxysuccinimide

[0748] Nmab: Natuximab

[0749] Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

[0750] PBS: Phosphate buffered saline

[0751] PEG: polyethylene glycol

[0752] PEG16 or Peg16: CO-CH2CH2O-(CH2CH2O) 16 -CH3

[0753] PF: CentriPure PF filter cartridge

[0754] PFF: Pentafluorophenyl

[0755] pH: acidity and alkalinity

[0756] Phe: Phenylalanine

[0757] quant.: quantitative

[0758] rt: room temperature

[0759] Rt: retention time

[0760] SMCC: 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester

[0761] Suc: succinic acid

[0762] TCEP: Tris(2-carboxyethyl)phosphine

[0763] TFA: trifluoroacetic acid

[0764] Tmab: trastuzumab

[0765] Tyr: tyrosine

[0766] UPLC: Ultra-Performance Liquid Chromatography

[0767] UV: Ultraviolet

[0768] μ: micro

[0769] 8.2 Starting Materials and Chemicals:

[0770] The main starting materials and chemicals used in the following examples are listed below:

[0771] >Solvents and deprotection reagents used for synthesis from Merck or Fischer Scientific AG (Switzerland);

[0772] >TFA, DIEA, N-hydroxysuccinimide, DBU, DPBS, 10xDPBS and H-Glu(OtBu)-OBn from Sigma-Aldrich (Switzerland);

[0773] >Bachem (Switzerland) Fmoc-Cit-OPFP, H-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, H-Tyr(OtBu)-OtBu, Fmoc-Lys(Mtt)-OH, Fmoc-Cit-OH, Fmoc-Phe-OH and Boc-Cit-OH;

[0774] >AMAS of Astatech Inc (USA);

[0775] >Biosolve (France) solvents and chemicals for high performance liquid chromatography (HPLC) and ultra-performance liquid chromatography mass spectrometry (UPLC-MS);

[0776] > PEG24-amine from BroadPharm (USA);

[0777] >HATU of Combi-Blocks (USA);

[0778] >Isatecan mesylate from Angel Pharmatech, Ltd. (China);

[0779] >LiOH.H2O from VWR (Switzerland);

[0780] >DM1-SMCC from Enovation Chemicals (USA);

[0781] >Boc-Gly-OH and dihydrofuran-2,5-dione from Fluka (USA);

[0782] >EDC.HCl from Apollo Chemical (USA);

[0783] >DM1 from Angel Pharmatech, Ltd. (USA);

[0784] >TCEP.HCl,Boc-Lys-OH from Fluorochem (UK);

[0785] > BrCH2 COOH, H-Tyr-OMe and Pd / C from Acros Organics (Belgium);

[0786] >H-Cit-Lys(PEG5-ma)-Tyr-OH from Ambiopharm (USA)

[0787] >Roche (Switzerland) (trastuzumab);

[0788] >Natuximab-antibody huCD37-3 described in WO2019 / 229677 (version 1.0);

[0789] >From PurePEG(mPEG 16 -OCH2CH2COO-NHS ester, m = methyl) mPEG16-NHS ester, LLC (USA);

[0790] >N-Succinimidyl-4-(maleimidomethyl)-cyclohexanecarboxylate (SMCC) from Sigma Aldrich Fine Chemicals.

[0791] 8.3 Methods:

[0792] The following methods were used to evaluate the compounds and conjugates of the invention:

[0793] 8.3.1 Purity determination

[0794] The purity of the compounds was determined on a UPLC-MS system:

[0795] Method 1: Waters Acquity UPLC system with CSH C18 column ( 1.7μm, 2.1mm x 50mm) Waters SQD mass spectrometer, the chromatographic column was heated to 40°C, solvent systems A (water + 0.1% FA) and B (ACN + 0.1% FA) were used, the flow rate was 0.9mL / min, and the gradient of B was 5-100% in 2.7min.

[0796] Method 2: Waters Acquity UPLC system equipped with a CSH fluorophenyl column ( The column was heated to 40°C, and solvent systems A (water + 0.1% FA) and B (ACN + 0.1% FA) were used. The flow rate was 0.9 mL / min, and the gradient of B was 5-100% in 2.9 min.

[0797] Method 3: A Waters Acquity UPLC system was coupled to a Waters SQD mass spectrometer equipped with a BEH C18 1.7 μm 50 x 2.1 mm column heated to 40°C and equipped with a 2 μm insert filter pre-columns (available from Waters), using solvent systems A1 (water + 0.1% FA) and B1 (ACN + 0.1% FA) at a flow rate of 0.9 mL / min and a B1 gradient of 5-100% in 2.9 min.

[0798] Method 4: Waters Acquity UPLC system with CSH C18 column ( The column was heated to 40°C, and solvent systems A (water + 0.1% FA) and B (ACN + 0.1% FA) were used. The flow rate was 0.9 mL / min, and the gradient of B was 5-85% in 5 min.

[0799] Method 5: Equipment: Shimadzu LCMS2020 mass spectrometer. Column: HALO C18 2.7μm, 3.0mm×30mm. Mobile phase: ACN (0.05% TFA)-water (0.05% TFA). Gradient: MeCN from 5% to 95% in 1.4min, hold for 0.6min, total run time 2.5min, flow rate: 1.8mL / min. Column temperature: 50℃. Wavelength: 214 and 254nm PDA.

[0800] Method 6: Equipment: Shimadzu LCMS2020 mass spectrometer. Chromatographic column: Kinetex C18 2.6μm, 4.6mm×50mm. Mobile phase: ACN (0.05% TFA)-water (0.05% TFA); Gradient: MeCN from 5% to 95% in 1.8 minutes, maintained for 0.7min, total running time of 3.0min, flow rate: 1.8mL / min. Chromatographic column temperature: 50℃. Wavelength: 214 and 254nmPDA.

[0801] 8.3.2 Aggregates: Size Exclusion Chromatography (SEC)

[0802] The amount of aggregates in the conjugate was determined using the following method:

[0803] Equipment 1UPLC Waters Acquity

[0804] Equipment 2UPLC Waters Acquity H-Class plus Bio

[0805] Detector Tunable UV detector (TUV)

[0806] Detector Cell Titanium

[0807] Pre-column Agilent AdvanceBio SEC 2.7μm 4.6*50mm

[0808] Column: Agilent AdvanceBio SEC 2.7μm 4.6*150mm

[0809] Mobile phase: Potassium phosphate de potassium 50mM pH 6.8 / 250mM KCl

[0810] Wavelength 280nm

[0811] Injection volume 10 μL

[0812] Column temperature Ambient temperature

[0813] Sample manager temperature 25°C

[0814] Running time 10min

[0815] Flow rate 0.35mL / min

[0816] Isocratique Mode

[0817] Weak wash H2O / ACN (90 / 10v / v)

[0818] Strong washing H2O / ACN (10 / 90v / v)

[0819] Mobile phase preparation: Potassium phosphate 50mM pH 6.8 / 250mM KCl

[0820] Weigh 3.61g KH2PO4 and 4.09g K2HPO4 into a 1L volumetric flask. Complete with milliQ water. If necessary, adjust pH with HCl or NaOH 1mol / L. Add 18.64g KCl.

[0821] Sample preparation: Prepare ADC solutions ranging from 1 to 2.5 mg / mL in milliQ water.

[0822] 8.3.3DAR: Reverse Phase Liquid Chromatography (RPLC)

[0823] Equipment UPLC Waters Acquity

[0824] Detector Adjustable UV detector (TUV)

[0825] Detector Cell Titanium

[0826] Column Waters Bioresolve 2.1 * 150 2.7um Part Number 186008946

[0827] Mobile phase A: water + TFA 0.1% (v / v)

[0828] Mobile phase B ACN + TFA 0.1% (v / v)

[0829] Wavelength 280nm

[0830] Injection volume 10 μL

[0831] Column temperature 90℃

[0832] Sample management temperature 25℃

[0833] Running time 10min

[0834] Flow rate 0.35mL / min

[0835] Weak washing H2O / ACN (90 / 10v / v)

[0836] Strong washing H2O / ACN (10 / 90v / v)

[0837] Gradient_Basic:

[0838] t(min) Flow rate (mL / min) ％A ％B 0.0 0.6 73 27 13.0 0.6 60 40 13.1 0.6 73 27 16.0 0.6 73 27

[0839] Gradient dev2:

[0840]

[0841]

[0842] Gradient dev7:

[0843] t(min) Flow rate (mL / min) ％A ％B 0 0.6 70 30 40 0.6 40 60 42 0.6 40 60 42.1 0.6 70 30 44 0.6 70 30

[0844] ·

[0845] Gradient dev7_short:

[0846] t(min) Flow rate (mL / min) ％A ％B 0 0.6 70 30 20 0.6 55 45 20.1 0.6 5 95 23 0.6 5 95 23.1 0.6 70 30 25 0.6 70 30

[0847] Mobile phase preparation:

[0848] ACN + 0.1% TFA (V / V): Use a measuring cylinder to measure 1000 mL of ACN and pour it into a 1L glass bottle. Use a 1mL pipette to add 1mL of TFA and shake vigorously.

[0849] Water + 0.1% TFA (V / V): Use a measuring cylinder to measure 1000 mL of ultrapure water and pour it into a 1L glass bottle. Use a 1mL pipette to add 1mL of TFA and shake vigorously.

[0850] Sample preparation:

[0851] mAb: Prepare a 2.5 mg / mL mAb solution in milliQ water. Dispense 45 μL of this solution and 5 μL of 1 mol / L DTT solution in a vial. Incubate at 45°C for 30 minutes.

[0852] ADC: Prepare a 2.5 mg / mL ADC solution in milliQ water. Dispense 45 μL of this solution and 5 μL of 0.1 mol / L DTT solution in a vial. Incubate at 30 °C for 1 hour

[0853] 8.3.4DAR: MS method

[0854] Sample preparation:

[0855] Samples were diluted twice with 50 mM ammonium acetate and 25 ug was injected per sample.

[0856] UPLC:

[0857] Using MAbPac TM Separation was performed using a SEC-1 column (Thermo Scientific) and 50 mM ammonium acetate (pH 7, flow rate 0.3 mL / min) as the mobile phase.

[0858] MS:

[0859] MS was performed using a QExactive HF Orbitrap operating in the high mass range. MS spectra were acquired in the range 1800-8000 m / z with a resolution setting of 15k and SID 50 eV. Mass spectra were deconvoluted using Protein Deconvolution (Thermo Scientific).

[0860] 8.3.5 Concentration: UV method

[0861] ·equipment:

[0862] UV spectrophotometer BioTek Synergy HT

[0863] Buffer preparation:

[0864] PBS pH 7.4: Weigh 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4.2H2O and 0.24g KH2PO4 into a 1L volumetric flask. Add 900mL milliQ water. Mix and when all salts are dissolved, adjust pH to between 7.35 and 7.44 with 1mol / L HCl. Add to 1L with milliQ water.

[0865] Sample preparation:

[0866] A 1 mg / mL ADC solution was prepared in PBS, pH 7.4. The absorbance at 252 nm and 280 nm was measured using a UV reader.

[0867] Calculation formula:

[0868]

[0869] A = absorbance

[0870] DM1 (drug) = relevant payload

[0871] L = path length [cm]

[0872] C = concentration [mol / L]

[0873] ε=extinction coefficient [L.mol-1.cm-1]

[0874] c mg / mL=c mol / L*MW ADC

[0875] MW ADC=MW mAb+(MW payload*DAR moyen)

[0876] 8.3.6 Purification

[0877] When prepared, the compounds were purified by preparative reverse phase HPLC on a Buchi C835 using a Waters column (XSelect CSH130 C18 5μm 19x150mm OBD or XBridge PrepC18 5μm OBD 19x150mm or XSELECT CSH Prep Fluoro-Phenyl 5μm 19x150mm) with the specified solvent system at a flow rate of 25mL / min. The elution was monitored by UV at a wavelength of 220nm and by ELSD. Alternatively, the compounds were purified on a Biotage Slekt system using a Sfar C18 column. The purity of the peptides was determined using the UPLC method described previously (see 9.3.1).

[0878] 8.3.7 pH8DPBS and pH810xDPBS

[0879] DPBS at pH 8 and 10x DPBS at pH 8 were obtained by adding 1 M NaOH to DPBS and 10x DPBS, respectively.

[0880] 8.3.8 Natuximab

[0881] Natuximab (also referred to herein as K7153A) refers to the antibody huCD37-3 (version 1.0) described in WO 2019 / 229677 (incorporated herein by reference).

[0882] 8.4 Preparation of Linker-Payload:

[0883] The linker-payloads were prepared using standard chemistry and a convergent strategy. The linker-payloads prepared in Examples 8.4.1-8.4.9 are shown in Table 1 below.

[0884]

[0885]

[0886]

[0887]

[0888] Table 1: Linker-Payload Example 8.4.1: Preparation of DM1-MCC-Cit-Lys(ma-PEG5)-Tyr-OH

[0889]

[0890] H-Cit-Lys(PEG5-ma)-Tyr-OH was purchased from Ambiopharm and prepared according to the following general procedure:

[0891] According to the general Fmoc / tBu strategy of solid phase peptide synthesis, peptide synthesis was carried out on 2-CTC resin, wherein carboxyl activation was carried out by diisopropylcarbodiimide / HOBT. Subsequently, each amino acid was coupled to the peptide chain in the form of an active ester starting from the C-terminal amino acid. The last amino acid in the sequence was coupled to the Boc group protected at the N-terminus. Lys derivatives were incorporated with side chain amino groups protected by ivDde, and ivDde was removed by 2% hydrazine in DMF. After removing ivDde, the side chain was derivatized with maleimide-PEG5-OH using an activated ester. Subsequently, the peptide was treated with a TFA-based acidolysis mixture, resulting in its cleavage from the resin and deprotection of the side chain groups. The peptide was then purified by liquid chromatography (RP-HPLC). The purified peptide TFA salt was lyophilized to obtain a purified peptide TFA salt as a white to off-white powder.

[0892] At room temperature, DIEA (20 μL, 0.11 mmol, 1.0 equiv) was added to a solution of H-Cit-Lys(PEG5-ma)-Tyr-OH (100 mg, 0.11 mmol, 1.0 equiv) and DM1-SMCC (131.8 mg, 0.12 mmol, 1.1 equiv) in DMF (1 mL). After stirring at room temperature for 4 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20 to 80% ACN+0.1% TFA in water+0.1% TFA) gave DM1-MCC-Cit-Lys(ma-PEG5)-Tyr-OH (138.5 mg, 74.8 μmol, 100% UV purity, 67% yield) as a white powder after freeze drying. UPLC-MS (method 4): Rt = 2.58 and 2.63 min, m / z = 1854 [M+H] + ,1852[MH] - .

[0893] Example 8.4.2: Preparation of DM1-Ac-Cit-Lys(ma-Lys(PEG16))-Tyr-OH

[0894]

[0895]

[0896] Step 1. DIEA (0.84 mL, 4.82 mmol, 5.0 equiv) was added to a mixture of mPEG16-NHS ester (900 mg, 0.96 mmol, 1.0 equiv) and Boc-Lys-OH (300 mg, 1.16 mmol, 1.2 equiv) in DMF (12 mL) at room temperature. After stirring at room temperature for 16 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20 to 80% ACN + 0.1% TFA in water + 0.1% TFA) gave Boc-Lys(PEG16)-OH (990 mg, 0.95 mmol, 100% UV purity, 99% yield) as a colorless oil after freeze drying. UPLC-MS (Method 1): Rt = 1.16 min, m / z = 1038 [M+H] + .

[0897] Step 2. TFA (6 mL) was added to a solution of Boc-Lys(PEG16)-OH (990 mg, 0.95 mmol, 1.0 equiv) in DCM (6 mL) at room temperature. After stirring at room temperature for 30 minutes, the reaction mixture was concentrated in vacuo. The residue was dissolved in a mixture of ACN / water (1:1, 16 mL) and then freeze-dried to give H-Lys(PEG16)-OH.TFA (1.00 g, 0.95 mmol, 100% UV purity, quantitative) as a colorless oil. UPLC-MS (Method 1): Rt = 0.89 min, m / z = 938 [M+H] + ,936[MH] - .

[0898] Step 3. DIEA (0.15 mL, 0.85 mmol, 4.0 equiv) was added to a mixture of H-Lys(PEG16)-OH (200 mg, 0.21 mmol, 1.0 equiv) and AMAS (56.5 mg, 0.22 mmol, 1.05 equiv) in DMF (2.5 mL) at room temperature. After stirring at room temperature for 1 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (10-40% ACN + 0.1% TFA in water + 0.1% TFA) gave ma-Lys(PEG16)-OH (220 mg, 0.20 mmol, 100% UV purity, 96% yield) as a colorless oil after freeze drying. UPLC-MS (Method 4): Rt = 1.74 min, m / z = 1075 [M+H] + ,1073[MH] - .

[0899] Step 4. DIEA (0.6 mL, 3.44 mmol, 3.7 equiv) was added to a mixture of Fmoc-Cit-OPFP (520.7 mg, 0.92 mmol, 1.0 equiv) and H-Lys(Boc)-OH (218.1 mg, 0.89 mmol, 0.96 equiv) in a mixture of DMF (6 mL) and water (3 mL) at room temperature. After stirring at room temperature for 1 h, TFA was added dropwise until an acidic pH was reached. Purification by C18 flash column chromatography (20-80% ACN + 0.1% TFA in water + 0.1% TFA) gave Fmoc-Cit-Lys(Boc)-OH (593.3 mg, 0.92 mmol, 97% UV purity, quantitative) as a white powder after freeze drying. UPLC-MS (Method 3): Rt = 1.80 min, m / z = 626 [M+H] + ,526[M-Boc+H] + ,624[MH]- .

[0900] Step 5. To a solution of Fmoc-Cit-Lys(Boc)-OH (400 mg, 0.64 mmol, 1.0 eq.) in DMF (10 mL) was added H-Tyr-OMe (139.2 mg, 0.71 mmol, 1.1 eq.) at room temperature, followed by HATU (334.2 mg, 0.88 mmol, 1.4 eq.) and DIEA (0.5 mL, 2.87 mmol, 4.5 eq.). After stirring at room temperature for 2 h, TFA was added until an acidic pH was reached. Purification by C18 flash column chromatography (20-80% ACN + 0.1% TFA in water + 0.1% TFA) gave H-Cit-Lys(Boc)-Tyr-H (513.3 mg, 0.57 mmol, 89% UV purity, 89% yield) as a white powder after freeze drying. UPLC-MS (method 3): Rt = 1.83 min, m / z = 803 [M+H] + ,703[M-Boc+H] + ,847[M+FA-H] - .

[0901] Step 6. Lithium hydroxide monohydrate (158.5 mg, 3.78 mmol, 4.2 equiv) was added to a solution of Fmoc-Cit-Lys(Boc)-Tyr-OMe (730.6 mg, 0.91 mmol, 1.0 equiv) in THF (8 mL) and water (5 mL) at room temperature. After stirring at room temperature for 40 min, TFA was added until a slightly acidic pH was reached, and the reaction mixture was then concentrated in vacuo. Purification by preparative HPLC (15 to 40% ACN + 0.1% TFA in water + 0.1% TFA) gave H-Cit-Lys(Boc)-Tyr-OH (510 mg, 0.90 mmol, 100% UV purity, 99% yield) as a white powder after freeze drying. UPLC-MS (Method 1): Rt = 0.80 min, m / z = 567 [M+H] + ,565[MH] - .

[0902] Step 7. DIEA (0.28 mL, 1.63 mmol, 6.0 equiv) was added to a solution of bromoacetic acid (64.8 mg, 0.47 mmol, 1.7 equiv) and DM1 (200 mg, 0.27 mmol, 1.0 equiv) in DMF (2 mL) at room temperature. After stirring at room temperature for 1 h, a mixture of HATU (113.3 mg, 0.30 mmol, 1.1 equiv) and 1-hydroxypyrrolidine-2,5-dione (34.3 mg, 0.30 mmol, 1.1 equiv) was added. After stirring at room temperature for 30 min, TFA was added until an acidic pH was reached. Purification on preparative HPLC (30 to 60% ACN + 0.1% TFA in water + 0.1% TFA) gave DM1-Ac-NHS ester (134.7 mg, 0.12 mmol, 80% UV purity, 45% yield) as a white powder after freeze drying. UPLC-MS (method 2): Rt = 1.57 min, m / z = 891 [MH] - .

[0903] Step 8. DIEA (0.22 mL, 1.24 mmol, 4.0 equiv) was added to a mixture of DM1-Ac-NHS ester (347 mg, 0.31 mmol, 1.0 equiv) and H-Cit-Lys(Boc)-Tyr-OH (222.1 mg, 0.33 mmol, 1.05 equiv) in DMF (4 mL) at room temperature. After stirring at room temperature for 1 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (30 to 60% ACN + 0.1% TFA in water + 0.1% TFA) gave DM1-Ac-Cit-Lys(Boc)-Tyr-OH (417 mg, 0.31 mmol, 100% UV purity, quantitative) as a white powder after freeze drying. UPLC-MS (Method 3): Rt = 1.67 min, m / z = 1344 [MH] - .

[0904] Step 9. A mixture of TFA (0.9 mL) and DCM (3.6 mL) was added to a mixture of DM1-Ac-Cit-Lys(Boc)-Tyr-OH (112.0 mg, 83.3 μmol, 1.0 equiv) at room temperature. After stirring at room temperature for 5 min, a mixture of ACN / water (1:1, 8 mL) was added to the reaction mixture. DCM was removed by vacuum concentration. Purification by preparative HPLC (2 to 40% ACN + 0.1% TFA in water + 0.1% TFA) gave DM1-Ac-Cit-Lys-Tyr-OH (80 mg, 64.3 μmol, 100% UV purity, 77% yield) as a white powder after freeze drying. UPLC-MS (Method 2): Rt = 1.03 min, m / z = 1242 [M+H] + ,1240[MH] - .

[0905] Step 10. 1-Hydroxypyrrolidine-2,5-dione (2.14 mg, 18.6 μmol, 1.0 equiv) and EDC.HCl (3.57 mg, 18.6 μmol, 1.0 equiv) were added to a solution of ma-Lys(PEG16)-OH (20.0 mg, 18.6 μmol, 1.0 equiv) in DCM (2 mL) at room temperature. After stirring at room temperature for 2.5 h, DM1-Ac-Cit-Lys-Tyr-OH (37.1 mg, 22.3 μmol, 1.2 equiv) and DIEA (13 μL, 74.5 μmol, 4.0 equiv) were added to the reaction mixture at room temperature. After stirring at room temperature for 10 min, TFA was added until an acidic pH was reached. Purification by preparative HPLC (5 to 100% ACN + 0.1% TFA in water + 0.1% TFA) gave DM1-Ac-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (17.2 mg, 7.50 μmol, 100% UV purity, 40% yield) as a white powder after freeze drying. UPLC-MS (Method 4): Rt = 2.59 min, m / z = 1173 [M+FA-H] - .

[0906] Example 8.4.3: Preparation of DM1-Ac-Cit-Lys(ma-PEG5)-Tyr-OH

[0907]

[0908] At room temperature, DIEA (31 μL, 0.18 mmol, 4.0 equiv) was added to a mixture of DM1-Ac-NHS ester (50.0 mg, 44.8 μmol, 1.0 equiv) and H-Cit-Lys(ma-PEG5)-Tyr-OH.TFA (45.2 mg, 44.8 μmol, 1.0 equiv) in DMF (1 mL). After stirring at room temperature for 30 minutes, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20 to 50% ACN+0.1% TFA in water+0.1% TFA) gave DM1-Ac-Cit-Lys(ma-PEG5)-Tyr-OH (74.9 mg, 44.8 μmol, 100% UV purity, quantitative) as a white powder after freeze drying. UPLC-MS (method 1): Rt = 1.31 min, m / z = 1672 [MH] -

[0909] Example 8.4.4: Preparation of Exatecan-Suc-Phe-Cit-Lys(ma-Lys(PEG16))-Tyr-OH

[0910]

[0911] Step 1. DIEA (2.0 mL, 11.8 mmol, 3.0 eq.) was added to a mixture of H-Tyr(OtBu)-OtBu (2.59 g, 7.85 mmol, 1.0 eq.), Fmoc-Lys(Mtt)-OH (2.51 g, 3.94 mmol, 1.0 eq.) and HATU (1.95 g, 5.12 mmol, 1.3 eq.) in DMF (40 mL) at 0-5°C. After stirring at room temperature for 50 min, the reaction mixture was diluted with EtOAc (300 mL) and washed with half-saturated brine (2 x 200 mL). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure to give Fmoc-Lys(Mtt)-Tyr(OtBu)-OtBu (7.07 g, 7.69 mmol, 98% UV purity, 98% yield) as a light brown oil. UPLC-MS (method 3): Rt = 2.42 min, m / z = 900 [M+H] + ,944[M+FA-H] - .

[0912] Step 2. A mixture of DMF / piperidine (9:1, 40 mL) was added to Fmoc-Lys(Mtt)-Tyr(OtBu)-OtBu (7.07 g, 7.69 mmol, 1.0 equiv) at room temperature. After stirring at room temperature for 10 min, the reaction mixture was concentrated to dryness. The residue was purified by flash chromatography (n-hexane, then DCM, and finally DCM: MeOH 95:5 to 90:10) to give H-Lys(Mtt)-Tyr(OtBu)-OtBu (5.10 g, 7.15 mmol, 95% UV purity, 93% yield) as an orange oil after vacuum concentration. UPLC-MS (Method 3): Rt=1.58 min, m / z=678 [M+H] + ,722[M+FA-H] - .

[0913] Step 3. DIEA (1.73 mL, 9.96 mmol, 3.0 eq) was added to a mixture of H-Lys(Mtt)-Tyr(OtBu)-OtBu (2.50 g, 3.32 mmol, 1.0 eq), Fmoc-Cit-OH (1.48 g, 3.65 mmol, 1.1 eq) and HATU (1.70 g, 4.31 mmol, 1.3 eq) in DMF (20 mL) at 0°C. After stirring at room temperature for 30 min, the reaction mixture was poured into 5% NaHCO3 aqueous solution (200 mL) and extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with brine (100 mL), dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash chromatography (heptane:EtOAc 80:20 to pure EtOAc, then 5-10% EtOAc:MeOH 95:5 to 90:10) gave Fmoc-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (3.40 g, 3.05 mmol, 95% UV purity, 92% yield) as a beige gum after concentration in vacuo. UPLC-MS (Method 3): Rt=2.15 min, m / z=1058 [M+H] + ,1102[M+FA-H] - .

[0914] Step 4. At room temperature, a mixture of DMF / piperidine (20 mL, 9:1 V / V) was added to Fmoc-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (3.40 g, 3.22 mmol, 1.0 equivalent). After stirring at room temperature for 10 min, the reaction mixture was concentrated to dryness. Purified by flash chromatography (heptane: EtOAc 80:20 to pure EtOAc, then 5-10% EtOAc: MeOH 95:5 to 90:10, then 25% NH3 in ACN 5-15%), H-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (2.55 g, 2.96 mmol, 97% UV purity, 92% yield) was obtained as a colorless oil after vacuum concentration. UPLC-MS (method 3): Rt = 1.54 min, m / z = 836 [M+H] + ,880[M+FA-H] - .

[0915] Step 5. DIEA (0.61 mL, 3.48 mmol, 3.0 eq) was added to a mixture of H-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (1.00 g, 1.16 mmol, 1.0 eq), Fmoc-Phe-OH (539 mg, 1.39 mmol, 1.2 eq) and HATU (594 mg, 1.51 mmol, 1.3 eq) in DMF (15 mL) at °C. After stirring at room temperature for 15 min, the reaction mixture was poured into 5% NaHCO3 aqueous solution (50 mL) and extracted with EtOAc (2 x 70 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and partially concentrated under reduced pressure. Precipitation with ether gave Fmoc-Phe-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (1.33 g, 1.06 mmol, 96% UV purity, 92% yield) as a white solid. UPLC-MS (Method 3): Rt = 2.29 min, m / z = 1205 [M+H] + ,1249[M+FA-H] - .

[0916] Step 6. A mixture of DMF / piperidine (10 mL, 9:1 V / V) was added to Fmoc-Phe-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (1.33 g, 1.06 mmol, 1.0 equiv) at room temperature. After stirring at room temperature for 10 min, the reaction mixture was concentrated to dryness. Purification by flash chromatography (heptane:EtOAc 80:20 to pure EtOAc, then 5-10% EtOAc:MeOH 90:10, then 25% aqueous NH3 in ACN 10-20%) gave H-Phe-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (0.93 g, 0.90 mmol, 95% UV purity, 81% yield) as a white powder after vacuum concentration. UPLC-MS (method 3): Rt = 1.57 min, m / z = 983 [M+H] + ,1027[M+FA-H] - .

[0917] Step 7. Dihydrofuran-2,5-dione (1.16 mg, 11.5 μmol, 1.0 equiv) was added to a mixture of exatecan mesylate (5.0 mg, 11.5 μmol, 1.0 equiv) and DIEA (0.02 mL, 0.14 mmol, 12.0 equiv) in DMF (0.5 mL) at room temperature. After stirring at room temperature for 15 min, H-Phe-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (11.28 mg, 11.5 μmol, 1.0 equiv) was added, followed by HATU (4.37 mg, 11.5 μmol, 1.0 equiv). After stirring at room temperature for 1 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (10 to 100% ACN + 0.1% TFA in water + 0.1% TFA) gave exatecan-Suc-Phe-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (8.1 mg, 5.30 mmol, 98% UV purity, 46% yield) as a yellow powder after freeze drying. UPLC-MS (Method 4): Rt = 3.70 min, m / z = 1501 [M+H] + ,1545[M+FA-H] - .

[0918] Step 8. A mixture of DCM (5 mL) and TFA (0.1 mL) was added to Exatecan-Suc-Phe-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (61.0 mg, 40.7 μmol, 1.0 equiv) at room temperature. The reaction mixture was stirred at room temperature for 1 h and then concentrated in vacuo. Exatecan-Suc-Phe-Cit-Lys-Tyr(OtBu)-OtBu (6.95 mg, 5.4 μmol, 97% UV purity, 13% yield) was obtained as a yellow powder after lyophilization by preparative HPLC (20 to 40% ACN+0.1% TFA in water+0.1% TFA). UPLC-MS (Method 4): Rt=2.82 min, m / z=1244 [M+H] + ,1288[M+FA-H] - .

[0919] Step 9. 1-Hydroxypyrrolidine-2,5-dione (9.0 mg, 78 μmol, 2.0 equiv) and EDC.HCl (15.0 mg, 78 μmol, 2.0 equiv) were added to a solution of ma-Lys(PEG16)-OH (41.9 mg, 39 μmol, 1.0 equiv) in DCM (2.0 mL) at room temperature. After stirring at room temperature for 1 h, exatecan-Suc-Phe-Cit-Lys-Tyr(OtBu)-OtBu (50.0 mg, 39 μmol, 1 equiv) and DIEA (27 μL, 0.16 mmol, 4.0 equiv) were added to the reaction mixture at room temperature. After stirring at room temperature for 20 min, TFA was added until an acidic pH was reached. Purification by preparative HPLC (30 to 80% ACN + 0.1% TFA in water + 0.1% TFA) gave exatecan-Suc-Phe-Cit-Lys(ma-Lys(Peg16))-Tyr(OtBu)-OtBu (10.0 mg, 4.30 mmol, 100% UV purity, 11% yield) as a white powder after freeze drying. UPLC-MS (Method 4): Rt = 3.15 min, m / z = 1151 [M+H] + .

[0920] Step 10. TFA (0.11 mL) was added to a solution of exatecan-Suc-Phe-Cit-Lys(ma-Lys(Peg16))-Tyr(OtBu)-OtBu (10.0 mg, 4.30 μmol, 1.0 equiv) in DCM (0.11 mL) at room temperature. After stirring at room temperature for 3 h, the reaction mixture was concentrated in vacuo. Purification by preparative HPLC (20 to 60% ACN+0.1% TFA in water+0.1% TFA) gave exatecan-Suc-Phe-Cit-Lys(ma-Lys(Peg16))-Tyr-OH (5.3 mg, 2.40 μmol, 100% UV purity, 56% yield) as a white powder after freeze drying. UPLC-MS (Method 4): Rt=2.44 min, m / z=1094 [M+H] + ,1092[M+FA-H] - .

[0921] Example 8.4.5: Preparation of ma-Gly-Glu(DM1-Ac-Cit-Lys-Tyr-OH)-Glu(DM1-Ac-Cit-Lys-Tyr-OH)-PEG24

[0922]

[0923] Step 1. DIEA (1.17 mL, 6.66 mmol, 3.0 equiv) was added to a mixture of Fmoc-Glu(OtBu)-OH.H2O (1.00 g, 2.22 mmol, 1.0 equiv), H-Glu(OtBu)-OBn.HCl (750 mg, 2.22 mmol, 1.0 equiv) and HATU (1.14 g, 2.99 mmol, 1.35 equiv) in DMF (15 mL) at 0°C. After stirring at 0°C for 15 min and at room temperature for 1 h, the reaction mixture was concentrated to dryness. The resulting brown oil was diluted with EtOAC and washed with aqueous NaHCO3. The organic phase was washed with brine, dried over MgSO4, filtered and concentrated to dryness. Flash chromatography (n-heptane:EtOAc 80:20 to 0:100) afforded Fmoc-Glu(OtBu)-Glu(OtBu)-OBn (1.46 g, 2.09 mmol, 100% Uv purity, 94% yield) as a white foam. UPLC-MS (Method 3): Rt = 2.62 min, m / z = 701 [M+H] + ,745[M+FA-H] - .

[0924] Step 2. DBU (0.3 mL, 2.00 mmol, 1.2 eq) was added to a solution of Fmoc-Glu(OtBu)-Glu(OtBu)-OBn (1.17 g, 1.67 mmol, 1.0 eq) in DMF (5 mL) at room temperature. After stirring at room temperature for 5 min, the reaction mixture was added dropwise to a mixture of Boc-Gly-OH (351 mg, 2.00 mmol, 1.2 eq), HATU (855.3 mg, 2.17 mmol, 1.3 eq) and DIEA (0.87 mL, 5.01 mmol, 3.0 eq) in DMF (12 mL) at room temperature over 3 min. After stirring at room temperature for 30 min, the reaction mixture was concentrated to dryness. The residue was diluted with EtOAc and then washed with 10% citric acid. The organic layer was dried over MgSO4 and concentrated to dryness. Flash chromatography (DCM:EtOAc 90:10 to 0:100) afforded Boc-Gly-Glu(OtBu)-Glu(OtBu)-OBn (982 mg, 1.47 mmol, 95% UV purity, 88% yield) as a white solid. UPLC-MS (Method 3): Rt = 2.22 min, m / z = 636 [M+H] + ,634[MH] - .

[0925] Step 3. Under nitrogen atmosphere, palladium (164.4 mg, 0.15 mmol, 0.1 eq) was added to a 25 mL flask for hydrogenation. A solution of Boc-Gly-Glu(OtBu)-Glu(OtBu)-OBn (982 mg, 1.54 mmol, 1.0 eq) in ethanol (9.8 mL) was added. The resulting suspension was stirred at room temperature under 1 bar of hydrogen for 30 min and then heated by The filtrate was concentrated under reduced pressure to give Boc-Gly-Glu(OtBu)-Glu(OtBu)-OH (898 mg, 1.65 mmol, 94% UV purity, quantitative) as white foam. UPLC-MS (Method 3): Rt = 1.73 min, m / z = 546 [M+H] + ,544[MH] - .

[0926] Step 4. DIEA (48 mL, 0.27 mmol, 3.0 equiv) was added to a mixture of Boc-Gly-Glu(OtBu)-Glu(OtBu)-OH (50.0 mg, 91.6 mmol, 1.0 equiv), PEG24-amine (120.5 mg, 0.11 mmol, 1.2 equiv) and HATU (59.0 mg, 0.15 mmol, 1.6 equiv) in DMF (0.5 mL) at room temperature. After stirring at room temperature for 10 min, the reaction mixture was acidified to pH 4-5 with TFA. Purification by preparative HPLC (10-100% ACN + 0.1% TFA in water + 0.1% TFA) gave Boc-Gly-Glu(OtBu)-Glu(OtBu)-PEG24 (100 mg, 61.9 mmol, 100% UV purity, 68% yield) as a white solid after freeze drying. UPLC-MS (method 3): Rt=1.82min, m / z=1614[MH] - ,1660[M+FA-H] - .

[0927] Step 5. TFA (0.5 mL) was added to a solution of Boc-Gly-Glu(OtBu)-Glu(OtBu)-PEG24 (100 mg, 61.9 mmol, 1.0 equiv) in DCM (0.5 mL). After stirring at room temperature for 30 min, the reaction mixture was concentrated to dryness to give H-Gly-Glu-Glu-PEG24.TFA (93 mg, 61.3 mmol, 99% yield) as a yellow oil. UPLC-MS (Method 1): Rt = 0.94 min, m / z = 1404 [M+H] + ,1402[MH] - .

[0928] Step 6. DIEA (53 μL, 0.31 mmol, 5.0 equiv) was added to a mixture of H-Gly-Glu-Glu-PEG24.TFA (93.0 mg, 61.3 μmol, 1.0 equiv) and AMAS (21.2 mg, 79.7 μmol, 1.3 equiv) in DMF (0.3 mL) at room temperature. After stirring at room temperature for 1 h, TFA (24 μL) was added. Purification by preparative HPLC (20-100% ACN + 0.1% TFA in water + 0.1% TFA) gave ma-Gly-Glu-Glu-PEG24 (94.0 mg, 58.0 mmol, 95% UV purity, 95% yield) as a colorless oil. UPLC-MS (Method 3): Rt = 1.20 min, m / z = 1539 [MH]- .

[0929] Step 7. DIEA (27 μL, 0.16 mmol, 8.0 equiv) was added to a mixture of ma-Gly-Glu-Glu-PEG24 (30.0 mg, 19.5 μmol, 1.0 equiv) and HATU (14.8 mg, 38.9 μmol, 2.0 equiv) in DMF (3 mL) at room temperature. After stirring at room temperature for 7 min, the reaction mixture was added to DM1-Ac-Cit-Lys-Tyr-OH (52.9 mg, 38.9 μmol, 2.0 equiv). After stirring at room temperature for 15 min, TFA was added until an acidic pH was reached. Purification by preparative HPLC (30 to 70% ACN + 0.1% TFA in water + 0.1% TFA) gave ma-Gly-Glu(DM1-Ac-Cit-Lys-Tyr-OH)-Glu(DM1-Ac-Cit-Lys-Tyr-OH)-PEG24 (15.1 mg, 3.40 μmol, 89% UV purity, 17% yield) as a white powder after freeze drying. UPLC-MS (Method 3): Rt = 1.70 min, m / z = 1996 [M-2H] 2- .

[0930] Example 8.4.6: Preparation of DM1-MCC-Cit-Lys(ma-Lys(PEG16))-Tyr-OH

[0931]

[0932] Step 1. DIEA (0.7 mL, 3.98 mmol, 3.0 equiv) was added to a mixture of H-Lys(Mtt)-Tyr(OtBu)-OtBu (1.00 g, 1.33 mmol, 1.0 equiv), HATU (680 mg, 1.73 mmol, 1.3 equiv) and Boc-Cit-OH (373 mg, 1.33 mmol, 1.0 equiv) in DMF (8 mL) at 0-5°C. After stirring at room temperature for 30 min, the reaction mixture was poured into EtOAc (200 mL), washed with brine (100 mL) and then with half-saturated brine (50 mL). The resulting emulsion was first evaporated under reduced pressure and then freeze-dried to give Boc-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (2.10 g, 1.80 mmol, 60% purity, quantitative) as a beige solid. UPLC-MS (method 3): Rt = 2.02 min, m / z = 936 [M+H] + ,980[M+FA-H] - .

[0933] Step 2. A mixture of DCM (65 mL) and TFA (2 mL) was added to Boc-Cit-Lys(Mtt)-Tyr(OtBu)-OtBu (1.24 g, 1.33 mmol, 1.0 equiv) at room temperature. After stirring at room temperature for 10 min, the reaction mixture was partially concentrated and then slowly added to cold ether. The oily product obtained by centrifugation was purified by preparative HPLC (25 to 80% ACN+0.1% TFA in water+0.1% TFA) to give Boc-Cit-Lys-Tyr(OtBu)-OtBu (602 mg, 0.76 mmol, 100% purity, 57% yield) as a white powder after freeze drying. UPLC-MS (Method 3): Rt=1.46 min, m / z=680 [M+H] + ,723[M+FA-H] - .

[0934] Step 3. DIEA (0.06 mL, 0.37 mmol, 4.0 equiv) was added to a mixture of ma-Lys(PEG16)-OH (100 mg, 93.1 μmol, 1.0 equiv), Boc-Cit-Lys-Tyr(OtBu)-OtBu (81.2 mg, 102.4 μmol, 1.1 equiv) and HATU (47.7 mg, 121 μmol, 1.3 equiv) in DMF (1.5 mL) at 5° C. After stirring at 5° C. for 10 min, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20 to 70% ACN + 0.1% TFA in water + 0.1% TFA) gave Boc-Cit-Lys(ma-Lys(PEG16))-Tyr(OtBu)-OtBu (158 mg, mmol, 96% UV purity, 94% yield) as a white powder after freeze drying. UPLC-MS (Method 3): Rt = 1.81 min, m / z = 1736 [M+-H] + ,1780[M+FA-H] - .

[0935] Step 4. A mixture of TFA (2.5 mL) and DCM (2.5 mL) was added to Boc-Cit-Lys(ma-Lys(PEG16))-Tyr(OtBu)-OtBu (158 mg, 87.2 μmol, 1.0 equiv) at room temperature. After stirring at 4°C for 16 h, the reaction mixture was concentrated in vacuo. Purification by preparative HPLC (10 to 50% ACN + 0.1% TFA in water + 0.1% TFA) gave H-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (132 mg, 79.6 μmol, 99% UV purity, 91% yield) as a colorless oil after freeze drying. UPLC-MS (Method 3): Rt = 1.04 min, m / z = 1523 [M+H] + ,1521[MH] - .

[0936] Step 5. DIEA (41 μL, 0.25 mmol, 4.0 equiv) was added to a mixture of DM1-SMCC (68.6 mg, 62.0 μmol, 1.0 equiv) and H-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (103 mg, 62.0 μmol, 1.0 equiv) in DMF (1.5 mL) at room temperature. After stirring at room temperature for 1.5 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20 to 90% ACN + 0.1% TFA in water + 0.1% TFA) gave DM1-MCC-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (73.4 mg, 29.4 μmol, 99% UV purity, 47% yield) as a white powder after freeze drying. UPLC-MS (method 3): Rt = 1.60 and 1.62 min, m / z = 827 [M+3H] 3+ ,870[M+FA-3H] 3-

[0937] Example 8.4.7: Preparation of DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH

[0938] Step 1.

[0939]

[0940] DIEA (0.14 mL, 0.83 mmol, 4.0 equiv) was added to a mixture of DM1-Ac-NHS ester (185 mg, 0.21 mmol, 1.0 equiv) and citrulline (72.6 mg, 0.41 mmol, 2.0 equiv) in DMSO (3.7 mL) at room temperature. After stirring at room temperature for 18 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (10 to 40% ACN + 0.1% TFA in water + 0.1% TFA) gave DM1-Ac-Cit (130 mg, 0.14 mmol, 100% UV purity, 66% yield) as a white powder after freeze drying. UPLC-MS (Method 1): Rt = 1.33 min, m / z = 952 [MH] - .

[0941] Step 2.

[0942]

[0943] Acetyl-L-cysteine ​​(0.32 mg, 2.17 μmol, 1.0 equiv) was added to a solution of DM1-Ac-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (5.0 mg, 2.17 μmol, 1.0 equiv) in DMF (1 mL) at room temperature. After stirring at room temperature for 40 min, TFA was added until an acidic pH was reached. Purification by preparative HPLC (5 to 100% ACN+0.1% TFA in water+0.1% TFA) gave DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH (4.59 mg, 1.86 μmol, 99% UV purity, 94% yield) as a white powder after freeze drying. UPLC-MS (method 4): Rt = 2.41 min, m / z = 1231 [M-2H] 2- .

[0944] Example 8.4.8: Preparation of Exatecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(Peg16))-Tyr-OH

[0945] Step 1.

[0946]

[0947] Step 1. DIEA (0.78 mL, 4.48 mmol, 4.0 equiv) was added to a mixture of citrulline (200 mg, 1.12 mmol, 1.0 equiv) and Boc-Phe-OSu (405 mg, 1.12 mmol, 1.0 equiv) in DMF (10 mL) at room temperature. After stirring at room temperature for 16 h, the reaction mixture was filtered and then concentrated under reduced pressure. A mixture of water / ACN / TFA (1:1:0.5%) was added. The mixture was filtered and then freeze-dried. Purification by preparative HPLC (10 to 60% ACN + 0.1% TFA in water + 0.1% TFA) gave Boc-Phe-Cit-OH (86 mg, 0.20 mmol, 100% UV purity, 18% yield) as a white powder after freeze-drying. UPLC-MS (Method 3): Rt = 1.27 min, m / z = 423 [M+H] + ,421[MH] - .

[0948] Step 2. A mixture of DCM (0.9 mL) and TFA (0.9 mL) was added to Boc-Phe-Cit-OH (60 mg, 0.14 mmol, 1.0 equiv) at room temperature. After stirring at room temperature for 20 min, the reaction mixture was concentrated. Water (10 mL) and ACN (10 mL) were added and the mixture was freeze-dried to give H-Phe-Cit-OH (30 mg, 83.8 μmol, 90% UV purity, 59% yield) as a white powder. UPLC-MS (Method 3): Rt = 0.35 min, m / z = 323 [M+H] + ,321[MH] - .

[0949] Step 3. Dihydrofuran-2,5-dione (8.4 mg, 83.5 μmol, 1.0 equiv) was added to a mixture of exatecan (36 mg, 83.5 μmol, 1.0 equiv) and DIEA (0.17 mL, 1.00 mmol, 12 equiv) in DMF (0.8 mL) at room temperature. After stirring at room temperature for 10 min, 1-hydroxypyrrolidine-2,5-dione (9.6 mg, 83.5 μmol, 1.0 equiv) was added, followed by HATU (31.7 mg, 83.5 μmol, 1.0 equiv). After stirring at room temperature for 15 min, H-Phe-Cit-OH (29.9 mg, 83.5 μmol, 1.0 equiv) was added to the reaction mixture. After stirring at room temperature for 1 h, H-Phe-Cit-OH (29.9 mg, 83.5 μmol, 1.0 equiv) was added to the reaction mixture. After stirring at room temperature for 1 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20 to 60% ACN + 0.1% TFA in water + 0.1% TFA) gave Exatecan-Suc-Phe-Cit (26.4 mg, 30.8 mmol, 98% UV purity, 37% yield) as a yellow powder after freeze drying. UPLC-MS (Method 2): Rt = 1.39 min, m / z = 840 [M+H] + ,838[MH] - .

[0950] Step 4

[0951]

[0952] DIEA (1.2 μL, 6.8 μmol, 4.0 eq) was added to a mixture of exatecan-Suc-Phe-Cit-Lys(ma-C1-Lys(Peg16))-Tyr-OH (3.71 mg, 1.7 mmol, 1.0 eq) and acetyl-L-cysteine ​​(0.28 mg, 1.7 μmol, 1.0 eq) in DMF (0.6 mL) at room temperature. After stirring at room temperature for 1 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20 to 60% ACN + 0.1% TFA in water + 0.1% TFA) gave Isanotecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(Peg16))-Tyr-OH (2.87 mg, 1.2 μmol, 100% UV purity, 72% yield) as a white powder after freeze drying. UPLC-MS (Method 4): Rt = 2.37 min, m / z = 1176 [M+2H] 2+ ,1174[M-2H] 2- .

[0953] Example 8.4.9: Preparation of DM1-Ac-Cit-Lys(D-Arg-ma)-Tyr-OH

[0954]

[0955] Step 1. DIEA (22.0 g, 0.17 mol, 4.0 equiv) was added to a mixture of Boc-Lys(Fmoc)-OH (20.0 g, 43.0 mmol, 1.0 equiv), H-Tyr-OMe (8.30 g, 43.0 mmol, 1.0 equiv) and HATU (21.0 g, 55.0 mmol, 1.3 equiv) in DMF (150 mL) at room temperature. After stirring at room temperature for 16 h, the reaction mixture was poured into water (400 mL) and extracted with EtOAc (3 x 80 mL). The combined organic phases were dried over sodium sulfate, concentrated and purified by silica gel column chromatography (eluted with MeOH / DCM, 0% to 5%) to give Boc-Lys(Fmoc)-Tyr-OMe (26.0 g, 32.6 mmol, 85% UV purity, 76% yield) as a white solid. LC-MS (Method 5): Rt = 0.71 min, m / z = 668.3 [M + Na] + .

[0956] Step 2. TFA (0.23 g, 2.00 mmol, 2.0 equiv) was added to a solution of Boc-Lys(Fmoc)-Tyr-OMe (1.00 g, 1.00 mmol, 1.0 equiv) in DCM (10 mL) at 0°C. After stirring at room temperature for 1 h, the reaction mixture was concentrated to give H-Lys(Fmoc)-Tyr-OMe (800 mg, 0.70 mmol, 85% UV purity, 70% yield), which was used directly in the next step without further purification. LC-MS (Method 5): Rt = 1.11 min, m / z = 546.3 [M+H] + .

[0957] Step 3. DIEA (11.3 g, 87.8 mmol, 2.4 eq) was added to a mixture of H-Lys(Fmoc)-Tyr-OMe (20.0 g, 36.6 mmol, 1.0 eq), Boc-Cit-OH (10.1 g, 36.6 mmol, 1.0 eq) and HATU (15.3 g, 40.2 mol, 1.1 eq) in DMF (100 mL) at room temperature. After stirring at room temperature for 16 h, the reaction mixture was poured into water (400 mL) and extracted with EtOAc (3 x 80 mL). The combined organic phases were dried over sodium sulfate, concentrated and purified by silica gel column chromatography (eluted with MeOH / DCM, 0% to 5%) to give Boc-Cit-Lys(Fmoc)-Tyr-OMe (12.6 g, 15.4 mmol, 90% Uv purity, 42% yield) as a white solid. LC-MS (Method 5): Rt=1.25 min, m / z=803.4 [M+H] + .

[0958] Step 4. Diethylamine (1.36 g, 18.6 mmol, 3.0 equiv) was added to a solution of Boc-Cit-Lys(Fmoc)-Tyr-OMe (5.00 g, 6.20 mmol, 1.0 equiv) in DCM (10 mL) at room temperature. After stirring at room temperature for 10 h, the reaction mixture was concentrated, washed with Et2O and triturated in Et2O to give Boc-Cit-Lys-Tyr-OMe (3.00 g, 4.59 mmol, 90% UV purity, 74% yield), which was used directly in the next step without further purification. LC-MS (Method 5): Rt = 1.26 min, m / z = 581.4 [M+H] + .

[0959]

[0960] Step 5. DIEA (0.80 g, 6.20 mol, 2.0 eq) was added to a mixture of Boc-Cit-Lys-Tyr-OMe (1.98 g, 3.40 mmol, 1.1 eq), Fmoc-D-Arg(Pbf)-OH (2.00 g, 3.10 mmol, 1.0 eq) and HATU (1.30 g, 3.40 mmol, 1.1 eq) in DMF (30 mL) at room temperature. After stirring at room temperature for 16 h, the reaction mixture was poured into water (300 mL) and extracted with EtOAc (3 x 80 mL). The combined organic phases were dried over sodium sulfate, concentrated, and purified by silica gel column chromatography (eluted with MeOH / DCM, 0% to 5%) to give Boc-Cit-Lys(Fmoc-D-Arg(Pbf))-Tyr-OMe (2.10 g, 2.26 mmol, 95% UV purity, 73% yield) as a white solid. LC-MS (Method 6): Rt=2.08 min, m / z=1211.7 [M+H] + .

[0961] Step 6. LiOH (50 mg, 1.20 mmol, 1.5 eq) was added to a solution of Boc-Cit-Lys(Fmoc-D-Arg(Pbf))-Tyr-OMe (1.00 g, 0.80 mmol, 1.0 eq) in THF / water (1:1, 10 mL) at room temperature. After stirring at room temperature for 10 h, the organic solvent was concentrated and the aqueous solution was then adjusted to pH 2-3 with citric acid. The precipitate was collected by filtration, washed with water, and purified on Biotage Isolera One (C18 column, eluted with 5% to 95% ACN / water containing 0.1% FA) to give Boc-Cit-Lys(D-Arg(Pbf))-Tyr-OH (0.40 g, 0.37 mmol, 90% UV purity, 46% yield) as a white solid. LC-MS (method 5): Rt = 0.98 min, m / z = 975.4 [M+H] + .

[0962] Step 7. Triethylamine (15 mg, 0.15 mmol, 1.5 eq) was added to a mixture of Boc-Cit-Lys(D-Arg(Pbf))-Tyr-OH (100 mg, 0.10 mmol, 1.0 eq), AMAS (28 mg, 0.11 mmol, 1.1 eq) in DMF (1 mL) at room temperature. After stirring at room temperature for 30 min, TFA was added until pH 5-6 was reached. Purification on Biotage Isolera One (C18 column, eluted with 5% to 95% ACN / water containing 0.1% TFA) gave Boc-Cit-Lys(D-Arg(Pbf)-ma)-Tyr-OH (38 mg, 29 μmol, 88% UV purity, 29% yield) as a white solid. LC-MS (method 6): Rt = 1.70 min, m / z = 1112.6 [M+H] + .

[0963] Step 8. Boc-Cit-Lys(D-Arg(Pbf)-ma)-Tyr-OH (10 mg, 9.0 μmol, 1.0 equiv) was dissolved in a mixture of TFA / DCM (1:1, 1 mL) at room temperature. After stirring at room temperature for 30 min, the mixture was concentrated, washed with Et2O and triturated in Et2O to give H-Cit-Lys(D-Arg-ma)-Tyr-OH (6.0 mg, 6.2 μmol, 90% UV purity, 69% yield), which was used directly in the next step without further purification. LC-MS (Method 6): Rt=0.97 min, m / z=758.4 [M+H] + .

[0964] Step 9. HOSu (1.6 mg, 13.8 μmol, 1.1 eq) was added to a mixture of DM1-Ac (10 mg, 12.6 μmol, 1.0 eq) and EDCI (2.6 mg, 13.8 μmol, 1.1 eq) in DMF (1 mL) at room temperature. After stirring at room temperature for 1 h, the reaction mixture was poured into water (40 mL) and extracted with EA (3 x 20 mL). The combined organic phases were dried over sodium sulfate and concentrated in vacuo. The residue was dissolved in DMF (1 mL). H-Cit-Lys(D-Arg-ma)-Tyr-OH (11.4 mg, 12.6 μmol, 1.0 eq) and DIEA (2.44 mg, 18.9 μmol, 1.5 eq) were added to the latter solution at room temperature. After stirring at room temperature for 1 h, TFA was added until pH 5-6 was reached. Purification on Biotage Isolera One (C18 column, eluted with 5% to 75% ACN / water containing 0.1% TFA) gave DM1-Ac-Cit-Lys(D-Arg-ma)-Tyr-OH (3.0 mg, 2.27 μmol, 88% UV purity, 18% yield) as a white solid. LC-MS (Method 5): Rt = 1.01 min, m / z = 1537.5 [M+H] + .

[0965] 8.5 Preparation and Characterization of Antibody-Drug Conjugates (ADCs):

[0966] 8.5.1 Preparation of ADC: General Procedure

[0967] All linker-payloads prepared in Section 8.4 were mixed with (Trastuzumab) was conjugated. Linker-payload DM1-Ac-Cit-Lys(ma-Lys(PEG16))-Tyr-OH and ixatecan-Suc-Phe-Cit-Lys(ma-Lys(PEG16))-Tyr-OH were also conjugated to natalizumab.

[0968] General procedure 1: For DOC 4Tmab conjugation:

[0969] At room temperature, a solution of TCEP.HCl (0.32 mg, 1.09 μmol, 2.3 equivalents) in DPBS (0.32 mL) was added to a solution of trastuzumab (70 mg, 0.45 μmol, 1.0 equivalents) in water (3.33 mL) and DPBS (3.33 mL). The reaction mixture was purged with nitrogen and then stirred at 40 ° C. After stirring at 40 ° C for 70 min, a solution of the linker-payload (3.55 μmol, 7.4 equivalents) in DMSO (0.7 mL) was added. The reaction mixture was stirred at room temperature for 70 min and then diluted to V = 10 mL with 10x pH8 DPBS. Purification was performed using a PF100 column and pH8 DPBS (as an eluent) to obtain a fraction (14 mL) containing the desired ADC. The fraction was stirred at room temperature for 16 h, then centrifuged (10 min, 4000 rpm), and finally the supernatant was transferred to an Amicon concentration pool (15 mL, 50 kDa). The mixture was concentrated to V = 1 mL by centrifugation (4500 rpm, 3800 G), DPBS buffer (14 mL) was added and the mixture was concentrated again (4500 rpm, 3800 G) to V = 1 mL. DPBS buffer (14 mL) was added and the mixture was concentrated again (4500 rpm, 3800 G) to V = 1 mL. The final volume was adjusted to V = 7.0 mL with DPBS buffer. The solution was filtered using a 25 mm PES 0.22 μm Millex filter, then aliquoted and stored at -80 ° C.

[0970] General operation 2: For DOC8Tmab conjugation:

[0971] At room temperature, a solution of TCEP.HCl (1.10 mg, 3.80 μmol, 8.0 equivalents) in DPBS (0.11 mL) was added to a solution of trastuzumab (70 mg, 0.45 μmol, 1.0 equivalents) in water (3.33 mL) and DPBS (3.33 mL). The reaction mixture was purged with nitrogen and then stirred at 40 ° C. After stirring at 40 ° C for 70 min, a solution of the linker-payload (9.60 μmol, 20.0 equivalents) in DMSO (0.7 mL) was added. The reaction mixture was stirred at room temperature for 70 min and then diluted to V = 10 mL with 10x pH8 DPBS. Purification was performed using a PF100 column and pH8 DPBS (as an eluent) to obtain a fraction (14 mL) containing the desired ADC. The fraction was stirred at room temperature for 16 h, then centrifuged (10 min, 4000 rpm), and finally the supernatant was transferred to an Amicon concentration pool (15 mL, 50 kDa). The mixture was concentrated to V = 1 mL by centrifugation (4500 rpm, 3800 G), DPBS buffer (14 mL) was added and the mixture was concentrated again (4500 rpm, 3800 G) to V = 1 mL. DPBS buffer (14 mL) was added and the mixture was concentrated again (4500 rpm.3800 G) to V = 1 mL. The final volume was adjusted to V = 7.0 mL with DPBS buffer. The solution was filtered using a 25 mm PES 0.22 μm Millex filter, then aliquoted and stored at -80 ° C.

[0972] General Procedure 3: For DOC8 Natuximab conjugation:

[0973] At room temperature, a solution of TCEP.HCl (1.10 mg, 3.80 μmol, 8.0 equivalents) in DPBS (0.11 mL) was added to a solution of natalizumab (70 mg, 0.48 μmol, 1.0 equivalents) in a buffer (7.00 mL, 50 mM potassium phosphate, 50 mM potassium chloride, 2 mM EDTA, pH 6.5). The reaction mixture was purged with nitrogen and then stirred at 40 ° C. After stirring at 40 ° C for 70 min, a solution of the linker-payload (9.60 μmol, 20.0 equivalents) in DMSO (0.7 mL) was added. The reaction mixture was stirred at room temperature for 70 min and then diluted to V = 10 mL with 10xpH8 DPBS. Purification was performed using a PF100 column and pH8 DPBS (as an eluent) to obtain a fraction (14 mL) containing the desired ADC. The fraction was stirred at room temperature for 16 h, then centrifuged (10 min, 4000 rpm), and the supernatant was finally transferred to an Amicon concentration pool (15 mL, 50 kDa). The mixture was concentrated to V = 1 mL by centrifugation (4500 rpm, 3800 G), DPBS buffer (14 mL) was added and the mixture was concentrated again (4500 rpm, 3800 G) to V = 1 mL. DPBS buffer (14 mL) was added and the mixture was concentrated again (4500 rpm.3800 G) to V = 1 mL. The final volume was adjusted to V = 7.0 mL with DPBS buffer. The solution was filtered using a 25 mm PES 0.22 μm Millex filter, then aliquoted and stored at -80 ° C.

[0974] 8.5.2 Overview of Characterization of Prepared ADCs

[0975]

[0976] Table 2: Overview of prepared ADCs All characteristics are given in Table 3 below.

[0977]

[0978]

[0979] Table 3: Characterization of prepared ADCs

[0980] The RPLC chromatograms of the prepared ADC are shown in Figures 3 to 6.

[0981] 8.5.3 Preparation of Naratuximab Emtansine (Debio 1562) (Comparative Example)

[0982] Natuximab was reacted with the heterobifunctional cross-linker SMCC and maytansine DM1 using the one-step method described in WO2012135517 A2.

[0983] The DAR is 3.5 (measured by UV at two different wavelengths, 280 nm and 252 nm).

[0984] 8.6 In vitro and in vivo studies

[0985] 8.6.1 In vitro cytotoxicity data

[0986] In vitro cytotoxicity assay: JIMT-1 (HER2 positive) cells were plated and after overnight rest (12 hours), serial dilutions of drugs (trastuzumab, natalizumab, ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, ADC-6, ADC-7 or ADC-9) were added to the cells. After 72 hours of incubation, CellTiter-Glo 2.0 (Promega kit (according to the manufacturer's instructions)) was added to each well and luminescence was read from the luminometer. Relative IC50 was calculated using GraphPad Prism. The results of the in vitro cytotoxicity assay are shown in Figure 2. Figure 7-Figure 11 shown.

[0987] 8.6.2 In vivo efficacy data

[0988] A) Briefly, Jimt-1 breast cancer cells (5x10 per mouse) were used to 6 When the tumor reached 150 mm 3 When the average size of the mice was 1.5, the mice were randomly divided into 3 groups, 8 mice in each group (day 0), and treatment was started on the second day (day 1). In the non-control group of mice, two (100ul) 5mg / kg ADC-2 or ADC-4 intravenous injections were performed at intervals of 1 week. The vehicle was phosphate buffered saline (PBS). The control group of mice received two (100ul) intravenous injections of the vehicle.

[0989] After inoculation of tumor cells, animals were routinely monitored as described below. The mean tumor volume and body weight results of the study are shown in Fig.12 and Fig.13 shown.

[0990] 8.6.3 Routine monitoring

[0991] After inoculation of tumor cells, animals were checked daily for morbidity and mortality. During routine monitoring, animals were checked for tumor growth and any effects of treatment on behavior (such as activity, food and water consumption, weight gain / loss (weight was measured twice a week after randomization), eye / hair matting, and any other abnormalities. Mortality and observed clinical symptoms were recorded in detail for each animal.

[0992] Tumor volume was measured twice weekly in two dimensions using calipers and expressed in mm using the following formula: 3 Volumes are expressed in units: “V = (L x W x W) / 22, where V is tumor volume, L is tumor length (longest tumor dimension), and W is tumor width (longest tumor dimension perpendicular to L). Dosing and tumor and body weight measurements were performed in a laminar flow cabinet.

[0993] B) Briefly, Jimt-1 breast cancer cells (5x10 per mouse) were used to 6 cells) were inoculated into NOD / SCID mice. When the tumor reached 150 mm 3 When the average size of the tumor cells was 1.5 mg / kg, the mice were randomly divided into 5 groups of 8 mice each and the treatment was started on the same day (day 0). In the non-control group of mice, 5 mg / kg ADC-1, ADC-2, ADC-3 or ADC-9 was injected intravenously twice at intervals of 1 week. The vehicle was PBS. The control group of mice received two (100 ul) intravenous injections of the vehicle. After tumor cell inoculation, the animals were routinely monitored as described above. The average tumor volume and body weight results of the study are shown in Figure 2. Fig.14 and Fig.15 shown.

[0994] 8.6.4 In vivo efficacy data

[0995] NMRI nude mice were inoculated with MV4;11 acute myeloid leukemia cells (5x10 per mouse). 6 When the tumor reaches 115 mm 3 When the average size of the tumor was 90.3 g / cm2, the mice were randomly divided into groups of 8 mice (for vehicle) (Group 1) or groups of 6 mice (for treatment conditions) (Groups 2 and 3) (D0), and treatment began on the second day (D1). In non-control mice, a single intravenous injection of 10 mg / kg of natalizumab or ADC-4 was performed. The vehicle was PBS. The control group mice received a single intravenous injection of vehicle (5 mL / kg). After inoculation of tumor cells, the animals were monitored routinely as described above. The average tumor volume results are shown in Figure 2. Fig.16 shown.

[0996] NOD / SCID mice were inoculated in the tail with THP-1 acute myeloid leukemia cells to allow tumor spread (1x10 per mouse). 7 cells) were irradiated with 200rad Co60 24 hours before. The mice were randomly divided (randomized according to the "matching distribution" method / "stratified" method (StudyDirectorTM software, version 3.1.399.19) / randomized block design) into 4 groups of 10 mice in each group (D0), and treatment was started 7 days later (D7). In non-control mice, a single intravenous injection of 5 mg / kg of natalizumab, ADC-2 or ADC-4 was performed. The vehicle was PBS. The control group mice received an injection of vehicle (5 mL / kg). After inoculation of tumor cells, the animals were monitored routinely as described above. The survival rate % results of this study are as follows Fig.17 shown.

[0997] NOD / SCID mice were inoculated into the tail with MOLM-13 luciferase-producing acute myeloid leukemia cells to allow tumor spread (2x10 per mouse). 6 cells). The mice were randomly divided into 4 groups of 8 mice each according to the total flow values ​​3 days after inoculation (D0), and treatment was started 7 days after inoculation (D4). In non-control mice, a single intravenous injection of 5 mg / kg of natalizumab, ADC-2 or ADC-4 was performed. The vehicle was PBS. The control group mice received an injection of vehicle (5 mL / kg). After inoculation of tumor cells, the animals were routinely monitored as described above, and tumor growth was imaged twice a week after Luciferine (using PerkinElmer's IVIS Spectrum BL). Tumor growth and survival rate% Results are shown in Fig.18a and Fig.18b shown.

[0998] NOD / SCID mice were inoculated into the tail with MOLM-13 luciferase-producing acute myeloid leukemia cells to allow tumor spread (2x10 per mouse). 6cells). Mice were randomly divided into 5 groups of 8 mice each according to the total flow values ​​7 days after inoculation, and treatment was started on the day of randomization (D0). In non-control mice, an intravenous injection of 3mg / kg ADC-4, 1mg / kg ADC-4, 0.3mg / kg ADC-4 or 3mg / kg Encinatuximab (Debio-1562) was performed. The vehicle was PBS. The control group mice received an injection of vehicle (5mL / kg). After inoculation of tumor cells, the animals were routinely monitored as described above, and tumor growth was imaged twice a week after administration of Luciferine (using PerkinElmer's IVISSpectrum BL). Tumor growth and survival rate% Results are shown in Fig.19a , Fig.19b , Fig.20a and Fig.20b shown.

[0999] 8.6.5 Cell Binding, Internalization, and Cytotoxicity in AML Cell Lines

[1000] AML cell lines MV-4-11, MOLM-13, HL-60, and THP-1 expressing CD37 were labeled with Live / Dead near IR (Thermo Fisher, L10119), Fc was blocked with HumanTruStain FcX (Biolegend, 422302), and incubated with 1 μg / mL of natuximab-PE (Biolegend, 99341, lot B304638, custom conjugate). Phycoerythrin fluorescent quantitative beads (BD, 340495) were used as a reference for estimating the natuximab antibody (ABC) bound per cell. Cells and beads were finally obtained on the Attune NxT flow cytometer. The CD37-negative ALL cell line MOLT-4 was included as a negative control. The number of natuximab antibodies bound per cell is shown in Figure 2. Fig.21a shown.

[1001] AML cell lines MV-4-11, MOLM-13, HL-60 and THP-1 cells expressing CD37 were labeled with Live / Dead near IR (Thermo Fisher, L10119) and then incubated with 0.5 μg / mL of anti-CD37-Alexa Fluor 488 (clone K7153A) on ice or at 37°C for 30 min or 2 h. The cells were washed twice and incubated with quenched anti-AlexaFluor 488 antibody (Thermo fisher, 710369) and then fixed (Biolegend, 422101). The fixed cells were analyzed by flow cytometry. The data are expressed as absolute internalization rate + / - SD (n = 3), which is defined as the fluorescence of the quenched sample corrected for incomplete surface quenching. The CD37-negative ALL cell line MOLT-4 was included as a negative control. The absolute internalization rate of natalizumab is as shown in Figure 21b shown.

[1002] In vitro cytotoxicity assay: MOLT-4, MV-4-11, MOLM-13, HL-60 and THP-1 cells were plated and after overnight standing (12 hours), serial dilutions of ADC-4 were added to the cells (8 10-fold dilutions, from 1 μM to 0.1 pM, in triplicate). After 72 hours of incubation, the plates were checked under an inverted microscope to ensure growth and sterile conditions of the control. Then, 100 μL CellTiter Glo 2.0 (Promega, G9242) was added to each well, and the luminescence values ​​were read according to the manufacturer's instructions. The relative IC50 for each cell line was calculated using GraphPad Prism. The percentage viability curve relative to the control (untreated cells) is shown in Figure 2. Fig.21c shown.

[1003] 8.6.6 In vitro cytotoxicity data

[1004] MV-4-11 cells were plated and after overnight rest (12 hours), serial dilutions of ADC-4, Debio 1562, or natalizumab were added to the cells (eight 10-fold dilutions, from 1 μM to 0.1 pM, in triplicate). After 72 hours of incubation, the plates were checked under an inverted microscope to ensure growth and sterile conditions of the control. Then, 100 μL CellTiter Glo 2.0 (Promega, G9242) was added to each well, and the luminescence values ​​were read according to the manufacturer's instructions. The relative IC50 for each cell line was calculated using GraphPadPrism. The viability percentage curve relative to the control (untreated cells) is shown in Figure 2. Fig.22a shown.

[1005] THP-1 cells were plated and after overnight rest (12 hours), serial dilutions of ADC-4 or Debio 1562 were added to the cells (9 10-fold dilutions, from 1 μM to 0.01 pM, in triplicate). After 72 hours of incubation, the plates were checked under an inverted microscope to ensure growth and sterile conditions of the control. Then, 100 μL CellTiter Glo2.0 (Promega, G9242) was added to each well, and the luminescence values ​​were read according to the manufacturer's instructions. The relative IC50 for each cell line was calculated using GraphPad Prism. The percentage of viability relative to the control (untreated cells) is shown in the curve. Figure 22b shown.

[1006] 8.6.7 In Vivo Efficacy Compared to Standard of Care for AML

[1007] NCG mice were inoculated with MV4;11-Luc AML cells via the tail vein to allow tumor dissemination (2x10 per mouse). 7 cells). Based on the bioluminescence results of each animal 14 days after inoculation (total flux, photons / second / cm 2 ), the mice were randomly divided into 4 groups, 8 mice in each group, and treatment was started immediately after random differentiation (D14). In non-control mice, an intravenous injection of 1 mg / kg or 5 mg / kg ADC-4 was performed, or on days 1 to 6 and 9 to 14, in addition to oral administration of 100 mg / kg Venetoclax, 5 consecutive daily intravenous injections of 3.5 mg / kg azacitidine were performed. The vehicle was PBS except Venetoclax, which was prepared in 60% phosal 50 propylene glycol, 30% polyethylene glycol 400 and 10% ethanol. The control group mice received an injection of the vehicle (10 μL / g). After inoculation of tumor cells, the clinical symptoms, morbidity and mortality of the animals were monitored daily, and body weight was measured twice a week. Twice a week, 15 minutes after the injection of D-luciferin (PerkinElmer, XenoLight D-Luciferin K+Salt), tumor growth was imaged using PerkinElmer's IVISSpectrum BL. Tumor growth results in luminescent signals were as follows Fig.23 shown.

[1008] 8.6.8 Pharmacokinetic profiles in mice

[1009] Three Swiss male mice were treated with 5 mg / kg natalizumab or ADC-4 (vehicle was PBS) by intravenous injection, and blood was collected by microsampling using K3EDTA microcapillaries at 5 min, 1 h, 6 h, 24 h, 48 h and 72 h after treatment. Microcapillary blood samples were centrifuged as quickly as possible (2000 g, 5 min, +4 ° C) to allow plasma processing. Plasma samples were frozen at -80 ° C and further analyzed using a qualified analytical immunoassay procedure to determine the concentration of total antibodies. Toxicokinetic parameters were estimated using Phoenix pharmacokinetic software. PK parameters and plasma concentrations were as follows Fig.24a shown.

[1010] Three CD1 female mice were treated with 5 mg / kg ADC-4 (vehicle was PBS) by intravenous injection, and blood was collected by microsampling using K2EDTA microcapillaries at 5 min, 1 h, 6 h, 24 h, 48 h and 96 h after treatment. Microcapillary blood samples were centrifuged as quickly as possible (1500 g, 10 min, +4 ° C) to allow plasma processing. Plasma samples were frozen at -80 ° C and further analyzed using two qualified analytical immunoassay procedures to determine the concentration of total antibodies (total Ab) and total ADC. Toxicokinetic parameters were estimated using Phoenix pharmacokinetic software. PK parameters and plasma concentrations were as follows Figure 24b And as shown in Table 4.

[1011]

[1012] Table 4

[1013] 8.6.9 Human and mouse plasma stability

[1014] ADC-2, ADC-4, Enhertu and Adcetris (Enhertu and Adcetris were used as controls) were spiked into human and mouse plasma at a concentration of 1 mg / mL. After incubation for 0, 24, 48 and 96 hours, ADC was captured with streptavidin magnetic beads (100 μL conjugate / 250 μL bead slurry) coated with biotin anti-IgG-Fc (human) conjugate. For ADC-2, 40 μL of plasma spiked with ADC was mixed with 60 μL PBS and incubated with 20 μL beads at room temperature at 900 rpm (Eppendorf thermomixer) for 1 hour. For ADC-4, Enhertu and Adcetris, 20 μL of plasma spiked with ADC was mixed with 80 μL PBS and incubated with 20 μL beads at room temperature at 900 rpm (Eppendorf thermomixer) for 1 hour. The beads were then washed 3 times with 500 μL PBS, eluted with 2 mM HCl solution, and neutralized with 0.5 M ammonium bicarbonate. ADC-2 and Adcetris were deglycosylated (PNGase F), and Adcetris was reduced with DTT. Samples were analyzed by LC-MS (Waters BioAccord). The DAR reduction of ADC was measured and is shown in Fig.25a (ADC-2) and Fig.25b (ADC-4).

[1015] 8.6.10 In vitro cytotoxicity against a panel of DLBCL cell lines

[1016] A3 / KAW, DOHH-2, HBL-1, KARPAS-422, OCI-LY19, OCI-LY3, OCI-LY7, Pfeiffer, U-DHL-2, SU-DHL-4, SU-DHL-5, SU-DHL-6, SU-DHL-8, Toledo, U-2932, WSU-DLCL2 and WSU-NHLL cells were plated on days 0 and 1, and propagation dilutions (9 10-fold dilutions, from 1 μM to 0.01 pM, in triplicate) with ADC-4, Debio 1562 or natalizumab were added to the cells. After incubation for 72 hours, the plates were checked under an inverted microscope to ensure growth and sterile conditions of the control. Then, 50 μL CellTiter Glo (Promega, G7572) was added, and luminescence readings were performed according to the manufacturer's instructions. Relative IC50 was calculated using GraphPad Prism. The IC50 values ​​of ADC-4, Debio 1562 and natalizumab are shown in Table 5.

[1017]

[1018] Table 5

[1019] 8.6.11 In vivo Efficacy in DLBCL Models

[1020] SCID beige female mice were inoculated with FarageDLBCL cells in the tail to allow tumor spread (2x10 per mouse). 7 cells). Mice were randomly divided into 6 groups of 10 mice each according to their body weight 7 days after inoculation, and treatment was started on the day of randomization (D0). In non-control mice, an intravenous injection of 1 mg / kg ADC-4 or 10 mg / kg Debio1562 was performed alone or in combination with 3 weekly intravenous injections of 10 mg / kg rituximab. The vehicle was PBS. Control mice received an injection of vehicle (5 mL / kg). After inoculation of tumor cells, animals were routinely monitored for clinical symptoms, morbidity, and mortality, and body weight was measured twice a week. Survival endpoint results are shown in Figure 2. Fig.26 shown.

[1021] 8.6.12 Screening Studies Using 5 and 50 mg / kg of ADC-4

[1022] The purpose of this study was to determine the potential toxicity of ADC-4 when administered intravenously (by bolus injection) as a single dose to female mice. In addition, the toxicokinetic characteristics of ADC-4 (as total antibody (TAb) and total ADC) were determined.

[1023] The study design is as follows:

[1024]

[1025] Table 6: Experimental design ( a Based on most recent weight measurement)

[1026] The following parameters and endpoints were evaluated in this study: mortality, clinical observations, body weight, food consumption, ophthalmology, clinical pathology parameters (hematology and clinical chemistry), toxicokinetic parameters, organ weights, and macroscopic and microscopic examinations.

[1027] Give IGS female mice were injected intravenously with a single dose of 5 mg / kg or 50 mg / kg of ADC-4. All animals administered a single dose of 5 or 50 mg / kg of ADC-4 survived to termination. The median values ​​of total ADC and TAb were observed at the first sampling time (5 minutes after administration). max Total ADC and TAb (average C0, C max and / or AUC tlastThe systemic exposure of ADC-04 increased in an approximately dose-proportional manner as the dose of ADC-04 increased from 5 mg / kg to 50 mg / kg. After administration of 5 and 50 mg / kg of ADC-4, the exposure to TAb in plasma (mean C0, C max and / or AUC tlast ) is slightly higher than the total ADC.

[1028] There were no clinical symptoms associated with ADC-4 in animals that had been administered a single dose of 5 mg / kg or 50 mg / kg of ADC-4. There were no ophthalmological findings at either of these two dose levels. Ten days after administration, an increase in reticulocyte counts (increase or non-increase in leukocyte, neutrophil, eosinophil, monocyte, and lymphocyte counts) was noted, as well as an increase in triglyceride, total protein, globulin concentration, and calcium and / or phosphorus levels.

[1029] Autopsy revealed that ADC-4 caused splenomegaly, the incidence of which was dose-related (which was associated with increased spleen weight and splenic extramedullary hematopoiesis (EMH)). No correlation was noted for increases in kidney and liver weights. There were no significant microscopic findings 10 days after injection, and local injections of ADC-4 were well tolerated.

[1030] 8.6.13 Dose Range Finding (DRF) ADC-4—Determination of the potential toxicity of ADC-4.

[1031] Towards IGS female mice were intravenously administered ADC-4 (two bolus injections of the same dose (25 mg, 50 mg or 100 mg / kg / adm) on days 1 and 8 (7 days apart), respectively) and the reversibility and / or delayed occurrence of any examined outcome during the 10-day observation period were evaluated. In addition, the toxicokinetic characteristics of ADC-4 (total ADC and total antibody) were determined.

[1032] A control group of IGS female mice received a control item: phosphate buffered saline (PBS).

[1033] The study design is as follows:

[1034]

[1035] Table 7: Experimental design ( a Based on most recent weight measurement)

[1036] The following parameters and endpoints were evaluated in this study: mortality, clinical observations, body weight, food consumption, ophthalmology, clinical pathology parameters (hematology and clinical chemistry), toxicokinetic parameters, organ weights, and macroscopic and microscopic examinations.

[1037] All animals that received 2 injections of ADC-4 at 100 mg / kg / adm were prematurely euthanized or found dead on day 10 due to severe weight loss and decreased food consumption accompanied by erected fur, hunched posture, and decreased activity. There were no relevant ADC-4-related clinical signs in animals that received 2 injections of ADC-4 at 25 mg / kg / adm or 2 injections of ADC-4 at 50 mg / kg / adm, and there were no premature deaths in these groups of animals.

[1038] In animals receiving 2 injections of ADC-4 at 25 mg / kg / ADM or 2 injections of ADC-4 at 50 mg / kg / ADM, higher mean body weight gains (when compared to mean control values) were noted between Days 1 and 17, with no effect on food consumption. No ophthalmic findings were found.

[1039] In mice receiving 2 injections of ADC-4 at 100 mg / kg / adm, a significant increase in total leukocyte counts was noted two days after the second injection (mainly due to increased neutrophil and monocyte counts). A reduction in erythrocyte, reticulocyte and platelet counts and hematocrit was also noted. Significant increases in enzyme activity (AST, ALT and ALP) were observed, with moderate increases in total bilirubin, total protein and especially globulin concentrations (therefore A / G ratios were reduced), and cholesterol, triglyceride and glucose concentrations were reduced. An increase in phosphorus concentration was also noted.

[1040] In mice that received two injections of ADC-4 at 25 mg / kg / adm or two injections of ADC-4 at 50 mg / kg / adm, an increase in total leukocyte counts (when compared to mean control values) was noted 10 days after the second injection (primarily due to increased neutrophil, lymphocyte, and monocyte counts). A decrease in platelet count was noted in animals that received two injections of ADC-4 at 50 mg / kg / adm (this was not observed in animals that received two injections of ADC-4 at 25 mg / kg / adm).

[1041] Significant changes in clinical chemistry consisted of increases in enzyme activities (AST, ALT, and ALP), total protein (particularly albumin concentration), and decreases in triglyceride concentrations (which were generally more pronounced in animals receiving 2 injections of ADC-4 at 50 mg / kg / ADM).

[1042] Under the microscope, the following findings were noted:

[1043] In animals receiving 2 injections of ≥25 mg / kg / adm, ADC-4-related findings were seen in the liver (diffuse hepatocellular hypertrophy, increased extramedullary hematopoiesis and mitosis, associated with higher weight and total volume increase), spleen (dose-related extramedullary hematopoiesis, associated with higher weight and total volume increase / pallor and single-cell necrosis), and duodenum (single-cell necrosis of epithelial cells of mucosa and submucosal glands). At the injection site, subacute perivascular inflammation reflected a mild local tissue effect of ADC-4.

[1044] In animals receiving 2 injections ≥ 50 mg / kg / adm, ADC-4-related findings occurred in the bone marrow (single cell necrosis, increased myeloid cellularity, decreased erythroid cellularity), liver (single cell necrosis and findings observed at 25 mg / kg / adm), heart (myocardial degeneration / necrosis and extramedullary hematopoiesis), and eye (single cell necrosis in the corneal epithelium).

[1045] In animals receiving 2 injections of 100 mg / kg / adm, ADC-4-related findings occurred in the liver (glycogen depletion), spleen (decreased white pulp cellularity), kidney (single cell necrosis of epithelial tubular and glomerular cells), digestive tract (single cell necrosis in stomach, jejunum, and ileum, erosions / ulcers in duodenum, jejunum, and ileum, epithelial hypertrophy / hyperplasia in duodenum and jejunum), iliac lymph nodes (single cell necrosis and extramedullary hematopoiesis), and skin (extramedullary hematopoiesis).

[1046] In summary:

[1047] · Mice receiving two 100 mg / kg / adm injections of ADC-4 (7 days apart) were not tolerated; all animals were prematurely euthanized or found dead on day 10 due to weight loss, obvious clinical signs, and decreased appetite. In addition, an increase in white blood cell counts and a decrease in red blood cell, reticulocyte, and platelet counts and hematocrit were noted, associated with necrosis and decreased cellularity of the spleen.

[1048] · Mice received two 25mg / kg / adm or 50mg / kg / adm ADC-4 injections (7 days apart) and were well tolerated clinically. Administration induced an increase in leukocyte counts and a decrease in platelet counts (associated with extramedullary hematopoiesis in the spleen and liver) after 18 days. There was no effect on food consumption or body weight, and no clinical signs.

[1049] Target organ effects were observed at all dose levels in the liver, spleen, digestive tract, kidneys, bone marrow, iliac lymph nodes, heart, eyes, and / or skin. The severity and extent of findings were dose-related.

[1050] Based on the above results, the no observed adverse effect level (NOAEL) of ADC-4 in mice was considered to be 25 mg / kg / adm.

[1051] 8.7 Cat B-induced cleavage studies

[1052] 8.7.1 Preparation of DM1-Ac-Cit and DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH

[1053] DM1-Ac-Cit

[1054]

[1055] DIEA (0.14 mL, 0.83 mmol, 4.0 equiv) was added to a mixture of DM1-Ac-NHS ester (185 mg, 0.21 mmol, 1.0 equiv) and citrulline (72.6 mg, 0.41 mmol, 2.0 equiv) in DMSO (3.7 mL) at room temperature. After stirring at room temperature for 18 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (10 to 40% ACN + 0.1% TFA in water + 0.1% TFA) gave DM1-Ac-Cit (130 mg, 0.14 mmol, 100% UV purity, 66% yield) as a white powder after freeze drying. UPLC-MS (Method 1): Rt = 1.33 min, m / z = 952 [MH] - .

[1056] DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH

[1057]

[1058] Acetyl-L-cysteine ​​(0.32 mg, 2.17 μmol, 1.0 equiv) was added to a solution of DM1-Ac-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (5.0 mg, 2.17 μmol, 1.0 equiv) in DMF (1 mL) at room temperature. After stirring at room temperature for 40 min, TFA was added until an acidic pH was reached. Purification by preparative HPLC (5 to 100% ACN+0.1% TFA in water+0.1% TFA) gave DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH (4.59 mg, 1.86 μmol, 99% UV purity, 94% yield) as a white powder after freeze drying. UPLC-MS (method 4): Rt = 2.41 min, m / z = 1231 [M-2H] 2- .

[1059] 8.7.2 Preparation of Exatecan-Suc-Phe-Cit and Exatecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(Peg16))-Tyr-OH

[1060] Exatecan-Suc-Phe-Cit

[1061]

[1062] Step 1. DIEA (0.78 mL, 4.48 mmol, 4.0 equiv) was added to a mixture of citrulline (200 mg, 1.12 mmol, 1.0 equiv) and Boc-Phe-OSu (405 mg, 1.12 mmol, 1.0 equiv) in DMF (10 mL) at room temperature. After stirring at room temperature for 16 h, the reaction mixture was filtered and then concentrated under reduced pressure. A mixture of water / ACN / TFA (1:1:0.5%) was added. The mixture was filtered and then freeze-dried. Purification by preparative HPLC (10 to 60% ACN + 0.1% TFA in water + 0.1% TFA) gave Boc-Phe-Cit-OH (86 mg, 0.20 mmol, 100% UV purity, 18% yield) as a white powder after freeze-drying. UPLC-MS (Method 3): Rt = 1.27 min, m / z = 423 [M+H] + ,421[MH] - .

[1063] Step 2. A mixture of DCM (0.9 mL) and TFA (0.9 mL) was added to Boc-Phe-Cit-OH (60 mg, 0.14 mmol, 1.0 equiv) at room temperature. After stirring at room temperature for 20 min, the reaction mixture was concentrated. Water (10 mL) and ACN (10 mL) were added and the mixture was freeze-dried to give H-Phe-Cit-OH (30 mg, 83.8 μmol, 90% UV purity, 59% yield) as a white powder. UPLC-MS (Method 3): Rt = 0.35 min, m / z = 323 [M+H] + ,321[MH] - .

[1064] Step 3. Dihydrofuran-2,5-dione (8.4 mg, 83.5 μmol, 1.0 equiv) was added to a mixture of exatecan (36 mg, 83.5 μmol, 1.0 equiv) and DIEA (0.17 mL, 1.00 mmol, 12 equiv) in DMF (0.8 mL) at room temperature. After stirring at room temperature for 10 min, 1-hydroxypyrrolidine-2,5-dione (9.6 mg, 83.5 μmol, 1.0 equiv) was added, followed by HATU (31.7 mg, 83.5 μmol, 1.0 equiv). After stirring at room temperature for 15 min, H-Phe-Cit-OH (29.9 mg, 83.5 μmol, 1.0 equiv) was added to the reaction mixture. After stirring at room temperature for 1 h, H-Phe-Cit-OH (29.9 mg, 83.5 μmol, 1.0 equiv) was added to the reaction mixture. After stirring at room temperature for 1 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20 to 60% ACN + 0.1% TFA in water + 0.1% TFA) gave Exatecan-Suc-Phe-Cit (26.4 mg, 30.8 mmol, 98% UV purity, 37% yield) as a yellow powder after freeze drying. UPLC-MS (Method 2): Rt = 1.39 min, m / z = 840 [M+H] + ,838[MH] - .

[1065] Exatecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(Peg16))-Tyr-OH

[1066]

[1067] DIEA (1.2 μL, 6.8 μmol, 4.0 eq) was added to a mixture of exatecan-Suc-Phe-Cit-Lys(ma-C1-Lys(Peg16))-Tyr-OH (3.71 mg, 1.7 mmol, 1.0 eq) and acetyl-L-cysteine ​​(0.28 mg, 1.7 μmol, 1.0 eq) in DMF (0.6 mL) at room temperature. After stirring at room temperature for 1 h, TFA was added until an acidic pH was reached. Purification by preparative HPLC (20 to 60% ACN + 0.1% TFA in water + 0.1% TFA) gave Isanotecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(Peg16))-Tyr-OH (2.87 mg, 1.2 mmol, 100% UV purity, 72% yield) as a white powder after freeze drying. UPLC-MS (Method 4): Rt = 2.37 min, m / z = 1176 [M+2H] 2+ ,1174[M-2H] 2- .

[1068] 8.7.3 Cat B-induced cleavage

[1069] Cat B-induced cleavage of linker payloads (DM1-Ac-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH and ixatecan-Suc-Phe-Cit-Lys(Ac-Cys-ma-Lys(PEG16))-Tyr-OH) (Ac-Cys-quenched linkers of the present invention) was evaluated according to an in vitro enzymatic cleavage assay using recombinant human cathepsin B (commercially available from R&D Systems, Bio-Techne AG, catalog number 953-CY-010, in precursor form) and UHPLC-MS / MS analysis.

[1070] The enzyme was reconstituted in 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer adjusted to pH 5.0 with 1 M NaOH solution and then activated with 20 nM dithiothreitol (DDT) solution at room temperature for at least 15 min. In the presence of 2 μg / mL of activated recombinant human cathepsin B enzyme in 25 mM MES buffer (pH 5.0), in vitro enzymatic assays were performed at 37°C with a concentration of 10 μM (2.5 μM when the test compound was an antibody-drug conjugate). At each determined time point, the enzymatic cleavage reaction was stopped by mixing a 1 / 10 ratio volume of acetonitrile + 0.1% formic acid (FA) containing an internal standard (0.5 μM warfarin). Analysis was performed using a Waters AcquityUPLC system coupled to a Waters Xevo TQ triple quadrupole mass spectrometer. UHPLC used an HSS T3 1.8m50 x 2.1mm column (Waters), the column temperature was 45°C, equipped with a 2m pre-filter (Waters), solvent system A1 (H2O + 0.1% FA) and B1 (acetonitrile + 0.1% FA), the flow rate was 0.6mL / min, and the B1 gradient was 5-95% in 2.0min. MS / MS was performed in positive ion mode using an electrospray ionization (ESI) interface, and specific MRM conversions were performed for each compound. The results are shown in Fig. 27 and Fig.28 shown.

[1071] 8.8DRF-DM1-Ac-Cit and DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH

[1072] The purpose of this study was to determine the potential toxicity of Multilink-DM1-Ac-Cit and DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH when administered intravenously (by bolus injection).

[1073] DM1-Ac-Cit was prepared as described above in Section 8.7.1.

[1074] Preparation of DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH

[1075]

[1076] Cysteine ​​(9.15 mg, 75.5 μmol, 2.0 equiv) was added to a solution of DM1-Ac-Cit-Lys(ma-Lys(PEG16))-Tyr-OH (86.9 mg, 37.8 μmol, 1.0 equiv) in DMF (7.6 mL) at room temperature. After stirring at room temperature for 5 h, the reaction mixture was filtered using a 33 mm 0.22 μm hydrophobic filter. Purified by preparative HPLC (10 to 50% ACN+0.1% TFA in water+0.1% TFA), DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH (62.4 mg, 25.8 μmol, 100% UV purity, 68% yield) was obtained as a white powder after freeze drying. UPLC-MS (method 1): Rt = 1.34 min, m / z = 1210 [M-2H] 2- .

[1077] Towards IGS mice were injected twice (on day 1 and day 8 (7 days apart) with the same dose of DM1-Ac-Cit (0.15, 0.68 or 1.4 mg / kg / adm (test item 1)) or twice with the same dose of DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH (3.5 mg / kg / adm (test item 2)), and the reversibility and / or delayed occurrence of any examination results were evaluated during the 10-day observation period. In addition, the toxicokinetic characteristics of DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH, DM1-Ac-Cit and the decomposition product DM1 were determined.

[1078] The control group received vehicle control 1: 8% ethanol / 8% polysorbate 80 / PBS 84%.

[1079] The study design is as follows:

[1080]

[1081] Table 8: Experimental design (adm = administration, a Based on most recent weight measurement)

[1082] The following parameters and endpoints were evaluated in this study: mortality, clinical observations, body weight, food consumption, ophthalmology, clinical pathology parameters (hematology and clinical chemistry), toxicokinetic parameters, organ weights, and macroscopic and microscopic examinations.

[1083] There were no DM1-Ac-Cit- or DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH-related mortality, clinical observations, or effects on body weight and food consumption.

[1084] There were no DM1-Ac-Cit- or DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH-related changes in ophthalmologic findings or clinical pathology parameters.

[1085] No DM1-Ac-Cit- or DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH-related changes in organ weights, nor macroscopic and microscopic findings were observed.

[1086] In summary:

[1087] Two intravenous (bolus) injections of DM1-Ac-Cit at 0.15, 0.68, and 1.4 mg / kg / adm given 7 days apart were given in females. Based on these results, the no observed adverse effect level (NOAEL) was considered to be 1.4 mg / kg / adm.

[1088] Likewise, 3.5 mg / kg / adm of DM1-Ac-Cit-Lys(Cys-ma-Lys(PEG16))-Tyr-OH was also well tolerated.

[1089] Reference Example: Cat B-induced cleavage studies

[1090] The reference compounds shown in Table 9 below were prepared in Reference Example according to the method described in WO 2019 / 096867 A1.

[1091] Compound structure 1 <![CDATA[AF-Arg-Lys(PEG4-Mal-Cys-Ac)-Phe-OH]]> 2 <![CDATA[AF-Arg-Lys(PEG4-Mal)-Phe-OH]]> 3 <![CDATA[AF-Arg-Phe-Lys(PEG4-Mal-Cys-Ac)-OH]]> 4 <![CDATA[AF-Arg-Phe-Lys(PEG4-Mal)-OH]]> 5 <![CDATA[DM1-Mal-Phe-Lys-Lys(PEG4-Mal-Cys-Ac)-Phe-OH]]> 6 <![CDATA[DM1-Mal-Phe-Cit-Lys(PEG4-Mal-Cys-Ac)-Phe-OH]]> 7 <![CDATA[DM1-Mal-Phe-Cit-Phe-Lys(PEG4-Mal-Cys-Ac)-OH]]> 8 <![CDATA[AF-Cit-Lys(PEG4-Mal-Cys-Ac)-Phe-OH]]> 9 <![CDATA[ACit-Lys(PEG4-Mal-Cys-Ac)-Phe-OH]]> 10 <![CDATA[ACit-Phe-Lys(PEG4-Mal-Cys-Ac)-OH]]> 11 <![CDATA[DM1-Mcc-Phe-Cit-Lys(PEG5-Ma-Cys-Ac)-Tyr-OH]]> 12 <![CDATA[DM1-Mcc-Cit-Lys(PEG5-Ma-Cys-Ac)-Tyr-OH]]> 13 <![CDATA[DM1-Mcc-Phe-Lys(PEG5-Ma-Cys-Ac)-Tyr-OH]]>

[1092] Table 9: Reference compounds containing linkers of formula (II) / (II')

[1093] The peptides were prepared by Fmoc-based SPPS using an Activo P-11 automated peptide synthesizer (available from Activotec) and Fmoc-Xxx-Wang resin (Xxx: C-terminal amino acid; loading: 0.60 mmol / g; Bachem).

[1094] In the presence of DIEA (7 eq.), 3 eq. of Fmoc-amino acid, Fmoc-NH-PEG4-COOH or Fmoc-NH-PEG5-COOH activated with HBTU (2.9 eq.) were used to perform the coupling reaction to form an amide bond at room temperature for more than 30 min. Fmoc deprotection was performed with 20% piperidine in DMF. Selective removal of the Mtt side chain protecting group (Lys) was performed using DCM / TFA / TIS (94 / 1 / 5, v / v / v).

[1095] For the synthesis of compounds 1 to 4 and 8, after removing Fmoc by fragment condensation (3 equivalents AF, 2.9 equivalents HBTU, 7 equivalents DIEA) within 30 min, auristatin F (AF) was coupled. For the synthesis of compounds 9 and 10, auristatin Cit (ACit) was coupled under the same conditions (3 equivalents ACit, 2.9 equivalents HBTU, 7 equivalents DIEA) after removing Fmoc.

[1096] For the synthesis of compounds 1 to 4 and 8 to 10, after removing Mtt by DCM / TFA / TIS (94 / 1 / 5, v / v / v), the derivative Mal-PEG4-NHS was added to the resin for 30 min (3 equivalents of Mal-PEG4-NHS, 7 equivalents of DIEA). Then, for compounds 1, 3, 8, 9 and 10, the maleimide residue on the PEG chain was reacted with acetyl-cysteine ​​(Ac-Cys-OH) on the resin by chemoselective ligation (3 equivalents of Ac-Cys-OH, DIEA, 7 equivalents) for 20 min. a / water (95 / 2.5 / 2.5, v / v / v; a The peptide was cleaved from the resin with simultaneous side chain deprotection by treatment with triisopropylsilane (TIS) for 60 min. After concentration of the cleavage mixture, the crude peptide was precipitated with cold ether and centrifuged.

[1097] For the synthesis of compounds 5 to 7, after removing Mtt by DCM / TFA / TIS (94 / 1 / 5, v / v / v), the derivative Mal-PEG4-NHS was added to the resin for 30min (3 equivalents of Mal-PEG4-NHS, 7 equivalents of DIEA). Then, the maleimide residue on the PEG chain was chemoselectively connected to the resin with acetyl-cysteine ​​(Ac-Cys-OH) for 20min by maleimide and thiol (3 equivalents of Ac-Cys-OH, DIEA, 7 equivalents). After Fmoc deprotection, the Mal-derivative was inserted by adding the Mal-NHS part to the N-terminus of Phe. By treating with TFA / TIS / water (95 / 2.5 / 2.5, v / v / v) for 60 minutes, the peptide was cut off from the resin under the simultaneous side chain deprotection. After concentrating the cleavage mixture, the crude peptide was precipitated with cold ether and centrifuged. Then, maytansine (DM1, 1.45 equivalents) was reacted with the terminal maleimide group by chemoselective ligation in PBS buffer at pH 7.4 and acetonitrile (ratio 2:1).

[1098] For the synthesis of compounds 11 to 13, after removing Fmoc, the derivative Ma-NHS was added to the resin for 30 min (3 equivalents of Mal-NHS, 7 equivalents of DIEA). Then, the maleimide residue was reacted with acetyl-cysteine ​​(Ac-Cys-OH) by chemical selective connection on the resin within 20 min (3 equivalents of Ac-Cys-OH, DIEA, 7 equivalents). In the case of simultaneous side chain deprotection, the peptide was cut off from the resin by treating with TFA / TIS / water (95 / 2.5 / 2.5, v / v / v) for 60 minutes. After concentrating the cleavage mixture, the crude peptide was precipitated with cold ether and centrifuged. After purification, the derivative DM1-smcc (1.1 equivalents) was reacted with the N-terminus of the joint in a DMF and 4-methylmorpholine (6 equivalents) solution for 4 h.

[1099] Solvent systems A (0.1% TFA in water) and B (0.1% TFA in acetonitrile) were used at a flow rate of 24 mL / min and a gradient of 20-60% B in 30 min on a column equipped with XSelect Peptide CSH C18 OBD Prep ( The peptides were purified on a Waters Autopurification HPLC system coupled to a SQD mass spectrometer (5 μm, 19 mm x 150 mm).

[1100] The appropriate fractions were concentrated and lyophilized. 1.7 μm, 2.1 mm x 50 mm) (using solvent system A (0.1% FA in water) and B (0.1% FA in acetonitrile) or CSH fluoro-phenyl column ( Purity was determined on a Waters Acquity UPLC system coupled to a SQD mass spectrometer (1.7 μm, 2.1 mm x 50 mm) (using solvent systems A (0.1% FA in water) and B (0.1% FA in acetonitrile) at a flow rate of 0.9 mL / min and a gradient of 5-95% B in 2.9 min).

[1101] MS analysis was performed using an electrospray ionization (ESI) interface in positive and negative modes. The analysis results of the compounds obtained in the reference examples are shown in Table 10 below.

[1102]

[1103]

[1104] Table 10: Analysis of Compounds 1-13

[1105] The propensity of Compounds 1-13 to be cleaved by cathepsin B was assessed based on an in vitro enzymatic cleavage assay using recombinant human cathepsin B and UHPLC-MS / MS analysis as described below.

[1106] Reference compounds Cys-MC-Val-Cit-PABC-MMAF and MMAF were used as positive controls. The enzyme was reconstituted in 25 mM MES buffer adjusted to pH 5.0 with 1 M NaOH solution and then activated with 20 nM DDT solution for at least 15 min at room temperature.

[1107] In vitro enzymatic assays were performed at 37° C. with test compounds at a concentration of 10 μM (2.5 μM when the test compound was an antibody-drug conjugate) in the presence of 2 μg / mL of activated recombinant human cathepsin B enzyme in 25 mM MES buffer pH 5.0. At each defined time point, the enzymatic cleavage reaction was stopped by mixing an equal volume of acetonitrile + 0.1% FA containing an internal standard (8 μM warfarin).

[1108] The analysis was performed using a Waters Acquity UPLC system coupled to a Waters Xevo TQ triple quadrupole mass spectrometer. UHPLC was performed using a BEH C8 1.7 μm 100 x 2.1 mm or BEH C18 1.7 μm 50 x 2.1 mm or HSST3 1.7 μm 50 x 2.1 mm column heated to 45°C or 50°C and equipped with a 2 μm plug-in filter pre-column (available from Waters), depending on the test compound, solvent systems A1 (H2O + 0.1% FA) and B1 (acetonitrile + 0.1% FA) at a flow rate of 0.6 mL / min and a gradient of 10-95% of B1 in 1.9 min.

[1109] MS / MS was performed using an electrospray ionization (ESI) interface in positive ion mode with specific MRM transitions for each compound tested.

[1110] The results are given in Table 11 below.

[1111]

[1112] Table 11: CatB-induced cleavage studies of compounds 1, 3, 5, 6-8, 11 and 12 (reference compound: Cys-MC-Val-Cit-PABC-MMAF)

[1113] It is evident from these results that exo-Cat B cleavage and drug release (AF-Arg, AF-Cit, ACit, DM1-Mal-Phe-Lys, DM1-Mal-Phe-Cit, DM1-Mcc-Phe-Cit) in compounds 1, 3, 5, 6-8, 11 and 12 occur simultaneously and very rapidly. For example, CatB-induced drug release from compound 5 is 20 times faster than the reference PABC compound Cys-MC-Val-Cit-PABC-MMAF. The rapid cleavage kinetics achieved by compounds 1-8 and 11-12 demonstrate that the compounds of the present invention comprising a linker of formula (II) or (II') exhibit high selectivity and binding affinity for the exopeptidase activity of Cat B. In addition, it was surprisingly found that the presence of the Ac-Cys-PEG4 moiety on the side chain of the Lys residue (corresponding to the residue Axx in formula (II) or (II')) had no adverse effect on the binding affinity of the compound to Cat B. These results also show that, by contrast, cleavage by an endopeptidase-based mechanism of Cat B achieved in the PABC linker system (e.g., as in the reference compound Cys-MC-Val-Cit-PABC-MMAF) occurs at a significantly slower rate. As particularly striking examples, compounds 11 and 12 are spontaneously cleaved by exo-Cat B (T 1 / 2<1 min), demonstrating very favorable binding properties for substrates based on linkers of formula (II) / (II'); it is believed that the favorable interaction between the C-terminal Tyr and the closed ring of Cat B strongly contributes to the fast cleavage rates observed in these compounds.

Claims

1. A compound represented by the general formula (I): in, D represents a moiety derived from a drug, the drug being selected from carboxyl-containing drugs such as auristatin F (AF), thiol-containing drugs such as maytansine (DM1) or ravtansine (DM4), amino-containing drugs such as monomethyl auristatin F (MMAF) or exatecan and hydroxyl-containing drugs such as Maaa-1181a, preferably selected from thiol-containing drugs, amino-containing drugs and hydroxyl-containing drugs; if more than one (D) is present, each (D) is independently selected from carboxyl-containing drugs, thiol-containing drugs, amino-containing drugs and hydroxyl-containing drugs, and the moieties (D) are preferably the same as each other; X represents a divalent group comprising 1 to 7, preferably 2 to 6, more preferably 2 to 5 backbone atoms independently selected from C, N, O and S; X is covalently attached to (D) via an atom selected from C, S, N and O derived from a carboxyl, thiol, amino or hydroxyl functional group contained in (D); Y is a divalent group containing one or more atoms selected from C, N, O, P and S, preferably a divalent group derived from a compound selected from maleimides, triazoles, hydrazones, carbonyl-containing compounds and derivatives thereof, more preferably a divalent group derived from maleimides and derivatives thereof such as ring-opening hydrolyzed maleimides, most preferably a divalent group derived from ring-opening hydrolyzed maleimide; L represents a linker that can be cleaved by cathepsin B; T represents a (2+n)-valent branched group; S represents a moiety derived from a compound comprising one or more, for example two, three or four, solubilizing groups; V represents a moiety derived from a carrier group capable of interacting with target cells; n is an integer from 1 to 4, preferably 1 or 2, more preferably 1; and m is an integer of 1-12, preferably 2-10, more preferably 4-8.

2. The compound of claim 1, wherein (L) is represented by the general formula (II) or (II'): in, Axx represents a moiety derived from a trifunctional amino acid, provided that Axx in formula (II) is not a moiety derived from an amino acid in the (D) configuration; Ayy represents a moiety derived from an amino acid selected from the group consisting of Phe, Ala, Trp, Tyr, phenylglycine (Phg), Met, Val, His, Lys, Arg, citrulline (Cit), 2-amino-butyric acid (Abu), ornithine (Orn), Ser, Thr, Leu and Ile; or Ayy in formula (II) represents a moiety derived from an amino acid selected from the group consisting of homotyrosine (homo-Tyr), homophenylalanine (homo-Phe), β-phenylalanine (β-Phe) and β-homophenylalanine (β-homo-Phe), Tyr (OR1) and homo-Tyr (OR1), wherein R1 is -(CH2CH2O)n1-R2, wherein R2 is a hydrogen atom or a methyl group, and n1 is an integer from 2 to 24; provided that Ayy in formula (II') is not a moiety derived from an amino acid in the (D) configuration; Dxx represents a single covalent bond or a moiety derived from an amino acid having a hydrophobic side chain, preferably a single covalent bond or a moiety derived from an amino acid selected from Phe, Val, Tyr, homo-Phe and Ala, more preferably a single covalent bond or a moiety derived from Phe or Val; Dyy represents a single covalent bond, a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain, preferably a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), more preferably a moiety derived from Arg or Cit; provided that if Dxx is a moiety derived from an amino acid with a hydrophobic side chain, Dyy is a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain, and if Dxx is a single covalent bond, Dyy is a single covalent bond, a moiety derived from Phe or a moiety derived from an amino acid with a basic side chain; Z represents a group selected from -OH and -N(H)(R) covalently bonded to the C-terminus of Ayy or Axx, wherein R represents a hydrogen atom, an alkyl group or a cycloalkyl group, preferably represents -OH; * indicates covalent attachment to (T); and ** indicates covalent attachment to (X).

3. The compound of claim 2, wherein at least one of Axx and Ayy is defined as follows: Axx represents a moiety derived from an amino acid selected from the group consisting of Glu, 2-amino-pimelic acid (Apa), 2-aminoadipic acid (Aaa), Dap, Dab, Lys, Orn, Ser, Ama, and homolysine (homo-Lys), preferably a moiety derived from an amino acid selected from the group consisting of Dap, Dab, Lys, Orn, and homo-Lys, more preferably a moiety derived from Orn or Lys, most preferably a moiety derived from Lys; Ayy in formula (II) represents a moiety derived from an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, homo-Tyr, Tyr(OR1) and homo-Tyr(OR1), wherein R1 is -(CH2CH2O)n1-R2, wherein R2 is a hydrogen atom or a methyl group and n1 is an integer from 2 to 24, preferably a moiety derived from Phe, homo-Phe, Tyr, homo-Tyr, Tyr(OR1) and homo-Tyr(OR1), more preferably a moiety derived from Phe or Tyr, most preferably a moiety derived from Tyr; Ayy in formula (II') represents a moiety derived from an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr and Ser, preferably a moiety derived from Phe, homo-Phe and Ser, more preferably a moiety derived from Phe or Ser, most preferably a moiety derived from Phe.

4. The compound according to any one of claims 1 to 3, wherein (X) represents a divalent carbonyl- or thiocarbonyl-containing group, preferably a group represented by one of the following formulae (IIIa) to (IIIf): ***-(CH2) n2 -(C=A)-**' (IIIa) ***-(C=A)-(CH2) n3 -**’ (IIIb) ***-(CH2) n2 -(C=A)-(CH2) n3 -**’ (IIIc) ***-(CH2) n2 -(C=A)-(C(CH3)2-(CH2) n3 -**' (IIId) ***-(CH2) n2 -(C(CH3)2)-(C=A)-(CH2) n3 -**' (III) ***-(C=A)-(NH) n4 -(CH2) n3 -(NH) n5 -(C=A)-**’ (IIIf) in n2 and n3 are each independently selected from 0 to 5, preferably 0, 1 or 2, more preferably 0 or 1; n4 and n5 are each selected from 0 or 1; Each A is independently selected from O and S, preferably O; *** represents covalent attachment to (D); and **' represents covalent attachment to (L).

5. The compound according to any one of claims 1 to 3, wherein (X) is represented by one of the following formulae (IVa) to (IVj'): in, *** represents covalent attachment to (D); **' represents covalent attachment to (L); Provided that if (X) is represented by Formula (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVp), (IVr), (IVt), (IVv), (IVw), (IVx), (IVy) or (IVz), then (D) in Formula (I) represents an amino-containing drug; if (X) is represented by Formula (IVj), (IVq), (IVs) or (IVu), then (D) in Formula (I) represents an amino-containing drug or a hydroxyl-containing drug; and if (X) is represented by Formula (IVa'), (IVb'), (IVc'), (IVd'), (IVe'), (IVf'), (IVg'), (IVh'), (IVi') or (IVj'), then (D) in Formula (I) represents a carboxyl-containing drug.

6. The compound of claim 5, wherein (X) is represented by formula (IVb'), (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), preferably represented by formula (IVc) or (IVm).

7. The compound of any one of claims 1 to 6, wherein each solubilizing group contained in (S) is independently selected from the group consisting of: a moiety comprising one or more ionic or ionizable groups, such as an ammonium, guanidinium, sulfate or sulfonate group, preferably a moiety derived from Arg, (D)-Arg, Dap, (D)-Dap, Dab, (D)-Dab, Orn, (D)-Orn, Lys, D-Lys or carnitine; - a sugar moiety selected from monosaccharides, disaccharides and linear or branched oligosaccharides, in particular linear or branched oligosaccharides having 3 to 10 monosaccharide units linked by glycosidic bonds, wherein each monosaccharide unit in the monosaccharide, disaccharide and oligosaccharide is independently selected from glucose, fructose, mannose, ribose and galactose; and - a polyoxyalkylene group, preferably a C2-3 polyoxyalkylene group, more preferably a C2-3 polyoxyalkylene group, which independently comprises 6 to 200, preferably 10 to 150, more preferably 12 to 80 repeating units.

8. The compound of any one of claims 1 to 7, wherein (S) is a moiety derived from a compound comprising one or more polyethylene oxide groups, wherein preferably each polyethylene oxide group independently comprises 6 to 200, more preferably 10 to 150, most preferably 12 to 80 repeating units; (S) is preferably a moiety represented by formula (V): ****-x 1 -(CH2CH2O) n3 -X 2 (V) in, n3 is an integer from 6 to 200, preferably from 10 to 150, more preferably from 12 to 80; **** indicates covalent attachment to (T); X 1 is selected from a single covalent bond, -(C=O)- and -N(R)-, wherein R represents a hydrogen atom, an alkyl group or a cycloalkyl group; X 2 represents an alkyl group having 1 to 6 carbon atoms, a carbonyl-containing group such as acetyl or a group of the formula -(CH2) n4 -CO2H group, thiocarbonyl-containing group, formula -(CH2) n4 OR group, formula -(CH2) n4 -SO3H group or amino group such as -(CH2) n4 -(C=A)-N(R)2 or -(CH2) n4 -N(R)2 group, wherein A is O or S, each R is independently selected from a hydrogen atom, an alkyl group and a cycloalkyl group, and n4 is an integer from 1 to 6; X 2 It is preferably -CH3, -CH2CH2OH or a group represented by the following formula (VI): -(CH2) n5 -(C=A)N(R)-(CH2) n6 -(C=A)N(H)(R) (VI) in, Each A is independently selected from O and S, preferably O; Each R is independently selected from a hydrogen atom, an alkyl group, and a cycloalkyl group; and n5 and n6 are each independently an integer from 1 to 6, preferably 1 or 2; X 2 Most preferably -CH3; and If more than one (S) is present, each (S) is preferably a moiety of formula (V) above.

9. The compound according to any one of claims 1 to 8, wherein (T) is represented by the following formula (VII): in, each AA independently represents a moiety derived from a trifunctional amino acid such as a diaminocarboxylic acid, an aminodicarboxylic acid, an azidoamino acid or an alkyne-containing amino acid, preferably a moiety derived from an amino acid selected from N-ε-propynyloxycarbonyl-L-lysine (Lys(Poc)), Asp, Glu, Orn, Lys, Dab and Dap, more preferably a moiety derived from Lys(Poc), Glu, Orn or Lys, most preferably a moiety derived from Lys; α represents covalent attachment to (Y); If n=1, the side chain derived from the trifunctional amino acid is covalently attached to (L) or (S), and the C-terminus is covalently attached to the further moiety (S) or (L), respectively; If n = 2, 3, or 4: *' indicates covalent attachment to (L); ****' indicates covalent attachment to (S); and n is as defined in claim 1.

10. The compound of any one of claims 1 to 8, wherein (T) is represented by formula (VIII) or (IX): in, Each AA 1 and AA 2 is independently a moiety derived from a trifunctional amino acid, such as a diaminocarboxylic acid, an aminodicarboxylic acid, an azido amino acid or an alkyne-containing amino acid, preferably a moiety derived from an amino acid selected from Lys(Poc), Asp, Glu, Orn, Lys, Dab and Dap, more preferably a moiety derived from Lys(Poc), Glu, Orn or Lys, most preferably a moiety derived from Lys; α represents covalent attachment to (Y); In formula (IX), the side chain derived from the trifunctional amino acid is covalently attached to (L) or (S), and the C-terminus is covalently attached to the other moiety (S) or (L), respectively; In formula (VIII), *' indicates covalent attachment to (L), and ****' indicates covalent attachment to (S).

11. The compound of any one of claims 1 to 10, wherein (D) is a moiety derived from a drug selected from: (i) Antineoplastic agents, such as o DNA alkylating agents, such as duocarmycin, o Topoisomerase inhibitors, such as doxorubicin, o RNA-polymerase II inhibitors, such as α-amanitin, o DNA cleaving agents, such as calicheamicin, o antimitotic or microtubule disrupting agents, such as taxanes, auristatins or maytansinoids, o antimetabolites, such as derivatives of gemcitabine, o Spindle kinesin inhibitors, such as filaments, o kinase inhibitors, such as patasertib or gefitinib, ο Nicotinamide phosphoribosyltransferase inhibitors, ο matrix metallopeptidase 9 inhibitors, o Phosphatase inhibitors such as Microcystis toxin-LR, (ii) Immunomodulators such as fluticasone (iii) anti-infective drugs, such as rifamycin, clindamycin or retapamulin, and (iv) radioactive isotopes, metabolites, pharmaceutically acceptable salts and / or prodrugs of any of the foregoing; Provided that the drug selected from (i) to (iv) is a carboxyl-containing drug, a thiol-containing drug, an amino-containing drug or a hydroxyl-containing drug; and If more than one (D) is present, each (D) is independently selected from the above-mentioned parts (i) to (iv), and the parts (D) are preferably identical to each other.

12. The compound of any one of claims 1 to 11, wherein (D) is a moiety derived from a drug selected from the group consisting of amanitin, duocarmycin, auristatin, auristatin F (AF), monomethyl auristatin F (MMAF), maytansine, maytansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, paclitaxel, daunorubicin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepine (PBD) and its dimer, indenobenzodiazepine (IBD) and its dimer, or its radioactive isotope and / or pharmaceutically acceptable salt; preferably a portion derived from a drug selected from auristatin, MMAF, exatecan, maytansinoid, DM1 and DM4; more preferably a portion derived from DM1 or DM4.

13. The compound according to any one of claims 1 to 12, which is represented by the general formula (X) or (X'): in, Axx in formula (X) and formula (X') represents a moiety derived from an amino acid selected from Glu, Apa, Aaa, Dap, Dab, Lys, Orn, Ser, Ama and homo-Lys, preferably a moiety derived from an amino acid selected from Dap, Dab, Lys, Orn and homo-Lys, more preferably a moiety derived from a moiety derived from Lys; Ayy in formula (X) represents a moiety derived from an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr, homo-Tyr, Tyr(OR1) and homo-Tyr(OR1), wherein R1 is -(CH2CH2O) n1 -R2, wherein R2 is a hydrogen atom or a methyl group, n1 is an integer from 2 to 24, preferably a moiety derived from Phe, homo-Phe, Tyr, homo-Tyr, Tyr(OR1) or homo-Tyr(OR1), more preferably a moiety derived from Tyr; Ayy in formula (X') represents a moiety derived from an amino acid selected from Phe, homo-Phe, Ala, Trp, Phg, Leu, Val, Tyr and Ser, preferably a moiety derived from Phe, homo-Phe or Ser, more preferably a moiety derived from Phe or Ser; D, Dxx, Dyy, X, Y, T, S, V, Z, m and n have the same meanings as specified in claims 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12; and wherein preferably at least one, for example two, three, four, five, six, seven or eight of D, Dxx, Dyy, X, Y, T, S and Z are defined as follows: (a) D is a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansinoids, DM1 and DM4, preferably a moiety derived from DM1 or DM4; (b) Dxx is a moiety derived from an amino acid selected from Phe, Val, Tyr, homo-Phe and Ala, preferably a moiety derived from Phe or Val; (c) Dyy is a covalent bond or a moiety derived from an amino acid selected from Arg, Lys, Cit, Orn, Dap and Dab, preferably a covalent bond or a moiety derived from Arg or Cit; (d) X is a group of formula (III) wherein n2 is 1 or 2, or a group represented by any one of formulae (IVa) to (IVz), preferably a group represented by any one of formulae (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm); (e) Y is a group derived from a compound selected from maleimides, triazoles, hydrazones, carbonyl-containing compounds and derivatives thereof, preferably selected from maleimides and derivatives thereof such as ring-opening hydrolyzed maleimide, and more preferably a group derived from ring-opening hydrolyzed maleimide; (f) T is a group of formula (VII), (VIII) or (IX); (g) S is a moiety of formula (V); and (h) Z is -OH.

14. The compound of claim 13, wherein in formula (X), each Dyy-Dxx-Axx-Ayy is independently selected from wherein Dyy is covalently bonded Arg-Lys-Phe, wherein Dyy is covalently bonded Arg-Lys-homo-Phe, wherein Dyy is covalently bonded Arg-Lys-Tyr, wherein Dyy is covalently bonded Cit-Lys-Phe, wherein Dyy is covalently bonded Cit-Lys-Tyr, wherein Dyy is covalently bonded wherein Dyy is covalently bonded Arg-Lys-homo-Tyr, wherein Dyy is covalently bonded Cit-Lys-homo-Tyr, Phe-Cit-Lys-Phe, Phe-Cit-Lys-Tyr, Phe-Arg-Lys-Tyr, Phe-Cit-Lys-homo-Tyr, Phe-Lys-Lys-Phe, homo-Phe-Arg-Lys-Phe, homo-Phe-Cit-Lys-Tyr; and In formula (X'), each Dyy-Dxx--Ayy-Axx is independently selected from Arg-Phe-Lys where Dyy is covalently bonded, Arg-Ser-Lys where Dyy is covalently bonded, Cit-Phe-Lys where Dyy is covalently bonded, Cit-Ser-Lys where Dyy is covalently bonded, Cit-homo-Phe-Lys where Dyy is covalently bonded, Phe-Cit-Phe-Lys, homo-Phe-Cit-Phe-Lys and Phe-Arg-Phe-Lys.

15. The compound according to any one of claims 1 to 14, which is represented by one of the following formulae: wherein D, X, Y, T, S, V, Z, m and n have the same meanings as specified in claim 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12; and wherein preferably at least one, for example two, three, four, five or six of D, X, Y, T, S and Z are defined as follows: (a) D is a moiety derived from a drug selected from auristatin, AF, MMAF, exatecan, maytansine, DM1 and DM4, preferably a moiety derived from DM1 or DM4; (d) X is a group of formula (III), wherein n2 is 1 or 2, or is a group represented by any one of formulae (IVa) to (IVz), preferably a group represented by any one of formulae (IVc), (IVm), (IVn), (IVo), (IVp), (IVs) or (IVt), more preferably a group represented by formula (IVc) or (IVm); (e) Y is a group derived from a compound selected from maleimides, triazoles, hydrazones, carbonyl-containing compounds and derivatives thereof, preferably selected from maleimides and derivatives thereof such as ring-opening hydrolyzed maleimide derivatives, and more preferably a group derived from ring-opening hydrolyzed maleimide; (f) T is a group of formula (VII), (VIII) or (IX); (g) S is a moiety of formula (V); and (h) Z is -OH.

16. The compound according to any one of claims 1 to 15, which is represented by one of the following formulae: wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; wherein the number of oxyethylene repeating units (17) may be replaced by 12 to 30, preferably 14 to 25, more preferably 15 to 19 oxyethylene groups; and / or wherein the maleimide attachment to (V) may be replaced by ring-opening hydrolyzed maleimide.

17. The compound of any one of claims 1 to 16, wherein (V) represents a moiety derived from a carrier group capable of interacting with a target cell, wherein the target cell is selected from tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, cells involved in autoimmune diseases, activated cells, myeloid cells, lymphoid cells, melanocytes and infectious agents including bacteria, viruses, mycobacteria, fungi; Preferably, the target cell is selected from lymphoma cells, myeloma cells, myeloid cells, lymphoid cells, renal cancer cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell cancer cells, small cell lung cancer cells, testicular cancer cells, skin cancer cells, pancreatic cancer cells, liver cancer cells and any cell that grows and divides at an unregulated and accelerated rate, thereby causing cancer.

18. The compound according to any one of claims 1 to 17, wherein (V) represents a moiety derived from a carrier group selected from antibodies, antibody fragments, proteins, peptides and non-peptide molecules; Preferred are moieties derived from antibodies or antibody fragments such as single chain antibodies, monoclonal antibodies, single chain monoclonal antibodies, monoclonal antibody fragments, chimeric antibodies, chimeric antibody fragments, domain antibodies or fragments thereof, cytokines, hormones, growth factors, colony stimulating factors, neurotransmitters or nutrient transport molecules.

19. A compound according to any one of claims 1 to 18, wherein (V) represents a moiety derived from: Monoclonal antibodies, preferably antibodies selected from the group consisting of adalimumab, aducanumab, alemtuzumab, atumomab triamine pentaacetic acid, ervantumab, atezolizumab, anetuzumab, avelumab, bapilumab, basiliximab, betumomab, belantamab, beramezumab, bevacizumab, belotuzumab, vebutin, vebutin, bodalumab, catumaxomab, cemiplimab, cetuximab, simpanumab, clituzumab, crenetuzumab, tetraxeta n, daclizumab, daratumumab, denosumab, dinutuximab, dotalimumab, durvalumab, edrecolomab, elotuzumab, epavacumab, enrofloxacin, enrofloxacin vedotin, icoretuzumab, epratuzumab, epratuzumab-SN-38, edamasumab, gemtuzumab, gemtuzumab ozogamicin), genmab, grofituzumab, gemtuzumab, gonelizumab, ibritumomab tiuxetan, inelizumab, infliximab, inotuzumab, ozogamicin inotuzumab, ipilimumab, isatuximab, ixetuzumab, J591 PSMA-antibodies, labetuzumab, lencanezumab, lentuximab tesirin, moglizumab, motuzumab, necituzumab, nimotuzumab, natalizumab, natalizumab, nasituximab, nivolumab, omezumab, ofatumumab, olaratumumab, ogovumab, panitumumab, pembrolizumab, pertuzumab, polotuzumab, vepotuzumab vedotin, putinumab, rituximab, ramucirumab, rituximab, certolizumab pegol Antibody, gosartan, ceritumomab, siltuximab, solanezumab, tetuzumab, tancitumomab, tetuximab, tipranezumab, tocilizumab, tositumomab, trastuzumab, detrastuzumab, emtansuzumab, TS23, ustekinumab, vedolizumab, futumomab, zagetelumab, zalumab, zalumumab, fragments and derivatives thereof; more preferably selected from atezolizumab, durvalumab, pembrolizumab, rituximab or trastuzumab; or - Antibody fragments introduced into Fc-fusion proteins, which are preferably selected from the group consisting of belatacept, aflibercept, Ziv-aflibercept, dulaglutide, rilocept, romiplostim, abatacept and alefacept.

20. The compound or salt of any one of claims 1 to 19, wherein (V) represents a moiety derived from an anti-HER2, anti-CD37, anti-PDL1 or anti-EGFR antibody, preferably derived from an antibody selected from trastuzumab, pembrolizumab, natalizumab, atezolizumab, durvalumab, avelumab, panitumumab and cetuximab, more preferably derived from natalizumab, trastuzumab and cetuximab, most preferably derived from natalizumab and trastuzumab.

21. The compound of claim 20, wherein (D) is a moiety derived from an antitumor agent, and preferably is a moiety derived from a drug selected from the group consisting of amanitin, duocarmycin, auristatin, auristatin F (AF), monomethyl auristatin F (MMAF), maytansine, maytansine (DM1), ravtansine (DM4), tubulysin, calicheamicin, camptothecin, SN-38, exatecan, Maaa-1181a, paclitaxel, daunorubicin, vinblastine, doxorubicin, methotrexate, pyrrolobenzodiazepine (PBD) and its dimer, indenobenzodiazepine (IBD) and its dimer, or its radioactive isotope and / or pharmaceutically acceptable salt.

22. The compound of claim 21, wherein (D) represents a moiety derived from a drug selected from auristatin, MMAF, exatecan, maytansinoid, DM1 and DM4; more preferably a moiety derived from DM1 or DM4.

23. The compound of claim 22, wherein (D) represents a moiety derived from DM1, and wherein the compound is represented by one of the following formulae: wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; and wherein the number of oxyethylene repeating units (17) may be replaced by 12 to 30, preferably from 14 to 25, more preferably from 15 to 19 oxyethylene groups, and / or wherein the maleimide attachment to (V) may be replaced by ring-opening hydrolyzed maleimide.

24. The compound of claim 23, which is represented by the formula: wherein m is an integer from 1 to 12, preferably from 2 to 10, more preferably from 4 to 8; and wherein the number of oxyethylene repeating units (17) may be replaced by 12 to 30, preferably from 14 to 25, more preferably from 15 to 19 oxyethylene groups, and / or wherein the maleimide attachment to (V) may be replaced by ring-opening hydrolyzed maleimide.

25. A composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 24 or a pharmaceutically acceptable salt thereof, and one or more components selected from carriers, diluents and other excipients.

26. A compound or composition according to any one of claims 1 to 25 for use in a method of treating or preventing cancer, an autoimmune disease and / or an infectious disease.

27. The compound or composition for use according to claim 26, wherein the method is a method of treating a cancer of the blood and bone marrow, preferably acute myeloid leukemia (AML).

28. The compound or composition for use according to claim 26, wherein the compound is as defined in any one of claims 20, 21, 22, 23 and 24, preferably as defined in claim 23 or 24, most preferably as defined in claim 24.

29. A compound or composition for use according to any one of claims 26 to 28, wherein the compound or composition is administered simultaneously with, before or after, one or more other therapeutic agents or therapies such as chemotherapeutic agents, radiotherapy, immunotherapeutic agents, autoimmune disease agents, anti-infective agents or other compounds of formula (I) in a method for treating or preventing cancer, autoimmune diseases and / or infectious diseases.

30. A compound represented by the general formula (XI): in, D, X, L, T, S and n have the same meanings as defined in claim 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12; Y' represents a moiety comprising a conjugating group capable of forming a covalent attachment with a molecule capable of interacting with a target cell (V'), such as a monoclonal antibody or an antibody fragment introduced into an Fc-fusion protein; Y' is preferably a moiety comprising a conjugating group selected from: - an optionally substituted maleimide, preferably capable of reacting with one or both thiol groups of (V'); - an optionally substituted haloacetamide, preferably capable of reacting with the thiol group of (V'); - an ester, preferably an ester capable of reacting with the side chain of the amino acid of (V'), such as an acyl halide, an N-hydroxysuccinimide ester or a phenolic ester; - a carbonate, preferably capable of reacting with the side chain of the amino acid of (V'), such as a haloformate, or a carbonate comprising a leaving group, such as N-hydroxysuccinimide or phenol; - an isocyanate or an isothiocyanate, preferably capable of reacting with the side chain of the amino acid of (V'); - an azide, preferably capable of reacting with the alkyne group contained in (V'); - an alkyne, preferably capable of reacting with the azide group contained in (V'); - an amino group, preferably capable of reacting with molecule (V') in the presence of an enzyme such as transglutaminase; The compound preferably has a structure as shown in claim 13 or 15, provided that (Y') replaces (Y) and m is 1.

31. A kit for modifying a molecule capable of interacting with a target cell, comprising a compound according to claim 30 and optionally a buffer, said buffer preferably having a pH of 6.0 to 10, more preferably a pH of 6.5 to 8.

0.

32. A method for modifying a molecule capable of interacting with a target cell, which comprises reacting a molecule capable of interacting with a target cell, such as a monoclonal antibody or an antibody fragment introduced into an Fc-fusion protein, with a compound according to claim 30.

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