TW201302799A - New binder-drug conjugates (ADCs) and use thereof - Google Patents

Abstract

The present application relates to new binder-drug conjugates (ADCs) of N, N-dialkylauristatins that are directed against the target mesothelin, to active metabolites of these ADCs, to processes for preparing these ADCs, to the use of these ADCs for treating and/or preventing illnesses, and also to the use of these ADCs for producing medicaments for treating and/or preventing illnesses, more particularly hyperproliferative and/or angiogenic diseases such as, for example, cancer diseases. Such treatments may be practised as a monotherapy or else in combination with other medicaments or further therapeutic measures.

Description

Novel binding agents - drug conjugates (ADCs) and their uses (1)

This application relates to a binding agent-drug conjugate (ADC) for N,N-dialkyl auristatin targeting target mesothelin, active metabolites of these ADCs, and preparation of these ADCs Methods, uses of these ADCs for the treatment and/or prevention of diseases, and the use of these ADCs for the manufacture of pharmaceuticals for the treatment and/or prevention of diseases, particularly hyperproliferative and/or angiogenic diseases, such as cancer diseases. Such treatments can be administered in a single treatment or in combination with other pharmaceuticals or additional treatments.

Cancer diseases are the result of the growth of uncontrolled cells in various tissues. In many cases these new cells pass through existing tissues (invasive growth) or they are transferred to distant organs. Cancer diseases occur in a wide variety of organs, and diseases are usually advanced in a tissue-specific manner. The term "cancer disease" is therefore a generic term that describes a defined disease of a large group of different organs, tissues and cell types.

Early tumors can be removed with surgery and radiation therapy. Metastatic tumors are generally only administerable to palliative treatment by chemotherapeutic agents. The purpose in this case is to achieve an optimal combination of improved quality of life and extended remaining life.

Currently, most of the chemotherapeutic agents administered parenterally are not targeted in tumor tissues or tumor cells, but instead they are replaced by Physical administration is non-specifically distributed throughout the body and therefore includes sites that are not desired to be exposed to the drug, such as healthy cells, tissues and organs. This can lead to unwanted side effects and even severe pan-toxic effects, which then often limit the therapeutically effective dose range of the drug or must completely stop medical treatment.

The improved and selective availability of these chemotherapeutic agents in tumor cells or in close proximity to tissues, and on the one hand, to enhance utility, and on the other hand to minimize toxic side effects, has therefore been the focus of development of new chemotherapeutic agents for many years. Many attempts have been made to date to develop an effective method for introducing drugs into target cells. For example, optimally linking drugs and intracellular targets to adjacent cells and minimizing drug distribution between cells continues to be a difficult problem.

Individual antibodies, for example, are suitable for targeted addressing of tumor tissues and tumor cells. Such drugs as trastuzumab (Herceptin), rituximab (Rituxan), cetuximab (Erbitux) and bevacizumab (Avastin) Based on the activity, which has been approved for the treatment of individual, specific tumor diseases, the importance of these antibodies for the treatment of clinical cancer diseases has been greatly enhanced in recent years [see, for example, GPAdams and LM Weiner, Nat. Biotechnol. 23, 1147-1157 (2005)]. Thus, there is also a significant increase in the benefits of so-called immunoconjugates, such as the aforementioned ADCs, in which an internalizing antibody directed against a tumor-associated antigen is covalently joined to a cytotoxic agent via a ligation unit (linker). After the introduction of the ADC into the tumor cells and subsequent cleavage of the conjugate, the cytotoxic agent itself or another cytotoxic activity metabolite formed by cytotoxicity is released intracellularly, wherein it can produce its effect directly or selectively. Compared to conventional cancer chemotherapy, in this method, damage to normal cells can be greatly limited as much as possible [see, for example, JMLambert, Curr. Opin. Pharmacol . 5 , 543-549 (2005); AMWu and PDSenter, Nat .Biotechnol. 23 , 1137-1146 (2005); PDSenter, Curr. Opin. Chem. Biol. 13 , 235-244 (2009); L. Ducry and B. Stump, Bioconjugate Chem. 21 , 5-13 (2010) ].

Substituting antibodies, conjugates from small molecule drug spheres can also be used as a conjugate that selectively binds to a specific position (palladium), such as a receptor, for example [see, for example, E. Ruoslahti et al, Science 279 , 377-380 ( 1998); D. Karkan et al., PLoS ONE 3(6), e2469 (June 25, 2008)]. Cytotoxic drugs and addressable ligands are also known which have a defined cleavage site between the ligand and the drug for release of the drug. Such "predetermined breakpoints", for example, may be present in the peptide chain, which may be selectively cleaved by a particular enzyme at the site of action at a particular position [see, for example, RA Firestone and LA Telan, US Patent Application US 2002/0147138] .

The activity of monoclonal antibodies is verified in various types of cancer. Therefore, HERCEPTIN® and Erbitux® can be successfully used to treat HER2-positive breast cancer and EGFR-positive colorectal cancer, respectively.

Coupling of cytotoxic compounds with antibodies additionally improves the likelihood of cancer treatment because these conjugates make tumor-specific toxic heaps Accumulate and reduce systemic toxicity at the same time. Related studies on activity and tolerability, with the clinical study of brothuximab vedotin in Hodgkin's lymphoma, and the clinical study of trastuzumab in breast cancer, have produced new antibodies and Highly promising results for the development of new ADCs against other tumor antigens

The mesothelin prodrug polypeptide is a glycophospholipid inositol (GPI) immobilized glycosylated cell surface protein that is proteolytically cleaved into a 30 kDa N-terminal secreted polypeptide (megakaryocyte promoting factor, MPF) and GPI-fixed (mesothelin) is present on the 40 kDa C-terminal polypeptide on the cell membrane (Chang, K. and I. Pastan, Proc. Natl. Acad. Sci. USA, (1996) 93(1): 136).

Because this surface manifestation and transition is manifested in many types of tumors, more specifically, ovarian cancer, prostate cancer, and mesothelioma, and because of the highly restricted performance in normal tissues, mesothelin is a suitable single plant. Antigens for antibody therapy (Argani, P. et al, Clin. Cancer Res. (2001) 7(12): 3862; Hassan, R., et al, Clin. Cancer Res. (2004) 10 (12 Pt 1): 3937). The exact function of mesothelin is still unknown; mesothelin-deficient mice do not exhibit any immediate anatomical, hematological or reproductive restriction traits (Bera, TK and I. Pastan, Mol. Cell. Biol. (2000) 20 ( 8): 2902). However, mesothelin is a highly variable protein that undergoes post-translational modifications such as proteinase breakdown and glycosylation (Hassan, R., et al, Clin. Cancer Res. (2004) 10 (12 Pt 1): 3937). Therefore, the therapeutic antibody should be able to recognize a constant epitope that is unrelated to individual mesothelin modification. Antibodies can be selected according to their desired properties. For example, constant affinity or internalization, for example in the use of monoclonal antibodies, on the one hand ensures that these properties are used for all antibodies involved, and on the other hand can also be produced consistently over a long period of time.

Auristatin E (AE) and monomethyl auristatin E (MMAE) are synthetic analogues of dolastatin, a specific linear pseudopeptide It was originally isolated from marine sources and, in some cases, has very potent cytotoxic activity against tumor cells [for a discussion, see, for example, GRPettit, Prog. Chem. Org. Nat. Prod. 70 , 1-79 (1997); GRPettit et al, Anti-Cancer Drug Design 10 , 529-544 (1995); GRPettit et al, Anti-Cancer Drug Design 13 , 243-277 (1998)].

However, MMAE has the disadvantage of being quite high in systemic toxicity. For improved tumor selectivity, MMAE is more specifically used with the enzyme-cleavable lysine-citrulline linker for ADC setup for more targeted tumor therapy [WO 2005/081711-A2; SODoronina et al. Bioconjugate Chem. 17 , 114-124 (2006)]. After protein cleavage, the MMAE is preferably released intracellularly from the corresponding ADC.

However, when a binder-drug conjugate (ADC) format is used, the MMAE is not compatible with the binding agent (linker) between the binding agent and the drug without a predetermined cleavable site of the enzyme cleavage [SODoronina et al ., Bioconjugate Chem. 17 , 114-124 (2006)].

Monomethyl auristatin F (MMAF) is an auristatin derivative having a C-terminal phenylalanine unit, which has only moderate antiproliferative activity compared to MMAE. This fact is most likely due to the free carboxylic acid group whose polarity and charge have a detrimental effect on the ability of this compound to enter the cell. In this regard, methyl ester of MMAF (MMAF-OMe) has been described as having a neutral-charged prodrug derivative with the ability to enter cells, which has in vitro cytotoxicity against various cancer cell lines that increase many orders of magnitude compared to MMAF [SODoronina] Et al, Bioconjugate Chem. 17 , 114-124 (2006)]. It can be assumed that this utility is caused by MMAF itself, which is rapidly released by intracellular ester hydrolysis after the prodrug is taken up by the cells.

However, drug compounds based on simple ester derivation are generally susceptible to chemical instability due to non-specific ester hydrolysis independent of the desired site of action, for example by esterases present in plasma; this non-specific Sexual hydrolysis may greatly limit the therapeutic effectiveness of such compounds.

Monomethyl auristatin F (MMAF) and its various ester derivatives and guanamine derivatives are disclosed in WO 2005/081711-A2. Other auristatin analogs having a C-terminal decylamine substituted amphetamine unit are described in WO 01/18032-A2. WO 02/088172-A2 and WO 2007/008603-A1 for MMAF analogs for side chain modification of phenylalanine, and WO 2007/008848-A2 for compounds having modified carboxyl groups of phenylalanine . The auristatin conjugate linked via the C-terminus has recently been described in WO 2009/117531-A1 [see also SO Doronina et al, Bioconjugate Chem. 19, 1960-1963 (2008)].

Furthermore, auristatin derivatives such as MMAE and MMAF are also substrates for transporters expressed by many tumor cells, and they may lead to the development of resistance to these drugs.

The problem addressed by the present invention is to provide a new binder-drug conjugate (ADC) that exhibits a very strong introduction via a novel N,N-dialkyl auristatin in combination with an innovative, suitable linker and binder. An attractive property, such as its specific tumor effect and/or a decrease in the potential for intracellular formation of metabolites as a transporter-associated matrix, and which is therefore suitable for the treatment and/or prevention of hyperproliferation and/or angiogenesis , for example, cancer disease.

The present invention provides a binding agent-drug conjugate of the general formula (Ia) Wherein n is a number from 1 to 50, AK is a binding agent, and the §-GL ¹ -BL ² -§§ group is a linker, wherein § is the position of the linkage to the AK group, and §§ is labeled with nitrogen The position of the atom, D is the group of the following formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen or methyl, and R ² is isopropyl, isobutyl, second butyl, tert-butyl, phenyl, benzyl, 1-hydroxy Ethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1 H -imidazol-4-yl Methyl or 1 H -indol-3-ylmethyl, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 of the following formula, 1-diyl group Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Wherein # ⁶ is a position to which a carbonyl group is bonded, R ⁶ is hydrogen, a hydroxyl group or a benzyloxy group, R ³ is hydrogen or a methyl group, and R ⁴ is an isopropyl group, an isobutyl group, a second butyl group, and a third group. Butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, two Benzyl, 1 H -imidazol-4-ylmethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form the formula (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a 4 a 7-membered heterocyclic ring, R ¹⁰ is a benzindenyl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group in the phenyl group, and R ⁵ is hydrogen, a methyl group or a group of the following formula group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. Methyl, R ³⁵ is a methyl or hydroxy group, as well as solvates of salts, solvates and salts thereof.

The compounds of the present invention are the compounds of the formulae (Ia) and (I) and the solvates of the salts, solvates and salts thereof, the same compounds as the formulae below, and the formulae (Ia) and (I) and salts thereof, solvents Solvates of the compounds and salts, and the same compounds as the working examples below, and encompassing the solvates of the formulae (Ia) and (I) and their salts, solvates and salts, which are the same as below and encompass the formula (Ia) and The compound of (I) is not yet a solvate of its salts, solvates and salts.

Depending on the structure, the compounds of the invention may exist in different stereoisomeric forms, ie, configuration isomers or others, if appropriate, as conformational isomers (mirroromers and/or diastereomers, Including such resistance Case of structure). The invention thus encompasses mirror image isomers and diastereomers and individual mixtures thereof. Stereoisomerically homogeneous constituents can be separated from such mirror image isomers and/or diastereomers by known methods; for this purpose, preferably for use on non-palphages or palms Chromatography, more specifically, HPLC chromatography.

When a compound of the invention can take the form of a tautomer, the invention encompasses all tautomeric forms.

The invention also encompasses all suitable isotopic variations of the compounds of the invention. It is to be understood that an isotopic variation of a compound of the invention is used herein to mean a compound in which at least one atom of the compound of the invention is exchanged with another atom of the same atomic number but different atomic weight than the normal or predominantly occurring atomic mass of nature. Examples of isotopes which may be incorporated into the compounds of the invention are isotopes of such hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, for example ² H(氘), ³ H(氚), ¹³ C , ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³² P, ³³ P, ³³ S, ³⁴ S, ³⁵ S, ³⁶ S, ¹⁸ F, ³⁶ Cl, ⁸² Br, ¹²³ I, ¹²⁴ I, ¹²⁹ I and ¹³¹ I. Particular isotopic variations of the compounds of the invention, for example, more particularly, those in which one or more radioisotopes are incorporated may be advantageous, for example, in the study of mechanisms of action or distribution of drugs in the body; It is easy to measure, and compounds calibrated with ³ H or ¹⁴ C isotope are particularly suitable for these purposes. Furthermore, the incorporation of isotopes, such as deuterium, may cause certain therapeutic benefits due to the greater metabolic stability of the compound, such as prolonging the half-life in the body or reducing the effective dosage required; such modifications of the compounds of the invention The effect therefore, if appropriate, also constitutes a preferred embodiment of the invention. Isotopic variations of the compounds of the invention can be prepared by methods known to those skilled in the art, for example, in accordance with the methods of production and operation examples described hereinafter, using individual reagents and/or corresponding compounds. Isotope modification.

Preferred salts of the invention are physiologically acceptable salts of the compounds of the invention. Also contemplated, although not in itself suitable for pharmaceutical applications, it can be used, for example, to isolate and purify salts of the compounds of the invention.

The physiologically acceptable salts of the compounds of the present invention include acid addition salts of inorganic acids, carboxylic acids and sulfonic acids, examples being hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluene. Salts of sulfonic acid, naphthalene disulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

Physiologically acceptable salts of the compounds of the invention also encompass salts of conventional bases, such as, and preferably, alkali metal salts (such as sodium and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts) or derived from ammonia. Or the organic amine has an ammonium salt of 1 to 16 C atoms, for example and preferably ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine , dimethylaminoethanol, 7 pcaine, benzhydrylamine, N-methylpiperidine, N-methylmorpholine, arginine, lysine and 1,2-ethanediamine.

The solvates in the context of the present invention are those forms of the compounds of the present invention which are coordinated to a solvent molecule to form a solid or liquid state complex. Hydrates are a specific form of solvate in which they are coordinated to water. Preferred solvates in the context of the present invention are hydrates.

Furthermore, the invention also encompasses prodrugs of the compounds of the invention. The term "prodrug" as used herein refers to a compound which is biologically activated or non-activated but which is converted to a compound of the invention (for example, by metabolism or hydrolysis) during its residence in the body.

In the context of the present invention, unless otherwise stated, the substituent has the following meaning: In the context of the present invention, (C ₁ -C ₄ )-alkyl is a straight or branched alkane having from 1 to 4 carbon atoms. base. Mention may be made, by way of example and preferably the following: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 1-methylpropyl and tert-butyl.

In the context of the present invention (C ₁ -C ₄ )-alkylcarbonyl is a straight or branched alkyl group having 1 to 4 carbon atoms which is bonded via a carbonyl group. Mention may be made, by way of example and preferably the following: methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, isobutylcarbonyl, 1-methylpropylcarbonyl and the third Butylcarbonyl.

In the context of the present invention, an alkylene group is a linear alpha, omega-divalent alkyl group having the specified number of carbon atoms. Mention may be made, by way of example, and preferably the following: methyl, ethyl-1,2-diyl (1,2-extended ethyl), propyl-1,3-diyl (1,3-propyl) ), 1,4-1,4-diyl (1,4-butylene), penta-1,5-diyl (1,5-exopenyl), hex-1,6-diyl (1,6) - hexyl), hept-1,7-diyl (1,7-exetylene), octane-1,8-diyl (1,8-exetylene), 壬-1,9-diyl ( 1,9-extension base), 癸-1,10-diyl (1,10-extension base).

In the context of the present invention, (C ₃ -C ₇ )-cycloalkyl and 3- to 7-membered carbocyclic ring are each a monocyclic, saturated cycloalkyl group having 3 to 7 carbon atoms. Mention may be made, by way of example and preferably the following: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

The pendant groups of the a-amino group in the definition of R ¹⁹ encompass not only the naturally occurring α-amino acid side groups, but also the analogs of these α-amino groups and the pendant groups of the isomers. The α-amino acid may be present in the L and D configurations or may be present in a mixture of L and D forms. Examples of pendant groups that can be administered are the following: methyl (alanine), prop-2-yl (proline), prop-1-yl (n-decyl), 2-methylprop-1-yl ( Amino acid), 1-methylpropan-1-yl (isoleucine), but-1-yl (normal leucine), tert-butyl (2-tert-butylglycine), benzene (2-phenylglycine), benzyl (phenylalanine), p-hydroxybenzyl (tyrosine), ind-3-ylmethyl (tryptophan), imidazole-4-yl (histidine), methylol (serine), 2-hydroxyethyl (homoserine), 1-hydroxyethyl (threonine), mercaptomethyl (cysteine), methyl Thiomethyl ( S -methylcysteine), 2-mercaptoethyl (homocysteine), 2-methylthioethyl (methionine), amine-methylmethyl group ( Aspartic acid, 2-aminoformylethyl (glutamic acid), carboxymethyl (aspartate), 2-carboxyethyl (glutamic acid), 4-aminobutyrate-1 -yl (isoamine), 4-amino-3-hydroxybutan-1-yl (hydroxyl-amino acid), 3-aminopropan-1-yl (ornithine), 2-aminoethyl ( 2,4-diaminobutyric acid), aminomethyl (2,3-diaminopropionic acid), 3-mercaptopropan-1-yl (arginine), 3-ureidopropan-1- Base (citrulline). In the definition of R ¹⁹ , preferred pendant α-amino acids are methyl (alanine), prop-2-yl (proline), 2-methylprop-1-yl (leucine), Benzyl (phenylalanine), imidazol-4-ylmethyl (histamine), methylol (serine), 1-hydroxyethyl (threonine), 4-aminobutan-1-yl (ionic acid), 3-aminopropan-1-yl (ornithine), 2-aminoethyl (2,4-diaminobutyric acid), amine methyl (2,3-diamino) Propionic acid), 3-mercaptopropan-1-yl (arginine). The L configuration is preferred in each case.

The 4- to 7-membered heterocyclic ring in the context of the present invention is a monocyclic, saturated heterocyclic ring having a total of 4 to 7 ring atoms, which contains one or two from N, O, S, SO and/or SO ₂ A series of heteroatoms are linked via a ring of carbon atoms or, if desired, a ring of nitrogen atoms. Preferably, a 5- to 7-membered heterocyclic ring having one or two ring heteroatoms from the N, O and/or S series is given, more preferably one or two rings from the N and/or O series. 5- to 6-membered heterocyclic ring of a hetero atom. Mention may be made, by way of example, of the following: tetrahydroindolyl, oxetanyl, pyrrolidinyl, pyrazolyl, tetrahydrofuranyl, thia Peptidyl, piperidine Base, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiafolamine, hexahydroindenyl and hexahydro-1,4-diazonium. Preferably, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperidine is administered Base, tetrahydropyranyl and morpholinyl.

In the chemical formulas of the groups represented by A, B, B ¹ , D, G, L ¹ , L ² , L ⁴ , L ⁶ , R ¹ , R ² , R ³ , R ⁴ and R ⁵ , respectively # ⁶ ,*,**,# ³ ,# ¹ ,# ² ,## ¹ ,## ² ,## ³ ,## ⁴ ,## ⁵ ,## ⁶ ,## ⁷ ,## ⁸ The endpoints of the line where the ***, ****, # ⁴ , # ⁵ , # ⁶ , # ⁷ , # ^8, or # ⁹ symbols are located are not carbon atoms or CH ₂ groups, but rather are assigned atoms. a portion of the phase bond, which is bonded to A, B, B ¹ , D, G, L ¹ , L ² , L ⁴ , L ⁶ , R ¹ , R ² , R ³ , R ⁴ or R ⁵ .

In the context of the present invention, all groups that occur multiple times have their own independent definitions. If a group on a compound of the invention is substituted, the groups may be substituted one or more times unless otherwise stated. One or more Substitution of the same or different substituents is preferred. A particularly preferred one is substituted with one substituent.

In the context of the present invention, the terminology used, unless otherwise indicated, has the following definition: the term "linker" is understood in the broadest sense to include a covalent bond or a series of chemical units linking the bonding agent to the atom of the drug. . The term "linker" is understood to be preferably a series of atoms that are covalently linked to a binder and a drug under the inventive concept. Further, the linker may be represented, for example, by a divalent chemical unit such as an alkylene group, an arylene group, a heteroarylene group, a heterocyclic group, a dicarboxylic acid ester, or a decylamine dicarboxylate.

The term "binder" is understood in the broadest sense to refer to a molecule that is present in a binding agent-drug conjugate in combination with a target molecule present in a particular target cell population. The term "junction" is to be understood in the broadest sense and encompasses, for example, lecithin, a protein or phospholipid binding protein that binds to a particular sugar chain. Such binding agents include, for example, high molecular weight proteins (binding proteins), polypeptides or peptides (binding peptides), non-peptides (for example, aptamer literature (US 5,270,163) Keefe AD., et al. Literature, Nat. Rev. Drug Discov. 2010; 9: 537-550), or vitamins) and all other cell binding molecules or substances. Binding proteins are, for example, antibodies and antibody fragments or antibody mimetics, such as affibody, adnectin, anticalin, DARPin, high affinity avimer, nanobodies (discussion literature) Gebauer M. et al., Curr. Opinion in Chem. Biol. 2009; 13: 245-255; Nuttall SD et al., Curr. Opinion in Pharmacology 2008; 8: 608-617). The binding peptide has, for example, a ligand-receptor pair ligand, such as a ligand-receptor to VEGF/KDR VEGF, such as a ligand-receptor to transferrin/transferrin receptor transferrin, Or a cytokine/cytokine receptor, such as a ligand-receptor to the TNFα/TNFα receptor ligand TNFα.

Preferred binding agents (more particularly human, monoclonal antibodies) according to the invention are antibodies or antigenic and antibody fragments that bind to mesothelin. In the case of the anti-mesothelin antibody, in other words, the number of toxophore molecules per antibody molecule is preferably in the range of from 1 to 10, more preferably from 2 to 8.

"Target molecule" is understood in the broadest sense to mean a molecule or a non-peptide molecule (eg, a sugar or a phospholipid) that is present in a target cell population and can be a protein (eg, a receptor for a growth factor). Preferably it is a receptor or antigen.

The term "extracellular" target molecule describes a portion of a target molecule that is linked to a cell and is located outside the cell or a portion of the target molecule that is external to the cell, that is, the binding agent binds to a target molecule of the intact cell. The extracellular target molecule can be immobilized in the cell membrane or can be part of the cell membrane. Those skilled in the art have learned how to identify extracellular target molecules. For proteins, this can be done by measuring the transmembrane region and the orientation of this protein in the membrane. This information is generally recorded in a protein database (eg SwissProt).

The term "cancer target molecule" describes a multiplex of one or more target molecules on a cancer cell type compared to non-cancer cells of the same tissue type. Cancer target score compared to non-cancer cells of the same tissue type Preferably, the species is selectively present in one or a type of cancer cell, wherein "selectivity" is compared to non-cancer cells of the same tissue type, describing at least two fold accumulation on cancer cells (selective cancer target molecules) ). Selective cancer cell therapy can be performed using the conjugate of the invention using a cancer target molecule.

The binding agent can be linked to the linker via a bond. The possibility of covalent bonding (joining) of various organic molecules to antibodies is known from the literature. The attachment of the binding agent can be carried out by the heteroatoms of the binding agent. The heteroatoms of the binding agents of the invention which can be used for attachment are sulfur (in one embodiment via a thiol group of a binder), oxygen (in accordance with the invention by a carboxyl or hydroxyl group of a binder) and nitrogen (in In one embodiment is a primary or secondary amine group or a guanamine group via a binder. Preferably, in accordance with the invention, the toxicant and antibody are joined via a sulfur atom of a cysteine residue of the antibody, and/or via an NH group of an amine acid residue of one or more antibodies. These heteroatoms may be present in the natural binding agent or may be introduced by chemical or molecular biological methods. According to the invention, the attachment of the binding agent to the toxicant has only minimal effect on the binding activity of the binding agent to the target molecule. In a preferred embodiment, this linkage has no effect on the binding activity of the binding agent to the target molecule.

In accordance with the present invention, the term "antibody" is understood in the broadest sense and encompasses immunoglobulin molecules, examples of which are intact or modified monoclonal antibodies, polyclonal antibodies or multi-specific antibodies (e.g., bispecific antibodies). Immunoglobulins preferably include molecules having four polypeptide chains, two heavy chains (H chains) and two light chains (L chains), which are typically linked by a disulfide bridge. Each heavy chain includes a heavy chain variable region (abbreviated VH) and a heavy chain constant Area. The heavy chain constant region can encompass, for example, three regions, CH1, CH2, and CH3. Each light chain includes a variable region (abbreviated VL) and a constant region. The constant region of the light chain includes a region (abbreviation CL). The VH and VL regions can be further subdivided into regions with hypervariability, also known as complementarity determining regions (abbreviated CDRs) and regions with low sequence variability ("architecture regions", abbreviated as FR). Each VH and VL region is typically composed of three CDRs and up to four FRs. For example, in the order of the following amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibody can be made from any species suitable for use as an antibody, such as a rabbit, alpaca, camel, mouse or rat. In one embodiment the antibody is of human or murine origin. The antibody, for example, can be human, humanized or chimeric.

The term "single-body" anti-system refers to antibodies derived from a population of substantially homogeneous antibodies, ie, the individual antibodies of the group are identical, but a few may undergo natural mutations. Individual antibodies recognize a single antigen binding site with high specificity. The term "monoclonal antibody" does not refer to a particular manufacturing method.

The term "intact" anti-system refers to an antibody that includes not only an antigen-binding region but also a light chain and heavy-chain constant region. The constant region can be a naturally occurring region or a variant in which a majority of the amino acid positions are altered.

The term "modified whole" anti-system refers to a whole antibody fused to another non-antibody-derived polypeptide or protein via a covalent bond (eg, a peptide link) via its amine or carboxy terminus. Furthermore, antibodies can be modified by introducing a reactive cysteine acid at a defined position to facilitate binding to the venom (see Junutula et al. Nat Biotechnol. 2008 Aug; 26(8): 925-32).

The term "human" antibody is an antibody obtainable from humans or a synthetic human antibody. A "synthetic" human antibody is an antibody obtained in part or in whole from a synthetic sequence by computer simulation based on human antibody sequence analysis. Human antibodies can be encoded, for example, by nucleic acids isolated from a library of antibody sequences of human origin. An example of such antibodies can be found in Söderlind et al, Nature Biotech. 2000, 18: 853-856.

The term "humanized" or "chimeric" anti-system describes antibodies consisting of non-human and human sequence components. Among these antibodies, a part of the human immunoglobulin sequence (receptor) is replaced by a sequence component (donor) of a non-human immunoglobulin. In many cases, the donor is a murine immunoglobulin. In the case of a humanized antibody, the amino acid of the acceptor CDR is substituted with the amino acid of the donor. In some cases, the structural amino acid is also replaced by the corresponding donor amino acid. In some cases, a humanized antibody contains an amino acid that is not present in the receptor or donor and is inserted during antibody optimization. In the case of inlays and antibodies, the variable regions of the donor immunoglobulin are fused to the constant regions of human antibodies.

As used herein, the term complementarity determining region (CDR) refers to the amino acid necessary for binding to an antigen in the variable region of an antibody. Each variable region typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each CDR region may comprise an amino acid according to the Kabat definition and/or an amino acid according to the hypermutation ring defined by Chotia. The definition of Kabat encompasses, for example, about 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) amine position of the light chain variable region and 31-35 (CDR1) of the heavy chain variable region. , 50-65 (CDR2) and 95-102 (CDR3) regions (Kabat Et al., Immulological Interest of Strategies of Sequences, 5th Ed. Public Health Service, Health of National Institutes, Bethesda, MD. (1991)). The definition of Chotia encompasses, for example, the approximately 26-32 (CDR1), 50-56 (CDR2) and 91-96 (CDR3) amine position of the light chain variable region and the heavy chain variable region 26-32 (CDR1) , regions of 53-55 (CDR2) and 96-101 (CDR3) (Chothia and Lesk; J Mol Biol 196:901-917 (1987)). In some cases, the CDRs can include an amino acid from a CDR region as defined by Kabat and Chotia.

Antibodies can be divided into different classes according to the amino acid sequence of the heavy chain constant region. There are five major full antibodies: IgA, IgD, IgE, IgG, and IgM, many of which can be subdivided into additional subspecies (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Corresponding to the different types of heavy chain constant regions were identified as [alpha/α], [delta/δ], [epsilon/ε], [gamma/γ], and [mu/μ]. Antibody ternary structures and subunit structures are known.

The term "functional fragment" or "antigen-binding antibody fragment" of an antibody/immunoglobulin is defined as a fragment of an antibody/immunoglobulin (eg, a variable region of IgG) that further encompasses the antigen-binding region of an antibody/immunoglobulin. . An "antigen binding region" of an antibody typically encompasses a hypervariable region of one or more antibodies, such as a CDR, CDR2 and/or CDR3 region. However, the "architecture" or "skeleton" region of an antibody may also be involved in the binding of an antibody to an antigen. The architectural region forms the skeleton of the CDR. The antigen binding region preferably comprises at least 4 to 103 amino acid chains of the light chain variable region and 5 to Amino acid of 109 chain variable regions, more preferably amino acid of 3 to 107 light chain variable region and amino acid of 4 to 111 heavy chain variable regions, particularly preferably given light weight Chain and heavy chain variable regions, i.e., 1-109 of VL and 1- to 113 amino acids of VH (numbering according to WO97/08320).

The "functional fragment" or "antigen-binding fragment" of the present invention encompasses (non-deterministic) Fab, Fab', F(ab') ₂ and Fv fragments, diabody, single-region antibody (DAb), linear antibody, antibody Individual chains (single-chain Fv, abbreviated as ScFv); and polyspecific antibodies, such as bi- and tri-specific antibodies, for example, by antibody fragment CAK Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press ; R. Kontermann & S. Duebel, editors (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag). Antibodies other than "multi-specific" or "multifunctional" antibodies are such antibodies having the same binding site. Multi-specific antibodies may be specific for different epitopes or may be specific for more than one antigen (see, for example, WO 93/17715; WO 92/08802; WO 91/00360; WO 92/ 05793; Tutt, et al., 1991, J. Immunol. 147: 60 69; U.S. Patent No. 4,474,893; 4,7,14,1 1,4,925,648; 5,573,920; 5,601,8 19; or Kostelny et al. Person, 1992, J. Immunol. 148: 1547 1553). The F(ab') ₂ or Fab molecule can be constructed to reduce or otherwise completely prevent the number of intramolecular disulfide interactions occurring in the Ch1 and CL intervals.

A "functional fragment" or "antigen-binding antibody fragment" can be fused to another polypeptide or protein that is not derived from an antibody via a covalent or carboxy terminus thereof by a covalent bond (eg, a peptide link). Furthermore, antibodies and antigen-binding fragments can be modified by introduction of reactive cysteine at defined positions to facilitate binding to the venom (see Junutula et al. Nat Biotechnol. 2008 Aug; 26(8): 925-32 ).

Multiple strains of antibodies can be prepared by methods known to those of ordinary skill in the art. Monoclonal antibodies can be prepared by methods known to those of ordinary skill in the art (Köhler and Milstein, Nature, 256, 495-497, 1975). Human and humanized monoclonal antibodies can be prepared by methods known to those of ordinary skill in the art (Olsson et al, Meth Enzymol. 92, 3-16 or Cabilly et al. US 4,816,567 or Boss et al. US 4,816,397).

A variety of methods for preparing human antibodies and fragments thereof are known to those of ordinary skill in the art, for example, by gene transfer mice (N Lonberg and D Huszar, Int Rev Immunol. 1995; 13(1): 65-93 Or phage display technology (Clackson et al, Nature. 1991 Aug 15; 352 (6336): 624-8). The antibodies of the invention can be obtained from a library of recombinant antibodies consisting of a large number of amino acid sequences of antibodies from a pool of healthy volunteers. Antibodies can also be made by known recombinant DNA techniques. The nucleic acid sequence of an antibody can be obtained by conventional sequencing or can be obtained from a public database.

An "isolated" antibody or binding agent is purified to remove additional cellular components. Composition of contaminated cells that may interfere with diagnostic or therapeutic utility There are, for example, peptides and non-peptidic components of enzymes, hormones or other cells. Preferred antibodies or binding agents are those which have been purified to more than 95% relative to the antibody or binding agent (for example, by the Lowry method, UV-Vis spectroscopy or by SDS capillary electrophoresis). Furthermore, the anti-system is purified to the extent that it is possible to determine the amino acid of at least 15 amine-terminated or internal amino acid sequences, or to homogeneity, which is based on SDS-PAGE in reducing or non-reducing conditions. Detection (detection can be by Coomassie Blau staining or preferably by silver). However, antibodies are typically prepared in one or more purification steps.

The term "specifically binds" refers to an antibody or binding agent that binds to a predetermined antigen/target molecule. The specific binding of the antibody or binding agent typically has an affinity of at least 10 ^-7 M (as a Kd value; that is, preferably has a Kd value of less than 10 ^-7 M), wherein the antibody or binding agent is for a predetermined The antigen/target molecule has at least twice the affinity for a non-specific antigen/target molecule (eg, bovine serum albumin or casein), wherein the non-specific antigen/target molecule is not a predetermined antigen/target molecule or Closely related antigen/target molecule.

The antibodies specific to the cancer cell antigen are preferably those of ordinary skill in the art by means of familiar methods (e.g., recombinant expression) or commercially available (e.g., from Merck KGaA) methods. preparation. Examples of known commercially available antibodies for cancer treatment are Erbitux® (Cetuximab, Merck KGaA), Avastin® (bevacizumab, Roche) and Herceptin® (trastuzumab, Genentech). Trastuzumab is a recombinant humanized monoclonal antibody of the IgG1 kappa type, which is in a cell The analysis (Kd = 5 nM) binds to the extracellular domain of human epidermal growth factor with high affinity. This anti-system is produced by recombination in CHO cells.

The compound of formula (I) represents a subgroup of compounds of formula (Ia).

A preferred subject of the invention is a binder-drug conjugate of formula (Ia) wherein n is a number from 1 to 50 and AK is AK ₁ or AK ₂ wherein AK ₁ is a binding agent, preferably anti- The mesothelin antibody) is bonded to the G group via a sulfur atom of the binding agent, and AK ₂ is a binding agent, preferably an anti-mesothelin antibody, which is bonded to the G group via a nitrogen atom of the binding agent. Knot, when K = AK ₁ , G is a group of the following formula, Wherein the # ¹ system designated connection position sulfur atom binding agent, the # ² line marked with connection position L ¹ groups of, or when AK = AK _2, a carbonyl group, L ¹ is a bond, a straight-chain (C ₁ - C ₁₀ )-alkylene group, group of the following formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and L ^1A is a linear (C ₂ -C ₁₀ )- An alkyl group, B ¹ is a group of the formula Where ## ⁵ indicates the position of attachment to the L ^1A group, ## ⁶ indicates the position of attachment to the L ^1B group, L ⁵ is a bond or (C ₂ -C ₄ )-alkylene group, and L ⁶ is One-key or one-group Where ## ⁷ indicates the position of attachment to the carbonyl group, ## ⁸ indicates the position of attachment to L ^1B , R ³³ is hydrogen, (C ₁ -C ₄ )-alkylcarbonyl, and the third butyloxycarbonyl group Or benzyloxycarbonyl, R ³⁴ is hydrogen or methyl, R ²⁹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²⁹ and R ³⁰ and its bonded atoms together form a 5- or 6-membered heterocyclic ring, R ³¹ is hydrogen or (C ₁ -C ₄ )-alkyl, and R ³² is hydrogen or (C ₁ -C ₄ )-alkane a group, or R ³¹ and R ^{32 together} with the atom to which they are bonded form a 5- or 6-membered heterocyclic ring, L ^1B is a linear (C ₂ -C ₁₀ )-alkylene group, and wherein (C ₁ -C ₁₀ )-alkylene group may be substituted by 1 to 4 substituents each independently selected from the group consisting of methyl, hydroxy and benzyl, and the intermediate alkylene chain thereof is 1, 2, 1, 3 or 1, 4 carbon atoms of the relationship, including any carbon atoms in between, can be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and B is a bond or a group of the formula Where * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , P is O or NH, L ³ is a bond or (C ₂ -C ₄ )-alkylene, and L ⁴ is a bond or a group of the following formula, Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is hydrogen or methyl, and R ²⁸ is hydrogen, (C ₁ -C ₄ )-alkyl a carbonyl group, a tert-butyloxycarbonyl group or a benzyloxycarbonyl group, Q ¹ is a 4- to 7-membered heterocyclic ring, Q ² is a 3- to 7-membered carbocyclic ring or a 4- to 7-membered heterocyclic ring, R ¹⁴ Is hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁵ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁴ and R ^{15 together} with the atoms to which they are bonded form a 5- or 6- a heterocyclic ring, R ¹⁶ is hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁶ and R ¹⁷ are bonded together with the atoms to which they are bonded a 5- or 6-membered heterocyclic ring, R ¹⁸ is hydrogen or (C ₁ -C ₄ )-alkyl, and R ¹⁹ is hydrogen or a pendant group of a natural alpha-amino acid or analog or isomer thereof, R ²⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁹ and R ^{20 together} with the atoms to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or (C ₁ -C ₄ )-alkyl , R ²² is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²¹ and R ^{22 together} with the atom to which they are bonded form a 3- to 7-membered carbocyclic ring, and R ²³ is (C ₁ -C ₄ ) - alkyl, R ²⁴ is hydrogen or (C ₁ -C ₄₎ - alkyl, R ²⁷ is hydrogen or (C ₁ -C ₄₎ - alkyl, R ³⁶ is , (C ₁ -C ₄₎ - alkyl carbonyl, tert-butoxy carbonyl or benzyloxy carbonyl group, R ³⁷ is hydrogen or methyl, or R ³⁶ and R ³⁷ phase of atoms bonded thereto together form a pyrrolidine a ring, L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be passed through 1 to 4 each independently substituted with a substituent selected from the group consisting of a methyl group, a hydroxyl group and a benzyl group, and two carbon atoms in which the intermediate alkylene chain is in a 1, 2, 1, 3 or 1, 4 relationship, and includes Any carbon atom in between may be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen or methyl, and R ² is isopropyl, isobutyl, second butyl, tert-butyl, phenyl, benzyl, 1-hydroxy Ethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1 H -imidazole-4- Methyl or 1 H -indol-3-ylmethyl, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 , 1-diyl group Wherein # ⁴ indicates the position of attachment to an adjacent nitrogen atom, and # ⁵ indicates the position of attachment to a carbonyl group, wherein the A ring of the NO group is present, which is a mono- or bicyclic ring of the following formula, which is substituted as needed. Heterocycle Wherein # ⁶ is a position to which a carbonyl group is bonded, R ⁶ is hydrogen, a hydroxyl group or a benzyloxy group, R ³ is hydrogen or a methyl group, and R ⁴ is an isopropyl group, an isobutyl group, a second butyl group, and a third group. Butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, two Phenylmethyl, 1 H -imidazol-4-ylmethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form the formula (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a 4 a 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, R ¹¹ is a benzyl group, which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, a methyl group or a group of the following formula group Wherein the line marked with # ⁹ -CHC (R ²⁶⁾ -T ² the connection position, R ¹² is phenyl, which may be, substituted carboxyl group or the formula -S (O) ₂ OH substituents of the group by methoxycarbonyl, R ¹³ is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indole-3- A methyl group, R ³⁵ is a methyl group or a hydroxyl group, and a solvate of a salt, a solvate thereof and a salt thereof.

A preferred subject of the invention is a binder-drug conjugate of formula (Ia) wherein n is a number from 1 to 50, AK is AK ₁ or AK ₂ wherein AK ₁ is a binding agent which is sulfur via a binding agent The atom is bonded to the G group, and AK ₂ is a binding agent which is bonded to the G group via a nitrogen atom of the bonding agent. When AK=AK ₁ , G is a group of the following formula Where # ¹ indicates the position of attachment to the sulfur atom of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , is a carbonyl group, and L ¹ is a bond, a straight chain (C ₁ -C ₁₀ )-alkylene group, group of the following formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and L ^1A is a linear (C ₂ -C ₁₀ )- An alkyl group, B ¹ is a group of the following formula, Where ## ⁵ indicates the position of attachment to the L ^1A group, ## ⁶ indicates the position of attachment to the L ^1B group, L ⁵ is a bond or (C ₂ -C ₄ )-alkylene group, and L ⁶ is One-key or one-group Where ## ⁷ indicates the position of attachment to the carbonyl group, ## ⁸ indicates the position of attachment to L ^1B , and R ³³ is hydrogen, (C ₁ -C ₄ )-alkylcarbonyl, third butoxycarbonyl or Benzyloxycarbonyl, R ³⁴ is hydrogen or methyl, R ²⁹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²⁹ and R ^{30 together} with the atoms bonded thereto form a 5- or 6-membered heterocyclic ring, R ³¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³² is hydrogen or (C ₁ -C ₄ )-alkyl Or R ³¹ and R ^{32 together} with the atoms to which they are bonded form a 5- or 6-membered heterocyclic ring, L ^1B is a linear (C ₂ -C ₁₀ )-alkylene group, and wherein (C ₁ -C ₁₀ The alkylene group may be substituted with 1 to 4 substituents each independently selected from the group consisting of methyl, hydroxy and benzyl, and the intermediate alkylene chain thereof is 1, 2, 1, 3 or 1, 4 The two carbon atoms of the relationship, including any carbon atoms in between, can be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and B is a bond or a group of the formula Where * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , P is O or NH, L ³ is a bond or (C ₂ -C ₄ )-alkylene, and L ⁴ is Key or group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is hydrogen or methyl, and R ²⁸ is hydrogen, (C ₁ -C ₄ )-alkyl a carbonyl group, a tert-butoxycarbonyl group or a benzyloxycarbonyl group, Q ¹ is a 4- to 7-membered heterocyclic ring, Q ² is a 3- to 7-membered carbocyclic ring or a 4- to 7-membered heterocyclic ring, and R ¹⁴ is Hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁵ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁴ and R ^{15 together} with the atoms to which they are bonded form a 5- or 6-member a heterocyclic ring, R ¹⁶ is hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁶ and R ^{17 together} with the atoms to which they are bonded form a a 5- or 6-membered heterocyclic ring, R ¹⁸ is hydrogen or (C ₁ -C ₄ )-alkyl, and R ¹⁹ is hydrogen or a pendant group of a natural a-amino acid or an analog or isomer thereof, R ²⁰ Is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁹ and R ^{20 together} with the atom to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ²² is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²¹ and R ^{22 together} with the atoms to which they are bonded form a 3- to 7-membered carbocyclic ring, and R ²³ is (C ₁ -C ₄ ) - alkyl, R ²⁴ is hydrogen or (C ₁ -C ₄₎ - alkyl, R ²⁷ is hydrogen or (C ₁ -C ₄₎ - alkyl, R ³⁶ is hydrogen (C ₁ -C ₄₎ - alkyl carbonyl, tert-butoxy carbonyl or benzyloxy carbonyl group, R ³⁷ is hydrogen or methyl, or R R ³⁷ bonded thereto of atoms and ³⁶ together form a pyrrolidine ring , L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the following formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be passed through 1 to 4 each independently substituted with a substituent selected from the group consisting of a methyl group, a hydroxyl group and a benzyl group, and two carbon atoms in which the intermediate alkylene chain is in a 1, 2, 1, 3 or 1, 4 relationship, and includes Any carbon atom in between may be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen or methyl, and R ² is isopropyl, isobutyl, second butyl, tert-butyl, phenyl, benzyl, 1-hydroxy Ethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1 H -imidazol-4-yl Methyl or 1 H -indol-3-ylmethyl, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 of the following formula, 1-diyl group Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Wherein # ⁶ is the position of the linkage to the carbonyl group, R ⁶ is hydrogen, hydroxy or benzyloxy R ³ is hydrogen or methyl, and R ⁴ is isopropyl, isobutyl, second butyl, third butyl Base, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenyl Methyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form (1 S , 2 R )- 2-phenylcyclopropane-1,1-diyl group # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a 4 a 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, a methyl group or a group of the formula group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. Methyl, R ³⁵ is a methyl or hydroxy group, as well as solvates of salts, solvates and salts thereof.

A preferred subject of the invention is a binding agent-drug conjugate of the formula (Ia) wherein n is a number from 1 to 50 and AK is AK ₁ or AK ₂ wherein AK ₁ is bound to mesothelin and via a binding agent An antibody or antigen-binding antibody fragment in which a sulfur atom of a cysteine residue is bonded to a G group, and AK ₂ is an NH side group and a G group bond which binds to mesothelin and the amino acid residue via a binding agent An antibody or antigen-binding antibody fragment, when AK = AK ₁ , G is a group of the formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , is a carbonyl group, and L ¹ is a bond, straight Chain (C ₂ -C ₆ )-alkylene group, group of the formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and L ^1A is a linear (C ₂ -C ₆ )-alkylene Base, B ¹ is a group of the formula Where ## ⁵ indicates the position of attachment to the L ^1A group, ## ⁶ indicates the position of attachment to the L ^1B group, L ⁵ is a bond, and L ⁶ is a bond or a group of the following formula Where ## ⁷ indicates the position of attachment to the carbonyl group, ## ⁸ indicates the position of attachment to L ^1B , R ³³ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, and R ³⁴ is hydrogen or methyl. R ²⁹ hydrogen, R ³⁰ hydrogen, R ³¹ hydrogen or methyl, R ³² hydrogen or methyl, L ^1B is a linear (C ₂ -C ₆ )-alkylene group, and (C ₂ -C ₆ )- An alkyl group may be substituted with 1 or 2 methyl substituents, and B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** is linked to the position of the carbonyl group, **** is linked to the position of L ² , R ²⁵ is hydrogen or methyl, and R ²⁸ is hydrogen, methylcarbonyl or tert-butoxycarbonyl. , Q ¹ is a 4- to 7-membered heterocyclic ring, R ¹⁴ is hydrogen, R ¹⁵ is hydrogen, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ¹⁷ are bonded thereto. Atoms together form a pipe a base ring, R ¹⁸ is hydrogen, R ¹⁹ is hydrogen, methyl, prop-2-yl, 2-methylprop-1-yl or 1-methylprop-1-yl, and R ²⁰ is hydrogen or methyl. Or R ¹⁹ and R ^{20 together} with the atoms to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or methyl, R ²² is hydrogen or methyl, or R ²¹ and R ²² are bonded together with the atoms to which they are bonded a cyclopropyl ring, R ²³ is methyl, R ²⁴ is hydrogen or methyl, R ²⁷ is hydrogen, R ³⁶ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, R ³⁷ is hydrogen or methyl, or R ³⁶ and R ^{37 together} with the atoms to which they are bonded form a pyrrolidine ring, L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be 1 or Substituted by two methyl substituents, D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-yl a methyl group, or R ¹ and R ^{2 together} with a carbon atom bonded thereto to form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Wherein # ⁶ indicates the position of attachment to a carbonyl group, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-benzene. Ethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 of the following formula, 1-diyl group # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a 4 a 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, R ¹¹ is a benzyl group, which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, a methyl group or a group of the following formula group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. Methyl, R ³⁵ is a methyl or hydroxy group, as well as solvates of salts, solvates and salts thereof.

A preferred subject of the invention is a binding agent-drug conjugate of formula (Ia) wherein n is a number from 1 to 20 and AK is AK ₁ or AK ₂ wherein AK ₁ is bound to mesothelin and via a binding agent An antibody or antigen-binding antibody fragment in which a sulfur atom of a cysteine residue is bonded to a G group, and AK ₂ is an NH side group and a G group bond which binds to mesothelin and the amino acid residue via a binding agent An antibody or antigen-binding antibody fragment, when AK = AK ₁ , G is a group of the formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , is a carbonyl group, L ¹ is a bond, straight Chain (C ₂ -C ₆ )-alkylene group, group of the formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and L ^1A is a linear (C ₂ -C ₆ )-alkylene Base, B ¹ is a group of the formula Where ## ⁵ indicates the position of attachment to the L ^1A group, ## ⁶ indicates the position of attachment to the L ^1B group, L ⁵ is a bond, and L ⁶ is a bond or a group of the following formula Where ## ⁷ indicates the position of attachment to the carbonyl group, ## ⁸ indicates the position of attachment to L ^1B , R ³³ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, and R ³⁴ is hydrogen or methyl. R ²⁹ is hydrogen, R ³⁰ is hydrogen, R ³¹ is hydrogen or methyl, R ³² is hydrogen or methyl, L ^1B is a linear (C ₂ -C ₆ )-alkylene group, and (C ₂ -C ₆ )-alkylene group may be substituted by 1 or 2 methyl substituents, and B is a bond or a group of the following formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is hydrogen or methyl, and R ²⁸ is hydrogen, methylcarbonyl or tert-butoxycarbonyl. , Q ¹ is a 4- to 7-membered heterocyclic ring, R ¹⁴ is hydrogen, R ¹⁵ is hydrogen, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ¹⁷ are bonded thereto. Atom together form a pipe a base ring, R ¹⁸ is hydrogen, R ¹⁹ is hydrogen, methyl, prop-2-yl, 2-methylprop-1-yl or 1-methylprop-1-yl, and R ²⁰ is hydrogen or methyl. Or R ¹⁹ and R ^{20 together} with the atoms to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or methyl, R ²² is hydrogen or methyl, or R ²¹ and R ²² are bonded together with the atoms to which they are bonded a cyclopropyl ring, R ²³ is methyl, R ²⁴ is hydrogen or methyl, R ²⁷ is hydrogen, R ³⁶ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, R ³⁷ is hydrogen or methyl, or R ³⁶ and R ^{37 together} with the atoms to which they are bonded form a pyrrolidine ring, L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be 1 or Substituted by two methyl substituents, D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-yl a methyl group, or R ¹ and R ^{2 together} with a carbon atom bonded thereto to form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Wherein # ⁶ indicates the position of attachment to a carbonyl group, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-benzene. Ethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 of the following formula, 1-diyl group # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a 4 a 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, a methyl group or a group of the formula group Wherein the line marked with # ⁹ -CHC (R ²⁶⁾ -T ² the connection position, R ¹² is phenyl which may be substituted by a carboxyl group or a methoxycarbonyl group of formula -S (O) ₂ OH the radical, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. Methyl, R ³⁵ is a methyl or hydroxy group, as well as solvates of salts, solvates and salts thereof.

A preferred subject of the invention is a binding agent-drug conjugate of formula (Ia) wherein n is a number from 1 to 10, AK is AK ₁ or AK ₂ wherein AK ₁ is an antibody comprising six MF-Ta The CDR sequence of the antibody, the variable light chain of the MF-Ta antibody, and the light and heavy chains of the variable heavy chain or MF-Ta antibody, and the sulfur atom and the G group of the cysteine residue via the binding agent Bonding, AK ₂ is an antibody comprising the CDR sequences of six MF-Ta antibodies, the variable light chain of the MF-Ta antibody, and the light and heavy chains of the variable heavy chain or MF-Ta antibody, and The NH pendant group of the lysine residue of the binding agent is bonded to the G group. When AK=AK ₁ , G is a group of the following formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , is a carbonyl group, L ¹ is a bond, a linear chain (C ₂ -C ₆ )-alkylene group, a group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₂ -C ₆ )-alkylene may be 1 or Substituted by two methyl substituents, B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is methyl, and R ²⁸ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, Q ¹ is piperidine-1,4-diyl, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ²¹ is hydrogen or methyl, R ²² is hydrogen or methyl, or R ²¹ and R ^{22 together} with the atom to which they are bonded form a cyclopropyl ring, R ²³ is a methyl group, and R ²⁴ is a hydrogen, R ³⁶ is hydrogen, methylcarbonyl or a third butoxycarbonyl group, R ³⁷ is hydrogen or methyl, and L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula Where p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is a group of the following formula, Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-yl a methyl group, or R ¹ and R ^{2 together} with a carbon atom bonded thereto to form a group of (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the following formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Wherein # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H - Ind-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl, and R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, R ¹¹ is benzyl, which may be substituted on the phenyl group by a methoxycarbonyl group or a carboxyl group, and R ⁵ is hydrogen, methyl or the following formula Group, Wherein # ⁹ is the position to which the -CHCH ₂ phenyl group is attached, and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, and R ¹³ is a phenyl group. It may be substituted by a methoxycarbonyl group or a carboxyl group, and R ³⁵ is a methyl group or a hydroxyl group, and a solvate of a salt, a solvate thereof and a salt thereof.

A preferred subject of the invention is a binding agent-drug conjugate of formula (Ia) wherein n is a number from 1 to 10, AK is AK ₁ or AK ₂ wherein AK ₁ is an antibody comprising six MF-Ta The CDR sequence of the antibody, the variable light chain of the MF-Ta antibody, and the light and heavy chains of the variable heavy chain or MF-Ta antibody, and the sulfur atom and the G group of the cysteine residue via the binding agent Bonding, AK ₂ is an antibody comprising the CDR sequences of six MF-Ta antibodies, the variable light chain of the MF-Ta antibody, and the light and heavy chains of the variable heavy chain or MF-Ta antibody, and The NH pendant group of the lysine residue of the binding agent is bonded to the G group. When AK=AK ₁ , G is a group of the following formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , is a carbonyl group, L ¹ is a bond, straight Chain (C ₂ -C ₆ )-alkylene group, group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₂ -C ₆ )-alkylene may be 1 or Substituted by two methyl substituents, B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to the L ² group, L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the formula Wherein *** is the position of the linkage to the carbonyl group, **** is the position of the linkage to L ¹ , R ²⁵ is a methyl group, and R ²⁸ is hydrogen, methylcarbonyl or a third butoxycarbonyl group, Q ¹ is piperidine-1,4-diyl, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ²¹ is hydrogen or methyl, R ²² is hydrogen or methyl, or R ²¹ and R ^{22 together} with the atom to which they are bonded form a cyclopropyl ring, R ²³ is methyl, R ²⁴ is hydrogen, L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula Where p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-yl a methyl group, or R ¹ and R ^{2 together} with a carbon atom bonded thereto to form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Wherein # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H - Ind-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl, and R ⁸ is hydrogen or methyl , R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, R ¹¹ is benzyl, which may be substituted on the phenyl group by a methoxycarbonyl group or a carboxyl group, and R ⁵ is hydrogen, methyl or the following formula Group Wherein # ⁹ is the position to which the -CHCH ₂ phenyl group is attached, and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, and R ¹³ is a phenyl group. It may be substituted by a methoxycarbonyl group or a carboxyl group, and R ³⁵ is a methyl group or a hydroxyl group, and a solvate of a salt, a solvate thereof and a salt thereof.

A preferred subject of the invention is a binding agent-drug conjugate of the formula (Ia) as shown above, wherein n is a number from 1 to 10, AK is AK ₂ , wherein AK ₂ is an antibody comprising six MF The CDR sequence of the -Ta antibody, the variable light chain of the MF-Ta antibody, and the light and heavy chains of the variable heavy chain or MF-Ta antibody, and the NH side group and G of the lysine residue via the binding agent The group is bonded, G is a carbonyl group, L ¹ is a bond, B is a bond, and L ² is a linear (C ₃ -C ₆ )-alkylene group or a group of the following formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Wherein # ³ is the position to which the nitrogen atom is bonded, R ¹ is hydrogen, R ² is benzyl, 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² are bonded thereto. The carbon atoms of the bond together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Wherein # ⁴ indicates the position of attachment to the nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, optionally substituted heterocyclic ring Where # ⁶ is the position to which the carbonyl group is attached, R ³ is hydrogen, R ⁴ is benzyl, 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 are} related thereto. The bonded carbon atoms together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula -C (= O) -OR ⁷ or -C (= O) -NR ⁸ a group of R ⁹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl, R ⁸ is hydrogen, R ⁹ is hydrogen or benzyl, R ³⁵ It is a methyl group, as well as a solvate of its salts, solvates and salts.

A preferred subject of the invention is a binding agent-drug conjugate of formula (Ia) wherein n is a number from 1 to 10 and AK is AK ₂ , wherein AK ₂ is an antibody comprising six MF-Ta antibodies a CDR sequence, a variable light chain of an MF-Ta antibody, and a light chain and a heavy chain of a variable heavy chain or MF-Ta antibody, and bonded to a G group via an NH side group of an amine acid residue of the binding agent , G is a carbonyl group, L ¹ is a bond, B is a bond, and L ² is a linear (C ₃ -C ₆ )-alkylene group or a group of the following formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Where # ³ is the position to which the nitrogen atom is bonded, R ¹ is hydrogen, R ² is benzyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² are bonded together with the carbon atom to which they are bonded (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Wherein # ⁶ is the position of the linkage to the carbonyl group, R ³ is hydrogen, R ⁴ is benzyl or 1 H -indol-3-ylmethyl, or R ³ and R ⁴ are bonded to the carbon atom to which they are bonded Formation of the (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula -C (= O) -OR ⁷ or -C (= O) -NR ⁸ a group of R ⁹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl, R ⁸ is hydrogen, R ⁹ is hydrogen or benzyl, R ³⁵ It is a methyl group, and a solvate of its salts, solvates and salts.

A preferred subject of the invention is a binding agent-drug conjugate of formula (Ia) as shown above, wherein n is a number from 1 to 10 and AK is AK ₁ , wherein AK ₁ is an antibody comprising six MF a CDR sequence of a -Ta antibody, a variable light chain of a MF-Ta antibody, and a light chain and a heavy chain of a variable heavy chain or MF-Ta antibody, and a sulfur atom and a G of a cysteine residue via a binding agent Group bonding, G is a group of the following formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, and L ¹ is a bond, linear (C ₃ -C ₅ )-alkylene Base or group Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₃ -C ₅ )-alkylene may be 1 or Substituted by two methyl substituents, B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** is linked to the position of the carbonyl group, **** is linked to the position of L ² , R ²⁵ is a methyl group, and R ²⁸ is hydrogen, methylcarbonyl or a third butoxycarbonyl group, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, L ² is a linear (C ₃ -C ₅ )-alkylene group or a group of the following formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Wherein # ³ is the position to which the nitrogen atom is bonded, R ¹ is hydrogen, R ² is benzyl, 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² are bonded thereto. The carbon atoms of the bond together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Where # ⁶ is the position to which the carbonyl group is attached, R ³ is hydrogen, R ⁴ is benzyl, 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 are} related thereto. The bonded carbon atoms together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula -C (= O) -OR ⁷ or -C (= O) -NR ⁸ a group of R ⁹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl, R ⁸ is hydrogen, R ⁹ is hydrogen or benzyl, R ³⁵ is a methyl group, as well as a solvate of its salts, solvates and salts.

A preferred subject of the invention is a binding agent-drug conjugate of formula (Ia) wherein n is a number from 1 to 10 and AK is AK ₁ , wherein AK ₁ is an antibody comprising six MF-Ta antibodies a CDR sequence, a variable light chain of a MF-Ta antibody, and a light chain and a heavy chain of a variable heavy chain or MF-Ta antibody, and bonded to a G group via a sulfur atom of a cysteine residue of the binding agent , G is a group of the following formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, and L ¹ is a bond, linear (C ₃ -C ₅ )-alkylene Base or group Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₃ -C ₅ )-alkylene may be 1 or Substituted by two methyl substituents, B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** is linked to the position of the carbonyl group, **** is linked to the position of L ² , R ²⁵ is a methyl group, and R ²⁸ is hydrogen, methylcarbonyl or a third butoxycarbonyl group, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, L ² is a linear (C ₃ -C ₅ )-alkylene group or a group of the following formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Where # ³ is the position to which the nitrogen atom is bonded, R ¹ is hydrogen, R ² is benzyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² are bonded together with the carbon atom to which they are bonded (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, and # ⁵ indicates the position of attachment to a carbonyl group, wherein the A ring of the NO group is present, which is a mono- or bicyclic ring of the following formula, which is optionally substituted Heterocycle Wherein # ⁶ is the position of the linkage to the carbonyl group, R ³ is hydrogen, R ⁴ is benzyl or 1 H -indol-3-ylmethyl, or R ³ and R ⁴ are bonded to the carbon atom to which they are bonded Formation of the (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ of formula -C (= O) -OR ⁷ or -C (= O) -NR ⁸ R ^a group of ⁹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl, R ⁸ is hydrogen, R ⁹ is hydrogen or benzyl, R ³⁵ It is a methyl group, as well as a solvate of its salts, solvates and salts.

Further, the present invention also provides a compound of the formula (XXXa) Wherein Cys is a cysteine residue bonded to a carbon atom of amber quinone via a sulfur atom of a pendant group, and L ¹ is a bond, a linear (C ₁ -C ₁₀ )-alkylene group, and the following formula Group Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and L ^1A is a linear (C ₂ -C ₁₀ )-alkylene Base, B ¹ is a group of the formula Where ## ⁵ indicates the position of attachment to the L ^1A group, ## ⁶ indicates the position of attachment to the L ^1B group, L ⁵ is a bond or (C ₂ -C ₄ )-alkylene group, and L ⁶ is a bond, R ²⁹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²⁹ and R ^{30 together} with the atoms to which they are bonded form a a 5- or 6-membered heterocyclic ring, R ³¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³² is hydrogen or (C ₁ -C ₄ )-alkyl, or R ³¹ and R ³² are bonded thereto The atoms of the knot together form a 5- or 6-membered heterocyclic ring, and L ^1B is a linear (C ₂ -C ₁₀ )-alkylene group in which the (C ₁ -C ₁₀ )-alkylene group can pass through 1 to 4 Each of which is independently substituted with a substituent selected from the group consisting of a methyl group, a hydroxyl group, and a benzyl group, and two carbon atoms in which the intermediate alkylene chain is in a 1, 2, 1, 3, or 1, 4 relationship, including any The carbon atom in between may be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and B is a bond or a group of the formula Where * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , P is O or NH, L ³ is a bond or (C ₂ -C ₄ )-alkylene, and L ⁴ is a bond, Q ¹ is a 4- to 7-membered heterocyclic ring, Q ² is a 3- to 7-membered carbocyclic ring or a 4- to 7-membered heterocyclic ring, and R ¹⁴ is hydrogen or (C ₁ -C ₄ )-alkyl , R ¹⁵ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁴ and R ^{15 together} with the atoms to which they are bonded form a 5- or 6-membered heterocyclic ring, R ¹⁶ is hydrogen or (C ₁ - C ₄ )-alkyl, R ¹⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a 5- or 6-membered heterocyclic ring, R ¹⁸ is Hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁹ is hydrogen or a side group of a natural alpha-amino acid or analogue or isomer thereof, R ²⁰ is hydrogen or (C ₁ -C ₄ )-alkane a group, or R ¹⁹ and R ^{20 together} with the atoms to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ²² is hydrogen or (C ₁ -C ₄ ) -alkyl, or R ²¹ and R ^{22 together} with the atoms to which they are bonded form a 3- to 7-membered carbocyclic ring, R ²³ is (C ₁ -C ₄ )-alkyl, R ²⁴ is hydrogen or (C ₁ -C ₄ )-alkyl, R ²⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be passed through 1 to 4 each independently substituted with a substituent selected from the group consisting of a methyl group, a hydroxyl group and a benzyl group, and two carbon atoms in which the intermediate alkylene chain is in a 1, 2, 1, 3 or 1, 4 relationship, and includes Any carbon atom in between may be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen or methyl, and R ² is isopropyl, isobutyl, second butyl, tert-butyl, phenyl, benzyl, 1-hydroxy Ethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1 H -imidazol-4-yl Methyl or 1 H -indol-3-ylmethyl, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 of the following formula, 1-diyl group Wherein # ⁴ indicates the position of attachment to an adjacent nitrogen atom, and # ⁵ indicates the position of attachment to a carbonyl group, wherein the A ring of the NO group is present, which is a monocyclic or bicyclic ring of the following formula, optionally substituted heterocyclic ring Wherein # ⁶ is a position to which a carbonyl group is bonded, R ⁶ is hydrogen, a hydroxyl group or a benzyloxy group, R ³ is hydrogen or a methyl group, and R ⁴ is an isopropyl group, an isobutyl group, a second butyl group, and a third group. Butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, two Benzyl, 1 H -imidazol-4-ylmethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form the formula (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group, # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a 4 a 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, R ¹¹ is a benzyl group, which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is a hydrogen group, a methyl group or a group of the formula Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. Methyl, R ³⁵ is a methyl or hydroxy group, as well as solvates of salts, solvates and salts thereof.

Further preferred in the context of the present invention are compounds of formula (XXXa) wherein Cys is a cysteine residue bonded to the carbon atom of amber quinone via a sulfur atom of a pendant group, L ¹ is a Bond, linear (C ₂ -C ₆ )-alkylene group, group of the following formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and L ^1A is a linear (C ₂ -C ₆ )-alkylene group. , B ¹ is a group of the following formula Where ## ⁵ indicates the position of attachment to the L ^1A group, ## ⁶ indicates the position of attachment to the L ^1B group, L ⁵ is a bond, L ⁶ is a bond, R ²⁹ is hydrogen, and R ³⁰ is hydrogen. , R ³¹ is hydrogen or methyl, R ³² is hydrogen or methyl, L ^1B is a linear (C ₂ -C ₆ )-alkylene group, and (C ₂ -C ₆ )-alkylene group thereof may be subjected to 1 Or substituted with 2 methyl substituents, B being a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond or B-1,2-diyl, L ⁴ is a bond, R ¹⁴ is hydrogen, R ¹⁵ Is hydrogen, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ²³ is a methyl group, R ²⁴ is hydrogen or a methyl group, and L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-yl a methyl group, or R ¹ and R ^{2 together} with a carbon atom to which they are bonded, form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula: Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Wherein # ⁶ indicates the position of attachment to a carbonyl group, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-benzene. Ethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 of the following formula, 1-diyl group, # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the atoms to which they are bonded form a 4- To a 7-membered heterocyclic ring, R ¹⁰ is a benzindenyl group, R ¹¹ is a benzyl group, which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is a hydrogen group, a methyl group or a group of the formula Wherein # ⁹ is the position to which the -CHCH ₂ phenyl group is attached, and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, and R ¹³ is a phenyl group. It may be substituted by a methoxycarbonyl group or a carboxyl group, and R ³⁵ is a methyl group or a hydroxyl group, and a solvate of a salt, a solvate thereof and a salt thereof.

Further particularly preferred in the context of the present invention are compounds of formula (XXXa) wherein Cys is a cysteine residue bonded to the carbon atom of amber quinone via a sulfur atom of a pendant group, L ¹ is a a bond or a linear (C ₂ -C ₆ )-alkylene group, and B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond, L ⁴ is a bond, R ¹⁶ is hydrogen or methyl, and R ¹⁷ is hydrogen or methyl. L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Where # ³ is the position to which the nitrogen atom is bonded, R ¹ is hydrogen, R ² is benzyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² are bonded together with the carbon atom to which they are bonded a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group, Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Wherein # ⁶ is the position of the linkage to the carbonyl group, R ³ is hydrogen, R ⁴ is benzyl or 1 H -indol-3-ylmethyl, or R ³ and R ⁴ are bonded to the carbon atom to which they are bonded Forming a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula -C (= O) -OR ⁷ or -C (= O) -NR ⁸ A group of R ⁹ wherein R ⁷ is hydrogen, R ⁸ is hydrogen, R ⁹ is hydrogen, R ³⁵ is methyl, and solvates of the salts, solvates and salts thereof.

A preferred subject of the invention is a compound of formula (XXXa) shown above wherein Cys is a cysteine residue bonded to the carbon atom of amber quinone via a sulfur atom of a pendant group, L ¹ is a bond or a linear (C ₂ -C ₆ )-alkylene group, B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond, L ⁴ is a bond, R ¹⁶ is hydrogen or methyl, and R ¹⁷ is hydrogen or methyl. L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, R is benzyl, 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² are bonded thereto. The carbon atoms together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula: Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Where # ⁶ is the position to which the carbonyl group is attached, R ³ is hydrogen, R ⁴ is benzyl, 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 are} related thereto. The bonded carbon atoms together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula -C (= O) -OR ^{7 or} -C (= O) -NR ⁸ A group of R ⁹ wherein R ⁷ is hydrogen, R ⁸ is hydrogen, R ⁹ is hydrogen, R ³⁵ is methyl, and solvates of the salts, solvates and salts thereof.

The invention further provides a compound of formula (XXXI) Wherein L ¹ is a bond, a linear (C ₁ -C ₁₀ )-alkylene group, a group of the formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and L ^1A is a linear (C ₂ -C ₁₀ )-alkylene Base, B ¹ is a group of the formula Where ## ⁵ indicates the position of attachment to the L ^1A group, ## ⁶ indicates the position of attachment to the L ^1B group, L ⁵ is a bond or (C ₂ -C ₄ )-alkylene group, and L ⁶ is a bond, R ²⁹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²⁹ and R ^{30 together} with the atoms to which they are bonded form a a 5- or 6-membered heterocyclic ring, R ³¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³² is hydrogen or (C ₁ -C ₄ )-alkyl, or R ³¹ and R ³² are bonded thereto The atoms of the knot together form a 5- or 6-membered heterocyclic ring, L ^1B is a linear (C ₂ -C ₁₀ )-alkylene group, and the (C ₁ -C ₁₀ )-alkylene group thereof may be 1 to 4 Each of which is independently substituted with a substituent selected from the group consisting of a methyl group, a hydroxyl group, and a benzyl group, and two carbon atoms in which the intermediate alkylene chain is in a 1, 2, 1, 3, or 1, 4 relationship, including any The carbon atom in between may be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to the L ² group, P is O or NH, Q ¹ is a 4- to 7-membered heterocyclic ring, and Q ² is 3- to 7- a carbocyclic ring or a 4- to 7-membered heterocyclic ring, R ¹⁸ being hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁹ being hydrogen or a side of hydrogen or a natural alpha-amino acid or analog or isomer thereof a group, R ²⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁹ and R ^{20 together} with the atom to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or (C ₁ -C ₄ ) -alkyl, R ²² is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²¹ and R ^{22 together} with the atoms to which they are bonded form a 3- to 7-membered carbocyclic ring, R ²⁷ is hydrogen or C ₁ -C ₄ )-alkyl, L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be passed through 1 to 4 each independently substituted with a substituent selected from the group consisting of a methyl group, a hydroxyl group and a benzyl group, and two carbon atoms in which the intermediate alkylene chain is in a 1, 2, 1, 3 or 1, 4 relationship, and includes Any carbon atom in between may be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen or methyl, and R ² is isopropyl, isobutyl, second butyl, tert-butyl, phenyl, benzyl, 1-hydroxy Ethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1 H -imidazol-4-yl Methyl or 1 H -indol-3-ylmethyl, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1 of the formula: 1-diyl group Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Wherein # ⁶ is the position of the linkage to the carbonyl group, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen or methyl, and R ⁴ is isopropyl, isobutyl, second butyl, third Butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, two Benzyl, 1 H -imidazol-4-ylmethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form the formula (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a 4 a 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, R ¹¹ is a benzyl group, which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, a methyl group or a group of the following formula group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. Methyl, R ³⁵ is a methyl or hydroxy group, as well as solvates of salts, solvates and salts thereof.

Also preferred in the context of the present invention are compounds of formula (XXXI) wherein L ¹ is a bond, a linear (C ₂ -C ₆ )-alkylene group or a group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₂ -C ₆ )-alkylene may be 1 or Substituted by two methyl substituents, B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , R ¹⁸ is hydrogen, and R ¹⁹ is methyl, prop-2-yl, 2-methylprop-1-yl or -Methylpropan-1-yl, R ²⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁹ and R ^{20 together} with the atoms to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or Methyl, R ²² is hydrogen or methyl, or R ²¹ and R ^{22 together} with the atom to which they are bonded form a cyclopropyl ring, R ²⁷ is hydrogen or methyl, and L ² is linear (C ₂ -C ₆ ) - an alkylene group or a group of the formula Wherein p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may pass 1 or 2 Substituted by a methyl substituent, and two carbon atoms in which the intermediate alkylene chain is in a 1,4 relationship with each other, including any carbon atom interposed therebetween, can be bridged to form a phenyl ring, and D is a group of the following formula group Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-yl a methyl group, or R ¹ and R ^{2 together} with a carbon atom bonded thereto to form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Wherein # ⁶ is the position of the linkage to the carbonyl group, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-benzene. Ethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 of the following formula, 1-diyl group # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a 4 a 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, R ¹¹ is a benzyl group, which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, a methyl group or a group of the formula group Wherein # ⁹ is the position to which the -CHCH ₂ phenyl group is attached, and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, and R ¹³ is a phenyl group. It may be substituted by a methoxycarbonyl group or a carboxyl group, and R ³⁵ is a methyl group or a hydroxyl group, and a solvate of a salt, a solvate thereof and a salt thereof.

Also preferred in the context of the present invention are compounds of formula (XXXI) wherein L ¹ is a bond, B is a bond, and L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the formula group Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Where # ³ is the position to which the nitrogen atom is bonded, R ¹ is hydrogen, R ² is benzyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² are bonded together with the carbon atom to which they are bonded (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Wherein # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, R ⁴ is benzyl or 1 H -indol-3-ylmethyl, or R ³ And R ⁴ and its bonded carbon atom together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula -C (= O) -OR ⁷ or -C (= O) -NR ⁸ A group of R ⁹ wherein R ⁷ is hydrogen, R ⁸ is hydrogen, R ⁹ is hydrogen, R ³⁵ is methyl, and solvates of the salts, solvates and salts thereof.

A preferred subject of the invention is a compound of formula (XXXI) as shown above, wherein L ¹ is a bond, B is a bond, and L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the formula group Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Wherein # ³ is the position to which the nitrogen atom is bonded, R ¹ is hydrogen, R ² is benzyl, 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² are bonded thereto. The carbon atoms of the bond together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Wherein # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is benzyl, 4-hydroxybenzyl or 1 H -indol-3-yl. a methyl group, or R ³ and R ^{4 together} with a carbon atom bonded thereto to form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula -C (= O) -OR ⁷ or -C (= O) -NR ⁸ A group of R ⁹ wherein R ⁷ is hydrogen, R ⁸ is hydrogen, R ⁹ is hydrogen, R ³⁵ is methyl, and solvates of the salts, solvates and salts thereof.

Particularly preferred are the following compounds of the formula (XXXa) and (XXXI) selected from the group: N -[6-(3-{[(2 R )-2-amino-2-carboxyethyl]indolyl}- 2,5-dioxapyrrolidin-1-yl)hexyl] -N -methyl-L-nonylamine fluorenyl, -N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(1 S )-1-carboxy-2-(1 H -indol-3-yl)ethyl]amino}-1-methoxy Benzyl-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L - amidoxime, N- [6-(3-{[(2 R )-2-amino-2-carboxyethyl]indolyl}-2,5-dioxapyrrolidin-1-yl)hexyl ] -N -Methyl-L-Amidinoinyl, -N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3- {[(2 S )-3-(1 H -indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine, N-(6-{[(5S)-5-amino-5-carboxyl Amyl]amino}-6-oxo-oxyhexyl)-N-methyl-L-nonylamine fluorenyl, -N-[(3R,4S,5S)-1-{(2S)-2-[(1R , 2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine guanamine trifluoroacetate, N-(6-{[(5S)-5-amino) -5-carboxypentyl]amino}-6-oxo-oxyhexyl)-N-methyl-L-nonylamine fluorenyl, -N-[(3R,4S,5S)-1-{(2S)-2 -[(1R,2R)-3-{[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl- 3-Phenoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine, and a solvate of salts, solvates and salts thereof.

The present invention further provides a binding agent-drug conjugate of the general formula (I) Wherein n is a number from 1 to 50, AK is a binder, and the §-GL ¹ -BL ² -§§ group is a linker, wherein § indicates the position of attachment to the AK group, and §§ indicates a nitrogen atom The connection position, D is the group of the following formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, or R ¹ And R ² and its bonded carbon atom together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Where # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H - Ind-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a 4 a 7-membered heterocyclic ring, R ¹⁰ is a benzindenyl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, methyl or the following formula Group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. A methyl group, as well as a solvate of its salts, solvates, and salts.

Preferred subject matter of the invention is a binder-drug conjugate of formula (I) wherein n is a number from 1 to 50, AK is AK ₁ or AK ₂ wherein AK ₁ is a binding agent which is sulfur via a binding agent The atom is bonded to the G group, and AK ₂ is a binding agent which is bonded to the G group via a nitrogen atom of the bonding agent. When AK=AK ₁ , G is a group of the following formula Where # ¹ indicates the position of attachment to the sulfur atom of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , is a carbonyl group, and L ¹ is a bond, a straight chain (C ₁ -C ₁₀ )-alkylene or a group of the formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₁ -C ₁₀ )-alkylene can pass through Substituted to four methyl substituents, and two carbon atoms in which the intermediate alkylene chain is 1, 2, 1, 3 or 1, 4 in relation to each other, including any carbon atoms in between, can be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or phenyl ring, B is a bond or a group of the formula Where * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , P is O or NH, L ³ is a bond or (C ₂ -C ₄ )-alkylene, and L ⁴ is a bond or a group of the formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is hydrogen or methyl, Q ¹ is a 4- to 7-membered heterocyclic ring, and Q ² is a 3- to 7-membered carbocyclic ring or a 4- to 7-membered heterocyclic ring, R ¹⁴ is hydrogen or (C ₁ -C ₄ )-alkyl, and R ¹⁵ is hydrogen or (C ₁ -C ₄ )-alkyl, Or R ¹⁴ and R ^{15 together} with the atoms to which they are bonded form a 5- or 6-membered heterocyclic ring, R ¹⁶ is hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁶ and R ^{17 together} with the atoms to which they are bonded form a 5- or 6-membered heterocyclic ring, R ¹⁸ is hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁹ is hydrogen Or a side group of a natural a-amino acid or an analog or isomer thereof, R ²⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁹ and R ²⁰ together form a bond with the atom to which they are bonded Pyrrolidinyl ring, R ²¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ²² is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²¹ and R ²² are bonded to the atom to which they are bonded Forming a 3- to 7-membered carbocyclic ring, R ²³ is (C ₁ -C ₄ )-alkyl, R ²⁴ is hydrogen or (C ₁ -C ₄ )-alkyl, R ²⁷ is hydrogen or (C ₁ - C ₄ )-alkyl, L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene can pass through Substituted to four methyl substituents, and two carbon atoms in which the intermediate alkylene chain is 1, 2, 1, 3 or 1, 4 in relation to each other, including any carbon atoms in between, can be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or phenyl ring, D having the definitions as defined above, as well as solvates of the salts, solvates and salts thereof.

Preferred subject of the invention is a binding agent-drug conjugate of formula (I) wherein n is a number from 1 to 50 and AK is AK ₁ or AK ₂ wherein AK ₁ is an antibody or antigen-binding antibody fragment and is via sulfur The atom is bonded to a G group, and AK ₂ is an antibody or antigen-binding antibody fragment and is bonded to a G group via a nitrogen atom, and G, L ¹ , B, L ² and D have the definitions as defined above, and a salt thereof Solvates of classes, solvates and salts.

Preferred subject matter of the invention is a binder-drug conjugate of formula (I) wherein n is a number from 1 to 20 and AK is AK ₁ or AK ₂ wherein AK ₁ is bound to mesothelin and via a binding agent An antibody or antigen-binding antibody fragment in which a sulfur atom of a cysteine residue is bonded to a G group, and AK ₂ is an NH side group and a G group bond which binds to mesothelin and the amino acid residue via a binding agent An antibody or antigen-binding antibody fragment, when AK = AK ₁ , G is a group of the formula # ¹ wherein the connection position designated based cysteine residues of the binding agent, and # ² connected to a position indicated based groups of T ^1, or when AK = ₂ when AK, a carbonyl group, L ¹ is a bond, a straight a chain (C ₂ -C ₆ )-alkylene group or a group of the formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₂ -C ₆ )-alkylene may pass through a group in which two methyl substituents are substituted for B as a bond or a formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is methyl, and Q ¹ is 4- to 6-membered carbocyclic or piperidine-1. 4-diyl, R ¹⁴ is hydrogen, R ¹⁵ is hydrogen, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ¹⁸ is hydrogen, R ¹⁹ is hydrogen, methyl, prop-2-yl, 2-methylprop-1-yl or 1-methylprop-1-yl, and R ²⁰ is hydrogen or methyl. Or R ¹⁹ and R ^{20 together} with the atoms to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or methyl, R ²² is hydrogen or methyl, or R ²¹ and R ²² are bonded together with the atoms to which they are bonded a cyclopropyl ring, R ²³ is a methyl group, R ²⁴ is hydrogen or methyl, and L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the adjacent nitrogen atom, wherein (C ₂ -C ₆ )-alkylene can be 1 to 2 methyl substituents substituted, D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, or R ¹ And R ² and its bonded carbon atom together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Where # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H - Ind-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a 4 a 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, R ¹¹ is a benzyl group, which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, a methyl group or a group of the following formula group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. A methyl group, as well as a solvate of its salts, solvates, and salts.

A particularly preferred subject of the invention is a binding agent-drug conjugate of formula (I) wherein n is a number from 1 to 10, AK is AK ₁ or AK ₂ wherein AK ₁ is an antibody comprising six MF-Ta The CDR sequence of the antibody, the variable light chain of the MF-Ta antibody, and the light and heavy chains of the variable heavy chain or MF-Ta antibody, and the sulfur atom and the G group of the cysteine residue via the binding agent Bonding, AK ₂ is an antibody comprising the CDR sequences of six MF-Ta antibodies, the variable light chain of the MF-Ta antibody, and the light and heavy chains of the variable heavy chain or MF-Ta antibody, and The NH pendant group of the lysine residue of the binding agent is bonded to the G group. When AK=AK ₁ , G is a group of the following formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , is a carbonyl group, L ¹ is a bond, straight a chain (C ₂ -C ₆ )-alkylene group or a group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₂ -C ₆ )-alkylene may pass through 1 to Substituted by two methyl substituents, B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is methyl, Q ¹ is piperidine-1,4-diyl, and R ¹⁶ is hydrogen. Or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ²¹ is hydrogen or methyl, R ²² is hydrogen or methyl, or R ²¹ and R ^{22 together} with the atom to which they are bonded form a cyclopropyl ring, R ²³ is methyl, R ²⁴ is hydrogen, L ² is a linear (C ₂ -C ₆ )-alkylene group, and D is a group of the following formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, or R ¹ And R ² and its bonded carbon atom together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Where # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is benzyl, 1-phenylethyl or 1 H -indol-3-yl a methyl group, or R ³ and R ^{4 together} with a carbon atom bonded thereto to form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, benzyl or adamantylmethyl, R ⁸ is hydrogen or methyl, and R ⁹ is hydrogen , methyl, ethyl, n-propyl or benzyl, R ¹¹ is benzyl, which may be substituted on the phenyl group via a methoxycarbonyl or carboxyl group, and R ⁵ is hydrogen or a group of the formula Wherein # ⁹ is a position to which a -CHC(R ²⁶ )phenyl group is bonded, and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, and R ¹³ is A phenyl group which may be substituted with a methoxycarbonyl group or a carboxyl group, and a solvate of a salt, a solvate thereof and a salt thereof.

The invention additionally provides a compound of formula (XXX) Wherein Cys is a cysteine residue bonded to a carbon atom of amber quinone via a sulfur atom of a pendant group, and L ¹ is a bond, a linear (C ₁ -C ₁₀ )-alkylene group or Group Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₁ -C ₁₀ )-alkylene can pass through Substituted to four methyl substituents, and two carbon atoms in which the intermediate alkylene chain is 1, 2, 1, 3 or 1, 4 in relation to each other, including any carbon atoms in between, can be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or phenyl ring, B is a bond or a group of the formula Wherein * indicates the position of attachment to the L ¹ group, ** indicates the position of attachment to L ² , P is O or NH, L ³ is a bond or (C ₂ -C ₄ )-alkylene, L ⁴ a group of one bond or the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is hydrogen or methyl Q ¹ is 3- to 7-membered carbon ring or 4- to 7 a nitrogen heterocycle, Q ² is a 3- to 7-membered carbocyclic ring or a 4- to 7-membered nitrogen heterocyclic ring, R ¹⁴ is hydrogen or (C ₁ -C ₄ )-alkyl, and R ¹⁵ is hydrogen or C ₁ -C ₄ )-alkyl, or R ¹⁴ and R ¹⁵ are taken together with the atoms to which they are bonded to form a 5- or 6-membered heterocyclic ring, and R ¹⁶ is hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁶ and R ^{17 together} with the atoms to which they are bonded form a 5- or 6-membered heterocyclic ring, R ¹⁸ is hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁹ is a pendant group of hydrogen or a natural alpha-amino acid or analogue or isomer thereof, R ²⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁹ and R ^{20 together} with the atoms to which it is bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ²² is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²¹ And R ²² and its bonded atom together form a 3- to 7-membered carbocyclic ring, R ²³ is (C ₁ -C ₄ )-alkyl, and R ²⁴ is hydrogen or (C ₁ -C ₄ )-alkyl , R ²⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the following formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene can pass through Substituted to four methyl substituents, and two carbon atoms in which the intermediate alkylene chain is 1, 2, 1, 3 or 1, 4 in relation to each other, including any carbon atoms in between, can be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or phenyl ring, D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, or R ¹ And R ² and its bonded carbon atom together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Where # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H - Ind-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁷ is the position of the connection with the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ⁸ a group of R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a 4 a 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, R ¹¹ is a benzyl group, which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, a methyl group or a group of the following formula group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. A methyl group, as well as a solvate of its salts, solvates, and salts.

Further particularly preferred in the context of the present invention are compounds of formula (XXX) wherein Cys is a cysteine residue bonded to the carbon atom of amber quinone via a sulfur atom of a pendant group, L ¹ is a a bond, a linear (C ₁ -C ₁₀ )-alkylene group or a group of the formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₂ -C ₆ )-alkylene may pass through Substituted to 2 methyl substituents, B is a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond or B-1,2-diyl, L ⁴ is a bond, R ¹⁴ is hydrogen, R ¹⁵ Is hydrogen, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ²³ is a methyl group, R ²⁴ is hydrogen or a methyl group, and L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, or R ¹ And R ² and its bonded carbon atoms together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, ## indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, optionally substituted ring Where # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is benzyl, 1-phenylethyl or 1 H -indol-3-yl a methyl group, or R ³ and R ^{4 together} with a carbon atom bonded thereto to form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines indicated the position adjacent to the nitrogen atom, # ⁸ and the connection position designated based groups of T ^1, T ¹ is the formula ^{-C (= O) -OR 7,} -C (= O) -NR 8 a group of R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, benzyl or adamantyl a group, R ⁸ is hydrogen or a methyl group, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, R ¹⁰ is a benzinyl group, and R ¹¹ is a benzyl group which can be subjected to a phenyl group. Substituted by methoxycarbonyl or carboxy, R ⁵ is hydrogen or a group of the formula Wherein # ⁹ is a position to which a -CHC(R ²⁶ )phenyl group is bonded, and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, and R ¹³ is A phenyl group which may be substituted with a methoxycarbonyl group or a carboxyl group, and a solvate of a salt, a solvate thereof and a salt thereof.

Particularly preferred in the context of the present invention are also compounds of formula (Ia) wherein n = 1-20, more preferably n = 1-10 and very preferably n = 2-8.

Particularly preferred in the context of the present invention are also compounds of formula (Ia) wherein AK is AK ₁ wherein AK ₁ is a sulfur atom and a G group bond which binds to mesothelin and a cysteine residue via a binding agent. An antibody or antigen-binding antibody fragment, and G is a group of the formula Wherein the connection position marked with lines # ¹ cysteine residue of binding agent, labeled with # l ² based connection position of ^a group, and ^{n, L 1, B, L} 2, D , and R ³⁵ have the above The definitions are shown, as well as the solvates of their salts, solvates and salts.

Preferred in the context of the present invention are also compounds of formula (Ia) wherein AK is AK ₂ wherein AK ₂ is a NH pendant group and a G group linkage to mesothelin and to the amine acid residue via a binding agent. The antibody or antigen-binding antibody fragment, G is a carbonyl group, and n, L ¹ , B, L ² , D and R ³⁵ have the definitions as defined above, as well as solvates of the salts, solvates and salts thereof.

Preferred in the context of the invention are also compounds of formula (Ia) wherein AK is AK ₁ wherein AK ₁ is an antibody comprising the CDR sequences of six MF-Ta antibodies, the variable light chain of the MF-Ta antibody And a light chain and a heavy chain of a variable heavy chain or an MF-Ta antibody, and are bonded to a G group via a sulfur atom of a cysteic acid residue of the binding agent, and G is a group of the following formula # ^1-based connection position wherein cysteine residues of the labeled binding agent, labeled # ² based connection position of the group to L ^1, and ^{n, L 1, B, L} 2, D , and R ³⁵ have the above The definitions are shown, as well as the solvates of their salts, solvates and salts.

Preferred in the context of the present invention is also a compound of formula (Ia) wherein AK is AK ₂ wherein AK ₂ is an antibody comprising a CDR sequence of six MF-Ta antibodies, a variable light chain of MF-Ta antibody And a light chain and a heavy chain of a variable heavy chain or MF-Ta antibody, and bonded to a G group via an NH pendant group of an amine acid residue of the binding agent, G is a carbonyl group, and n, L ¹ , B And L ² , D and R ³⁵ have the definitions as indicated above, as well as solvates of the salts, solvates and salts thereof.

Preferred in the context of the present invention is also a compound of formula (Ia) wherein AK is AK ₂ wherein AK ₂ is an antibody comprising the CDR sequences of six MF-Ta antibodies, the variable light chain of the MF-Ta antibody And a light chain and a heavy chain of a variable heavy chain or MF-Ta antibody, and bonded to a G group via an NH pendant group of an amine acid residue of the binding agent, G is a carbonyl group, and L ¹ is a bond, B Is a bond, L ² is a linear (C ₃ -C ₆ )-alkylene group or a group of the following formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and n, D, and R ³⁵ have the definitions as defined above, and salts thereof. Solvates of solvates and salts.

Preferred in the context of the invention are also compounds of formula (Ia) wherein AK is AK ₁ wherein AK ₁ is an antibody comprising the CDR sequences of six MF-Ta antibodies, the variable light chain of the MF-Ta antibody And a light chain and a heavy chain of a variable heavy chain or an MF-Ta antibody, and are linked to a G group via a sulfur atom of a cysteic acid residue of the binding agent, and G is a group of the following formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, and L ¹ is a bond, linear (C ₃ -C ₅ )-alkylene Base or group Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₃ -C ₅ )-alkylene may pass through 1 to Substituted by two methyl substituents, B is a bond or a group of the formula Where * is the position to which L ^{1 is} attached, ** is the position to which L ² is attached, L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** is linked to the position of the carbonyl group, **** is linked to the position of L ² , R ²⁵ is a methyl group, and R ²⁸ is hydrogen, methylcarbonyl or a third butoxycarbonyl group, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, L ² is a linear (C ₃ -C ₅ )-alkylene group or a group of the formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and n, D and R ³⁵ have the definitions as defined above, and the salt thereof Solvates of classes, solvates and salts.

Preferred in the context of the present invention are also compounds of the formulae (Ia), (XXXa) and (XXXI) wherein L ¹ is a bond, B is a bond, and L ² is a linear chain (C ₃ -C ₆ )- An alkylene group or a group of the formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and n, AK, Cys, G, D, and R ³⁵ have the above The definition, as well as the solvates of its salts, solvates and salts.

Also preferred in the context of the present invention are compounds of formula (Ia) wherein L ¹ is a linear (C ₁ -C ₁₀ )-alkylene group or a group of the formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₁ -C ₁₀ )-alkylene can pass through Up to 4 substituents each independently selected from the group consisting of a methyl group, a hydroxyl group, and a benzyl group, and B is a bond or a group of the following formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ¹ , L ³ is a bond or (C ₂ -C ₄ )-alkylene, and L ⁴ is a group of the formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is hydrogen or methyl, and R ²⁸ is hydrogen, (C ₁ -C ₄ )-alkyl a carbonyl group, a third butoxycarbonyl group or a benzyloxycarbonyl group, Q ¹ is a 4- to 7-membered heterocyclic ring, R ¹⁶ is hydrogen or (C ₁ -CC ₄ )-alkyl, and R ¹⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁶ and R ^{17 together} with the atoms to which they are bonded form a 5- or 6-membered heterocyclic ring, R ²³ is (C ₁ -C ₄ )-alkyl, and R ²⁴ is hydrogen. Or (C ₁ -C ₄ )-alkyl, R ³⁶ is hydrogen, (C ₁ -C ₄ )-alkylcarbonyl, tert-butoxycarbonyl or benzyloxycarbonyl, R ³⁷ is hydrogen or methyl, or R ³⁶ and R ^{37 together} with the atoms to which they are bonded form a pyrrolidine ring, and L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the following formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be passed through 1 to 4 each independently substituted with a substituent selected from the group consisting of a methyl group, a hydroxyl group and a benzyl group, and n, AK, G, D and R ³⁵ have the definitions as defined above, as well as salts, solvates and salts thereof Solvate.

Also preferred in the context of the present invention are compounds of formula (Ia) wherein L ¹ is a linear (C ₂ -C ₆ )-alkylene group or a group of the formula Where m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and B is a group of one or the following formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a group of the formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is hydrogen or methyl, and R ²⁸ is hydrogen, methylcarbonyl or tert-butoxycarbonyl. , R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ³⁶ is hydrogen, methylcarbonyl or a third butoxycarbonyl group, R ³⁷ is hydrogen or methyl, or R ³⁶ and R ^{37 together} with the atoms bonded thereto form a pyrrolidine ring, and L ² is straight a chain (C ₂ -C ₆ )-alkylene group or a group of the formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and n, AK, G, D, and R ³⁵ have the definitions shown above And solvates of salts, solvates and salts thereof.

Preferred in the context of the present invention are also compounds of formula (Ia) wherein L ¹ is a bond, a linear (C ₂ -C ₆ )-alkylene group or a group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₂ -C ₆ )-alkylene may pass through 1 to Substituted by two methyl substituents, B being a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is methyl, and R ²⁸ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, Q ¹ is piperidine-1,4-diyl, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ²¹ is hydrogen or methyl, R ²² is hydrogen or methyl, or R ²¹ and R ^{22 together} with the atom to which they are bonded form a cyclopropyl ring, R ²³ is a methyl group, and R ²⁴ is a hydrogen, R ³⁶ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, R ³⁷ is hydrogen or methyl, L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula Where p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, as well as solvates of salts, solvates, and salts thereof.

Also preferred in the context of the present invention are compounds of formula (Ia) wherein L ¹ is a linear (C ₂ -C ₆ )-alkylene group wherein (C ₂ -C ₆ )-alkylene may be passed through Or substituted with 2 methyl substituents, B being a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond, and L ⁴ is a group of the following formula Wherein *** is the position of the linkage to the carbonyl group, **** is the position of the linkage to L ² , R ²⁵ is a methyl group, and R ^2w is hydrogen, methylcarbonyl or a third butoxycarbonyl group, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, R ²³ is methyl, R ²⁴ is hydrogen, R ³⁶ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, and R ³⁷ is hydrogen or methyl. L ² is a linear (C ₂ -C ₆ )-alkylene group, and a solvate of the salts, solvates and salts thereof.

Preferred in the context of the present invention are also compounds of formula (Ia) wherein G is a group of the formula # ¹ wherein the connection position designated based cysteine residues of the binding agent, labeled # ² and the connection lines L ¹ position of the group, L ¹ is a straight-chain (C ₃ -C ₅₎ - or lower alkylene Group Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₃ -C ₅ )-alkylene may be 1 or Substituted by two methyl substituents, B is a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond or B-1,2-diyl, L ⁴ is a bond, and L ² is a linear chain (C _3- C ₅ )-alkylene or a group of the formula Wherein p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and n, AK ₁ , Cys, D, R ¹⁶ and R ¹⁷ have the above The definitions shown, as well as the solvates of their salts, solvates and salts.

Preferred in the context of the present invention are also compounds of formula (Ia) and (XXXa) wherein B is a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ¹ , L ³ is a bond or B-1,2-diyl, L ⁴ is a bond, n, AK, Cys, G And L ¹ , L ² , D, R ¹⁶ , R ¹⁷ and R ³⁵ have the definitions as defined above, as well as solvates of the salts, solvates and salts thereof.

Preferred in the context of the present invention are also compounds of the formulae (Ia), (XXXa) and (XXXI) wherein L ¹ is a bond, a linear (C ₃ -C ₅ )-alkylene group or is of the formula Group Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₃ -C ₅ )-alkylene may be 1 or Substituted by two methyl substituents, B is a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond, L ⁴ is a bond, R ¹⁶ is hydrogen, R ¹⁷ is hydrogen, and L ² is linear ( C ₃ -C ₆ )-alkylene or a group of the formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and n, AK, Cys, G, D, and R ³⁵ have the above Definitions, as well as solvates of their salts, solvates and salts.

Preferred in the context of the present invention are also compounds of the formulae (Ia), (XXXa) and (XXXI) wherein L ¹ is a bond, B is a bond, and L ² is a linear chain (C ₃ -C ₆ )- An alkylene group or a group of the formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and n, AK, Cys, G, D, and R ³⁵ have the above Definitions, as well as solvates of their salts, solvates and salts.

Also preferred in the context of the present invention are compounds of formula (Ia), (XXXa) and (XXXI) wherein L ¹ is a linear (C ₃ -C ₅ )-alkylene group or a group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₃ -C ₅ )-alkylene may be 1 or Substituted by two methyl substituents, B is a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond, L ⁴ is a bond, R ¹⁶ is hydrogen, R ¹⁷ is hydrogen, and L ² is linear ( C ₃ -C ₆ )-alkylene or a group of the formula Wherein p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and n, AK, Cys, G, D, and R ³⁵ have the above Definitions, as well as solvates of their salts, solvates and salts.

Preferred in the context of the present invention are also compounds of formula (Ia) wherein n is a number from 2 to 8, preferably from 2 to 5, and AK is AK ₁ or AK ₂ wherein AK ₁ is associated with mesothelin and An antibody or antigen-binding antibody fragment conjugated to a G group via a sulfur atom of a cysteine residue of a binding agent, AK ₂ being an NH side group that binds to mesothelin and a lysine residue via a binding agent G group-bound antibody or antigen-binding antibody fragment, when AK=AK ₁ , G is a group of the following formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , is a carbonyl group, L ¹ is a bond, straight Chain (C ₃ -C ₅ )-alkylene or a group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₃ -C ₅ )-alkylene may be 1 or Substituted by two methyl substituents, B is a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond, L ⁴ is a bond, R ¹⁶ is hydrogen, R ¹⁷ is hydrogen, and L ² is linear ( C ₃ -C ₆ )-alkylene or a group of the formula Wherein p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D and R ³⁵ have the definitions as defined above, and their salts, Solvates of solvates and salts.

Preferred in the context of the present invention are also compounds of formula (Ia) wherein n is a number from 2 to 8, preferably from 2 to 5, and AK is AK ₁ or AK ₂ wherein AK ₁ is associated with mesothelin. And an antibody or antigen-binding antibody fragment bound to the G group via a sulfur atom of a cysteic acid residue of the binding agent, AK ₂ being a NH pendant group that binds to mesothelin and the lysine residue via the binding agent An antibody or antigen-binding antibody fragment bound to a G group, when AK=AK ₁ , G is a group of the following formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , is a carbonyl group, and L ¹ is a bond, B Is a bond, L ² is a linear (C ₃ -C ₆ )-alkylene group or a group of the following formula Wherein p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D and R ³⁵ have the definitions as defined above, as well as salts and solvents thereof. Solvates of the compounds and salts.

Preferred in the context of the present invention are also compounds of formula (Ia) wherein n is a number from 2 to 8, preferably from 2 to 5, and AK is AK ₁ wherein AK ₁ is bonded to mesothelin and via The antibody or antigen-binding antibody fragment to which the sulfur atom of the cysteine residue of the agent binds to the G group, and G is a group of the following formula # ¹ wherein the connection position designated based cysteine residues of the binding agent, labeled # ² and the connection lines L ¹ position of the group, L ¹ is a straight-chain (C ₃ -C ₅₎ - or lower alkylene Group Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₃ -C ₅ )-alkylene may be 1 or Substituted by two methyl substituents, B is a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond, L ⁴ is a bond, R ¹⁶ is hydrogen, R ¹⁷ is hydrogen, and L ² is linear ( C ₃ -C ₆ )-alkylene or a group of the formula Wherein p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D and R ³⁵ have the definitions as defined above, as well as salts and solvents thereof. Solvates of the compounds and salts.

Also preferred in the context of the present invention are compounds of formula (Ia) wherein L ¹ is a bond, a linear (C ₃ -C ₅ )-alkylene group, a group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and L ^1A is a linear (C ₃ -C ₆ )-alkylene group. , B ¹ is a group of the following formula Where ## ⁵ indicates the position of attachment to the L ^1A group, ## ⁶ indicates the position of attachment to the L ^1B group, L ⁵ is a bond or B-1,2-diyl, and L ⁶ is a bond or Group of the following formula Where ## ⁷ indicates the position of attachment to the carbonyl group, ## ⁸ indicates the position of attachment to L ^1B , and R ³³ is hydrogen, (C ₁ -C ₄ )-alkylcarbonyl or tert-butoxycarbonyl. R ³⁴ is hydrogen or methyl, R ²⁹ is hydrogen or methyl, R ³⁰ is hydrogen or methyl, R ³¹ is hydrogen or methyl, R ³² is hydrogen or methyl, and L ^1B is linear (C ₃ -C ₆ )-alkylene, B is a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , P is O, L ³ is a bond or B-1,2-diyl, and L ⁴ is a group of the following formula Wherein *** is the position of the linkage to the carbonyl group, **** is the position of attachment to L ² , R ²⁵ is hydrogen or methyl, R ²⁸ is hydrogen, (C ₁ -C ₄ )-alkyl a carbonyl or a tert-butoxycarbonyl group, Q ¹ is a 4- to 7-membered heterocyclic ring, Q ² is a 3- to 7-membered carbocyclic ring or a 4- to 7-membered heterocyclic ring, and R ¹⁶ is hydrogen or methyl. R ¹⁷ is hydrogen or methyl, R ²³ is (C ₁ -C ₄ )-alkyl, R ²⁴ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³⁶ is hydrogen, (C ₁ -C ₄ ) -alkylcarbonyl or tert-butoxycarbonyl, R ³⁷ is hydrogen or methyl, or R ³⁶ and R ^{37 together} with the atoms to which they are bonded form a pyrrolidine ring, L ² is linear (C ₂ -C ₆ )-alkylene or a group of the formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and the solvate of its salts, solvates and salts.

Also preferred in the context of the present invention are compounds of formula (Ia) wherein L ¹ is a linear (C ₃ -C ₅ )-alkylene group or a group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of the connection with the G group, ## ² indicates the position of the connection with the B group, and B is the group of the following formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a group of the formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is methyl, R ²⁸ is hydrogen, methylcarbonyl or third butoxycarbonyl, Q ¹ is piperidine-1,4-diyl, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, R ²³ is methyl, R ²⁴ is hydrogen, R ³⁶ is hydrogen, methylcarbonyl or the third Butoxycarbonyl, R ³⁷ is or methyl, L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and the solvate of its salts, solvates and salts.

Preferred in the context of the present invention are also compounds of the formulae (Ia), (XXXa) and (XXXI) wherein D is a group of the formula

Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen or methyl, and R ² is isopropyl, isobutyl, second butyl, tert-butyl, phenyl, benzyl, 1-hydroxy Ethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazole-4-yl a group or a 1H-indol-3-ylmethyl group, or a combination of R ¹ and R ² with a carbon atom bonded thereto to form (1 S , 2 R )-2-phenylcyclopropane-1,1- Dibasic group Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, is optionally substituted Heterocycle Wherein # ⁶ is a position to which a carbonyl group is bonded, R ⁶ is hydrogen, a hydroxyl group or a benzyloxy group, R ³ is hydrogen or a methyl group, and R ⁴ is an isopropyl group, an isobutyl group, a second butyl group , and a third group . Butyl , phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, two Benzyl, 1 H -imidazol-4-ylmethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form the formula (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a a 4- to 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, methyl or the following formula Group Wherein the line marked with # ⁹ -CHC (R ²⁶⁾ -T ² the connection position, R ¹² is phenyl, which may be, substituted carboxyl group or the formula -S (O) ₂ OH the radical R ¹³ substituted by methoxycarbonyl Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-ylmethyl group. And n, AK, Cys, G, L ¹ , B, L ² , D and R ³⁵ have the definitions as indicated above, as well as solvates of the salts, solvates and salts thereof.

Preferred in the context of the present invention are also compounds of the formulae (Ia), (XXXa) and (XXXI) wherein D is a group of the formula

Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, in which A ring of NO group, which is a heterocyclic ring of the following formula

Wherein # ⁶ is the position of the linkage to the carbonyl group, R ³ is hydrogen, and R ⁴ is benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, T ¹ Is a group of the formula -C(=O)-OR ⁷ or -C(=O)-NR ⁸ R ⁹ wherein R ⁷ is hydrogen, R ⁸ is hydrogen, R ⁹ is hydrogen, n, AK, Cys, G And L ¹ , B, L ² , D and R ³⁵ have the definitions as defined above, as well as solvates of the salts, solvates and salts thereof.

Preferred in the context of the present invention are also compounds of the formulae (Ia), (XXXa) and (XXXI) wherein D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, in which A ring of NO group, which is a heterocyclic ring of the following formula

Where # ⁶ is the position of the linkage to the carbonyl group, R ³ is hydrogen, and R ⁴ is benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, T ¹ Is a group of the formula -C(=O)-NR ⁸ R ⁹ wherein R ⁸ is hydrogen, R ⁹ is hydrogen, and n, AK, Cys, G, L ¹ , B, L ² , D and R ³⁵ have the above The definitions shown, as well as the solvates of their salts, solvates and salts.

Preferred in the context of the present invention are also compounds of the formulae (Ia), (XXXa) and (XXXI) wherein D is a group of the formula Wherein # ³ is the position of the connection to the nitrogen atom, R ¹ is hydrogen, and R ² is 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, wherein the A ring of the NO group is present, Heterocycle Where # ⁶ is the position of the linkage to the carbonyl group, R ³ is hydrogen, R ⁴ is 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, and T ¹ is of the formula -C(=O)- a group of NR ⁸ R ⁹ wherein R ⁸ is hydrogen, R ⁹ is hydrogen, and n, AK, Cys, G, L ¹ , B, L ² , D and R ³⁵ have the definitions as defined above, and salts thereof Solvates of solvates and salts.

Preferred in the context of the present invention are also compounds of the formulae (Ia), (XXXa) and (XXXI) wherein D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ³ is hydrogen, R ⁴ is 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, and T ¹ is a formula -C(=O)-OR ^{a group of 7} or -C(=O)-NR ⁸ R ⁹ wherein R ⁷ is hydrogen, R ⁸ is hydrogen, R ⁹ is hydrogen, n, AK, Cys, G, L ¹ , B, L ² , D And R ³⁵ have the definitions as indicated above, as well as solvates of the salts, solvates and salts thereof.

Preferred in the context of the present invention are also compounds of the formulae (Ia), (XXXa) and (XXXI) wherein D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ³ is hydrogen, R ⁴ is 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, and T ¹ is a formula -C(=O)-NR ^{a group of 8} R ⁹ wherein R ⁸ is hydrogen, R ⁹ is hydrogen, and n, AK, Cys, G, L ¹ , B, L ² , D and R ³⁵ have the definitions as defined above, and salts thereof, Solvates of solvates and salts.

Preferred in the context of the present invention are also compounds of the formulae (Ia), (XXXa) and (XXXI) wherein D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen or methyl, and R ² is isopropyl, isobutyl, second butyl, tert-butyl, phenyl, benzyl, 1-hydroxy Ethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazole-4-yl Or a 1H-indol-3-ylmethyl group, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1- Dibasic group Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Wherein # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, and n, AK, Cys, G, L ¹ , B, L ² and R ³⁵ have the definitions shown above, And solvates of salts, solvates and salts thereof.

Preferred in the context of the present invention are also compounds of the formulae (Ia), (XXXa) and (XXXI) wherein D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, or R ¹ And R ² and its bonded carbon atom together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Where # ⁶ is the position of the linkage to the carbonyl group, and n, AK, Cys, G, L ¹ , B, L ² and the definitions as defined above, as well as the solvates of the salts, solvates and salts thereof. .

Also preferred in the context of the present invention are compounds of formula (Ia), (XXXa) and (XXXI) wherein R ³⁵ is hydroxy, and n, AK, Cys, G, L ¹ , B, L ² , D and R ³⁵ has the definitions as indicated above, as well as solvates of the salts, solvates and salts thereof.

Also preferred in the context of the present invention are compounds of formula (Ia), (XXXa) and (XXXI) wherein R ³⁵ is methyl, and n, AK, Cys, G, L ¹ , B, L ² , D And R ³⁵ have the definitions as indicated above, as well as solvates of the salts, solvates and salts thereof.

Particularly preferred in the context of the present invention are also compounds of formula (XXXa) wherein Cys is an L-cysteine residue bonded via a sulfur atom of a pendant group via a carbon atom of amber quinone imine, and a salt thereof Solvates of solvates and salts.

Preferred in the context of the present invention are also compounds of formula (I) and (XXX) wherein D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, R ² is benzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² are bonded to each other. The carbon atoms of the knot form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Wherein # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, and n, AK, Cys, G, L ¹ , L ² and B have the definitions as defined above, and salts thereof. Solvates of solvates and salts.

Particularly preferred in the context of the present invention are also compounds of formula (I) and (XXX) wherein D is a group of the formula Wherein # ³ is the position to which the nitrogen atom is bonded, R ¹ is hydrogen, R ² is benzyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² are bonded together with the carbon atom to which they are bonded (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group

Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to a carbonyl group, and the A ring in which the NO group is present is a heterocyclic ring of the following formula Wherein # ⁶ is the position to which the carbonyl group is attached, and n, AK, Cys, G, L ¹ , L ² and B have the definitions as defined above, as well as solvates of the salts, solvates and salts thereof.

Also preferred in the context of the present invention are compounds of formula (I) wherein n = 1-20, more preferably n = 1-10 and very preferably n = 2-8.

Preferred in the context of the present invention are also compounds of formula (I) and (XXX) wherein B is a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond or B-1,2-diyl, L ⁴ is a bond, n, AK, Cys, G And L ¹ , L ² , D, R ¹⁶ and R ¹⁷ have the definitions as defined above, as well as solvates of the salts, solvates and salts thereof.

Particularly preferred in the context of the present invention are also compounds of formula (I) and (XXX) wherein B is a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ and L ⁴ are a bond, n, AK, Cys, G, L ¹ , L ² , D, R ¹⁶ and R ¹⁷ has the definitions as indicated above, as well as solvates of the salts, solvates and salts thereof.

Preferred in the context of the present invention is also a binding agent-drug conjugate of the general formula (I) wherein AK is AK ₁ , wherein AK ₁ is a binding agent which is a sulfur of a cysteine residue via a binding agent. The atom is bonded to the G group, and G is a group of the following formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, and L ¹ is a bond, linear (C ₁ -C ₁₀ )-alkylene Base or group Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₁ -C ₁₀ )-alkylene can pass through 1 The substitution of four methyl substituents and two carbon atoms in which the intermediate alkylene chains are 1, 2, 1, 3 or 1, 4 in relation to each other, including any carbon atoms in between, can be bridged to form one ( C ₃ -C ₆ )-cycloalkyl ring or phenyl ring, B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond or (C ₂ -C ₄ )-alkylene, and L ⁴ is a bond or a base of the formula group Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is hydrogen or methyl, Q ¹ is a 4- to 7-membered heterocyclic ring, and R ¹⁴ is Hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁵ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁴ and R ^{15 together} with the atoms to which they are bonded form a 5- or 6-member a heterocyclic ring, R ¹⁶ is hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁶ and R ^{17 together} with the atoms to which they are bonded form a 5- or 6-membered heterocyclic ring, L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be passed through 1 to Four methyl substituents substituted, and two carbon atoms in which the intermediate alkylene chain is 1, 2, 1, 3 or 1, 4 in relation to each other, including any carbon atoms in between, can be bridged to form one ( C ₃ -C ₆ )-cycloalkyl ring or phenyl ring, and solvates of the salts, solvates and salts thereof.

Preferred in the context of the present invention is also a binding agent-drug conjugate of the general formula (I) wherein AK is AK ₂ , wherein AK ₂ is a binding agent which is via the NH side of the lysine residue of the binding agent. The group is bonded to the G group, G is a carbonyl group, and L ¹ is a bond, a linear (C ₁ -C ₁₀ )-alkylene group or a group of the following formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₁ -C ₁₀ )-alkylene can pass through The substitution of four methyl substituents and two carbon atoms in which the intermediate alkylene chains are 1, 2, 1, 3 or 1, 4 in relation to each other, including any carbon atoms in between, can be bridged to form one ( C ₃ -C ₆ )-cycloalkyl ring or phenyl ring, B is a bond or a group of the formula Where * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , P is O or NH, and Q ² is 3- to 7-membered carbocyclic or 4- to 7-membered heterocyclic ring, R ¹⁸ is hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁹ is hydrogen or a pendant group of a natural α-amino acid or an analog or isomer thereof, R ²⁰ is hydrogen or (C ₁ -C ₄ -alkyl, or R ¹⁹ and R ^{20 together} with the atoms to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ²² is hydrogen or (C ₁ - C ₄ )-alkyl, or R ²¹ and R ^{22 together} with the atoms to which they are bonded form a 3- to 7-membered carbocyclic ring, R ²³ is (C ₁ -C ₄ )-alkyl, R ²⁴ is hydrogen or (C ₁ -C ₄ )-alkyl, L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be passed through 1 to The four methyl substituents are substituted with two carbon atoms in which the intermediate alkylene chains are 1, 2, 1, 3 or 1, 4, and any carbon atoms in between may be bridged to form one (C). _{3- C6} )-cycloalkyl or phenyl ring, and solvates of salts, solvates and salts thereof.

Particularly preferred in the context of the present invention is a binding agent-drug conjugate of the general formula (I) wherein n is a number from 2 to 8, and AK is AK ₁ or AK ₂ , wherein AK ₁ is associated with mesothelin and a human or humanized antibody or antigen-binding antibody fragment linked to a G group via a sulfur atom of a cysteine residue of a binding agent, AK ₂ being an acyl acid residue bound to mesothelin and via a binding agent a human or humanized antibody or antigen-binding antibody fragment to which a NH pendant group is bonded to a G group, and when AK = AK ₁ , G is a group of the following formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , is a carbonyl group, L ¹ is a bond, straight Chain (C ₂ -C ₆ )-alkylene or a group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₂ -C ₆ )-alkylene may be 1 or Two methyl substituents replacing B as a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is methyl, and R ²⁸ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, Q ¹ is piperidine-1,4-diyl, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ²¹ is hydrogen or methyl, R ²² is hydrogen or methyl, or R ²¹ and R ^{22 together} with the atom to which they are bonded form a cyclopropyl ring, R ²³ is methyl, R ²⁴ is hydrogen, L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula Where p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is a group of the formula Wherein # ³ is a position to which a nitrogen atom is bonded, R ¹ is hydrogen, and R ² is a 4-hydroxybenzyl group or a 1 H -indol-3-ylmethyl group in which an A ring of an NO group is present. Where # ⁶ is the position of the linkage to the carbonyl group, R ³ is hydrogen, R ⁴ is 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, and T ¹ is of the formula -C(=O)- A group of NR ⁸ R ⁹ wherein R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl or ethyl, R ³⁵ is methyl, and solvates of the salts, solvates and salts thereof.

Particularly preferred in the context of the present invention is a binding agent-drug conjugate of the formula (Ia) wherein n is a number from 2 to 8, preferably from 2 to 5, and AK is AK ₁ or AK ₂ , wherein AK ₁ is a human or humanized antibody or antigen-binding antibody fragment that binds to mesothelin and is bonded to the G group via a sulfur atom of a cysteic acid residue of the binding agent, AK ₂ is bound to mesothelin and via a binding agent a human or humanized antibody or antigen-binding antibody fragment to which an NH side group of an amine acid residue is bonded to a G group, and when AK=AK ₁ , G is a group of the following formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , is a carbonyl group, and L ¹ is a bond, B Is a bond R ¹⁷ is hydrogen, L ² is pent-1,5-diyl, hex-1,6-diyl or a group of the formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Wherein # ³ is a position to which a nitrogen atom is bonded, R ¹ is hydrogen, and R ² is a 4-hydroxybenzyl group or a 1 H -indol-3-ylmethyl group in which an A ring of an NO group is present. Where # ⁶ is the position of the linkage to the carbonyl group, R ³ is hydrogen, R ⁴ is 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, and T ¹ is of the formula -C(=O)- A group of NR ⁸ R ⁹ wherein R ⁸ is hydrogen, R ⁹ is hydrogen, R ³⁵ is methyl, and solvates of the salts, solvates and salts thereof.

A preferred subject of the invention is a binder-drug conjugate of formula (Ia) wherein n is a number from 2 to 8, preferably from 2 to 5, AK is AK ₁ or AK ₂ , wherein AK ₁ is Mesothelin binding and human or humanized antibody or antigen-binding antibody fragment linked to a G group via a sulfur atom of a cysteine residue of a binding agent, AK ₂ is bound to mesothelin and via a binding agent A human or humanized antibody or antigen-binding antibody fragment in which the NH side group of the amine acid residue is bonded to the G group, and when AK=AK ₁ , G is a group of the following formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , is a carbonyl group, and L ¹ is a linear chain (C) ₂ -C ₆ )-alkylene, B is a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond, and L ⁴ is a group of the following formula Wherein *** is linked to the position of the carbonyl group, **** is linked to the position of L ¹ , R ²⁵ is a methyl group, and R ²⁸ is hydrogen, methylcarbonyl or a third butoxycarbonyl group, R ¹⁶ is hydrogen, R ¹⁷ is hydrogen, R ³⁶ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, R ³⁷ is methyl, L ² is linear (C ₂ -C ₄ )-alkylene, and D is Group of the following formula Wherein # ³ is a position to which a nitrogen atom is bonded, R ¹ is hydrogen, and R ² is a 4-hydroxybenzyl group or a 1 H -indol-3-ylmethyl group in which an A ring of an NO group is present. Where # ⁶ is the position of the linkage to the carbonyl group, R ³ is hydrogen, R ⁴ is 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, and T ¹ is of the formula -C(=O)- A group of NR ⁸ R ⁹ wherein R ⁸ is hydrogen, R ⁹ is hydrogen, R ³⁵ is a methyl group or a hydroxyl group, and a solvate of a salt, a solvate thereof and a salt thereof.

Particularly preferred in the context of the present invention is a binder-drug conjugate selected from the following compounds:

Wherein, in each case, n is a number from 2 to 8, preferably from 2 to 5, and AK ₁ is bonded to mesothelin and bonded to the G group via a sulfur atom of a cysteine residue of the binding agent. Human or humanized antibody or antigen-binding antibody fragment, AK ₂ is a human or humanized antibody or antigen that binds to mesothelin and binds to the G group via the NH side group of the amine acid residue of the binding agent. The antibody fragment, more particularly preferred in the context of the present invention, is also a binding agent-drug conjugate selected from the following compounds:

Wherein n is a number from 2 to 8, preferably 2 to 5, and AK ₁ is an antibody comprising CDR sequences of six MF-Ta antibodies, variable light chains of MF-Ta antibodies and variable The light chain and the heavy chain of the heavy chain or MF-Ta antibody, and are bonded to the G group via a sulfur atom of a cysteine residue of the binding agent, and AK ₂ is an antibody comprising six MF-Ta antibodies a CDR sequence, a variable light chain of a MF-Ta antibody, and a light chain and a heavy chain of a variable heavy chain or MF-Ta antibody, and a NH group and a G group bond of an amino acid residue via a binding agent Knot.

The individual group definitions referred to in the respective group combinations or preferred combinations may also be arbitrarily substituted in other combinations, as indicated by separate individual group combinations.

Particularly preferred are combinations of two or more of the above preferred ranges.

The invention further provides a process for the preparation of a compound of the invention of formula (Ia), characterized in that a binder solution is dissolved in PBS buffer

[A] is mixed with a suitable reducing agent such as dithiothreitol or hydrazine (2-carboxyethyl)phosphine hydrochloride, followed by reaction with a compound of formula (IIa), Wherein D, L ¹ , B, L ² and R ³⁵ each have the definition as defined above, to give a compound of formula (IA) Wherein n, AK ₂ , D, L ¹ , B, L ² and R ³⁵ each have the definitions shown above, or [B] reacts with a compound of formula (IIIa) (IIIa), wherein each of D, L ¹ , B, L ² and R ³⁵ has the definition as defined above, to give a compound of formula (Ia-B) Wherein n, AK ₂ , D, L ¹ , B, L ² and R ³⁵ each have the definitions as indicated above.

The invention further provides a process for the preparation of a compound of the invention of formula (I), characterized in that a binder solution is dissolved in PBS buffer

[A] is mixed with a suitable reducing agent such as dithiothreitol or hydrazine (2-carboxyethyl)phosphine hydrochloride, followed by reaction with a compound of formula (II), Wherein D, L ¹ , B and 'L ² each have the definitions as defined above, to give a compound of formula (IA) Wherein n, AK ₁ , D, L ¹ , B and L ² each have the definition as defined above, or [B] reacts with a compound of formula (III) Wherein D, L ¹ , B and L ² each have the definition as defined above, to give a compound of formula (IB) Wherein n, AK ₂ , D, L ¹ , B and L ² each have the definitions as indicated above.

Cysteine coupling:

Partial reduction of the antibody and subsequent attachment of the (partially) reduced antibody to the compound of formula (II) or (IIa) is carried out according to methods known to those skilled in the art, for example, see, for example, Ducry et al, Bioconj. Chem. 2010, 21, 5 and references herein, Klussman et al, Bioconj. Chem. 2004, 15(4), 765-773. The modest reduction of the antibody is preferably carried out by adding 2-6 equivalents of TCEP to the antibody, the TCEP being present in a suitable buffer, preferably a phosphate buffer, at a temperature between 15 and 40 ° C, preferably It is stirred at room temperature for 30-180 minutes. Next, a solution of the compound of the formula (II) or (IIa) dissolved in DMSO, acetonitrile or DMF is added to the (partially) reduced antibody solution in PBS buffer, and then at a temperature of 0 ° C to +40 ° C. More specifically, +10 ° C to +30 ° C, the reaction is carried out for a period of 30 minutes to 6 hours, more specifically 1 to 2 hours.

Coupling with aminic acid:

The compound of formula (III) or (IIa) or the equivalent activated carboxyl component is first prepared by conventional peptide chemistry. It is then treated in an inert solvent such as DMSO or DMF and the antibody is added, preferably in the presence of a neutral pH phosphate buffer. The solution is stirred at a temperature between 15 and 40 ° C, preferably at RT, for 1-16 hours.

The above preparation method is illustrated by an example using the following processes (flows 1 and 2):

Process 1 [a): 1. AK (antibody), TCEP, PBS buffer, RT; 2. Addition of maleic imide derivative dissolved in DMSO, RT].

[a): AK (antibody), PBS buffer, RT mixed with the activated carboxyl derivative of the linker-drug component].

a compound of formula (II) wherein L ¹ and B are a bond, which can be obtained by formulating a compound of formula (IV) Wherein D has the definition as defined above, and reductive amination with a compound of formula (V) in an inert solvent Wherein L ^2A has the definition of L ² above, but one carbon atom is less in the length of the alkyl chain, and PG ¹ is an amine-protecting group such as (9 H -fluoren-9-ylmethoxy)carbonyl, third Butoxycarbonyl or benzyloxycarbonyl to give a compound of formula (VI) Wherein D, L ² and PG ¹ have the definitions indicated above, the PG ¹ protecting group is removed from the compound by methods known to those skilled in the art, and the deprotected compound is in an inert solvent at a suitable base. Reaction with methyl 2,5-di-oxo-2,5-dihydro-1H-pyrrole-1-carboxylate in the presence of the compound to give a compound of formula (II-A) Wherein D and L ² each have the definition as shown above.

a compound of formula (II) wherein B is a group of formula (B ¹ ) Wherein *, **, R ¹⁴ and R ¹⁵ each have the definitions indicated above, the PG ¹ protecting group can be removed from the compound of formula (VI) by methods known to those skilled in the art, and the deprotected compound is Prepared by reacting with a compound of formula (VII) in the presence of a suitable base in an inert solvent Wherein each of L ¹ has the definition shown above to give a compound of formula (II-B) Wherein D, L ¹ and L ² each have the definitions as indicated above.

a compound of formula (II) wherein B is a group of formula (B ² ) Wherein *, **, L ³ , R ¹⁶ and R ¹⁷ each have the definitions indicated above, by reductive amination of a compound of formula (VIII) with a compound of formula (VIII) in an inert solvent, Wherein L ^2A has the definition of L ² above, but one carbon atom is less in length of the alkyl chain to give a compound of formula (IX) Wherein D and L ² are as defined above, and the compound is reacted with a compound of formula (X) in the presence of a suitable coupling agent and a base in an inert solvent. Wherein L ¹ and L ³ each have the definitions shown above to give a compound of formula (II-C) Wherein D, L ¹ , L ² and L ³ each have the definitions as indicated above.

a compound of formula (II) wherein B is a group of formula (B ³ ) Wherein *, **, L ³ , R ¹⁶ and R ¹⁷ each have a definition as defined above and L ^4A is a group of the formula Wherein *** is the position of the linkage to the carbonyl group, **** is the position of the linkage to L ² , and R ²⁵ is hydrogen or methyl, which can be obtained by dissolving the compound of formula (IX) in an inert solvent. Prepared by reacting a suitable base with a suitable coupling agent with a compound of formula (XI-A) or (XI-B), Wherein R ²⁵ and PG ¹ each have the definitions indicated above, and PG ² is a suitable carboxy-protecting group, more particularly a benzyl group, to give a compound of formula (XII-A) or (XII-B) or Wherein D, PG ¹ , PG ² and L ² have the definitions indicated above, the PG ² protecting group is subsequently removed from the compound by methods known to those skilled in the art, and the deprotected compound is in an inert solvent. Reacting with a compound of formula (X) in the presence of a suitable coupling agent and a suitable base, and finally removing the PG1 protecting group from the compound by methods known to those skilled in the art to provide formula (II-DA) or (II- DB) compound or Wherein D, L ¹ , L ² and L ³ have the definitions as shown above.

a compound of formula (II) wherein B is a group of (B ⁴ ) Wherein * ** each have the definitions indicated above, and Q ^1A is an N-linked 4- to 7-membered heterocyclic ring, which may be employed in a suitable base by using a compound of formula (IX) in an inert solvent. Prepared by reacting with a compound of formula (XXI) in the presence of a coupling agent, Wherein PG ¹ and Q ^1A each have the definitions shown above to give a compound of formula (XXII) Wherein PG ¹ , Q ^1A , D and L ² have the definitions indicated above, the PG ¹ protecting group is removed from the compound by a method known to the skilled artisan and the deprotected compound is subsequently dissolved in an inert solvent. Reacting with a compound of formula (XXIII) in the presence of a suitable coupling agent and a suitable base, Wherein L ¹ has the definition as defined above to give a compound of formula (II-D) Wherein Q ^1A , D, L ¹ and L ² have the definitions as shown above.

A compound of formula (III) wherein L ¹ and B are a bond which can be reacted with N-hydroxysuccinimide by reacting a compound of formula (IX) in an inert solvent in the presence of a suitable coupling reagent and a suitable base. To prepare a compound of formula (III-A) Wherein D and L ² each have the definition as shown above.

a compound of formula (III) wherein L ¹ is a bond and B is a group of formula (B ^5A ) Wherein *, ** and P each have the definitions indicated above, and Q ^2A is a 3- to 7-membered carbocyclic ring, by using a compound of formula (IX) in an inert solvent, in a suitable coupling agent and suitable Prepared by reacting with a compound of formula (XIII) in the presence of a base, Wherein P, Q ^2A and PG ² each have the definitions shown above to give a compound of formula (XIV) (XIV) wherein each of D, P, Q ^2A , L ² and PG ² has the definitions indicated above, the PG ² protecting group is removed from the compound by methods known to those skilled in the art, and subsequently deprotected a compound of the formula (III-B) in an inert solvent in the presence of a suitable base with N-hydroxysuccinimide Wherein D, P, Q ^2A and L ² each have the definitions as indicated above.

a compound of formula (III) wherein L ¹ is a bond and B is a group of formula (B ⁶ ) Wherein *, **, R ¹⁸ , R ¹⁹ and R ²⁰ are each as defined above, by formulating the compound of formula (IX) in an inert solvent in the presence of a suitable coupling agent and a suitable base. (XV) compound reaction to prepare, Wherein R ¹⁸ , R ¹⁹ , R ²⁰ and PG ² each have the definitions shown above to give a compound of formula (XVI) Wherein D, R ¹⁸ , R ¹⁹ , R ²⁰ , L ² and PG ² each have the definitions indicated above, the PG ² protecting group is removed from the compound by methods known to those skilled in the art, and subsequently deprotected Compounds of formula (III-C) are reacted with N-hydroxysuccinimide in an inert solvent in the presence of a suitable coupling reagent and a suitable base. Wherein D, R ¹⁸ , R ¹⁹ , R ²⁰ and L ² each have the definitions as indicated above.

a compound of formula (III) wherein L ¹ is a bond and B is a group of formula (B ⁷ ) Wherein *, **, R ²¹ and R ²² each have the definitions indicated above, the PG ¹ protecting group can be removed from the compound of formula (VI) by methods known to those skilled in the art and the resulting deprotected compound Prepared by reacting with a compound of formula (XVII) in the presence of a suitable base in an inert solvent, Wherein R ²¹ and R ²² each have the definitions shown above to give a compound of formula (III-D) Wherein D, R ²¹ , R ²² and L ² each have the definitions as indicated above.

a compound of formula (III) wherein B is a group of formula (B ⁸ ) Wherein *, **, R ²³ and R ²⁴ are each as defined above, by formula (XVIII) in the presence of a suitable coupling agent and a suitable base in an inert solvent. Reaction to prepare Wherein R ²³ , R ²⁴ and PG ¹ each have the definition as defined above, to give a compound of formula (XIX) Wherein D, R ²³ , R ²⁴ , L ² and PG ¹ each have the definitions indicated above, the PG ¹ protecting group is removed from the compound by methods known to those skilled in the art, and the deprotected compound is subsequently Reacting with a compound of formula (XX) in an inert solvent in the presence of a suitable coupling agent and a suitable base Wherein L ^1A is a linear (C ₁ -C ₁₀ )-alkylene group or a group of the formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₁ -C ₁₀ )-alkylene can pass through Substituted to four methyl substituents, and two carbon atoms in which the intermediate alkylene chain is 1, 2, 1, 3 or 1, 4 in relation to each other, including any carbon atoms in between, can be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or phenyl ring to give a compound of formula (III-E) Wherein D, R ²³ , R ²⁴ , L ^1A and L ² each have the definitions as indicated above.

a compound of formula (III) wherein B is a group of formula (B ^5B ) Wherein * and ** each have the definitions as defined above, and Q ^2B is an N-linked 4- to 7-membered heterocyclic ring, which may be employed in a suitable base by using a compound of formula (IX) in an inert solvent. Prepared by reacting with a compound of formula (XXIV) in the presence of a coupling agent, Wherein PG ¹ and Q ^2B each have the definitions shown above to give a compound of formula (XXV) Wherein PG ¹ , Q ^2B , D and L ² have the definitions indicated above, the PG ¹ protecting group is removed from the compound by methods known to those skilled in the art, and the subsequently deprotected compound is in an inert solvent, Conversion of a compound of formula (XX) to a compound of formula (III-F) in the presence of a suitable base Wherein Q ^2B , D, L ^1A and L ² each have the definitions as indicated above.

(IV) + (V) → (VI) and (IV) + (VIII) → (IX) are used in solvents which are conventionally used for reductive amination and are inert under the reaction conditions, if necessary in acid and / Or the dehydrating agent is carried out as a catalyst. The solvent includes, for example, an alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol or tert-butanol, an ether such as tetrahydrofuran, 1,4-two Alkane, 1,2-dimethoxyethane or bis(2-methoxyethyl)ether, or other solvents such as dichloromethane, 1,2-dichloroethane, N,N -dimethyl Guanamine or water. Mixtures of these solvents can also be used. Preferably, 1,4-two is used. The alkane/water mixture is used as a solvent, and acetic acid or dilute hydrochloric acid is added as a catalyst.

Reducing agents suitable for this reaction, in particular, borohydride complexes such as sodium borohydride, sodium cyanoborohydride, triethoxy hydrazine hydroboration Sodium, tetra-n-butylammonium borohydride or borane-pyridine complex. Preferably, sodium cyanoborohydride or a borane-pyridine complex is used.

(IV) + (V) → (VI) and (IV) + (VIII) → (IX) reactions are generally in the temperature range of 0 ° C to +120 ° C, preferably from +50 ° C to +100 ° C get on. The reaction can be carried out at atmospheric pressure, elevated or reduced pressure (e.g., 0.5 to 5 bar); it is typically operated at atmospheric pressure.

The above coupling reaction (IX) + (X) → (II-C), (XII-A) or (XII-B) + (X) → (II-DA) or (II-DB), (IX) + ( XIII)→(XIV), (IX)+(XV)→(XVI) and (XXII)+(XXIII)→(II-D) (formation of guanamine by amine component and carboxylic acid component, respectively) Peptide chemistry is carried out [see, for example, M. Bodanszky, Principles of peptide Synthesis , Springer-Verlag, Berlin, 1993; M. Bodanszky and A. Bodanszky, The Practice of peptide Synthesis , Springer-Verlag, Berlin, 1984; H.-D. Jakubke and H. Jeschkeit, aminosäuren, peptide, Proteine , Verlag Chemie, Weinheim, 1982].

Examples of the inert solvent used in these coupling reactions are ethers such as diethyl ether, diisopropyl ether, tert-butyl methyl ether, tetrahydrofuran, 1,4-two. Alkane, 1,2-dimethoxyethane or bis(2-methoxyethyl)ether, hydrocarbons such as benzene, toluene, xylene, pentane, hexane, heptane, cyclohexane or petroleum distillation a halogenated hydrocarbon such as dichloromethane, chloroform, tetrachloromethane, 1,2-dichloroethane, trichloroethane or chlorobenzene, or a dipolar-aprotic solvent such as acetone or methyl ethyl ketone. , acetonitrile, ethyl acetate, pyridine, dimethyl hydrazine (DMSO), N,N -dimethylformamide (DMF), N,N -dimethylacetamide (DMA), N,N' - Dimethyl propylene urea (DMPU) or N -methyl pyrrolidone (NMP). Mixtures of these solvents can also be used. Preferably, N,N -dimethylformamide is used.

Suitable activating/condensing agents for these coupling reactions include carbodiimides such as N,N' -diethyl-, N,N' -dipropyl-, N,N' -diisopropyl-, N , N '- dicyclohexyl carbodiimide (DCC) or N - (3- dimethylamino-isopropyl) - N' - ethylcarbodiimide hydrochloride (EDC), phosgene (phosgene a derivative such as N,N' -carbonyldiimidazole (CDI) or isobutyl chloroformate, 1,2- An oxazolium compound such as 2-ethyl-5-phenyl-1,2- Azathioprine 3-sulfate or 2-tert-butyl-5-methyliso An oxazolidine perchlorate, a mercaptoamine compound such as 2-ethoxy-1-ethoxycarbonyl-1,2-dichloroquinoline, a phosphorus compound such as n-propylphosphoric anhydride, diethyl cyanophosphate, Double (2-sided oxygen-3- Pyrazinyl)phosphonium chloride, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yloxytris(pyrrolidino)hexafluorophosphate鏻 (PyBOP) or Compounds such as O- (benzotriazol-1-yl) -N,N,N',N' -tetramethyltetrafluoroboric acid (TBTU), O- (benzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate (HBTU), 2-(2-oxo-l-( 2H )-pyridyl)-1,1,3,3-tetramethyltetrafluoroboric acid (TPTU), O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate (HATU) or O- (1 H -6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyltetrafluoroboric acid (TCTU), if necessary in combination with an additional adjuvant such as 1-hydroxybenzotriazole (HOBt) or N -hydroxysuccinimide (HOSu), and with a base, an alkali metal carbonate such as sodium or potassium carbonate, Or a quaternary amine base such as triethylamine, N -methylmorpholine, N -methylpiperidine, N,N -diisopropylethylamine, pyridine or 4- N,N -dimethylamino Pyridine combination.

Within the context of the invention, such a coupling activator for the reaction / condensation agent used is preferably based N - (3- dimethylamino-isopropyl) - N '- ethylcarbodiimide hydrochloride ( EDC) in combination with 1-hydroxybenzotriazole (HOBt) and N,N -diisopropylethylamine, or O- (7-azabenzotriazol-1-yl) -N,N, N',N' -tetramethylhexafluorophosphate (HATU) is also combined with N,N -diisopropylethylamine.

Coupling reaction (IX) + (X) → (II-C), (XII-A) or (XII-B) + (X) → (II-DA) or (II-DB), (IX) + (XIII →(XIV), (IX)+(XV)→(XVI) and (XXII)+(XXIII)→(II-D) are generally carried out in a temperature range of -20 ° C to +60 ° C, preferably 0 ° C to +40 ° C. These reactions can be carried out at atmospheric pressure, elevated or reduced pressure (e.g., 0.5 to 5 bar); they are typically operated at atmospheric pressure.

Esterification (IX) + (XVIII) → (XII) and (IX) + (XI-A) or (XI-B) → (XII-A) or (XII-B), (IX) + (XXIV) →(XXV) and (IX)+(XXI)→(XXII) are similarly carried out in the above-described indole coupling reaction. These reactions are preferably based in dichloromethane, using N - (3- dimethylamino-isopropyl) - N '- ethylcarbodiimide hydrochloride (EDC) and 4-dimethylamine The pyridine is carried out at a temperature of from +50 ° C to 100 ° C under atmospheric pressure.

Functional groups present in the compound as desired - for example, especially amine groups, hydroxyl groups and carboxyl groups - may be present in a temporary protective form during the above treatment steps, if useful or desired. In these cases, such protecting groups can be introduced and removed according to conventional methods known for peptide chemistry [see, for example, TW Greene and PGM Wuts, Protective Groups in Organic Synthesis , Wiley, New York, 1999; M. Bodanszky and A. Bodanszky, The Practice of Peptide Synthesis , Springer-Verlag, Berlin, 1984]. When two or more protecting groups are present, they may be simultaneously re-released in a single container reaction as needed, or otherwise released again in a separate reaction.

Using a third preferred line-butoxycarbonyl (Boc), benzyloxycarbonyl (Z) or (9 H - fluorenyl-9-ylmethoxy) carbonyl (Fmoc) as PG ¹ amino - protecting group; As the hydroxyl or carboxyl functional group, a third butyl group or a benzyl group is preferably used as the PG ² protecting group. Removal of the third butyl or third butoxycarbonyl group is typically carried out as a strong acid such as hydrochloric acid, hydrobromic acid or trifluoroacetic acid in an inert solvent such as diethyl ether, 1,4-di The treatment is carried out in an alkane, dichloromethane or acetic acid; this reaction can also be carried out without adding an inert solvent, if necessary. In the case of a benzyl or benzyloxycarbonyl group as a protecting group, this group is preferably removed by hydrogenolysis in the presence of a suitable palladium catalyst such as palladium on activated carbon. (9 H - fluorenyl-9-ylmethoxy) carbonyl group is generally based secondary amines using base such as diethylamine or piperidine to remove.

The (VI)→(II-A) reaction is carried out in a solvent which is inert under the reaction conditions, such as ethers such as tetrahydrofuran, 1,4-two Alkane, 1,2-dimethoxyethane or bis(2-methoxyethyl)ether, alcohols such as methanol, ethanol, isopropanol, n-butanol or tert-butanol, or dipolar-non- Protic solvents such as acetone, methyl ethyl ketone, acetonitrile, ethyl acetate, pyridine, dimethyl hydrazine (DMSO), N,N -dimethylformamide (DMF), N,N -dimethyl B Indoleamine (DMA), N,N' -dimethylpropenylurea (DMPU) or N -methylpyrrolidone (NMP) or water. Mixtures of these solvents can also be used. Preferably, 1,4-two is used. a mixture of alkane and water.

Suitable bases for the (VI)→(II-A) reaction are, for example, alkali metal carbonates such as potassium carbonate, sodium carbonate or lithium carbonate, alkali metal hydrogencarbonates such as sodium hydrogencarbonate or potassium hydrogencarbonate, or alkali metals. Alkanols such as sodium methoxide, sodium ethoxide or potassium butoxide. Preferably sodium bicarbonate is used.

The (VI)→(II-A) reaction is carried out at a temperature ranging from 0 ° C to +50 ° C, preferably from +10 ° C to +30 ° C. These reactions can be carried out at atmospheric pressure, elevated or reduced pressure (e.g., 0.5 to 5 bar); they are typically operated at atmospheric pressure.

The (VI) + (VII) → (II-B) reaction is carried out in a solvent which is inert under the reaction conditions, such as ethers such as tetrahydrofuran, 1,4-di Alkane, 1,2-dimethoxyethane or bis(2-methoxyethyl)ether, alcohols such as methanol, ethanol, isopropanol, n-butanol or tert-butanol, or dipolar-non- Protic solvents such as acetone, methyl ethyl ketone, acetonitrile, ethyl acetate, pyridine, dimethyl hydrazine (DMSO), N,N -dimethylformamide (DMF), N,N -dimethyl B Indoleamine (DMA), N,N' -dimethylpropenylurea (DMPU) or N -methylpyrrolidone (NMP) or water. Mixtures of these solvents can also be used. Preferably, DMF is used.

Suitable bases for (VI) + (VII) → (II-B) are, for example, quaternary amine bases such as triethylamine, N -methylmorpholine, N -methylpiperidine, N,N- Diisopropylethylamine, pyridine or 4- N,N -dimethylaminopyridine. Preferably, N,N -diisopropylethylamine is used.

The (VI) + (VII) → (II-B) reaction is carried out at a temperature ranging from 0 ° C to +50 ° C, preferably from +10 ° C to +30 ° C. This reaction can be carried out at atmospheric pressure, elevated or reduced pressure (e.g., 0.5 to 5 bar); it is typically operated at atmospheric pressure.

(IX)→(III-A), (XIV)→(III-B) and (XVI)→(III-C), and (VI)+(XVII)→(III-D), (XIX)+( The XX)→(III-E) and (XXV)+(XX)→(III-F) reactions are carried out in a solvent which is inert under the reaction conditions. Examples of suitable solvents are ethers such as diethyl ether, diisopropyl ether, tert-butyl methyl ether, tetrahydrofuran ether, 1,4-two Alkenyl ether, 1,2-dimethoxyethane or bis(2-methoxyethyl)ether, hydrocarbons such as benzene, toluene, xylene, pentane, hexane, heptane, cyclohexane or petroleum Fractions, halogenated hydrocarbons such as dichloromethane, chloroform, tetrachloromethane, 1,2-dichloroethane, trichloroethane or chlorobenzene, or dipolar-aprotic solvents such as acetone, methylethyl Ketone, acetonitrile, ethyl acetate, pyridine, dimethyl hydrazine (DMSO), N,N -dimethylformamide (DMF), N,N -dimethylacetamide (DMA), N, N ' -Dimethyl propylene urea (DMPU) or N -methylpyrrolidone (NMP). Mixtures of these solvents can also be used. Preferably, N,N -dimethylformamide is used.

Suitable bases for these reactions are, for example, quaternary amines such as triethylamine, N -methylmorpholine, N -methylpiperidine, N,N -diisopropylethylamine, pyridine or 4- N,N -dimethylaminopyridine. Preferably, N,N -diisopropylethylamine is used, and 4- N,N -dimethylaminopyridine is added as needed.

(IX)→(III-A), (XIV)→(III-B) and (XVI)→(III-C), And (VI)+(XVII)→(III-D) and (XIX)+(XX)→(III-E) reactions are carried out at a temperature ranging from 0 ° C to +50 ° C, preferably +10 ° C to +30 ° C. These reactions can be carried out at atmospheric pressure, elevated or reduced pressure (e.g., 0.5 to 5 bar); they are typically operated at atmospheric pressure.

The compounds of formula (II), (III), (IA) and (IB) are subcomponents of the compounds of formula (IIa), (IIIa), (Ia-A) and (Ia-B), respectively, wherein R ³⁵ is methyl . The preparation of the compounds of formula (IIa) and (IIIa) is carried out analogously to the preparation of the compounds of formula (II) and (III) above.

The above method is illustrated by the following synthetic procedure (flows 3 to 13, 18) by way of example: [a): 1. Water / II Alkane, 1N HCl, 100 ° C; 2. H ₂ , Pd/C, methanol, RT; b): NaHCO ₃ , H ₂ O, II Alkane, RT].

[a): Diisopropylethylamine, DMF, RT].

[a): Water / II Alkane, 1N HCl, 100 ° C; b): HATU, diisopropylamine, DMF, RT].

[a): 1. EDCI, DMAP, dichloromethane, RT; 2. H ₂ , methanol, RT; b): 1. EDCI, HOBt, diisopropylamine, DMF, RT; 2. dichloromethane, RT] .

[a): 1. EDCI, DMAP, dichloromethane, RT; 2. H ₂ , methanol, RT; b): HATU, diisopropylamine, DMF, RT].

[a): EDCI, dichloromethane, RT].

[a): HATU, diisopropylethylamine, DMF, RT; 2. H ₂ , methanol, RT; b): EDCI, DMAP, dichloromethane, RT].

[a): 1. HATU, diisopropylethylamine, DMF, RT; 2. dichloromethane, RT; b): EDCI, DMAP, dichloromethane, RT].

[a): Diisopropylethylamine, DMF, RT].

[a): 1. EDCI, DMAP, dichloromethane, RT; 2. dichloromethane, RT; b): diisopropylamine, DMAP, dichloromethane, RT].

[a): DMAP, diisopropylamine, dichloromethane, RT].

[a): 1. Water / II Alkane, 1N HCl, 100 ° C; 2. H ₂ , Pd/C, methanol, RT; b): HATU, diisopropylethylamine, RT].

Compounds of formula (IV) may be from commercially available amino acid building blocks or those known in the literature (see, for example, Pettit et al, Synthesis 1996, 719; Shioiri et al, Tetrahedron Lett. 1991, 32 , 931; Shioiri et al., Tetrahedron 1993, 49 , 1913; Koga et al., Tetrahedron Lett. 1991, 32 , 2395; Vidal et al., Tetrahedron 2004, 60 , 9715; Poncet et al., Tetrahedron 1994, 50 , 5345. Pettit et al. J. Org. Chem. 1994, 59 , 1796) was prepared in a manner similar to that known in the literature, according to the conventional methods of peptide chemistry and the experimental part of the present invention. The following synthetic schemes (Schemes 14 to 16) were prepared by way of example.

[a): Hydroxylamine hydrochloride, KOH, MeOH, 0 ° C → RT; b): BrCH ₂ (CH ₂ ) ₂ CH ₂ Br, K ₂ CO ₃ , acetone, reflux].

[a): 1. Diisopropylethylamine, BEP, dichloromethane, -10 ° C → RT; 2. MeOH].

[a): 1. Diisopropylethylamine, BEP, DMF, RT; 2. Dichloromethane; b): 1. HATU, diisopropylethylamine, DMF, RT; , RT; c): 1. HATU, diisopropylethylamine, DMF, RT; 2. piperidine, DMF, RT].

Compounds of formula (XI), (XIII), (XV), (XVII) and (XXI) include, if appropriate, palm or diastereomeric forms, available from the literature It is known, or can be prepared by methods similar to those disclosed in the literature, by means of a path apparent to those skilled in the art. A wide range of teaching and literature materials for the preparation of starting materials are also given in the relevant sections of the experimental section, the preparation of the starting compounds and the intermediates.

Formulas (V), (VII), (VIII), (X), (XVIII), (XX) and (XXIII) include, if appropriate, palm or diastereomeric forms, commercially available or described in In the literature, or by methods apparent to those skilled in the art, it can be prepared in a manner similar to that disclosed in the literature. A wide range of teaching and literature materials for the preparation of starting materials are also given in the relevant sections of the experimental section, the preparation of the starting compounds and the intermediates.

Additionally, the individual steps of the preparation procedure can be performed in a different order. This is illustrated by the following synthetic scheme by way of example (Schemes 17, 19 and 20).

[a): borane-pyridine complex, acetic acid, MeOH; b): 1. HOBt, EDCI, diisopropylethylamine, DMF, RT; 2. TFA, dichloromethane, RT; c): HATU , diisopropylethylamine, DMF, RT; d): 1. Pd/C, MeOH, RT; 2. NaHCO ₃ , II Alkane, water].

[a): 1. HATU, diisopropylethylamine, DMF, RT; 2. TFA, dichloromethane, RT; 3. ((H ₃ C) ₃ C(C=O)) ₂ O, DMF , diisopropylethylamine; b): diisopropylethylamine, BEP, DMF, RT; c): 1. H ₂ , Pd/C (10%), methanol, RT; 2. HATU, Diisopropylethylamine, DMF, RT; 3. TFA, dichloromethane, RT].

[a): borane-pyridine complex, acetic acid, MeOH; b): 1. HOBt, EDCI, diisopropylethylamine, DMF, RT; 2. FA, dichloromethane, RT; c): 1 H ₂ , Pd/C, MeOH, RT; 2. NaHCO ₃ , II Alkane, water; d): HATU, diisopropylethylamine, DMF, RT;

In addition, the ADC can be prepared by an example according to the following method: [a): Diisopropylethylamine, DMF, RT].

[a): 1. EDCI, DMAP, dichloromethane, RT; 2. H ₂ , methanol, RT; b): HATU, diisopropylamine, DMF, RT; In one embodiment, the binder is There is binding of the target molecule on the cancer cell. In a preferred embodiment, the binding agent binds to the cancer target molecule.

In another preferred embodiment, the target molecule is a selective cancer target molecule.

In a particularly preferred embodiment, the target molecule is a protein.

In one embodiment, the target molecule is an extracellular target molecule. In a preferred embodiment, the extracellular target molecule is a protein.

Cancer target molecules have been known to those skilled in the art. The example series is below.

Examples of cancer target molecules are as follows:

(1) EGF receptor (NCBI reference sequence NP_005219.2) sequence (1210 amino acids): >gi|29725609|ref|NP_005219.2|Epidermal growth factor receptor isoform-precursor [Homo sapiens]

MRPSGTAGAALLALLAALCPASRA LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPV AFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAYVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS

The extracellular zone is indicated by the bottom line.

(2) Sequence of mesothelium (SwissProt reference number Q13421-3) (622 amino acids): >sp|Q13421-3|MSLN_HUMAN mesothelin 2nd isoform OS=Homo sapiens GN=MSLN

MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA

Mesothelin is encoded by amino acids 296-598. The amino acid 37-286 code is a "megakaryocyte promoting factor". The mesothelin is immobilized in the cell membrane by GPI anchors and localized to the extracellular.

(3) Carbonic anhydrase IX (SwissProt reference number Q16790) sequence (459 amino acids): >sp|Q16790|CAH9_HUMAN carbonic anhydrase 9 OS=Homo GN=CA9 PE=1 SV=2

MAPLCPSPWLPLLIPAPAPGLTVQLLLSLLLLVPVHP QRLPRMQEDSPLGGGSSGEDDPLGEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKPKSEEEGSLKLEDLPTVEAPGDPQEPQNNAHRDKEGDDQSHWRYGGDPPWPRVSPACAGRFQSPVDIRPQLAAFCPALRPLELLGFQLPPLPELRLRNNGHSVQLTLPPGLEMALGPGREYRALQLHLHWGAAGRPGSEHTVEGHRFPAEIHVVHLSTAFARVDEALGRPGGLAVLAAFLEEGPEENSAYEQLLSRLEEIAEEGSETQVPGLDISALLPSDFSRYFQYEGSLTTPPCAQGVIWTVFNQTVMLSAKQLHTLSDTLWGPGDSRLQLNFRATQPLNGRVIEASFPAGVDSSPRAAEPVQLNSCLAAGD ILALVFGLLFAVTSVAFLVQMRRQHRRGTKGGVSYRPAEVAETGA

The extracellular zone is indicated by the bottom line.

(4) C4.4a (NCBI reference sequence NP_055215.2; Synonym LYPD3) sequence (346 amino acids): >gi|93004088|ref|NP_055215.2|containing ly6/PLAUR region protein 3 precursor [Homo sapiens]

MDPARKAGAQAMIWTAGWLLLLLLRGGAQA LECYSCVQKADDGCSPNKMKTVKCAPGVDVCTEAVGAVETIHGQFSLAVRGCGSGLPGKNDRGLDLHGLLAFIQLQQCAQDRCNAKLNLTSRALDPAGNESAYPPNGVECYSCVGLSREACQGTSPPVVSCYNASDHVYKGCFDGNVTLTAANVTVSLPVRGCVQDEFCTRDGVTGPGFTLSGSCCQGSRCNSDLRNKTYFSPRIPPLVRLPPPEPTTVASTTSVTTSTSAPVRPTSTTKPMPAPTSQTPRQGVEHE ASRDEEPRLTGGAAGHQDRSNSGQYPAKGGPQQPHNKGC VAPTAGLAALLLAVAAGVLL

The mature extracellular zone is indicated by the bottom line. (SEQ ID NO: 1).

(5) CD52 (NCBI reference sequence NP_001794.2)>gi|68342030|ref|NP_001794.2|CAMPATH-1 antigen precursor [Homo sapiens]

MKRFLFLLLTISLLVMVQIQTGLSGQNDTSQTSSPSASSNISGGIFLFFVANAIIHLFCFS

(6) Her2 (NCBI reference sequence NP_004439.2)>gi|54792096|ref|NP_004439.2|Receptor tyrosine-protein kinase erbB-2 isoform [Homo sapiens]

MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFG PEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV

(7) CD20 (NCBI reference sequence NP_068769.2)>gi|23110987|ref|NP_068769.2|B-lymphocyte antigen CD20[智人]

MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVE NEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP

(8) lymphocyte activation antigen CD30 (SwissProt ID P28908)>gi|68348711|ref|NP_001234.2|Tumor apoptotic factor receptor superfamily member 8 isomer type 1 precursor [Homo sapiens]

MRVLLAALGLLFLGALRAFPQDRPFEDTCHGNPSHYYDKAVRRCCYRCPMGLFPTQQCPQRPTDCRKQCEPDYYLDEADRCTACVTCSRDDLVEKTPCAWNSSRVCECRPGMFCSTSAVNSCARCFFHSVCPAGMIVKFPGTAQKNTVCEPASPGVSPACASPENCKEPSSGTIPQAKPTPVSPATSSASTMPVRGGTRLAQEAASKLTRAPDSPSSVGRPSSDPGLSPTQPCPEGSGDCRKQCEPDYYLDEAGRCTACVSCSRDDLVEKTPCAWNSSRTCECRPGMICATSATNSRARCVPYPICAAETVTKPQDMAEKDTTFEAPPLGTQPDCNPTPENGEAPASTSPTQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVGSSAFLLCHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK

(9) lymphocyte adhesion molecule CD22 (SwissProt ID P20273 )>gi|157168355|ref|NP_001762.2|B-cell receptor CD22 isoform 1 precursor [Homo sapiens]

MHLLGPWLLLLVLEYLAFSDSSKWVFEHPETLYAWEGACVWIPCTYRALDGDLESFILFHNPEYNKNTSKFDGTRLYESTKDGKVPSEQKRVQFLGDKNKNCTLSIHPVHLNDSGQLGLRMESKTEKWMERIHLNVSERPFPPHIQLPPEIQESQEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQAAVTSTSLTIKSVFTRSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKHTPKLEIKVTPSDAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVTKDQSGKYCCQVSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPAVEGSQVEFLCMSLANPLPTNYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAENILGTGQRGPGAELDVQYPPKKVTTVIQNPMPIREGDTVTLSCNYNSSNPSVTRYEWKPHGAWEEPSLGVLKIQNVGWDNTTIACAACNSWCSWASPVALNVQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEVQFFWEKNGRLLGKESQLNFDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSMSPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSLPYHSQKLRLEPVKVQHSGAYWCQGTNSVGKGRSPLSTLTVYYSPETIGRRVAVGLGSCLAILILAICGLKLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLGCYNPMMEDGISYTTLRFPEMNIPRTGDAESSEMQRPPPDCDDTVTYSALHKRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYVILKH

(10) bone marrow cell surface antigen CD33 (SwissProt ID P20138 )>gi|130979981|ref|NP_001763.3|Bone marrow cell surface antigen CD33 isoform 1 precursor [Homo sapiens]

MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

(11) Transmembrane glycoprotein NMB (SwissProt ID Q14956)>gi|52694752|ref|NP_001005340.1|Transmembrane glycoprotein NMB isoform precursor [Homo sapiens]

MECLYYFLGFLLLAARLPLDAAKRFHDVLGNERPSAYMREHNQLNGWSSDENDWNEKLYPVWKRGDMRWKNSWKGGRVQAVLTSDSPALVGSNITFAVNLIFPRCQKEDANGNIVYEKNCRNEAGLSADPYVYNWTAWSEDSDGENGTGQSHHNVFPDGKPFPHHPGWRRWNFIYVFHTLGQYFQKLGRCSVRVSVNTANVTLGPQLMEVTVYRRHGRAYVPIAQVKDVYVVTDQIPVFVTMFQKNDRNSSDETFLKDLPIMFDVLIHDPSHFLNYSTINYKWSFGDNTGLFVSTNHTVNHTYVLNGTFSLNLTVKAAAPGPCPPPPPPPRPSKPTPSLATTLKSYDSNTPGPAGDNPLELSRIPDENCQINRYGHFQATITIVEGILEVNIIQMTDVLMPVPWPESSLIDFVVTCQGSIPTEVCTIISDPTCEITQNTVCSPVDVDEMCLLTVRRTFNGSGTYCVNLTLGDDTSLALTSTLISVPDRDPASPLRMA NSALISVGCLAIFVTVISLLVYKKHKEYNPIENSPGNVVRSKGLSVFLNRAKAVFFPGNQEKDPLLKNQEFKGVS

(12) Adhesion molecule CD56 (SwissProt ID P13591)>gi|94420689|ref|NP_000606.3|Neutral cell adhesion molecule 1 Isomer 1 [Homo sapiens]

MLQTKDLIWTLFFLGTAVSLQVDIVPSQGEISVGESKFFLCQVAGDAKDKDISWFSPNGEKLTPNQQRISVVWNDDSSSTLTIYNANIDDAGIYKCVVTGEDGSESEATVNVKIFQKLMFKNAPTPQEFREGEDAVIVCDVVSSLPPTIIWKHKGRDVILKKDVRFIVLSNNYLQIRGIKKTDEGTYRCEGRILARGEINFKDIQVIVNVPPTIQARQNIVNATANLGQSVTLVCDAEGFPEPTMSWTKDGEQIEQEEDDEKYIFSDDSSQLTIKKVDKNDEAEYICIAENKAGEQDATIHLKVFAKPKITYVENQTAMELEEQVTLTCEASGDPIPSITWRTSTRNISSEEKTLDGHMVVRSHARVSSLTLKSIQYTDAGEYICTASNTIGQDSQSMYLEVQYAPKLQGPVAVYTWEGNQVNITCEVFAYPSATISWFRDGQLLPSSNYSNIKIYNTPSASYLEVTPDSENDFGNYNCTAVNRIGQESLEFILVQADTPSSPSIDQVEPYSSTAQVQFDEPEATGGVPILKYKAEWRAVGEEVWHSKWYDAKEASMEGIVTIVGLKPETTYAVRLAALNGKGLGEISAASEFKTQPVQGEPSAPKLEGQMGEDGNSIKVNLIKQDDGGSPIRHYLVRYRALSSEWKPEIRLPSGSDHVMLKSLDWNAEYEVYVVAENQQGKSKAAHFVFRTSAQPTAIPANGSPTSGLSTGAIVGILIVIFVLLLVVVDITCYFLNKCGLFMCIAVNLCGKAGPGAKGKDMEEGKAAFSKDESKEPIVEVRTEEERTPNHDGGKH TEPNETTPLTEPEKGPVEAKPECQETETKPAPAEVKTVPNDATQTKENESKA

(13) Surface molecule CD70 (SwissProt ID P32970)>gi|4507605|ref|NP_001243.1|CD70 antigen [Homo sapiens]

MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP

(14) Surface molecule CD74 (SwissProt ID P04233)>gi|10835071|ref|NP_004346.1|HLA class II histocompatibility antigen γ chain isomerism b [Homo sapiens]

MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPVPM

(15) B-lymphocyte antigen CD19 (SwissProt ID P15391)>gi|296010921|ref|NP_001171569.1|B-lymphocyte antigen CD19 isoform 1 precursor [Homo sapiens]

MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWN VSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLAGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR

(16) surface protein mucin-1 (mucin-1) (SwissProt ID P15941)>gi|65301117|ref|NP_002447.4|mucin-1 isomer 1 precursor [Homo sapiens]

MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNALSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAATSANL

(17) Surface protein CD138 (SwissProt ID P18827)>gi|29568086|ref|NP_002988.3|syndecan-1 precursor [Homo sapiens]

MRRAALWLWLCALALSLQPALPQIVATNLPPEDQDGSGDDSDNFSGSGAGALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLE ATAASTSTLPAGEGPKEGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHQASTTTATTAQEPATSHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQLPAAEGSGEQDFTFETSGENTAVVAVEPDRRNQSPVDQGATGASQGLLDRKLGGVIAGGLVGLIFAVCLVGFMLYRMKKKDEGSYSLEEPKQANGGAYQKPTKQEEFYA

(18) Integrin αV (Genbank accession number: NP_002201.1)>gi|4504763|ref|NP_002201.1|Integrin α-V isomer type 1 precursor [Homo sapiens]

MAFPPRRRLRLGPRGLPLLLSGLLLPLCRAFNLDVDSPAEYSGPEGSYFGFAVDFFVPSASSRMFLLVGAPKANTTQPGIVEGGQVLKCDWSSTRRCQPIEFDATGNRDYAKDDPLEFKSHQWFGASVRSKQDKILACAPLYHWRTEMKQEREPVGTCFLQDGTKTVEYAPCRSQDIDADGQGFCQGGFSIDFTKADRVLLGGPGSFYWQGQLISDQVAEIVSKYDPNVYSIKYNNQLATRTAQAIFDDSYLGYSVAVGDFNGDGIDDFVSGVPRAARTLGMVYIYDGKNMSSLYNFTGEQMAAYFGFSVAATDINGDDYADVFIGAPLFMDRGSDGKLQEVGQVSVSLQRASGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGFNDIAIAAPYGGEDKKGIVYIFNGRSTGLNAVPSQILEGQWAARSMPPSFGYSMKGATDIDKNGYPDLIVGAFGVDRAILYRARPVITVNAGLEVYPSILNQDNKTCSLPGTALKVSCFNVRFCLKADGKGVLPRKLNFQVELLLDKLKQKGAIRRALFLYSRSPSHSKNMTISRGGLMQCEELIAYLRDESEFRDKLTPITIFMEYRLDYRTAADTTGLQPILNQFTPANISRQAHILLDCGEDNVCKPKLEVSVDSDQKKIYIGDDNPLTLIVKAQNQ GEGAYEAELIVSIPLQADFIGVVRNNEALARLSCAFKTENQTRQVVCDLGNPMKAGTQLLAGLRFSVHQQSEMDTSVKFDLQIQSSNLFDKVSPVVSHKVDLAVLAAVEIRGVSSPDHIFLPIPNWEHKENPETEEDVGPVVQHIYELRNNGPSSFSKAMLHLQWPYKYNNNTLLYILHYDIDGPMNCTSDMEINPLRIKISSLQTTEKNDTVAGQGERDHLITKRDLALSEGDIHTLGCGVAQCLKIVCQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSLKSSASFNVIEFPYKNLPIEDITNSTLVTTNVTWGIQPAPMPVPVWVIILAVLAGLLLLAVLVFVMYRMGFFKRVRPPQEEQEREQLQPHENGEGNSET

(19) teratoma-derived growth factor 1 protein TDGF1 (Genbank accession number: NP_003203.1)>gi|4507425|ref|NP_003203.1| teratomas-derived growth factor 1 isoform 1 precursor [Homo sapiens]

MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYLAFRDDSIWPQEEPAIRPRSSQRVPPMGIQHSKELNRTCCLNGGTCMLGSFCACPPSFYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQAFLPGCDGLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY

(20) Prostate specific membrane antigen PSMA (Swiss Prot ID: Q04609)> gi|4758398|ref|NP_004467.1|bromoproline carboxypeptidase 2 isomer 1 [Homo sapiens]

MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPH LAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA

(21) tyrosine protein kinase EPHA2 (Swiss Prot ID: P29317)>gi|32967311|ref|NP_004422.2|A type ephrin receptor 2 precursor [Homo sapiens]

MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCPEL LQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIGGVAVGVVLLLVLAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTYVDPHTYEDPNQAVLKFTTEIHPSCVTRQKVIGAGEFGEVYKGMLKTSSGKKEVPVAIKTLKAGYTEKQRVDFLGEAGIMGQFSHHNIIRLEGVISKYKPMMIITEYMENGALDKFLREKDGEFSVLQLVGMLRGIAAGMKYLANMNYVHRDLAARNILVNSNLVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWTAPEAISYRKFTSASDVWSFGIVMWEVMTYGERPYWELSNHEVMKAINDGFRLPTPMDCPSAIYQLMMQCWQQERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSGSEGVPFRTVSEWLESIKMQQYTEHFMAAGYTAIEKVVQMTNDDIKRIGVRLPGHQKRIAYSLLGLKDQVNTVGIPI

(22) Surface protein SLC44A4 (Genbank Accession No: NP_001171515)>gi|295849282|ref|NP_001171515.1|Cholinergic transporter protein 4 isomer 2 [Homo sapiens]

MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGDPRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQTVITSLQQELCPSFLLPSAPALGRCFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVALVLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLSAYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLVTFVLLLICIAYWAMTALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCPGLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQDIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCCFKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDLLLFFGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFSVFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK

(23) Surface protein BMPR1B (SwissProt: O00238)

(24) Transporter SLC7A5 (SwissProt: Q01650)

(25) Epithelial prostate antigen STEAP1 (SwissProt: Q9UHE8)

(26) Ovarian cancer antigen MUC16 (SwissProt: Q8WXI7)

(27) Transporter SLC34A2 (SwissProt: O95436)

(28) Surface protein SEMA5b (SwissProt: Q9P283)

(29) Surface protein LYPD1 (SwissProt: Q8N2G4)

(30) Endothelial receptor type B EDNRB (SwissProt: P24530)

(31) Ring finger protein RNF43 (SwissProt: Q68DV7)

(32) Prostate cancer connexin STEAP2 (SwissProt: Q8NFT2)

(33) cation channel TRPM4 (SwissProt: Q8TD43)

(34) Complement receptor CD21 (SwissProt: P20023)

(35) B-cell antigen receptor complex-associated protein CD79b (SwissProt: P40259)

(36) Cell adhesion antigen CEACAM6 (SwissProt: P40199)

(37) Dipeptidase DPEP1 (SwissProt: P16444)

(38) Interleukin receptor IL20Ralpha (SwissProt: Q9UHF4)

(39) Proteoglycan BCAN (SwissProt: Q96GW7)

(40) ephrin receptor EPHB2 (SwissProt: P29323)

(41) Prostate stem cell connexin PSCA (Genbank accession number: NP_005663.2)

(42) Surface protein LHFPL3 (SwissProt: Q86UP9)

(43) Receptor protein TNFRSF13C (SwissProt: Q96RJ3)

(44) B-cell antigen receptor complex-associated protein CD79a (SwissProt: P11912)

(45) Receptor protein CXCR5 (SwissProt: P32302)

(46) Ion channel P2X5 (SwissProt: Q93086)

(47) Lymphocyte antigen CD180 (SwissProt: Q99467)

(48) Receptor protein FCRL1 (SwissProt: Q96LA6)

(49) Receptor protein FCRL5 (SwissProt: Q96RD9)

(50) MHC class II molecule Ia antigen HLA-DOB (Genbank accession number: NP_002111.1)

(51) T-cell protein VTCN1 (SwissProt: Q7Z7D3). In a preferred subject matter of the invention, the cancer target molecule is selected from the group consisting of cancer target molecules (1)-(51).

In a further preferred subject matter of the invention, the binding agent binds to an extracellular cancer target molecule selected from the group consisting of cancer target molecules (1)-(51).

In a further preferred subject of the invention, the binding agent specificity binds to an extracellular cancer target molecule selected from the group consisting of cancer target molecules (1)-(51).

In a particularly preferred subject of the invention, the cancer target molecule is selected from the group consisting of EGF receptor (NP_005219.2), mesothelin (Q13421-3), C4.4a (NP_055215.2), and Carboxylic acid anhydride IX (CA IX; NP_001207.2).

In a further preferred subject of the invention, the binding agent binds to an extracellular cancer target molecule selected from the group consisting of EGF receptor (NP_005219.2), mesothelin (Q13421-3), C4. 4a (NP_055215.2) and carboxylic anhydride enzyme IX (CA IX; Q16790).

In a further preferred subject of the invention, the binding agent specificity is combined with an extracellular cancer target molecule selected from the group consisting of: EGF receptor (NP_005219.2), mesothelin (Q13421-3), C4.4a (NP_055215.2) and carboxylic anhydride enzyme IX (CA IX; Q16790).

In a preferred embodiment, the binding agent is internalized by binding after binding to an extracellular target molecule on its target cell. This effect is a binder-drug conjugate, which can be an immunoconjugate or ADC, which is targeted Cell absorption.

In one embodiment, the cement is a binding protein. In a preferred embodiment, the binding agent is an antibody, an antigen binding antibody fragment, a multi-specific antibody or an antibody mimetic.

Preferred antibody mimetics are affibodies, Adnectin, anticalin, DARPin, high affinity avimers, and nanobodies. Preferred multi-specific antibodies are bispecific and trispecific antibodies.

In a preferred embodiment, the binding agent is an antibody, an antigen binding antibody fragment, a more isolated antibody or an isolated antigen binding antibody fragment.

Preferred antigen-binding antibody fragments are Fab, Fab', F(ab') ₂ and Fv fragments, diabodies, DAb, linear antibodies and scFv. Particularly preferred are Fab, diabody and scFv.

In a particularly preferred embodiment, the binding agent is an antibody. Particularly preferred is a monoclonal antibody or an antigen-binding antibody fragment thereof. Further preferred are human, humanized or chimeric antibodies or antigen-binding antibody fragments thereof.

The antibody or antigen-binding antibody fragment that binds to the cancer target molecule can be prepared by one of ordinary skill in the art using known methods, such as chemical synthesis or recombinant expression. Binding agents for cancer target molecules can be obtained commercially or can be prepared by one of ordinary skill in the art using known methods, such as chemical synthesis or recombinant expression. Further methods of preparing antibodies or antigen-binding antibody fragments are described in WO 2007/070538 (see "Antibodies" on page 22). It is known to those skilled in the art how to assemble, for example Methods of phage display libraries (e.g., Morphosys HuCAL Gold) and for the search for antibodies or antigen-binding antibody fragments (see WO 2007/070538, page 24 ff and page 70 examples, page 72 example 2). Additional methods for preparing antibodies using DNA libraries of B cells are described, for example, on page 26 (WO 2007/070538). Methods for humanizing antibodies are described on pages 30-32 of WO2007070538 and in Queens et al, Pros. Natl. Acad. Sci. USA 86: 10029-10033, 1989 or in WO 90/0786. Furthermore, recombinant expression methods for general proteins and specific antibodies are known to those skilled in the art (see, for example, Berger and Kimrnel (Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc.). ); Sambrook et al, (Molecular Cloning: A Laboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY; 1989) Vol. 1-3); Current Protocols in Molecular Biolony, (FMAusabel et al. [Eds.], Current Protocols, Green Publishing Associates, Inc./John Wiley & Sons, Inc.; Harlow et al, (Monoclonal Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (19881, Paul [Ed.]) ; Fundamental Immunology, (Lippincott Williams & Wilkins (1998)); and Harlow, et al, (Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1998)). Those skilled in the art are aware of proteins/antibodies for expression. Corresponding vector, promoter And the message peptide. The general method is also described on pages 41-45 of WO 2007/070538. Methods of preparing antibodies are described, for example, in Example 6 of page 74 ff of WO 2007/070538. Methods for measuring antibody internalization after binding of an antibody to its antigen are known to those skilled in the art and are described, for example, on page 80 of WO 2007/070538. Those skilled in the art will be able to prepare antibodies having specificity of different target molecules using the methods described in WO 2007/070538, which has been used to prepare carbonic anhydrase IX (Mn) antibodies.

EGFR antibody

Examples of antibodies that bind to the cancer target molecule EGFR are cetuximab (INN No. 7906), panitumumab (INN No. 8499), and nimotuzumab (INN No. 8545). Cetuximab (Drug Bank Accession No. DB00002) is a chimeric anti-EGFR1 antibody produced in SP2/0 mouse myeloma cells and sold by ImClone Systems, Inc./Merck KgaA/Bristol-Myers Squibb. Cetuximab is suitable for the treatment of colorectal cancer with metastatic, EGFR expression of the wild-type K-Ras gene. It has an affinity of 10 ^-10 M.

sequence: Cetuximab light chain (κ):

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTTLSTLSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Cetuximab heavy chain:

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Panitumumab (INN No. 8499) (Pharmaceutical Bank Accession No. DB01269) is a recombinant human monoclonal IgG2 antibody that specifically binds to human EGF receptor 1 and is sold by Abgenix/Amgen. The panitumumab is derived from immunization of a genetically transformed mouse (XenoMouse). These mice are capable of producing human immunoglobulins (light and heavy chains). A specific B-cell selection strain producing an anti-EGFR antibody was selected, and the selected strain was immortalized in CHO cells (Chinese hamster uterus cells). These cells were used to make 100% human antibodies. Pani antibody is suitable for treatment Therapeutic treatment of fluoropyrimidine, oxaliplatin and irinotecan is resistant to EGFR-expressing and metastatic colorectal cancer. It has an affinity of 10-11M.

sequence: Panitumab light chain (kappa):

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Panitumab heavy chain:

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Nimotuzumab (INN No. 8545) (EP 00586002, EP 00712863) is a humanized monoclonal IgG1 antibody that binds to the human EGF receptor and is sold by YM BioScienecs (Mississauga, Canada). It is produced in non-secreting NSO cells (mammalian cells). Nimotuzumab is approved for the treatment of head and neck tumors, highly malignant astrocytoma and polymorphic glioblastoma (EU and US unapproved) and pancreatic cancer (rare drugs, EMA). It has an affinity of 10 ^-8 M.

Additional examples of EGFR antibodies are as follows:

Zalumumumab/2F8/HuMax-EGFr from Genmab A/S (WO 02/100348, WO 2004/056847, INN No. 8605)

Necitumumab/11F8, ImClone/IMC-11F8, from ImClone Systems, Inc. [Eli Lilly & Co] (WO 2005/090407 (EP 01735348-A1, US 2007/0264253-A1, US 7,598,350, WO) 2005/090407-A1), INN No. 9083)

Matuzumab/anti-EGFR MAb, Merck KGaA/anti-EGFR MAb, Takeda/EMD 72000/EMD-6200/EMD-72000 and EMD-55900/MAb 425/single antibody 425 from Merck KGaA/Takeda (WO 92/15683, INN No. 8103 (Matuzumab))

̇RG-7160/GA-201/GA201/R-7160/R7160/RG7160/RO-4858696/RO-5083945/RO4858696/RO5083945 from Glycart Biotechnology AG (Roche Holding AG) (WO 2010/112413-A1, WO 2010 /115554)

̇ GT-MAB 5.2-GEX/CetuGEX from Glycotope GmbH (WO 2008/028686-A2 (EP 01900750-A1, EP 01911766-A1, EP 02073842-A2, US 2010/0028947-A1)

̇ISU-101 from Isu Abxis Inc (ISU Chemical Co Ltd)/Scancell (WO 2008/004834-A1)

̇ABT-806/mAb-806/ch-806/anti-EGFR monoclonal antibody 806 from Ludwig Institute for Cancer Research/Abbott/Life Science Pharmaceuticals (WO 02/092771, WO 2005/081854 and WO 2009/023265)

̇SYM-004 (containing two chimeric IgG1 antibodies (992 and 1024)) from Symphonogen A/S (WO 2010/022736-A2)

̇MR1-1/MR1-1KDEL from IVAX Corp (Teva Pharmaceutical Industries Ltd) (Duke University), (Patent: WO2001/062931-A2)

̇Anti-deletion mutant antibody, EGFRvIII, from Amgen/Abgenix (WO 2005/010151, US 7,628,986)

̇SC-100 from Scancell Ltd (WO 01/088138-A1)

̇MDX-447/EMD 82633/BAB-447/H 447/MAb, EGFR, Medarex/Merck KgaA, from Bristol-Myers Squibb (US)/Merck KGaA(DE)/Takeda (JP), (WO 91/05871, WO 92/15683)

̇Anti-EGFR-Mab from Xencor (WO 2005/056606)

̇DXL-1218/anti-EGFR monoclonal antibody (cancer), InNexus, from InNexus Biotechnology Inc., Pharmaprojects PH048638.

In a preferred embodiment, the anti-EGFR anti-system is selected from the group consisting of cetuximab, panitumumab, nimotuzumab, zarulimumab, naproximumab , Martuzumab, RG-716, GT-MAB 5.2-GEX, ISU-101, ABT-806, SYM-004, MR1-1, SC-100, MDX-447 and DXL-1218.

In a particularly preferred embodiment, the anti-EGFR anti-system is selected from the group consisting of cetuximab, zarulimumab, naxycizumab, and matuzumab.

Those skilled in the art will appreciate, from sequence changes in the CDR regions of the above antibodies, methods useful for the preparation of additional antibodies which have similar or better affinity and/or specificity for the target molecule.

In a further embodiment, the anti-EGFR antibody or antigen-binding antibody fragment is selected from the group consisting of an antibody or antigen comprising three light chain CDR regions and three heavy chain CDR regions of any of the following antibodies Binding antibody fragments: cetuximab, panitumumab, nimotuzumab, zalumuzumab, naxylimumab, matuzumab, RG-716, GT-MAB 5.2-GEX, ISU-101, ABT-806, SYM-004, MR1-1, SC-100, MDX-447 and DXL-1218.

In another embodiment, the anti-EGFR antibody or antigen-binding antibody fragment is selected from the group consisting of: An antibody or antigen-binding antibody fragment comprising three light chain CDR regions and three heavy chain CDR regions of any of the following antibodies: cetuximab, panitumumab, nimotuzumab, zalumuzumab, Naximizumab, Martuzumab.

Carbonic anhydrase IX antibody

An example of an antibody that binds to the cancer target molecule carbonic anhydrase IX is described in WO 2007/070538-A2 (for example, Patent Application Nos. 1-16).

In a preferred embodiment, the anti-carbonic anhydrase IX antibody or antigen-binding antibody fragment is selected from the group consisting of the following anti-carbonic anhydrase IX antibodies or antigen-binding antibody fragments: 3ee9 (WO 2007/070538-A2 Patent Application No. 4 (a)), 3ef2 (WO 2007/070538-A2, Patent Application No. 4 (b)), 1e4 (WO 2007/070538-A2, Patent Application No. 4 (c) ), 3a4 (WO 2007/070538-A2 patent application scope item 4 (d)), 3ab4 (WO 2007/070538-A2 patent application scope item 4 (e)), 3ah10 (WO 2007/070538-A2) Patent Application No. 4 (f)), 3bb2 (WO 2007/070538-A2, Patent Application No. 4 (g)), 1aa1 (WO 2007/070538-A2, Patent Application No. 4 (h) ), 5a6 (the patent application scope 4 (i) of WO 2007/070538-A2) and 5aa3 (the patent application scope 4 (j) of WO 2007/070538-A2).

In a preferred embodiment, the anti-carbonic anhydrase IX antibody or antigen-binding antibody fragment is selected from the group consisting of: An anti-carbonic anhydrase IX antibody or antigen-binding antibody fragment thereof (from WO 2007/070538-A2) comprising three light chain CDR region sequences of the 3ee9 antibody and three heavy chain CDR region sequences, including three lightly of the 3ef2 antibody An anti-carbonic anhydrase IX antibody or an antigen-binding antibody fragment thereof (from WO 2007/070538-A2) comprising a chain CDR region sequence and three heavy chain CDR region sequences, comprising three light chain CDR region sequences of the 1e4 antibody and three An anti-carbonic anhydrase IX antibody or an antigen-binding antibody fragment thereof (from WO 2007/070538-A2) of the heavy chain CDR region sequence, comprising three light chain CDR region sequences of the 3a4 antibody and three heavy chain CDR region sequences a carbonic anhydrase IX antibody or antigen-binding antibody fragment thereof (from WO 2007/070538-A2), comprising three light chain CDR region sequences of the 3ab4 antibody and three heavy chain CDR region sequences of an anti-carbonic anhydrase IX antibody or An antigen-binding antibody fragment thereof (from WO 2007/070538-A2), comprising three light chain CDR region sequences of the 3ah10 antibody and three heavy chain CDR region sequences of an anti-carbonic anhydrase IX antibody or antigen-binding antibody fragment thereof (from WO 2007/070538-A2), comprising three light chain CDR region sequences and three heavy chain CDR region sequences of the 3bb2 antibody An anti-carbonic anhydrase IX antibody or antigen-binding antibody fragment thereof (from WO 2007/070538-A2), comprising three light chain CDR region sequences of a 1aa1 antibody and three heavy chain CDR region sequences of an anti-carbonic anhydrase IX antibody Or an antigen-binding antibody fragment thereof (from WO 2007/070538-A2), An anti-carbonic anhydrase IX antibody comprising the three light chain CDR region sequences of the 5a6 antibody and three heavy chain CDR region sequences or an antigen-binding antibody fragment thereof (from WO 2007/070538-A2) and three lightly comprising 5aa3 antibodies An anti-carbonic anhydrase IX antibody or an antigen-binding antibody fragment thereof (from WO 2007/070538-A2) having a chain CDR region sequence and three heavy chain CDR region sequences. The CDR region sequences referred to herein are disclosed in Figures 2a-2c on pages 128-130 of WO 2007/070538-A2.

In a preferred embodiment, the anti-carbonic anhydrase IX antibody or antigen-binding antibody fragment is selected from the group consisting of an antibody comprising a variable light chain of a 3ee9 antibody and an amino acid sequence of a variable heavy chain. Or an antigen-binding antibody fragment, as shown in Figure 4b on page 137 of WO 2007/070538-A2, comprising an antibody or antigen-binding antibody fragment of the variable light chain of the 3ef2 antibody and the amino acid sequence of the variable heavy chain, such as WO 2007/070538-A2 Figure 4c on page 138 and Figure 4b on page 137, including antibodies or antigen-binding antibody fragments of the variable light chain and variable heavy chain amino acid sequences of the 1e4 antibody, such as WO 2007 /070538-A2, shown in Figure 4a on page 136, comprising an antibody or antigen-binding antibody fragment of the variable light chain of the 3a4 antibody and the amino acid sequence of the variable heavy chain, as described in WO 2007/070538-A2, page 136 Figure 4a shows an antibody or antigen-binding antibody fragment comprising the variable light chain of the 3ab4 antibody and the amino acid sequence of the variable heavy chain, as described in WO 2007/070538-A2 An antibody or antigen-binding antibody fragment comprising the amino acid sequence of the variable light chain and variable heavy chain of the 3ah10 antibody, as shown in Figure 4a on page 136 of WO 2007/070538-A2, is shown in Figure 4a on page 136. An antibody or antigen-binding antibody fragment comprising a variable light chain of a 3bb2 antibody and an amino acid sequence of a variable heavy chain, as shown in Figure 4b on page 137 of WO 2007/070538-A2, including 1aa1 antibody An antibody or antigen-binding antibody fragment that changes the amino acid sequence of the light chain and the variable heavy chain, as shown in Figure 4a on page 136 of WO 2007/070538-A2, including the variable light chain and variable weight of the 5a6 antibody An antibody or antigen-binding antibody fragment of a chain amino acid sequence, as shown in Figure 4b on page 137 of WO 2007/070538-A2, and an amino acid sequence comprising a variable light chain and a variable heavy chain of a 5aa3 antibody Antibody or antigen-binding antibody fragment, as shown in Figure 4b on page 137 of WO 2007/070538-A2.

In a particularly preferred embodiment, the anti-carbonic anhydrase IX antibody is a 3ee9 antibody fragment from WO 2007/070538-A2.

In a particularly preferred embodiment, the anti-carbonic anhydrase IX antibody or antigen-binding antibody fragment comprises an amino acid sequence of a variable heavy chain CDR region of a 3ee9 antibody (VH3-CDR1: GFTFSSYGMS; VH3-CDR2: GISSLGSTTYYADSVKG; VH3-CDR3: TGSPGTFMHGDH, see Figure 2a on page 128 of WO 2007/070538-A2 and the amino acid sequence of the variable light chain CDR regions of the 3ee9 antibody Columns (VLk1-CDR1: RASQDINNYLS; VLk1-CDR2: YGASNLQS; VLk1-CDR3: QQYYGRPT, see Figure 2b on page 129 of WO 2007/070538-A2). In a particularly preferred embodiment, the anti-carbonic anhydrase IX antibody or antigen-binding antibody fragment comprises the amino acid sequence of the variable heavy chain of the 3ee9 antibody (VH3: ELVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSGISSLGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTGSPGTFMHGDHWGQGTLVTVSS, see WO 2007/070538-A2 page 137 Figure 4b) and the amino acid sequence of the variable light chain of the 3ee9 antibody (VLk1: DIQMTQSPSSLSASVGDRVTITCRaSQDINNYLSWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYYGRPTTFGQGTKVEIKRT, see Figure 4b on page 137 of WO 2007/070538-A2).

In a preferred embodiment, the anti-carbonic anhydrase IX antibody 3ee9 is an IgG antibody.

In a particularly preferred embodiment, the anti-carbonic anhydrase IX antibody 3ee9 is an IgG1 antibody (3ee9-IgG1), wherein the heavy chain amino acid sequence comprises the following sequence: QVELVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSGISSLGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTGSPGTFMHGDHWGQGTLVTVSSA Amino acid sequences are STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK and light chains comprise the following sequences: DIQMTQSPSSLSASVGDRVTITCRASQDINNYLSWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYYGRPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Anti-Carnitic Anhydrase IX Antibody 3ee9-IgG1:

In another aspect of the invention, an anti-carbonic anhydrase IX antibody 3ee9-IgG1 is provided.

C4.4a antibody:

Examples of C4.4a antibodies and antigen-binding antibody fragments are described below. The sequences of the antibodies are shown in Table 1, wherein each column produces the CDR amino acid sequences of the individual variable light and variable heavy chains of the antibodies listed in column 1, respectively. The CDR amino acid sequence and nucleic acid sequence of the variable light chain and variable heavy chain of the antibody shown in the first column of each example are also indicated.

In one embodiment, the anti-C4.4a antibody or antigen-binding antibody fragment binds to the S1 region S1 of C4.4a (amino acid position 1-85 of SEQ ID NO: 1).

In one embodiment, the anti-C4.4a antibody or antigen-binding antibody fragment cross-reacts with human C4.4a (SEQ ID NO: 1) and mouse C4.4a (SEQ ID NO: 2).

In one embodiment, the anti-C4.4a antibody or antigen-binding antibody fragment thereof is internalized by the cell after binding to cells expressing C4.4a.

In additional embodiments, the anti-C4.4a antibody or antigen-binding antibody fragment competes with the M31-B01 antibody and/or the M20-D02-S-A antibody and binds to C4.4a. The M31-B01 and M20-D02-S-A anti-system competitions are combined with C4.4a. The B01-1 to B01-12 anti-system was prepared from M31-B01 by affinity maturation and competed with M31-B01 and C4.4a. The D02-1 to D02-13 anti-system was prepared from M20-D02-S-A by affinity maturation and competed with M20-D02-S-A and C4.4a.

In additional embodiments, the anti-C4.4a antibody or antigen-binding antibody fragment comprises at least one, two or three CDR amino acid sequences given in Table 1 or Table 2.

In additional embodiments, the anti-C4.4a antibody or antigen-binding antibody fragment comprises at least one, two or three CDR amino acid sequences of the antibodies given in Table 1 or Table 2.

In an additional embodiment, an anti-C4.4a antibody or antigen binding antibody The fragment comprises at least one, two or three CDR amino acid sequences of the variable heavy chain of the antibody given in Table 1 or Table 2.

In additional embodiments, the anti-C4.4a antibody or antigen-binding antibody fragment comprises at least 50 of the CDR amino acid sequence of the variable light chain and the CDR amino acid sequence of the variable heavy chain of Table 1 or Table 2. %, 60%, 70%, 80%, 90% or 95% consistency.

In additional embodiments, the anti-C4.4a antibody or antigen-binding antibody fragment comprises: CDR sequences SEQ ID NO: 297 (CDR H1), SEQ ID NO: 298 (CDR H2) and SEQ ID NO: 299 (CDR) The heavy chain CDR sequence of H3), and the light chain CDR sequences of CDR sequences SEQ ID NO: 300 (CDR L1), SEQ ID NO: 22 (CDR L2) and SEQ ID NO: 301 (CDR L3), or CDR The heavy chain CDR sequences of SEQ ID NO: 302 (CDR H1), SEQ ID NO: 303 (CDR H2) and SEQ ID NO: 304 (CDR H3), and the CDR sequence SEQ ID NO: 305 (CDR L1), Light chain CDR sequences of SEQ ID NO: 306 (CDR L2) and SEQ ID NO: 307 (CDR L3).

In a further embodiment, the anti-C4.4a antibody or antigen-binding antibody fragment comprises at least 50%, 60%, 70 of the variable light and variable heavy chains of the antibodies given in Table 1 or Table 2. %, 80%, 90% or 95% consistency.

In additional embodiments, the anti-C4.4a antibody or antigen-binding antibody fragment comprises three variable light chain CDRs of the antibodies given in Table 1 or Table 2. An amino acid sequence and three variable heavy chain CDR amino acid sequences.

In additional embodiments, the anti-C4.4a antibody or antigen-binding antibody fragment comprises one of the variable light chains and/or a variable heavy chain of an antibody given in Table 1 or Table 2.

In additional embodiments, the anti-C4.4a antibody or antigen-binding antibody fragment comprises a variable light chain and a variable heavy chain of an antibody given in Table 1 or Table 2.

In a preferred embodiment, the C4.4a antibody and the antigen-binding antibody fragment are selected from the group consisting of the variable heavy chain CDR sequences represented by the sequences of SEQ ID NOs: 75-77, and including SEQ. ID NO: an antibody (B01-10) of the variable light chain CDR sequence represented by the 78-80 sequence, comprising the variable heavy chain CDR sequences represented by the sequences of SEQ ID NOs: 5, 9 and 13, and comprising SEQ ID NO An antibody (M31-B01) of the variable light chain CDR sequences represented by the sequences of 17, 21 and 25, comprising the variable heavy chain CDR sequences represented by the sequences of SEQ ID NOs: 6, 10 and 14, and comprising SEQ ID NO An antibody (M20-D02-SA) of the variable light chain CDR sequences represented by the sequences of 18, 22 and 26, comprising the variable heavy chain CDR sequences represented by the sequences of SEQ ID NOs: 7, 11 and 15, and including SEQ ID NO: an antibody (M60-G03) of the variable light chain CDR sequence represented by the 19, 23 and 27 sequences, comprising the variable heavy CDR sequences represented by the sequences of SEQ ID NOs: 8, 12 and 16, and including SEQ ID NO: an antibody (36-H02) of the variable light chain CDR sequence represented by the 20, 24 and 28 sequences, Included are the variable heavy chain CDR sequences represented by the sequences of SEQ ID NOs: 45-47, and the antibodies (B01-3) comprising the variable light chain CDR sequences represented by the sequence of SEQ ID NO: 48-50, including SEQ ID NO: a variable heavy chain CDR sequence represented by the 55-57 sequence, and an antibody (B01-5) comprising the variable light chain CDR sequence represented by the sequence of SEQ ID NO: 58-60, including SEQ ID NO: 65- a sequence of variable heavy chain CDR sequences, and an antibody (B01-7) comprising the variable light chain CDR sequences represented by the sequence of SEQ ID NO: 68-70, comprising the sequence represented by SEQ ID NO: 85-87 Variable heavy chain CDR sequences, and antibodies (B01-12) comprising the variable light chain CDR sequences represented by the sequences of SEQ ID NOs: 88-90, comprising the variable heavy chain represented by the sequence of SEQ ID NOs: 95-97 a CDR sequence, and an antibody (D02-4) comprising the variable light chain CDR sequence represented by the sequence of SEQ ID NO: 98-100, comprising the variable heavy CDR sequence represented by the sequence of SEQ ID NOs: 105-107, and An antibody (D02-6) comprising the variable light chain CDR sequence represented by the sequence of SEQ ID NO: 108-110, comprising the variable heavy chain CDR sequence represented by the sequence of SEQ ID NO: 115-117, and comprising the SEQ ID NO: 118-120 The antibody (D02-7) of the variable light chain CDR sequence represented by the column, comprising the variable heavy chain CDR sequences represented by the sequence of SEQ ID NO: 125-127, and comprising the sequence represented by SEQ ID NO: 128-130 An antibody that changes the light chain CDR sequence (D02-11), Including a variable heavy CDR sequence represented by the sequence of SEQ ID NO: 135-137, and an antibody (D02-13) comprising the variable light chain CDR sequence represented by the sequence of SEQ ID NO: 138-140, preferably In an embodiment, the anti-C4.4a antibody and the antigen-binding antibody fragment are selected from the group consisting of: an amino acid sequence comprising a variable heavy chain represented by the sequence of SEQ ID NO: 81, and comprising the SEQ ID The antibody (B01-7) of the amino acid sequence of the variable light chain represented by the NO:82 sequence, comprising the amino acid sequence of the variable heavy chain represented by the sequence of SEQ ID NO: 33, and comprising SEQ ID NO: An antibody (M31-B01) comprising the amino acid sequence of the variable light chain of the sequence 29, comprising the amino acid sequence of the variable heavy chain represented by the sequence of SEQ ID NO: 34, and comprising the sequence of SEQ ID NO: An antibody (M20-D02 SA) representing the amino acid sequence of the variable light chain, comprising the amino acid sequence of the variable heavy chain represented by the sequence of SEQ ID NO: 35, and comprising the sequence represented by SEQ ID NO: 31 An antibody (M60-G03) of the amino acid sequence of the variable light chain, comprising the amino acid sequence of the variable heavy chain represented by the sequence of SEQ ID NO: 36, and comprising S EQ ID NO: an antibody (M36-H02) of the amino acid sequence of the variable light chain represented by the sequence of 32, comprising the amino acid sequence of the variable heavy chain represented by the sequence of SEQ ID NO: 51, and including SEQ ID An antibody (B01-3) of the amino acid sequence of the variable light chain represented by the NO: 52 sequence, comprising the amino acid of the variable heavy chain represented by the sequence of SEQ ID NO: 61 a sequence, and an antibody (B01-5) comprising an amino acid sequence of the variable light chain represented by the sequence of SEQ ID NO: 62, comprising the amino acid sequence of the variable heavy chain represented by the sequence of SEQ ID NO: 71, And an antibody (B01-7) comprising an amino acid sequence of the variable light chain represented by the sequence of SEQ ID NO: 72, comprising the amino acid sequence of the variable heavy chain represented by the sequence of SEQ ID NO: 91, and comprising An antibody (B01-12) of the amino acid sequence of the variable light chain represented by the sequence of SEQ ID NO: 92, comprising the amino acid sequence of the variable heavy chain represented by the sequence of SEQ ID NO: 101, and including SEQ ID NO: an antibody (D02-4) of the amino acid sequence of the variable light chain represented by the sequence 102, comprising the amino acid sequence of the variable heavy chain represented by the sequence of SEQ ID NO: 111, and comprising SEQ ID NO An antibody (D02-6) of the amino acid sequence of the variable light chain represented by the sequence 112, comprising the amino acid sequence of the variable heavy chain represented by the sequence of SEQ ID NO: 121, and comprising SEQ ID NO: 122 An antibody (D02-7) comprising the amino acid sequence of the variable light chain of the sequence, comprising the amino acid sequence of the variable heavy chain represented by the sequence of SEQ ID NO: 131, and comprising SEQ I D NO: 132 sequence of an antibody (D02-11) of the amino acid sequence of the variable light chain, comprising the amino acid sequence of the variable heavy chain represented by the sequence of SEQ ID NO: 141, and comprising SEQ ID NO : 142 sequence of the antibody (D02-13) of the amino acid sequence of the variable light chain.

In a further embodiment, the anti-C4.4a anti-system comprises the Light and heavy chains of antibodies.

In a preferred embodiment, the anti-C4.4a anti-system comprises the light and heavy chains of the antibodies given in Table 2.

In a particularly preferred embodiment, the C4.4a anti-system is selected from the group consisting of: an amino acid sequence comprising the light chain represented by the sequence of SEQ ID NO: 346, and comprising the sequence represented by SEQ ID NO: 347 The antibody (M31-B01) of the amino acid sequence of the heavy chain, comprising the amino acid sequence of the light chain represented by the sequence of SEQ ID NO: 352, and the amino group comprising the heavy chain represented by the sequence of SEQ ID NO: 353 The acid sequence antibody (B01-3), comprising the amino acid sequence of the light chain represented by the sequence of SEQ ID NO: 364, and the antibody comprising the amino acid sequence of the heavy chain represented by the sequence of SEQ ID NO: 365 (B01) And 10), and an amino acid sequence comprising the light chain represented by the sequence of SEQ ID NO: 382, and an antibody (D02-6) comprising the amino acid sequence of the heavy chain represented by the sequence of SEQ ID NO: 383.

Anti-C4.4a Antibody IgG:

Another aspect of the invention provides anti-C4.4a antibody IgG comprising the amino acid sequences of the light and heavy chains of the antibodies given in Table 2.

An example of an antibody that binds to the cancer target molecule Her2 is trastuzumab (Genentech). Trastuzumab is a humanized antibody for the treatment of breast cancer (among other diseases). An example of an antibody that binds to the cancer target molecule CD20 is rituximab (Genentech). Rituximab (CAS number: 174722-31-7) is a chimeric antibody for the treatment of non-Hodgkin's lymphoma. An example of an antibody that binds to the cancer target molecule CD52 is alemtuzumab (Genzyme). Alemtuzumab (CAS No.: 216503-57-0) is a humanized antibody for the treatment of chronic lymphocytic leukemia.

Mesothelin antibody

A particularly preferred binding agent in accordance with the invention is an anti-mesothelin antibody, more particularly a human or humanized anti-mesothelin antibody. Preferably, the antibody has an affinity of at least 10 ^-7 M (which is a Kd value; in other words, preferably has a Kd value of less than 10 ^-7 M), preferably at least 10 ^-8 M, more preferably 10 ^-9 M to 10 ^-11 M range. The Kd value can be determined, for example, by surface plasma resonance spectroscopy.

The antibody-drug conjugates of the invention also have affinities within these ranges. This affinity is preferably substantially unaffected by drug binding (generally, the affinity decreases by less than one order, in other words, for example, from 10 ^-8 M to 10 ^-7 M).

The antibodies used in accordance with the invention are preferably known for their high selectivity. When the antibody of the present invention exhibits affinity for a target protein, it is preferably at least 2 times, preferably 5 times or more preferably 10 times higher than other anti-sources unrelated to the pair, such as human serum albumin. Sex (affinity, for example, can be determined by surface electrical resonance spectrum).

Furthermore, the antibodies of the invention used are preferably cross-reactive. In order to facilitate and enhance the clarification of preclinical studies, such as toxicology or activity studies (eg, in xenograft mice), if the antibodies used in the present invention bind not only to human target proteins but also to the species used in the study species Protein binding is advantageous. In one embodiment, an antibody used in the invention, in addition to a human target protein, cross-reacts with a target protein of at least one additional species. For toxicology and activity studies, it is preferred to use rodent families, dogs and non-human primate species. Preferred rodent species are mice and rats. Preferred non-human primate species are rhesus monkeys, chimpanzees, and long-tailed macaques.

In one embodiment, the antibody used in the present invention, in addition to the human target protein, cross-reacts with a target protein of at least one other species selected from the group consisting of: mouse, rat, and long-tailed macaque (Macaca) Fascicularis). Particularly preferred antibodies for use in the present invention, in addition to the human target protein, are at least cross-reactive with the mouse target protein. Preferably, the cross-reactive antibody is administered with a 50-fold, and more preferably no more than 10-fold, difference in affinity for a target protein of another non-human species than for a human target protein.

The antibodies used in the present invention are additionally preferably distinguished by a constant binding to mesothelin. Invariant binding, for example, is due to the fact that the antibodies of the invention used to bind to the epitope of mesothelin are not obscured by additional extracellular proteins. This additional extracellular protein is, for example, the protein ovarian cancer antigen 125 (CA125). Preferably, the antibody used is not attracted by CA125 because it binds to mesothelin.

Anti-mesothelin anti-systems are described, for example, in WO 2009/068204. These antibodies can be used in the present invention.

Another aspect of the invention provides a novel anti-mesothelin antibody (MF-Ta), the amino acid sequence comprising SEQ ID NO: 398 (HCDR1), SEQ ID NO: 399 (HCDR2) and SEQ ID NO: 400 (HCDR3) sequence represents the CDR sequence of the variable heavy chain, and the variable light chain represented by the sequences of SEQ ID NO: 401 (LCDR1), SEQ ID NO: 402 (LCDR2) and SEQ ID NO: 403 (LCDR3) CDR sequence.

In a preferred embodiment, the amino acid sequence of the anti-mesothelin antibody MF-Ta or antigen-binding antibody fragment comprises the sequence of the variable heavy chain represented by the sequence of SEQ ID NO: 404, and SEQ ID NO: The 405 sequence represents the sequence of the variable light chain. In a preferred embodiment, the anti-interval The amino acid sequence of the dermatan antibody MF-Ta or antigen-binding antibody fragment comprises the sequence of the variable heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 406, and the variable encoded by the nucleic acid sequence of SEQ ID NO: 407 The sequence of the light chain.

In a particularly preferred embodiment, the amino acid sequence of the anti-mesothelin antibody MF-Ta comprises a heavy chain sequence represented by the sequence of SEQ ID NO: 408, and a light chain represented by the sequence of SEQ ID NO: 409 sequence.

In a particularly preferred embodiment, the amino acid sequence of the anti-mesothelin antibody MF-Ta comprises a heavy chain sequence encoded by the nucleic acid sequence of SEQ ID NO: 410, and encoded by the nucleic acid sequence of SEQ ID NO: 411 Light chain sequence.

Examples of antibodies that bind to the cancer target molecule mesothelin are known to those skilled in the art and are described, for example, in WO 2009/068204, and can be used in the present invention as a binding agent-drug conjugate.

In an embodiment of a binding agent-drug conjugate, the binding agent is an anti-mesothelin antibody or an antigen-binding antibody fragment, wherein the anti-system binds to mesothelin and has a constant binding.

In an embodiment of a binding agent-drug conjugate, the anti-mesothelin antibody or antigen-binding antibody fragment comprises three light chains of the antibodies described in WOv2009/068204-A1 (Table 7; pages 61-63) The amino acid sequence of the CDR regions, and the amino acid sequence of the three heavy chain CDR regions.

In a preferred embodiment, the anti-mesothelin antibody or antigen-binding antibody fragment is selected from the group consisting of: an amino acid sequence comprising three light chain CDR regions of the MF-Ta antibody, and An anti-mesothelin antibody or antigen-binding antibody fragment thereof of an amino acid sequence of three heavy chain CDR regions, comprising three light chain CDR region sequences of MF-J antibody (WO2009068204-A1; Table 7; page 61), And three heavy chain CDR region sequences of anti-mesothelin antibodies or antigen-binding antibody fragments thereof, including three light chain CDR region sequences of MOR06640 antibody (WO2009/068204-A1; Table 7; page 61), and three Anti-mesothelin antibody or antigen-binding antibody fragment thereof of heavy chain CDR region sequence, including three light chain CDR region sequences of MF-226 antibody (WO2009/068204-A1; Table 7; page 61), and three heavy Anti-mesothelin antibody or antigen-binding antibody fragment thereof having a CDR region sequence, and three light chain CDR region sequences including MOR06626 antibody (WO2009/068204-A1; Table 7; page 61), and three heavy chain CDR sequences A sequence of anti-mesothelin antibodies or antigen-binding antibody fragments thereof.

In a particularly preferred embodiment, the anti-mesothelin antibody or antigen-binding antibody fragment is selected from the group consisting of: a variable light chain sequence comprising a MF-Ta antibody and a variable heavy chain sequence of anti-mesothelin Antibody or antigen-binding antibody fragment thereof, including the variable light chain sequence of MF-J antibody (WO2009/068204-A1; Table 7; page 61) and the variable heavy chain sequence anti-mesothelin antibody or antigen-binding antibody thereof a fragment comprising a variable light chain sequence of the MOR06640 antibody (WO 2009/068204-A1; Table 7; page 61) and a variable heavy chain sequence anti-mesothelin antibody or antigen-binding antibody fragment thereof, Anti-mesothelin antibodies or antigen-binding antibody fragments thereof comprising the variable light chain sequence of the MF-226 antibody (WO 2009/068204-A1; Table 7; page 61) and the variable heavy chain sequence, including MOR06626 (WO2009/068204 -A1; Table 7; page 61) Anti-mesothelin antibodies or antigen-binding antibody fragments thereof, of the variable light chain sequence of the antibody and the variable heavy chain sequence.

Other antibodies:

An example of an antibody that binds to the cancer target molecule Her2 is trastuzumab (Genentech). Trastuzumab is a humanized antibody for the treatment of (especially) breast cancer. An example of an antibody that binds to the cancer target molecule CD20 is rituximab (Genentech). Rituximab (CAS number: 174722-31-7) is a chimeric antibody for the treatment of non-Hodgkin's lymphoma. An example of an antibody that binds to the cancer target molecule CD52 is alemtuzumab (Genzyme). Alemtuzumab (CAS No.: 216503-57-0) is a humanized antibody for the treatment of chronic lymphocytic leukemia.

In addition to trastuzumab (INN 7637, CAS No: RN: 180288-69-1) and pertuzumab (Cas No: 380610-27-5), examples of other antibodies that bind to HER2 are An antibody as disclosed in WO 2009/123894-A2, WO 200/8140603-A2 or WO 2011/044368-A2. An example of an anti-HER2 conjugate is trastuzumab-emtansine (INN No. 9295).

Binds to the cancer target molecule CD30 and can be used to treat cancer, for example Examples of antibodies to Hodgkin's lymphoma include brutuximab, iratumumab, and antibodies disclosed in WO 2008/092117, WO 2008/036688 or WO 2006/089232. An example of an anti-CD30 conjugate is brentuximab vedotine (INN No. 9144).

An example of an antibody that binds to the cancer target molecule CD22 and can be used to treat cancer, lymphoma, is intuzumab or epratuzumab. An example of an anti-CD22 conjugate is intuzumab ozagamycin (INN No. 8574), or anti-CD22-MMAE and anti-CD22-MC-MMAE (CAS RN: 139504-50) -0 and 474645-27-7).

An example of an antibody that binds to the cancer target molecule CD33 and can be used to treat cancer, such as lymphoma, is gemtuzumab or lintuzumab (INN 7580). An example of an anti-CD33 conjugate is gemtuzumab-ozomymid.

An example of an antibody that binds to the cancer target molecule NMB and can be used to treat cancer, such as melanoma or breast cancer, is glembatumumab (INN 9199). An example of an anti-NMB conjugate is glembatumum vedotine (CAS RN: 474645-27-7).

An example of an antibody that binds to the cancer target molecule CD56 and can be used to treat cancer, such as multiple myeloma, small cell lung cancer MCC or ovarian cancer, is lofotuzumab. An example of an anti-CD56 conjugate is lofomuzumab mertansine (CAS) RN: 139504-50-0).

An example of an antibody that binds to the cancer target molecule CD70 and can be used to treat cancer, such as non-Hodgkin's lymphoma or renal cell carcinoma, is disclosed in WO 2007/038637-A2 or WO 2008/070593-A2. An example of an anti-CD70 conjugate is SGN-75 (CD70 MMAF).

An example of an antibody that binds to the cancer target molecule CD74 and can be used to treat cancer, such as multiple myeloma, is milatuzumab. An example of an anti-CD74 conjugate is milatuzumab-doxorubicin (CAS RN: 23214-92-8).

An example of an antibody that binds to the cancer target molecule CD19 and can be used to treat cancer, such as non-Hodgkin's lymphoma, is disclosed in WO 2008/031056-A2. Further examples of antibodies or anti-CD19 conjugates (SAR3419) are disclosed in WO 2008/047242-A2.

An example of an antibody that binds to the cancer target molecule mucin-1 and can be used to treat cancer, such as non-Hodgkin's lymphoma, is clivatuzumab or is disclosed in WO 2003/106495-A2. , WO 2008/028686-A2 antibodies. Examples of anti-mucin conjugates are disclosed in WO 2005/009369-A2.

Examples of antibodies useful for the treatment of cancer, such as multiple myeloma and the cancer target molecule CD138, and conjugates thereof are disclosed in WO 2009/080829-A1, WO 2009/080830-A1.

An example of an antibody that binds to the cancer target molecule integrin αV and can be used to treat cancer, such as myeloma, sarcoma or malignancy, is intetumumab (Cas RN: 725735-28-4), axidan anti- (abciximab) (Cas-RN: 143653-53-6), iriracizumab (Cas-RN: 892553-42-3) or as disclosed in US 7,465,449, EP 19859-A1, WO 2002/012501 -A1 or an antibody in WO 2006/062779-A2. Examples of anti-conjugates are intimuzumab-DM4 and other ADCs disclosed in WO 2007/024536-A2.

Examples of antibodies that bind to the cancer target molecule TDGF1 and are useful in the treatment of cancer are disclosed in WO 02/077033-A1, US 7,318,924, WO 2003/083041-A2, and WO 2002/088170-A2. Examples of anti-TDGF1 conjugates are disclosed in WO 2002/088170-A2.

Examples of antibodies that bind to the cancer target molecule PSMA and which are useful in the treatment of cancer, such as prostate cancer, are disclosed in WO 97/35616-A1, WO 99/47554-A1 and WO 01/009192-A1. Examples of anti-PSMA conjugates are disclosed in WO 2009/026274-A1.

An example of an antibody that can be used in the preparation of conjugates and useful in the treatment of cancer, in combination with the cancer target molecule EPHA2, is disclosed in WO 2004/091375-A2.

An example of an antibody that binds to the cancer target molecule SLC44A4, which can be used to prepare a conjugate and can be used to treat cancer, such as pancreatic cancer or prostate cancer, is disclosed in WO 2009/033094-A2 and US 2009/0175796-A1.

An example of an antibody that binds to the cancer target molecule HLA-DOB is a lym-1 antibody (Cas-RN: 301344-99-0), which is useful for treating cancer, For example, non-Hodgkin's lymphoma. Examples of anti-HLA-DOB conjugates are disclosed, for example, in WO 2005/081711-A2.

An example of an antibody that binds to the cancer target molecule VTCN1, which can be used to prepare a conjugate and can be used to treat cancer, such as ovarian cancer, is disclosed in WO 2006/074418-A2.

The compounds of the present invention have valuable pharmacological properties and are useful in the prevention and treatment of diseases in humans and animals.

The binding agent-drug conjugate (ADC) of the invention of formula (Ia) has a highly and specific cytotoxicity against tumor cells as shown by the analysis presented in the experimental part (C-1. to C-7e.) of the present invention. active. This highly and specific cytotoxic active moiety of the conjugate-drug (ADC) conjugate of the invention of formula (Ia), via a suitable novel N,N-dialkyl auristatin derivative and binding agent, Enzymatic, hydrolytical or reducing cleavage can be achieved by cleavage of a predetermined breaking point for the release of the venom and a combination of linkers without this breaking point. More specifically, by using a non-enzymatic, hydrolytical or reductive cleavable predetermined breakpoint for the release of a stable linker of the venom, and after inhaling the ADC into the tumor cell and in the complete cell of the antibody, the enzyme After sexual lysis, intact or partially intact remains, and this activity is very specifically restricted to tumor cells. The compatibility between ACD and a stable linker is presupposed that the metabolite formed in the cell can form sufficient potency to reach its target and at that point to produce anti-proliferative activity against the target with sufficient potency. It is not necessary to perform tumor cells again with a transporter in advance. Metabolites formed in the cell, after inhalation of the compound of the formula (Ia) of the present invention, exhibit the possibility of reduction to a transporter matrix Sex, thereby inhibiting its redistribution into the systemic circulation, and thus triggering potential side effects of the venom itself. Furthermore, the innovative N-alkyl linkage maintains the basic nature of the amine-based end of the monomethyl-oratinin peptide. Particularly in the case of a binding agent-drug conjugate of the formula (Ia) of the present invention (ADC) linked via an amine acid side chain, the overall antibody charge is still constant, and the number of carrier-ligands carried Nothing.

The compatibility of the ADC with a stable linker chemistry and the indicated target, combined with a metabolite representing a relatively low transporter matrix, increases the therapeutic window.

More specifically, the binding agent-drug conjugate of the formula (Ia) of the present invention has high and specific cytotoxic activity against tumor cells expressing mesothelin. At the same time, the activity of non-expressing mesothelin was significantly weaker.

Based on this characteristic property, in general, the compounds of the invention are therefore particularly suitable for the treatment of hyperproliferative diseases in humans and mammals. This compound inhibits, blocks, reduces or reduces cell proliferation and cell division on the one hand, and increases apoptosis on the other hand.

Hyperproliferative diseases that can be treated using the compounds of the invention include, inter alia, a population of cancers and neoplastic diseases. In the context of the present invention. These should be understood to mean, in particular, but not limited to, breast cancer and breast tumors (in the form of ductal and lobular, also in situ), respiratory tumors (small and non-small cell carcinoma, bronchial carcinoma). , brain tumors (such as tumors of the brainstem and hypothalamus, astrocytoma, medulloblastoma, ependymoma and neuroepithelial neoplasia and pineal tumor), tumors of the digestive organs (esophagus, stomach, gallbladder, Small intestine, large intestine, rectum), liver tumor (including hepatocellular carcinoma, bile duct) Cell carcinoma and mixed hepatocytes and cholangiocarcinoma), tumors in the head and neck (throat, hypopharynx, nasopharynx, oropharynx, lips and mouth), skin tumors (squamous carcinoma, Kaposi's sarcoma, malignant melanin) Tumor, Meckel cell skin cancer and non-melanoma skin cancer), soft tissue tumors (including soft tissue cancer, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma and rhabdomyosarcoma), tumors of the eye (including intraocular melanoma and retina) Tumors of the endocrine and exocrine glands (such as tumors of the thyroid and parathyroid gland, pancreas and salivary glands), tumors of the urinary tract (tumors of the bladder, penis, kidney, renal pelvis and ureter) and tumors of the reproductive organs (female) The tumor of the endometrium, cervix, ovary, vagina, vulva and uterus, and the tumor of the male prostate and testicular). These also include solid forms of proliferative blood diseases and circulating blood cells such as lymphoma, leukemia and myeloproliferative diseases such as acute myeloid, acute lymphoblastic, chronic lymphocytic, chronic granulocyte and cell leukemia. And AIDS-related lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt's lymphoma, and lymphoma of the central nervous system.

Preferred hyperproliferative disease of anti-CA9 binding agent-drug conjugate

The hyperproliferative diseases to which the compounds of the invention are preferably administered are CA9-overexpressing tumors, breast and breast tumors (in the form of ductal and lobular, also in situ), respiratory tumors (small and non-small cell carcinoma) , bronchial carcinoma), including preferably non-small cell lung cancer; brain tumors (eg, tumors of the brainstem and hypothalamus, astrocytoma, medulloblastoma, ependymoma, and/or neuroepithelial neoplasia and pineal Body tumor), digestive organs Tumors (esophagus, stomach, gallbladder, small intestine, large intestine, rectum), including better gastric and small intestine tumors; liver tumors (including hepatocellular carcinoma, cholangiocarcinoma, and mixed hepatocytes and cholangiocarcinoma), head and neck regions Tumors (eg, larynx, hypopharynx, nasopharynx, oropharynx, lips, mouth, tongue, and esophagus), tumors of the urinary tract (tumors of the bladder, penis, kidney, renal pelvis, and ureter), including better kidneys and bladders Tumors; and/or tumors of the reproductive organs (tumors of the female endometrium, cervix, ovaries, vagina, vulva and uterus, and/or tumors of the male prostate and testicles), including better cervix and uterus Tumor.

Preferred hyperproliferative disease of anti-EGFR binding agent-drug conjugate

Preferably, the hyperproliferative diseases to which the compounds of the invention are applied are EGFR-overexpressing tumors, respiratory tumors (small cell and non-small cell carcinoma, bronchial carcinoma), including preferably non-small cell lung cancer; tumors of the digestive organs (esophagus, stomach, gallbladder, small intestine, large intestine, rectum), including especially small intestine tumors; tumors of endocrine and exocrine glands (such as tumors of the thyroid and parathyroid gland, pancreas and salivary glands), including preferred pancreas; head and neck Tumors of the area (eg, throat, hypopharynx, nasopharynx, oropharynx, lips, mouth, tongue, and esophagus); and/or glioma.

Preferred hyperproliferative disease of anti-mesothelin binding agent-drug conjugate

Preferably, the hyperproliferative disease to which the compound of the present invention is applied is a mesothelin-excessive tumor, a tumor of the reproductive organs (a tumor of the female endometrium, the cervix, the ovary, the vagina, the vulva, and the uterus, and/or Or male prostate and testicular tumors, including preferably ovarian cancer; endocrine and exocrine gland tumors (eg thyroid and parathyroid, pancreas Tumors of the visceral and salivary glands, including preferably the pancreas; respiratory tumors (small and non-small cell carcinoma, bronchial carcinoma), including preferably non-small cell lung cancer; and/or mesothelioma.

Preferred hyperproliferative disease of anti-C4.4a binding agent-drug conjugate

Preferably, the hyperproliferative disease to be treated with the compound of the present invention is a tumor or squamous epithelioma with excessive expression of C4.4a (for example, cervical, vulva, vaginal tumor, tumor of the anal canal, endometrium, fallopian tube, penis) , esophageal tumors, breasts, bladder tumors, bile duct tumors, endometrium, uterus and ovaries; breast and breast tumors (milk and lobular forms, also in situ), respiratory tumors (small cells and non-small) Cell carcinoma, bronchial carcinoma, including preferably non-small cell lung cancer, lung squamous cell carcinoma, and lung adenocarcinoma; tumors in the head and neck region (eg, larynx, hypopharynx, nasopharynx, oropharynx, lips, mouth, tongue, and Squamous epithelial cancer of the esophagus, head and neck area; tumor of the urinary tract (tumor, penis, kidney, pelvis and ureteral tumor, squamous cell carcinoma of the bladder), including better tumors of the kidney and bladder; skin tumors (scale Epithelial carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer and non-melanoma skin cancer), including better melanoma; endocrine and exocrine gland tumors (eg thyroid and parathyroid, pancreas) Tumors of the salivary glands, including the preferred pancreas; tumors of the digestive organs (esophagus, stomach, gallbladder, small intestine, large intestine, rectum), including especially colorectal cancer; and/or tumors of the reproductive organs (in female uterus) Membranes of the membrane, cervix, ovary, vagina, vulva and uterus, and/or tumors of the male prostate and test capsules, including better cervical cancer.

The diseases described in these humans can also occur similarly to the cause It can be treated with other compounds and with the compounds of the invention.

In the context of the present invention, the term "treatment" or "treat" encompasses inhibiting, delaying, ameliorating, attenuating, limiting, suppressing, suppressing or curing a condition, a disease, a disease, an injury or a health damage, such symptoms and/or The onset, process, or progression of symptoms such as symptoms. The term "therapy" should be understood to mean the same as "treatment".

The terms "prevention", "prophylaxis", and "prevention" are used synonymously in the context of the present invention and refer to avoiding or reducing the acquisition, experience, experience or illness, illness, disease. , injury or health damage, the risk of occurrence, process or progression of such symptoms and/or symptoms of such symptoms.

The treatment or prevention of a condition, illness, disease, injury or health damage can be partial or complete.

The invention therefore further provides the use of the compounds of the invention for the treatment and/or prophylaxis of diseases, in particular the diseases mentioned above.

The invention further provides the use of a compound of the invention for the preparation of a medicament for the treatment and/or prevention of a disease, in particular a disease as described above.

The invention further provides the use of a compound of the invention in a method of treating and/or preventing a disease, in particular a disease as described above.

The invention further provides a method of using an effective amount of at least one compound of the invention for the treatment and/or prophylaxis of a disease, particularly a disease as described above.

The anti-mesothelin-binding agent-drug conjugate of the present invention is preferably used for treating cancer of a patient, wherein the cancer cell line of the patient to be treated is There is mesothelin performance. More preferably, the treatment is administered to a patient whose cancer cell mesothelin is higher than healthy cells.

A method of identifying a patient having a favorable response to the treatment of an anti-mesothelin-drug conjugate of cancer comprises determining the expression of mesothelin in a cancer cell of a patient. In one embodiment, the mesothelin expression is determined by a mesothelin gene expression assay. Those skilled in the art are aware of methods for gene expression analysis, such as RNA detection, quantification or qualitative polymerase chain reaction or in situ fluorescence hybridization (FISH). In another preferred embodiment, mesothelin expression is determined by immunohistochemistry using an anti-mesothelin antibody. Immunohistochemistry is preferably carried out on formaldehyde-fixed tissue. The anti-system used for immunohistochemistry is the same antibody used in the conjugate. The antibody for immunohistochemistry is a second antibody - preferably specifically identifying the mesothelin target protein.

The compounds of the invention may be used as such or in combination with one or more other pharmacologically active substances as long as the combination does not cause undesirable and unacceptable side effects. The invention thus additionally provides a medicament comprising at least one compound of the invention and one or more additional medicaments, in particular for the treatment and/or prevention of the above mentioned diseases.

For example, the compounds of the invention can be used in combination with known anti-hyperproliferation, cytostatic or cytotoxic substances for the treatment of cancer. Drugs suitable for combination may be mentioned, for example the following: aldesleukin, alendronic acid, alfoferone, litretinoin, allopurinol , aloprim, alooxi, Altretamine, glutethimide, amifostine, amrubicin, amsacrine, anastrozole, anzmet , aranesp, arglabin, arsenic trioxide, aromasin, 5-azacytidine, azathioprine Azathioprine), BCG or tice-BCG, bestatin, betamethasone acetate, betamethasone sodium phosphate, bexarotene, bleomycin sulfate Bleomycin sulphate, broxuridine, bortezomib, busulfan, calcitonin, campath, capecitabine, card Carboplatin, casodex, cefesone, celmoleukin, cerubidin, chlorambucil, cisplatin, clapper Cladribin, clodronic acid, cyclophosphamide, cytarabine (cytar) Abine), dacarbazine, dactinomycin, daunoxome, decadron, decadron phosphate, estradiol valerate Delestrogen), denileukin diftitox, depomedrol, deslorelin, dexrazoxane, diethylstilbestrol, diflucan, dosi It is docetaxel, deoxyfluorouridine (doxifluridine), doxorubicin, dronabinol, DW-166HC, eligard, elitek, ellence, aprepitant (emend ), epirubicin, epoetin-alfa, epogen, eptaplatin, ergamisol, estrace, estradiol, estra Estramustine sodium phosphate, ethinylestradiol, ethyol, etidronic acid, etopophos, etoposide, fadrozole, farstone, filgrastim (filgrastim), finasteride, fligrastim, floxuridine, fluconazole, fludarabine, 5-fluorodeoxyuridine 5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU), fluoxymesterone, flutamide, formestane, fosteabine , fotemustine, fulvestrant, gammagard, gemcitabine, jitrozine (gemtuzumab), gleevec, gliadel, goserelin, granisetron hydrochloride (granisetron hydrochloride), histrelin, hycamtin, hydrogenation Hydrocortone, erythro-hydroxydecyl adenine, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, interferon-α, interference -α-2, interferon-α-2α, interferon-α-2β, interferon-α-n1, interferon-α-n3, stem Interferon-β, interferon-γ-1α, interleukin-2, (intron A, iressa, irinotecan, kytril, lentinan sulfate) Lentinan sulphate), letrozole, leucovorin, leuprolide, leuprolide acetate, levamisole, levofolic acid Calcium salt), levothroid, levoxyl, lomustine, lonidamine, marinol, mechlorethamine, mecobalamin, memcoxyprogesterone Acetate), megestrol acetate, melphalan, menest, 6-mercaptopurine, mesna, methotrexate, metvix, miltefosine Miltefosine), minocycline, mitomycin C, mitotane, mitoxantrone, modrenal, myocet, nedaplatin, neulasta, neumega, neupogen, Nilutamide, nolvadex, NSC-631570, OCT-4 3. Octreotide, ondansetron hydrochloride, onapred, oxaliplatin, paclitaxel, pediapred, pegaspargase, pegasys, whipped Pentostatin, picibanil, pilocarpine hydrochloride (pilocarpine hydrochloride), pirarubicin, plicamycin, porfimer sodium, splash Prednimustine, prednisolone, prednisone (prednisone), premarin, procarbazine, procrit, raltitrexed, ribitoxib (rebif, 铼-186 etidronate, rituximab, roferon -A, romurtide, salagen, sandostatin, sargramostim, semustine, sizofiran, sobuzoxane ), solu-medrol, streptozocin, strontium chloride-89, (synthroid), tamoxifen, tamsulosin, tasonermin , testostatin (tastolactone), taxoter, teceleukin, temozolomide, teniposide, testosterone propionate, testred, thioguanine Thioguanine), thiotepa, thyrotropin, tiludronic acid, topotecan, toremifen, tositumomab , tastuzumab (tastuzumab), teosulfan, tretinoin, trexall, trimethyltrimerization Cyanamide, trimetrexate, triptorelin acetate, triptorelin pamoate, UFT, uridine, valrubicin, Vesnarinone, vinblastine, vincristine, vindesine, vinorelbine, virulizin, zinecard, net statin ester (zinostatin-stimalamer), zofran; ABI-007, Acolbifen, actimmune, (affinitak, aminopterin, arzoxifen, asoprisnil, atamestane, atrasentan , avastin, BAY 43-9006, sorafenib, CCI-779, CDC-501, celebrex, cetuximab, crest (crisnatol), cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dSLIM, dutasteride, autelin (edotecarin), eflornithine, exatecan, fenretinide, histamine dihydrochloride, histamine hydrogel implant (histrelin hydrogel) Implant), 鈥-166 DOTMP, ibandronic acid, interferon-γ, intron-PEG, ixabepilone, keyhole limpet hemocyanine, L-651582, Lanreotide, lasofoxifen, libra, lonafarnib, miproxifen, minodronate, MS-20 9. Microplasty MTP-PE, MX-6, nafarelin, nemorubicin, neovastat, nolatrexed, oblimersen ), onko-TCS, osidem, paclitaxel polyglutamate, pamidronate disodium, PN-401, QS-21, quazepam, R-1549 , raloxifen, ranpirnas, 13-cis-retic Acid, satraplatin, seocalcitol, T-138067, tarceva, taxoprexin, thymosin-α-1, thiazole Tiazofurin, tipifarnib, tirapazamine, TLK-286, toremifen, anti-MID-107R, valspodar, cutting Vaporotide, vatalanib, verteporfin, vinflunin, Z-100, zoledronic acid, and combinations of these.

In a preferred embodiment, the compounds of the invention may be combined with an anti-hyperproliferative agent, for example - this list is not critical, as shown below: Amine Glumet, L-Aspartate, Azin Azathioprine, 5-azacytidine, bleomycin, busulfan, carboplatin, carmustine, phenylbutyric acid Chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, Daunorubicin, diethylstilbestrol, 2',2'-difluorodeoxycytidine, docetaxel, doxorubicin (adriamycin), Epirubicin, epothilone and its derivatives, erythro-hydroxydecyl adenine, ethinyl-estradiol, etoposide, fludarabine phosphate (fludarabin phosphate), 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil, futuron (fluo) Xymesterone), flutamide, hexamethyl melamine, hydroxyurea, hydroxyprogesterone caproate, idarubicin, ifosfamide, interferon, Elano dicken Irinotecan), leucovorin, lomustine, mechlorethamine, medroxyprogesterone acetate, megestrol acetate, melphalan , 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitotane, mitoxantrone, paclitaxel, spray Pentostatin, N -phosphonium L-aspartate (PALA), procamycin, prednisolone, prednisone, procarbazine (prednisone) Procarbazine), raloxifen, semustine, streptozocin, tamoxifen, teniposide, testosterone propionate , thioguanine, thiotepa, topotecan, top three , Uridine (uridine), vinblastine (vinblastine), vincristine (vincristine), vindesine (vindesine), and vinorelbine (vinorelbine).

The compounds of the invention may also be in a very promising manner with biological therapeutic agents such as antibodies (e.g., avastin, rituxan, erbitux, herceptin). The compounds of the invention may also achieve a positive effect on the therapeutic combination of angiogenesis effects, for example Such as avastin (axitinib), centrein, regorafenib, sorafenib or sunitinib. It is particularly suitable in combination with proteasome and inhibitors of mTOR and with anti-hormone and steroid metabolic enzyme inhibitors because of its advantageous side-effect properties.

In general, the combination of a compound of the invention and other agents having cytostatic or cytotoxic effects can achieve the following objectives: ̇ is associated with slowing tumor growth, reducing its size, or even completely abolishing improved activity compared to treatment with individual drugs. ̇ is more likely to use a lower dose of chemotherapeutic agent than a single treatment; ̇ is more tolerant to treatment with fewer side effects than individual administration; ̇ may treat a wider range of neoplastic diseases; High response rate; patients have a longer survival time than current standard treatment.

The compounds of the invention may also be used in combination with radiation therapy and/or surgery.

The invention further provides pharmaceuticals comprising at least one compound of the invention and one or more inert, non-toxic, pharmaceutically acceptable excipients conventionally employed, and the use thereof for the above purposes.

The compounds of the invention may act systemically and/or topically. It may be administered in a manner suitable for this purpose, such as parenteral, possibly by inhalation or as an implant or stent.

The compounds of the invention may be adapted for the administration form of these routes of administration Come to vote.

Parenteral administration can be carried out by absorbing the shunting of the steps (e.g., intravenous, intraarterial, intracardiac, intraspinal or intralumbar) or including absorption (e.g., intramuscular, subcutaneous, intradermal, transdermal, or intraperitoneal). Administration forms suitable for parenteral administration include injection and infusion formulations in the form of solutions, suspensions, emulsions or lyophilizates. Parenteral administration is preferred, especially intravenous administration.

In general, it has proven to be advantageous to administer from about 0.001 to 1 mg/kg body weight, preferably from 0.01 to 0.5 mg/kg body weight, in the case of parenteral administration, in order to achieve an effect.

However, it may be necessary to deviate from the above amounts, and in particular, depending on the body weight, the route of administration, the orientation of the individual to the active compound, the nature of the formulation, and the time or interval of administration. Therefore, in some cases, it is sufficient to be lower than the minimum amount, and in other cases, the upper limit must be exceeded. Where a substantial amount is given, the appropriate system divides these amounts into individual doses throughout the day.

The following examples illustrate the invention. The invention is not limited to these examples.

The percentages in the following tests and examples are calculated as weight percent; the components are parts by weight unless otherwise stated. In each case, the solvent ratio, dilution ratio, and concentration data for the liquid/liquid solution refer to the volume.

A. Example Abbreviations and acronyms:

A431NS human cell line

HPLC and LC-MS methods: Method 1 (LC-MS):

Instrument: Waters Acquity SQD UPLC System; Column: Waters Acquity UPLC HSS T3 1.8μ 50 mm x 1 mm; Eluent A: 1 l water + 0.25 ml 99% concentrated formic acid, dissolving agent B: 1 l acetonitrile + 0.25 ml 99 % concentrated formic acid; gradient: 0.0 min 90% A→1.2 min 5% A→2.0 min 5% A; flow rate: 0.40 ml/min; oven: 50 ° C; UV detection: 210-400 nm.

Method 2 (LC-MS):

Instrument: Micromass QuattroPremier with Waters UPLC Acquity; Column: Thermo Hypersil GOLD 1.9μ 50 mm x 1 mm; Eluent A: 1 l water + 0.5 ml 50% concentrated formic acid, dissolving agent B: 1 l acetonitrile + 0.5 ml 50 % concentrated formic acid; gradient: 0.0 min 90% A→0.1 min 90% A→1.5 min 10% A→2.2 min 10% A; flow Speed: 0.33 ml/min; oven: 50 ° C; UV detection: 210 nm.

Method 3 (LC-MS):

Instrument: Micromass Quattro Micro MS with HPLC Agilent Series 1100; Column: Thermo Hypersil GOLD 3μ 20 mm x 4 mm; Eluent A: 1 l water + 0.5 ml 50% concentrated formic acid, eluent B: 1 l acetonitrile + 0.5 Ml 50% concentrated formic acid; gradient: 0.0 min 100% A→3.0 min 10% A→4.0 min 10% A→4.01 min 100% A (flow rate 2.5 ml/min)→5.00 min 100% A; oven: 50°C; Flow rate: 2 ml/min; UV detection: 210 nm.

Method 4 (LC-MS):

MS instrument: Micromass ZQ; HPLC instrument: HP 1100 Series; UV DAD; column: Phenomenex Gemini 3μ 30 mm x 3.00 mm; dissolving agent A: 1 l water + 0.5 ml 50% concentrated formic acid, dissolving agent B: 1 l acetonitrile +0.5 ml 50% concentrated formic acid; Gradient: 0.0 min 90% A→2.5 min 30% A→3.0 min 5% A→4.5 min 5% A; Flow rate: 0.0 min 1 ml/min→2.5 min/3.0 min/4.5 Min 2 ml/min; oven: 50 ° C; UV detection: 210 nm.

Method 5 (HPLC):

Instrument: HP 1090 Series II; Column: Merck Chromolith SpeedROD RP-18e, 50 mm x 4.6 mm; Prepared column: Merck Chromolith Guard Cartridge set RP-18e, 5 mm x 4.6 mm; Injection volume: 5 μl; Dissolution Agent A: 70% aqueous solution of HClO ₄ (4 ml / liter), dissolving agent B: acetonitrile; gradient: 0.00 min 20% B → 0.50 min 20% B → 3.00 min 90% B → 3.50 min 90% B → 3.51 min 20% B→4.00 min 20% B; flow rate: 5 ml/min; column temperature: 40 °C.

Method 6 (HPLC):

Instrument: Waters 2695 with DAD 996; column: Merck Chromolith SpeedROD RP-18e, 50 mm x 4.6 mm; Ord. No.: 1.51450.0001, preparatory column: Merck Chromolith Guard Cartridge set RP-18e, 5 mm x 4.6 mm; ordering number: 1.51470.0001, dissolving agent A: 70% aqueous solution of HClO ₄ (4 ml / liter), dissolving agent B: acetonitrile; gradient: 0.00 min 5% B → 0.50 min 5% B → 3.00 min 95% B→4.00 min 95% B; flow rate: 5 ml/min.

Method 7 (LC-MS):

MS instrument: Waters ZQ; HPLC instrument: Agilent 1100 Series; UV DAD; column: Thermo Hypersil GOLD 3μ 20 mm x 4 mm; dissolving agent A: 1 l water + 0.5 ml 50% concentrated formic acid, dissolving agent B: 1 l Acetonitrile + 0.5 ml 50% concentrated formic acid; Gradient: 0.0 min 100% A→3.0 min 10% A→4.0 min 10% A→4.1 min 100% A (flow rate 2.5 ml/min); oven: 55 ° C; flow rate: 2 Ml/min; UV detection: 210 nm.

Method 8 (LC-MS):

MS instrument: Waters ZQ; HPLC instrument: Agilent 1100 Series; UV DAD; column: Thermo Hypersil GOLD 3μ 20 mm x 4 mm; dissolving agent A: 1 l water + 0.5 ml 50% concentrated formic acid, dissolved Reagent B: 1 l acetonitrile + 0.5 ml 50% concentrated formic acid; Gradient: 0.0 min 100% A→2.0 min 60% A→2.3 min 40% A→3.0 min 20% A→4.0 min 10% A→4.2 min 100 % A (flow rate 2.5 ml/min); oven: 55 ° C; flow rate: 2 ml/min; UV detection: 210 nm.

Method 9 (LC-MS):

Instrument: Waters Acquity SQD UPLC System; Column: Waters Acquity UPLC HSS T3 1.8μ 50 mm x 1 mm; Eluent A: 1 l water + 0.25 ml 99% concentrated formic acid, dissolving agent B: 1 l acetonitrile + 0.25 ml 99 % concentrated formic acid; Gradient: 0.0 min 95% A→6.0 min 5% A→7.5 min 5% A; oven: 50 ° C; flow rate: 0.35 ml/min; UV detection: 210-400 nm.

Method 10 (HPLC):

Instrument: Agilent 1200 Series; Column: Agilent Eclipse XDB-C18 5μ 4.6 mm x 150 mm; Prepared column: Phenomenex KrudKatcher disposable preparation-column; Injection volume: 5 μl; Eluent A: 1 l water + 0.01 % trifluoroacetic acid; dissolving agent B: 1 l acetonitrile + 0.01% trifluoroacetic acid; gradient: 0.00 min 10% B→1.00 min 10% B→1.50 min 90% B→5.5 min 10% B; flow rate: 2 ml/ Min; column temperature: 30 ° C.

For all reactants or reagents, the preparation thereof is not explicitly described below and is obtained from commercially available sources. For other reactants or reagents, the preparation thereof is likewise not described below, and is not commercially available or obtained from sources that are not generally available, The disclosure of the disclosure in which it is described is incorporated by reference.

Method 11 (LC-MS):

Instrument: Waters ACQUITY SQD UPLC System; Column: Waters Acquity UPLC HSS T3 1.8 μ 30 x 2 mm; Eluent A: 1 l water + 0.25 ml 99% concentrated formic acid, eluent B: 1 l acetonitrile + 0.25 ml 99% Concentrated formic acid; Gradient: 0.0 min 90% A→1.2 min 5% A→2.0 min 5% A oven: 50 ° C; flow rate: 0.60 ml/min; UV detection: 208-400 nm.

Method 12 (HPLC):

Instruments: Agilent 1200 Series with column, oven and DAD; column: Merck Chromolith SpeedROD RP-18e, 50 mm x 4.6 mm; Ord. No.: 1.51450.0001; preparatory column: Merck Chromolith Guard Cartridge set RP -18e, 5 mm x 4.6 mm; Ord. No.: 1.51470.0001; dissolving agent A: 70% aqueous solution of HClO ₄ (4 ml/liter), dissolving agent B: acetonitrile; gradient: 0.00 min 5% B→0.50 Min 5% B→3.00 min 95% B→4.00 min 95% B; flow rate: 5 ml/min; column temperature: 30 °C.

Method 13 (LC-MS):

MS instrument: Waters (Micromass) Quattro Micro; instrument HPLC: Agilent 1100 Series; column: YMC-Triart C18 3 μ 50 x 3 mm; dissolving agent A: 1 l water + 0.01 mol ammonium carbonate, dissolving agent B: 1 l Acetonitrile; Gradient: 0.0 min 100% A→2.75 min 5% A→4.5 min 5% A; oven: 40°C; flow rate: 1.25 ml/min; UV detection: 210 nm.

Starting compounds and intermediates: Starting compound 1 (2 R, 3 R) -3 - [(2 S) -1- ( tert-butoxy-carbonyl) pyrrolidin-2-yl] -3-methoxy-2-methylpropanoic acid (Boc-dolaproine )

The title compound can be prepared in a variety of ways according to literature methods; see, for example, Pettit et al, Synthesis 1996, 719; Shioiri et al, Tetrahedron Lett. 1991, 32 , 931; Shioiri et al, Tetrahedron 1993, 49 , 1913; Koga et al. Man, Tetrahedron Lett. 1991, 32 , 2395; Vidal et al, Tetrahedron 2004, 60 , 9715; Poncet et al, Tetrahedron 1994, 50 , 5345. It can be a free acid or a 1:1 salt with dicyclohexylamine.

Starting compound 2a (3 R , 4 S , 5 S )-3-methoxy-5-methyl-4-(methylamino)heptanoic acid tert-butyl ester hydrochloride (dolaisoleucine-OtBu x HCl)

The title compound can be prepared in a variety of ways according to literature methods; see, for example, Pettit et al, J. Org. Chem. 1994, 59 , 1796; Koga et al, Tetrahedron Lett. 1991, 32 , 2395; Shioiri et al, Tetrahedron Lett .1991, 32 , 931; Shioiri et al., Tetrahedron 1993, 49 , 1913.

Starting compound 2b (3 R , 4 S , 5 S )-3-methoxy-5-methyl-4-(methylamino)heptanoic acid tert-butyl ester (dolaisoleucine-O ^t Bu)

The title compound was prepared analogous to starting compound 2a, but hydrogenation was carried out without the addition of 1N hydrochloric acid.

Starting compound 3 N α-(Tertibutoxycarbonyl) -N -hydroxy-L-phenylpropylamine decylamine

The title compound was prepared by literature methods (A. Ritter et al., J. Org. Chem. 1994, 59 , 4602).

Yield: 750 mg (75% of theory)

LC-MS (Method _{3): R t = 1.67 min} ; MS (ESIpos): m / z = 281 (M + H) +.

Starting compound 4 1,2- Azoleidine hydrochloride

The title compounds were prepared by literature methods (see, for example, H. King, J. Chem. Soc. 1942, 432); they are also commercially available.

Starting compound 5 1,2- Alkane hydrochloride

The title compound was prepared by literature methods (see, for example, H. King, J. Chem. Soc . 1942, 432).

Starting compound 6 2-oxa-3-azabicyclo[2.2.2]oct-5-ene

The title compound can be prepared in a Boc-protected form by literature methods (see, for example, C. Johnson et al., Tetrahedron Lett. 1998, 39 , 2059); deprotection is carried out in a conventional manner by treatment with trifluoroacetic acid and subsequent And come on.

Yield: 149 mg (89% of theory)

Starting compound 7 (1 S , 2 R )-1-(hydroxylaminocarbamido)-2-phenylcyclopropylaminecarboxylic acid tert-butyl ester

The title compound from commercially available (1 S, 2 R) -1 - [( tert-butoxy carbonyl) amino] -2-phenyl-cyclopropanecarboxylic acid to literature procedures (A.Ritter et al., J. Org. Chem. 1994, 59 , 4602) Preparation by treatment (C. Cativiela et al, Chirality 1999, 11, 583).

Yield: 339 mg (59% of theory)

LC-MS (Method _{1): R t = 0.82 min} ; MS (ESIpos): m / z = 293 (M + H) +.

Intermediate 1 (3 R ,4 S ,5 S )-4-[{ N -[(benzyloxy)carbonyl]-L-nonylamine fluorenyl}(methyl)amino]-3-methoxy-5- Tert-butyl heptanoate

10.65 g (41.058 mmol) of (3 R , 4 S , 5 S )-3-methoxy-5-methyl-4-(methylamino)heptanoic acid tert-butyl ester (starting compound 2b) Treat with 250 ml of dichloromethane and cool the solution to -10 °C. Then, 10.317 g (41.058 mmol) of N-[(benzyloxy)carbonyl]-L-proline and 16.866 g (61.586 mmol) of 2-bromo-1-ethyltetrafluoroborate pyridinium were added with stirring ( BEP) and 28.6 ml of N,N -diisopropylethylamine, and the mixture was stirred at RT for 20 h. The reaction mixture was then diluted with dichloromethane and EtOAc (EtOAc)EtOAc The residue was purified by flash chromatography on silica gel eluting with 4:1 petroleum ether/ethyl acetate as solvent. The corresponding dissolved fractions were concentrated and the residue was dried under high vacuum overnight. The title compound was obtained as a light yellow oil.

HPLC (Method 5): R _t =2.3 min; LC-MS (Method 2): R _t = 1.59 min; MS (ESI): m/z = 493 (M+H) ⁺ .

Intermediate 2 (3 R , 4 S , 5 S )-3-methoxy-5-methyl-4-[methyl(L-decylguanidino)amino]heptanoic acid tert-butyl ester

500 mg (1 mmol) of (3 R , 4 S , 5 S )-4-[{ N -[(benzyloxy)carbonyl]-L-nonylamine decyl}(methyl)amino]-3 -Methoxy-5-methylheptanoic acid tert-butyl ester (Intermediate 1) is dissolved in 50 ml of methanol, and after adding 100 mg of 10% activated carbon on palladium, hydrogenation is carried out under standard hydrogen pressure and RT. 1 h. The catalyst was then filtered off and the solvent was removed under reduced pressure. 370 mg (quant.) of the title compound are obtained as a colourless oil.

HPLC (method 5): R _t = 1.59 min; LC-MS (method 1): R _t = 0.74 min; MS (ESIs): m/z = 359 (M+H) ⁺ .

Intermediate 3 N -[(9 H -茀-9-ylmethoxy)carbonyl] -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1- third -3-methoxy-5-methyl-1-oxo-side heptan-4-yl] - N - methyl amine Amides -L- valine

The 4.64 g (13.13 mmol) of N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valine dissolved in 20 ml of DMF and successively with 4.28 g (11.94 (3 R , 4 S , 5 S )-3-methoxy-5-methyl-4-[methyl(L-decylguanidino)amino]heptanoic acid tert-butyl ester (intermediate) 2), 2.75 g (14.33 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2.2 g (14.33 mmol) of 1-hydroxy-1 H - The benzotriazole hydrate is mixed. The mixture was stirred at RT until overnight. The reaction mixture was then poured into a mixture of a half saturated aqueous ammonium chloride solution and ethyl acetate. The organic layer was removed, washed successively with a saturated aqueous solution of sodium hydrogen carbonate and saturated sodium chloride, dried over magnesium sulfate, filtered and concentrated. The residue was used directly in the next step without further purification.

Yield: 9.1 g (quantitative, 60% pure)

HPLC (Method 5): R _t = 2.7 min; LC-MS (Method 2): R _t = 1.99 min; MS (ESIs): m/z = 694 (M+H) ⁺ .

Intermediate 4 N -[(9 H -茀-9-ylmethoxy)carbonyl] -N -methyl-L-nonylamine fluorenyl- N -[(2 R ,3 S ,4 S )-1-carboxy-2 -methoxy-4-methylhex-3-yl] -N -methyl-L-nonylamine decylamine

The crude product was 9.1 g of N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N - [(3 R, 4 S, 5 S) -1-Tertioxy-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine amide (Intermediate 3) was placed at 56.6 Treated with ml of dichloromethane, 56.6 ml of trifluoroacetic acid was added, and the mixture was stirred at RT for 2 h. Subsequently, the reaction mixture was concentrated under reduced pressure and the residue was purifiedjjjjjjjjjjjj Methanol was purified as a dissolving agent. After purification and concentration of the corresponding fractions, 5.8 g (yield: 86%)

HPLC (Method 5): R _t = 2.2 min; LC-MS (Method 1): R _t = 1.3 min; MS (ESIs): m/z = 638 (M+H) ⁺ .

Intermediate 5 (2 S )-1-(1,2- Alkyl-2-yl)-1-oxo-oxo-3-phenylpropan-2-ylaminecarboxylic acid tert-butyl ester

500 mg (1.9 mmol) of N- (t-butoxycarbonyl)-L-phenylalanine was dissolved in 10 ml of DMF and continuously with 466 mg (3.8 mmol) of 1,2- Alkyl hydrochloride (starting compound 5), 433 mg (2.3 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 382 mg (2.8 mmol) The 1-hydroxy-1 H -benzotriazole hydrate was mixed with 731 mg (5.7 mmol) of N,N -diisopropylethylamine. The mixture was stirred at RT until overnight. The reaction mixture was then poured into a mixture of a half-saturated aqueous solution of ammonium chloride and ethyl acetate. The organic layer was removed, washed successively with saturated aqueous sodium hydrogen carbonate and saturated sodium chloride, dried over magnesium sulfate, filtered and concentrated. 620 mg (98% of theory) of title compound.

HPLC (Method 5): R _t = 1.8 min; LC-MS (Method 2): R _t = 1.62 min; MS (ESI pos): m/z = 235 (MC ₄ H ₈ -CO ₂ + H) ⁺ .

Intermediate 6 (2 S )-2-amino-1-(1,2- Alkan-2-yl)-3-phenylpropan-1-one trifluoroacetate

620 mg (1.85 mmol) of (2 S )-1-(1,2- Alkyl-2-yl)-1-oxo-3-phenylpropan-2-ylaminecarboxylic acid tert-butyl ester (Intermediate 5) was treated with 5 ml of dichloromethane and 10 ml of trifluoroacetic acid was added. The mixture was stirred at RT for 30 min. The reaction mixture was concentrated under reduced pressure and the residual residue was lyophilized from water / acetonitrile. In this way, 779 mg (91% of theory) of title compound was obtained as colorless foam.

HPLC (Method 5): R _t = 0.45 min; LC-MS (Method 3): R _t = 1.09 min; MS (ESIs): m/z = 235 (M+H) ⁺ .

Intermediate 7 (2 R ,3 R )-3-methoxy-2-methyl- N -[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]-3-[(2 S )-pyrrolidin-2-yl]propanamide trifluoroacetate

360 mg (1.25 mmol) of (2 R , 3 R )-3-[(2 S )-1-( t- butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2- Methylpropionic acid (starting compound 1) was treated in 10 ml of DMF and continuously with 579.2 mg (1.25 mmol) of ( 2S )-2-amino-1-(1,2- Alkan-2-yl)-3-phenylpropan-1-one trifluoroacetate (intermediate 6), 714.5 mg (1.88 mmol) of O- (7-azabenzotriazol-1-yl)- N,N,N',N' -tetramethylhexafluorophosphate (HATU) and 655 μl of N,N -diisopropylethylamine. The mixture was stirred at RT for 16 h. The reaction mixture was then concentrated and the residue was taken in ethyl acetate eluting with EtOAc EtOAc EtOAc. The organic layer was concentrated and the residue was purified by flash chromatography eluting eluting The corresponding dissolved fractions were combined and the solvent was removed under reduced pressure. After drying the residue in vacuo, 503.5 mg (yield: 74%) of Boc-protected intermediate ( 2S )-2-[(1 R ,2 R )-1-methoxy-2-methyl- 3-{[(2 S )-1-(1,2- Alkyl-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidine-1-carboxyl tert-butyl ester.

HPLC (Method 12): R _t = 2.0 min; LC-MS (Method 1): R _t = 1.12 min; MS (ESIs): m/z = 504 (M+H) ⁺ .

503 mg (1 mmol) of this intermediate was taken in 20 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated under reduced pressure and diluted with dichloromethane. The remaining residue was precipitated from ethyl acetate as n-propane and the solvent was decanted. The residue obtained was dissolved in water and extracted with shaking with ethyl acetate, and then the aqueous layer was lyophilized. In this way, 462 mg (89% of theory) of title compound was obtained as colorless foam.

HPLC (Method 12): R _t = 1.53 min; LC-MS (Method 11): R _t = 0.57 min; MS (ESIs): m/z = 404 (M+H) ⁺ .

Intermediate 8 N-(Tertibutoxycarbonyl)-N-methyl-L-decylamine-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexyl- 3-yl]-N-methyl-L-amidamine

The 51 mg (0.08 mmol) of N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N - [(2 R, 3 S, 4 S )-1-carboxy-2-methoxy-4-methylhex-3-yl] -N -methyl-L-decylamine (Intermediate 4) is dissolved in 10 ml of DMF and added to 0.5 Methyl piperidine. After stirring at RT for 10 min, the~~~~~~~~~~ The insoluble fractions were filtered off and washed repeatedly with diethyl ether. Then place the filter residue in 5 ml of two Treat in alkane/water and adjust the solution to pH 11 with 1N sodium hydroxide solution. After treatment under ultrasonic waves, a total of 349 mg (1.6 mmol) of di-tert-butyl dicarbonate was added in several portions, during which the pH of the solution was maintained at 11. After the reaction is over, evaporate two The alkane was adjusted to pH 2-3 with citric acid. The mixture was extracted twice with 50 ml of ethyl acetate. The organic layers were combined, dried over magnesium sulfate and evaporated. The residue was taken up in diethyl ether. The solvent was removed by decantation. The residue was re-digested several times with pentane and finally dried under high vacuum. This gave 40 mg (97% of theory) of the title compound.

HPLC (Method 6): R _t = 2.2 min; LC-MS (Method 2): R _t = 1.32 min; MS (ESI): m/z = 516 (M+H) ⁺ .

Intermediate 9 (2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidine-1-carboxylic acid tert-butyl ester

The title compound is similar to the synthesis of intermediates 5, 6 and 7 by the commercially available (1 S , 2 R )-1-[(t-butoxycarbonyl)amino]-2-phenyl group in three stages. Cyclopropanecarboxylic acid with 1,2- The alkanoic acid salt (starting compound 5) was coupled, followed by deprotection with trifluoroacetic acid and coupling with starting compound 1. The final product was purified by preparative HPLC.

HPLC (Method 5): R _t = 2.12 min; LC-MS (Method 2): R _t = 1.25 min; MS (ESIs): m/z = 516 (M+H) ⁺ .

Intermediate 10 N -[(9 H -茀-9-ylmethoxy)carbonyl]- N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxanthene 4-yl] -N -methyl-L-amidamine

The 315 mg (0.494 mmol) of N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N - [(2 R, 3 S, 4 S )-1-carboxy-2-methoxy-4-methylhex-3-yl] -N -methyl-L-decylamine (Intermediate 4) is dissolved in 12 ml of DMF with 104 mg (0.543 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 83 mg (0.543 mmol) of 1-hydroxy-1 H -benzotriazole hydrate The mixture was mixed and the mixture was stirred at RT for 90 min. Subsequently, 112 μl of N,N -diisopropylethylamine and 149 mg (0.494 mmol) (2 R , 3 R ) previously prepared by removing the protecting group from the starting compound 1 by trifluoroacetic acid were added. 3-methoxy-2-methyl-3-[( 2S )-pyrrolidin-2-yl]propionic acid trifluoroacetate. The mixture was stirred at RT for 2 h and then concentrated under high vacuum. The remaining residue was purified twice by preparative HPLC. The title compound was obtained as a colorless foam in the form of 140 mg (35% of theory).

HPLC (Method 5): R _t = 2.40 min; LC-MS (Method 1): R _t = 1.38 min; MS (ESIs): m/z = 807 (M+H) ⁺ .

Intermediate 11 N -[(benzyloxy)carbonyl] -N -methyl-L-threonyl- N -[(2 R ,3 S ,4 S )-1-carboxy-2-methoxy-4-methyl Hexyl-3-yl] -N -methyl-L-nonylamine amide

First, N -[(benzyloxy)carbonyl] -N -methyl-L-threonine was extracted from its 237 mg (0.887 mmol) by treatment with ethyl acetate and shaking extraction with 5% sulfuric acid solution. Released from the cyclohexylamine salt. The organic layer was dried with MgSO4, filtered and evaporated. The residue was taken up in 16 ml of DMF and continuously with 365 mg (1 mmol) of ( 3R , 4S , 5S )-3-methoxy-5-methyl-4-[methyl (L- Amidoxime)amino]heptanoic acid tert-butyl ester (Intermediate 2), 185 mg (0.967 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt The acid salt was mixed with 148 mg (0.967 mmol) of 1-hydroxy-1 H -benzotriazole hydrate. The mixture was stirred at RT for 2 h. The reaction mixture was then poured into a mixture of a half saturated aqueous ammonium chloride solution and ethyl acetate. The organic layer was removed, washed successively with saturated aqueous sodium bicarbonate and saturated sodium chloride, dried over magnesium sulfate, filtered and concentrated. The residue was purified by preparative HPLC. Thus 283 mg (53% of theory) of the third butyl ester intermediate N -[(benzyloxy)carbonyl] -N -methyl-L-threonyl- N -[(3 R ,4) S , 5 S )-1-tert-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine.

HPLC (Method 5): R _t = 2.17 min.

283 mg (0.466 mmol) of this intermediate was taken in 5 ml of dichloromethane, 5 ml of anhydrous trifluoroacetic acid was added and the mixture was stirred at RT for 2 h. Subsequently, the reaction mixture was concentrated under high vacuum and the residue was purified by preparative HPLC. This gave 156 mg (61% of theory) of title compound as colorless foam.

HPLC (method 5): R _t = 1.50 min; LC-MS (Method 2): R _t = 1.09 min; MS (ESI): m/z = 552 (M+H) ⁺ .

Intermediate 12 N -{(2 R ,3 R )-3-methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propenyl}-L-phenylalanine benzyl ester III Fluoroacetate

In the first step, 600 mg (1.28 mmol) of the corresponding dicyclohexylammonium salt is dissolved in 100 ml of ethyl acetate and then 50 ml of 0.5% sulfuric acid is then used to extract the shock with a saturated sodium chloride solution. The starting compound is released from this salt. The organic layer was then dried over magnesium sulfate, filtered, concentrated and then reacted with benzyl phenylalanamine as a similar intermediate 7 and then deprotected.

Yield: 650 mg (94% in 2 stages)

HPLC (Method 6): R _t = 1.76 min; LC-MS (Method 2): R _t = 1.68 min; MS (ESIs): m/z = 425 (M+H) ⁺ .

Intermediate 13 (β S )- N -{(2 R ,3 R )-3-methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propanyl}-β- benzyl-L-phenylalanine benzyl ester trifluoroacetate

First, (2 R , 3 R )-3-[(2 S )-1-(t-butoxycarbonyl)pyrrolidine--by treatment with ethyl acetate and extraction with a 5% aqueous solution of potassium hydrogensulfate. 2-Methoxy]-3-methoxy-2-methylpropanoic acid was released from its 351 mg (0.75 mmol) dicyclohexylamine salt (starting compound 1). The organic layer was dried with MgSO4, filtered and evaporated. The residue was taken up in 10 ml of DMF is treated continuously with 373 mg (0.75 mmol) of (β S) -β- methyl -L- phenylalanine benzyl ester trifluoroacetate [prepared from commercially available (β S ) - N - (tert-butoxy carbonyl) -L- [beta phenyl-alanine methyl esterification to EDC / DMAP- media and benzyl alcohol, followed by separation of the Boc protecting group with trifluoroacetic acid Preparation], 428 mg (1.125 mmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate (HATU) was mixed with 392 μl of N,N -diisopropylethylamine. The mixture was stirred at RT for 20 h. The reaction mixture was then poured into a mixture of a half saturated aqueous ammonium chloride solution and ethyl acetate. The organic layer was removed, washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, and then concentrated. The residue was purified by preparative HPLC. This gave 230 mg (57% of theory) of Boc-protected intermediate (β S )- N -{(2 R ,3 R )-3-[(2 S )-1-(t-butoxy) Carbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanyl}-β-methyl-L-phenylalanine benzyl ester.

HPLC (Method 6): R _t =2.3 min; LC-MS (Method 1): R _t = 1.36 min; MS (ESIs): m/z = 539 (M+H) ⁺ .

230 mg (0.42 mmol) of this intermediate was taken up in 5 ml of dichloromethane, 5 ml of trifluoroacetic acid was added, and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated under reduced pressure. The remaining residue was further dried under reduced pressure and then lyophilized with acetonitrile / water. In this way, 230 mg (quant.) of the title compound was obtained.

HPLC (Method 6): R _t = 1.6 min.

Intermediate 14 N -methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R ) 1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-decylamine guanamine trifluoroacetate

The 143 mg (0.223 mmol) of N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N - [(2 R, 3 S, 4 S )-1-carboxy-2-methoxy-4-methylhex-3-yl] -N -methyl-L-decylamine (Intermediate 4) was treated in 15 ml of DMF and Continuous with 141 mg (0.22 mmol) of (2 R , 3 R )-3-methoxy-2-methyl- N -[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]-3-[( 2S )-pyrrolidin-2-yl]propanamide trifluoroacetate (intermediate 7) , 102 mg (0.27 mmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethyltetramethylhexafluorophosphate (HATU) was mixed with 128 μl (0.74 mmol) of N,N -diisopropylethylamine. The mixture was stirred at RT for 3 h. The mixture was then poured into a mixture of a half saturated aqueous ammonium chloride solution and ethyl acetate. The organic layer was removed, washed successively with saturated aqueous sodium bicarbonate and saturated sodium chloride, dried over magnesium sulfate, filtered and concentrated. Thereby obtaining 275 mg (quant.) Fmoc- protected intermediate N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N - [( 3 R , 4 S , 5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[ (2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-decylamine.

HPLC (method 5): R _t = 2.73 min; LC-MS (method 4): R _t = 3.19 min; MS (ESI pos): m/z = </RTI> (M+H) ⁺ .

46 mg (0.045 mmol) of this intermediate was dissolved in 4 ml of DMF. After addition of 1 ml of piperidine, the reaction mixture was stirred at RT for 1 h. Subsequently, the reaction mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC (solvent: acetonitrile: &lt;RTI ID=0.0&gt;&gt; The title compound was obtained as a colorless foam.

HPLC (Method 5): R _t = 1.68 min; LC-MS (Method 2): R _t = 1.03 min; MS (ESIs): m/z = 801 (M+H) ⁺

¹ H NMR (600 MHz, DMSO-d ₆ ): δ = 8.8 (m, 2H), 8.7 (m, 1H), 8.42 and 8.15 (2d, 1H), 7.3-7.1 (m, 5H), 5.12 and 4.95 (2m, 1H), 4.70 and 4.62 (2m, 1H), 4.62 and 4.50 (2t, 1H), 4.1-3.9 (m, 3H), 3.85 (m, 1H), 3.75-3.6 (m, 2H), 3.23 , 3.18, 3.17, 3.14, 3.02 and 2.96 (6s, 9H), 3.1-2.9 and 2.75 (2m, 2H), 2.46 (m, 3H), 2.4-2.1 (m, 2H), 2.05 (br.m, 2H) ), 1.85-1.55 (br.m, 6H), 1.5-1.2 (br.m, 3H), 1.1-0.8 (m, 18H), 0.75 (t, 3H) [More signals are hidden in the H ₂ O peak under].

Intermediate 15 N -methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R ) 1-methoxy-2-methyl-3-{[(2 S ,3 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-decylamine guanamine trifluoroacetate

The 126 mg (0.198 mmol) of N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N - [(2 R, 3 S, 4 S )-1-carboxy-2-methoxy-4-methylhex-3-yl] -N -methyl-L-decylamine (Intermediate 4) was treated in 10 ml of DMF and continued And 105 mg (0.198 mmol) of (2 R , 3 R )-3-methoxy-2-methyl- N -[(2 S ,3 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylbutan-2-yl]-3-[( 2S )-pyrrolidin-2-yl]propanamide trifluoroacetate (intermediate 17) 41.6 mg (0.217 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 33 mg (0.217 mmol) of 1-hydroxy-1 H -benzo The triazole hydrate was mixed with 79 μl (0.454 mmol) of N,N -diisopropylethylamine. The mixture was stirred at RT until overnight. The reaction mixture was then poured into a mixture of a half saturated aqueous ammonium chloride solution and ethyl acetate. The organic layer was removed, washed successively with saturated aqueous sodium bicarbonate and saturated sodium chloride, dried over magnesium sulfate, filtered and concentrated. Thereby obtaining 220 mg (quant.) Fmoc- protected intermediate N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N - [( 3 R , 4 S , 5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[ (2 S ,3 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-decylamine.

HPLC (Method 5): R _t = 2.77 min; LC-MS (Method 1): R _t = 1.5 min; MS (ESI s): m/z = 1037 (M+H) ⁺ .

220 mg (0.212 mmol) of this intermediate was dissolved in 5 ml of DMF. After adding 1 ml of piperidine, the reaction mixture was stirred at RT for 1 h. Subsequently, the reaction mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC. (Dissolving agent: acetonitrile + 0.01% TFA / water + 0.01% TFA). The title compound was obtained as a colorless foam.

HPLC (method 5): R _t = 1.71 min; LC-MS (method 1): R _t = 0.9 min; MS (ESI pos): m/z = 815 (M+H) ⁺

¹ H NMR (600 MHz, DMSO-d ₆ ): δ = 8.87 and 8.80 (2d, 2H), 8.75 (m, 1H), 8.40 and 7.98 (2d, 1H), 7.3-7.1 (m, 5H), 5.45 And 5.2 (2t, 1H), 4.78 and 4.62 (2m, 1H), 4.73 and 4.58 (2t, 1H), 4.2-4.0 (m, 3H), 3.7-3.6 (m, 1H), 3.35, 3.20, 3.18, 3.14, 3.12 and 3.00 (6s, 9H), 3.1 and 2.95 (2m, 2H), 2.46 (m, 3H), 2.4-2.0 (m, 4H), 1.9-1.6 (m, 4H), 1.6-1.2 (m) , 5H), 1.1-0.75 (m, 21H), 0.80 (t, 3H) [more signals are hidden under the H ₂ O peak].

Intermediate 16 N -methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R ) 1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-Amidoxime trifluoroacetate

617 mg (1.2 mmol) of (2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-( 1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidine-1-carboxylic acid tert-butyl ester (intermediate 24) was placed in 44 ml of dichloro Treated in methane, 4.4 ml of trifluoroacetic acid was added and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated under reduced pressure and the remaining residue was taken from The alkane/water is freeze dried. 702 mg (quant.) deprotected compound (2 R , 3 R )-3-methoxy-2-methyl- N -[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]-3-[( 2S )-pyrrolidin-2-yl]propanamide trifluoroacetate as crude product, which is used in the next stage No further purification.

The 470 mg (0.74 mmol) of N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N - [(2 R, 3 S, 4 S )-1-carboxy-2-methoxy-4-methylhex-3-yl] -N -methyl-L-decylamine (Intermediate 4) was treated in 57 ml of DMF and continued With 390 mg (about 0.74 mmol) of the above (2 R , 3 R )-3-methoxy-2-methyl- N -[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]-3-[( 2S )-pyrrolidin-2-yl]propanamide trifluoroacetate, 336 mg (0.88 mmol) of O - ( 7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate (HATU) was mixed with 423 μl (2.4 mmol) of N,N -diisopropylethylamine. The mixture was stirred at RT for 2 h. The reaction mixture was then poured into a mixture of a half saturated aqueous ammonium chloride solution and ethyl acetate. The organic layer was removed, washed successively with saturated aqueous sodium bicarbonate and saturated sodium chloride, dried over magnesium sulfate, filtered and concentrated. The residue was purified by preparative HPLC. Thereby obtaining 453 mg (59% of theory) of the Fmoc-protected intermediate N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3 -{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-decylamine.

HPLC (method _{5): R t = 2.58 min} ; LC-MS ( Method _{1): R t = 3.10 min} ; MS (ESIpos): m / z = 1035 (M + H) +.

453 mg (0.438 mmol) of this intermediate was dissolved in 24 ml of DMF. After adding 2.4 ml of piperidine, the reaction mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC (solvent: acetonitrile / 0.1% TFA in water). The title compound was obtained as 260 mg (64% of theory).

HPLC (method 5): R _t = 1.64 min; LC-MS (method 1): R _t = 0.86 min; MS (ESI): m/z = 813 (M+H) ⁺

¹ H NMR (400 MHz, DMSO-d ₆ ): δ = 8.8 (m, 2H), 8.65 (m, 2H), 7.3-7.1 (m, 5H), 4.8-4.05 (m, 2H), 4.0 and 3.82 (2m, 2H), 3.8-3.5 (m, 8H), 3.32, 3.29, 3.20, 3.19, 3.12 and 3.00 (6s, 9H), 2.65 (t, 1H), 2.5-2.45 (m, 3H), 2.4- 1.3 (m, 15H), 1.15-0.85 (m, 18H), 0.8 and 0.75 (2d, 3H) [more signals are hidden under the H ₂ O peak].

Intermediate 17 N -benzyl- N -methyl-L-phenylpropylamine decylamine trifluoroacetate

1000 mg (3.77 mmol) of N- (t-butoxycarbonyl)-L-phenylalanine was dissolved in 10 ml of DMF and with 457 mg (3.77 mmol) of N -methylbenzylamine, 2150 mg (5.65 mmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate It was mixed with 657 μl of N,N -diisopropylethylamine. The reaction mixture was stirred at RT for 30 min then concentrated EtOAc. The residue was taken up in dichloromethane and extracted three times with water. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash chromatography on silica gel eluting with 3:1 petroleum ether/ethyl acetate as solvent. The product was concentrated and the residue was dried under high vacuum. Thus, 1110 mg (75% of theory) of the Boc-protected intermediate N -benzyl- N[ ^alpha] -(t-butoxycarbonyl) -N -methyl-L-phenylpropylamine decylamine was obtained.

HPLC (method 6): _Rt = 2.1 min; LC-MS (Method 1): R _t = 1.14 min; MS (ESIs): m/z = 369 (M+H) ⁺ .

1108 mg (3,007 mmol) of this intermediate was taken up in 30 ml of dichloromethane, 10 ml of trifluoroacetic acid was added and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated under reduced pressure. The residue was stirred twice more with pentane, and the solvent was evaporated, and then the title compound was dried under high vacuum. Thus, 1075 mg (93% of theory) of the title compound was obtained as a resin.

HPLC (Method 6): R _t = 1.6 min; LC-MS (Method 1): R _t = 0.6 min; MS (ESIs): m/z = 269 (M+H) ⁺ .

Intermediate 18 N -benzyl- N ^α -{(2 R ,3 R )-3-methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propanyl}- N - methyl-L-phenylpropylamine guanamine trifluoroacetate

First, (2 R , 3 R )-3-[(2 S )-1-(t-butoxycarbonyl)pyrrolidine-2- was treated by ethyl acetate treatment and shaking extraction with 5% aqueous sulfuric acid. 3-methoxy-2-methylpropanoic acid (starting compound 1) was released from its 141 mg (0.491 mmol) dicyclohexylamine salt. The organic layer was dried over magnesium sulfate, filtered and concentrated. The residue was taken up in 10 mL of DMF and 187.6 mg (0.49 mmol) of N -benzyl- N -methyl-L-phenyl propylamine decylamine trifluoroacetate (Intermediate 9), 190.3 mg (1.47) mmol) of O - (7- aza-benzotriazol-1-yl) - N, N, N ' , N' - tetramethyl hexafluorophosphate (HATU) and 256 μl of N,N -diisopropylethylamine. The mixture was stirred at RT for 1 h. The reaction mixture was then concentrated and the residue was taken in ethyl acetate and then filtered and evaporated th The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash chromatography on silica gel eluting with 30:1 acetonitrile/water as solvent. The product was concentrated and the residue was dried under high vacuum. This gave 168 mg (64% of theory) of Boc-protected intermediate ( 2S )-2-[(1 R ,2 R )-3-({( 2S )-1-[benzyl (A Amino]-1-yloxy-3-phenylpropan-2-yl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1-carboxylic acid Third butyl ester.

HPLC (method 6): _Rt = 2.2 min; LC-MS (Method 2): R _t = 1.22 min; MS (ESIs): m/z = 538 (M+H) ⁺ .

168 mg (0.312 mmol) of this intermediate was taken in 15 ml of dichloromethane, 3 ml of trifluoroacetic acid was added and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated under reduced pressure. The remaining residue was stirred with dichloromethane and then stirred with diethyl ether. After drying under high vacuum, 170 mg (yield: 99%) of title compound was obtained as a resin.

HPLC (Method 6): R _t = 1.7 min; LC-MS (Method 1): R _t = 0.73 min; MS (ESIs): m/z = 438 (M+H) ⁺ .

Intermediate 19 N -{(2 R ,3 R )-3-methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propanyl}-L-phenylalanine methyl ester trifluoro Acetate

The title compound is synthesized analogous to the intermediate 18 to give ( 2R , 3R )-3-[( 2S )-1-(t-butoxycarbonyl)pyrrolidine which is liberated from the dicyclohexylamine salt. Prepared by 2-yl]-3-methoxy-2-methylpropanoic acid (starting compound 1) and L-phenylalanine methyl ester hydrochloride.

HPLC (Method 5): R _t = 0.6 min; LC-MS (Method 3): R _t = 1.17 min; MS (ESI): m/z = 349 (M+H) ⁺ .

Intermediate 20 N -{(2 R ,3 R )-3-methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propanyl}-L-tryptophan benzyl ester Trifluoroacetate

The title compound is similar to the synthesis of intermediate 18, ( 2R , 3R )-3-[( 2S )-1-(t-butoxycarbonyl)pyrrolidin-2 which is liberated from dicyclohexylamine salt. -Methoxy-3-methyl-2-methylpropanoic acid (starting compound 1) and L-tryptophan benzyl ester were prepared initially.

HPLC (Method 6): R _t = 2.0 min; LC-MS (Method 1): R _t = 0.8 min; MS (ESI): m/z = 464 (M+H) ⁺ .

Intermediate 21 (1 S , 2 R )-1-({(2 R ,3 R )-3-methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propanyl} Amino)-2-phenylcyclopropanecarboxylic acid benzyl ester trifluoroacetate

The title compound is similar to the synthesis of intermediate 18, ( 2R , 3R )-3-[( 2S )-1-(t-butoxycarbonyl)pyrrolidin-2 which is liberated from dicyclohexylamine salt. -Based on the preparation of 3-methoxy-2-methylpropionic acid (starting compound 1) and (1 S , 2 R )-1-amino-2-phenylcyclopropanecarboxylic acid benzyl ester . (1 S , 2 R )-1-Amino-2-phenylcyclopropanecarboxylic acid benzyl ester is previously prepared by standard methods by using commercially available (1 S , 2 R )-1-[(third Butyloxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid was prepared by esterification with a benzyl alcohol and subsequent separation of Boc with trifluoroacetic acid.

HPLC (Method 5): R _t = 1.5 min; LC-MS (Method 2): R _t = 0.93 min; MS (ESI): m/z = 437 (M+H) ⁺ .

Intermediate 22 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-N'-methylhexane fluorene trifluoroacetate

100 mg (473 μmol) of 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexanoic acid was dissolved in 71 μl of DMF and then with 139 mg (947 Molmol) of 1-butylindolecarboxylic acid tert-butyl ester, 182 mg (947 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 145 mg (947 μmol) of 1-hydroxy-1 H -benzotriazole hydrate was mixed. The mixture was stirred at RT overnight and then concentrated under reduced pressure. The remaining residue was purified by preparative HPLC. From two After lyophilization of the alkane/water, 129 mg (80% of theory) of the intermediate was obtained as a colorless foam.

Subsequently, 129 mg (380 μmol) was dissolved in 2 ml of trifluoroacetic acid in 8 ml of dichloromethane. After the mixture was stirred at RT for 1 h, the reaction mixture was evaporated. The residue was lyophilized with EtOAc / EtOAc (EtOAc:EtOAc)

LC-MS (method 1): R _t =0.38 min; MS (ESI pos): m/z = 240 (M+H) ⁺

Intermediate 23 N-(2-Aminoethyl)-4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-N-methylbutyramine trifluoroacetate

First, 35 mg (164 μmol) of 2-(methylamino)ethylaminecarboxylic acid tert-butyl ester hydrochloride trifluoroacetate, 30 mg (164 μmol) of 4-(2,5-two-sided) Oxy-2,5-dihydro-1 H -pyrrol-1-yl)butyric acid, 75 mg (197 μmol) of O-(7-azabenzotriazol-1-yl) -N,N,N ',N' -tetramethylhexafluorophosphate Mix with 57 μl of N,N -diisopropylethylamine with 5 ml of DMF and stir at RT until overnight. Subsequently, the solvent was removed under reduced pressure and the residue was purified using preparative HPLC. Concentrate the corresponding dissolved fractions from two The alkane/water was lyophilized to give 35 mg (63% of theory) of intermediate.

HPLC (Method 12): R _t = 1.6 min; LC-MS (Method 1): R _t = 0.71 min; MS (ESI): m/z = 340 (M+H) ⁺ .

Subsequently, the total amount of the protected intermediate was deprotected with 1 ml of trifluoroacetic acid in 5 ml of dichloromethane to give 28 mg (77% of theory) of the title compound.

LC-MS (Method _{3): R t = 0.75 min} ; MS (ESIpos): m / z = 240 (M + H) +.

Intermediate 24 4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-N-[2-(methylamino)ethyl]butanamine trifluoroacetate

First, 35 mg (164 μmol) of (2-aminoethyl)methylaminecarboxylic acid tert-butyl ester hydrochloride trifluoroacetate, 30 mg (164 μmol) of 4-(2,5-two side Oxy-2,5-dihydro-1 H -pyrrol-1-yl)butyric acid, 75 mg (197 μmol) of O-(7-azabenzotriazol-1-yl) -N,N,N ',N' -tetramethylhexafluorophosphate Mix with 57 μl of N,N -diisopropylethylamine in 5 ml of DMF and stir at RT. Subsequently, the solvent was removed under reduced pressure and the residue was purified using preparative HPLC. Concentrate the corresponding dissolved fractions from two The alkane/water was lyophilized to give 51 mg (91% of theory) of intermediate.

HPLC (Method 12): R _t = 1.6 min; LC-MS (Method 1): R _t = 0.77 min; MS (ESI): m/z = 340 (M+H) ⁺ .

Subsequently, the total amount of the protected intermediate was deprotected with 1 ml of trifluoroacetic acid in 5 ml of dichloromethane to give 45 mg (yield: 69%) of the title compound.

LC-MS (Method _{1): R t = 0.19 min} ; MS (ESIpos): m / z = 240 (M + H) +.

Intermediate 25 (2 R , 3 R )-3-methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propionic acid benzyl ester trifluoroacetate

First, (2 R , 3 R )-3-[(2 S )-1-(t-butoxycarbonyl)pyrrolidine was treated by ethyl acetate treatment and shaking extraction with 100 ml of 5% aqueous sulfuric acid solution. 2-yl]-3-methoxy-2-methylpropanoic acid was released from its 1.82 g (388 mmol) dicyclohexylamine salt. The organic layer was dried over magnesium sulfate, filtered and concentrated. Place the residue in 10 ml of two The alkane was treated with 10 ml of water, and 1517 mg (4.66 mmol) of cesium carbonate was added, and the mixture was treated in an ultrasonic bath for 5 min, concentrated under reduced pressure and then distilled again with DMF. The residue was then taken up in 15 mL of dichloromethane and EtOAc (1. The mixture was treated with an ultrasonic bath for 15 min and then concentrated under reduced pressure. The residue was partitioned between ethyl acetate and water, and the organic layer was taken and evaporated and evaporated and evaporated. The residue was then purified by preparative HPLC. This gave 1170 mg (80% of theory) of Boc-protected intermediate.

Subsequently, these protected 1170 mg were deprotected by dissolving 5 ml of trifluoroacetic acid in 15 ml of dichloromethane. After stirring at RT for 15 min, the reaction mixture was concentrated under reduced pressure. Residue from two The alkane is freeze dried. After drying under high vacuum, 1333 mg (84% of theory) of title compound

HPLC (Method 6): R _t = 1.5 min; LC-MS (Method 1): R _t = 0.59 min; MS (ESI): m/z = 278 (M+H) ⁺ .

Intermediate 26 N-(Tertibutoxycarbonyl)-N-methyl-L-decylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N - methyl-L-melamine

1200 mg (2.33 mmol) of N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(2 R ,3 S ,4 S )-1-carboxy-2 - methoxy-4-methyl-3-yl] - N - methyl amine Amides -L- valine (intermediate 5) and 910.8 mg (2.33 mmol) of (2 R, 3 R) -3- Methoxy-2-methyl-3-[( 2S )-pyrrolidin-2-yl]propionic acid benzyl ester trifluoroacetate (intermediate 14), 1327 mg (3.49 mmol) of O- (7 -azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate 2027 μl of N,N -diisopropylethylamine was mixed in 50 ml of DMF, and the mixture was stirred at RT for 5 min. After that, the solvent was removed under reduced pressure. The remaining residue was taken up in ethyl acetate and extracted successively with 5% aqueous citric acid and saturated sodium hydrogen carbonate. The organic layer was removed and concentrated. The residue was purified by preparative HPLC. The product fractions were combined and concentrated, and the residue was dried under high vacuum. This gave 1000 mg (55% of theory) of the benzyl ester intermediate N -(t-butoxycarbonyl) -N -methyl-L-decyl fluorenyl- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-(benzyloxy)-1-methoxy-2-methyl-3-oxopropyl] Pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine is a resin.

LC-MS (Method _{1): R t = 1.56 min} ; MS (ESIpos): m / z = 775 (M + H) +.

The resulting total amount of this intermediate is placed in 25 ml of methanol and dichloro The methane (20:1) mixture was treated with hydrogenation under standard hydrogen pressure to remove the benzyl ester group using 10% activated carbon on palladium as a catalyst. After stirring at RT for 30 min, the catalyst was filtered and the filtrate was concentrated. This gave 803 mg (91% of theory)

HPLC (Method 6): R _t = 2.1 min; LC-MS (Method 1): R _t = 1.24 min; MS (ESIs): m/z = 685 (M+H) ⁺ .

Intermediate 27 (1 S , 2 R )-1-Amino-2-phenyl-n-propylcyclopropanecarbamidamine trifluoroacetate

The title compound is obtained by using commercially available ( 1S , 2R )-1-[(t-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid as n-propylamine in O- (7). -azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of (HATU), and subsequent separation of Boc with trifluoroacetic acid (yield: 85% of theory in two stages).

HPLC (Method 6): R _t = 1.2 min; LC-MS (Method 1): R _t = 0.52 min; MS (ESIs): m/z = 219 (M+H) ⁺ .

Intermediate 28 (1 S , 2 R )-1-Amino-2-phenylcyclopropanecarboxylic acid ethyl ester trifluoroacetate

The title compound is esterified with ethanol by a commercially available method of ( 1S , 2R )-1-[(t-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid, and This was followed by separation of Boc with trifluoroacetic acid.

LC-MS (Method _{1): R t = 0.50 min} ; MS (ESIpos): m / z = 206 (M + H) +.

Intermediate 29 4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-2,2-dimethylbutyric acid

To a solution of 1.39 g (8.95 mmol) of N-methoxycarbonyl maleic acid in 44 ml of saturated sodium bicarbonate, 1.5 g (8.95 mmol) of 4-amino-2,2-dimethyl was added at 0 °C. Butyric acid was added and the mixture was stirred for 40 min. Subsequently, the cooling bath was removed and the reaction mixture was stirred for an additional 1 h. After cooling with ice, the reaction mixture was adjusted to pH 3 by adding sulfuric acid and extracted with ethyl acetate. The combined organic layers were dried over magnesium sulfate and concentrated. The title compound was obtained in 1.17 g (yield: 79%, 49%).

LC-MS (method 1): R _t = 0.64 min; m/z = 212 (M+H) ⁺ .

Intermediate 30 2-[4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-2,2-dimethylbutylidene]carboxylic acid tert-butyl ester

50 mg (237 μmol) of 4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-2,2-dimethylbutyric acid in 2 ml of THF In the solution, 26 μl (237 μmol) of 4-methylmorpholine was added first at 0 ° C and then 31 μl (237 μmol) of isobutyl chloroformate was added. After removing the cooling bath and stirring for another 1 h at RT, 31.3 mg (237 μmol) of the third butoxycarbonyl hydrazine was added. The reaction mixture was stirred overnight and then concentrated. The residue was purified by preparative HPLC. The title compound was obtained in 50.8 mg (66% of theory).

LC-MS (Method 1): R _t = 0.71 min; m/z = 324 (MH) ^- .

Intermediate 31 4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-2,2-dimethylbutane fluorene trifluoroacetate

50 mg (154 mmol) of 2-[4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-2,2-dimethylbutylidene]pyrene The acid tert-butyl ester was dissolved in 2 ml of dichloromethane and 0.4 ml of trifluoroacetic acid was added. The reaction mixture was stirred at RT for 30 min and then concentrated. The title compound was obtained in 55.2 mg (93% purity, 99% of theory).

LC-MS (method 1): R _t =0.36 min; m/z = 226 (M+H) ⁺ .

Intermediate 32 N- (Tertibutoxycarbonyl)-L-phenylalanine adamantyl-1-ylmethyl ester

Add 1192 mg (6.2 mmol) of EDC, 578 μl (4.1 mmol) of triethylamine, 345 to a solution of 500 mg (1.89 mmol) of N- Boc-L-phenylalanine in 25 ml of dichloromethane. Mg (2.8 mmol) of DMAP and 345 mg (2.1 mmol) of 1-adamantyl methanol. The reaction mixture was stirred overnight, then diluted with 50 mL of dichloromethane and washed successively with 10% aqueous citric acid and saturated sodium chloride. The organic layer was dried over MgSO.sub.4, then evaporated. This gave 769 mg (90% of theory) of title compound.

LC-MS (method 2): _rt = 1.84 min; m/z = 414 (M+H) ⁺ .

Intermediate 33 L -phenylalanine adamantyl-1-ylmethyl ester hydrochloride

769 mg (1.86 mmol) of N-(t-butoxycarbonyl) -L -phenylalanine adamantyl-1-ylmethyl ester (intermediate 13) was dissolved in 25 ml of 4 N hydrochloric acid Stir in the alkane solution for 1 h at RT. Subsequently, the reaction mixture was concentrated and the residue was dried under reduced pressure. This gave 619 mg (95% of theory) of title compound.

LC-MS (method 1): R _t = 0.82 min; m/z = 314 (M+H) ⁺ .

Intermediate 34 N -(Tertibutoxycarbonyl)-N-methyl- L -decylamine-N-[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-(adamantan-1-ylmethoxy)-1-oxo-3-phenylpropan-2-yl]amino}-1- Methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl - L - amidoxime

At 20 mg (29 μmol) N -(t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxo-heptane- Add 1-5.3 μl (88 μmol) of N,N -diisopropylethylamine, 6.7 mg (44) to a solution of 4-Methyl- N -methyl-L-amidamine in 1 ml of DMF. Μmol) of HOBt and 6.7 mg (35 μmol) of EDC, and the mixture was stirred for 30 min. Subsequently, 10.1 mg (32 μmol) of adamantyl-1-yl L -phenylalanine hydrochloride was added. After stirring until overnight, the reaction mixture was directly separated into components by preparative HPLC. 27.5 mg (93% of theory) of the title compound are obtained.

LC-MS (method 1): R _t = 1.70 min; m/z = 980 (M+H) ⁺ .

Intermediate 35 N -methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[( 2 S )-1-(adamantan-1-ylmethoxy)-1-oxooxy-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3- Sideoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine guanamine trifluoroacetate

27.5 mg (28 μmol) of N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(adamantan-1-ylmethoxy)-1-oxo-3-phenylpropan-2- Amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4 -yl] -N -methyl-L-decylamine was dissolved in 1.8 ml of dichloromethane and added to 361 μl of TFA. The reaction mixture was changed for 30 min and then concentrated. The residue was taken up in water and lyophilized. 22.7 mg (81% of theory) of the title compound are obtained.

LC-MS (method 1): R _t = 1.14 min; m/z = 880 (M+H) ⁺ .

Intermediate 36 (2 S )-1-(benzyloxy)-3-phenylpropan-2-ylaminecarboxylic acid tert-butyl ester

500 mg (1.99 mmol) of N- Boc- L -amphetamine was dissolved in 5 ml of DMF under argon pressure and cooled to 0 °C. Subsequently, 159 mg (3.98 mmol) of a 60% suspension of sodium hydride in paraffin oil was added. The reaction mixture was stirred until the end of the gas evolution and then 260 μl (2.19 mmol) of benzyl bromide was added. The cooling bath was removed and the reaction mixture was stirred at RT for 2 h. After that time, the reaction mixture was concentrated, the residue was taken in ice water and the mixture was extracted with dichloromethane. The organic layer was washed with a saturated sodium chloride solution, dried over magnesium sulfate and evaporated. The residue was purified by preparative HPLC. This gave 226 mg (33% of theory) of title compound.

LC-MS (Method 1): _rt = 1.28 min; m/z = 342 (M+H) ⁺ .

Intermediate 37 (2 S )-1-(benzyloxy)-3-phenylpropan-2-amine hydrochloride

220 mg (644 μmol) of ( 2S )-1-(benzyloxy)-3-phenylpropan-2-ylaminecarboxylic acid tert-butyl ester was dissolved in 11 ml of 4 N hydrochloric acid Alkane solution and stirred at RT for 1 h. The reaction mixture was then concentrated and the residue dried under reduced pressure. 138 mg (77% of theory) of title compound.

LC-MS (method 1): R _t = 0.65 min; m/z = 242 (M+H) ⁺ .

Intermediate 38 N - (tert-butoxy-carbonyl) - N - methyl -L- valinamide acyl - N - [(3 R, 4 S, 5 S) -1 - {(2 S) -2 - [(1 R , 2 R )-3-{[(2 S )-1-(benzyloxy)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3- Sideoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

20 mg (29 μmol) of N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxanthene Add 15.3 μl (88 μmol) of N,N -diisopropylethylamine, 6.7 mg (RT) to 1 ml of DMF solution of -4-yl] -N -methyl-L-amidamine. 44 μmol) of HOBt and 6.7 mg (35 μmol) of EDC, and the mixture was stirred for 30 min. Subsequently, 7.8 mg (32 μmol) of ( 2S )-1-(benzyloxy)-3-phenylpropan-2-amine hydrochloride was added. After stirring until overnight, the reaction mixture was directly separated from the components by preparative HPLC. This gave 26 mg (98% of theory) of the title compound.

LC-MS (Method 1): Rt = 1.51 min; m/z = 909 (M+H)+.

Intermediate 39 N -methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[( 2 S )-1-(benzyloxy)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl }-3-Methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine guanamine trifluoroacetate

26 mg (29 μmol) of N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzyloxy)-3-phenylpropan-2-yl]amino}-1-methoxy -2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L- Amidoxime was dissolved in 1.8 ml of dichloromethane and 370 μl of TFA was added. The reaction mixture was stirred at RT and then concentrated. The residue was taken up in water and lyophilized. 26.4 mg (quant.) of the title compound are obtained.

LC-MS (Method _{1): R t = 0.97 min} ; m / z = 809 (M + H) +.

Intermediate 40 N-methyl-L-Amidoxime-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1 -hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5 -methyl-1-oxoheptan-4-yl]-N-methyl-L-amidamine

50 mg (70 μmol) of intermediate 26 and 11 mg (70 μmol) of (1S,2R)-2-amino-1-phenylpropan-1-ol in 10 ml of DMF with 42 mg (0.11) Molmol) of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylhexafluorophosphate Mix with 25 μl of N,N -diisopropylethylamine and stir the reaction mixture at RT for 5 min. It was then concentrated and the residue was purified by preparative HPLC. The corresponding dissolved fractions were combined, concentrated and dried under high vacuum to give 49 mg (81%). Subsequently, it was deprotected with trifluoroacetic acid in dichloromethane by known conditions. The title compound was purified by preparative EtOAc (EtOAc)

HPLC (Method 12): R _t = 1.65 min; LC-MS (Method 1): R _t = 0.77 min; MS (ESI): m/z = 718 (M+H) ⁺ .

Intermediate 41 3-{2-[2-(2-Aminoethoxy)ethoxy]ethoxy}propionic acid trifluoroacetate

150 mg (541 μmol) of 3-butyl 2-(2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoate was dissolved in 3 ml of dichloromethane and added to 1.5 Trifluoroacetic acid in ml and the reaction mixture was stirred at RT for 1 h then concentrated. 181 mg (100% of theory) of the title compound.

MS (EI): m/z 222 (M+H) ⁺

Intermediate 42 3-(2-{2-[2-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)ethoxy]ethoxy}ethoxy)propanoic acid

186 mg (555 μmol) of 3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionic acid trifluoroacetate was dissolved in 2.6 ml of saturated sodium bicarbonate solution And at 0 ° C with 86 mg (555 μmol) of N-methoxycarbonyl maleimide. The reaction mixture was stirred at 0&lt;0&gt;C for 40 min and at RT for 1 h then cooled again to 0 &lt;0&gt;C, EtOAc (EtOAc) The combined organic layers were dried over magnesium sulfate and concentrated. 126 mg (75% of theory) of the title compound.

LC-MS (method 1): R _t = 0.53 min; m/z = 302 (M+H) ⁺ .

Intermediate 43 15-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-4-oxo-7,10,13-trioxo-2,3-diaza Alkyl-1-acid tert-butyl ester

125 mg (417 μmol) of 3-(2-{2-[2-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)ethoxy]ethoxy The base ethoxy)propionic acid was dissolved in 2.1 ml of THF at 0 ° C and mixed with 46 μl (417 mmol) of 4-methylmorpholine and 54.5 μl (417 μmol) of isobutyl chloroformate. The ice bath was removed and the reaction mixture was stirred at RT for 30 min. Subsequently, 55 mg (417 μmol) of a third butoxycarbonyl hydrazine was added at 0 °C. The reaction mixture was warmed to RT until overnight, concentrated and purified by preparative HPLC. 60 mg (33% of theory) of the title compound.

LC-MS (method 1): R _t = 0.66 min; m/z = 416 (M+H) ⁺ .

Intermediate 44 3-(2-{2-[2-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)ethoxy]ethoxy}ethoxy)propane oxime Trifluoroacetate

60 mg (145 μmol) of base 15-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-4-sideoxy-7,10,13-trioxy- The 2,3-diazapentadecan-1-carboxylic acid tert-butyl ester was dissolved in 1 ml of dichloromethane and 0.2 ml of trifluoroacetic acid was added. The reaction mixture was stirred at RT for 30 min and then concentrated. 62 mg (100% of theory) of the title compound.

LC-MS (Method 1): _rt = 0.35 min; m/z = 316 (M+H) ⁺ .

Intermediate 45 (1 S , 2 R )-1-Amino-2-phenylcyclopropanecarboxylic acid benzyl ester trifluoroacetate

The title compound is esterified with a benzyl alcohol by a commercially available method of ( 1S , 2R )-1-[(t-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid. And subsequently prepared by separating Boc with trifluoroacetic acid.

LC-MS (Method _{1): R t = 0.72 min} ; MS (ESIpos): m / z = 268 (M + H) +.

Intermediate 46 N -(Tertibutoxycarbonyl) -N -methyl-L-decylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(1 S )-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine- 1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

383 mg (0.743 mmol) of N- ( t- butoxycarbonyl) -N -methyl-L-nonylamine- N -[(2 R ,3 S ,4 S )-1-carboxy-2 -Methoxy-4-methylhex-3-yl] -N -methyl-L-decylamine (Intermediate 8) with 485 mg (0.743 mmol) of N -{(2 R ,3 R ) 3-methoxy-2-methyl-3-[( 2S )-pyrrolidin-2-yl]propanyl}-L-phenylalanine benzyl trifluoroacetate (intermediate 12), 424 Mg (1.114 mmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate 388 μl of N,N -diisopropylethylamine was mixed in 15 ml of DMF, and the mixture was stirred at RT for 10 min. Subsequently, the solvent was removed under reduced pressure. The remaining residue was taken up in ethyl acetate and extracted successively with 5% aqueous citric acid and saturated sodium hydrogen carbonate. The organic layer was removed and concentrated, and the residue was purified by preparative HPLC. The product fractions were combined, concentrated and the residue was dried under high vacuum. 335 mg (48% of theory) of the benzyl ester intermediate was obtained as a foam.

LC-MS (Method _{1): R t = 1.49 min} ; MS (ESIpos): m / z = 922 (M + H) +.

100 mg (0.11 mmol) of this intermediate was taken up in 15 ml of methanol and the benzyl ester group was removed by hydrogenation under standard hydrogen pressure with 10% activated carbon on palladium as catalyst. After stirring at RT for 1 h, the catalyst was filtered and the filtrate was concentrated. From two After lyophilization of the alkane, 85 mg (94% of theory)

HPLC (Method 12): R _t = 2.4 min; LC-MS (Method 1): R _t = 1.24 min; MS (ESIs): m/z = 832 (M+H) ⁺ .

Intermediate 47 N -benzyl-L-tryptamine guanamine trifluoroacetate

Dissolve 202 mg (0.5 mmol) of 2,5-dioxapyrrolidin-1-yl N- (t-butoxycarbonyl)-L-tryptophanate and 45 mg (0.42 mmol) of benzylamine In 10 ml of DMF and adding 110 μl (630 μmol) of N,N -diisopropylethylamine. The reaction mixture was stirred at RT for 3 h. Subsequently, the reaction mixture was concentrated under reduced pressure and the residue was purified crystalljjjjjjjjjj The corresponding dissolved fractions were combined and concentrated. The resulting residue was treated with diethyl ether and then dried under high vacuum. Subsequently, this residue was taken up in 10 ml of dichloromethane and 3 ml of anhydrous trifluoroacetic acid was added. After stirring at RT for 45 min, the mixture was concentrated and the residue was purified by preparative HPLC. After drying under high vacuum, 117 mg (yield: 57%)

HPLC (method 12): _Rt = 1.6 min; LC-MS (Method 1): R _t = 0.66 min; MS (ESIs): m/z = 294 (M+H) ⁺ .

Intermediate 48 (1 S , 2 R )-1-Amino-2-phenylcyclopropanecarbamidamine trifluoroacetate

50 mg (180 μmol) of commercially available (1 S , 2 R )-1-[(tatabutoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid was dissolved in 5 ml of DMF and added 94 μl (541 μmol) of N,N -diisopropylethylamine, 31 mg (270 μmol) of N -hydroxysuccinimide and 41.5 mg (216 μmol) of EDC, and then the mixture was stirred at RT Until overnight. The reaction mixture is then concentrated and the residue is placed in two Treatment with alkane, 71 mg (901 μmol) of ammonium hydrogencarbonate were added and the reaction mixture was then placed at RT for 3 days. The reaction mixture was diluted with a 1:1 mixture of ethyl acetate and water. The organic layer was removed, dried over magnesium sulfate and concentrated. The resulting residue was then taken up in 3 ml of dichloromethane and 3 ml of anhydrous trifluoroacetic acid was added. Stir at RT for 1 h and concentrate. The residue is stirred with pentane, filtered with suction and from two The alkane is freeze dried. From this method, 32 mg (62% of theory in two phases) of title compound was obtained.

HPLC (Method 6): R _t =0.38 min; LC-MS (Method 1): R _t =0.20 min; MS (ESI): m/z = 177 (M+H) ⁺ .

Intermediate 49 N ^α -{(2 R ,3 R )-3-methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propanyl}-L-tryptamine Fluoroacetate

The title compound was prepared in a similar analog of intermediate 13 from starting compound 1 and L-tryptamine hydrochloride.

HPLC (Method 5): R _t = 1.4 min; LC-MS (Method 1): R _t = 0.92 min; MS (ESIs): m/z = 473 (M+H) ⁺ .

Intermediate 50 4-(2-, 2,2-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)ethylaminecarboxylic acid 4-nitrophenyl ester

813 mg (3.1 mmol) of triphenylphosphine was dissolved in 25 ml of THF and cooled to -70 ° C under argon. After adding 627 mg (3.1 mmol) of diisopropyl azide dicarboxylate dropwise, the mixture was stirred for 5 min. Subsequently, 500 mg (3.1 mmol) of 2-aminoethylaminocarbamic acid tert-butyl ester dissolved in 5 ml of THF was added dropwise, and the reaction mixture was further stirred at -70 ° C for further 5 min. Then add 136.6 mg (1.55) dissolved in 1 ml of THF. Methyl) 2,2-dimethyl-1-propanol and 301 mg (3.1 mmol) of maleimide, the reaction mixture was stirred at -70 °C for an additional 10 min and then the mixture was taken to RT. After stirring at RT for additional 16 h, the solvent was removed under reduced pressure and the residue was purified by preparative HPLC. 463 mg (62%) of the protected intermediate was obtained.

After removal of the Boc protecting group under standard conditions, 652 mg of 1-(2-aminoethyl)-1 H -pyrrole-2,5-dione as trifluoroacetic acid salt was obtained.

112.9 mg (543 μmol) of nitrophenyl chloroformate was dissolved in 30 ml of THF and 100 mg (271.6 μmol) of 1-(2-aminoethyl)-1 H -pyrrole-2,5 was added. After the diketone trifluoroacetate, the mixture was stirred at RT for 30 min. The mixture was filtered and the filtrate was concentrated to dryness and then taken to diethyl ether. After suction filtration and drying, 60 mg (95% of theory) of title compound was obtained.

HPLC (method 5): Rt = 0.65 min; LC-MS (method 1): Rt = 0.74 min; MS (ESIs): m/z = 306 (M+H)+.

Intermediate 51 ( 1S )-2-phenyl-1-(5-phenyl-1,3,4-di Zin-2-yl)ethylamine trifluoroacetate

Initially 200 mg (0.75 mmol) of N- (t-butoxycarbonyl)-L-phenylalanine was charged to 5.5 ml of dichloromethane at 0 ° C and 128 mg (0.79 mmol) of 1, 1'-carbonyldiimidazole. After 30 min, 103 mg (0.75 mmol) of benzamidine was added. After another 45 min at 0 °C, 500 mg (1.5 mmol) of carbon tetrabromide and 395 mg (1.5 mmol) of triphenylphosphine were added last. The reaction mixture was stirred at 0 °C for 2 h and then stirred at rt overnight. The mixture was then concentrated on a rotary evaporator and the residue was dried under high vacuum. The crude product thus obtained was purified by preparative HPLC. Obtained 217 mg (78% of theory) of Boc-protected intermediate ( 1S )-2-phenyl-1-(5-phenyl-1,3,4-di T-butyl ester of oxazol-2-yl)ethylaminecarboxylate.

LC-MS (method 12): R _t = 1.15 min; MS (ESI): m/z = 366 (M+H) ⁺

217 mg (0.59 mmol) of this intermediate was taken up in 3 ml of dichloromethane, then 0.6 ml of trifluoroacetic acid was added and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated under reduced pressure. The remaining residue is further dried under reduced pressure as a reaction mixture and then from two The alkane is freeze dried. In this way, 214 mg (90% of theory) of title compound.

LC-MS (Method 11): R _t = 0.62 min; MS (ESI): m/z = 266 (M+H) ⁺

Intermediate 52 ( 1R )-2-phenyl-1-(5-phenyl-1,3,4-di Zin-2-yl)ethylamine trifluoroacetate

Initially 200 mg (0.75 mmol) of N- (t-butoxycarbonyl)-D-phenylalanine was charged to 5.5 ml of dichloromethane at 0 ° C and 128.3 mg (0.79 mmol) of 1, 1'-carbonyldiimidazole. After 30 min, 103 mg (0.75 mmol) of benzamidine was added. After another 45 min at 0 °C, 500 mg (1.5 mmol) of carbon tetrabromide and 395 mg (1.5 mmol) of triphenylphosphine were added last. The reaction mixture was stirred at 0 °C for 2 h and then stirred at rt overnight. The mixture was then concentrated on a rotary evaporator and the residue was dried under high vacuum. The crude product thus obtained was purified by preparative HPLC. 219 mg (80% of theory) of Boc-protected intermediate (1 R )-2-phenyl-1-(5-phenyl-1,3,4-di T-butyl ester of oxazol-2-yl)ethylaminecarboxylate.

LC-MS (Method 2): R _t = 1.36 min; MS (ESI): m/z = 366 (M+H) ⁺

219 mg (0.6 mmol) of this intermediate was taken in 3 ml of dichloromethane, then 0.6 ml of trifluoroacetic acid was added and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated under reduced pressure. The remaining residue is further dried under reduced pressure as a reaction mixture and then from two The alkane is freeze dried. In this way, 196 mg (86% of theory) of title compound was obtained as a solid.

HPLC (Method 10): R _t =2.41 min

Intermediate 53 ( 2S )-1-(Benzylsulfonyl)-3-phenylpropan-2-amine

Initially 200 mg (1.13 mmol) of (4 S )-4-benzyl-1,3- The oxazolidine-2-one was charged to 3 ml of the third butanol and 280 mg (2.26 mmol) of benzyl mercaptan was added. The mixture was then heated to reflux for 2 days. Thereafter, the reaction mixture was concentrated on a rotary evaporator and the resulting ( 2S )-1-(benzylmercapto)-3-phenylpropan-2-amine intermediate was directly further converted without subsequent workup.

HPLC (Method 10): R _t = 2.63 min

LC-MS (Method 1): R _t = 0.67 min; MS (ESI): m/z = 258 (M+H) ⁺

The crude intermediate obtained above was dissolved in 2 ml of 30% hydrogen peroxide solution and 5 ml of formic acid and the mixture was stirred at RT for 12 h. The reaction mixture was then added to a saturated sodium sulfate solution and extracted three times with ethyl acetate. The organic layer was dried with MgSO4 and evaporated. The obtained crude product was purified by preparative HPLC. This gave 343 mg (61% of theory) of the title compound.

HPLC (method 10): R _t = 2.40 min; LC-MS (method 12): R _t = 0.65 min; MS (ESIs): m/z = 290 (M+H) ⁺

Intermediate 54 ( 2S,3E )-1,4-diphenylbut-3-en-2-amine

552.7 mg (9.85 mmol) of potassium hydroxide was dissolved in methanol, adsorbed onto 1.1 g of neutral alumina and then dried under high vacuum. 240 mg (0.82 mmol) of ( 2S )-1-(benzylsulfonyl)-3-phenylpropan-2-amine prepared in 6.2 ml of n-butanol and potassium hydroxide on 1.56 g of alumina Into this solution, 307 μl (3.3 mmol) of dibromodifluoromethane was added dropwise at 5-10 °C. The reaction mixture was stirred at RT for 2 h then EtOAc (EtOAc)EtOAc. The filtrate was concentrated and the residue obtained was dried under reduced pressure. The crude product thus obtained was purified by preparative HPLC. The title compound was obtained in a 98 mg (35% of theory) yield with a 4:1 ratio of E/Z diastereomers.

HPLC (Method 10): R _t =2.46 min; LC-MS (Method 12): R _t = 0.75 min; MS (ESIs): m/z = 224 (M+H) ⁺

The E/Z diastereomer obtained above was dissolved in 2 ml of ethanol and 0.2 ml of N,N -diisopropylethylamine, and separated by HPLC on the palm phase [column: DaicCl Chiralpak AD-H, 5 μm 250 mm x 20 mm, dissolving agent: hexane/(ethanol + 0.2% diethylamine) 50:50 v/v; UV detection: 220 nm; temperature: 30 °C]. The appropriate fractions were concentrated on a rotary evaporator and the residue dried under reduced pressure. This gave 45 mg of the title compound.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ [ppm] = 2.62 - 2.83 (m, 2 H) 3.52-3.71 (m, 1 H) 6.18-6.30 (m, 1 H) 6.34-6.46 (m, 1 H) 6.98-7.57 (m, 10 H) [More signals are hidden under the H ₂ O peak].

Intermediate 55 N-methyl- L -carbamicinyl-N-[( 3R, 4 S, 5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )- 1-methoxy-2-methyl-3-oxo-3-{[(1 S )-2-phenyl-1-(5-phenyl-1,3,4-di Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl- L -decylamine Trifluoroacetate

20 mg (29 μmol) of N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxanthene -4-yl] -N -methyl-L-decylamine is dissolved in 1 ml of DMF, and 13.3 mg (35 μmol) of HATU and 15.3 μl (88 μmol) of N,N -diisopropyl B are added. The amine was stirred and the mixture was stirred at RT for 30 min. Subsequently, 12.2 mg (32 μmol) of (1 S )-2-phenyl-1-(5-phenyl-1,3,4-di was added. Zin-2-yl)ethylamine trifluoroacetate. The reaction mixture was stirred at rt overnight and then separated by preparative HPLC. Thus, 22 mg (81% of theory) of N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R , 4 S , 5 S )- 3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 S )- 2-phenyl-1-(5-phenyl-1,3,4-di Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine .

LC-MS (Method 12): _rt = 1.45 min; MS (ESI): m/z = 933 (M+H) ⁺

The BOC protecting group was subsequently isolated in trifluoroacetic acid to afford 22.4 mg (yield: 98%) of title compound.

LC-MS (Method 11): R _t = 0.85 min; MS (ESI s): m/z = 833 (M+H) ⁺

Intermediate 56 N -methyl-L-Amidinoinyl- N -[(3 R, 4 S, 5 S )-3-methoxy-1-{(2 S )-2-[(1 R, 2 R ) 1-methoxy-2-methyl-3-oxo-3-{[(1 R )-2-phenyl-1-(5-phenyl-1,3,4-di Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine Trifluoroacetate

N -(Tertibutoxycarbonyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R, 4 S, 5 S )-3-methoxy-1-{(2 S ) -2-[(1 R, 2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 R )-2-phenyl-1-(5-phenyl- 1,3,4-two Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine By the synthesis of a similar intermediate 55, by 20 mg (29 μmol) of N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R, 4 S, 5 S )-1-{(2 S )-2-[(1 R, 2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy -5-Methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine amide with 12.2 mg (32 μmol) of (1 R )-2-phenyl-1-(5 -phenyl-1,3,4-di Prepared by the reaction of oxazol-2-yl)ethylamine trifluoroacetate.

Yield: 17 mg (64% of theory)

HPLC (method 10): R _t = 3.74 min; LC-MS (method 1): R _t = 1.45 min; MS (ESI): m/z = 933 (M+H) ⁺

The BOC protecting group was subsequently isolated in trifluoroacetic acid to afford 17.1 mg (yield:

HPLC (method 10): R _t =2.55 min; LC-MS (method 11): R _t = 0.85 min; MS (ESIs): m/z = 833 (M+H) ⁺

Intermediate 57 N-methyl- L -Amidinoinyl-N-[(3 R, 4 S, 5 S )-1-{(2 S) -2-[(1 R, 2 R )-3-{[( 2 S )-1-(benzylsulfonyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1 -yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl- L -decylamine guanamine trifluoroacetate

N -(Tertibutoxycarbonyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R, 4 S, 5 S )-1-{(2 S )-2-[(1 R, 2 R )-3-{[(2 S )-1-(benzylsulfonyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl- 3-Phenoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine amide Similar to the synthesis of intermediate 55 by 20 mg (29 μmol) of N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R, 4 S, 5 S )-1-{(2 S )-2-[(1 R, 2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5 -methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine amide with 9.3 mg (20 μmol) of ( 2S )-1-(benzylsulfonyl)-3 Prepared by a -phenylpropan-2-amine reaction.

Yield: 19.2 mg (68% of theory)

HPLC (Method 10): R _t = 3.5 min; LC-MS (Method 12): R _t = 1.41 min; MS (ESIs): m/z = 957 (M+H) ⁺

The BOC protecting group was subsequently isolated in trifluoroacetic acid to afford 19.3 mg (yield:

HPLC (method 10): R _t =2.52 min; LC-MS (method 1): R _t =0.86 min; MS (ESIs): m/z=857 (M+H) ⁺

Intermediate 58 N -methyl-L-Amidinoinyl- N -[(3 R, 4 S, 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[( 2 S, 3 E )-1,4-Diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1 -yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine guanamine trifluoroacetate

N -(Tertibutoxycarbonyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R, 4 S, 5 S )-1-{(2 S )-2-[(1 R, 2 R )-3-{[(2 S, 3 E )-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl- 3-Phenoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine amide Similar to the synthesis of intermediate 55 by 20 mg (29 μmol) of N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[( 3R,4S,5S )- 1-{( 2S )-2-[( 1R,2R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-side Oxyheptan-4-yl] -N -methyl-L-decylamine amide with 7.1 mg (32 μmol) of (2 S, 3 E )-1,4-diphenylbut-3-en-2- Prepared by an amine reaction.

Yield: 15.1 mg (58% of theory)

HPLC (method 10): R _t =4.2 min; LC-MS (method 12): R _t = 1.51 min; MS (ESIs): m/z = 891 (M+H) ⁺

The BOC protecting group was subsequently isolated in trifluoroacetic acid to afford 15.7 mg (yield: 99%) of the title compound.

HPLC (method 10): R _t = 2.62 min; LC-MS (method 12): R _t = 0.97 min; MS (ESI): m/z = 791 (M+H) ⁺

Intermediate 61 N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )- 2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

50 mg (0.054 mmol) of N -methyl-L-amidino- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2- [(1 R , 2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-decylamine trifluoroacetate (intermediate 16) is dissolved in 8 ml of two Aq./water was added with 70 ml (0.108 mmol) of 15% aqueous solution of 4-oxobutyric acid. The reaction mixture was then stirred at 100 ° C for 1 h. After cooling to RT, 3.7 mg (0.059 mmol) of sodium cyanoborohydride was added and the mixture was added to about 300 μl of 0.1 N hydrochloric acid to adjust to pH 3. The reaction mixture was then stirred at 100 ° C for an additional 2 h. After cooling, 70 ml (0.108 mmol) of a 15% 4-oxobutyric acid solution was added and the reaction mixture was again stirred at 100 ° C for 1 h. Then another 3.7 mg (0.059 mmol) of sodium cyanoborohydride was added, and then the pH was adjusted back to 3 using about 300 μl of 0.1 N hydrochloric acid. The reaction mixture was then stirred at 100 ° C for an additional 2 h. Repeat this step a third time if the conversion is still not complete. The reaction mixture was finally concentrated and the residue was purified by preparative HPLC. This method gave 32 mg (65% of theory) of the title compound as a colorless foam.

HPLC (method 5): R _t = 1.64 min; LC-MS (method 9): R _t = 4.76 min; MS (ESIs): m/z = 899 (M+H) ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ): δ = 8.95 and 8.8 (2m, 1H), 8.88 and 8.65 (2 s, 1H), 7.4-7.1 (m, 5H), 5.0, 4.78, 4.65 and 4.55 ( 4m, 2H), 4.1-3.7 (m, 5H), 3.32, 3.29, 3.20, 3.12, 3.1 and 3.0 (6s, 9H), 2.75 (m, 2H), 2.63 (t, 1H), 2.4-2.2 (m , 4H), 2.1-1.2 (m, 12H), 1.2-0.8 (m, 16H), 0.75 (m, 3H) [more signals are hidden under H ₂ O and DMSO peaks].

Intermediate 62 N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )- 2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-amidamine

The title compound is analogous to the synthesis of intermediate 61, with 50 mg of N - methyl -L- valinamide acyl - N - [(3 R, 4 S, 5 S) -3- methoxy-1 {(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-decylamine guanamine trifluoroacetate (Intermediate 14) was prepared by reaction with 4-oxobutyric acid.

Yield: 34 mg (70% of theory)

HPLC (Method 5): R _t = 1.64 min; LC-MS (Method 9): R _t = 4.77 min; MS (ESIs): m/z = 887 (M+H) ⁺ .

Intermediate 63 N- (4-carboxybenzyl) -N -methyl-L-decylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )- 2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

The title compound is analogous to the synthesis of intermediate 61, with 15 mg of N - methyl -L- valinamide acyl - N - [(3 R, 4 S, 5 S) -3- methoxy-1 {(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - Preparation of N -methyl-L-guanamine guanamine trifluoroacetate (Intermediate 16) in reaction with 4-methyl benzoic acid.

Yield: 7.5 mg (48% of theory)

HPLC (method 5): R _t = 1.75 min; LC-MS (Method 1): R _t = 0.97 min; MS (ESI): m/z = 947 (M+H) ⁺ .

Intermediate 64 N- (5-carboxypentyl) -N -methyl-L-decylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )- 2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

10 mg (0.011 mmol) of N -methyl-L-amidino- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2- [(1 R , 2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-decylamine trifluoroacetate (intermediate 16) is dissolved in 2 ml of two In alkane/water, 2.8 mg (0.022 mmol) of 6-oxohexanoic acid was added. The reaction mixture was then stirred at 100 ° C for 1 h. After cooling to RT, 0.75 mg (0.012 mmol) of sodium cyanoborohydride was added and the mixture was added to 0.1 N hydrochloric acid to adjust to pH 3. The reaction mixture was then stirred at 100 ° C for an additional hour. After cooling, another 2.8 mg (0.022 mmol) of 6-oxohexanoic acid was added and the reaction mixture was again stirred at 100 ° C for 1 h. An additional 0.75 mg (0.012 mmol) of sodium cyanoborohydride was added, and then the pH was adjusted back to 3 using about 0.1 N hydrochloric acid. The reaction mixture was then stirred at 100 ° C for an additional 1 h. Then repeat this step a third time. The reaction mixture was finally concentrated and the crude material was purified using preparative HPLC. This gave 6.4 mg (64% of theory) of the title compound as a colourless foam.

HPLC (Method 5): R _t = 1.68 min; LC-MS (Method 9): R _t = 4.86 min; MS (ESIs): m/z = 927 (M+H) ⁺ .

Intermediate 65 N- (2-Aminoethyl) -N -methyl-L-decylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S ) -2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-decylamine, bistrifluoroacetate

The title compound is 68 mg of N -methyl-L-amidino- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[ (1 R , 2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Prepared by the reaction of hept-4-yl] -N -methyl-L-decylamine guanamine trifluoroacetate (intermediate 14) with 2-butoxyethylamine carboxylic acid tert-butyl ester, and subsequently with trifluoro Acetic acid separates the BOC protecting group.

Yield: 49 mg (62% of the theoretical value in the second stage)

HPLC (method 5): R _t = 1.58 min; LC-MS (method 2): R _t = 1.05 min; MS (ESIs): m/z = 844 (M+H) ⁺

¹ H NMR (600 MHz, DMSO-d ₆ ): δ = 8.25 (m, 1H), 8.45 and 8.15 (2d, 1H), 7.65-7.55 (m, 3H), 7.23-7.1 (m, 5H), 5.12 And 4.95 (2m, 1H), 4.72 and 4.62 (2m, 1H), 4.6 and 4.52 (2t, 1H), 4.2-3.8 (m, 4H), 3.7 (d, 1H), 3.23, 3.20, 3.19, 3.18, 3.03 and 2.98 (6s, 9H), 3.0-2.7 (m, 6H), 2.4-1.2 (m, 15H), 1.05, 1.0, 0.88 and 0.82 (4d, 6H), 0.92 (m, 6H), 0.73 (m) , 6H) [More signals are hidden under the H ₂ O peak].

Intermediate 66 N- (3-Aminopropyl) -N -methyl-L-decylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S ) -2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

The title compound was synthesized as a similar intermediate 66 by using 25 mg (0.027 mmol) of N -methyl-L-indoleamine- N -[(3 R , 4 S , 5 S )-3-methoxy Base-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2 - Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-Amidoxime trifluoroacetate (intermediate 16) is prepared by reaction with 3-benzyloxypropylamine benzyl acrylate, and then the Z protecting group is separated by hydrogenolysis ( Palladium on 10% activated carbon was used as a catalyst and ethanol was used as a solvent.

Yield: 11 mg (41% of the theoretical value in the second stage)

HPLC (Method 5): R _t = 1.53 min; LC-MS (Method 1): R _t = 0.72 min; MS (ESI): m/z = 870 (M+H) ⁺ .

Intermediate 67 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-(adamantan-1-ylmethoxy)-1-oxooxy-3-phenylpropan-2-yl]amino}-1-A Oxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl- L-Amidoxime

26 mg (26 μmol) of N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(adamantan-1-ylmethoxy)-1-oxooxy-3-phenylpropan-2-yl]amino}-1-methoxy -2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L- Amidoxime trifluoroacetate and 33.9 μl of 15% succinic acid aqueous solution (53 μmol) dissolved in 957 μl of 1:1-two The alkane/water mixture was heated to 100 ° C for 1 h. After brief cooling, 1.81 mg (29 μmol) of sodium cyanoborohydride was added. The reaction mixture was adjusted to pH 3 by addition of 0.1 N hydrochloric acid and the mixture was further warmed to 100 ° C for 2 h. The same amount of succinic acid solution, sodium cyanoborohydride and hydrochloric acid were added again, and the mixture was again heated to 100 ° C for 2 h. The reaction mixture was then directly separated into its components by preparative HPLC. This gave 18.5 mg (73% of theory) of the title compound.

LC-MS (Method _{1): R t = 1.17 min} ; m / z = 967 (M + H) +.

Intermediate 68 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-(benzyloxy)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3- side Oxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

24 mg (26 μmol) of N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzyloxy)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropane Pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine guanamine trifluoroacetate and 33.7 μl A 15% aqueous solution of succinic acid (52 μmol) dissolved in 953 μl of 1:1-two The alkane/water mixture was heated to 100 ° C for 1 h. After brief cooling, 1.81 mg (29 μmol) of sodium cyanoborohydride was added. The reaction mixture was adjusted to pH 3 by addition of 0.1 N hydrochloric acid and the mixture was further warmed to 100 ° C for 2 h. The same amount of succinic acid solution, sodium cyanoborohydride and hydrochloric acid were again added, and the mixture was again heated to 100 ° C for 2 h. The reaction mixture was then directly separated into its components by preparative HPLC. This gave 15.2 mg (65% of theory) of the title compound.

LC-MS (Method 1): R _t = 1.01 min; m/z = 895 (M+H) ⁺

Intermediate 69 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-(benzyloxy)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl 3-yloxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

53 mg (84 μmol) of N -[(9 H -茀-9-ylmethoxy)carbonyl] -N -methyl-L-nonylamine- N -[(2 R , 3 S , 4 S )-1-carboxy-2-methoxy-4-methylhexan-3-yl] -N -methyl-L-decylamine (intermediate 4) and 45 mg (84 μmol) of N- {(2 R ,3 R )-3-methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propanyl}-L-phenylalanine benzyl trifluoroacetic acid The salt (intermediate 12) was treated in 2 ml of DMF, and 19 μl of N,N -diisopropylethylamine, 14 mg (92 μmol) of HOBt and 17.6 mg (92 μmol) of EDC were added and then The mixture was stirred at RT until overnight. Subsequently, the reaction mixture was concentrated and the residue was purified by preparative HPLC. Thereby obtaining 59 mg (68% of theory) of the Fmoc-protected intermediate N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzyloxy)-1 -Phenoxy-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy- 5-Methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine.

LC-MS (Method _{1): R t = 1.55 min} ; m / z = 1044 (M + H) +.

57 mg (0.055 mmol) of this intermediate was treated with a solution of 1.2 ml of piperidine in 5 ml of DMF to isolate the Fmoc protecting group. Concentrated and purified to formula HPLC, to give 39 mg (76% of theory) of the free amine intermediate N - methyl -L- valinamide acyl - N - [(3 R, 4 S, 5 S) - 1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzyloxy)-1-oxo-3-phenylpropan-2-yl Amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4- The base] -N -methyl-L-guanamine amine is a trifluoroacetate salt.

HPLC (Method 5): R _t = 1.9 min; LC-MS (Method 1): R _t = 1.01 min; m/z = 822 (M+H) ⁺ .

Dissolve 37 mg (0.045 mmol) of this intermediate in 5 ml of two The preparation of the compound in alkane/water, and similar intermediate 66, was carried out with 15% aqueous 4-oxobutanoic acid in the presence of sodium cyanoborohydride. The title compound was obtained as a colorless foam in 16 mg (yield 39%).

HPLC (method 6): _Rt = 2.1 min; LC-MS (Method 1): R _t = 1.01 min; MS (ESIs): m/z = 908 (M+H) ⁺ .

Intermediate 70 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S ,3 S )-1-(benzyloxy)-1-oxo-3-phenylbutan-2-yl]amino}-1-methoxy- 2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-indole Amine

First, the intermediates 4 and 26 were treated in a similar manner to that described in Intermediate 14, to prepare the amine compound N -methyl-L-amidino- N -[(3 R , 4 S , 5 S )-1 -{(2 S )-2-[(1 R ,2 R )-3-{[(2 S ,3 S )-1-(benzyloxy)-1-oxo-3-phenylbutene-2 -yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxo-heptane- 4-yl] -N -methyl-L-decylamine.

30 mg (0.032 mmol) of this compound was dissolved in 6 ml of two In alkane/water, 41 μl (0.063 mmol) of 15% aqueous 4-oxobutanoic acid solution was added. The reaction mixture was then stirred at 100 ° C for 1 h. After cooling to RT, 2.2 mg (0.035 mmol) of sodium cyanoborohydride was added and the mixture was added to about 300 μl of 0.1 N hydrochloric acid to adjust to pH 3. The reaction mixture was then stirred at 100 ° C for an additional 2 h. After cooling, 41 μl (0.063 mmol) of a 15% 4-oxobutyric acid solution was further added, and the reaction mixture was further stirred at 100 ° C for 1 h. Then another 2.2 mg (0.035 mmol) of sodium cyanoborohydride was added and then the pH was adjusted back to 3 using about 300 μl of 0.1 N hydrochloric acid. The reaction mixture was stirred at 100 ° C for an additional 2 h. Repeat this step a third time if the conversion is still not complete. The reaction mixture was finally concentrated and the crude material was purified using preparative HPLC. This gave 24 mg (82% of theory) of the title compound as a colourless foam.

HPLC (method 5): R _t = 1.9 min; LC-MS (Method 9): R _t = 5.15 min; MS (ESIs): m/z = 922 (M+H) ⁺ .

Intermediate 71 N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )- 2-[(1 R ,2 R )-1-methoxy-3-{[(2 S )-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino} -2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

First, the intermediates 4 and 39 were treated in a similar manner to that described in Intermediate 14, to prepare the amine compound N -methyl-L-amidino- N -[(3 R , 4 S , 5 S )-3. -Methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-3-{[(2 S )-1-methoxy-1-sideoxy- 3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]- N -methyl-L-decylamine. Then 7 mg (0.009 mmol) of this compound was used, similar to the preparation in Intermediate 61, to give 2 mg (22% of theory) by reaction with 4-oxobutyric acid in the presence of sodium cyanoborohydride. The title compound is in the form of a colorless foam.

HPLC (Method 6): R _t = 1.9 min; LC-MS (Method 2): R _t = 1.06 min; MS (ESIs): m/z = 832 (M+H) ⁺ .

Intermediate 72 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-(benzyloxy)-3-(1 H -indol-3-yl)-1-oxopropan-2-yl]amino}- 1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N - methyl-L-melamine

212 mg (411 μmol) of N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(2 R ,3 S ,4 S )-1-carboxy-2 -Methoxy-4-methylhex-3-yl] -N -methyl-L-decylamine (Intermediate 8) and 237 mg (411 μmol) of benzyl- N -{(2 R , 3 R )-3-Methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propanyl}-L-Tryptophan Trifluoroacetate (Intermediate 20) Treated in 30 ml of DMF and added 188 mg (493 μmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluoro Phosphate And 215 μl of N,N -diisopropylethylamine. The reaction mixture was stirred at RT for 20 h then concentrated EtOAc. The product fractions were combined and concentrated, and the residue was dried under high vacuum. This gave 315 mg (80% of theory) of Boc-protected intermediate N- (t-butoxycarbonyl) -N -methyl-L-decylamine- N -[(3 R , 4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzyloxy)-3-(1 H -吲哚- 3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy -5-Methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine is a colorless foam.

LC-MS (method 1): R _t = 1.45 min; m/z = 961 (M+H) ⁺ .

50 mg (52 μmol) of this intermediate was treated with 1 ml of trifluoroacetic acid in 9 ml of dichloromethane to isolate the Boc protecting group. Concentrated and purified to formula HPLC, to give 29 mg (57% of theory) of the free amine intermediate N - methyl -L- valinamide acyl - N - [(3 R, 4 S, 5 S) - 1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzyloxy)-3-(1 H -indol-3-yl)- 1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl -1-Sideoxyhept-4-yl] -N -methyl-L-decylamine amide is trifluoroacetate.

LC-MS (Method _{1): R t = 0.99 min} ; m / z = 861 (M + H) +.

Dissolve 29 mg (0.03 mmol) of this intermediate in 6 ml of two Alkane/water was added with 39 μl (0.059 mmol) of 15% aqueous 4-oxobutanoic acid. The reaction mixture was then stirred at 100 ° C for 1 h. After cooling to RT, 2 mg (0.033 mmol) of sodium cyanoborohydride was added and the mixture was added to about 300 μl of 0.1 N hydrochloric acid to adjust to pH 3. The reaction mixture was then stirred at 100 ° C for an additional 2 h. After cooling, another 39 μl (0.059 mmol) of a 15% 4-oxobutyric acid solution was added, and the reaction mixture was further stirred at 100 ° C for 1 h. Then 2 mg (0.033 mmol) of sodium cyanoborohydride was added and then the pH was adjusted back to 3 using about 300 μl of 0.1 N hydrochloric acid. The mixture was then stirred at 100 ° C for an additional 2 h. Thereafter, the reaction mixture was poured into a mixture of a 1:1 half-saturated aqueous ammonium chloride solution and ethyl acetate. The organic layer was removed, washed with a saturated sodium chloride solution, dried over magnesium sulfate and evaporated. The residue was lyophilized with water / acetonitrile. This gave 27 mg (94% of theory) of the title compound as a colourless foam.

HPLC (Method 5): R _t = 2.2 min; LC-MS (Method 9): R _t = 5.04 min; MS (ESIs): m/z = 947 (M+H) ⁺ .

Intermediate 73 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-({(2 S )-1-[benzyl(methyl)amino]-1-yloxy-3-phenylpropan-2-yl}amino)-1-methoxy Benzyl-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L - amidoxime

First, intermediates 4 and 38 were treated in a similar manner to that described in Intermediate 14, to prepare the amine compound N -methyl-L-amidino- N -[(3 R , 4 S , 5 S )-1 -{(2 S )-2-[(1 R ,2 R )-3-({(2 S )-1-[benzyl)amino]-1-oxo-3-phenylpropane -2-yl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-decylamine. Then 25 mg (0.026 mmol) of this compound was used, similar to the preparation of intermediate 61, by reaction with 4-oxobutyric acid in the presence of sodium cyanoborohydride to give 13 mg (54% of theory) The title compound is in the form of a colorless foam.

HPLC (method 12): _Rt = 2.2 min; LC-MS (Method 9): R _t = 5.01 min; MS (ESIs): m/z = 921 (M+H) ⁺ .

Intermediate 74 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-({(1 S ,2 R )-1-[(benzyloxy)carbonyl]-2-phenylcyclopropyl}amino)-1-methoxy-2-methyl -3-Siloxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

50 mg (73 μmol) of N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxanthene 4-yl] -N -methyl-L-decylamine (intermediate 26) and 28 mg (73 μmol) of (1 S , 2 R )-1-amino-2-phenylcyclopropanecarboxylate The acid benzyl ester trifluoroacetate (intermediate 45) was treated in 5 ml of DMF and 42 mg (110 μmol) of O- (7-azabenzotriazol-1-yl) -N was added. N,N',N' -tetramethylhexafluorophosphate And 38 μl of N,N -diisopropylethylamine. The reaction mixture was stirred at rt for 5 h then concentrated EtOAc. The product fractions were combined and concentrated. From two After lyophilization of the alkane/water, 35 mg (51% of theory) of Boc-protected intermediate N- (t-butoxycarbonyl) -N -methyl-L-decylamine- N -[( 3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-({(1 S ,2 R )-1-[(benzyloxy)carbonyl) ]-2-phenylcyclopropyl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl -1-Sideoxyheptan-4-yl] -N -methyl-L-decylamine amide is a colorless foam.

LC-MS (Method _{1): R t = 1.52 min} ; m / z = 934 (M + H) +.

The Boc protecting group was isolated by dissolving 35 mg of this intermediate in 1 ml of trifluoroacetic acid in 5 ml of dichloromethane. Concentrated and from two After dioxane / water and lyophilized to give 34 mg (97% of theory) of the free amine intermediate N - methyl -L- valinamide acyl - N - [(3 R, 4 S, 5 S) -1- {(2 S )-2-[(1 R ,2 R )-3-({(1 S ,2 R )-1-[(benzyloxy)carbonyl]-2-phenylcyclopropyl}amino) )-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]- N -methyl-L-guanamine amine is a trifluoroacetate salt.

LC-MS (Method _{1): R t = 0.91 min} ; m / z = 834 (M + H) +.

Then 11 mg (0.011 mmol) of this intermediate was used, similar to the preparation of intermediate 66, by reaction with 4-oxobutyric acid in the presence of sodium cyanoborohydride to give 2.5 mg (24% of theory) The title compound is in the form of a colorless foam.

HPLC (Method 12): R _t = 2.2 min; LC-MS (Method 9): R _t = 5.1 min; MS (ESIs): m/z = 920 (M+H) ⁺ .

Intermediate 75 N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )- 2-[(1 R ,2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 S ,2 R )-2-phenyl-1-(propylamine) Mercapto)cyclopropyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

First, by a synthesis similar to that described in Intermediate 14, by N- (t-butoxycarbonyl) -N -methyl-L-amidino-yl- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5- Keith-1-oxoheptan-4-yl] -N -methyl-L-decylamine amide (intermediate 26) and (1 S , 2 R )-1-amino-2-phenyl-n-propyl Cyclopropyrylamine trifluoroacetate (intermediate 27) in O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of and, followed by separation of the Boc protecting group with trifluoroacetic acid to prepare the amine compound N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy -1{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 S ,2 R )-2 -Phenyl-1-(propylaminemethanyl)cyclopropyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N - Methyl-L-guanamine indoleamine is a trifluoroacetate salt. This was then used to prepare 11.3 mg (83% of theory) using 14 mg (0.016 mmol) of this compound, which was obtained from the intermediate compound 61, by reacting with 4-oxobutyric acid in the presence of sodium cyanoborohydride. ) the title compound.

HPLC (Method 6): R _t = 1.9 min; LC-MS (Method 2): R _t = 1.27 min; MS (ESIs): m/z = 871 (M+H) ⁺ .

Intermediate 76 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(1 S ,2 R )-1-(ethoxycarbonyl)-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3 -Phenoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

First, by intermediate 46 ( N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxanthene -4-yl] -N -methyl-L-decylamine amide and intermediate 48 ((1 S , 2 R )-1-amino-2-phenylcyclopropanecarboxylate ethyl ester trifluoroacetate ) in O-(7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of, and subsequent separation of Boc, preparation of the starting compound N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2- [(1 R , 2 R )-3-{[(1 S ,2 R )-1-(ethoxycarbonyl)-2-phenylcyclopropyl]amino}-1-methoxy-2- Methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-amidamine Amine trifluoroacetate. Then 70 mg (0.079 mmol) of the title compound was obtained using EtOAc (EtOAc):

HPLC (method 6): R _t = 1.9 min; LC-MS (method 2): R _t = 1.28 min; MS (ESIs): m/z = 858 (M+H) ⁺

Intermediate 77 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-amino-1-one-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3 -Phenoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

First, by a synthesis similar to that described in Intermediate 75, by N-(t-butoxycarbonyl)-N-methyl-L-nonylamine-N-[(3R,4S,5S)-1 -{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-sideoxy Hept-4-yl]-N-methyl-L-decylamine (Intermediate 26) and L-Phenylalanine indoleamine hydrochloride in O-(7-azabenzotriazol-1-yl) )-N,N,N',N'-tetramethylhexafluorophosphate Coupling in the presence of the same, followed by separation of the Boc protecting group with trifluoroacetic acid to prepare the amine compound N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-1-{(2S)-2- [(1R,2R)-3-{[(2S)-1-amino-1-one-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl- 3-Phenoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine is three Fluoroacetate. Then, 47 mg (0.049 mmol) of this compound was used in a similar analogy to intermediate 61 to give 39 mg (96% of theory) by reaction with 4-oxobutyric acid in the presence of sodium cyanoborohydride. The title compound.

HPLC (method 6): R _t = 1.7 min; LC-MS (method 9): R _t = 4.44 min; MS (ESIs): m/z = 817 (M+H) ⁺

¹ H NMR (500 MHz, DMSO-d ₆ ): δ = 8.95 and 8.8 (2m, 1H), 8.25 and 8.0 (2d, 1H), 7.45, 7.35 and 7.0 (3s, broad, 2H), 7.3-7.1 ( m, 5H), 4.8-4.4 (2m, 3H), 3.95 (m, 1H), 3.82 (m, 1H), 3.72 (d, 1H), 3.22, 3.18, 3.15, 3.05 and 3.00 (5s, 9H), 2.85-2.7 (m, 4H), 2.45-1.6 (m, 12H), 1.5-1.2 (m, 3H), 1.1-0.7 (m, 21H) [more signals are hidden under the solvent peak].

Intermediate 78 N- (6-Aminohexyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )- 2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-amidamine

This compound was prepared in a two-stage phase similar to intermediate 66 starting from 20 mg (16 μmol) of compound from intermediate 14 and benzyl 6-oxohexylaminecarboxylate, and hydrogenation with methanol as solvent.

Yield: 7.6 mg (55% of theoretical value in 2 stages)

HPLC (Method 6): R _t = 1.8 min; LC-MS (Method 1): R _t = 0.7 min; MS (ESI): m/z = 901 (M+H) ⁺ .

Intermediate 79 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-(benzylamino)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2- Methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-amidoxime amine

36 mg (43 μmol) of N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(1 S )-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3- Sideoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine amide (Intermediate 46 And 4.6 mg (43 μmol) of benzylamine were treated in 5 ml of DMF, and 7.5 μl (88 μmol) of N,N -diisopropylethylamine, 10 mg (65 μmol) of HOBt and 10 mg (52 μmol) of EDC, and then the mixture was stirred at RT until overnight. Subsequently, the reaction mixture was concentrated and the residue was purified by preparative HPLC. Obtained 29 mg (73% of theory) of Boc-protected intermediate N -(t-butoxycarbonyl) -N -methyl-L-decylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzylamino)-1-sideoxy-3-phenylpropane -2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-decylamine.

LC-MS (method 1): R _t = 1.43 min; m/z = 921 (M+H) ⁺ .

29 mg of this intermediate was dissolved in 6 ml of dichloromethane in 1 ml of trifluoroacetic acid to isolate the Boc protecting group. Concentrate and from two After lyophilization of the alkane/water, 30 mg (quant.) of the free amine intermediate N -methyl-L-decyl fluorenyl- N -[(3 R , 4 S , 5 S )-1-{(2) S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzylamino)-1-yloxy-3-phenylpropan-2-yl]amino} 1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -Methyl-L-Amidoxime is a trifluoroacetate salt.

LC-MS (method 1): R _t = 0.95 min; m/z = 821 (M+H) ⁺ .

Then 17 mg (0.018 mmol) of this intermediate was used, similar to the preparation of intermediate 61, by reaction with 4-oxobutyric acid in the presence of sodium cyanoborohydride to give 13 mg (80% of theory) The title compound is in the form of a colorless foam.

HPLC (Method 5): R _t = 1.7 min; LC-MS (Method 9): R _t = 4.97 min; MS (ESI): m/z = 907 (M+H) ⁺ .

Intermediate 80 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-(benzylamino)-3-(1 H -indol-3-yl)-1-oxopropan-2-yl]amino} 1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-amidamine

First, by a synthesis similar to that described in Intermediate 74, by N- (Tertilybutoxycarbonyl) -N -methyl-L-amidoxime- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5- Keith-1-oxoheptan-4-yl] -N -methyl-L-decylamine amide (intermediate 26) and N -benzyl-L-tryptamine guanamine trifluoroacetate (intermediate 47) O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of the same, and subsequent separation of the Boc protecting group with trifluoroacetic acid to prepare the amine compound N -methyl-L-decyl fluorenyl- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzylamino)-3-(1 H -indol-3-yl)-1-yloxy Prop-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-side Oxyheptan-4-yl] -N -methyl-L-nonylamine decylamine is a trifluoroacetate salt. Using 10 mg (0.01 mmol) of this compound, similar to intermediate 61, was obtained by reaction with 4-oxobutyric acid in the presence of sodium cyanoborohydride to give 2.5 mg (26% of theory). Title compound.

HPLC (Method 5): R _t = 1.7 min; LC-MS (Method 2): R _t = 1.13 min; MS (ESIs): m/z = 946 (M+H) ⁺ .

Intermediate 81 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(1 S ,2 R )-1-amine-methylmethyl-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3- side Oxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

First, by a synthesis similar to that described in Intermediate 74, by N- (Tertilybutoxycarbonyl) -N -methyl-L-amidoxime- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5- Keith-1-oxoheptan-4-yl] -N -methyl-L-decylamine (intermediate 26) and (1 S , 2 R )-1-amino-2-phenylcyclopropane Indole trifluoroacetate (intermediate 48) in O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of the same, and subsequent separation of the Boc protecting group with trifluoroacetic acid to prepare the amine compound N -methyl-L-decyl fluorenyl- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(1 S ,2 R )-1-Aminomethylamino-2-phenylcyclopropyl]amino}-1-methoxy -2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L- The amidoxime is a trifluoroacetate salt. Using 14 mg (0.0163 mmol) of this compound, similar to intermediate 61, was obtained by reaction with 4-oxobutyric acid in the presence of sodium cyanoborohydride to give 8 mg (57% of theory) Title compound.

HPLC (Method 5): R _t = 1.6 min; LC-MS (Method 9): R _t = 4.64 min; MS (ESIs): m/z = 829 (M+H) ⁺ .

Intermediate 82 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2-yl]amino}-1-yl Oxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl- L-Amidoxime

First, in a manner similar to the synthesis of the intermediate 69, with N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N - [( 2 R , 3 S , 4 S )-1-carboxy-2-methoxy-4-methylhex-3-yl]- N -methyl-L-decylamine (Intermediate 4) and N ^α -{(2 R ,3 R )-3-methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propanyl}-L-tryptamine guanamine trifluoroacetic acid Salt (Intermediate 49) in O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of the same, and then separating the Fmoc protecting group with piperidine to prepare the amine compound N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2-yl Amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4- The base] -N -methyl-L-guanamine amine is a trifluoroacetate salt. Then 78 mg (0.088 mmol) of this compound was used in a similar analogy to intermediate 61 to afford 68 mg (yield: 90%) from 4-oxobutyric acid in the presence of sodium cyanoborohydride. The title compound.

HPLC (Method 5): R _t = 1.8 min; LC-MS (Method 9): R _t = 4.49 min; MS (ESIs): m/z = 856 (M+H) ⁺ .

Intermediate 83 N- (5-carboxypentyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2-yl]amino}-1-yl Oxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl- L-Amidoxime

This compound is similar to the compound in intermediate 82, consisting of 20 mg (26 μmol) of N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2- Amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4 Starting from -N -methyl-L-guanamine indole trifluoroacetate, prepared by reaction with 4-oxobutyric acid in the presence of sodium cyanoborohydride.

Yield: 5 mg (25% of theory)

HPLC (method 5): _Rt = 1.6 min; LC-MS (Method 11): R _t = 0.72 min; MS (ESIs): m/z = 884 (M+H) ⁺ .

Intermediate 84 N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )- 2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(morpholine-4-yl)-1-oxo-3- Phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L- Amidoxime

First, by a synthesis similar to that described in Intermediate 79, by N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(1 S )-1-carboxy-2-phenylethyl]amino}-1-methoxy- 2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-indole Amine amide (intermediate 46) and morpholine, coupled in the presence of EDC and HOBT, and subsequent separation of the Boc protecting group with trifluoroacetic acid to prepare the amine compound N -methyl-L-nonylamine fluorenyl- N- [( 3R , 4 S , 5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{ [(2 S )-1-(morpholine-4-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl }-5-Methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine is a trifluoroacetate salt. Then 30 mg (0.033 mmol) of this compound was used, similar to intermediate 61, to give 22 mg (76% of theory) by reaction with 4-oxobutyric acid in the presence of sodium cyanoborohydride. The title compound.

HPLC (Method 5): R _t = 1.6 min; LC-MS (Method 9): R _t = 4.58 min; MS (ESIs): m/z = 887 (M+H) ⁺ .

Intermediate 85 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S ,3 R )-1-(benzylamino)-3-hydroxy-1-oxobutan-2-yl]amino}-1-methoxy- 2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-indole Amine

First, by a synthesis similar to that described in Intermediate 79, by N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(1 S )-1-carboxy-2-phenylethyl]amino}-1-methoxy- 2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-indole Amine amine (Intermediate 46) and N -benzyl-L-thretonamide trifluoroacetate, coupled in the presence of HATU, and subsequent separation of the Boc protecting group with trifluoroacetic acid to prepare the amine compound N -methyl -L-Amidoxime- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S ,3 R )-1-(benzylamino)-3-hydroxy-1-oxobutan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine 1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine is a trifluoroacetate salt. Then 21 mg (0.024 mmol) of this compound was used in a similar analogy to intermediate 61 to afford 20 mg (97% of theory) by reaction with 4-oxobutyric acid in the presence of sodium cyanoborohydride. The title compound.

HPLC (Method 5): R _t = 1.54 min; LC-MS (Method 9): R _t = 4.49 min; MS (ESIs): m/z = 861 (M+H) ⁺ .

Intermediate 86 N-(3-carboxypropyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3 -{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropane Pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

First, by a synthesis similar to that described in Intermediate 74, by N- (Tertilybutoxycarbonyl) -N -methyl-L-amidoxime- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5- Base-1-oxoheptan-4-yl] -N -methyl-L-decylamine (intermediate 26) and tert-butyl-L-phenylalanine hydrochloride, at O- (7- Azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of the same, and subsequent separation of the Boc protecting group with trifluoroacetic acid to give the third butyl ester (with trifluoroacetic acid in dichloromethane for 40 minutes) to prepare the amine compound N -methyl-L-amidamine -N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1 -Tertidinoxy Keto- 1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine is a trifluoroacetate salt. Then 22 mg (0.02 mmol) of this compound was used, similar to intermediate 61, to give 16 mg (94% of theory) by reaction with 4-oxobutyric acid in the presence of sodium cyanoborohydride. The title compound.

HPLC (Method 5): R _t = 2.0 min; LC-MS (Method 9): R _t = 5.05 min; MS (ESI): m/z = 874 (M+H) ⁺ .

Intermediate 87 N-(3-carboxypropyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3 -{[(2S)-1-tert-butoxy-3-(1H-indol-3-yl)-1-oxooxypropan-2-yl]amino}-1-methoxy-2- Methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-amidamine amine

This compound was synthesized as described in Intermediate 86, from 230 mg (336 μmol) of N -(t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[( 3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3 -Methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine amide (Intermediate 26) and tert-butyl-L-tryptophanate The acid salt was prepared starting in 3 stages.

Yield: 95 mg (31% of theoretical value in 3 stages)

HPLC (Method 5): R _t = 2.0 min; LC-MS (Method 9): R _t = 5.05 min; MS (ESIs): m/z = 913 (M+H) ⁺ .

Intermediate 88 N- (6-Aminohexyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2-yl]amino}-1-yl Oxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl- L-Amidoxime

First, by a synthesis similar to that described in Intermediate 69, by N -[(9H-茀-9-ylmethoxy)carbonyl] -N -methyl-L-nonylamine- N -[(2) R , 3 S , 4 S )-1-carboxy-2-methoxy-4-methylhex-3-yl]- N -methyl-L-decylamine (Intermediate 4) and N ^α - {(2 R ,3 R )-3-methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propanyl}-L-tryptamine guanamine trifluoroacetate (Intermediate 49) in O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of the same, and subsequent separation of the Fmoc protecting group with piperidine to prepare the amine compound N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2-yl Amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4- The base] -N -methyl-L-guanamine amine is a trifluoroacetate salt. Then 30 mg (0.03 mmol) of this compound was used, in the preparation of a compound analogous to intermediate 61, by oxidizing with benzyl 6-xyloxyhexylamine (which previously oxidized 6-hydroxyhexylamine benzyl ester) The reaction was carried out in the presence of sodium cyanoborohydride to give 17 mg (45% of theory) of Z-protective compound. Subsequent hydrogenolysis on 10% palladium on activated carbon in methanol gave the title compound.

Yield: 14 mg (95% of theory)

HPLC (Method 5): R _t = 1.5 min; LC-MS (Method 1): R _t = 0.73 min; MS (ESIs): m/z = 869 (M+H) ⁺ .

Intermediate 89 N- (6-Aminohexyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1 -tert-butoxy- 3-(1 H -indol-3-yl)-1-oxopropan-2-yl]amino}- 1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N - methyl-L-melamine

First, by a synthesis similar to that described in Intermediate 86, by N- ( t- butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5- Keith-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine (intermediate 26) and tert-butyl-L-tryptophanate hydrochloride at O- (7 -azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of the same, and subsequent separation of the Boc protecting group with trifluoroacetic acid to give the third butyl ester (agitated with 1:10 trifluoroacetic acid / dichloromethane for 30 min) to prepare the amine compound N -methyl-L-indole Aminyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1- third Butoxy- 3-(1 H -indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl] Pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine is a trifluoroacetate salt. Then 71 mg (0.075 mmol) of this compound was used, similar to the preparation of intermediate 61, by oxidizing with benzyl 6-oxohexylamineamine which was previously oxidized with benzyl 6-hydroxyhexylaminecarboxylate. The reaction was carried out in the presence of sodium cyanoborohydride to give 35 mg (44% of theory) of Z-protective compound. Subsequent hydrogenolysis on 10% palladium on activated carbon in methanol gave the title compound.

Yield: 30 mg (98% of theory)

HPLC (method 5): Rt = 1.9 min; LC-MS (method 1): Rt = 0.77 min; MS (ESI): m/z = 926 (M+H)+.

Intermediate 90 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[2-(1 H -indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine- 1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

First, similar to the synthesis of intermediate 74, with N - (tert-butoxy-carbonyl) - N - methyl -L- valinamide acyl - N - [(3 R, 4 S, 5 S )-1-{(2 S }2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl -1-Sideoxyheptan-4-yl] -N -methyl-L-decylamine amide (Intermediate 26) and 2-( 1H -indol-3-yl)ethylamine in O- (7 -azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of the same, and subsequent separation of the Boc protecting group with trifluoroacetic acid to prepare the amine compound N -methyl-L-decyl fluorenyl- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[2-(1 H -indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3 -Phenoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine is trifluoro Acetate. Then 100 mg (0.119 mmol) of this compound was used, similar to the preparation of intermediate 61, by reacting with 4-oxobutyric acid in the presence of sodium cyanoborohydride to give 50 mg (49% of theory) The title compound. The title compound was purified by flash chromatography on silica gel using dichloromethane/methanol/17% aqueous ammonia as solvent. During this time, the mixture ratio was changed from the original 15/2/02 to 15/4/0.5.

HPLC (Method 6): R _t = 1.8 min; LC-MS (Method 1): R _t = 0.87 min; MS (ESI): m/z = 813 (M+H) ⁺ .

Intermediate 91 N- (3-carboxypropyl) -N -methyl-L-decylamine- N -{(3 R ,4 S ,5 S )-3-methoxy-1-[(2 S )- 2-{(1 R ,2 R )-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl] -5-methyl-1-oxoheptan-4-yl}- N -methyl-L-nonylamine decylamine

First, by a synthesis similar to that described in Intermediate 74, by N- (Tertilybutoxycarbonyl) -N -methyl-L-amidoxime- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5- Base-1-one-oxyheptan-4-yl] -N -methyl-L-decylamine amide (intermediate 26) and phenethylamine in O- (7-azabenzotriazole-1- Base) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of the same, and subsequent separation of the Boc protecting group with trifluoroacetic acid to prepare the amine compound N -methyl-L-decyl fluorenyl- N -{(3 R , 4 S , 5 S )-3-methoxy -1-[(2 S )-2-{(1 R ,2 R )-1-methoxy-2-methyl-3-oxo-3-yl([2-phenylethyl)amino]propane The base pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl} -N -methyl-L-nonylamine decylamine is a trifluoroacetate salt. Then 57 mg (0.071 mmol) of this compound was used, similar to intermediate 61, to give 44 mg (80% of theory) by reaction with 4-oxobutyric acid in the presence of sodium cyanoborohydride. The title compound. The title compound can also be purified by flash chromatography on silica gel eluting with dichloromethane/methanol/17% aqueous ammonia as solvent (15/2/02->15/4/0.5).

HPLC (Method 5): R _t = 1.7 min; LC-MS (Method 9): R _t = 4.64 min; MS (ESI): m/z = 774 (M+H) ⁺ .

Intermediate 92 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(1 S ,2 R )-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-lateral oxygen Propyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

Using 100 mg (0.139 mmol) of N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[( 1R ,2 R )-3-{[(1 S ,2 R )-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl] Pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine amide (intermediate 40), similar to the middle The title compound was obtained by the reaction of 4-oxo-oxybutyric acid in the presence of sodium cyanoborohydride to give 94 mg (yield: 84%) of the title compound. The title compound was purified by preparative HPLC.

HPLC (Method 5): R _t = 1.5 min; LC-MS (Method 9): R _t = 4.46 min; MS (ESI): m/z = 804 (M+H) ⁺ .

Intermediate 93 N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R, 4 S, 5 S )-3-methoxy-1-{(2 S )- 2-[(1 R, 2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 S )-2-phenyl-1-(5-phenyl-1 , 3,4-two Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

22.4 mg (24 μmol) of N -methyl-L-amidino- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2- [(1 R , 2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 S )-2-phenyl-1-(5-phenyl-1,3 , 4-two Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine Trifluoroacetate dissolved in 1.4 ml of two Alkane/water, and similar intermediates 61 were prepared and reacted with 15% aqueous 4-oxobutanoic acid in the presence of sodium cyanoborohydride. From two After lyophilization of the alkane, 8.2 mg (yield: 38%) of the title compound was obtained as white solid.

HPLC (Method 10): R _t =2.54 min

LC-MS (Method 12): R _t = 0.94 min; MS (ESIs): m/z = 919 (M+H) ⁺

Intermediate 94 N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R, 4 S, 5 S )-3-methoxy-1-{(2 S )- 2-[(1 R, 2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 R )-2-phenyl-1-(5-phenyl-1 , 3,4-two Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

17.1 mg (18 μmol) of N -methyl-L-nonylamine- N -[(3 R, 4 S , 5 S )-3-methoxy-1-{(2 S )-2- [(1 R , 2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 R )-2-phenyl-1-(5-phenyl-1,3 , 4-two Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine Trifluoroacetate dissolved in 1.1 ml of two The alkane/water, and similar intermediate 61 was prepared by reaction with 15% aqueous 4-oxobutanoic acid in the presence of sodium cyanoborohydride. From two After lyophilization of the alkane, 14.8 mg (yield: 89%) of the title compound was obtained as white solid.

HPLC (method 10): R _t =2.54 min; LC-MS (method 12): R _t = 0.92 min; MS (ESIs): m/z = 919 (M+H) ⁺

Intermediate 95 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R, 4 S, 5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-(benzylsulfonyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3 -Phenoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

19.3 mg (20 μmol) N -methyl-L-nonylamine- N -[(3 R, 4 S, 5 S )-1-{(2 S )-2-[(1 R, 2 R )-3-{[(2 S )-1-(benzylsulfonyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxo Propyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine guanamine trifluoroacetate is soluble 1.2 ml of two The alkane/water, and similar intermediate 61 was prepared by reaction with 15% aqueous 4-oxobutanoic acid in the presence of sodium cyanoborohydride. From two After lyophilization of the alkane, 8.6 mg (45% of theory)

LC-MS (Method 11): R _t = 0.85 min; MS (ESI): m/z = 943 (M+H) ⁺

Intermediate 96 N- (3-carboxypropyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R, 4 S, 5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S, 3 E )-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3 -Phenoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

15.5 mg (10 μmol) of N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S ,3 E )-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3- side Oxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine guanamine trifluoroacetic acid salt In 1.0 ml of two The alkane/water, and similar intermediates 61 were prepared by reaction with 15% aqueous 4-oxobutanoic acid in the presence of sodium cyanoborohydride. From two After lyophilization of the alkane, 10.3 mg (yield: 68%) of title compound was obtained as white solid.

HPLC (method 10): R _t = 2.59 min; LC-MS (Method 11): R _t = 0.94 min; MS (ESIs): m/z = 877 (M+H) ⁺

Intermediate 97 N- (6-Aminohexyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )- 2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

The title compound was synthesized as a similar intermediate 66 by using 200 mg (0.108 mmol) of N -methyl-L-indoleamine- N -[(3 R , 4 S , 5 S )-3-methoxy Base-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2 - Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - reaction of N -methyl-L-guanamine guanamine trifluoroacetate (intermediate 16) with benzyl 6-oxohexylamine amide, and subsequent hydrogenolysis of the Z protecting group (at 5% activated carbon) Palladium was used as a catalyst and methanol was used as a solvent.

Yield: 69 mg (65% of the theoretical value in the second stage)

HPLC (Method 5): R _t = 1.7 min; LC-MS (Method 1): R _t = 0.76 min; MS (ESI): m/z = 912 (M+H) ⁺ .

Intermediate 98 N- (5-carboxypentyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-(benzylamino)-3-(1 H -indol-3-yl)-1-oxopropan-2-yl]amino} 1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-amidamine

This compound was prepared in a similar manner to that described in Intermediate 80. Purification was carried out by preparative HPLC.

Yield: 40 mg (29% of theoretical value in three stages)

HPLC (method 5): R _t = 1.9 min; LC-MS (Method 1): R _t = 0.92 min; MS (ESI): m/z = 974 (M+H) ⁺ .

Intermediate 99 (2 S )-2-amino-3-(1 H -indol-3-yl)-1-(1,2- Alkan-2-yl)propan-1-one trifluoroacetate

324 mg (0.81 mmol) of 2,5-dioxapyrrolidin-1-yl N- (t-butoxycarbonyl)-L-tryptophanate was dissolved in 20 ml of DMF and added to 200 mg ( 1.62-mmol) 1,2- Alkanoic acid hydrochloride (starting compound 5) and 850 μl of N,N -diisopropylethylamine. The reaction mixture was stirred at 50 ° C overnight and then concentrated under reduced pressure. The residue was taken up in dichloromethane and extracted with water. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by flash chromatography on silica gel eluting with 4:1 dichloromethane/ethyl acetate as solvent. The product was concentrated and the residue was dried under high vacuum. This gave 147.5 mg (48% of theory) of Boc-protected intermediate.

HPLC (Method 12): R _t = 1.9 min; LC-MS (Method 1): R _t = 1.03 min; MS (ESI): m/z = 374 (M+H) ⁺ .

Using 166 mg (444.5 μmol) of this intermediate, the Boc protecting group was separated under standard conditions with 3 ml of trifluoroacetic acid in 20 ml of dichloromethane. After purification by HPLC, 155 mg (86% theory) Value) of the title compound.

HPLC (Method 12): R _t = 1.43 min; LC-MS (Method 11): R _t = 0.56 min; MS (ESIs): m/z = 274 (M+H) ⁺ .

Intermediate 100 N- (6-{[(benzyloxy)carbonyl]amino}hexyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{( 2 S )-2-[(1 R ,2 R )-3-{[(2 S )-3-(1 H -吲哚-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

177 mg (260 μmol) of N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxanthene 4-yl] -N -methyl-L-decylamine (Intermediate 26) and 100 mg (260 μmol) of ( 2S )-2-amino-3-( 1H -indole-3) -base)-1-(1,2- Alkyl-2-yl)propan-1-one trifluoroacetate (Intermediate 99) was taken up in 15 ml of DMF and added 118 mg (310 μmol) of O- (7-azabenzotriazole- 1-yl) -N,N,N',N' -tetramethylhexafluorophosphate And 140 μl of N,N -diisopropylethylamine. The reaction mixture was stirred at RT for 30 min then concentrated over EtOAc. The product fractions were combined and concentrated. From two After lyophilization of the alkane, 170 mg (68% of theory) of Boc-protected intermediate was obtained.

LC-MS (method 1): R _t = 1.36 min; m/z = 940 (M+H) ⁺ .

170 mg of this intermediate was treated with a solution of 3 ml of trifluoroacetic acid in 30 ml of dichloromethane to isolate the Boc protecting group. The reaction mixture was then concentrated under reduced pressure and purified title purified mjjjjjjjjjjjjjjjjjjjj ,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine intermediate.

HPLC (Method 12): R _t = 1.85 min; LC-MS (Method 1): R _t =0.86 min; MS (ESIs): m/z = 840 (M+H) ⁺ .

Then 50 mg (0.052 mmol) of this intermediate was used, similar to the preparation of intermediate 97, with benzyl 6-oxohexylaminecarboxylate in the presence of sodium cyanoborohydride, and subsequent hydrogenolysis of the Z protecting group. The title compound was prepared by separation (palladium on 5% activated carbon) and methanol as solvent.

Yield: 21 mg (37% of theory)

HPLC (method _{12): R t = 2.1 min} ; LC-MS ( Method _{1): R t = 1.02 min} ; MS (ESIpos): m / z = 1073 (M + H) +.

Intermediate 101 N- (6-Aminohexyl) -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-3-(1 H -indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

26.7 mg (24.87 μmol) of intermediate 100 was dissolved in 10 ml of methanol and hydrogenated on palladium on activated carbon (5%) under standard hydrogen pressure for 30 min. The catalyst was filtered off and the solvent was evaporated under reduced pressure. The residue was dried under high vacuum to give 22.5 mg (yiel.

HPLC (Method 5): R _t = 1.7 min; LC-MS (Method 1): R _t = 0.76 min; MS (ESI): m/z = 939 (M+H) ⁺ .

Intermediate 102 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(morpholine-4-yl)-1-oxo-3-phenylpropan-2-yl] Amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

This compound was synthesized as described in Intermediate 157 from N- (3-carboxypropyl) -N -methyl-L-decylamine- N -[(3 R , 4 S , 5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-( Morpholine-4-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1- Bis-oxyheptan-4-yl] -N -methyl-L-decylamine and commercially available 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl ) Prepared by hexane.

Yield: 8 mg (71% of theory)

HPLC (Method 12): R _t = 1.9 min; LC-MS (Method 1): R _t = 0.87 min; MS (ESIs): m/z=1094 (M+H) ⁺ .

Intermediate 103 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(2 S ,3 R )-1-(benzylamino)-3-hydroxy-1-oxobutan-2-yl]amino}-1-methoxy-2-methyl-3- side Oxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

This compound was synthesized as described in Intermediate 157 from N- (3-carboxypropyl) -N -methyl-L-decylamine- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S ,3 R )-1-(benzylamino)-3-hydroxy-1-sided oxygen But-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-side Oxyheptan-4-yl] -N -methyl-L-decylamine and commercially available 6-(2,5-di- oxo-2,5-dihydro-1 H -pyrrol-1-yl) Prepared by hexane.

Yield: 3 mg (22% of theory)

HPLC (method _{5): R t = 1.6 min} ; LC-MS ( Method _{1): R t = 0.78 min} ; MS (ESIpos): m / z = 1069 (M + H) +.

Intermediate 104 N -{4-[(trans-4-{[(2,5-dioxapyrrolidin-1-yl)oxy)carbonyl}cyclohexyl)amino]-4-oxobutyl}- N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3- side Oxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

First, trans-4-aminocyclohexanecarboxylic acid benzyl ester III was prepared from trans-4-aminocyclohexanecarboxylic acid by introducing a Boc protecting group and subsequently separating the Boc protecting group by conventional peptide chemical method. Fluoroacetate.

Then 15 mg (18 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2- Amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4 -yl] -N -methyl-L-amidamine dissolved in 5 ml of dimethylformamide and subsequently with 13 mg (35 μmol) of O- (7-azabenzotriazole-1- Base) -N,N,N',N' -tetramethylhexafluorophosphate 9 μl of N,N -diisopropylethylamine was mixed with 15 mg (44 μmol) of trans-4-aminocyclohexanecarboxylic acid benzyl ester trifluoroacetate. The mixture was stirred at RT for 1 h and then concentrated under reduced pressure. The remaining residue was purified by preparative HPLC. The product fractions were combined and the solvent was evaporated under reduced pressure. After drying the residue under high vacuum, 14.7 mg (yield: 78% of theory) of intermediates were obtained as a colourless foam.

HPLC (Method 6): R _t = 2.0 min; LC-MS (Method 1): R _t = 0.95 min; MS (ESIs): m/z=1072 (M+H) ⁺ .

The intermediate thus protected first removes the benzyl ester by hydrogenolysis and yields a free carboxyl component of a certain yield. 14 mg (14 μmol; 1 equiv.) of the deprotected compound was placed in 5 ml of DMF and treated with 3.3 mg (29 μmol; 2.1 equiv.) of N -hydroxysuccinimide at 4.1 mg (21 μmol; 1.5 equiv.) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 7.5 μl (44 μmol; 3.1 equiv.) of N,N -diisopropyl The ethylamine was mixed with 0.9 mg (7 μmol; 0.5 equiv.) of 4-dimethylaminopyridine, and the mixture was stirred at RT overnight. Then add 10 equivalents of N -hydroxysuccinimide, 5 equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 5 equivalents of N,N - Diisopropylethylamine and 0.5 equivalents of 4-dimethylaminopyridine were used and the reaction mixture was treated with an ultrasonic bath for 5 h. Subsequently, the solvent is evaporated, the residue is purified by preparative HPLC and the corresponding fractions are combined and concentrated, and the residue is taken from After lyophilization of the alkane, 9.7 mg (yield: 62%)

HPLC (method 6): _Rt = 1.8 min; LC-MS (Method 11): R _t = 0.77 min; MS (ESI s): m/z = 1078 (M+H) ⁺ .

Intermediate 105 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(1 S )-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy 5--5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine amide

This compound was prepared as described in Intermediate 157, from 4-{[( 2S )-1-{[( 2S )-1-{[(3 R ,4 S ,5 S )- 1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl Amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4- (methyl)amino}-3-methylbutan-2-yl]amino}-3-methyl-1-oxobutan-2-yl](methyl)amino}butyric acid and the city The preparation of 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane oxime was started. An ester intermediate of 42% yield was obtained. In the second step, 6 mg (6 μmol) of this intermediate was cleaved with trifluoroacetic acid to cleave the third butyl ester. After purification by HPLC, 3.4 mg (yield: 48%)

HPLC (method _{5): R t = 1.66 min} ; LC-MS ( Method _{2): R t = 1.04 min} ; MS (ESIpos): m / z = 1025 (M + H) +.

Intermediate 106 N -[6-(2,5-di- oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] -N -methyl-L-nonylamine fluorenyl- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-Amino-3-(1 H -吲哚) -3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy 5--5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine amide

14 mg (16 μmol) of N- (6-aminohexyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2-yl Amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4- Base] -N -methyl-L-decylamine (intermediate 88) placed in 750 μl of two Treated with alkane and with 1.5 ml of saturated sodium bicarbonate solution and then with 3.2 mg (21 μmol) of 2,5-di-oxo-2,5-dihydro-1 H -pyrrole-1-carboxylic acid methyl ester mixing. The reaction mixture was stirred at RT for 1 h then concentrated EtOAc. The remaining residue was purified by preparative HPLC. After lyophilization, 5.5 mg (36% of theory) of title compound.

HPLC (Method 5): R _t = 1.7 min; LC-MS (Method 1): R _t = 0.84 min; MS (ESIs): m/z = 949 (M+H) ⁺ .

Intermediate 107 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [2-( 1H -indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

38 mg (47 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[2-(1 H -indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3- Side oxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine amide is dissolved in 37 ml DMF, and then with 71 mg (187 μmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate And 33 μl of N,N -diisopropylethylamine and 37 mg (140 μmol) of commercially available 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrole-1 -Base) Hexane oxime is mixed. The mixture was stirred at RT for 1 h. It was then concentrated under high vacuum and the residue was purified using preparative HPLC. This gave 12.2 mg (26% of theory) of title compound as colourless foam.

HPLC (Method 5): R _t = 1.6 min; LC-MS (Method 1): R _t = 0.85 min; MS (ESIs): m/z = 020 (M+H) ⁺ .

Intermediate 108 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -{(3 R ,4 S ,5 S )-3-methoxy-1-[(2 S )-2-{(1 R ,2 R )-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1- Side oxyheptan-4-yl}- N -methyl-L-nonylamine decylamine

This compound was prepared in a similar intermediate 107.

Yield: 2.5 mg (30% of theory)

HPLC (Method 12): R _t = 1.9 min; LC-MS (Method 1): R _t = 0.9 min; MS (ESI): m/z = 981 (M+H) ⁺ .

Intermediate 109 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(1 S , 2 R )-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1- -3-}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

This compound, similar to intermediate 107, was prepared from intermediate 92.

Yield: 35 mg (65% of theory)

HPLC (Method 5): R _t = 1.9 min; LC-MS (Method 11): R _t = 0.76 min; MS (ESIs): m/z=1011 (M+H) ⁺ .

Intermediate 110 N -{6-[(2,5-di-oxaxopyridin-1-yl)oxy]-6-oxo-oxyhexyl}- N -methyl-L-nonylamine fluorenyl- N -[(3 R , 4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-Amino-3-(1 H -吲哚-3 -yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy- 5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

This compound is similar to intermediate 147, from intermediate 83 The compound was prepared.

Yield: 2.4 mg (24% of theory)

HPLC (Method 6): R _t = 1.8 min; LC-MS (Method 1): R _t = 0.87 min; MS (ESIs): m/z = 981 (M+H) ⁺ .

Intermediate 111 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl]-1-methylindenyl}-4 - side oxybutyl) -N -methyl-L-decylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R -3{[(2 S )-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2 -methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine Guanamine

This compound, similar to intermediate 140, was prepared from intermediate 82 and intermediate 22.

Yield: 6.5 mg (51% of theory)

HPLC (Method 6): R _t = 1.8 min; LC-MS (Method 1): R _t = 4.71 min; MS (ESI s): m/z=1077 (M+H) ⁺ .

Intermediate 112 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(1 S , 2 R )-1-Aminomethylguanidino-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1 -yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

This compound, similar to intermediate 157, was prepared from the compound in intermediate 81.

Yield: 5.7 mg (57% of theory)

HPLC (method 5): _Rt = 1.6 min; LC-MS (Method 1): R _t = 0.87 min; MS (ESIs): m/z = 1 036 (M+H) ⁺ .

Intermediate 113 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(1 S )-1-carboxy-2-(1 H -indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine -1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

95 mg (104 μmol) of 4-{[(2 S )-1-{[(2 S )-1-{[(3 R ,4 S ,5 S )-1-{(2 S )-2 -[(1 R ,2 R )-3-{[(2 S )-1-tert-butoxy-3-(1 H -indol-3-yl)-1-oxopropan-2-yl Amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4- (M)aminomethyl}-3-methylbutan-2-yl]amino}-3-methyl-1-oxobutan-2-yl](methyl)amino}butyric acid DMF, and then with 79.5 mg (209 μmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate 73 μl of N,N -diisopropylethylamine and 68 mg (261 μmol) of commercially available 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrole-1 -Base) Hexane oxime is mixed. The mixture was stirred at RT for 2 h. It was then concentrated under high vacuum and the residue was purified using preparative HPLC. The title compound of 104 mg (89% of theory) of yt.

HPLC (Method 5): R _t = 2.0 min; LC-MS (Method 1): R _t = 0.93 min; MS (ESIs): m/z=1121 (M+H) ⁺ .

The intermediate was placed in 33.4 ml of dichloromethane and added to 17 Trifluoroacetic acid in ml and the mixture was stirred at RT for 1 h. Subsequently, the reaction mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC.

Thus 61 mg (62% of theory) of title compound was obtained as colourless foam.

HPLC (method _{5): R t = 1.7 min} ; LC-MS ( Method _{1): R t = 0.86 min} ; MS (ESIpos): m / z = 1064 (M + H) +.

Intermediate 114 N -[6-({[2-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)ethyl]amine-carbamoyl}amino)hexyl] -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3- side Oxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

5 mg (5 μmol) of N- (6-aminohexyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2-yl Amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4- Base] -N -methyl-L-decylamine in 885 μl of DMF and with 5.3 mg (8 μmol) of 4-nitrophenyl 2-(2,5-di-oxo-2 , 5-Dihydro-1 H -pyrrol-1-yl)ethylamine formate and 2.8 μl of N,N -diisopropylethylamine were mixed. The reaction mixture was stirred at RT for 2 h then concentrated to dryness. The residue was purified by preparative HPLC.

Yield: 0.58 mg (11% of theory) of colorless foam.

HPLC (method _{5): R t = 1.6 min} ; LC-MS ( Method _{1): R t = 0.83 min} ; MS (ESIpos): m / z = 1035 (M + H) +.

Intermediate 115 N -{4-[(2,5-Dioxapyrrolidin-1-yl)oxy]-4-oxobutylbutyl}- N -methyl-L-nonylamine fluorenyl- N -[(3 R , 4 S , 5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[( 1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

This compound was prepared in a similar analog of intermediate 147 from 8 mg (9 μmol) of N- (3-carboxypropyl) -N -methyl-L-indoleamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S , 2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-guanamine amine. After concentration, the activated ester was purified by preparative HPLC and immediately after removal of the solvent under reduced pressure.

Yield: 3 mg (27% of theory) (hydrolysis-sensitivity)

HPLC (Method 5): R _t = 1.7 min; LC-MS (Method 1): R _t = 0.87 min; MS (ESIs): m/z = 996 (M+H) ⁺ .

Intermediate 116 N -{4-[(2,5-Dioxapyrrolidin-1-yl)oxy]-4-oxobutylbutyl}- N -methyl-L-nonylamine fluorenyl- N -[(3 R , 4 S , 5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[( 2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-amidamine

This compound was prepared in a similar manner to the compound in Intermediate 147, from 5 mg (6 μmol) of N- (3-carboxypropyl) -N -methyl-L-indoleamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-guanamine amide is used. After concentration, the activated ester was purified by preparative HPLC and immediately after removal of the solvent under reduced pressure.

Yield: 3.2 mg (43% of theory) (hydrolysis-sensitivity)

HPLC (method 5): R _t = 1.7 min; LC-MS (Method 1): R _t = 0.92 min; MS (ESIs): m/z = 984 (M+H) ⁺ .

Intermediate 117 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(2 S )-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl Pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

This compound, similar to intermediate 157, was prepared from the compound in intermediate 86.

Yield: 7 mg (42% of theory)

HPLC (method 5): _Rt = 1.6 min; LC-MS (Method 1): R _t = 0.94 min; MS (ESI s): m/z=1081 (M+H) ⁺ .

Intermediate 118 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [( 2R )-1-(Benzyloxy)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1 -yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

The title compound, analogous to intermediate 157, was prepared from 7 mg (7.8 μmol) of intermediate 68 compound. Yield: 6.3 mg (53% of theory)

LC-MS (Method _{1): R t = 1.00 min} ; MS (ESIpos): m / z = 1102 (M + H) +.

Intermediate 119 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 S )-2-phenyl-1-(5-phenyl-1,3,4-di Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

7.4 mg (8.1 mmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R, 4 S, 5 S )-3-methoxy- 1-{(2 S )-2-[(1 R, 2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 S )-2-phenyl-1 -(5-phenyl-1,3,4-di Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine It was treated with 6.3 mg (24.2 mmol) of 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane oxime hydrochloride in combination with an intermediate 157. This gave 1.6 mg (13% of theory) of title compound as a solid.

LC-MS (Method 11): R _t = 0.89 min; MS (ESIs): m/z=1126 (M+H) ⁺

Intermediate 120 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 R )-2-phenyl-1-(5-phenyl-1,3,4-di Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

12.8 mg (13.9 mmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R, 4 S, 5 S )-3-methoxy- 1-{(2 S )-2-[(1 R, 2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 R )-2-phenyl-1 -(5-phenyl-1,3,4-di Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine It was treated with 10.9 mg (41.8 mmol) of 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane hydrazine hydrochloride in an analogous manner to intermediate 157. The title compound was obtained as a solid, 10.8 mg (yield: 59%).

LC-MS (Method 11): R _t =0.90 min; MS (ESI): m/z=1126 (M+H) ⁺

Intermediate 121 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(2 S )-1-(benzylsulfonyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine -1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

7.4 mg (7.9 mmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R, 4 S, 5 S )-1-{(2 S )-2-[(1 R, 2 R )-3-{[(2 S )-1-(benzylsulfonyl)-3-phenylpropan-2-yl]amino}-1-methoxy Benzyl-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L - amidoxime and 6.2 mg (23.5 mmol) of 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane hydrazine hydrochloride The object 157 is processed. The title compound was obtained as a solid 6.9 mg (yield: 74%).

LC-MS (Method 11): R _t = 0.87 min; MS (ESIs): m/z=1150 (M+H) ⁺

Intermediate 122 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(2 S ,3 E )-1,4-Diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine -1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

8 mg (9.1 mmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R, 4 S, 5 S )-1-{(2 S )-2-[(1 R, 2 R )-3-{[(2 S, 3 E )-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy Benzyl-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L - amidoxime and 7.2 mg (27.4 mmol) of 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane oxime hydrochloride The object 157 is processed. The title compound was obtained as a white solid.

LC-MS (Method 11): _rt = 0.95 min; MS (ESIs): m/z = 1083 (M+H) ⁺

Intermediate 123 N -[6-(2,5-di- oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] -N -methyl-L-nonylamine fluorenyl- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1 -Tertoxy- 3-(1 H -Indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3 -Methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

Place 30 mg (30 μmol) of intermediate 89 in 2 ml of 1,4-two Treated in alkane and mixed with 4 ml of saturated sodium bicarbonate solution, and then with 7.5 mg (50 μmol) of 2,5-di-oxo-2,5-dihydro-1 H -pyrrole-1-carboxylic acid Methyl ester is mixed. The reaction mixture was stirred at RT for 1 h then concentrated EtOAc. The remaining residue was purified by preparative HPLC. After lyophilization, 24 mg (74% of theory) of title compound was obtained.

HPLC (method _{5): R t = 2.2 min} ; LC-MS ( Method _{1): R t = 1.01 min} ; MS (ESIpos): m / z = 1006 (M + H) +.

Intermediate 124 N -[6-(2,5-di- oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] -N -methyl-L-nonylamine fluorenyl- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(1 S )-1-carboxy-2-(1 H -吲哚- 3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1- Side oxyheptan-4-yl] -N -methyl-L-nonylamine amide

22 mg (20 μmol) of intermediate 123 was reacted with 4 ml of trifluoroacetic acid in 8 ml of dichloromethane at RT for 1 h, then the reaction mixture was concentrated under reduced pressure. The remaining residue was purified by preparative HPLC. After lyophilization, 11 mg (54% of theory) of title compound.

HPLC (Method 5): R _t = 1.8 min; LC-MS (Method 11): R _t = 0.85 min; MS (ESIs): m/z = 950 (M+H) ⁺ .

Intermediate 125 N -[6-(2,5-di- oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] -N -methyl-L-nonylamine fluorenyl- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-3-(1 H -吲哚-3-yl) -1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

22.5 mg (20 μmol) of intermediate 101 was placed in 2 ml of 1:1 Treated in alkane/water, and then with 5.6 mg (40 μmol) of 2,5-di-oxo-2,5-dihydro-1 H -pyrrole-1-carboxylic acid methyl ester and with 0.25 ml of saturated hydrogen carbonate The sodium solution is mixed. The reaction mixture was stirred at RT for 30 min. Then 0.25 ml of a saturated sodium bicarbonate solution was added and the reaction mixture was stirred at RT for 15 min and then concentrated under reduced pressure. The remaining residue was purified by preparative HPLC. After lyophilization, 12.8 mg (50% of theory) of title compound was obtained as colourless foam.

HPLC (Method 5): R _t = 1.9 min; LC-MS (Method 1): R _t = 0.95 min; MS (ESIs): m/z=1019 (M+H) ⁺ .

Intermediate 126 N -[6-(2,5-di- oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] -N -methyl-L-nonylamine fluorenyl- N -[(3 R , 4 S , 5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[( 1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

64 mg (70 μmol) of N- (6-aminohexyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-decylamine (intermediate 97) placed in 3 ml of 1:1 Treated in alkane/water, then adjusted to pH 9 with 4 ml of saturated sodium bicarbonate solution and subsequently with 16.3 mg (110 μmol) of 2,5-di- oxo-2,5-dihydro-1 H -pyrrole Methyl-1-carboxylate was mixed. The reaction mixture was stirred at RT for 1 h then concentrated EtOAc. Then add 8 mg (55 μmol) of 2,5-di-oxo-2,5-dihydro-1 H -pyrrole-1-carboxylic acid methyl ester, and adjust the reaction mixture to pH 9 and RT again. Stir for 1 hour. Concentration followed by purification of the remaining residue by preparative HPLC. First, 31 mg of the intermediate that has not been cyclized is obtained. Place 27 mg of this intermediate in 2 ml of 1:1 Treat in alkane/water and then mix with 250 μl of saturated sodium bicarbonate solution. After stirring for 2 h at RT, the reaction mixture was concentrated and the residue was purified mjjj. After lyophilization, 20 mg (29% of theory) of title compound was obtained.

HPLC (Method 5): R _t = 1.96 min; LC-MS (Method 1): R _t = 0.97 min; MS (ESIs): m/z = 992 (M+H) ⁺ .

Intermediate 127 N -{6-[(2,5-dioxapyrrolidin-1-yl)oxy]-6-oxo-oxyhexyl}- N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzylamino)-3-(1 H -Indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3 -Methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

17 mg (18 μmol) of N- (5-carboxypentyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzylamino)-3-(1 H -indol-3-yl)-1-oxopropane -2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-decylamine (Intermediate 98) was dissolved in 2.8 ml of dichloromethane and with 20 mg (174 mmol) of 1-hydroxypyrrolidine-2,5- The diketone was mixed and then mixed with 10 mg (52 μmol) of 1-(3 dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.21 mg (0.17 μmol) of DMAP. After stirring at RT for 4 h, the reaction mixture was evaporated. The remaining residue was purified by preparative HPLC. After lyophilization, 8.2 mg (43% of theory) of title compound.

HPLC (method 5): _Rt = 2.0 min; LC-MS (Method 1): R _t = 0.98 min; MS (ESIs): m/z = 071 (M+H) ⁺ .

Intermediate 128 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-amidamine

5 mg (5.6 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-guanamine indole is dissolved in 845 μl of DMF and then with 3.2 mg (17 μmol) of 1-(3-dimethylaminopropyl)-3- Ethylcarbodiimide hydrochloride, 2.6 mg (17 μmol) of 1-hydroxy-1 H -benzotriazole hydrate, 1.96 μl of N,N -diisopropylethylamine and with 5.9 mg ( 22.5 μmol) of commercially available 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane was mixed. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. This gave 2.2 mg (36% of theory) of title compound as colorless foam.

HPLC (method 5): _Rt = 1.7 min; LC-MS (Method 1): R _t = 0.98 min; MS (ESIs): m/z = 1094 (M+H) ⁺ .

Intermediate 129 N -(6-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-6-oxo-oxyhexyl) - N -methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

4 mg (4.3 μmol) of N- (5-carboxypentyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-guanamine in 646 μl of DMF and then with 2.5 mg (13 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride, 2.0 mg (13 μmol) of 1-hydroxy-1 H -benzotriazole hydrate, 2.25 μl of N,N -diisopropylethylamine and commercially available with 4.5 mg (17 μmol) 6-(2,5-Di- oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane oxime was mixed. The mixture was stirred at RT for 3 h and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. This gave 1.9 mg (39% of theory) of title compound as colourless foam.

HPLC (Method 5): R _t = 1.7 min; LC-MS (Method 9): R _t = 4.9 min; MS (ESIs): m/z=1134 (M+H) ⁺ .

Intermediate 130 N -(4-{[(2 R )-1-({5-[(2,5-di-oxaxopyridin-1-yl)oxy]-5-oxo-oxopentyl}amino)propane -2-yl]oxy}-4-oxobutyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1 -{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

10.5 mg (11.7 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-guanamine in 3.7 ml of dichloromethane, and then with 6.7 mg (35 μmol) of 1-(3-dimethylaminopropyl)-3-ethyl carbon Diimine hydrochloride, 0.7 mg (5.8 μmol) of 4-dimethylaminopyridine and mixed with 8.2 mg (47 μmol) of commercially available (3 R )-2-hydroxypropylaminecarboxylic acid tert-butyl ester . The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. This gave 7.5 mg (61% of theory) of the Boc-protected intermediate as a colorless foam.

HPLC (method _{5): R t = 2.0 min} ; LC-MS ( Method _{1): R t = 1.03 min} ; MS (ESIpos): m / z = 1056 (M + H) +.

Subsequently, the Boc protecting group was isolated as trifluoroacetic acid. Then 4.9 mg (0.005 mmol) of the deprotected crude product was taken up in 1.8 ml of dichloromethane without purification and with 3.7 mg (0.011 mmol) of 1,1 '-[(1,5-di-side oxygen) Penta-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione, 2.4 μl (0.014 mmol) of N,N -diisopropylethylamine and 0.6 mg (5 μmol) 4-dimethylaminopyridine. The mixture was stirred at RT for 2 h and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. This gave 0.77 mg (15% of theory) of title compound as colorless foam.

HPLC (Method 5): R _t = 1.8 min; LC-MS (Method 1): R _t = 0.93 min; MS (ESIs): m/z=1167 (M+H) ⁺ .

Intermediate 131 N -{4-[(1-{5-[(2,5-dioxapyrrolidin-1-yl)oxy]-5-oxooxopentyl}piperidin-4-yl)oxy] 4--4-oxobutyl} -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2 -[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

10 mg (11 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-Amidoxime is dissolved in 2 ml of dichloromethane and then with 4.3 mg (22 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbamate The imine hydrochloride, 0.88 mg (6 μmol) of 4-dimethylaminopyridine was mixed with 5.2 mg (22 μmol) of commercially available 4-hydroxypiperidine-1-carboxylic acid benzyl ester. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. This gave 5 mg (40% of theory) of the Z-protected intermediate as a colorless foam.

HPLC (Method 5): R _t = 2.1 min; LC-MS (Method 1): R _t = 1.04 min; MS (ESIs): m/z=1116 (M+H) ⁺ .

Subsequently, the Z protecting group is separated from the palladium on activated carbon by hydrogenolysis in ethanol. Then 4.6 mg (0.005 mmol) of the deprotected crude product was taken in 1.8 ml of dichloromethane without further purification and with 3.8 mg (0.012 mmol) of 1,1 '-[(1,5- 2-oxopenta-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione, 0.8 μl (0.005 mmol) of N,N -diisopropylethylamine and 0.6 mg ( 5 μmol) of 4-dimethylaminopyridine was mixed. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. Thus, 0.96 mg (16% of theory) of title compound was obtained as colorless foam.

HPLC (Method 5): R _t = 1.8 min; LC-MS (Method 1): R _t = 0.94 min; MS (ESIs): m/z=1193 (M+H) ⁺ .

Intermediate 132 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

15 mg (16.7 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-guanamine in 2500 μl of DMF and then with 9.6 mg (50 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride, 7.6 mg (50 μmol) of 1-hydroxy-1 H -benzotriazole hydrate, 5.8 μl of N,N -diisopropylethylamine and commercially available as 17.4 mg (67 μmol) 6-(2,5-Di- oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane oxime was mixed. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. This gave 11.2 mg (52% of theory) of title compound as colorless foam.

HPLC (method 5): _Rt = 1.7 min; LC-MS (Method 2): R _t = 1.09 min; MS (ESIs): m/z=1106 (M+H) ⁺ .

Intermediate 133 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(2 S ,3 S )-1-(benzyloxy)-1-oxooxy-3-phenylbutan-2-yl]amino}-1-methoxy-2-methyl-3- side Oxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

5.8 mg (6.3 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S ,3 S )-1-(benzyloxy)-1-oxo-3-phenylbutan-2-yl]amino }-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]- N -methyl-L-melamine indole is dissolved in 943 μl of DMF and then with 3.6 mg (19 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt Acid salt, 2.9 mg (19 μmol) of 1-hydroxy-1 H -benzotriazole hydrate, 2.2 μl of N,N -diisopropylethylamine and commercially available with 6.6 mg (25 μmol) 6 -(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane oxime was mixed. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. This gave 4.5 mg (64% of theory) of title compound as colorless foam.

HPLC (Method 5): R _t = 2.0 min; LC-MS (Method 1): R _t = 1.03 min; MS (ESIs): m/z=1129 (M+H) ⁺ .

Intermediate 134 N -[3-({[2-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)ethyl]amine-methylamino}amino)propyl]- N -methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R ) 1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

First, 4-nitrophenyl 2-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)ethylaminecarboxylate is prepared under standard conditions and is commercially available. The 1-(2-aminoethyl)-1 H -pyrrole-2,5-dione trifluoroacetate and 4-nitrophenyl chlorocarbonate are used.

5 mg (6 μmol) of N- (3-aminopropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy -1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-Amidoxime is dissolved in 1000 μl of DMF and then with 2 μl of N,N -diisopropylethylamine and with 2.2 mg (9 μmol) of 2-(2,5 - Mixture of 4-nitrophenyl 2,5-dihydro-1 H -pyrrol-1-yl)ethylaminecarboxylate. The mixture was stirred at RT for 1 h and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. This gave 1.6 mg (23% of theory) of title compound as colorless foam.

HPLC (Method 5): R _t = 1.7 min; LC-MS (Method 2): R _t = 1.09 min; MS (ESI s): m/z = 1 036 (M+H) ⁺ .

Intermediate 135 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(2 S )-1-(benzyloxy)-1-oxooxy-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl Pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

10 mg (11 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzyloxy)-1-oxo-3-phenylpropan-2-yl]amino}-1 -Methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -A The base-L-melamine indole is dissolved in 4000 μl of DMF and then with 6.3 mg (33 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4.5 mg (33 μmol) of 1-hydroxy-1 H -benzotriazole hydrate, 5.7 μl of N,N -diisopropylethylamine and commercially available 6-(2) with 11.5 mg (44 μmol) , 5-dioxaxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane oxime was mixed. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. This gave 2.6 mg (14% of theory) of title compound as colorless foam.

HPLC (Method 6): R _t = 2.1 min; LC-MS (Method 1): R _t = 1.01 min; MS (ESIs): m/z=1115 (M+H) ⁺ .

Intermediate 136 N -(4-{4-[4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)butanyl]piperidin -1-yl}-4-oxobutyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{( 2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

First, prepare 1-[4- side oxy-4- (piperider -1-yl)butyl]-1 H -pyrrole-2,5-dione trifluoroacetate is used under standard conditions by The tert-butyl 1-carboxylic acid and 4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)butyric acid were carried out in two stages.

5 mg (5.6 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-Amidoxime is dissolved in 1000 μl of DMF and then with 2.1 mg (11 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride, 1.7 mg (11 μmol) of 1-hydroxy-1 H -benzotriazole hydrate, 2 μl of N,N -diisopropylethylamine and with 3.5 mg (5.6 μmol) of 1- [4- Side Oxygen-4-(Phosphate) Mixing of -1-yl)butyl]-1 H -pyrrole-2,5-dione trifluoroacetate. The mixture was stirred at RT until overnight. Then add 2.1 mg (5.6 μmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate The reaction mixture was stirred at RT for a further 3 h. Subsequently, the solvent was removed under reduced pressure and the residue was purified using preparative HPLC. The corresponding fractions were lyophilized with water to give 0.6 mg (10% of theory)

HPLC (Method 6): R _t = 1.9 min; LC-MS (Method 1): R _t = 0.9 min; MS (ESIs): m/z=1132 (M+H) ⁺ .

Intermediate 137 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl]-1-methylindenyl}-4 -Phenoxybutyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[ (1 R , 2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-amidamine

First, the preparation of 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl) -N '-methylhexane fluorene trifluoroacetate is carried out under standard conditions. Commercially available 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexanoic acid and 1-butylindolecarboxylic acid tert-butyl ester were carried out in two stages.

6.9 mg (8 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-amidamine is dissolved in 2540 μl of DMF and then with 3.6 mg (9 μmol) of O- (7-azabenzotriazol-1-yl) - N,N,N',N' -tetramethylhexafluorophosphate 3 μl of N,N -diisopropylethylamine and 4.1 mg (12 μmol) of 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl ) -N '-methylhexane fluorene trifluoroacetate mixed. The mixture was stirred at RT until overnight. Subsequently, the solvent was evaporated under reduced pressure and the residue was purified by preparative HPLC. This gave 3.9 mg (45% of theory) of title compound as colorless foam.

HPLC (Method 5): R _t = 1.8 min; LC-MS (Method 1): R _t = 0.93 min; MS (ESIs): m/z=1108 (M+H) ⁺ .

Intermediate 138 N -{4-[(2-{[4-(2,5-dioxyloxy-2,5-dihydro-1 H -pyrrol-1-yl)butanyl](methyl)amino}ethyl) (Methyl)amino]-4-oxobutyl} -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1- {(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

Starting from 2-butylmethyl 2-(methylamino)ethylaminecarboxylic acid and 4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)butyric acid, In the second stage, 4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl) -N -methyl- N- [2-(methylamino) is prepared first. Ethyl]butanamine trifluoroacetate.

6.6 mg (7.3 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-guanamine in 2000 μl of DMF and then with 5.6 mg (14.7 μmol) of O- (7-azabenzotriazol-1-yl) -N,N,N ',N' -tetramethylhexafluorophosphate 2.6 μl of N,N -diisopropylethylamine and 4.1 mg (9 μmol) of 4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl) ) -N -Methyl- N- [2-(methylamino)ethyl]butanamine trifluoroacetate is mixed. After stirring at RT for 3 h, the same amount of HATU and N,N -diisopropylethylamine were again added, and then the reaction mixture was stirred at RT overnight. Subsequently, the solvent was evaporated under reduced pressure and the residue was purified by preparative HPLC. This gave 4 mg (44% of theory) of the title compound as a colourless foam.

HPLC (method 6): _Rt = 2.0 min; LC-MS (Method 1): R _t = 0.91 min; MS (ESIs): m/z=1134 (M+H) ⁺ .

Intermediate 139 (2 R , 3 S )-3-Amino-4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexanyl] Mercapto}-4-oxobutan-2-yl (3 R , 4 S , 7 S , 10 S )-4-[(2 S )-but-2-yl]-7,10-diisopropyl -3-(2-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5 ,11-Dimethyl-6,9-di-oxo-2-oxo-5,8,11-triazadecane-15-erate

13 mg (14.7 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-amidamine is dissolved in 10 ml of dichloromethane and then with 8.4 mg (44 μmol) of 1-(3-dimethylaminopropyl)- 3-ethylcarbodiimide hydrochloride, 5.4 mg (44 μmol) of 4-dimethylaminopyridine and 9 mg (29.3 μmol) of commercially available N- (t-butoxycarbonyl)-L - Benzyl benzyl ester is mixed. The mixture was stirred at RT for 5 h. Subsequently, the reaction mixture was extracted twice with water and the organic layer was dried over magnesium sulfate and evaporated. The remaining residue was purified by preparative HPLC. From two After lyophilization of the alkane/water, 14 mg (81% of theory) of the intermediate was obtained as a colorless foam.

HPLC (Method 12): _Rt = 2.3 min; LC-MS (Method 1): R _t = 1.13 min; MS (ESIs): m/z=1178 (M+H) ⁺ .

Subsequently, the Z protecting group was separated by hydrogenolysis in methanol on 10% palladium on activated carbon. Then 9.5 mg (0.0087 mmol) of the deprotected crude product was taken up in 5 ml of DMF without further purification and with 5 mg (26.2 μmol) of 1-(3-dimethylaminopropyl)- 3-ethylcarbodiimide hydrochloride, 4 mg (26.2 μmol) of 1-hydroxy-1 H -benzotriazole hydrate, 54.6 μl of N,N -diisopropylethylamine and with 9.1 Mg (34.9 μmol) of commercially available 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane oxime was mixed. The mixture was stirred at RT for 1 h and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. From two After lyophilization of the alkane, 9.5 mg (84% of theory) of Boc-protected intermediate was obtained.

HPLC (Method 12): R _t = 2.1 min; LC-MS (Method 1): R _t =0.97 min; MS (ESI s): m/z=1295 (M+H) ⁺ .

Subsequently, 9.5 mg (7.3 μmol) of the Boc-protected intermediate was deprotected in 0.5 ml of trifluoroacetic acid in 2 ml of dichloromethane, from After lyophilization of the alkane, 9 mg (yield: 82%)

HPLC (method 12): _Rt = 2.1 min; LC-MS (Method 1): R _t = 0.84 min; MS (ESIs): m/z=1195 (M+H) ⁺ .

Intermediate 140 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl]-1-methylindenyl}-4 -Phenoxybutyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[ (1 R , 2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

4.1 mg (12 μmol) of 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl) -N '-methylhexane fluorene trifluoroacetate (middle 22) co-dissolved in 2.5 ml of DMF with 6.9 mg (8 μmol) of compound from intermediate 61, and then with 3.5 mg (9 μmol) of O- (7-azabenzotriazol-1-yl) ) -N,N,N',N' -tetramethylhexafluorophosphate Mix with 3 μl of N,N -diisopropylethylamine. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. From two After lyophilization of the alkane, 2.6 mg (30% of theory)

HPLC (Method 5): R _t = 1.8 min; LC-MS (Method 1): R _t =0.90 and 0.91 min; MS (ESI pos): m/z=1120 (M+H) ⁺ .

Intermediate 141 N -[4-({1-[4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)butanyl]piperidin-4-yl}oxy)- 4-sided oxybutyl] -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2- [(1 R , 2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

44 mg (49 μmol) of N-(3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-Amidoxime is dissolved in 2 ml of dichloromethane and then with 18.8 mg (98 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbamate Imine hydrochloride, 3.8 mg (24 μmol) of 4-dimethylaminopyridine and mixed with 23 mg (98 μmol) of commercially available 4-hydroxypiperidine-1-carboxylic acid benzyl ester. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. This gave 22 mg (40% of theory) of the Z-protected intermediate as a colorless foam.

HPLC (Method 5): R _t = 2.1 min; LC-MS (Method 1): R _t = 1.04 min; MS (ESIs): m/z=1116 (M+H) ⁺ .

Subsequently, the Z protecting group is separated from the palladium on activated carbon by hydrogenolysis in ethanol.

Then 19 mg (19 μmol) of the deprotected crude product was taken up in 4 ml of DMF without further purification and with 7 mg (39 μmol) of 4-(2,5-di- oxy-2,5 -dihydro-1 H -pyrrol-1-yl)butyric acid, 11 mg (29 μmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' - Tetramethyl hexafluorophosphate Mix with 5 μl of N,N -diisopropylethylamine. The mixture was stirred at RT for 1 h and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. From two After lyophilization of the alkane, 7.5 mg (34% of theory)

HPLC (Method 5): R _t = 1.8 min; LC-MS (Method 1): R _t = 0.94 min; MS (ESIs): m/z=1147 (M+H) ⁺ .

Intermediate 142 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(2 S )-1-(benzyloxy)-3-(1 H -indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2- Methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-amidoxime amine

9 mg (9.5 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzyloxy)-3-(1 H -indol-3-yl)-1-oxopropane- 2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxo-heptane 4-yl] -N -methyl-L-decylamine (Intermediate 72) is dissolved in 1000 μl of DMF and then with 10 mg (38 μmol) of commercially available 6-(2,5-di-side oxygen -2,5-dihydro-1 H -pyrrol-1-yl)hexane oxime, 7.2 mg (19 μmol) of O- (7-azabenzotriazol-1-yl) -N,N,N ',N'- t tetramethylhexafluorophosphate Mix with 8 μl of N,N -diisopropylethylamine and stir the reaction mixture at RT for 1 h. Subsequently, the solvent was evaporated under reduced pressure and the residue was purified by preparative HPLC. The corresponding fractions were concentrated and lyophilized to give 6.4 mg (yield: 58%).

HPLC (method _{5): R t = 1.9 min} ; LC-MS ( Method _{1): R t = 0.99 min} ; MS (ESIpos): m / z = 1154 (M + H) +.

Intermediate 143 N -(4-{2-[4-(2,5-di-sideoxy-2,5-dihydro-1 H -pyrrol-1-yl)-2,2-dimethylbutanyl]indenyl}- 4-sided oxybutyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2- [(1 R , 2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

6 mg (6.7 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-decylamine (intermediate 61) with 3 mg (8.7 μmol) of 4-(2,5-di- oxo-2,5-dihydro-1 H -pyrrole-1- 2,2-Dimethylbutane fluorenium trifluoroacetate, similar to intermediate 142, gave 2 mg (27% of theory) of title compound.

HPLC (method 12): _Rt = 2.1 min; LC-MS (Method 3): R _t = 1.92 min; MS (ESIs): m/z=1106 (M+H) ⁺ .

Intermediate 144 N -(4-{2-[4-(2,5-di-sideoxy-2,5-dihydro-1 H -pyrrol-1-yl)-2,2-dimethylbutanyl]indenyl}- 4-sided oxybutyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2- [(1 R , 2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-amidamine

5 mg (5.6 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Add 7.65 mg (22.5 μmol) of 4-(2,5-di-oxo-2,5-dihydrogen) to 1 ml of DMF solution of hept-4-yl] -N -methyl-L-amidamine. -1H-pyrrol-1-yl)-2,2-dimethylbutane fluorene trifluoroacetate, 3.2 mg (16.9 μmol) of EDC, 1.96 μl (11.3 μmol) of diisopropylethylamine and 2.6 Mg (16.9 μmol) of HOBT. The reaction mixture was stirred at RT for 3 h. Subsequently, 0.95 mg (2.8 μmol) of 4-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutane fluorene was added. Acetate. After stirring until overnight, the reaction mixture was concentrated and purified using preparative HPLC. The title compound was obtained in 3.5 mg (85% purity, 48% of theory).

LC-MS (Method _{3): R t = 1.86 min} ; m / z = 1094 (M + H) +.

Intermediate 145 N -[3-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)propyl]- N -methyl-L-nonylamine fluorenyl- N -[( 3 R , 4 S , 5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[ (1 S , 2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

12 mg (14 μmol) of N- (3-aminopropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy -1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-decylamine (intermediate 66) placed in 750 μl of two Treated with alkane and with 1.5 ml of saturated sodium bicarbonate solution and then with 3.2 mg (21 μmol) of 2,5-di-oxo-2,5-dihydro-1 H -pyrrole-1-carboxylic acid methyl ester mixing. The reaction mixture was stirred at RT for 1 h then concentrated EtOAc. The remaining residue was purified by preparative HPLC. After lyophilization, 4.2 mg (32% of theory) of title compound.

HPLC (Method 5): R _t = 1.7 min; LC-MS (Method 1): R _t = 0.94 min; MS (ESI): m/z = 950 (M+H) ⁺ .

Intermediate 146 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-( {(2 S )-1-[benzyl(methyl)amino]-1-oxo-3-phenylpropan-2-yl}amino)-1-methoxy-2-methyl-3 -Phenoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

9 mg (9.8 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-({(2 S )-1-[benzyl)amino]-1-oxo-3-phenylpropan-2-yl} Amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl ] -N -methyl-L-decylamine (Intermediate 73), similar to intermediate 133 with 10 mg (39 μmol) of 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane oxime gave 1.8 mg (15% of theory).

HPLC (Method 12): R _t = 2.2 min; LC-MS (Method 9): R _t = 5.11 min; MS (ESI pos): m/z=1128 (M+H) ⁺ .

Intermediate 147 N -{4-[(2,5-Dioxapyrrolidin-1-yl)oxy]-4-oxobutylbutyl}- N -methyl-L-nonylamine fluorenyl- N -[(3 R , 4 S , 5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S ,3 S )-1-(benzyloxy)-1- Side oxy-3-phenylbutan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5 -methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine amide

16 mg (17 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S ,3 S )-1-(benzyloxy)-1-oxo-3-phenylbutan-2-yl]amino }-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]- N -methyl-L-decylamine (Intermediate 70) is dissolved in 2 ml of dichloromethane and with 2.6 mg (23 mmol) of 1-hydroxypyrrolidine-2,5-dione and then with 4 mg (21 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was mixed. After stirring at RT for 2 h, the same amount of 1-hydroxypyrrolidine-2,5-dione and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added again. . It was then stirred at RT overnight until the reaction mixture was concentrated under reduced pressure. The remaining residue was purified by preparative HPLC. After lyophilization, 10 mg (56% of theory) of title compound was obtained.

HPLC (Method 5): R _t =2.0 min;

Intermediate 148 N -{4-[(2-{[4-(2,5-dioxyloxy-2,5-dihydro-1 H -pyrrol-1-yl)butanyl](methyl)amino}ethyl) Amino]-4-oxobutyl} -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

6 mg (7 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-decylamine (intermediate 61) with 2.8 mg (8 μmol) of N- (2-aminoethyl)-4-(2,5-di-oxo-2,5 -Dihydro-1 H -pyrrol-1-yl) -N -methylbutyramine trifluoroacetate, 10.1 mg (27 μmol) of O- (7-azabenzotriazol-1-yl)- N,N,N',N' -tetramethylhexafluorophosphate Mix with 5 μl of N,N -diisopropylethylamine in 2 ml of DMF and stir at RT until overnight. Then add 5 mg (23.5 μmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate And 3 μl of N,N -diisopropylethylamine. After stirring at RT for 5 h, the solvent was removed under reduced pressure and the residue was purified by preparative HPLC. Concentrate the corresponding dissolved fraction from two After lyophilization of the alkane, 1.3 mg (15% of theory)

HPLC (Method 12): R _t = 2.1 min; LC-MS (Method 2): R _t = 1.21 min; MS (ESI pos): m/z = 1120 (M+H) ⁺ .

Intermediate 149 N -{4-[(2-{[4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)butanyl]amino}ethyl)(methyl) Amino]-4-oxobutyl} -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

6 mg (7 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-decylamine (intermediate 61) and 3.1 mg (9 μmol) of 4-(2,5-di-oxo-2,5-dihydro-1 H -pyrrole-1- yl) - N - [2- (methylamino) ethyl] butoxy acyl amine trifluoroacetate, 10.1 mg (27 μmol) of O- (7- aza-benzotriazol-1-yl) - N ,N,N',N' -tetramethylhexafluorophosphate 5 μl of N,N -diisopropylethylamine was mixed in 2 ml of DMF, and the mixture was stirred at RT for 4 h. The solvent was then removed under reduced pressure and the residue was purified using preparative HPLC. Concentrate the corresponding dissolved fraction from two After lyophilization of the alkane, 1 mg (13.4% of theory) of title compound was obtained.

HPLC (Method 12): R _t = 2.1 min; LC-MS (Method 1): R _t =0.89 min; MS (ESIs): m/z=1121 (M+H) ⁺ .

Intermediate 150 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 S ,2 R )-2-phenyl-1-(propylaminecarbamimidyl)cyclopropyl] Amino]propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

7.9 mg (9 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 S ,2 R )-2-benzene 1-(propylamine-mercapto)cyclopropyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl -L-Amidoxime is dissolved in 3 ml of DMF and then with 10.4 mg (54 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 8.3 Mg (54 μmol) of 1-hydroxy-1 H -benzotriazole hydrate, 9 μl of N,N -diisopropylethylamine and commercially available 6-(2, with 9.5 mg (36 μmol) 5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane oxime was mixed. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. This gave 4.3 mg (22% of theory) of title compound as colourless foam.

HPLC (method 6): _Rt = 1.9 min; LC-MS (Method 9): R _t = 4.93 min; MS (ESI s): m/z = 1078 (M+H) ⁺ .

Intermediate 151 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(1 S , 2 R )-1-Aminomethylguanidino-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1 -yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

This compound was prepared to be similar to intermediate 150 and was carried out from the compound of intermediate 81.

HPLC (Method 5): R _t = 1.7 min; LC-MS (Method 1): R _t = 0.87 min; MS (ESI s): m/z = 1 036 (M + H) ⁺ .

Intermediate 152 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(1 S , 2 R )-1-(ethoxycarbonyl)-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine -1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

10 mg (12 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(1 S ,2 R )-1-(ethoxycarbonyl)-2-phenylcyclopropyl]amino}-1-methoxy Benzyl-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L - Amidoxime is dissolved in 3 ml of DMF and then with 8.9 mg (23 μmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetra Hexafluorophosphate 10 μl of N,N -diisopropylethylamine and 12 mg (47 μmol) of commercially available 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrole-1 -Base) Hexane oxime is mixed. The mixture was stirred at RT for 1 h. It was then concentrated under high vacuum and the residue was purified using preparative HPLC. This gave 5.8 mg (37% of theory) of title compound as colorless foam.

HPLC (Method 6): R _t = 2.0 min; LC-MS (Method 9): R _t = 4.49 min; MS (ESI s): m/z = 066 (M+H) ⁺ .

Intermediate 153 N- [1-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-12,15-di-oxo-3,6,9-trioxo-13, 14-diazooctadecane-18-yl] -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl] -N -methyl-L-amidamine

5 mg (5.6 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo 9.7 mg (22.5 μmol) of 3-(2-{2-[2-(2,5-II) was added to 1 ml of DMF solution of hept-4-yl] -N -methyl-L-amidamine. Side oxy-2,5-dihydro-1 H -pyrrol-1-yl)ethoxy]ethoxy}ethoxy)propanium guanidine trifluoroacetate, 3.2 mg (16.9 μmol) EDC, 1.96 μl ( 11.3 μmol) of N,N-diisopropylethylamine and 2.6 mg (16.9 μmol) of HOBT. The reaction mixture was stirred at RT for 3 h. Subsequently, an additional 1.2 mg (2.8 μmol) of 3-(2-{2-[2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy group was added. Ethoxy}ethoxy)propane fluorene trifluoroacetate. The reaction mixture was stirred at rt overnight and then purified by preparative HPLC. 3.6 mg (51% of theory) of the title compound.

LC-MS (Method _{1): R t = 0.90 min} ; m / z = 1185 (M + H) +.

Intermediate 154 (2 R , 3 S )-3-Amino-4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexanyl] Mercapto}-4-oxobutan-2-yl (3 R , 4 S , 7 S , 10 S )-4-[(2 S )-but-2-yl]-7,10-diisopropyl -3-(2-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1) ,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,11-dimethyl -6,9-di-oxo-2-oxo-5,8,11-triazadecane-15-erate

15 mg (17 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S )-1-(1,2- Alkan-2-yl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]- N -methyl-L-amidamine dissolved in 10 ml of dichloromethane and then with 12.8 mg (67 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiam Amine hydrochloride, 10 mg (83 μmol) of 4-dimethylaminopyridine and 10.3 mg (33 μmol) of commercially available N- (t-butoxycarbonyl)-L-threonate benzyl ester mixing. The mixture was heated to reflux for 4 h. The same amount of coupling agent and 4-dimethylaminopyridine were then added again and the reaction mixture was heated to reflux overnight. Subsequently, the reaction mixture was diluted with dichloromethane and extracted once with water, and organic layer was removed and concentrated under high vacuum. The remaining residue was purified by preparative HPLC. This gave 7.7 mg (37% of theory) of the protected intermediate as a colorless foam.

HPLC (Method 12): _Rt = 2.5 min; LC-MS (Method 1): R _t = 1.13 min; MS (ESIs): m/z=1190 (M+H) ⁺ .

Subsequently, the benzyl ester protecting group is removed by hydrogenation under standard hydrogen pressure on 10% palladium on activated carbon in methanol, as described in Intermediate 151, and the acid thus obtained is 6-(2). , 5-dioxaxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane oxime was mixed. In the final step, the Boc protecting group is separated with trifluoroacetic acid. The remaining residue was purified by preparative HPLC. Thus, 0.22 mg (2.5% of theory) of the title compound was obtained as a colorless foam.

HPLC (Method 12): R _t = 2.0 min; LC-MS (Method 1): R _t = 0.81 min; MS (ESI): m/z = 207 (M+H) ⁺ .

Intermediate 155 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(2 S )-1-Amino-1-oxooxy-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine -1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

This compound was synthesized as described in Intermediate 152, from N- (3-carboxypropyl) -N -methyl-L-decylamine- N -[(3 R , 4 S , 5 S ) -1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-Amino-1-yloxy-3-phenylpropan-2-yl]amine }-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-decylamine and commercially available 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane.

HPLC (Method 5): R _t = 1.6 min; LC-MS (Method 1): R _t = 0.82 min; MS (ESI+): m/z = 1024 (M+H) ⁺ .

Intermediate 156 N -(3-{[(1-{[(2,5-di-oxaxopyridin-1-yl)oxy]carbonyl}cyclopropyl)carbonyl]amino}propyl) -N -methyl- L-Amidoxime- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy Benzyl-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

This compound was synthesized as described in the last stage of intermediate 131, from N- (3-aminopropyl) -N -methyl-L-nonylamine- N -[(3 R , 4 S , 5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-decylamine and 1,1'-[cyclopropane-1,1-diylbis(carbonyloxy)]dipyrrolidine-2,5-dione (previously corresponding Prepared by the carboxylic acid.

HPLC (Method 12): R _t = 2.0 min; LC-MS (Method 1): R _t = 0.92 min; MS (ESI+): m/z = 1080 (M+H) ⁺ .

Intermediate 157 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl- 3-Phenoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

15 mg (18 μmol) of ( N -(3-carboxypropyl)- N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2- Amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4 -yl] -N -methyl-L-amidamine dissolved in 3.8 ml of DMF and then with 27 mg (70 μmol) of O- (7-azabenzotriazol-1-yl) -N, N,N',N' -tetramethylhexafluorophosphate 12 μl of N,N -diisopropylethylamine and 14 mg (53 μmol) of commercially available 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrole-1 -Base) Hexane oxime is mixed. The reaction mixture was stirred at RT for 1 h. It was then concentrated under high vacuum and the residue was purified using preparative HPLC. This gave 6.2 mg (33% of theory) of title compound as colorless foam.

HPLC (method 5): _Rt = 1.6 min; LC-MS (Method 1): R _t = 0.85 min; MS (ESI+): m/z=1063 (M+H) ⁺ .

¹ H-NMR (500 MHz, DMSO-d ₆ , characteristic signal): δ = 10.8 (d, 1H), 9.8-9.7 (m, 2H), 9.6 and 9.4 (2m, 1H), 8.9, 8.88, 8.78 and 8.75 (4d, 1H), 8.08 and 7.85 (2d, 1H), 7.6-6.9 (m, 9H), 4.7-4.4 (m, 3H), 3.4 (t, 2H), 3.23, 3.2, 3.18, 3.0, and 2.99 (5s, 9H), 2.8 (m, 3H), 2.1 (t, 2H), 1.06 and 1.01 (2d, 3H), 0.95-0.8 (m, 15H), 0.8-0.75 (dd, 3H).

Intermediate 158 N -[4-({(2 R )-1-[(2,5-Di-Phenylpyrrolidin-1-yl)oxy]-4-methyl-1-oxomethoxy-2-yl}amine) 4-)oxybutyl] -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R , 2 R )-3-{[(2 S )-1-(benzylamino)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2 -methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine Guanamine

13 mg (14.7 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzylamino)-1-yloxy-3-phenylpropan-2-yl]amino}- 1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N - Methyl-L-guanamine in 4 ml of dimethylformamide and then with 9.4 mg (25 μmol) of O- (7-azabenzotriazol-1-yl) -N,N ,N',N' -tetramethylhexafluorophosphate 6 μl of N,N -diisopropylethylamine was mixed with 7 mg (31 μmol) of commercially available D-leucine tartrate hydrochloride. The mixture was stirred at RT for 5 h and then concentrated under reduced pressure. The remaining residue was purified by preparative HPLC. From two After lyophilization of the alkane/water, 6.5 mg (49% of theory) of intermediate was obtained.

HPLC (method 5): _Rt = 2.2 min; LC-MS (Method 1): R _t = 1.21. min; MS (ESI+): m/z = 1076 (M+H) ⁺ .

The Boc protecting group was first isolated using trifluoroacetic acid in the thus protected intermediate in dichloromethane to afford 6.2 mg (yield: 99% of theory). 5.2 mg (5 μmol) of this intermediate was placed in 1.5 ml of dichloromethane and treated with 0.8 mg (7 μmol) of N -hydroxysuccinimide at 1.2 mg (6 μmol) of 1-(3-di The reaction was carried out in the presence of methylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.16 mg (1 μmol) of 4-dimethylaminopyridine. After stirring at RT for 2 h, the reaction mixture was concentrated and purified using preparative HPLC. 1.3 mg of the title compound are obtained, some of which are hydrolyzed.

Intermediate 159 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-4-oxobutyl ) -N -Methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{ [(2 S )-1-(benzylamino)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropane Pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

This compound was synthesized as described in Intermediate 157, from N- (3-carboxypropyl) -N -methyl-L-decylamine- N -[(3 R , 4 S , 5 S ) -1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzylamino)-1-oxo-3-phenylpropan-2- -yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxo-heptane- 4-yl] -N -methyl-L-decylamine and commercially available 6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexane preparation.

Yield: 6 mg (53% of theory)

HPLC (Method 5): R _t = 1.9 min; LC-MS (Method 1): R _t = 0.94 min; MS (ESI+): m/z=1114 (M+H) ⁺ .

Intermediate 160 N -(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl} 4-oxobutyl) - N -methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[ ( 2S )-1-(Benzylamino)-3-( 1H -indol-3-yl)-1-oxooxypropan-2-yl]amino}-1-methoxy-2- Methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-amidoxime amine

This compound was synthesized as described in Intermediate 157, from 20 mg (21 μmol) of N- (3-carboxypropyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzylamino)-3-(1 H -Indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3 -Methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine and commercially available 6-(2,5-di-oxo-2,5- Prepared by dihydro-1 H -pyrrol-1-yl)hexane.

Yield: 13 mg (52% of theory)

HPLC (Method 5): R _t = 1.9 min; LC-MS (Method 1): R _t = 0.92 min; MS (ESI+): m/z=1153 (M+H) ⁺ .

Intermediate 161 N -(6-{2-[6-(2,5-di-oxo-2,5-dihydro-1 H -pyrrol-1-yl)hexyl] fluorenyl}-6-oxo-oxyhexyl) - N -methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[ (2 S )-1-Amino-3-(1 H -indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3 -Phenoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine

This compound was synthesized as described in Intermediate 157, from N- (5-carboxypentyl) -N -methyl-L-nonylamine- N -[(3 R , 4 S , 5 S ) -1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1- 2-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1 -Sideoxyhept-4-yl] -N -methyl-L-decylamine and commercially available 6-(2,5-di- oxo-2,5-dihydro-1 H -pyrrol-1-yl ) Prepared by hexane.

Yield: 0.8 mg (16% of theory)

HPLC (method 5): _Rt = 1.6 min; LC-MS (Method 1): R _t =0.78 min; MS (ESI+): m/z=1092 (M+H) ⁺ .

Intermediate 162 N -{6-[(2,5-dioxapyrrolidin-1-yl)oxy]-6-oxo-oxyhexyl}- N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S , 2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-amidamine

18 mg (20 μmol) of N- (5-carboxypentyl) -N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy- 1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(1 S ,2 R )-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] - N -methyl-L-decylamine (Intermediate 64) is dissolved in 3.2 ml of dichloromethane and then mixed with 22 mg (190 mmol) of 1-hydroxypyrrolidine-2,5-dione, and It was then mixed with 11 mg (60 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.24 mg (0.17 μmol) of DMAP. After stirring for 2 h at RT, an additional 22 mg (190 mmol) of 1-hydroxypyrrolidine-2,5-dione and 11 mg (60 μmol) of 1-(3-dimethylaminopropyl)- 3-ethylcarbodiimide hydrochloride and 0.24 mg (0.17 μmol) of DMAP and the reaction mixture was stirred at RT for an additional hour. It was then concentrated under reduced pressure. The remaining residue was purified by preparative HPLC. After lyophilization, 8.2 mg (41% of theory).

HPLC (Method 5): R _t = 2.0 min; LC-MS (Method 11): R _t = 0.9 min; MS (ESI+): m/z = 1024 (M+H) ⁺ .

Intermediate 163 [(1 S , 2 R )-1-Amino-2-phenylcyclopropyl](1,4-dihydro-3 H -2,3-benzo -3-yl)methanone trifluoroacetate

First, starting from 265 mg (0.82 mmol) of (1 S , 2 R )-1-(hydroxylaminomethyl)-2-phenylcyclopropylaminecarboxylic acid tert-butyl ester (starting compound 7), borrow Preparation of Boc-protected (1S, 2R)-1-(1) by reaction with 1,2-bis(bromomethyl)benzene in a similar literature method (see H. King, J. Chem. Soc. 1942, 432) 1,4-dihydro-3H-2,3-benzo a tert-butyl intermediate of -3-ylcarbonyl)-2-phenylcyclopropylaminecarboxylate.

Yield: 108 mg (34% of theory)

LC-MS (Method _{2): R t = 1.3 min} ; MS (ESI +): m / z = 395 (M + H) +.

108 mg (0.27 mmol) of this intermediate was taken up in 3.7 ml of dichloromethane, 1.8 ml of trifluoroacetic acid was added and the mixture was stirred at RT for 15 min. Then concentrate under reduced pressure and the remaining residue from the second The alkane is freeze dried. A quantitative yield of 112 mg of the title compound was obtained as a colorless foam.

LC-MS (Method _{1): R t = 0.7 min} ; MS (ESI +): m / z = 295 (M + H) +.

Intermediate 164 N -methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[( 1 S , 2 R )-1-(1,4-dihydro-3 H -2,3-benzo -3-ylcarbonyl)-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy -5-methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine guanamine trifluoroacetate

The 166 mg (0.196 mmol) of N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N - [(3 R, 4 S, 5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5- Methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine (Intermediate 10) was treated in 40 ml of DMF and continuously with 80 mg (0.196 mmol) of [( 1 S , 2 R )-1-Amino-2-phenylcyclopropyl](1,4-dihydro-3 H -2,3-benzo 3-yl)methanone trifluoroacetate (intermediate 163), 112 mg (0.294 mmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate (HATU) was mixed with 682 μl (3.9 mmol) of N,N -diisopropylethylamine. The mixture was then stirred at RT until overnight. The reaction mixture was then concentrated under reduced pressure. The organic layer was dried with MgSO4, filtered and evaporated. The residue was finally purified by preparative HPLC. In this method, to give 19 mg (9% of theory) of the Fmoc-protected intermediate N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl acyl -L- valinamide - N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(1 S ,2 R )-1-(1 ,4-dihydro-3 H -2,3-benzo -3-ylcarbonyl)-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy 5-5-Methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine.

HPLC (method _{5): R t = 1.68 min} ; LC-MS ( Method _{1): R t = 1.51 min} ; MS (ESI +): m / z = 1083 (M + H) +.

19 mg (0.015 mmol) of this intermediate was dissolved in 4 ml of DMF. After 817 μl of piperidine was added, the reaction mixture was stirred at RT for 5 min. Concentration was then carried out under reduced pressure and the residue was purified eluting with diethyl ether and then purified by preparative HPLC (solvent: acetonitrile: &lt;RTIgt;&lt;/RTI&gt; Combine the corresponding dissolved fractions, remove the solvent under reduced pressure and then remove the residue from the second The alkane/water is freeze dried. The title compound was obtained as a colorless foam, 12 mg (yield: 92%).

HPLC (Method 6): R _t = 2.0 min; LC-MS (Method 1): R _t = 0.94 min; MS (ESI+): m/z = 861 (M+H) ⁺ .

Intermediate 165 N-(6-Aminohexyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3 -{[(1S,2R)-1-(1,4-dihydro-3H-2,3-benzo -3-ylcarbonyl)-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy -5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine amide

Using 20 mg (0.021 mmol) of intermediate 164, similar to the preparation of intermediate 97, benzyl 6-oxohexylamine amide, in the presence of sodium cyanoborohydride, followed by hydrogenolysis of the Z protecting group (The title compound was prepared by using palladium on 5% activated carbon as a catalyst and methanol as a solvent).

Yield: 4.5 mg (23% of theoretical value in 2 stages)

HPLC (Method 12): R _t = 1.9 min; LC-MS (Method 1): R _t = 0.9 min; MS (ESI+): m/z = 960 (M+H) ⁺ .

Intermediate 166 N-[6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-nonylamine-yl-N-[(3R, 4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-(1,4-Dihydro-3H-2,3-benzo) -3-ylcarbonyl)-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy -5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine amide

Place 4.4 mg (4.5 μmol) of intermediate 165 in 1 ml of 1:1 Treated in alkane/water and then mixed with 1 mg (6.8 μmol) of 2,5-di-oxo-2,5-dihydro-1H-pyrrole-1-carboxylic acid methyl ester and 50 μl of saturated aqueous sodium bicarbonate . The reaction mixture was stirred at RT for 30 min. Then 50 μl of a saturated aqueous solution of sodium hydrogencarbonate was added and the mixture was stirred at RT for 15 min and then concentrated under reduced pressure. The remaining residue was purified by preparative HPLC. After lyophilization, 1 mg (21% of theory) of title compound was obtained as colourless foam.

HPLC (Method 12): R _t = 2.1 min; LC-MS (Method 1): R _t = 1.08 min; MS (ESI+): m/z = 1040 (M+H) ⁺ .

Intermediate 167 3-{2-[2-(2-Sideoxyethoxy)ethoxy]ethoxy}propanoic acid benzyl ester

The title compound consists of 6 g (21.55 mmol) of commercially available 3-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}propanoic acid under standard conditions, starting with benzyl chloride and Ethyl carbonate is esterified and subsequently prepared by oxidation of a sulfur trioxide-pyridine complex.

Yield: 611 mg (10% theoretical value in 2 stages)

LC-MS (Method _{2): R t = 1.69 min} ; MS (ESI +): m / z = 311 (M + H) +.

Intermediate 168 N-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S) ,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-yloxy Prop-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-side Oxyheptan-4-yl]-N-methyl-L-nonylamine decylamine

First, in a manner similar to the synthesis of intermediate 69, with N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N - [( 2 R , 3 S , 4 S )-1-carboxy-2-methoxy-4-methylhex-3-yl]- N -methyl-L-decylamine (Intermediate 4) and N ^α -{(2 R ,3 R )-3-methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propanyl}-L-tryptamine guanamine trifluoroacetic acid Salt (Intermediate 49) in O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of the same, and subsequent separation of the Fmoc protecting group with piperidine to prepare the amine compound N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-amino-3-(1 H -indol-3-yl)-1-oxopropan-2-yl Amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4- The base] -N -methyl-L-guanamine amine is a trifluoroacetate salt.

25 mg (0.028 mmol) of this compound and 17.5 mg (0.06 mmol) of intermediate 167 were mixed with 2 ml of methanol and mixed with 12.6 mg (0.14 mmol) of borane-pyridine complex and 2.5 ml of acetic acid. The reaction mixture was stirred at RT until overnight. The same amount of borane-pyridine complex and acetic acid were then added again and the reaction mixture was stirred at RT for an additional 24 h. Concentration was then carried out under reduced pressure and the residue was purified by preparative HPLC. Concentrate the corresponding dissolved fractions and take 1:1 After lyophilization of the alkane/water, 26.5 mg (yield: 88%) of the title compound.

HPLC (Method 12): R _t = 2.04 min; LC-MS (Method 1): R _t =0.97 min; MS (ESI+): m/z=1064 (M+H) ⁺ .

25 mg (0.024 mmol) of this intermediate was taken up in 10 ml of methanol and hydrogenated on 10% activated carbon under standard hydrogen pressure for 45 min. The catalyst was then filtered off and the solvent was removed under reduced pressure. From two After lyophilization of the alkane, 19.7 mg (85% of theory)

HPLC (Method 12): R _t = 1.8 min; LC-MS (Method 1): R _t = 0.83 min; MS (ESI+): m/z = 974 (M+H) ⁺ .

Intermediate 169 N-{2-[2-(2-{3-[(2,5-dioxaxopyridin-1-yl)oxy]-3-oxooxypropoxy}ethoxy)ethoxy] Ethyl}-N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S) 1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl Pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

10 mg (10 μmol) of intermediate 168 was dissolved in 3 ml of DMF and mixed with 3.5 mg (30 mmol) of 1-hydroxypyrrolidine-2,5-dione, and then with 2.4 mg (10 μmol) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was mixed with 5 μl of N,N-diisopropylethylamine. After stirring for 20 h at RT, 8 mg (0.02 mmol) of HATU was added and the mixture was stirred again at RT overnight and then concentrated under reduced pressure. The remaining residue was purified by preparative HPLC. From two After lyophilization of the alkane, 8.6 mg (64% of theory)

HPLC (method 12): Rt = 1.9 min; LC-MS (Method 11): Rt = 0.81 min; MS (ESI+): m/z = 071 (M+H)+.

Intermediate 170 N-(6-Aminohexyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[( 1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl]-N-methyl-L-amidamine

This compound was prepared in a similar phase from intermediate 101 starting from 26 mg (0.028 mmol) of Intermediate 15.

Yield: 16.7 mg (63% of theory in 2 stages)

HPLC (method 12): _Rt = 1.9 min; LC-MS (Method 1): R _t = 0.81 min; MS (ESI+): m/z = 914 (M+H) ⁺ .

Intermediate 171 N-(6-{[4-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)butanyl]amino}hexyl)-N-methyl-L-decylamine Mercapto-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{ [(2S,3S)-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl]-N-methyl-L-amidamine

6.7 mg (7.3 μmol) of compound formed from intermediate 170 and 3 mg (14.7 μmol) of commercially available 4-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl) Butyric acid was treated in 2 ml of DMF with 5.6 mg (14.7 μmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethyl Hexafluorophosphate (HATU) was mixed with 2 μl of N,N -diisopropylethylamine. The mixture was stirred at RT for 30 min. The reaction mixture was concentrated and the residue was purified using EtOAc. Combine the corresponding dissolved fractions, and then remove the residue from the second The alkane is freeze dried. This gave 4.5 mg (56% of theory) of the title compound.

HPLC (method _{12): R t = 2.0 min} ; LC-MS ( Method _{1): R t = 1.12 min} ; MS (ESI +): m / z = 1079 (M + H) +.

Intermediate 172 2-{2-[2-(2-Sideoxyethoxy)ethoxy]ethoxy}ethylamine benzyl benzoate

The title compound is obtained from commercially available 2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethanol under standard conditions, first introducing a Z protecting group and then with sulfur trioxide - The pyridine complex is prepared by oxidation.

HPLC (method 12): _Rt = 1.4 min; LC-MS (Method 11): R _t = 0.65 min; MS (ESI+): m/z = 326 (M+H) ⁺ .

Intermediate 173 Benzyl {2-[2-(2-oxoethoxyethoxy)ethoxy]ethylaminecarboxylate

The title compound is similar to intermediate 172, from commercially available 2-[2-(2-aminoethoxy)ethoxy]ethanol under standard conditions, first introduced with a Z protecting group and then with a sulfur trioxide-pyridine complex. Oxidation preparation.

HPLC (Method 12): R _t = 1.3 min; LC-MS (Method 11): R _t =0.68 min; MS (ESI+): m/z = 282 (M+H) ⁺ .

Intermediate 174 N-(2-{2-[2-(2-Aminoethoxy)ethoxy]ethoxy}ethyl)-N-methyl-L-nonylamine-yl-N-[(3R, 4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1- (1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-amidamine

47 mg (0.05 mmol) of intermediate 16 was prepared in analogy to intermediate 167 with benzyl 2-{2-[2-(2-o-ethoxyethoxy)ethoxy]ethoxy}ethylamine. The base ester is subjected to reductive amination in a borane-pyridine complex. Subsequently, the Z protecting group was removed by hydrogenolysis using palladium on 5% activated carbon as a solvent and methanol as a solvent to prepare 38 mg (66% of theory).

HPLC (Method 5): R _t = 1.7 min; LC-MS (Method 1): R _t = 0.8 min; MS (ESI+): m/z = 988 (M+H) ⁺ .

Intermediate 175 N-[2-(2-{2-[2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy) Ethyl]-N-methyl-L-decylamine-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1 -Methoxy-2-methyl-3-{[(1S,2R)-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-amidamine

This preparation was similar to intermediate 166, starting with 34 mg (0.03 mmol) of intermediate 174.

Yield: 8.3 mg (23% of theory)

HPLC (method 5): _Rt = 1.9 min; LC-MS (Method 1): R _t = 0.97 min; MS (ESI+): m/z = 10.68 (M+H) ⁺ .

Intermediate 176 N-(2-{2-[2-(2,5-di-sideoxy-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethyl)-N-A --L-Amidoxime-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-Amino-3 -(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl 3-Benzyloxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

This preparation is similar to intermediates 174 and 175, starting with reductive amination of intermediate 16 with intermediate 173, followed by deprotection and formation of maleimide.

HPLC (Method 12): R _t = 1.8 min; LC-MS (Method 11): R _t = 0.8 min; MS (ESI+): m/z = 981 (M+H) ⁺ .

Intermediate 177 N-[2-(2-{2-[2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy) Ethyl]-N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S) 1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl Pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

This preparation is similar to intermediates 174 and 175, starting with reductive amination of intermediate 16 with intermediate 172, followed by deprotection and formation of maleimide.

HPLC (method _{12): R t = 1.9 min} ; LC-MS ( Method _{1): R t = 0.86 min} ; MS (ESI +): m / z = 1025 (M + H) +.

Intermediate 178 N-{4-[(2,5-dioxapyrrolidin-1-yl)oxy]-4-oxobutylbutyl}-N-methyl-L-nonylamine fluorenyl-N-[(3R ,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1- 2-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1 -Sideoxyhept-4-yl]-N-methyl-L-nonylamine amide

This preparation was similar to intermediate 162, starting with 6 mg of intermediate 82.

LC-MS (Method _{1): R t = 0.82 min} ; MS (ESI +): m / z = 953 (M + H) +.

Intermediate 179 4-[(1E,3S)-3-amino-4-phenylbut-1-en-1-yl]benzenesulfonic acid trifluoroacetate

13.6 mg (0.06 mmol) of palladium(II) acetate, 469 mg (1.46 mmol) of potassium 4-iodobenzenesulfonate, 300 mg (1.21 mmol) of (S)-t-butyl 1-phenylbutene- A mixture of 3-alken-2-ylcarbamate, 16.5 mg (0.12 mmol) of phenylurea and 167.6 mg (1.21 mmol) of potassium carbonate in 7.5 ml of DMF was heated in a microwave to 160 ° C for 15 min. The crude product was then purified directly by preparative HPLC. A mixture of 312 mg of 31% Boc-protected compound and 69% free amine was obtained.

The mixture was then taken up in 30 ml of dichloromethane, mixed with 1 ml of trifluoroacetic acid and stirred at RT for 20 h. After concentration under reduced pressure, the~~~~~~~~~~ This gave 200 mg (62% of theory) of the title compound.

LC-MS (method _{11): R t = 0.44 min} ; MS (ESI +): m / z = 304 (M + H) +.

Intermediate 180 4-[(3R)-3-amino-4-phenylbutyl]benzenesulfonic acid

100 mg (0.25 mmol) of 4-[(1E,3S)-3-amino-4-phenylbut-1-en-1-yl]benzenesulfonic acid trifluoroacetate was suspended in 10 ml of acetic acid and A few drops of DMF and water were mixed with 70 mg (0.07 mmol) of palladium on carbon (10%) and hydrogenated at 2.2 bar (bar) for 24 h. The solution was filtered and the filtrate was purified by preparative HPLC. 29 mg (76% purity, 21% of theory) product.

LC-MS (Method _{1): R t = 0.46 min} ; MS (ESI +): m / z = 306 (M + H) +.

Intermediate 181 N-(Tertibutoxycarbonyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[ (1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulfophenyl)but-3- Alken-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

90 mg (0.13 mmol) of N-(t-butoxycarbonyl)-N-methyl-L-nonylamine-N-[(3R,4S,5S)-1-{(2S)-2 -[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-side To 4 ml of DMF solution of oxyheptan-4-yl]-N-methyl-L-decylamine was added 60 mg (0.16 mmol) of HATU and 69 μl of (0.39 mmol) Hünig base. The reaction mixture was stirred at RT for 30 h and then with 60 mg (0.15 mmol) 60.3 mg (0.13 mmol) of 4-[(1E,3S)-3-amino-4-phenylbut-1-ene-1- Mixture of benzenesulfonic acid trifluoroacetate. After stirring until overnight, the reaction mixture was purified by preparative HPLC. This gave 127 mg of the title compound and a 44:56 mixture of the deprotected amine.

LC-MS (Method _{1): R t = 1.21 min} ; MS (ESI +): m / z = 971 (M + H) +; R t = 0.84 min; MS (ESI +): m / z = 871 (M + H) ⁺ deprotected compounds.

Intermediate 182 N-methyl-L-Amidoxime-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy -2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulfophenyl)but-3-en-2-yl]amino}propyl Pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-decylamine guanamine trifluoroacetate

Dissolve 90 mg of intermediate 180 in 4.6 ml of dichloromethane and add 0.92 ml of trifluoroacetic acid was added. The reaction mixture was stirred at RT and then concentrated. The obtained crude product was purified by preparative HPLC.

This gave 91 mg (98% of theory) of desired compound.

LC-MS (method 1): R _t = 0.85 min; MS (ESI+): m/z = 871 (M+H) ⁺

Intermediate 183 N-(3-carboxypropyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[( 1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulfophenyl)but-3-ene -2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

16.7 μl (0.03 mmol) of 15% aqueous succinic acid was first charged to 943 μl of methanol and with 17 mg (0.02 mmol) of N-methyl-L-amidoxime-N-[(3R, 4S, 5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E) 1-phenyl-4-(4-sulfophenyl)but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxo-heptane- 4-yl]-N-methyl-L-amidoxime trifluoroacetate (intermediate 181) and 1.1 μl (0.02 mmol) of acetic acid was mixed. The reaction mixture was stirred at RT for 5 min and then 2.9 μl (0.02 mmol) of borane-pyridine complex was added. After 1 h, 2 equivalents of each of succinaldehyde, acetic acid and borane-pyridine complex were added, and the mixture was stirred at RT for 20 h. The reaction mixture was then purified by preparative HPLC. This gave 20 mg (83% purity, 80% of theory) of the title compound.

LC-MS (Method 1): R _t = 0.87 min; MS (ESI+): m/z = 957 (M+H) ⁺

Intermediate 184 N-(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]]yl}-4-oxobutyl) -N-methyl-L-Amidoxime-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy 2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulfophenyl)but-3-en-2-yl]amino}propyl Pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

8 mg (7.5 μmol) of N-(3-carboxypropyl)-N-methyl-L-nonylamine-N-[(3R,4S,5S)-3-methoxy-1-{ (2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulfonate) Phenyl)but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- Amidoxime, 2.8 mg (8.2 μmol) of 6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexanetrifluoroacetate, 3.4 mg (9 The HATU of μmol) and 3.9 μl of Hünig's base were stirred in 0.77 ml of DMF at RT for 20 h. Subsequently, the reaction mixture was purified by preparative HPLC.

The title compound was obtained in 3 mg (31% of theory).

LC-MS (method 1): R _t =0.90 min; MS (ESI+): m/z=1164 (M+H) ⁺

Intermediate 185 N-{4-[(2,5-dioxapyrrolidin-1-yl)oxy]-4-oxobutylbutyl}-N-methyl-L-nonylamine fluorenyl-N-[(3R ,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S , 3E)-1-phenyl-4-(4-sulfophenyl)but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-side Oxyheptan-4-yl]-N-methyl-L-nonylamine decylamine

8 mg (7.5 μmol) of N-(3-carboxypropyl)-N-methyl-L-nonylamine-N-[(3R,4S,5S)-3-methoxy-1-{ (2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulfonate) Phenyl)but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- To a solution of 2 ml of DMF of amidoxime, 8.6 mg (74.8 μmol) of N-hydroxysuccinimide, 8.5 mg (22.4 μmol) of EDCI and 0.1 mg (0.75 μmol) of DMAP were added. The reaction mixture was stirred at RT for 20 h. Subsequently, 1.3 μl (7.5 μmol) of Hünig's base was added and the mixture was stirred at RT for 1 h. The reaction mixture was then purified by preparative HPLC. The title compound was obtained in 2.6 mg (72% purity, 21%).

LC-MS (method 1): R _t = 0.89 min; MS (ESI+): m/z=1054 (M+H) ⁺

Intermediate 186 N-(Tertibutoxycarbonyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[ (1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulfophenyl)butan-2-yl] Amino]propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

43 mg (0.06 mmol) of N-(t-butoxycarbonyl)-N-methyl-L-nonylamine-N-[(3R,4S,5S)-1-{(2S)-2 -[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]- To a solution of N-methyl-L-guanamine in 1.9 ml of DMF was added 29 mg (0.07 mmol) of HATU and 33 μl (0.19 mmol) of Hünig base. The reaction mixture was stirred at RT for 30 min and then mixed with 29 mg (0.07 mmol) of 4-[(3R)-3-amino-4-phenylbutyl]benzenesulfonic acid trifluoroacetate. After stirring at RT until overnight, the reaction mixture was purified using preparative HPLC. This gave 58 mg of the title compound and a 45:55 mixture of the deprotected amine.

LC-MS (Method _{1): R t = 1.09 min} ; MS (ESI +): m / z = 973 (M + H) +; R t = 0.87 min; MS (ESI +): m / z = 873 (M + H) ⁺ deprotected compounds.

Intermediate 187 N-methyl-L-Amidoxime-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy -2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulfophenyl)butan-2-yl]amino}propyl]pyrrole Pyridin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-decylamine guanamine trifluoroacetate

58 mg of intermediate 186 was dissolved in 4.1 ml of dichloromethane, 0.41 ml of trifluoroacetic acid was added and the mixture was stirred at RT for 30 min. After concentration under reduced pressure, the crude material was purified by preparative HPLC.

The title compound was obtained in 50 mg (90% purity, 85% of theory).

LC-MS (method 1): R _t = 0.87 min; MS (ESI+): m/z = 873 (M+H) ⁺

Intermediate 188 N-(3-carboxypropyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[( 1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulfophenyl)butan-2-yl]amine }]propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

171 μl (0.26 mmol) of 15% aqueous succinic acid was first charged to 2.5 ml of methanol and 50 mg (0.05 mmol) of N-methyl-L-amidoxime-N-[(3R,4S, 5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1 -Phenyl-4-(4-sulfophenyl)butan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N Methyl-L-Amidoxime trifluoroacetate was mixed with 11.6 μl (0.2 mmol) of acetic acid. The reaction mixture was stirred at RT for 5 min and then 30 μl (0.24 mmol) of borane-pyridine complex was added. After stirring for 24 hours, another equivalent of borane-pyridine complex was added and the mixture was stirred for a further 2 h. The reaction mixture was then purified by preparative HPLC.

40 mg (90% purity, 66% of theory) of title compound.

LC-MS (method 1): R _t = 0.91 min; MS (ESI+): m/z = 959 (M+H) ⁺

Intermediate 189 N-(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]]yl}-4-oxobutyl) -N-methyl-L-amidamine -N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo- 3-{[(2R)-1-phenyl-4-(4-sulfophenyl)butan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-side Oxyheptan-4-yl]-N-methyl-L-nonylamine decylamine

10 mg (9.3 μmol) of N-(3-carboxypropyl)-N-methyl-L-nonylamine-N-[(3R,4S,5S)-3-methoxy-1-{ (2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulfophenyl) Butyr-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine, 3.5 Mg (10.3 μmol) of 6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexane fluorene trifluoroacetate, 4.3 mg (11.2 μmol) of HATU and 4.9 Μl (28 μmol) of Hünig's base was stirred in 1 ml of DMF at RT for 20 h. Subsequently, the reaction mixture was purified by preparative HPLC. 4.2 mg (92% purity, 33% of theory) of title compound.

LC-MS (method 1): R _t = 0.91 min; MS (ESI+): m/z=1166 (M+H) ⁺

Intermediate 190 N-{4-[(2,5-dioxapyrrolidin-1-yl)oxy]-4-oxobutylbutyl}-N-methyl-L-nonylamine fluorenyl-N-[(3R , 4S, 5S)-3-methoxy -1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4) - sulfophenyl)butan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-decylamine Guanamine

10 mg (9.3 μmol) of N-(3-carboxypropyl)-N-methyl-L-nonylamine-N-[(3R,4S,5S)-3-methoxy-1-{ (2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulfophenyl) )but-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine 2.5 10.7 mg (93 μmol) of N-hydroxysuccinimide, 10.6 mg (28 μmol) of EDCI and 0.12 mg (0.9 μmol) of DMAP were added to the DMF solution of ml. The reaction mixture was stirred at RT 20 and then purified by preparative HPLC.

The title compound was obtained in 3.8 mg (72% purity, 25%).

LC-MS (Method _{1): R t = 0.90 min} ; MS (ESI +): m / z = 1055 (M + H) +

Intermediate 191 (2 R ,3 R )- N -[(2 S )-3-(1 H -吲哚-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]-3-methoxy-2-methyl-3-[( 2S )-pyrrolidin-2-yl]propanamide trifluoroacetic acid salt

The title compound is similar to the synthesis of intermediate 7 in the second stage from starting compound 1 and ( 2S )-2-amino-3-( 1H -indol-3-yl)-1-(1,2- Alk-2-yl)propan-1-trifluoroacetate (Intermediate 99) was prepared.

Yield in 2 stages: 62 mg (67% of theory)

HPLC (Method 6): R _t = 1.65 min; LC-MS (Method 1): R _t = 0.7 min; MS (ESI+): m/z = 443 (M+H) ⁺ .

Intermediate 192 N-methyl-L-Amidinoinyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-( 1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

The 1015 mg (1.59 mmol) of N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N - [(2 R, 3 S, 4 S )-1-carboxy-2-methoxy-4-methylhex-3-yl] -N -methyl-L-decylamine (Intermediate 4) is treated in 50 ml of DMF, 654 mg (2.39 mmol) of 2-bromo-1-ethyltetrafluoroborate pyrene (BEP) was mixed with 2.8 ml of N,N -diisopropylethylamine and stirred at RT. Then add 1083 mg (1.75 mmol) of (2 R , 3 R )- N -[(2 S )-3-(1 H -indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]-3-methoxy-2-methyl-3-[( 2S )-pyrrolidin-2-yl]propanamide trifluoroacetic acid Salt (Intermediate 191) and then the mixture was treated in an ultrasonic bath at RT for 30 min. The reaction mixture was then concentrated under reduced pressure and the residue was crystallised eluted elut The organic layer was washed successively with 5% aqueous citric acid and 5% aqueous sodium hydrogen carbonate, dried over magnesium sulfate, filtered and concentrated. The crude product (1684 mg) thus obtained was taken up in 20 ml of acetonitrile without further purification, 2 ml of piperidine was added and then the mixture was stirred at RT for 10 min. The mixture was then concentrated under reduced pressure and the residue was combined with diethyl ether. The solvent was again concentrated by evaporation and the residue was purified by flash chromatography on silica gel (solvent: 15:1:0.1→15:2:0.2 dichloromethane/methanol/17% aqueous ammonia). The corresponding dissolved fractions were combined, the solvent was removed under reduced pressure and the residue was lyophilized from acetonitrile/water. This gave 895 mg (67% in 2) of title compound.

HPLC (Method 12): R _t = 1.8 min; LC-MS (Method 1): R _t = 0.84 min; MS (ESI+): m/z = 840 (M+H) ⁺ .

¹ H NMR (500 MHz, DMSO-d ₆ ): δ = 10.8 (d, 1H), 8.3 and 8.05 (2d, 1H), 8.0 (d, 1H), 7.5 (m, 1H), 7.3 (m, 1H) ), 7.15 and 7.08 (2s, 1H) 7.05-6.9 (m, 2H), 5.12 and 4.95 (2m, 1H), 4.65 (m, 1H), 4.55 (m, 1H), 4.1-3.8 (m, 4H) , 3.75 (d, 1H), 3.23, 3.18, 3.17, 3.12, 2.95 and 2.88 (6s, 9H), 3.1-3.0 and 2.85 (2m, 2H), 2.65 (d, 1H), 2.4-2.2 (m, 3H) ), 2.15 (m, 3H), 1.95 (br.m, 2H), 1.85-0.8 (br.m, 11H), 1.08 and 1.04 (2d, 3H), 0.9-0.75 (m, 15H), 0.75-0.65 (dd, 3H) [More signals are hidden under the H ₂ O peak].

Intermediate 193 N-(3-carboxypropyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3 -{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

50 mg (0.052 mmol) of N-methyl-L-amidino-yl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{ [(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-decylamine (intermediate 192) and 204 μl of 15% aqueous 4-oxobutanoic acid in 2 Mol was mixed in methanol and mixed with 23.4 mg (0.252 mmol) of borane-pyridine complex and 6 μl of acetic acid. The reaction mixture was stirred at RT until overnight. Concentration was then carried out under reduced pressure and the residue was purified by preparative HPLC. After concentrating the corresponding fractions, 38 mg (78% of theory)

HPLC (Method 5): R _t = 1.7 min; LC-MS (Method 9): R _t = 4.7 min; MS (ESI+): m/z = 926 (M+H) ⁺ .

Intermediate 194 N-(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]]yl}-4-oxobutyl) -N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3- (1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

This compound was synthesized as described in Intermediate 157, from 10 mg (11 μmol) of N-(3-carboxypropyl)-N-methyl-L-decylamine-N-[(3R, 4S) ,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-decylamine and commercially available 6-(2,5-di-oxo-2,5-dihydro -1H-pyrrol-1-yl)hexane oxime was prepared.

Yield: 4.4 mg (35% of theory)

HPLC (method 5): _Rt = 1.8 min; LC-MS (Method 1): R _t =0.90 min; MS (ESI+): m/z=1133 (M+H) ⁺ .

Intermediate 195 N-[6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-nonylamine-yl-N-[(3R, 4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1- (1,2- Alkan-2-yl)-1-oxooxy-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl]-N-methyl-L-amidamine

This compound was similar to intermediate 166, prepared from 9 mg (0.010 mmol) of intermediate 170.

Yield: 1.1 mg (10% of theory)

HPLC (Method 12): R _t = 2.0 min; LC-MS (Method 1): R _t = 0.99 min; MS (ESI+): m/z = 994 (M+H) ⁺ .

Intermediate 196 ( 2S )-2-amino-1-(2-oxo-3-azabicyclo[2.2.2]oct-5-en-3-yl)-3-phenylpropan-1-one trifluoroacetic acid salt

41 mg (0.37 mmol) of 2,5-dioxapyrrolidin-1-yl N- (t-butoxycarbonyl)-L-phenylalanine was treated in 10 ml of DMF with 149 mg ( 0.41 mmol) of 2-oxo-3-azabicyclo[2.2.2]oct-5-ene (starting compound 6) and 72 μl (0.41 mmol) of N,N -diisopropylethylamine were combined. The mixture was stirred at RT for 1 h. The solvent was removed under reduced pressure and the~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ The organic layer was concentrated and the residue was purified by flash chromatography on silica gel eluting with 10:1 toluene/ethanol as solvent. The corresponding fractions were dissolved and the solvent was removed under reduced pressure. After drying the residue under high vacuum, 69 mg (yield: 47%) of Boc-protected intermediate ( 2S )-1-(2-oxo-3-azabicyclo[2.2.2] s- The third butyl 3-en-3-yl)-1-oxo-3-phenylpropan-2-ylaminecarboxylate is a mixture of diastereomers.

LC-MS (Method _{1): R t = 1.1 min} ; MS (ESI +): m / z = 359 (M + H) +.

64 mg (0.18 mmol) of this intermediate was taken in 10 ml of dichloromethane, 1 ml trifluoroacetic acid was added and the mixture was stirred at RT for 30 min. Then concentrate under reduced pressure and the remaining residue from water / two The alkane is freeze dried. From this method, 66 mg (quant.) of the title compound was obtained as a foam.

HPLC (method 6): _Rt = 1.45 min; LC-MS (Method 3): R _t = 1.12 min; MS (ESI+): m/z = 259 (M+H) ⁺ .

Intermediate 197 (2 R ,3 R )-3-Methoxy-2-methyl- N -[(2 S )-1-(2-oxo-3-azabicyclo[2.2.2]oct-5-ene- 3-yl)-1-oxo-3-phenylpropan-2-yl]-3-[(2 S )-pyrrolidin-2-yl]propanamide trifluoroacetate

First, (2 R , 3 R )-3-[(2 S )-1-(t-butoxycarbonyl)pyrrolidine--by treatment with ethyl acetate and extraction with a 5% aqueous solution of potassium hydrogensulfate. 2-Methoxy]-3-methoxy-2-methylpropanoic acid (starting compound 1) was released from its 83 mg (0.18 mmol) dicyclohexylamine salt. The organic layer was dried with MgSO4, filtered and evaporated. The residue was taken up in 10 ml of DMF is treated continuously with 66 mg (0.18 mmol) of (2 S) -2- amino-1- (2-oxo-3-azabicyclo [2.2.2] oct - 5-en-3-yl)-3-phenylpropan-1-one trifluoroacetate (intermediate 196), 101 mg (0.266 mmol) of O- (7-azabenzotriazol-1-yl) ) -N,N,N',N' -tetramethylhexafluorophosphate (HATU) was mixed with 93 μl (0.53 mmol) of N,N -diisopropylethylamine. The mixture was stirred at RT for 30 min. The reaction mixture was then concentrated and the residue was purified by preparative HPLC. This gave 52 mg (56% of theory) of Boc-protected intermediate ( 2S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[( 2 S )-1-(2-oxo-3-azabicyclo[2.2.2]oct-5-en-3-yl)-1-oxo-3-phenylpropan-2-yl]amino} 3-tert-oxypropyl]pyrrolidine-1-carboxylic acid tert-butyl ester.

HPLC (Method 6): R _t = 2.13 min; LC-MS (Method 1): R _t = 1.13 min; MS (ESI+): m/z = 528 (M+H) ⁺ .

52 mg (0.1 mmol) of this intermediate was taken in 10 ml of dichloromethane, 1 ml trifluoroacetic acid was added and the mixture was stirred at RT for 20 min. It was then concentrated under reduced pressure and the residue was stirred with 20 ml of diethyl ether. After 10 min, the mixture was filtered and the filtrate dried under high vacuum. 39 mg (72% of theory) of the title compound was obtained from this procedure.

HPLC (Method 6): R _t = 1.62 min; LC-MS (Method 1): R _t =0.68 min; MS (ESI+): m/z = 428 (M+H) ⁺ .

Intermediate 198 N -methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R ) 1-methoxy-2-methyl-3-{[(2 S )-1-(2-oxo-3-azabicyclo[2.2.2]oct-5-en-3-yl)-1 -Phenoxy-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-Amidoxime Trifluoroacetate

The 44.5 mg (0.071 mmol) of N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N - [(2 R, 3 S, 4 S )-1-carboxy-2-methoxy-4-methylhex-3-yl] -N -methyl-L-decylamine (Intermediate 4) was treated in 10 ml of DMF and continued and 38.6 mg (0.071 mmol) of (2 R, 3 R) -3- methoxy-2-methyl - N - [(2 S) -1- (2- oxo-3-azabicyclo [2.2. 2]oct-5-en-3-yl)-1-oxooxy-3-phenylpropan-2-yl]-3-[(2 S )-pyrrolidin-2-yl]propanamide trifluoroacetic acid Salt (Intermediate 197), 32.5 mg (0.086 mmol) of O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate (HATU) was mixed with 41 μl (0.235 mmol) of N,N -diisopropylethylamine. The mixture was stirred at RT for 1 h. The reaction mixture was then concentrated under reduced pressure and the residue was crystallised The organic layer was washed successively with aq. 5% EtOAc EtOAc. Thereby obtaining 73 mg (98% of theory) of the Fmoc-protected intermediate N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3 -{[(2 S )-1-(2-oxo-3-azabicyclo[2.2.2]oct-5-en-3-yl)-1-oxo-3-phenylpropan-2-yl Amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine.

HPLC (Method 6): R _t = 2.78 min; LC-MS (Method 3): R _t = 2.96 min; MS (ESI+): m/z=1047 (M+H) ⁺ .

73 mg (0.071 mmol) of this intermediate was dissolved in 5 ml of DMF. After the addition of 0.5 ml of piperidine, the reaction mixture was stirred at RT for 10 min then concentrated over EtOAc. After the diethyl ether was decanted, the residue was purified by preparative HPLC (solvent: acetonitrile / 0.1% aq. TFA). Get 16 mg (26% of theory) The title compound is a foam.

HPLC (method 6): R _t = 1.94 min; LC-MS (method 3): R _t = 1.71 min; MS (ESI+): m/z = 825 (M+H) ⁺

¹ H NMR (400 MHz, DMSO-d ₆ ): δ = 8.9-8.6 (m, 3H), 8.4, 8.3, 8.1 and 8.0 (4d, 1H), 7.3-7.1 (m, 5H), 6.7-6.5 ( m, 2H), 5.2-4.8 (m, 3H), 4.75-4.55 (m, 3H), 4.05-3.95 (m, 1H), 3.7-3.4 (m, 4H), 3.22, 3.17, 3.15, 3.05, 3.02 And 2.95 (6s, 9H), 3.0 and 2.7 (2 br.m, 2H), 2.46 (m, 3H), 2.4-1.2 (br.m, 13H), 1.1-0.85 (m, 18H), 0.75 (m) , 3H) [More signals are hidden under the H ₂ O peak].

Intermediate 199 N-(4-{2-[6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]]yl}-4-oxobutyl) -N-methyl-L-Amidoxime-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy 2-methyl-3-{[(2S)-1-(2-oxo-3-azabicyclo[2.2.2]oct-5-en-3-yl)-1-sideoxy-3- Phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- Amidoxime

The title compound is similar to intermediates 193 and 194 from 23 mg (24 μmol) of N-methyl-L-decylamine-N-[(3R,4S,5S)-3-methoxy-1-{ (2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(2-oxo-3-azabicyclo[2.2.2] octyl -5-en-3-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1 -Sideoxyheptan-4-yl]-N-methyl-L-decylamine guanamine trifluoroacetate (Intermediate 198) was prepared.

HPLC (Method 12): Rt = 1.9 min; LC-MS (Method 2): Rt = 2.1 min; MS (ESI+): m/z=1118 (M+H)+.

Intermediate 200 N-[2-(2-{2-[2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy) Ethyl]-N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S) -3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

This preparation is similar to intermediates 174 and 175, starting with the alkylation of intermediate 192 with intermediate 172, followed by deprotection and formation of Malay The imine is carried out.

HPLC (method 12): Rt = 1.9 min; LC-MS (Method 1): Rt = 0.86 min; MS (ESI+): m/z = 10.25 (M+H)+.

Intermediate 201 N-{6-[(bromoethenyl)amino]hexyl}-N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2- [(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

22 mg (0.023 mmol) of N-(6-aminohexyl)-N-methyl-L-nonylamine-N-[(3R,4S,5S)-1-{(2S)-2- [(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-decylamine amide (Intermediate 101) is dissolved in 9.5 ml of THF at 0 ° C with 4.2 μl of three Mix ethylamine. A solution of bromoacetic chloride in THF was added dropwise and the mixture was stirred at 0 ° C for 30 min. The reaction mixture was concentrated and the residue was purified using EtOAc. This gave 6.9 mg (26% of theory) of the title compound as a foam.

HPLC (method _{5): R t = 1.8 min} ; LC-MS ( method _{11): R t = 0.9 min} ; MS (ESI +): m / z = 1059 and 1061 (M ⁺ H) +.

Intermediate 202 N-{2-[2-(2-{3-[(2,5-dioxaxopyridin-1-yl)oxy]-3-oxooxypropoxy}ethoxy)ethoxy] Ethyl}-N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S) -3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

This preparation is first followed by a similar intermediate 168, starting with reductive alkylation of intermediate 192 with intermediate 167, and subsequent hydrogenolysis cleavage of N-(2-{2-[2-(2-carboxyethoxy)) Oxy]ethoxy}ethyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)- 3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A The benzyl ester of oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-decylamine is carried out.

13 mg (10 μmol) of this intermediate was dissolved in 5 ml of DMF with 2.1 mg (20 mmol) of 1-hydroxypyrrolidine-2,5-dione, 6.5 μl of N,N -diisopropyl Ethylamine was mixed with 7.1 mg (0.02 mmol) of HATU. The reaction mixture was stirred at rt overnight then concentrated EtOAc. The remaining residue was purified by preparative HPLC. After lyophilization with acetonitrile / water, 9.2 mg (yield: 62%)

HPLC (Method 12): _rt = 2.0 min; LC-MS (Method 2): R _t = 2.1 min; MS (ESI+): m/z=1141 (M+H) ⁺ .

Intermediate 203 6-mercapto-6-oxo-oxyhexylaminecarboxylic acid tert-butyl ester

This compound is coupled by standard peptide chromatography by the coupling of 6-[(t-butoxycarbonyl)amino]hexanoic acid with a benzyl hydrazine carboxylate in the presence of EDCI and HOBT, and subsequent hydrogenolysis. Prepared by cleaving the benzyloxycarbonyl protecting group.

LC-MS (method _{11): R t = 0.59 min} ; MS (ESI +): m / z = 246 (M + H) +.

Intermediate 204 N-{4-[2-(6-Aminohexylidene)indolyl]-4-sideoxybutyl}-N-methyl-L-decylamine-N-[(3R,4S,5S) )-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropane- 2-yl]amino}-1-methoxy -2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- Amidoxime trifluoroacetate

146 mg (50 μmol) of (N-(3-carboxypropyl)-N-methyl-L-nonylamine-N-[(3R,4S,5S)-1-{(2S)-2 -[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1- Methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl -L-Amidoxime is dissolved in 5 ml of DMF and then with 30.6 mg (80 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N',N'- Tetramethyl hexafluorophosphate 19 μl of N,N-diisopropylethylamine was mixed with 22.4 mg (60 μmol) of 6-mercapto-6-oxo-oxyhexylaminecarboxylic acid tert-butyl ester. The reaction mixture was stirred at RT for 1.5 h. It was then concentrated under high vacuum and the residue was purified using preparative HPLC. This gave 43 mg (68% of theory) of protected intermediate which was then taken up in 10 ml of dichloromethane and deprotected with 1 ml of trifluoroacetic acid. The reaction mixture was concentrated and the residue was crystallised from dichloromethane, This gave 45 mg (68% of theory in two phases) of title compound.

HPLC (Method 12): _Rt = 1.6 min; LC-MS (Method 11): R _t = 0.66 min; MS (ESI+): m/z = 983 (M+H) ⁺ .

Intermediate 205 N-(4-{2-[6-({[2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amine)醯 肼 肼 肼 } } -4- -4- -4- -4- -4- -4- -4- -4- -4- -4- -4- -4- -4- -4- -4- -4- -4- -4- -4- -4- -4- - - - - - - - - - - - - - - - - - - - (1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy Benzyl-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L - amidoxime

This compound is similar to intermediate 114 and is prepared starting from intermediates 50 and 204.

Yield: 4 mg (78% of theory)

HPLC (Method 12): _Rt = 1.7 min; LC-MS (Method 11): R _t = 0.73 min; MS (ESI+): m/z=1149 (M+H) ⁺ .

Intermediate 206 N-(6-{[3-({3-[(2,5-dioxapyrrolidin-1-yl)oxy)-3-yloxypropyl}didecyl)propanyl]amino} Hexyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)- 3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

8 mg (10 μmol) of intermediate 101 was dissolved in 2 ml of DMF and with 8.6 mg (20 μmol) of 1,1'-{di-p-aminophenylsulfonyl bis[(1-oxo-propane-3, 1-Diyl)oxy]}dipyrrolidine-2,5-dione and 3.7 μl of N,N -diisopropylethylamine were mixed. The reaction mixture was stirred at RT for 2 h then EtOAc was evaporated. This gave 7.2 mg (68% of theory) of title compound.

HPLC (Method 5): R _t = 1.9 min; LC-MS (Method 11): R _t = 0.94 min; MS (ESI+): m/z = 615 [1⁄2 (M+2H ⁺ ]

Intermediate 207 (1S,2R)-1-amino-2-phenylcyclopropanecarboxylic acid trifluoroacetate

210 mg (0.76 mmol) of commercially available (1 S , 2 R )-1-[(t-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid was deprotected with trifluoroacetic acid to give The title compound in mass yield.

LC-MS (Method _{1): R t = 0.23 min} ; MS (ESI +): m / z = 178 (M + H) +.

Intermediate 208 9H-fluoren-9-ylmethyl-6-oxohexylamine carbamate

The title compound was prepared from 1 g (2.95 mmol) of commercially available 9H-indole-9-ylmethyl 6-hydroxyhexylamine formate under standard conditions, oxidized with sulfur trioxide-pyridine complex. This gave 840 mg (85% of theory) of title compound.

HPLC (Method 12): R _t = 2.0 min; LC-MS (Method 1): R _t = 1.1 min; MS (ESI+): m/z = 338 (M+H) ⁺ .

Intermediate 209 N-[6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-nonylamine-yl-N-[(3R, 4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-carboxy-2-phenylcyclopropyl]amino}-1-A Oxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl- L-Amidoxime

First, a synthesis similar to that described in Intermediate 75, by N-(t-butoxycarbonyl)-N-methyl-L-nonylamine-N-[(3R,4S,5S)-1 -{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-sideoxy Hept-4-yl]-N-methyl-L-decylamine (intermediate 26) and (1S,2R)-1-amino-2-phenylcyclopropanecarboxylic acid trifluoroacetate (intermediate) 207) O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylhexafluorophosphate Coupling in the presence of the same, followed by separation of the Boc protecting group with trifluoroacetic acid to prepare the amine compound N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-1-{(2S)-2- [(1R,2R)-3-{[(1S,2R)-1-carboxy-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl Pyrrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine is a trifluoroacetate salt.

Then, 22 mg (0.026 mmol) of this compound in 10 ml of methanol was added 17 mg (0.05 mmol) of 9H-indol-9-ylmethyl 6-oxohexylamine carbamate (intermediate 208) and 2.3 mg of acetic acid and 11.4 mg (0.12 mmol) of borane-pyridine complex. The reaction mixture was stirred at RT until overnight. Then add the same amount of borane-pyridine complex and acetic acid and 8 mg of indole-9-ylmethyl-6-oxohexylaminecarboxylic acid The ester was stirred and the reaction mixture was stirred at RT for a further 24 h. Concentration was then carried out under reduced pressure and the residue was purified by preparative HPLC. This product was used in the next stage immediately after concentration of the corresponding dissolved fraction.

33 mg of the intermediate with impurities was placed in 5 ml of DMF and 1 ml of piperidine was added. After stirring at RT for 15 min, the reaction mixture was concentrated and residue was purified using preparative HPLC. This gave 11 mg (55% of theory in two stages) of the aminocarboxylic acid intermediate.

HPLC (Method 12): _Rt = 1.7 min; LC-MS (Method 11): _Rt = 0.7 min; MS (ESI+): m/z = 843 (M+H) ⁺ .

6 mg (7.12 μmol) of this intermediate was placed in 1 ml of two It was treated with an alkane and then mixed with 6.6 mg (42.7 μmol) of methyl 2,5-di-oxo-2,5-dihydro-1H-pyrrole-1-carboxylate and with 5 μl of a saturated aqueous solution of sodium hydrogencarbonate. The reaction mixture was stirred at RT for 1 h. Then another 3 portions of each 50 μl of a saturated aqueous solution of sodium hydrogencarbonate were added and the mixture was stirred at RT for additional 30 min. The reaction mixture was then acidified to pH 2 with trifluoroacetic acid and then concentrated under reduced pressure. The remaining residue was purified by preparative HPLC. After lyophilization with acetonitrile/water, 4 mg (60% of theory) of title compound was obtained as a foam.

HPLC (Method 12): _Rt = 1.9 min; LC-MS (Method 11): R _t = 0.98 min; MS (ESI+): m/z = 923 (M+H) ⁺ .

Intermediate 210 N-{6-[(2,5-Dioxapyrrolidin-1-yl)oxy]-6-oxo-oxyhexyl}-N-methyl-L-nonylamine fluorenyl-N-[(3R, 4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

First, 6-oxohexanoic acid was prepared by a literature method (J. Org. Chem. 58, 1993, 2196-2200).

80 mg (0.08 mmol) of N-methyl-L-nonylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{ [(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-decylamine (Intermediate 192) and 65.4 mg (0.5 mmol) of 6-oxohexanoic acid 9 ml of methanol was mixed and mixed with 10 μl of acetic acid and 37.4 mg (0.4 mmol) of borane-pyridine complex. The reaction mixture was stirred at RT until overnight. Concentration was then carried out under reduced pressure and the residue was taken in 1:1 acetonitrile/water and adjusted to pH 2 with trifluoroacetic acid. The reaction mixture was concentrated again and the residue was purified by preparative HPLC. After concentrating the corresponding dissolved fraction, 70 mg (86% of theory) of N-(5-carboxypentyl)-N-methyl-L-decylamine-N-[(3R,4S,5S) was obtained. )-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine is a trifluoroacetate salt.

HPLC (Method 12): R _t = 1.9 min; LC-MS (Method 1): R _t = 0.87 min; MS (ESI+): m/z = 955 (M+H) ⁺ .

¹ H NMR (500 MHz, DMSO-d ₆ , characteristic signal): δ = 12.0 (br. M, 1H), 10.8 (s, 1H), 9.4 (m, 1H), 8.9 and 8.8 (2d, 1H), 8.3 and 8.02 (2d, 1H), 7.5 (m, 1H), 7.3 (m, 1H), 7.15 and 7.1 (2s, 1H) 7.05-6.9 (m, 2H), 5.12 and 4.95 (2m, 1H), 4.7 -4.5 (m, 2H), 4.1-3.8 (m, 4H), 3.75 (d, 1H), 3.25, 3.2, 3.18, 3.13, 2.98 and 2.88 (6s, 9H), 2.8 (m, 3H), 1.08 and 1.04 (2d, 3H), 0.95-0.8 (m, 15H), 0.8-0.65 (dd, 3H).

22 mg (23 μmol) of this intermediate was dissolved in 1.8 ml of dichloromethane and with 13.2 mg (70 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride, 26.5 mg (230 μmol) of 1-hydroxypyrrolidine-2,5-dione and 0.28 mg (2 μmol) of dimethylaminopyridine were combined and the reaction mixture was stirred at RT for 2 h. Subsequently, the reaction mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC purification. After lyophilization from acetonitrile/water, 21.3 mg (yield: 88%) of title compound.

HPLC (method _{12): R t = 1.9 min} ; LC-MS ( Method _{1): R t = 0.94 min} ; MS (ESI +): m / z = 1052 (M + H) +.

Intermediate 211 N-{6-[(2,5-Dioxapyrrolidin-1-yl)oxy]-6-oxo-oxyhexyl}-N-methyl-L-nonylamine fluorenyl-N-[(3R, 4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1- (1,2- Alkan-2-yl)-1-oxooxy-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl]-N-methyl-L-amidamine

Similar to intermediate 210, 15 mg (20 μmol) of N -methyl-L-nonylamine- N -[(3 R ,4 S ,5 S )-3-methoxy-1-{(2 S )-2-[(1 R ,2 R )-1-methoxy-2-methyl-3-{[(2 S ,3 S )-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidinyl}-5-methyl-1-oxo-heptane- The 4-alkyl] -N -methyl-L-guanamine guanamine trifluoroacetate (Intermediate 15) is reductively alkylated with 6-oxohexanoic acid.

Yield: 9.2 mg (61% of theory)

HPLC (Method 12): R _t = 1.9 min; LC-MS (Method 1): R _t = 0.87 min; MS (ESI+): m/z = 929 (M+H) ⁺ .

9 mg (10 μmol) of this intermediate was dissolved in 3 ml of DMF with 5.6 mg (48 μmol) of 1-hydroxypyrrolidine-2,5-dione, 5 μl of N,N -diisopropyl Ethylamine was mixed with 5.5 mg (0.015 mmol) of HATU and the reaction mixture was treated in an ultrasonic bath for 6 h. During this time, 5.5 mg of HATU was added per hour. Subsequently, the reaction mixture was concentrated under reduced pressure, and the residue was taken and taken to acetonitrile/water and adjusted to pH with trifluoroacetic acid. After concentration again under reduced pressure, the remaining residue was purified by preparative HPLC. After lyophilization from acetonitrile / water, 5.8 mg (yield: 57%)

HPLC (Method 12): R _t = 2.0 min; LC-MS (Method 1): R _t = 0.95 min; MS (ESI+): m/z = 127 (M+H) ⁺ .

Intermediate 212 N-{2-[2-(2-{3-[(2,5-dioxapyrrolidin-1-yl)oxy]-3-yloxypropyl}ethoxy)ethoxy]B }--N-methyl-L-decyl fluorenyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- Methoxy-2-methyl-3-{[(2S,3S)-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo Hept-4-yl]-N-methyl-L-amidamine

This preparation begins with a similar intermediate 168, starting with the reductive alkylation of intermediate 15 with intermediate 167, and subsequent hydrogenolysis cleavage of N-(2-{2-[2-(2-carboxyethoxy)) Oxy]ethoxy}ethyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[ (1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2- Alkan-2-yl)-1-oxooxy-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxo The benzyl ester of hept-4-yl]-N-methyl-L-decylamine is used.

8.4 mg (8 μmol) of this intermediate was dissolved in 3 ml of DMF with 9.5 mg (80 μmol) of 1-hydroxypyrrolidine-2,5-dione, 10 μl of N,N -diisopropyl Ethylamine and 9.4 mg (25 μmol) of HATU were combined and the mixture was stirred at RT overnight and then concentrated under reduced pressure. Subsequently, the reaction mixture was concentrated under reduced pressure. After concentration under reduced pressure, the remaining residue was purified by preparative HPLC. After lyophilization from acetonitrile / water, 4 mg (32% of theory)

HPLC (Method 12): _Rt = 2.0 min; LC-MS (Method 1): R _t = 0.96 min; MS (ESI+): m/z=1117 (M+H) ⁺ .

Intermediate 213 N-{6-[(trans-4-{[(2,5-dioxaxopyridin-1-yl)oxy]carbonyl}cyclohexyl)amino]-6-oxohexyl}-N- Methyl-L-Amidoxime-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-吲哚-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

This compound is similar to intermediate 104 from N-(5-carboxypentyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2 -[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine (the synthesis of which is described in Intermediate 210) was prepared initially. 9.3 mg of the title compound are obtained (37% of theory in three phases).

HPLC (Method 12): R _t = 1.9 min; LC-MS (Method 1): R _t = 0.9 min; MS (ESI+): m/z=1177 (M+H) ⁺ .

Intermediate 214 N-{4-[(2,5-dioxapyrrolidin-1-yl)oxy]-4-oxobutylbutyl}-N-methyl-L-nonylamine fluorenyl-N-[(3R ,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}- 1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-melamine

This compound is similar to intermediate 210 and is prepared by converting intermediate 92 to the active ester.

HPLC (Method 5): R _t = 1.6 min; LC-MS (Method 11): R _t = 0.82 min; MS (ESI+): m/z = 901 (M+H) ⁺ .

Intermediate 215 N-{6-[(2,5-Dioxapyrrolidin-1-yl)oxy]-6-oxo-oxyhexyl}-N-methyl-L-nonylamine fluorenyl-N-[(3R, 4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1 -Methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-A ke-L-amide amine

First, an intermediate 40, similar to the intermediate 183, was used to prepare N-(5-carboxypentyl)-N-methyl-L-decylamine-N-[(3R, 4S) with a borane-pyridine complex. ,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1- Methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl -L-Amidoxime. From this compound, similar to intermediate 210, an active ester is produced. The title compound was obtained as 34 mg (36% of theory).

HPLC (method 5): _Rt = 1.6 min; LC-MS (Method 1): R _t = 0.85 min; MS (ESI+): m/z = 930 (M+H) ⁺ .

Intermediate 216 N-(4-{[(2,5-Dioxaxopyridin-1-yl)oxy]carbonyl}benzyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S) ,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

First, similar to the preparation of the intermediate 183, the intermediate 192 is reacted with 4-methylmercaptobenzoic acid and a borane-pyridine complex to obtain N-(4-carboxybenzyl)-N-methyl-L-decylamine. Mercapto-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl) -1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine. This compound was then used, similar to intermediate 210, to yield 11 mg (yield: 68%) of the title compound.

HPLC (method 5): _Rt = 1.8 min; LC-MS (Method 1): R _t = 1.13 min; MS (ESI+): m/z = 1072 (M+H) ⁺ .

Intermediate 217 N-(5-carboxypentyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3 -{[(2S)-1-(benzyloxy)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropane Pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

53 mg (84 μmol) of N -[(9 H -茀-9-ylmethoxy)carbonyl] -N -methyl-L-nonylamine- N -[(2 R , 3 S , 4 S )-1-carboxy-2-methoxy-4-methylhex-3-yl] -N -methyl-L-decylamine (Intermediate 4) and 45 mg (84 μmol) of N- {(2 R ,3 R )-3-methoxy-2-methyl-3-[(2 S )-pyrrolidin-2-yl]propanyl}-L-phenylalanine benzyl trifluoroacetic acid The salt (Intermediate 12) was treated with 2 ml of DMF and 19 μl of N,N -diisopropylethylamine, 14 mg (92 μmol) of HOBt and 17.6 mg (92 μmol) of EDC were added. And the mixture was then stirred at RT until overnight. Subsequently, the reaction mixture was concentrated and the residue was purified by preparative HPLC. Thereby obtaining 59 mg (68% of theory) of the Fmoc-protected intermediate N - [(9 H - fluorenyl-9-ylmethoxy) carbonyl] - N - methyl -L- valinamide acyl - N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzyloxy)-1 -Phenoxy-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy- 5-Methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine.

LC-MS (Method _{1): R t = 1.55 min} ; m / z = 1044 (M + H) +.

57 mg (0.055 mmol) of this intermediate was treated with 1.2 ml piperidine in 5 ml of DMF to isolate the Fmoc protecting group. Concentrated and purified to formula HPLC, to give 39 mg (76% of theory) of the free amine intermediate N - methyl -L- valinamide acyl - N - [(3 R, 4 S, 5 S) - 1-{(2 S )-2-[(1 R ,2 R )-3-{[(2 S )-1-(benzyloxy)-1-oxo-3-phenylpropan-2-yl Amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4- The base] -N -methyl-L-guanamine amine is a trifluoroacetate salt.

HPLC (Method 5): R _t = 1.9 min; LC-MS (Method 1): R _t = 1.01 min; m/z = 822 (M+H) ⁺ .

60 mg (0.06 mmol) of this intermediate, similar to intermediate 210, was reacted with 6-oxohexanoic acid in the presence of a borane-pyridine complex. The title compound was obtained as a foam of 45 mg (75% of theory).

HPLC (method 5): _Rt = 1.9 min; LC-MS (Method 1): R _t = 0.97 min; MS (ESI+): m/z=9936 (M+H) ⁺ .

Intermediate 218 N-{6-[(2,5-Dioxapyrrolidin-1-yl)oxy]-6-oxo-oxyhexyl}-N-methyl-L-nonylamine fluorenyl-N-[(3R, 4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzyloxy)-1-oxo-3-phenylpropan-2- Amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4 -yl]-N-methyl-L-amidamine

This compound is obtained by using 42 mg (0.05 mmol) of intermediate 217 Prepared by conversion to an active ester.

Yield: 26 mg (54%)

HPLC (method 5): _Rt = 2.1 min; LC-MS (Method 1): R _t = 1.01 min; MS (ESI+): m/z=1034 (M+H) ⁺ .

Intermediate 219 N-{6-[(2,5-Dioxapyrrolidin-1-yl)oxy]-6-oxo-oxyhexyl}-N-methyl-L-nonylamine fluorenyl-N-[(3R, 4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2 -methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-decylamine Guanamine

20 mg (0.02 mol) of the compound from intermediate 218 was treated with 2.4 ml of methanol and palladium on 5% activated carbon under standard hydrogen pressure and hydrogenated at RT for 30 min. The catalyst was then filtered off and the solvent was removed under reduced pressure. The residue was lyophilized from 1:1 acetonitrile / water. This gave 14 mg (92% of theory) of title compound as colourless foam.

HPLC (method 5): _Rt = 1.7 min; LC-MS (Method 1): R _t = 0.96 min; MS (ESI+): m/z = 944 (M+H) ⁺ .

Intermediate 220 N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1- (1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

0.5 g (1.01 mmol) of the intermediate in 10 ml of dichloromethane was mixed with 1 ml of trifluoroacetic acid. After 30 min in an ultrasonic bath, the mixture was concentrated and re-evaporated with DCM, then evaporated with diethyl ether and dried under high vacuum. The oily residue was used in the next stage without further purification.

500 mg of this intermediate was dissolved in 20 ml of DMF and with 466 mg (3.8 mmol) of intermediate 191, 382 mg (1.01 mmol) of O-(7-azabenzotriazol-1-yl)- N,N,N',N'-tetramethylhexafluorophosphate (HATU) was mixed with 440 μl (2.5 mmol) of N,N-diisopropylethylamine. The mixture was stirred at RT for 1 h and stirred at rt until concentrated overnight. The residue was taken up in dichloromethane and extracted with aq. EtOAc EtOAc. The organic layer was concentrated and the residue was purified by flash chromatography eluting eluting The corresponding dissolved fractions were combined and the solvent was removed under reduced pressure. After the residue was dried under high vacuum, 562 mg (yield: 65% of theory).

562 mg (0.57 mmol) of this intermediate was taken up in 50 ml of methanol and hydrogenated on 155 mg of 10% activated carbon on palladium under standard hydrogen pressure for 20 min. The catalyst was then filtered off and the solvent was removed under reduced pressure. The residue was purified by preparative HPLC. The corresponding dissolved fractions are combined, the solvent is evaporated under reduced pressure and the residue is taken from The alkane is freeze dried. This gave 361 mg (87% of theory) of title compound as foam.

HPLC (method 5): double peak at Rt = 1.75 and 1.86 min; LC-MS (method 1): double peak at Rt = 0.84 min and 0.91 min with the same mass; MS (ESI+): m/z = 944 (M) +H) ⁺ .

Intermediate 221 N-{(2S)-2-[(Tertibutoxycarbonyl)amino]-3-phenylpropyl}-N-methyl-L-proline

100 mg (0.76 mmol) of commercially available N-methyl-L-proline and 285 mg (1.14 mmol) of commercially available (2S)-1-oxo-3-phenylpropan-2-ylaminecarboxylic acid The third butyl ester was mixed in 22 ml of methanol and mixed with 340 mg (3.66 mmol) of borane-pyridine complex and 70 μl of acetic acid. The reaction mixture was stirred at RT until overnight. Concentration was then carried out under reduced pressure and the residue was purified by flash chromatography on silica gel eluting with methylene chloride / methanol / 17% aqueous ammonia as solvent. Concentrate the corresponding dissolved fractions from 1:1 After lyophilization of the alkane/water, 259 mg (93% of theory)

HPLC (Method 12): _Rt = 1.6 min; LC-MS (Method 11): R _t = 0.76 min; MS (ESI+): m/z = 365 (M+H) ⁺ .

Intermediate 222 N-[(2S)-2-Amino-3-phenylpropyl]-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-1-{(2S)- 2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine guanamine trifluoroacetate

40 mg (0.11 mmol) of N-{(2S)-2-[(tatabutoxycarbonyl)amino]-3-phenylpropyl}-N-methyl-L-proline (middle 221) dissolved in 5 ml of DMF and with 80 mg (0.11 mmol) of N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[( 2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-decylamine (Intermediate 220), 50 mg (0.13 mmol) of O- (7-aza Benzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate (HATU) was mixed with 57 μl (2.5 mmol) of N,N -diisopropylethylamine. The mixture was stirred at RT for 1 h and then concentrated. The residue was taken up in ethyl acetate and taken and washed with 5% aqueous citric acid and then water. The organic layer was concentrated and the residue was purified by preparative HPLC. The corresponding dissolved fractions were combined and the solvent was removed under reduced pressure. From two After lyophilization of the alkane, 60 mg (50% of theory) of protected intermediate was obtained.

HPLC (method _{12): R t = 2.2 min} ; LC-MS ( Method _{1): R t = 1.17 min} ; MS (ESI +): m / z = 1073 (M + H) +.

60 mg (0.05 mmol) of this intermediate was taken in 10 ml of dichloromethane, 2 ml of trifluoroacetic acid was added and the mixture was stirred at RT for 1.5 h. Subsequently, the reaction mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC. Combine the corresponding dissolved fractions, remove the solvent under reduced pressure and remove the residue from the second The alkane is freeze dried. There was obtained 25 mg (42% of theory) of the title compound as a foam.

HPLC (Method 12): _Rt = 1.9 min; LC-MS (Method 1): R _t = 0.95 min; MS (ESI+): m/z = 974 (M+H) ⁺ .

Intermediate 223 N-[(2S)-2-({[2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amine)-amino)amino)- 3-phenylpropyl]-N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{ [(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

This preparation was similar to intermediate 134, starting with 5 mg (4.6 μmol) of intermediate 222. This gave 3.4 mg (65% of theory) of title compound.

HPLC (Method 12): R _t = 2.0 min; LC-MS (Method 1): R _t = 0.99 min; MS (ESI+): m/z=1140 (M+H) ⁺ .

Intermediate 224 N-[(2S)-2-({[2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amine-methyl)amino)propyl ]]-N-methyl-L-Amidinoindolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)- 3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

This preparation is carried out analogously to the synthesis of intermediate 223.

HPLC (method _{12): R t = 1.9 min} ; LC-MS ( Method _{1): R t = 0.92 min} ; MS (ESI +): m / z = 1064 (M + H) +.

Intermediate 225 N-(2-Aminoethyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)- 3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine guanamine trifluoroacetate

100 mg (0.76 mmol) of commercially available N-methyl-L-proline and 182 mg (1.14 mmol) of commercially available tert-butyl 2-oxoethoxyethylamine in 20 ml of methanol were mixed. It was mixed with 340 mg (3.66 mmol) of borane-pyridine complex and 65 μl of acetic acid. The reaction mixture was stirred at RT until overnight. Concentration was then carried out under reduced pressure and the residue was purified by flash chromatography on silica gel eluting with methylene chloride / methanol / 17% aqueous ammonia as solvent. Concentrate the corresponding dissolved fractions and take 1:1 After lyophilization of the alkane/water, 190 mg, 39% purity (35% of theory) of intermediate was obtained which was converted without further purification.

50 mg (0.07 mmol) of this intermediate was dissolved in 10 ml of DMF and with 52 mg (0.07 mmol) of N-[(3R,4S,5S)-1-{(2S)-2-[(1R, 2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-decylamine (Intermediate 220), 32 mg (0.09 mmol) of O- (7-aza Benzotriazol-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate (HATU) was mixed with 37 μl (0.2 mmol) of N,N -diisopropylethylamine. The mixture was stirred at RT overnight and then concentrated. The residue was taken up in ethyl acetate and extracted with aq. The organic layer was concentrated and the residue was purified by preparative HPLC. The corresponding dissolved fractions were combined and the solvent was removed under reduced pressure. From two After lyophilization of the alkane, 53 mg (76% of theory) of intermediate was obtained.

HPLC (Method 12): _rt = 2.0 min; LC-MS (Method 1): R _t = 1.02 min; MS (ESI+): m/z = 984 (M+H) ⁺ .

53 mg (0.05 mmol) of this intermediate was taken up in 10 ml of dichloromethane, 2 ml of trifluoroacetic acid was added and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC. Combine the corresponding dissolved fractions, remove the solvent under reduced pressure and remove the residue from the second The alkane/water is freeze dried. This gave 21 mg (40% of theory) of the title compound.

HPLC (Method 12): R _t = 1.7 min; LC-MS (Method 1): R _t = 0.87 min; MS (ESI+): m/z = 884 (M+H) ⁺ .

Intermediate 226 N-[2-({[2-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amine-carbamoyl}amino)ethyl]-N -Methyl-L-Amidoxime-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H -吲哚-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

This preparation is similar to the synthesis of intermediate 134, starting with intermediate 225. 11.6 mg (59% of theory) of the title compound.

HPLC (Method 12): R _t = 1.9 min; LC-MS (Method 1): R _t =0.90 min; MS (ESI+): m/z=1050 (M+H) ⁺ .

Intermediate 227 N-{6-[(2,5-dioxapyrrolidin-1-yl)oxy]-6-oxohexyl}-N-A --L-Amidinoinyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzyloxy) --3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine- 1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

This compound is similar to intermediate 218 and is prepared by conversion to the active ester.

Yield: 18 mg (51% of theory)

HPLC (method _{5): R t = 2.1 min} ; LC-MS ( Method _{1): R t = 0.98 min} ; MS (ESI +): m / z = 1073 (M + H) +.

Intermediate 228 (2R,3S)-3-[(Tertibutoxycarbonyl)amino]-4-{2-[6-(2,5-di-oxo-2,5-dihydro-1H-pyrrole-1 -yl)hexyl]mercapto}-4-oxobutan-2-yl (3R,4S,7S,10S)-4-[(2S)-but-2-yl]-7,10-diiso Propyl-3-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,11-dimethyl -6,9-di-oxo-2-oxo-5,8,11-triazadecane-15-erate

The title compound is a Boc-protected intermediate obtained by synthesizing the intermediate 154 with a commercially available 6-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl). Prepared by hexane oxime coupling.

HPLC (method 12): _Rt = 2.1 min; LC-MS (Method 1): R _t = 0.97 min; MS (ESI+): m/z=1308 (M+H) ⁺ .

Intermediate 229 (2R,3S)-3-acetamido-4-{2-[6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexanyl]anthracene }--4-oxobutan-2-yl (3R,4S,7S,10S)-4-[(2S)-but-2-yl]-7,10-diisopropyl-3-(2- {(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,11-dimethyl -6,9-di-oxo-2-oxo-5,8,11-triazadecane-15-erate

The title compound is from 7.5 mg (2.5 μmol) of intermediate 154 by using 2.3 μl of acetic anhydride in 1 ml of DMF in the presence of 0.4 μl of N,N -diisopropylethylamine. Chemical preparation

Yield: 1.4 mg (40% of theory)

HPLC (Method 12): R _t = 1.9 min; LC-MS (Method 1): R _t =0.86 min; MS (ESI+): m/z=1250 (M+H) ⁺ .

Intermediate 230 (2R,3S)-3-[(Tertibutoxycarbonyl)amino]-4-{2-[6-(2,5-di-oxo-2,5-dihydro-1H-pyrrole-1 -yl)hexyl]mercapto}-4-oxobutan-2-yl (3R,4S,7S,10S)-4-[(2S)-but-2-yl]-3-(2-{ (2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-side Oxyethyl)-7,10-diisopropyl-5,11-dimethyl-6,9-di-oxo-2-oxo-5,8,11-triazadecane-15-erate

This compound is similar to intermediate 228 and is prepared starting from intermediate 193. The title compound was obtained in 16 mg (30% of theory).

HPLC (Method 12): _rt = 2.0 min; LC-MS (Method 1): R _t = 1.02 min; MS (ESI+): m/z=1335 (M+H) ⁺ .

Intermediate 231 (2R,3S)-3-acetamido-4-{2-[6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexanyl]anthracene }}-4-oxobutan-2-yl (3R,4S,7S,10S)-4-[(2S)-but-2-yl]-3-(2-{(2S)-2-[( 1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-side Oxyethyl)-7,10-diisopropyl-5,11-dimethyl-6,9-di-oxo-2-oxo-5,8,11-triazadecane-15-erate

This compound is deprotected with 8 mg (6 μmol) of intermediate 230, first with trifluoroacetic acid, and subsequently acetylated with acetic anhydride in DMF in the presence of N,N -diisopropylethylamine. Prepared. The title compound was obtained in 2 mg (37% of theory).

HPLC (Method 12): _Rt = 1.9 min; LC-MS (Method 1): R _t = 0.98 min; MS (ESI+): m/z=1277 (M+H) ⁺ .

Intermediate 232 N-[(4-nitrophenoxy)carbonyl]-β-alanine benzyl ester

200 mg (0.57 mmol) of commercially available 4-methylbenzenesulfonic acid-benzyl β-alanine ester and 229 mg (1.14 mmol) of 4-nitrophenyl chlorocarbonate were placed in 15 ml of tetrahydrofuran. The reaction mixture was then heated to reflux for 30 min. Subsequently, the reaction mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC. The corresponding fractions were concentrated and dried <RTI ID=0.0>

HPLC (Method 12): R _t = 1.8 min; LC-MS (Method 1): R _t = 1.07 min; MS (ESI+): m/z=345 (M+H) ⁺ .

Intermediate 233 N-{2-[({3-[(2,5-dioxaxopyridin-1-yl)oxy]-3-oxopropyl}amine)-amino)amino]-N -Methyl-L-Amidoxime-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H -吲哚-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

13 mg (10 μmol) of intermediate 225 and 6.7 mg (20 μmol) of intermediate 232 were dissolved in 3 ml of DMF and then 7 μl of N,N-diisopropylethylamine was added. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by preparative HPLC. The corresponding soluble fractions were concentrated and the residue was dried under high vacuum to afford 5.4 mg (yield: 38%) of intermediate.

HPLC (method 5): _Rt = 2.1 min; LC-MS (Method 1): R _t = &lt;RTI ID=0.0&^gt;

5.4 mg (5 μmol) of this intermediate was dissolved in 5 ml of methanol and 2 mg of 10% activated carbon on palladium was added and hydrogenated at RT under standard hydrogen pressure for 20 min. The catalyst was then filtered off and the solvent was removed under reduced pressure. After drying the residue under high vacuum, a 5 mg (quant.) acid intermediate was obtained.

HPLC (Method 12): R _t = 1.8 min; LC-MS (Method 1): R _t = 0.84 min; MS (ESI+): m/z = 999 (M+H) ⁺ .

5 mg (10 μmol) of this intermediate was dissolved in 1 ml of DMF and mixed with 5.8 mg (50 mmol) of 1-hydroxypyrrolidine-2,5-dione and then with 2.6 μl of N,N -di Mix propyl ethylamine with 3.8 mg (10 μmol) of HATU. After stirring at RT for 20 h, the reaction mixture was evaporated. The remaining residue was purified by preparative HPLC. From 1:1 After lyophilization of the alkane/water, 1.1 mg (20% of theory) of title compound.

HPLC (method _{12): R t = 1.9 min} ; LC-MS ( Method _{1): R t = 0.87 min} ; MS (ESI +): m / z = 1096 (M + H) +.

Intermediate 234 N-(6-{[(benzyloxy)carbonyl]amino}hexyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-3-methoxy-1 -{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-benzene) Base-1,3,4-two Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

25 mg (30 μmol) of N-methyl- L -amidino-N-[( 3R, 4 S, 5 S )-3-methoxy-1-{(2 S )-2-[ (1 R , 2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 S )-2-phenyl-1-(5-phenyl-1,3, 4-two Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl- L -decylamine (Intermediate 55) and 45 mg (180 μmol) of benzyl 6-oxohexylaminecarboxylate were treated in 3 ml of methanol and acidified with acetic acid. 15 μl (144 μmol; 9.4 M) of the borane-pyridine complex was then added at room temperature. The mixture was then stirred at RT for 24 h and acetic acid and 15 μl (144 μmol; 9.4 M) of borane-pyridine complex were added again after 8 h. The reaction mixture was then adjusted to pH 2 with TFA and purified using preparative HPLC. The product fractions were combined and concentrated, and the residue was dried under high vacuum. This gave 15 mg (46% of theory) of the title compound as a foam.

LC-MS (Method _{1): R t = 1.03 min} ; m / z = 1066 (M + H) +.

Intermediate 235 N-(6-Aminohexyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[( 1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-di Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

15 mg (14 μmol) of N-(6-{[(benzyloxy)carbonyl]amino}hexyl)-N-methyl-L-nonylamine-N-[(3R,4S,5S) -3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-benzene Base-1-(5-phenyl-1,3,4-di Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine (Intermediate 234) was treated with 3 ml of methanol and 1.8 mg of palladium on carbon (5%) was added. The reaction mixture was then hydrogenated at RT for 2 h under standard hydrogen pressure. The catalyst was then filtered off and the solvent was removed under reduced pressure. The residue was lyophilized in 1:1 acetonitrile / water. The title compound was obtained as a foam (11 mg, 86%).

LC-MS (Method 1): R _t = 0.81 min; m/z = 932 (M+H) ⁺ .

Intermediate 236 N-[6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-nonylamine-yl-N-[(3R, 4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S) -2-phenyl-1-(5-phenyl-1,3,4-di Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

11 mg (12 μmol) of N-(6-aminohexyl)-N-methyl-L-nonylamine-N-[(3R,4S,5S)-3-methoxy-1-{ (2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl- 1,3,4-two Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine (Intermediate 235) placed in 500 μl of 1:1 Treated in alkane/water and mixed with 253 μl of 1 M aqueous sodium bicarbonate and then with 2.8 mg (18 μmol) of 2,5-di-oxo-2,5-dihydro-1H-pyrrole-1-carboxylate . The reaction mixture was stirred at RT for 30 min and then acidified with trifluoroacetic acid. The reaction mixture was purified by preparative HPLC. After lyophilization, 0.8 mg (7% of theory) of title compound was obtained.

LC-MS (Method _{1): R t = 1.01 min} ; m / z = 1012 (M + H) +.

Intermediate 237 N-(5-carboxypentyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[( 1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-di Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

25 mg (30 μmol) of N-methyl- L -amidino-N-[( 3R, 4 S, 5 S )-3-methoxy-1-{(2 S )-2-[ (1 R , 2 R )-1-methoxy-2-methyl-3-oxo-3-{[(1 S )-2-phenyl-1-(5-phenyl-1,3, 4-two Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl- L -decylamine (Intermediate 55) and 23 mg (180 μmol) of 6-sided oxyhexanoic acid were treated in 3 ml of methanol and acidified with acetic acid. 15 μl (144 μmol; 9.4 M) of the borane-pyridine complex was then added at room temperature. The reaction mixture was then stirred at RT for 20 h, and after 8 h, acetic acid and 15 μl (144 μmol; 9.4 M) of borane-pyridine complex were added again. The reaction mixture was then adjusted to pH 2 with trifluoroacetic acid and purified using preparative HPLC. The product fractions were combined and concentrated, and the residue was lyophilized. This gave 21 mg (74% of theory) of the title compound as a foam.

LC-MS (method 1): R _t = 0.91 min; m/z = 947 (M+H) ⁺ .

Intermediate 238 N-{6-[(2,5-Dioxapyrrolidin-1-yl)oxy]-6-oxo-oxyhexyl}-N-methyl-L-nonylamine fluorenyl-N-[(3R, 4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S) -2-phenyl-1-(5-phenyl-1,3,4-di Zin-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

21 mg (22 μmol) of intermediate 237 was dissolved in 1 ml of DMF with 38 mg (333 μmol) of 1-hydroxypyrrolidine-2,5-dione and then with 2.4 mg (10 μmol) of O- (7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylhexafluorophosphate (HATU) was mixed with 19 μl of N,N-diisopropylethylamine. After stirring at RT for 2 h, the reaction mixture was purified by preparative HPLC. From two After lyophilization of the alkane, 22 mg (96% of theory)

LC-MS (Method _{1): R t = 0.95 min} ; m / z = 1044 (M + H) +.

Intermediate 239 N-methyl-L-threonyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-( 1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine guanamine trifluoroacetate

First, N -[(benzyloxy)carbonyl] -N -methyl-L-threonine was extracted from its 237 mg (0.887 mmol) by treatment with ethyl acetate and shaking with 5% aqueous sulfuric acid. Released from the cyclohexylamine salt. The organic layer was dried over magnesium sulfate, filtered and concentrated. 14.7 mg (0.055 mmol) of N -[(benzyloxy)carbonyl] -N -methyl-L-threonine was treated in 3 ml of DMF and continuously with 40 mg (0.055 mmol) of intermediate 220 12.7 mg (0.066 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mg (0.066 mmol) of 1-hydroxy-1 H -benzo The triazole hydrate is mixed. The mixture was then stirred at RT for 2 h. The solvent was removed under reduced pressure and the residue was purified by preparative HPLC. This gave 29 mg (54% of theory) of the Z-protected intermediate.

LC-MS (Method _{1): R t = 1.15 min} ; MS (ESI +): m / z = 976 (M + H) +.

29 mg (0.003 mmol) of this intermediate was dissolved in 5 ml of methanol and hydrogenated on 5 mg of 5% palladium on activated carbon under RT and standard hydrogen pressure for 1 h. The catalyst was then filtered off and the solvent was evaporated. The remaining residue was purified by preparative HPLC. This gave 17 mg (54% of theory) of title compound.

LC-MS (Method _{1): R t = 0.77 min} ; MS (ESI +): m / z = 842 (M + H) +.

Intermediate 240 N-{6-[(2,5-dioxapyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-threonyl-N-[(3R, 4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

This compound was similar to intermediate 210 and was prepared from 15.6 mg (0.016 mmol) of Intermediate 239. The title compound was obtained in 10.8 mg (67% of theory).

HPLC (method _{5): R t = 1.7 min} ; LC-MS ( Method _{1): R t = 0.85 min} ; MS (ESI +): m / z = 1053 (M + H) +.

Intermediate 241 N-methyl-L-Amidinoinyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-( 4-hydroxyphenyl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine guanamine trifluoroacetate

First, similar to intermediate 5, preparation of trifluoroacetic acid-(2S)-2-amino-3-(4-hydroxyphenyl)-1-(1,2- Alk-2-yl)propan-1-one (1:1). This reagent is then used to synthesize similarly as described in Intermediate 75, by reacting with N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R ,4) S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy -5-Methyl-1-oxoheptan-4-yl] -N -methyl-L-decylamine amide (Intermediate 26) in O-(7-azabenzotriazol-1-yl) ) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of, and subsequent separation of the Boc protecting group with trifluoroacetic acid afforded the title compound.

HPLC (Method 12): _Rt = 1.7 min; LC-MS (Method 1): R _t = 0.85 min; MS (ESI+): m/z=817 (M+H) ⁺ .

Intermediate 242 N-{6-[(2,5-Dioxapyrrolidin-1-yl)oxy]-6-oxo-oxyhexyl}-N-methyl-L-nonylamine fluorenyl-N-[(3R, 4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-hydroxyphenyl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

50 mg (0.05 mmol) of intermediate 241, similar to intermediate 210, was reacted with 6-oxohexanoic acid in the presence of a borane-pyridine complex. Subsequently, 22.5 mg (0.02 mmol) of the resulting acid was converted to the activated ester. The title compound was obtained in an amount of 13.5 mg (36% of theory).

HPLC (method _{12): R t = 1.8 min} ; LC-MS ( Method _{1): R t = 0.86 min} ; MS (ESI +): m / z = 1028 (M + H) +.

Intermediate 243 N-(6-Aminohexyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3 -{[(2S)-3-(4-hydroxyphenyl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

This preparation is similar to intermediate 78 by reductive alkylation of intermediate 241 with benzyl 6-oxo-oxyhexylamine and borane-pyridine complexes, followed by hydrogenation with methanol as solvent.

Yield: 17.5 mg (34% of theoretical value in 2 stages)

HPLC (Method 12): R _t = 1.7 min; LC-MS (Method 1): R _t = 0.63 min; MS (ESI+): m/z = 916 (M+H) ⁺ .

Intermediate 244 N-[6-(2,5-di-oxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-nonylamine-yl-N-[(3R, 4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-hydroxyphenyl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

This preparation is similar to intermediate 166, starting with intermediate 243.

Yield: 1.3 mg (12% of theory)

HPLC (Method 12): R _t = 1.9 min; LC-MS (Method 1): R _t =0.89 min; MS (ESI+): m/z = 996 (M+H) ⁺ .

Intermediate 245 2,5-dioxapyrrolidin-1-yl O-[(3R,4S,7S,10S)-4-[(2S)-but-2-yl]-3-(2-{(2S)- 2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-side Oxyethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,15-trilateral oxy-2-oxo-5,8,11-triazadecane-15- ]---(t-butoxycarbonyl)-L-threonyl-β-alanine

First, an intermediate 193, as described for intermediate 154, is reacted with N-(t-butoxycarbonyl)-L-threonate benzyl ester, and then the benzyl ester is removed by hydrogenolysis. The thus obtained 30 mg (0.027 mmol) of N-[4-({(1S,2R)-1-[(t-butoxycarbonyl)amino]-1-carboxypropan-2-yl}oxy) 4--4-oxobutyl]-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3 -{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A The presence of oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-decylamine and 4-methylbenzenesulfonic acid-benzyl β-alaninate in HATU The lower coupling was carried out and the benzyl ester was removed by hydrogenolysis (yield: 24 mg (71% of theory in 2 stages)). Finally, 10 mg (0.008 mmol) of the resulting acid was converted to the activated ester. After purification by HPLC, 2.7 mg (yield: 23%)

HPLC (method 5): R _t = 1.9 min; LC-MS (method 1): R _t = 1.01 min; MS (ESI+): m/z=1295 (M+H) ⁺

Intermediate 246a (2S)-2-amino-1-(4-hydroxy-1,2- Zoxadin-2-yl)-3-(1H-indol-3-yl)propan-1-one trifluoroacetate (diastereomer 1)

1.6 g (3.982 mmol) of 2,5-dioxapyrrolidin-1-yl N-(t-butoxycarbonyl)-L-tryptophanate was dissolved in 15 ml of DMF with 500 mg (3.982) Ment) 1,2- The oxazolidine-4-ol was mixed with 100 μl of N,N -diisopropylethylamine. The reaction mixture was stirred at RT until overnight. Then, another 100 μl of N,N -diisopropylethylamine was added, and the mixture was first treated with an ultrasonic bath for 5 h, then stirred at RT overnight, and then concentrated under reduced pressure. The remaining residue was taken up in ethyl acetate and extracted twice with 5% aqueous EtOAc EtOAc. The organic layer was concentrated and the residue was crystallised eluted eluted elute The corresponding dissolved fractions of the two diastereomers were combined and the solvent was removed under reduced pressure. After the residue was dried under high vacuum to afford 272 mg (18% of theory) of diastereomer product _{(R f = 0.18 (95 1} : 5 dichloromethane / methanol) and 236 mg (16% of Theoretical value) of diastereomer 2 ( _Rf = 0.13 (95:5 dichloromethane / methanol), and 333 mg (22% of theory) of a mixture of Boc-protected intermediates.

The Boc protecting group is isolated from the diastereomer 1 of 272 mg (725 μmol) of this intermediate using 5 ml of trifluoroacetic acid in 20 ml of dichloromethane under standard conditions. After hexane/water lyophilization afforded 290 mg (yield) of the title compound, 75% purity and used for the next stage without further purification.

HPLC (Method 12): R _t = 1.1 min; LC-MS (Method 13): R _t = 1.80 min; MS (ESI+): m/z = 276 (M+H) ⁺

Intermediate 246b (2S)-2-amino-1-(4-hydroxy-1,2- Zoxadin-2-yl)-3-(1H-indol-3-yl)propan-1-one trifluoroacetate (diastereomer 2)

The Boc protecting group was isolated and concentrated using 5 ml of trifluoroacetic acid in 20 ml of dichloromethane from 236 mg (630 μmol) of the intermediate diastereomer 2 as described in 246a. The title compound was obtained after EtOAc (EtOAc m.

LC-MS (method 13): R _t = 1.84 min; MS (ESI+): m/z = 276 (M+H) ⁺

Intermediate 247a N-{6-[(2,5-Dioxapyrrolidin-1-yl)oxy]-6-oxo-oxyhexyl}-N-methyl-L-nonylamine fluorenyl-N-[(3R, 4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(4-hydroxy-1,2- Azolidin-2-yl)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxo Propyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine (diastereomer Object 1)

Coupling of intermediates 26 and 246a is first carried out as described for intermediate 74, followed by separation of the Boc protecting group to synthesize the compound. Subsequently, the acid is alkylated with 6-oxohexanoic acid in the presence of a borane-pyridine complex, and then the acid is converted to the active ester as described for intermediate 210. The title compound was purified by preparative HPLC.

HPLC (method _{12): R t = 1.8 min} ; LC-MS ( Method _{1): R t = 0.86 min} ; MS (ESI +): m / z = 1053 (M + H) +

Intermediate 247b N-{6-[(2,5-Dioxapyrrolidin-1-yl)oxy]-6-oxo-oxyhexyl}-N-methyl-L-nonylamine fluorenyl-N-[(3R, 4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(4-hydroxy-1,2- Azolidin-2-yl)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxo Propyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine (diastereomer Object 2)

Coupling of intermediates 26 and 246b is first carried out as described for intermediate 74, followed by separation of the Boc protecting group to synthesize the compound. Subsequently, the acid is alkylated with 6-oxohexanoic acid in the presence of a borane-pyridine complex, and then the acid is converted to the active ester as described for intermediate 210. The title compound was purified by preparative HPLC.

HPLC (method _{12): R t = 1.8 min} ; LC-MS ( Method _{1): R t = 0.86 min} ; MS (ESI +): m / z = 1053 (M + H) +

Intermediate 248 N-(5-carboxypentyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3 -{[(2S)-1-tert-butoxy-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl- 3-sided oxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxo Hept-4-yl]-N-methyl-L-amidamine

First, a synthesis similar to that described in Intermediate 86, by N- (t-butoxycarbonyl) -N -methyl-L-nonylamine- N -[(3 R , 4 S , 5 S ) -1-{(2 S )-2-[(1 R ,2 R )-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl -1-Sideoxyheptan-4-yl] -N -methyl-L-decylamine amide (Intermediate 26) and L-tyrosine tert-butyl ester in O- (7-azabenzotriene) Zin-1-yl) -N,N,N',N' -tetramethylhexafluorophosphate Coupling in the presence of the same, and then separating the Boc protecting group with trifluoroacetic acid to give the third butyl ester (with trifluoroacetic acid in dichloromethane for 40 min) to prepare the amine compound N-[(2R,3R)-3- Methoxy-3-{(2S)-1-[(3R,4S,5S)-3-methoxy-5-methyl-4-(methyl{(2S)-3-methyl-2-) [(N-Methyl-L-carbamicinyl)amino]butyl}amino)heptinyl]pyrrolidin-2-yl}-2-methylpropanyl]-L-tyrosine Tributyl ester is trifluoroacetate. Then 38 mg (0.04 mmol) of this compound was used, similar to the preparation of intermediate 210, by reaction with 6-oxohexanoic acid in the presence of a borane-pyridine complex to give 31 mg (99% of theory) The title compound.

HPLC (Method 12): R _t = 1.8 min; LC-MS (Method 1): R _t = 0.98 min; MS (ESI+): m/z = 918 (M+H) ⁺ .

B: Preparation of antibody-drug conjugate (ADC)

The above intermediate is linked to, for example, an anti-mesothelin antibody MF-Ta, wherein the linkage is carried out alternatively via the cysteine or amido acid side chain of the antibody protein according to the methods described below. The anti-mesothelin antibody MF-Ta was prepared in a manner similar to that described in WO 2009/068204-A1. The antibody MF-Ta line was expressed in eukaryotic CHO cells (stable cell line) and purified by filtration through Protein A and gel, followed by ligation in D-PBS buffer.

B-1. General Procedure 1 (via cysteine coupling):

A solution of 3 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in PBS buffer was added to the corresponding antibody in a concentration range of 1 mg/ml and 15 mg/ml in PBS buffer. The solution was stirred and the mixture was stirred at RT for 1 hour. Subsequently, depending on the desired loading, 2 to 10 equivalents of the maleimide precursor compound or halide precursor compound for the coupling (intermediate 102, 103, 105-109, 111-114, 117) are added. -126, 128, 129, 132-146, 148-155, 157, 159-161, 166, 171, 175-177, 184, 189, 194-195, 199-201, 205, 209, 223-224, 226 228-231, 236 and 244) DMSO solutions. The amount of DMSO should not exceed 10% of the total volume. This was stirred for 60-120 minutes in this document RT, and then administered by the balance of the PD 10 column PBS ^{(Sephadex ® G-25, GE} Healthcare), and eluting with PBS buffer. The concentration procedure is additionally carried out by ultracentrifugation as needed. If necessary, the low molecular weight composition is more effectively removed, and after further dilution with PBS buffer, it is repeatedly concentrated by ultrafiltration.

In general, unless otherwise indicated, reduction and subsequent coupling were carried out using 5 mg of the corresponding antibody in PBS buffer. After purification through a PD10 column, a solution in which each ADC was dissolved in 3.5 ml of PBS buffer was obtained. The specific protein concentration referred to by these solutions is then determined. Further, the antibody loading (drug/mAb ratio) was measured in accordance with the method described in B-4.

This method is used to prepare examples 1, 2, 5-19, 21-28, 30, 31, 33-37, 39-46, 48, 51-57, 59, 60, 71, 73-76, 78-80. Immunoconjugates represented by 82-84, 86-87, 96, 98-103, 108 and 112.

In the structural formula shown, AK ₁ is defined as follows AK ₁ =MF-Ta (partially reduced)-S§ ¹ wherein § ¹ refers to the amber quinone imine group and MF-Ta (partial reduction) to the moiety The reduced MF-Ta antibody (heavy chain SEQ ID NO: 408 and light chain SEQ ID NO: 409) and the S line represent the sulfur atom of the cysteine residue of the partially reduced antibody.

B-2. General Procedure 2 (coupled via an amine acid side chain):

Between 2 and 5 equivalents of coupled precursor compound (intermediate 104, 110, 115, 116, 127, 130, 131, 147, 156, 158, 162, 169, 178, 185, 190), depending on the desired loading. DMSO solutions of 202, 206, 210-216, 218, 219, 227, 233, 238, 240, 242, 245, 247a and 247b) were added to the concentration range of 1 mg/ml and 15 mg/ml. The antibody is in a solution in PBS buffer. After stirring at RT for 30 minutes, the same amount of the precursor compound in DMSO was added again. Alternatively, 4-10 equivalents of the coupled precursor compound can be added in one portion. Another was stirred at RT for 30 minutes, the batch was administered by eluting the balance of the PD 10 column PBS ^{(Sephadex ® G-25, GE} Healthcare), and with PBS buffer. The concentration procedure is additionally carried out by ultracentrifugation as needed. If necessary, the low molecular weight composition is more effectively removed, and after further dilution with PBS buffer, it is repeatedly concentrated by ultrafiltration.

In general, unless otherwise indicated, 5 mg of the corresponding antibody in PBS buffer was used for coupling. After purification through a PD10 column, a solution of each of the corresponding ADCs dissolved in 3.5 ml of PBS buffer was obtained. The specific protein concentration referred to by these solutions was then determined and the antibody loading (drug/mAb ratio) was determined according to the method described in B-4.

This method is used to prepare Examples 3, 4, 20, 29, 32, 38, 47, 49, 50, 58, 61, 72, 77, 81, 85, 88-95, 97, 104, 109-111 and 113 The immunoconjugate represented by -115.

In the structural formula shown, AK ₂ is defined as follows AK ₂ =MF-Ta-NH§ ² wherein § ² represents a linkage to a carbonyl group and MF-Ta is a non-reduced MF-Ta antibody (heavy chain SEQ ID NO: 408 and light chain SEQ ID NO: 409) and the NH line represents a side chain amine group of an amino acid residue of the antibody.

B-3a. General method for preparing cysteine adducts:

10 μmol of the above-described maleimide precursor compound was treated in 3 ml of DMF and mixed with 2.1 mg (20 μmol) of L-cysteine. The reaction mixture was stirred at RT for 2 h then concentrated under reduced pressure and then purified by preparative HPLC.

In the structural formula shown, the definition of Cys is as follows Where § ³ refers to the connection to the linker-band toxic unit.

B-3b. General method for preparing an amide acid adduct:

10 μmol of the above active ester precursor compound was treated in 5 ml of DMF and in the presence of 30 μmol of N,N-diisopropylethylamine, with α-amino-protected L-lysine mixing. The reaction mixture was stirred at RT for 2 h then concentrated under reduced pressure and then purified by preparative HPLC. The protecting group is then removed in a known manner.

Further purification and characterization of the conjugate of the invention

After the reaction has taken place, in a specific case the reaction mixture is concentrated, for example by ultrafiltration, and then desalted and purified by chromatography, for example using Sephadex® G-25. For example, the dissolution is carried out in phosphate-buffered saline (PBS). This solution was then sterile filtered and frozen. Another option is to freeze dry the conjugate.

B-4. Determination of toxic load

The toxic load of the resulting solution of the conjugate as described in the Examples of Operation, in PBS buffer, was measured as follows: The toxic load of the amine-linked ADC was determined by mass spectrometry to determine the molecular weight of the individual conjugate species. To measure. In this case, the antibody conjugate was initially deglycosylated with PNGaseF, and the sample was acidified, followed by HPLC separation, and analyzed by mass spectrometry using ESI-MicroTofQ (Bruker Daltonik). All spectra were added via the TIC (Total Ion Chromatogram) signal and the molecular weights of the various conjugates were calculated based on MaxEnt Deconvolution. After integrating different types of signals, then calculate DAR (drug/antibody ratio).

For protein identification, in addition to molecular weight determination, trypsinization is performed after deglycosylation and/or denaturation, and this digestion is based on the detected trypsin peptide after denaturation, reduction and derivatization. To confirm the properties of the protein.

The toxic load of the cysteine-linked conjugate was determined by reverse phase chromatography of the reduced and denatured ADC. The ADC solution (1 mg/mL, 50 μL) was mixed with guanidine hydrochloride (GuHCl) (28.6 mg) and with DL-dithiothreitol (DTT) (500 mM, 3 μL). The mixture was incubated at 55 ° C for 1 hour and analyzed by HPLC.

HPLC analysis was performed on an Agilent 1260 HPLC system with 220 nm detection. The column used was a Polymer Laboratories PLRP-S polymeric reverse phase column (model PL 1912-3802) (2.1 x 150 mm, 8 μm particle size, 1000 Å) at a flow rate of 1 mL/min using the following ladder Degree: 0 min, 25% B; 3 min, 25% B; 28 min, 50% B. Eluent A consisted of an aqueous solution of 0.05% trifluoroacetic acid (TFA) and Eluent B consisted of 0.05% trifluoroacetic acid in acetonitrile.

The detected peaks were compared with the light chain (L0) and heavy chain (H0) of the conjugate antibody, and were assigned by residence time. The peaks detected separately in the conjugate sample are assigned to the light chain, containing one toxicant (L1), while the heavy chain contains one, two and three venoms (H1, H2, H3).

The average load of the antibody containing the venom is calculated as follows: First, the light chain load is calculated from the peak area - determined by integration - the L0 and L1 peaks belonging to the light chain, which are the weighted integration results of the L0 and L1 venom numbers. The sum of these is divided by the sum of the singularly weighted integration results of L0 and L1. In the same way, the heavy chain load is calculated from the wave front area - measured by integral - the H0, H1, H2 and H3 peaks belonging to the heavy chain, which are the weighted integration results of the number of poisons of H0, H1, H2 and H3. The sum, divided by the sum of the singularly weighted integration results of H0, H1, H2, and H3. DAR is expressed as light chain loading and heavy chain loading, which is the sum of the light chain loading and the heavy chain loading. The 2-fold is a fact that the antibody is composed of two light chains and two heavy chains. In certain individual cases, the toxic load may not be accurately measured due to co-elution of specific peaks.

B-5. Experimental antigen binding of ADC

The ability of the binding agent to bind to the palladium molecule is tested after binding occurs. Those skilled in the art that various known for performing the method of testing, for example, ELISA techniques or engagement surface plasmon resonance analysis of the affinity thereof (BIAcore ^TM measurement) test. Conjugate concentrations can be determined by the skilled artisan using conventional methods, such as for antibody conjugates, by protein assays (see also Doronina et al; Nature Biotechnol. 2003; 21: 778-784 and Polson et al, Blood 2007; 1102: 616-623) to measure.

Operational example - immunoconjugate Example 1

Protein concentration: 0.96 mg/ml

Drug / mAb ratio: 3.1

Example 2

Protein concentration: 0.44 mg/ml

Drug / mAb ratio: 4.6

Example 3

Protein concentration: 1.09 mg/ml

Drug / mAb ratio: 2.1

Example 4

Protein concentration: 0.87 mg/ml

Drug / mAb ratio: 3.8

Example 5

Protein concentration: 0.45 mg/ml

Drug / mAb ratio: 6.5

Example 6

Protein concentration: 0.15 mg/ml

Drug / mAb ratio: 3.1

Example 7

Protein concentration: 0.94 mg/ml

Drug / mAb ratio: 2.8

Example 8

Protein concentration: 0.45 mg/ml

Drug / mAb ratio: 0.9

Example 9

Protein concentration: 0.51 mg/ml

Drug / mAb ratio: 6.6

Example 10

Protein concentration: 0.47 mg/ml

Drug / mAb ratio: 4.2

Example 11

Protein concentration: 0.45 mg/ml

Drug / mAb ratio: 5.9

Example 12

Protein concentration: 0.47 mg/ml

Drug / mAb ratio: 3.3

Example 13

Protein concentration: 0.53 mg/ml

Drug / mAb ratio: 2.8

Example 14

Protein concentration: 0.92 mg/ml

Drug / mAb ratio: 3.5

Example 15

Protein concentration: 0.09 mg/ml

Drug/mAb ratio: nd

Example 16

Protein concentration: 0.62 mg/ml

Drug / mAb ratio: 1.8

Example 17

Protein concentration: 0.55 mg/ml

Drug / mAb ratio: 3.8

Example 18

Protein concentration: 0.54 mg/ml

Drug / mAb ratio: 4.4

Example 19

Protein concentration: 0.56 mg/ml

Drug / mAb ratio: 4.0

Example 20

Protein concentration: 1.1 mg/ml

Drug / mAb ratio: 0.3

Example 21

Protein concentration: 0.61 mg/ml

Drug / mAb ratio: 0.9

Example 22

Protein concentration: 0.57 mg/ml

Drug / mAb ratio: 1.2

Example 23

This was carried out by coupling with 100 mg of MF-Ta in PBS, and after Sephadex purification, the reaction mixture was concentrated by ultracentrifugation.

Protein concentration: 11.2 mg/ml

Drug / mAb ratio: 3.4

Example 24

Protein concentration: 1.56

Drug / mAb ratio: 2.8

Example 25

Protein concentration: 0.60 mg/ml

Drug / mAb ratio: 2.4

Example 26

Protein concentration: 0.58 mg/ml

Drug/mAb-Ratio: 2.6

Example 27

Protein concentration: 0.39 mg/ml

Drug / mAb - ratio: 0.8

Example 28

This was carried out by coupling with 70 mg of MF-Ta in PBS, and after Sephadex purification, the reaction mixture was concentrated by ultracentrifugation.

Protein concentration: 13.2 mg/ml

Drug / mAb ratio: 4.6

Example 29

Protein concentration: 0.98 mg/ml

Drug / mAb ratio: 1.1

Example 30

Protein concentration: 0.55 mg/ml

Drug/mAb ratio: undetectable

Example 31

This was carried out by coupling with 40 mg of MF-Ta in PBS, and after Sephadex purification, the reaction mixture was concentrated by ultracentrifugation.

Protein concentration: 10.6 mg/ml

Drug / mAb ratio: 4.1

Example 32

Protein concentration: 0.96 mg/ml

Drug / mAb ratio: 0.4

Example 33

This was carried out by coupling with 70 mg of MF-Ta in PBS, and after Sephadex purification, the reaction mixture was concentrated by ultracentrifugation.

Protein concentration: 12.7 mg/ml

Drug / mAb ratio: 3.6

Example 34

Protein concentration: 1.1 mg/ml

Drug / mAb ratio: 2.7

Example 35

Protein concentration: 1.24 mg/ml

Drug / mAb ratio: 2.6

Example 36

Protein concentration: 0.99 mg/ml

Drug / mAb ratio: 2.3

Example 37

Protein concentration: 1.22 mg/ml

Drug / mAb ratio: 3.3

Example 38

Protein concentration: 1.34 mg/ml

Drug / mAb ratio: 1.2

Example 39

Protein concentration: 1.28 mg/ml

Drug / mAb ratio: 3.2

Example 40

This was carried out by coupling with 70 mg of MF-Ta in PBS, and after Sephadex purification, the reaction mixture was concentrated by ultracentrifugation.

Protein concentration: 10.9 mg/ml

Drug / mAb ratio: 5.1

Example 41

This was carried out by coupling with 100 mg of MF-Ta in PBS, and after Sephadex purification, the reaction mixture was concentrated by ultracentrifugation.

Protein concentration: 10.3 mg/ml

Drug / mAb ratio: 4.3

Example 42

Protein concentration: 1.08 mg/ml

Drug / mAb ratio: 2.8

Example 43

Protein concentration: 1.24 mg/ml

Drug / mAb ratio: 2.8

Example 44

Protein concentration: 1.28 mg/ml

Drug / mAb ratio: 3.8

Example 45

Protein concentration: 1.07 mg/ml

Drug / mAb ratio: 3.0

Example 46

Protein concentration: 1.35 mg/ml

Drug / mAb ratio: 4.0

Example 47

This was carried out by coupling with 100 mg of MF-Ta in PBS, and after Sephadex purification, the reaction mixture was concentrated by ultracentrifugation.

Protein concentration: 12.2 mg/ml

Drug / mAb ratio: 5.6

Example 48

Protein concentration: 1.32 mg/ml

Drug / mAb ratio: 3.2

Example 49

Protein concentration: 1.01 mg/ml

Drug / mAb ratio: 0.9

Example 50

Protein concentration: 1.03 mg/ml

Drug / mAb ratio: 0.3

Example 51

Protein concentration: 0.62 mg/ml

Drug / mAb ratio: 3.1

The ADC was concentrated by voraspin centrifugation, re-diluted and concentrated and diluted again.

Example 52

Protein concentration: 1.26 mg/ml

Drug/mAb ratio: undetectable

Example 53

This was coupled using 5 mg of MF-Ta in PBS, and after Sephadex purification, the batch was concentrated by ultracentrifugation and re-diluted.

Protein concentration: 1.21 mg/ml

Drug/mAb ratio: cannot be measured

Example 54

This was coupled using 5 mg of MF-Ta in PBS, and after Sephadex purification, the batch was concentrated by ultracentrifugation and re-diluted.

Protein concentration: 0.8 mg/ml

Drug / mAb ratio: 2.8

Example 55

Protein concentration: 1.44 mg/ml

Drug / mAb ratio: 4.1

Example 56

Protein concentration: 0.92 mg/ml

Drug / mAb ratio: 3.5

Example 57

This was coupled using 5 mg of MF-Ta in PBS, and after Sephadex purification, the batch was concentrated by ultracentrifugation and re-diluted.

Protein concentration: 0.77 mg/ml

Drug/mAb ratio: >1.5 (cannot be measured)

Example 58

Protein concentration: 1.3 mg/ml

Drug / mAb ratio: 2.0

Example 59

This was carried out by coupling with 150 mg of MF-Ta in PBS, and after Sephadex purification, the reaction mixture was concentrated by ultracentrifugation.

Protein concentration: 11.2 mg/ml

Drug / mAb ratio: 3.7

Example 60

This was carried out by coupling with 100 mg of MF-Ta in PBS, and after Sephadex purification, the reaction mixture was concentrated by ultracentrifugation.

Protein concentration: 11.4 mg/ml

Drug / mAb ratio: 3.9

Example 61

This was carried out by coupling with 60 mg of MF-Ta in PBS, and after Sephadex purification, the reaction mixture was concentrated by ultracentrifugation.

Protein concentration: 10.5 mg/ml

Drug / mAb ratio: 4.4

Example 62 N-(4-{2-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]indolyl}-2,5-dioxapyrrolidin-1-yl)hexanyl) Alkyl}-4-yloxybutyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R, 2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2 -methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-decylamine Guanamine

10 mg (10 μmol) of intermediate 157 was treated in 5.2 ml of DMF and mixed with 2.28 mg (20 μmol) of L-cysteine. The reaction mixture was stirred at RT for 2 h then concentrated under reduced pressure and then purified by preparative HPLC. This gave 5.8 mg (48% of theory) of the title compound.

HPLC (Method 5): R _t = 1.45 min; LC-MS (Method 1): R _t = 0.74 min; MS (ESI+): m/z=1184 (M+H) ⁺ .

Example 63 N-(4-{2-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]indolyl}-2,5-dioxapyrrolidin-1-yl)hexanyl) Alkyl}-4-yloxybutyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R, 2R)-3-{[(1S)-1-carboxyl 2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

10 mg (10 μmol) of intermediate 113 was placed in 5.2 ml of DMF and mixed with 2.28 mg (20 μmol) of L-cysteine. The reaction mixture was stirred at RT for 2 h then concentrated under reduced pressure and then purified by preparative HPLC. This gave 6 mg (54% of theory) of the title compound.

HPLC (method 5): _Rt = 1.5 min; LC-MS (Method 1): R _t = 0.77 min; MS (ESI+): m/z=1185 (M+H) ⁺ .

Example 64 N-(4-{2-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]indolyl}-2,5-dioxapyrrolidin-1-yl)hexanyl) Alkyl}-4-yloxybutyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-3-methoxy-1-{(2S)- 2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-amidamine

9 mg (8.3 μmol) of intermediate 132 was treated in 4 ml of DMF and mixed with 3 mg (24.4 μmol) of L-cysteine. The reaction mixture was stirred at rt over rt then concentrated over EtOAc EtOAc. This gave 6.8 mg (68% of theory) of the title compound.

HPLC (Method 12): R _t = 1.8 min; LC-MS (Method 1): R _t =0.78 min; MS (ESI+): m/z=1227 (M+H) ⁺ .

Example 65 N-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]indolyl}-2,5-dioxapyrrolidin-1-yl)hexyl]-N-methyl- L-Amidoxime-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-Amino-3-( 1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}- 3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

10 mg (10 μmol) of intermediate 106 was placed in 5.8 ml of DMF and mixed with 2.5 mg (20 μmol) of L-cysteine. The reaction mixture was stirred at RT for 2 h then concentrated under reduced pressure and then purified by preparative HPLC. This gave 5.2 mg (46% of the title compound).

HPLC (method _{5): R t = 1.5 min} ; LC-MS ( method _{11): R t = 0.71 min} ; MS (ESI +): m / z = 1070 (M + H) +.

Example 66 N-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]indolyl}-2,5-dioxapyrrolidin-1-yl)hexyl]-N-methyl- L-Amidoxime-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(1H) -Indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-A Keith-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

10 mg (10 μmol) of intermediate 124 was placed in 4 ml of DMF and mixed with 2.5 mg (20 μmol) of L-cysteine. The reaction mixture was stirred at RT for 2 h then concentrated under reduced pressure and then Purified by preparative HPLC. This gave 7.2 mg (64% of the title compound).

HPLC (method 5): _Rt = 1.6 min; LC-MS (Method 1): R _t = 0.8 min; MS (ESI+): m/z = 071 (M+H) ⁺ .

Example 67 N-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]indolyl}-2,5-dioxapyrrolidin-1-yl)hexyl]-N-methyl- L-Amidoxime-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-吲哚- 3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine

10 mg (10 μmol) of intermediate 125 was placed in 4 ml of DMF and mixed with 2.4 mg (20 μmol) of L-cysteine. The reaction mixture was stirred at RT for 2 h then concentrated under reduced pressure and then purified by preparative HPLC. This gave 7.7 mg (69% of the title compound).

HPLC (method 5): _Rt = 1.7 min; LC-MS (Method 2): R _t = 1.91 min; MS (ESI+): m/z=1140 (M+H) ⁺ .

Example 68 N-(4-{2-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]indolyl}-2,5-dioxapyrrolidin-1-yl)hexanyl) Alkyl}-4-yloxybutyl)-N-methyl-L-decylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R, 2R)-3-{[(2S)-1-(benzylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-A Oxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl- L-Amidoxime

10 mg (10 μmol) of intermediate 160 was placed in 3 ml of DMF and mixed with 2.1 mg (20 μmol) of L-cysteine. The reaction mixture was stirred at RT for 2 h then concentrated under reduced pressure and then purified by preparative HPLC. This gave 8.1 mg (73% of the title compound).

HPLC (method _{5): R t = 1.7 min} ; LC-MS ( Method _{1): R t = 0.86 min} ; MS (ESI +): m / z = 1274 (M + H) +.

Example 69 N-(4-{2-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]indolyl}-2,5-dioxapyrrolidin-1-yl)hexanyl) Alkyl}-4-yloxybutyl)-N-methyl-L-indole Aminyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-1- Side oxy-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5 -methyl-1-oxoheptan-4-yl]-N-methyl-L-amidamine

3.5 mg (3 μmol) of intermediate 159 was treated in 1 ml of DMF and mixed with 0.76 mg (6 μmol) of L-cysteine. The reaction mixture was stirred at RT for 2 h then concentrated under reduced pressure and then purified by preparative HPLC. This gave 2.6 mg (65% of the title compound).

HPLC (Method 5): R _t = 1.75 min; LC-MS (Method 1): R _t = 0.85 min; MS (ESI+): m/z=1235 (M+H) ⁺ .

Example 70 N-(6-{2-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]indolyl}-2,5-dioxapyrrolidin-1-yl)hexanyl肼] 肼}}-6-oxo-oxyhexyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2 -[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2- Alkyl-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-amidamine

3.6 mg (3 μmol) of intermediate 129 was treated in 1 ml of DMF and mixed with 0.77 mg (6 μmol) of L-cysteine. The reaction mixture was stirred at RT for 2 h then concentrated under reduced pressure and then purified by preparative HPLC. This gave 1.55 mg (39% of the title compound).

HPLC (Method 5): R _t = 1.6 min; LC-MS (Method 1): R _t = 0.87 min; MS (ESI+): m/z=1255 (M+H) ⁺ .

Example 71

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 0.9 mg/ml

Drug / mAb ratio: 1

Example 72

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.86 mg/ml

Drug / mAb ratio: 2.9

Example 73

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.05 mg/ml

Drug / mAb ratio: 4.4

Example 74

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.13 mg/ml

Drug / mAb ratio: 2.8

Example 75

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.41 mg/ml

Drug / mAb ratio: 3.9

Example 76

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.38 mg/ml

Drug / mAb ratio: 4.3

Example 77

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.32 mg/ml

Drug / mAb ratio: 1

Example 78

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.14 mg/ml

Drug / mAb ratio: 5.3

Example 79

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.25 mg/ml

Drug / mAb ratio: 4.8

Example 80

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.12 mg/ml

Drug / mAb ratio: 1.7

Example 81

This was coupled using 150 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation, diluted again with PBS and concentrated again.

Protein concentration: 12.2 mg/ml

Drug / mAb ratio: 4.1

Example 82

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 0.86 mg/ml

Drug / mAb ratio: 3.4

Example 83

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.43 mg/ml

Drug / mAb ratio: 3.7

Example 84

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 0.8 mg/ml

Drug/mAb ratio: 0.7

Example 85

This was coupled using 50 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation, diluted again with PBS and concentrated again.

Protein concentration: 9.5 mg/ml

Drug / mAb ratio: 2.9

Example 86

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.52 mg/ml

Drug / mAb ratio: 3.2

Example 87

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.25 mg/ml

Drug / mAb ratio: 4.6

Example 88

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.47 mg/ml

Drug / mAb ratio: 1.6

Example 89

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 0.99 mg/ml

Drug / mAb ratio: 5.5

Example 90

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.02 mg/ml

Drug / mAb ratio: 4.0

Example 91

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.63 mg/ml

Drug / mAb ratio: 3.8

Example 92

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.27 mg/ml

Drug / mAb ratio: 3.0

Example 93

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.58 mg/ml

Drug / mAb - ratio: 0.6

Example 94

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.31 mg/ml

Drug / mAb ratio: 6.6

Example 95

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.75 mg/ml

Drug / mAb ratio: 1.8

Example 96

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.44 mg/ml

Drug / mAb ratio: 2.5

Example 97

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.96 mg/ml

Drug / mAb ratio: 5.6

Example 98

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.58 mg/ml

Drug / mAb ratio: 4.2

Example 99

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.48 mg/ml

Drug / mAb ratio: 4.6

Example 100

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.5 mg/ml

Drug / mAb ratio: 3.1

Example 101

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.3 mg/ml

Drug / mAb ratio: 4.3

Example 102

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.62 mg/ml

Drug / mAb ratio: 2.2

Example 103

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.37 mg/ml

Drug / mAb ratio: 2.8

Example 104

This was carried out by coupling with 5 mg of MF-Ta in PBS, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted.

Protein concentration: 1.43 mg/ml

Drug / mAb ratio: 4.0

Example 105 N-(6-{[(5S)-5-Amino-5-carboxypentyl]amino}-6-oxo-oxyhexyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R ,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine guanamine trifluoroacetate

15.5 mg (15 μmol) of intermediate 210 was placed in 5 ml of DMF and treated with 4.4 mg (18 μmol) of N ² -(t-butoxycarbonyl)-L-lysine and 7.7 μL (44 Μmol) of N,N -diisopropylethylamine is mixed. The reaction mixture was stirred at rt overnight then concentrated EtOAc. The residue was then purified by preparative HPLC. This gave 14 mg (81% of theory) of the title compound as the title compound, which was then taken up in 1 ml of dichloromethane and deprotected with 1 ml of trifluoroacetic acid. The reaction mixture was concentrated, and EtOAc EtOAcjjjjjj

HPLC (method _{12): R t = 1.8 min} ; LC-MS ( Method _{1): R t = 0.79 min} ; MS (ESI +): m / z = 1083 (M + H) +.

Example 106 N-(6-{[(5S)-5-Amino-5-carboxypentyl]amino}-6-oxo-oxyhexyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R ,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]amine }-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N-methyl-L-amidamine

40 mg (40 μmol) of intermediate 227 was treated in 5 ml of DMF with 11.5 mg (40 μmol) of N ² -[(benzyloxy)carbonyl]-L-isoamine and 13 μL (80 Μmol) of N,N -diisopropylethylamine is mixed. The reaction mixture was stirred at rt over rt then concentrated over EtOAc EtOAc. This gave an intermediate of 32.5 mg (70% of theory) of title compound.

This 32.5 mg intermediate was dissolved in 10 ml of methanol and 2 mg of 10% activated carbon on palladium was added and hydrogenated at RT under standard hydrogen pressure for 30 minutes. The catalyst was removed by filtration and the solvent was removed under reduced pressure. Residue from two After hexane/water 1:1 lyophilization, 26 mg (99% of theory)

HPLC (Method 12): R _t = 1.7 min; LC-MS (Method 1): R _t = 0.76 min; MS (ESI+): m/z = 011 (M+H) ⁺ .

Example 107 N-[(18S)-18-Amino-18-carboxy-12-sideoxy-3,6,9-trioxa-13-azaoctadecan-1-yl]-N-methyl-L- Amidoxime-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-吲哚-3- Base)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine guanamine trifluoroacetate

3.5 mg (3 μmol) of intermediate 202 was placed in 2 ml of DMF and treated with 0.8 mg (3 μmol) of N ² -(t-butoxycarbonyl)-L-isoamine and 1.6 μL (10) Μmol) of N,N -diisopropylethylamine is mixed. The reaction mixture was stirred at rt overnight then concentrated EtOAc. The residue was taken up in acetonitrile / water: 1:1, pH was taken to 2 with trifluoroacetic acid and then purified by preparative HPLC. This gave 1 mg (25% of theory) of the title compound as the title compound, which was then taken up in 500 <RTIgt; The reaction mixture was concentrated and EtOAcqqqqqqq

HPLC (method 12): _Rt = 1.9 min; LC-MS (Method 1): R _t = 0.82 min; MS (ESI+): m/z=1173 (M+H) ⁺ .

Example 108

This was coupled using 5 mg of MF-Ta in PBS, and the fraction was purified by Sephadex, concentrated by ultracentrifugation and re-diluted.

Protein concentration: 0.81 mg/ml

Drug / mAb ratio: 2.5

Example 109

This was coupled using 5 mg of MF-Ta, and the batch was purified by Sephadex, concentrated by ultracentrifugation and re-diluted.

Protein concentration: 1.06 mg/ml

Drug / mAb ratio: 1.8

Example 110

This was coupled using 5 mg of MF-Ta, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted with PBS.

Protein concentration: 1.36 mg/ml

Drug/mAb ratio: 7.2

Example 111

This was coupled using 5 mg of MF-Ta, and the reaction mixture was purified by Sephadex, concentrated by ultracentrifugation and diluted with PBS.

Protein concentration: 1.57 mg/ml

Drug / mAb ratio: 2.9

Example 112

This was coupled using 5 mg of MF-Ta in PBS, and the fraction was purified by Sephadex, concentrated by ultracentrifugation and re-diluted.

Protein concentration: 1.44 mg/ml

Drug / mAb ratio: 2.5

Example 113

This was coupled using 5 mg of MF-Ta in PBS, and the fraction was purified by Sephadex, concentrated by ultracentrifugation and re-diluted.

Protein concentration: 1.74 mg/ml

Drug / mAb ratio: 3.6

Example 114 (diastereomer 1)

This was coupled using intermediate 247a and 5 mg of MF-Ta in PBS, and the fraction was purified by Sephadex, concentrated by ultracentrifugation and re-diluted with PBS.

Protein concentration: 1.57 mg/ml

Drug / mAb ratio: 4.2

Example 115 (diastereomer 2)

This was coupled using intermediate 247b and 5 mg of MF-Ta in PBS, and the fraction was purified by Sephadex, concentrated by ultracentrifugation and re-diluted with PBS.

Protein concentration: 1.42 mg/ml

Drug / mAb ratio: 4.0

Example 116 N-(6-{[(5S)-5-Amino-5-carboxypentyl]amino}-6-oxo-oxyhexyl)-N-methyl-L-threonyl-N-[(3R ,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine guanamine trifluoroacetate

8.6 mg (8 μmol) of intermediate 240 was placed in 5 ml of DMF and treated with 4.0 mg (16 μmol) of N ² -(t-butoxycarbonyl)-L-isoamine and 2 μL (16 Μmol) of N,N -diisopropylethylamine is mixed. The reaction mixture was stirred at RT for 4 hours and then mixed again with the same amount of N ² -(t-butoxycarbonyl)-L-isoamine and N,N -diisopropylethylamine, and stirred at RT. Until overnight. The batch was then concentrated under reduced pressure. The residue was purified by preparative HPLC. This gave 7 mg (72% of theory) of the title compound as the title compound, which was taken in 1 ml of dichloromethane and deprotected with 0.5 ml of trifluoroacetic acid. The batch was concentrated and the residue was purified using preparative HPLC. Drying under high vacuum gave 3.3 mg (47% of theory).

HPLC (Method 5): R _t = 1.5 min; LC-MS (Method 1): R _t = 0.8 min; MS (ESI+): m/z=1084 (M+H) ⁺ .

Example 117 N-(6-{[(5S)-5-Amino-5-carboxypentyl]amino}-6-oxo-oxyhexyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R ,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-hydroxyphenyl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine guanamine trifluoroacetate

8 mg (8 μmol) of intermediate 242 was treated in 3 ml of DMF with 2.9 mg (12 μmol) of N ² -(t-butoxycarbonyl)-L-isoamine and 2.7 μL (16 Μmol) of N,N -diisopropylethylamine is mixed. The reaction mixture was stirred overnight at RT until then again with the same amount of ² N - (tertiary butoxycarbonyl) -L- lysine, and N, N - diisopropyl ethyl amine mixed and stirred at RT 4 hours. The batch was then concentrated under reduced pressure. The residue was purified by preparative HPLC. After lyophilization with acetonitrile/water, 6.5 mg (yield: 72%) of the title compound of the title compound was obtained, which was taken in 5 ml of dichloromethane and deprotected with 0.75 ml of trifluoroacetic acid. Concentrate this batch and remove the residue from the second After lyophilization of the alkane/water, 5 mg (76% of theory) of title compound.

HPLC (Method 12): R _t = 1.7 min; LC-MS (Method 1): R _t =0.69 min; MS (ESI+): m/z = 159 (M+H) ⁺ .

Example 118 N-(6-{[(5S)-5-Amino-5-carboxypentyl]amino}-6-oxo-oxyhexyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R ,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(4-hydroxyphenyl)ethyl]amino}- 1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N- Methyl-L-Amidoxime Trifluoroacetate

38 mg (41 μmol) of intermediate 248 was first converted to N-hydroxysuccinimide. The resulting 72 mg crude product was taken up in 5 ml of DMF and combined with 24 mg (100 μmol) of N ² -(t-butoxycarbonyl)-L-lysine and 23 μL of N,N - II Isopropyl ethylamine is mixed. The reaction mixture was stirred at RT until overnight, and then again mixed with 16 mg of N ² -(t-butoxycarbonyl)-L-isoamine and 12 μL of N,N -diisopropylethylamine. And then further treatment in the ultrasonic bath for 2 hours. This batch was then concentrated under reduced pressure and the residue was purified using preparative HPLC. Freeze-drying in acetonitrile / water afforded 20 mg (50% of theory)

15 mg (12 μmol) of this intermediate was then taken up in 3 ml of dichloromethane and mixed with 1 ml of trifluoroacetic acid. After stirring for 40 minutes at RT, an additional 1.5 ml of trifluoroacetic acid was added and the batch was further treated in an ultrasonic bath for an additional 1 hour. Then concentrate the batch and remove the residue from the second The alkane/water was lyophilized to give 13 mg (yield: 90%).

HPLC (Method 12): _Rt = 1.5 min; LC-MS (Method 1): R _t = 0.68 min; MS (ESI+): m/z = 990 (M+H) ⁺ .

C: Assessment of biological activity:

The biological utility of the compounds of the invention can be verified by the following analysis:

C-1. In vitro cell proliferation assay

The cytotoxic activity of the conjugates of the invention is tested in an in vitro cell proliferation assay. For this purpose, mammalian cells that endogenously or recombinantly express the target molecule of the binding agent are cultured with the conjugate of the invention. After several hours to several days of culture, cell proliferation was measured on the basis of the cell count as compared with the control group to which no conjugate was added. For further control, it may be added separately to the unconjugated toxicant, or to cells that are not the target molecule of the binding agent. The number of cells is determined by methods known to those skilled in the art, such as counting sets of cells (e.g., ATPlite ^(TM) , Perkin-Elmer) that are calculated by ATP measurements, either by counting or via use. In this way, the IC _{50 of the} conjugate of the present invention is determined. Target molecule carrying a cell binding agent conjugates of the measured IC ₅₀ compared with the non-carrying cells can be determined selectively engaging the object.

C-2. Determination of anti-proliferative activity of anti-mesothelin ADC in human colorectal cancer cell line HT29

A defined amount of human colorectal cancer cell line HT29wt (2500 cells/well, wild type) was seeded into 96-well MTP complete medium (10% FCS-RPMI) and cultured at 37 ° C / 5% carbon dioxide. Overnight. Parallel to this, HT29 cells stably expressing mesothelin-transfected cells were seeded in 96-well MTP complete medium and cultured overnight (2500 cells/well, 37 ° C / 5% carbon dioxide).

After 18 hours, the seeding medium was changed to fresh medium containing 10% FCS. Treatment begins with the addition of a compound of the invention. The transfected cells were treated in the same manner as HT29wt cells.

For the substances studied, the dose-utility curve was determined over a concentration range of 10 ^-5 M -10 ^-14 M (1:10 serial dilution).

The incubation time of 48 h-96 h was selected.

Proliferation was detected using MTT assay (ATCC, Manassas, Virginia, USA, model 30-1010K). At the end of the selected incubation time, HT29wt cells were cultured for 4 h in MTT, followed by the addition of detergent to dissociate the cells overnight.

The dye formed was detected at 570 nm.

Proliferation of cells that are not treated with the test substance but otherwise treated The system is defined as a 100% value. The data obtained from this experiment are three replicate measurements and at least two independent experiments were performed.

Table 3 series shows the IC ₅₀ values for representative examples of this analysis ¹⁾ : ¹⁾ The proposed activity data is an example of the operation described in the experimental part of the invention, with the drug/mAb ratio shown. These values may deviate for different drug/mAB ratios.

C-3. Determination of the effect of tubulin polymerization

Cancer cells are degenerative cells that often lead to tumor formation due to increased cell division. Microtubules form the spindle of the spindle and are an essential component of the cell cycle. The regular construction and destruction of microtubules allows precise division of chromosomes in progeny cells and constitutes a continuous dynamic process. Interrupting this dynamic process leads to incorrect cell division and eventual cell death. However, increased cell division of cancer cells also makes them particularly sensitive to spindle toxin, which constitutes a fixed component of chemotherapy. Spindle toxins, such as paclitaxel or epothilone, cause polymerization of microtubules The rate is drastically increased, while vinblastine or other monomethyl auristatin E (MMAE) causes a dramatic decrease in the polymerization rate of microtubules. In both cases, this necessary cell cycle dynamics is severely disrupted. The compounds studied in the context of the present invention reduce the rate of polymerization of microtubules.

Tubulin polymerization was evaluated using a "fluorescent microtubule polymerization assay kit" from Cytoskeleton (Denver, Colorado, USA; order number: BK011). From this analysis, the addition of GTP to unpolymerized tubulin causes the polymerization to occur spontaneously. This analysis was based on the binding of fluorophore 4',6-dimethylhydrazino-2-phenylindole (DAPI) to tubulin. Free and bound DAPI can be distinguished based on different emission spectra. Because DAPI has a relatively high affinity for polymerized tubulin compared to unpolymerized tubulin, tubulin polymerization can be tracked by increased fluorescence of the bound DAPI fluorophore.

For this analysis, a DMSO solution of the compound of the invention was diluted with water from the initial concentration of 10 mM to 1 μM. In addition to the buffer control group, paclitaxel having an increased polymerization effect and vinblastin having a polymerization inhibitory effect were additionally used as an analysis control group. The measurement was performed using a 96-well plate with a half of the substrate area. The kinetics of tubulin polymerization was monitored by a Fluorimeter for 1 hour at 37 °C. The excitation light wavelength is 355 nm, and the emission light is monitored at 460 nm. The area of linear increase in the first 10 minutes was calculated as the change in fluorescence per minute (ΔF/min), which represents the polymerization rate of the microtubes. The potency of the test substance is quantified based on its individual rate of polymerization reduction.

The decrease in tubulin polymerization rate is dependent on the concentration of test substance added. The addition of 100 μM MMAF completely stopped the polymerization. Adding 1 μM MMAF reduced the polymerization rate by 40-45%, and the best line showed the activity comparison of different substances. The compound of Example 64 inhibited the polymerization rate to an extent of 41% at 1 μM. The compounds of Examples 62-70 and 105-107 have tubulin polymerization inhibition comparable to MMAF.

The inhibition value of MMAF at a concentration of 1 μM was set to 100%.

Table 6 is data for inhibition of tubulin polymerization for representative example manipulations.

The MMAF venom and manipulation examples inhibit tubulin polymerization as a function of its concentration. 100 μM MMAF, tubulin polymerization was completely inhibited. In Example 1, 105, the inhibition of tubulin polymerization was 45% of the 1 μM MMAF measurement at 1 μM.

C-4. Determination of cell permeability in vitro

The cellular permeability of the substance can be assessed by flux analysis using Caco-2 cells by in vitro assays [MD Troutman and DRThakker, Pharm . Res . 20 (8) , 1210-1224 (2003)]. For this purpose, the cells were incubated on a 24-well filter plate for 15-16 days. For the measurement of the osmosis, an operation example was applied to the cells in the HEPES buffer as the apex (A) or the base (B), and cultured for 2 hours. After 0 hours and 2 hours, the sample was taken out from the chute and the back chute. The sample was separated by HPLC (Agilent 1200, Böblingen, Germany) using a reverse phase column. The HPLC system was combined with a Triple Quadropol mass spectrometer API 4000 (Applied Biosystems Applera, Darmstadt, Germany) via a Turbo ion spray interface. Permeability was evaluated on the basis of the P _app value, which was calculated using the formula published by Schwab et al. [D. Schwab et al., J. Med . Chem . 46, 1716-1725 (2003)].

Very important for intracellular release of venom is permeability from B to A [P _app (BA)]: the lower the permeability, the longer the operating example stays in the cell after intracellular release, and Therefore, the longer it takes to interact with the biochemical target (in this case: tubulin).

Table 4 below shows the permeability data for representative operational examples of this analysis:

The example of the operation has a low permeability of B to A [P _app (BA), and thus has a long-term retention in CaCo-2 cells. In comparison, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) have a P _app (BA) value of 73 nm/s in this test, and therefore have a significantly shorter time to stay in In Caco-2 cells.

C-5 Determination of the in vitro nature of the substance properties of P-glycoprotein (P-gp)

Many tumor cells display transport proteins to drugs, and this is often accompanied by resistance to cytostatics. Substances other than such transporters, such as P-glycoprotein (P-gp) or BCRP, may therefore have improved active properties.

The matrix properties of P-gp (ABCB1) substances were determined by flux analysis using LLC-PK1 cells (L-MDR1 cells) expressing P-gp [AHSchinkel et al., J. Clin . Invest . 96, 1698-1705 (1995)]. For this purpose, LLC-PK1 cells or L-MDR1 cells were cultured on a 96-well filter disk for 3-4 days. For the measurement of permeability, individual test substances are either apical (A) or base in HEPES buffer alone or in the presence of inhibitors (eg, Ivermectin or Verapamil). (B) Administration to cells and incubation for 2 hours. After 0 hours and 2 hours, the sample was taken out from the chute and the back chute. The sample was separated by HPLC using a reverse phase column. The HPLC system was combined with a Triple Quadropol mass spectrometer API 3000 (Applied Biosystems Applera, Darmstadt, Germany) via a Turbo ion spray interface. Permeability was evaluated on the basis of the P _app value, which was calculated using the formula published by Schwab et al. [D. Schwab et al., J. Med . Chem . 46, 1716-1725 (2003)].

Very important for intracellular release of venom is permeability from B to A [P _app (BA)]: the lower the permeability, the longer the operating example stays in the cell after intracellular release, and Therefore, the longer it takes to interact with the biochemical target (in this case: tubulin).

Table 5 below shows the permeability data for a representative example of the operation of this assay (which is performed in L-MDR1 cells):

The example of the operation has a low permeability of B to A [P _app (BA), and thus has a long stay in the L-MDR1 cells.

C-6. Pharmacokinetics in HT29 tumor model of mesothelin-transfected and non-transfected HT29 cells

After intravenous administration of Example 5 at 16 mg/kg, Example 5 and possible metabolites, such as the plasma concentration and tumor concentration of Example 64, were measured. Example 64 in animals with mesothelin-transfected tumors The area under the curve (AUC) was about 0.50 mg*h/l in plasma; in tumors, the exposure of the compound of Example 64 was about 400 times higher (AUC = 203 mg*h/L). In animals with non-transfected tumors, the exposure of Example 64 in plasma was the same as that in the plasma of animals bearing transfected tumors. In contrast, AUC in tumors of non-transfected animals was approximately 8 times lower than that of transfected animals. This line shows a clear target effect in tumors in the presence of antigen.

Quantitative analysis of the compound of Example 64

The measurement of the compound of Example 64 in plasma and tumor was performed after precipitation of the protein with methanol by high pressure liquid chromatography (HPLC) in combination with a tandem mass spectrometer (MS).

For 100 μL plasma, mix it with 400 μL of methanol and 10 μL of internal standard (ISTD, 50 ng/mL in methanol) and shake for 10 seconds. After centrifugation at 16 000 g for 5 minutes, 250 μL of the supernatant was transferred to an auto-sort bottle, which was added to 250 μL of ammonium acetate buffer (AAC, 10 mM, pH 6.8) and shaken again.

For the treatment of tumors, it was mixed with 4 times the amount of methanol. In a tissue mill Tissuelyser II (Quiagen), the sample was pulverized for 6 minutes with 30 impacts per minute, and then centrifuged at 16 000 g for 5 minutes. 50 μL of the supernatant was transferred to an autosampler vial and 50 μL of ammonium acetate buffer (10 mM, pH 6.8) and 5 μL of ISTD were added. After the shock again, the tumor sample is ready for measurement.

Measurement of two matrix samples, and finally, HPLC-bound atmosphere Press free/tandem mass spectrometry was performed on a SW4000 EXP4000 instrument using a Turbo ion spray interface (TISP). The measured m/z transition is as follows: Example 64 (meter) 614.652 → 570.9

Example 64 (meter 1) 614.652 → 555.0

Example 64 (meter 2) 614.652 → 500.4

Internal Standard (ISTD) 726.665 → 694.5 HPLC/LC-MSMS (TISP) analysis was performed on an HP1100 pump (Agilent) equipped with a Gemini column (5 μm C18 110 A, 50 x 3 mm, Phenomenex). Under the following gradient conditions: flow rate 0.4 mL/min; gradient: 0.0 min-1.0 min 10% acetonitrile/90% AAC, 1.0 min-3.0 min 10% acetonitrile/90% AAC→50% acetonitrile/50% AAC, 3.0 min - 5.5 min 50% acetonitrile / 50% AAC, 5.5 min - 5.6 min 50% acetonitrile / 50% AAC → 10% acetonitrile / 90% AAC, 5.6 min - 6.0 min 10% acetonitrile / 90% AAC.

For calibration, plasma samples were mixed at a concentration of 0.5-2000 μg/L. The detection limit (LOQ) is 2 μg/L. The linear range is from 2 to 1000 μg/L.

For tumor sample correction, untreated tumor supernatant was mixed with a concentration of 0.5-200 μg/L. The detection limit is 5 μg/L. The linear range is from 5 to 200 μg/L.

The qualitative control of the effective control contained 5 and 50 μg/L with 500 μg/L in plasma. The concentration of these samples was found to deviate from the desired value (data not attached) by 20%.

C-7. Activity test in vivo

The activity of the conjugate of the present invention is in vivo and tested, for example, in a xenograft model. Methods for testing the activity of the compounds of the invention in the prior art are known to those skilled in the art (see, for example, WO 2005/081711; Polson et al, Cancer Res. 2009 Mar 15; 69(6): 2358-64). For this purpose, for example, rodents (e.g., mice) are implanted into a tumor cell line that exhibits a binding agent target molecule. The implanted animal is then administered to the conjugate or control antibody or isotonic saline solution of the invention. Administration can be carried out singly or more frequently. After several days of incubation time, tumor size was determined and conjugate treated animals were compared to controls. The conjugate treated animals showed smaller tumors.

C-7a. Test anti-mesothelin ADC in mouse experimental tumors

Human tumor cells expressing mesothelin are injected subcutaneously into immunosuppressive mice, such as the flank of nude mice or SCID mice. One million to ten million cells were isolated from the cell culture, centrifuged and resuspended in 100 μl of medium or 1:1 medium/matrigel. The cell suspension was injected under the skin of the mouse.

The tumor is allowed to grow within a few days. Treatment begins after the tumor is greater than 20 mm ² . In order to assess the utility of established tumors, treatment can also begin only when the tumor size is 50-100 mm ² .

The treatment of the ADC is carried out via a venous route into the tail vein of the mouse. The ADC was dissolved in PBS and administered in an amount of 5 ml/kg.

The course of treatment is governed by the pharmacokinetics of the antibody. The treatment was performed three times every three days as a standard. However, treatment is also sustainable, Or at a later point in time, follow the second round of three days of treatment.

Eight animals were used as a baseline for each treatment group. This amount can be higher if there is a particularly large fluctuation after tumor growth or expected treatment. As with the group receiving the active substances, a group as a control group was treated with only a buffer according to the same procedure.

Tumor area was measured periodically using a two-dimensional (length/width) vernier scale during the experiment.

At the end of the experiment, the tumor was removed and weighed. The ratio of the average tumor weight of the treatment group (T) to the control group (C) is expressed as T/C.

C-7b. Activity of mesothelin-transfected HT29 cells in HT29 colorectal cancer tumor model

One million HT29 cells (stable transfection with mesothelin) were subcutaneously inoculated into the flank of NMRI nude mice. On the sixth day, when the average tumor size was 30 mm ² , intravenous treatment was started (days 6, 10, and 14). After treatment, the growth of the tumor was monitored until the 26th day. Based on the animal protection law, the control animals must be appropriately euthanized on the 26th day due to the large tumor.

The ADC caused significant inhibition of tumor growth. On day 26, the experiment was final and the tumors of all animals were weighed. Thus, T/C was calculated to be the tumor weight of the treatment group relative to the control group.

C-7c. Activity in Ovcar3 ovarian cancer tumor model

3 million Ovcar3 cells were subcutaneously inoculated into the flank of NMRI nude mice.

At the mean tumor size of 40 mm ² on day 49, treatment with an intravenous dose of 5 mg/kg was started (days 38, 42, 46 and days 70, 74). After treatment, the growth of the tumor was monitored until the 78th day. At the end of the experiment, the tumor was removed and weighed.

The ADC caused significant inhibition of tumor growth. On day 78, the experiment was final and the tumors of all animals were weighed. Thus, T/C was calculated to be the tumor weight of the treatment group relative to the control group.

C-7d. Activity in endogenous NCI-H322 lung cancer tumor model expressing endothelin. Treatment process q4dx3.

5 million NCI-H322 cells were subcutaneously inoculated into the flank of NMRI nude mice. On the 21st day, when the average tumor size was 50 mm ² , intravenous treatment was started (days 21, 25, and 29). After treatment, the growth of the tumor was monitored until the 42nd day. Based on the animal protection law, the control animals must be appropriately euthanized on the 42nd day due to the large tumor.

The ADC caused significant inhibition of tumor growth. On day 42, the experiment was final and the tumors of all animals were weighed. Thus, T/C was calculated to be the tumor weight of the treatment group relative to the control group.

C-7e. Activity in endogenous NCI-H322 lung cancer tumor model expressing endothelin. Single treatment

5 million NCI-H322 cells were subcutaneously inoculated into the flank of NMRI nude mice. On day 22, when the tumor size was about 45 mm ² , treatment with ACD or vehicle was performed intravenously. After treatment, monitor the growth of the tumor. Based on the animal protection law, the control animals must be appropriately euthanized on the 50th day due to the large tumor.

The ADC caused significant inhibition of tumor growth. On day 50, tumor area (length x width) was used to determine T/C.

D. Operational examples of pharmaceutical compositions

The compounds of the invention can be converted to pharmaceutical preparations as follows:

I.v. solution:

The compound of the present invention is dissolved in a physiologically tolerable solvent at a concentration lower than the saturation solubility (for example, an isotonic aqueous solution of saline, D-PBS or sorbitol with glycerin and sodium chloride dissolved in a citrate buffer) Formulation of ester 80). The solution is sterile filtered and dispersed in sterile and disease free injection containers.

I.v. solution:

The compounds of the invention can be converted to the form of administration described. It can be carried out by a known method by "mixing" or "dissolving" in an inert, non-toxic pharmaceutically suitable excipient (for example, a buffer substance, a stabilizer, a solubilizer, a preservative). For example, the following substances may be present: amino acids (glycine, histidine, methionine, arginine, lysine, leucine, isoleucine, threonine, glutamic acid, amphetamine Acids and other), sugars and related compounds (glucose, sucrose, mannitol, trehalose, sucrose, gan Caramel, lactose, sorbitol), glycerol, sodium, potassium, ammonium and calcium salts (such as sodium chloride, potassium chloride or disodium hydrogen phosphate and many others), acetate/acetate buffer system, phosphate buffer system , citric acid and citric acid buffer systems, trometamol (TRIS and TRIS salts), polysorbates (eg Polysorbate 80 and Polysorbate 20), Poloxamer (eg poloxamer 188) And poloxamer 171), Polyglycol (Macrogol) (PEG, eg 3350), Triton X-100, EDTA salt, glutathione, albumin (eg human), benzyl alcohol, phenol, chlorocresol , m-cresol, benzalkonium chloride and many others.

For lyophilizates that are subsequently converted to i.v., s.c. or i.m. solutions:

In addition, the compounds of the present invention can be converted into stable lyophilizates (with the aid of the above-mentioned excipients), and reconstituted and administered in a suitable solvent (e.g., injection grade water, isotonic saline solution) prior to administration. .

<110> Bayer Pharmaceutical Co., Ltd. Bayer Wisdom Property Co., Ltd.

<120> Novel binding agents - drug conjugates (ADCs) and their uses (1)

<130> BHC 09 1 029

<160> 411

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<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 56

<210> 57

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 57

<210> 58

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 58

<210> 59

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 59

<210> 60

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 60

<210> 61

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 61

<210> 62

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 62

<210> 63

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 63

<210> 64

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 64

<210> 65

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 65

<210> 66

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 66

<210> 67

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 67

<210> 68

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 68

<210> 69

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 69

<210> 70

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 70

<210> 71

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 71

<210> 72

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 72

<210> 73

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 73

<210> 74

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 74

<210> 75

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 75

<210> 76

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 76

<210> 77

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 77

<210> 78

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 78

<210> 79

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 79

<210> 80

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 80

<210> 81

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 81

<210> 82

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 82

<210> 83

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 83

<210> 84

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 84

<210> 85

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 85

<210> 86

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 86

<210> 87

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 87

<210> 88

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 88

<210> 89

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 89

<210> 90

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 90

<210> 91

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 91

<210> 92

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 92

<210> 93

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 93

<210> 94

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 94

<210> 95

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 95

<210> 96

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 96

<210> 97

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 97

<210> 98

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 98

<210> 99

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 99

<210> 100

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 100

<210> 101

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 101

<210> 102

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 102

<210> 103

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 103

<210> 104

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 104

<210> 105

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 105

<210> 106

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 106

<210> 107

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 107

<210> 108

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 108

<210> 109

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 109

<210> 110

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 110

<210> 111

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 111

<210> 112

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 112

<210> 113

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 113

<210> 114

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 114

<210> 115

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 115

<210> 116

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 116

<210> 117

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 117

<210> 118

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 118

<210> 119

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 119

<210> 120

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 120

<210> 121

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 121

<210> 122

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 122

<210> 123

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 123

<210> 124

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 124

<210> 125

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 125

<210> 126

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 126

<210> 127

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 127

<210> 128

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 128

<210> 129

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 129

<210> 130

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 130

<210> 131

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 131

<210> 132

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 132

<210> 133

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 133

<210> 134

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 134

<210> 135

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 135

<210> 136

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 136

<210> 137

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 137

<210> 138

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 138

<210> 139

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 139

<210> 140

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 140

<210> 141

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 141

<210> 142

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 142

<210> 143

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 143

<210> 144

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 144

<210> 145

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 145

<210> 146

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 146

<210> 147

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 147

<210> 148

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 148

<210> 149

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 149

<210> 150

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 150

<210> 151

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 151

<210> 152

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 152

<210> 153

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 153

<210> 154

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 154

<210> 155

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 155

<210> 156

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 156

<210> 157

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 157

<210> 158

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 158

<210> 159

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 159

<210> 160

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 160

<210> 161

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 161

<210> 162

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 162

<210> 163

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 163

<210> 164

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 164

<210> 165

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 165

<210> 166

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 166

<210> 167

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 167

<210> 168

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 168

<210> 169

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 169

<210> 170

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 170

<210> 171

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 171

<210> 172

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 172

<210> 173

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 173

<210> 174

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 174

<210> 175

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 175

<210> 176

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 176

<210> 177

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 177

<210> 178

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 178

<210> 179

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 179

<210> 180

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 180

<210> 181

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 181

<210> 182

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 182

<210> 183

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 183

<210> 184

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 184

<210> 185

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 185

<210> 186

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 186

<210> 187

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 187

<210> 188

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 188

<210> 189

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 189

<210> 190

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 190

<210> 191

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 191

<210> 192

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 192

<210> 193

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 193

<210> 194

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 194

<210> 195

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 195

<210> 196

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 196

<210> 197

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 197

<210> 198

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 198

<210> 199

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 199

<210> 200

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 200

<210> 201

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 201

<210> 202

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 202

<210> 203

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 203

<210> 204

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 204

<210> 205

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 205

<210> 206

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 206

<210> 207

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 207

<210> 208

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 208

<210> 209

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 209

<210> 210

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 210

<210> 211

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 211

<210> 212

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 212

<210> 213

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 213

<210> 214

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 214

<210> 215

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 215

<210> 216

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 216

<210> 217

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 217

<210> 218

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 218

<210> 219

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 219

<210> 220

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 220

<210> 221

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 221

<210> 222

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 222

<210> 223

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 223

<210> 224

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 224

<210> 225

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 225

<210> 226

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 226

<210> 227

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 227

<210> 228

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 228

<210> 229

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 229

<210> 230

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 230

<210> 231

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 231

<210> 232

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 232

<210> 233

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 233

<210> 234

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 234

<210> 235

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 235

<210> 236

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 236

<210> 237

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 237

<210> 238

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 238

<210> 239

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 239

<210> 240

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 240

<210> 241

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 241

<210> 242

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 242

<210> 243

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 243

<210> 244

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 244

<210> 245

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 245

<210> 246

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 246

<210> 247

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 247

<210> 248

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 248

<210> 249

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 249

<210> 250

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 250

<210> 251

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 251

<210> 252

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 252

<210> 253

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 253

<210> 254

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 254

<210> 255

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 255

<210> 256

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 256

<210> 257

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 257

<210> 258

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 258

<210> 259

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 259

<210> 260

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 260

<210> 261

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 261

<210> 262

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 262

<210> 263

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 263

<210> 264

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 264

<210> 265

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 265

<210> 266

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 266

<210> 267

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 267

<210> 268

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 268

<210> 269

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 269

<210> 270

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 270

<210> 271

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 271

<210> 272

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 272

<210> 273

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 273

<210> 274

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 274

<210> 275

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 275

<210> 276

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 276

<210> 277

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 277

<210> 278

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 278

<210> 279

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 279

<210> 280

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 280

<210> 281

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 281

<210> 282

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 282

<210> 283

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 283

<210> 284

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 284

<210> 285

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 285

<210> 286

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 286

<210> 287

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 287

<210> 288

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 288

<210> 289

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 289

<210> 290

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 290

<210> 291

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 291

<210> 292

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 292

<210> 293

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 293

<210> 294

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 294

<210> 295

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 295

<210> 296

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 296

<210> 297

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> M20 D02 S-A derived CDR H1 consensus sequence

<220>

<221> variant

<222> (3)..(3)

<223> X=D or S

<400> 297

<210> 298

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> M20 D02 S-A derived CDRH2 consensus sequence

<220>

<221> variant

<222> (4)..(4)

<223> X=V or I

<220>

<221> variant

<222> (9)..(9)

<223> X=A or G

<220>

<221> variant

<222> (10)..(10)

<223> X=R or S

<400> 298

<210> 299

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> M20 D02 S-A derived CDRH3 consensus sequence

<220>

<221> variant

<222> (9)..(9)

<223> X=S, K, or R

<220>

<221> variant

<222> (10)..(10)

<223> X=A, S or R

<220>

<221> variant

<222> (14)..(14)

<223> X=D, K, E or R

<220>

<221> variant

<222> (15)..(15)

<223> X=S or Y

<400> 299

<210> 300

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Derived from M20 D02 S-A CDRL1 Consistent Sequence

<220>

<221> variant

<222> (7)..(7)

<223> X=V or I

<400> 300

<210> 301

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Consensus sequence derived from CDR L3 derived from M20 D02 S-A

<220>

<221> variant

<222> (3)..(3)

<223> X=A, Q or R

<220>

<221> variant

<222> (5)..(5)

<223> X=D or G

<220>

<221> variant

<222> (7)..(7)

<223> X=R or S

<220>

<221> variant

<222> (9)..(9)

<223> X=N, W or S

<220>

<221> variant

<222> (12)..(12)

<223> X=V, A or G

<400> 301

<210> 302

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Consistent sequence derived from M31 B01 CDR H1

<220>

<221> variant

<222> (3)..(3)

<223> X=N or S

<220>

<221> variant

<222> (4)..(4)

<223> X=A or Y

<400> 302

<210> 303

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> A consensus sequence derived from M31 B01 CDR H2

<220>

<221> variant

<222> (10)..(10)

<223> X=T or S

<220>

<221> variant

<222> (11)..(11)

<223> X=I or T

<400> 303

<210> 304

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Consistent sequence derived from M31 B01 CDR H3

<220>

<221> variant

<222> (10)..(10)

<223> X=Y, K, G, W or N

<400> 304

<210> 305

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Consistent sequence of derived M31 B01 CDR L1

<220>

<221> variant

<222> (1)..(1)

<223> X=T or S

<400> 305

<210> 306

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Consistent sequence derived from M31 B01 CDR L2

<220>

<221> variant

<222> (4)..(4)

<223> X=K or Q

<400> 306

<210> 307

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Consistent sequence of derived M31 B01 CDR L3

<220>

<221> variant

<222> (4)..(4)

<223> X=W, E, F or Y

<220>

<221> variant

<222> (7)..(7)

<223> X=R, S or M

<220>

<221> variant

<222> (9)..(9)

<223> X=N, K or S

<220>

<221> variant

<222> (10)..(10)

<223> X=G or R

<220>

<221> variant

<222> (11)..(11)

<223> X=P or A

<400> 307

<210> 308

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 308

<210> 309

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 309

<210> 310

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 310

<210> 311

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 311

<210> 312

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 312

<210> 313

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 313

<210> 314

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 314

<210> 315

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 315

<210> 316

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 316

<210> 317

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 317

<210> 318

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 318

<210> 319

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 319

<210> 320

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 320

<210> 321

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 321

<210> 322

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 322

<210> 323

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 323

<210> 324

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 324

<210> 325

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 325

<210> 326

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 326

<210> 327

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 327

<210> 328

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 328

<210> 329

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a

<400> 329

<210> 330

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 330

<210> 331

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 331

<210> 332

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 332

<210> 333

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 333

<210> 334

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 334

<210> 335

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 335

<210> 336

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 336

<210> 337

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 337

<210> 338

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 338

<210> 339

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 339

<210> 340

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 340

<210> 341

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 341

<210> 342

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 342

<210> 343

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 343

<210> 344

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 344

<210> 345

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> C4.4a bonding agent

<400> 345

<210> 346

<211> 217

<212> PRT

<213> Homo sapiens

<400> 346

<210> 347

<211> 447

<212> PRT

<213> Homo sapiens

<400> 347

<210> 348

<211> 217

<212> PRT

<213> Homo sapiens

<400> 348

<210> 349

<211> 446

<212> PRT

<213> Homo sapiens

<400> 349

<210> 350

<211> 217

<212> PRT

<213> Homo sapiens

<400> 350

<210> 351

<211> 446

<212> PRT

<213> Homo sapiens

<400> 351

<210> 352

<211> 217

<212> PRT

<213> Homo sapiens

<400> 352

<210> 353

<211> 446

<212> PRT

<213> Homo sapiens

<400> 353

<210> 354

<211> 217

<212> PRT

<213> Homo sapiens

<400> 354

<210> 355

<211> 446

<212> PRT

<213> Homo sapiens

<400> 355

<210> 356

<211> 217

<212> PRT

<213> Homo sapiens

<400> 356

<210> 357

<211> 446

<212> PRT

<213> Homo sapiens

<400> 357

<210> 358

<211> 217

<212> PRT

<213> Homo sapiens

<400> 358

<210> 359

<211> 446

<212> PRT

<213> Homo sapiens

<400> 359

<210> 360

<211> 217

<212> PRT

<213> Homo sapiens

<400> 360

<210> 361

<211> 446

<212> PRT

<213> Homo sapiens

<400> 361

<210> 362

<211> 217

<212> PRT

<213> Homo sapiens

<400> 362

<210> 363

<211> 446

<212> PRT

<213> Homo sapiens

<400> 363

<210> 364

<211> 217

<212> PRT

<213> Homo sapiens

<400> 364

<210> 365

<211> 446

<212> PRT

<213> Homo sapiens

<400> 365

<210> 366

<211> 217

<212> PRT

<213> Homo sapiens

<400> 366

<210> 367

<211> 446

<212> PRT

<213> Homo sapiens

<400> 367

<210> 368

<211> 217

<212> PRT

<213> Homo sapiens

<400> 368

<210> 369

<211> 446

<212> PRT

<213> Homo sapiens

<400> 369

<210> 370

<211> 216

<212> PRT

<213> Homo sapiens

<400> 370

<210> 371

<211> 452

<212> PRT

<213> Homo sapiens

<400> 371

<210> 372

<211> 216

<212> PRT

<213> Homo sapiens

<400> 372

<210> 373

<211> 451

<212> PRT

<213> Homo sapiens

<400> 373

<210> 374

<211> 216

<212> PRT

<213> Homo sapiens

<400> 374

<210> 375

<211> 451

<212> PRT

<213> Homo sapiens

<400> 375

<210> 376

<211> 216

<212> PRT

<213> Homo sapiens

<400> 376

<210> 377

<211> 451

<212> PRT

<213> Homo sapiens

<400> 377

<210> 378

<211> 216

<212> PRT

<213> Homo sapiens

<400> 378

<210> 379

<211> 451

<212> PRT

<213> Homo sapiens

<400> 379

<210> 380

<211> 216

<212> PRT

<213> Homo sapiens

<400> 380

<210> 381

<211> 451

<212> PRT

<213> Homo sapiens

<400> 381

<210> 382

<211> 216

<212> PRT

<213> Homo sapiens

<400> 382

<210> 383

<211> 451

<212> PRT

<213> Homo sapiens

<400> 383

<210> 384

<211> 216

<212> PRT

<213> Homo sapiens

<400> 384

<210> 385

<211> 451

<212> PRT

<213> Homo sapiens

<400> 385

<210> 386

<211> 216

<212> PRT

<213> Homo sapiens

<400> 386

<210> 387

<211> 451

<212> PRT

<213> Homo sapiens

<400> 387

<210> 388

<211> 216

<212> PRT

<213> Homo sapiens

<400> 388

<210> 389

<211> 451

<212> PRT

<213> Homo sapiens

<400> 389

<210> 390

<211> 216

<212> PRT

<213> Homo sapiens

<400> 390

<210> 391

<211> 451

<212> PRT

<213> Homo sapiens

<400> 391

<210> 392

<211> 216

<212> PRT

<213> Homo sapiens

<400> 392

<210> 393

<211> 451

<212> PRT

<213> Homo sapiens

<400> 393

<210> 394

<211> 216

<212> PRT

<213> Homo sapiens

<400> 394

<210> 395

<211> 451

<212> PRT

<213> Homo sapiens

<400> 395

<210> 396

<211> 216

<212> PRT

<213> Homo sapiens

<400> 396

<210> 397

<211> 451

<212> PRT

<213> Homo sapiens

<400> 397

<210> 398

<211> 10

<212> PRT

<213> Homo sapiens

<400> 398

<210> 399

<211> 20

<212> PRT

<213> Homo sapiens

<400> 399

<210> 400

<211> 11

<212> PRT

<213> Homo sapiens

<400> 400

<210> 401

<211> 14

<212> PRT

<213> Homo sapiens

<400> 401

<210> 402

<211> 11

<212> PRT

<213> Homo sapiens

<400> 402

<210> 403

<211> 10

<212> PRT

<213> Homo sapiens

<400> 403

<210> 404

<211> 120

<212> PRT

<213> Homo sapiens

<400> 404

<210> 405

<211> 113

<212> PRT

<213> Homo sapiens

<400> 405

<210> 406

<211> 360

<212> DNA

<213> Homo sapiens

<400> 406

<210> 407

<211> 339

<212> DNA

<213> Homo sapiens

<400> 407

<210> 408

<211> 450

<212> PRT

<213> Homo sapiens

<400> 408

<210> 409

<211> 217

<212> PRT

<213> Homo sapiens

<400> 409

<210> 410

<211> 1353

<212> DNA

<213> Homo sapiens

<400> 410

<210> 411

<211> 654

<212> DNA

<213> Homo sapiens

<400> 411

Claims (55)

a binding agent-drug conjugate of the general formula (Ia), Where n is a number from 1 to 50, AK is a binder, and §-GL ¹ -BL ² -§§ is a linker, where § is the position to which the AK is attached, and §§ is labeled with a nitrogen atom. Connection position, D is a group of the following formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen or methyl, and R ² is isopropyl, isobutyl, second butyl, tert-butyl, phenyl, benzyl, 1-hydroxy Ethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1 H -imidazole-4- Methyl or 1 H -indol-3-ylmethyl, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 , 1-diyl group Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Wherein # ⁶ is a position to which a carbonyl group is bonded, R ⁶ is hydrogen, a hydroxyl group or a benzyloxy group, R ³ is hydrogen or a methyl group, and R ⁴ is an isopropyl group, an isobutyl group, a second butyl group, and a third group. Butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, two Phenylmethyl, 1 H -imidazol-4-ylmethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form the formula (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a a 4- to 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, a methyl group or the following formula Group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. Methyl, R ³⁵ is a methyl or hydroxy group, as well as solvates of salts, solvates and salts thereof. A binding agent-drug conjugate of the general formula (Ia) of claim 1 wherein n is a number from 1 to 50 and AK is AK ₁ or AK ₂ wherein AK ₁ is a binding agent which is via a binding agent The sulfur atom is bonded to the G group, and AK ₂ is a binding agent which is linked to the G group via a nitrogen atom of the binding agent. When AK=AK ₁ , G is a group of the following formula Where # ¹ indicates the position of attachment to the sulfur atom, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , G is a carbonyl group, and L ¹ is a bond, a straight chain (C ₁ -C) ₁₀ )-alkylene group, group of the following formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and L ^1A is a linear (C ₂ -C ₁₀ )-alkylene Base, B ¹ is a group of the formula Where ## ⁵ indicates the position of attachment to the L ^1A group, ## ⁶ indicates the position of attachment to the L ^1B group, L ⁵ is a bond or (C ₂ -C ₄ )-alkylene group, and L ⁶ is One-key or one-group Where ## ⁷ indicates the position of attachment to the carbonyl group, ## ⁸ indicates the position of attachment to the L ^1B group, R ³³ is hydrogen, (C ₁ -C ₄ )-alkylcarbonyl, and the third butoxy group Carbonyl or benzyloxycarbonyl, R ³⁴ is hydrogen or methyl, R ²⁹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²⁹ And R ³⁰ and its bonded atom together form a 5- or 6-membered heterocyclic ring, R ³¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³² is hydrogen or (C ₁ -C ₄ )- An alkyl group, or R ³¹ and R ^{32 together} with the atom to which they are bonded form a 5- or 6-membered heterocyclic ring, L ^1B is a linear (C ₂ -C ₁₀ )-alkylene group, and wherein (C ₁ - The C ₁₀ )-alkylene group may be substituted with 1 to 4 substituents each independently selected from the group consisting of a methyl group, a hydroxyl group and a benzyl group, and the intermediate alkylene chain thereof is 1, 2, 1, 3 or 1 The two carbon atoms of the 4 relationship (including any carbon atoms in between may be bridged) to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and B is a bond or a base of the formula group Wherein * indicates the position of attachment to the L ¹ group, ** indicates the position of attachment to the L ² group, P is O or NH, and L ³ is a bond or (C ₂ -C ₄ )-alkylene group, L ⁴ is a bond or a group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position to which L ¹ is attached, R ²⁵ is hydrogen or methyl, and R ²⁸ is hydrogen, (C ₁ -C ₄ )-alkyl a carbonyl group, a tert-butoxycarbonyl group or a benzyloxycarbonyl group, Q ¹ is a 4- to 7-membered heterocyclic ring, Q ² is a 3- to 7-membered carbocyclic ring or a 4- to 7-membered heterocyclic ring, and R ¹⁴ is Hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁵ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁴ and R ^{15 together} with the atoms to which they are bonded form a 5- or 6-member a heterocyclic ring, R ¹⁶ is hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁶ and R ^{17 together} with the atoms to which they are bonded form a a 5- or 6-membered heterocyclic ring, R ¹⁸ is hydrogen or (C ₁ -C ₄ )-alkyl, and R ¹⁹ is hydrogen or a pendant group of a natural a-amino acid or an analog or isomer thereof, R ²⁰ Is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁹ and R ^{20 together} with the atom to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ²² is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²¹ and R ^{22 together} with the atoms to which they are bonded form a 3- to 7-membered carbocyclic ring, and R ²³ is (C ₁ -C ₄ ) - alkyl, R ²⁴ is hydrogen or (C ₁ -C ₄₎ - alkyl, R ²⁷ is hydrogen or (C ₁ -C ₄₎ - alkyl, R ³⁶ is hydrogen (C ₁ -C ₄₎ - alkyl carbonyl, tert-butoxy carbonyl or benzyloxy carbonyl group, R ³⁷ is hydrogen or methyl, or R R ³⁷ bonded thereto of atoms and ³⁶ together form a pyrrolidine ring , L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the following formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be passed through 1 to 4 each independently substituted by a substituent selected from the group consisting of a methyl group, a hydroxyl group, and a benzyl group, and two carbon atoms in which the intermediate alkylene chain is in a 1, 2, 1, 3 or 1, 4 relationship (including Any carbon atom in between may be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen or methyl, and R ² is isopropyl, isobutyl, second butyl, tert-butyl, phenyl, benzyl, 1-hydroxy Ethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1 H -imidazole-4- Methyl or 1 H -indol-3-ylmethyl, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 , 1-diyl group Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Wherein # ⁶ is a position to which a carbonyl group is bonded, R ⁶ is hydrogen, a hydroxyl group or a benzyloxy group, R ³ is hydrogen or a methyl group, and R ⁴ is an isopropyl group, an isobutyl group, a second butyl group, and a third group. Butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, two Phenylmethyl, 1 H -imidazol-4-ylmethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form the formula (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a 4 a 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, a methyl group or a group of the formula group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. Methyl, R ³⁵ is a methyl or hydroxy group, as well as solvates of salts, solvates and salts thereof. A binding agent-drug conjugate of the general formula (Ia) of claim 1 or 2, wherein n is a number from 1 to 20, and AK is AK ₁ or AK ₂ wherein AK ₁ is bound to mesothelin and via The antibody or antigen-binding antibody fragment in which the sulfur atom of the cysteine residue of the binding agent binds to the G group, and the AK ₂ is the NH side group and G which bind to mesothelin and the amino acid residue via the binding agent. a group-bound antibody or antigen-binding antibody fragment, when AK=AK ₁ , G is a group of the formula Wherein # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK=AK ₂ , G is a carbonyl group, and L ¹ is a bond. Linear (C ₂ -C ₆ )-alkylene group, a group of the formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and L ^1A is a linear (C ₂ -C ₆ )-alkylene Base, B ¹ is a group of the formula Where ## ⁵ indicates the position of attachment to the L ^1A group, ## ⁶ indicates the position of attachment to the L ^1B group, L ⁵ is a bond, and L ⁶ is a bond or a group of the following formula Where ## ⁷ indicates the position of attachment to the carbonyl group, ## ⁸ indicates the position of attachment to the L ^1B group, R ³³ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, and R ³⁴ is hydrogen or a group, R ²⁹ is hydrogen, R ³⁰ is hydrogen, R ³¹ is hydrogen or methyl, R ³² is hydrogen or methyl, L ^1B is a linear (C ₂ -C ₆ )-alkylene group, and (C ₂ -C ₆ )-alkylene group may be substituted by 1 or 2 methyl substituents, and B is a bond or a group of the following formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is hydrogen or methyl, and R ²⁸ is hydrogen, methylcarbonyl or tert-butoxycarbonyl. , Q ¹ is a 4- to 7-membered heterocyclic ring, R ¹⁴ is hydrogen, R ¹⁵ is hydrogen, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ¹⁷ are bonded thereto. Atom together form a pipe a base ring, R ¹⁸ is hydrogen, R ¹⁹ is hydrogen, methyl, prop-2-yl, 2-methylprop-1-yl or 1-methylprop-1-yl, and R ²⁰ is hydrogen or methyl. Or R ¹⁹ and R ^{20 together} with the atoms to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or methyl, R ²² is hydrogen or methyl, or R ²¹ and R ²² are bonded together with the atoms to which they are bonded a cyclopropyl ring, R ²³ is methyl, R ²⁴ is hydrogen or methyl, R ²⁷ is hydrogen, R ³⁶ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, R ³⁷ is hydrogen or methyl, or R ³⁶ and R ^{37 together} with the atoms to which they are bonded form a pyrrolidine ring, L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be 1 or Substituted by two methyl substituents, D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-yl Methyl, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Wherein # ⁶ indicates the position of attachment to a carbonyl group, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-benzene. Ethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 of the following formula, 1-diyl group Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a a 4- to 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, a methyl group or the following formula Group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. Methyl, R ³⁵ is a methyl or hydroxy group, as well as solvates of salts, solvates and salts thereof. A binding agent-drug conjugate of the formula (Ia) according to any one of claims 1 to 3, wherein n is a number from 1 to 10, and AK is AK ₁ or AK ₂ wherein AK ₁ is an antibody, It comprises a CDR sequence of six MF-Ta antibodies, a variable light chain of MF-Ta antibody and a light chain and a heavy chain of a variable heavy chain or MF-Ta antibody, and is a cysteine residue via a binding agent. The sulfur atom is bonded to the G group, and AK ₂ is an antibody comprising the CDR sequences of six B01-3, B01-10, M31-B01 or D02-6 antibodies, B01-3, B01-10, M31- The light and heavy chains of the B01 or D02-6 antibodies or the light and heavy chains of the B01-3, B01-10, M31-B01 or D02-6 antibodies, and are lysine residues via the binding agent The NH pendant group of the group is bonded to the G group, and when AK=AK ₁ , G is a group of the following formula Wherein # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK=AK ₂ , G is a carbonyl group, and L ¹ is a bond. Linear (C ₂ -C ₆ )-alkylene group, a group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₂ -C ₆ )-alkylene may be 1 or Substituted by two methyl substituents, B is a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is methyl, R ²⁸ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, Q ¹ is piperidine-1,4-diyl, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ²¹ is hydrogen or methyl, R ²² is hydrogen or methyl, or R ²¹ and R ^{22 together} with the atom to which they are bonded form a cyclopropyl ring, R ²³ is a methyl group, and R ²⁴ is a hydrogen, R ³⁶ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, R ³⁷ is hydrogen or methyl, L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula Where p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-yl Methyl, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Wherein # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H - Ind-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl, R ⁸ is hydrogen or a group, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, methyl Or a group of the following formula Wherein # ⁹ is the position to which the -CHCH ₂ phenyl group is attached, and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, and R ¹³ is a phenyl group. It may be substituted by a methoxycarbonyl group or a carboxyl group, and R ³⁵ is a methyl group or a hydroxyl group, and a solvate of a salt, a solvate thereof and a salt thereof. A binding agent-drug conjugate of the general formula (Ia) according to any one of claims 1 to 4, wherein n is a number from 1 to 10, and AK is AK ₂ wherein AK ₂ is an antibody comprising six CDR sequences of MF-Ta antibodies, variable light chains of MF-Ta antibodies and light and heavy chains of variable heavy or MF-Ta antibodies, and NH side groups of lysine residues via binding agents Bonded to a G group, G is a carbonyl group, L ¹ is a bond, B is a bond, L ² is a linear (C ₃ -C ₆ )-alkylene group or a group of the formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Wherein # ³ is the position to which the nitrogen atom is bonded, R ¹ is hydrogen, R ² is benzyl, 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² are bonded thereto. The carbon atoms of the knot together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Where # ⁶ is the position to which the carbonyl group is attached, R ³ is hydrogen, R ⁴ is benzyl, 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 are} related thereto. The bonded carbon atoms together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines marked with the adjacent nitrogen atom of the position, indicated with the # ⁸ line connecting the position of the groups T ^1, T ¹ is the formula -C (= O) -OR ⁷ or -C (= O) -NR ^{a group of 8} R ⁹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl, R ⁸ is hydrogen, R ⁹ is hydrogen or benzyl, R ³⁵ is a methyl group, as well as a solvate of its salts, solvates and salts. A binding agent-drug conjugate of the general formula (Ia) according to any one of claims 1 to 4, wherein n is a number from 1 to 10, and AK is AK ₁ , wherein AK ₁ is an antibody, which comprises CDR sequences of six MF-Ta antibodies, variable light chains of MF-Ta antibodies and light and heavy chains of variable heavy or MF-Ta antibodies, and sulfur via cysteine residues of binding agents The atom is bonded to the G group, and G is a group of the following formula Where # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, and L ¹ is a bond, linear (C ₃ -C ₅ )-alkylene Base or group Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₃ -C ₅ )-alkylene may be 1 or Substituted by two methyl substituents, B is a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is methyl, and R ²⁸ is hydrogen, methylcarbonyl or tert-butoxycarbonyl, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, L ² is a linear (C ₃ -C ₅ )-alkylene group or a group of the formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Wherein # ³ is the position of the linkage to the carbonyl group, R ¹ is hydrogen, R ² is benzyl, 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, or R ¹ and R ^{2 are} related thereto. The bonded carbon atoms together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Where # ⁶ is the position to which the carbonyl group is attached, R ³ is hydrogen, R ⁴ is benzyl, 4-hydroxybenzyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 are} related thereto. The bonded carbon atoms together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines marked with the adjacent nitrogen atom of the position, indicated with the # ⁸ line connecting the position of the groups T ^1, T ¹ is the formula -C (= O) -OR ⁷ or -C (= O) -NR ^{a group of 8} R ⁹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl, R ⁸ is hydrogen, and R ⁹ is hydrogen or benzyl, R ³⁵ is a methyl group, and a solvate of a salt, a solvate thereof and a salt thereof. a compound of formula (XXXa), Wherein Cys is a cysteine residue bonded to a carbon atom of amber succinimide via a sulfur atom of a pendant group, and L ¹ is a bond, a linear (C ₁ -C ₁₀ )-alkylene group, Group of the following formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and L ^1A is a linear (C ₂ -C ₁₀ )-alkylene Base, B ¹ is a group of the formula Where ## ⁵ indicates the position of attachment to the L ^1A group, ## ⁶ indicates the position of attachment to the L ^1B group, L ⁵ is a bond or (C ₂ -C ₄ )-alkylene group, and L ⁶ is a bond, R ²⁹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²⁹ and R ^{30 together} with the atoms to which they are bonded form a a 5- or 6-membered heterocyclic ring, R ³¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³² is hydrogen or (C ₁ -C ₄ )-alkyl, or R ³¹ and R ³² are bonded thereto The atoms of the knot together form a 5- or 6-membered heterocyclic ring, L ^1B is a linear (C ₂ -C ₁₀ )-alkylene group, and the (C ₁ -C ₁₀ )-alkylene group thereof may be 1 to 4 Each of which is independently substituted with a substituent selected from the group consisting of a methyl group, a hydroxyl group, and a benzyl group, and two carbon atoms in which the intermediate alkylene chain is in a 1, 2, 1, 3, or 1, 4 relationship (including any The carbon atom in between may be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , P is O or NH, L ³ is a bond or (C ₂ -C ₄ )-alkylene, and L ⁴ is a bond, Q ¹ is a 4- to 7-membered heterocyclic ring, Q ² is a 3- to 7-membered carbocyclic ring or a 4- to 7-membered heterocyclic ring, and R ¹⁴ is hydrogen or (C ₁ -C ₄ )-alkyl , R ¹⁵ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁴ and R ^{15 together} with the atoms to which they are bonded form a 5- or 6-membered heterocyclic ring, R ¹⁶ is hydrogen or (C ₁ - C ₄ )-alkyl, R ¹⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a 5- or 6-membered heterocyclic ring, R ¹⁸ is Hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁹ is hydrogen or a side group of a natural alpha-amino acid or analogue or isomer thereof, R ²⁰ is hydrogen or (C ₁ -C ₄ )-alkane a group, or R ¹⁹ and R ^{20 together} with the atoms to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ²² is hydrogen or (C ₁ -C ₄ ) -alkyl, or R ²¹ and R ^{22 together} with the atoms to which they are bonded form a 3- to 7-membered carbocyclic ring, R ²³ is (C ₁ -C ₄ )-alkyl, R ²⁴ is hydrogen or (C ₁ -C ₄ )-alkyl, R ²⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the carbonyl group, wherein (C ₂ -C ₁₀ )-alkylene can pass through Up to 4 substituents each independently selected from the group consisting of methyl, hydroxy and benzyl, and two carbon atoms in which the intermediate alkylene chain is 1, 2, 1, 3 or 1, 4 in relation to each other (at the same time) Including any carbon atom in between may be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen or methyl, and R ² is isopropyl, isobutyl, second butyl, tert-butyl, phenyl, benzyl, 1-hydroxy Ethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1 H -imidazole-4- Methyl or 1 H -indol-3-ylmethyl, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 , 1-diyl group Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Wherein # ⁶ is a position to which a carbonyl group is bonded, R ⁶ is hydrogen, a hydroxyl group or a benzyloxy group, R ³ is hydrogen or a methyl group, and R ⁴ is an isopropyl group, an isobutyl group, a second butyl group, and a third group. Butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, two Phenylmethyl, 1 H -imidazol-4-ylmethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form the formula (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a a 4- to 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, methyl or the following formula Group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. Methyl, R ³⁵ is a methyl or hydroxy group, as well as solvates of salts, solvates and salts thereof. A compound of the formula (XXXa) of claim 7 wherein Cys is a cysteine residue bonded via a sulfur atom of a pendant group via a carbon atom of amber quinone imine, and L ¹ is a bond , a linear (C ₂ -C ₆ )-alkylene group, a group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and L ^1A is a linear (C ₂ -C ₆ )-alkylene group. B ¹ is a group of the following formula Where ## ⁵ indicates the position of attachment to the L ^1A group, ## ⁶ indicates the position of attachment to the L ^1B group, L ⁵ is a bond, L ⁶ is a bond, R ²⁹ is hydrogen, and R ³⁰ is hydrogen. , R ³¹ is hydrogen or methyl, R ³² is hydrogen or methyl, L ^1B is a linear (C ₂ -C ₆ )-alkylene group, and (C ₂ -C ₆ )-alkylene group thereof may be subjected to 1 Or substituted with 2 methyl substituents, B being a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond or B-1,2-diyl, L ⁴ is a bond, R ¹⁴ is hydrogen, R ¹⁵ Is hydrogen, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ²³ is a methyl group, R ²⁴ is hydrogen or a methyl group, and L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-yl Methyl, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Wherein # ⁶ indicates the position of attachment to a carbonyl group, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-benzene. Ethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 of the following formula, 1-diyl group Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the atoms to which they are bonded form a 4 a 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, a methyl group or a group of the formula group Wherein # ⁹ is the position to which the -CHCH ₂ phenyl group is attached, and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, and R ¹³ is a phenyl group. It may be substituted by a methoxycarbonyl group or a carboxyl group, and R ³⁵ is a methyl group or a hydroxyl group, and a solvate of a salt, a solvate thereof and a salt thereof. A compound of the formula (XXXa) according to claim 7 or 8, wherein Cys is a cysteine residue bonded via a sulfur atom of a pendant group via a carbon atom of amber quinone imine, and L ¹ is a a bond or a linear (C ₂ -C ₆ )-alkylene group, and B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , L ³ is a bond, L ⁴ is a bond, R ¹⁶ is hydrogen or methyl, and R ¹⁷ is hydrogen or methyl. L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Wherein # ³ is the position to which the nitrogen atom is bonded, R ¹ is hydrogen, R ² is benzyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² are bonded together with the carbon atom to which they are bonded (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Wherein # ⁶ is the position of the linkage to the carbonyl group, R ³ is hydrogen, R ⁴ is benzyl or 1 H -indol-3-ylmethyl, or R ³ and R ⁴ are bonded to the carbon atom to which they are bonded Formation of the (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines marked with the adjacent nitrogen atom of the position, indicated with the # ⁸ line connecting the position of the groups T ^1, T ¹ is the formula -C (= O) -OR ⁷ or -C (= O) -NR A group of ⁸ R ⁹ wherein R ⁷ is hydrogen, R ⁸ is hydrogen, R ⁹ is hydrogen, R ³⁵ is methyl, and solvates of the salts, solvates and salts thereof. a compound of the formula (XXXI), Wherein L ¹ is a bond, a linear (C ₁ -C ₁₀ )-alkylene group, a group of the formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, ## ² indicates the position of attachment to the B group, and L ^1A is a linear (C ₂ -C ₁₀ )-alkylene Base, B ¹ is a group of the formula Where ## ⁵ indicates the position of attachment to the L ^1A group, ## ⁶ indicates the position of attachment to the L ^1B group, L ⁵ is a bond or (C ₂ -C ₄ )-alkylene group, and L ⁶ is a bond, R ²⁹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²⁹ and R ^{30 together} with the atoms to which they are bonded form a a 5- or 6-membered heterocyclic ring, R ³¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ³² is hydrogen or (C ₁ -C ₄ )-alkyl, or R ³¹ and R ³² are bonded thereto The atoms of the knot together form a 5- or 6-membered heterocyclic ring, L ^1B is a linear (C ₂ -C ₁₀ )-alkylene group, and the (C ₁ -C ₁₀ )-alkylene group thereof may be 1 to 4 Each of which is independently substituted with a substituent selected from the group consisting of a methyl group, a hydroxyl group, and a benzyl group, and two carbon atoms in which the intermediate alkylene chain is in a 1, 2, 1, 3, or 1, 4 relationship (including any The carbon atom in between may be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and B is a bond or a group of the formula Where * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , P is O or NH, Q ¹ is 4- to 7-membered heterocyclic ring, and Q ² is 3- to 7-membered carbon. a ring or a 4- to 7-membered heterocyclic ring, R ¹⁸ is hydrogen or (C ₁ -C ₄ )-alkyl, and R ¹⁹ is hydrogen or a pendant group of a natural a-amino acid or an analog or isomer thereof, R ²⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁹ and R ^{20 together} with the atom to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or (C ₁ -C ₄ )-alkane a group, R ²² is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²¹ and R ^{22 together} with the atoms to which they are bonded form a 3- to 7-membered carbocyclic ring, R ²⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be passed through 1 to 4 each independently substituted by a substituent selected from the group consisting of a methyl group, a hydroxyl group, and a benzyl group, and two carbon atoms in which the intermediate alkylene chain is in a 1, 2, 1, 3 or 1, 4 relationship (including Any carbon atom in between may be bridged to form a (C ₃ -C ₆ )-cycloalkyl ring or a phenyl ring, and D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen or methyl, and R ² is isopropyl, isobutyl, second butyl, tert-butyl, phenyl, benzyl, 1-hydroxy Ethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1 H -imidazole-4- Methyl or 1 H -indol-3-ylmethyl, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 , 1-diyl group Where # ⁴ is the position of the connection with the adjacent nitrogen atom, # ⁵ is the position of the connection with the carbonyl group, and the A ring of the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Wherein # ⁶ is a position to which a carbonyl group is bonded, R ⁶ is hydrogen, a hydroxyl group or a benzyloxy group, R ³ is hydrogen or a methyl group, and R ⁴ is an isopropyl group, an isobutyl group, a second butyl group, and a third group. Butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, two Phenylmethyl, 1 H -imidazol-4-ylmethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form the formula (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a a 4- to 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, methyl or the following formula Group # ⁹ is the position of the linkage to -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, and R ¹³ is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group Further, R ³⁵ is a methyl group or a hydroxyl group, and a solvate of a salt, a solvate thereof and a salt thereof. A compound of the formula (XXXI) according to claim 10, wherein L ¹ is a bond, a linear (C ₂ -C ₆ )-alkylene group or a group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₂ -C ₆ )-alkylene may be 1 or Substituted by two methyl substituents, B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , R ¹⁸ is hydrogen, and R ¹⁹ is methyl, prop-2-yl, 2-methylprop-1-yl or -Methylpropan-1-yl, R ²⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁹ and R ^{20 together} with the atoms to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or Methyl, R ²² is hydrogen or methyl, or R ²¹ and R ^{22 together} with the atom to which they are bonded form a cyclopropyl ring, R ²⁷ is hydrogen or methyl, and L ² is linear (C ₂ -C ₆ ) - an alkylene group or a group of the formula Wherein p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may pass 1 or 2 Substituted by a methyl substituent, and two carbon atoms in which the mesoalkylene chain is in a 1,4 relationship with each other (including any carbon atoms in between may be bridged) to form a phenyl ring, and D is a group of the following formula group Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1 H -indol-3-yl Methyl, or R ¹ and R ^{2 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to an adjacent nitrogen atom, and # ⁵ indicates the position of attachment to a carbonyl group, wherein the A ring of the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Wherein # ⁶ indicates the position of attachment to a carbonyl group, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-benzene. Ethyl or 1 H -indol-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form (1 S , 2 R )-2-phenylcyclopropane-1 of the following formula, 1-diyl group Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a a 4- to 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, methyl or the following formula Group Wherein # ⁹ is the position to which the -CHCH ₂ phenyl group is attached, and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, and R ¹³ is a phenyl group. It may be substituted by a methoxycarbonyl group or a carboxyl group, and R ³⁵ is a methyl group or a hydroxyl group, and a solvate of a salt, a solvate thereof and a salt thereof. A compound of the formula (XXXI) according to claim 10 or 11, wherein L ¹ is a bond, B is a bond, and L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula group Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and D is the group of the following formula Wherein # ³ is the position to which the nitrogen atom is bonded, R ¹ is hydrogen, R ² is benzyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² are bonded together with the carbon atom to which they are bonded (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Wherein # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, R ⁴ is benzyl or 1 H -indol-3-ylmethyl, or R ³ And R ⁴ and its bonded carbon atom together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula # ⁷ wherein the connecting lines marked with the adjacent nitrogen atom of the position, indicated with the # ⁸ line connecting the position of the groups T ^1, T ¹ is the formula -C (= O) -OR ⁷ or -C (= O) -NR A group of ⁸ R ⁹ wherein R ⁷ is hydrogen, R ⁸ is hydrogen, R ⁹ is hydrogen, R ³⁵ is methyl, and solvates of the salts, solvates and salts thereof. A compound of the formula (XXXa) and (XXXI) is selected from the group consisting of N- [6-(3-{[(2 R )-2-amino-2-carboxyethyl]indolyl}-2 , 5-dioxapyrrolidin-1-yl)hexyl] -N -methyl-L-nonylamine fluorenyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )- 2-[(1 R ,2 R )-3-{[(1 S )-1-carboxy-2-(1 H -indol-3-yl)ethyl]amino}-1-methoxy- 2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-indole Amine amide, N- [6-(3-{[(2 R )-2-amino-2-carboxyethyl]indolyl}-2,5-dioxapyrrolidin-1-yl)hexyl]- N -methyl-L-Amidinoinyl- N -[(3 R ,4 S ,5 S )-1-{(2 S )-2-[(1 R ,2 R )-3-{[( 2 S )-3-(1 H -indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl] -N -methyl-L-nonylamine decylamine, N-(6-{[(5S)-5-amino-5-carboxyl Amyl]amino}-6-oxo-oxyhexyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R, 2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2- Alkan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-A Oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine guanamine trifluoroacetate, N-(6-{[(5S)-5-amino) -5-carboxypentyl]amino}-6-oxo-oxyhexyl)-N-methyl-L-nonylamine fluorenyl-N-[(3R,4S,5S)-1-{(2S)-2- [(1R,2R)-3-{[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3 -Phenoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-nonylamine decylamine, and Solvates of salts, solvates and salts. A binding agent of formula (I) - drug conjugate Where n is a number from 1 to 50, AK is a binder, and §-GL ¹ -BL ² -§§ is a linker, where § indicates the position to which the AK group is attached, and §§ indicates a nitrogen atom The connection position, D is the group of the following formula Where# is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, or R ¹ and R ² and its bonded carbon atoms together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Where # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H - Ind-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a a 4- to 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, methyl or the following formula Group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H -indol-3-yl group. A methyl group, as well as a solvate of its salts, solvates, and salts. A binding agent-drug conjugate of the general formula (I) according to claim 14 wherein n is a number from 1 to 50, and AK is AK ₁ or AK ₂ wherein AK ₁ is a binding agent which is via a binding agent The sulfur atom is bonded to the G group, and AK ₂ is a binding agent which is linked to the G group via a nitrogen atom of the binding agent. When AK=AK ₁ , G is a group of the following formula Where # ¹ indicates the position of attachment to the sulfur atom of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK = AK ₂ , G is a carbonyl group, and L ¹ is a bond, a straight chain (C) ₁ -C ₁₀ )-alkylene or a group of the formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₁ -C ₁₀ )-alkylene can pass through 1 Substituted to 4 methyl substituents, and two carbon atoms in which the intermediate alkylene chains are 1, 2, 1, 3 or 1, 4 in relation to each other (including any carbon atoms in between may be bridged) to form a carbon atom (C ₃ -C ₆ )-cycloalkyl ring or phenyl ring, B is a bond or a group of the formula Wherein * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , P is O or NH, L ³ is a bond or (C ₂ -C ₄ )-alkylene, and L ⁴ is Key or group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is hydrogen or methyl, Q ¹ is a 4- to 7-membered heterocyclic ring, and Q ² is a 3- to 7-membered carbocyclic ring or a 4- to 7-membered heterocyclic ring, R ¹⁴ is hydrogen or (C ₁ -C ₄ )-alkyl, and R ¹⁵ is hydrogen or (C ₁ -C ₄ )-alkyl, Or R ¹⁴ and R ^{15 together} with the atoms to which they are bonded form a 5- or 6-membered heterocyclic ring, R ¹⁶ is hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁶ and R ^{17 together} with the atoms to which they are bonded form a 5- or 6-membered heterocyclic ring, R ¹⁸ is hydrogen or (C ₁ -C ₄ )-alkyl, R ¹⁹ is hydrogen Or a side group of a natural a-amino acid or an analog or isomer thereof, R ²⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁹ and R ²⁰ together form a bond with the atom to which they are bonded Pyrrolidinyl ring, R ²¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ²² is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²¹ and R ²² are bonded to the atom to which they are bonded Forming a 3- to 7-membered carbocyclic ring, R ²³ is (C ₁ -C ₄ )-alkyl, R ²⁴ is hydrogen or (C ₁ -C ₄ )-alkyl, R ²⁷ is hydrogen or (C ₁ - C ₄ )-alkyl, L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be passed through 1 to Four methyl substituents substituted, and two carbon atoms in which the intermediate alkylene chain is 1, 2, 1, 3 or 1, 4 in relation to each other (including any carbon atoms in between may be bridged) form one ( C ₃ -C ₆ )-cycloalkyl ring or phenyl ring, D has the definitions as shown in item 14 of the patent application, as well as solvates of the salts, solvates and salts thereof. A binding agent-drug conjugate of the general formula (I) according to claim 14 or 15, wherein n is a number from 1 to 50, and AK is AK ₁ or AK ₂ wherein AK ₁ is an antibody or antigen-binding antibody fragment And bonding to a G group via a sulfur atom, AK ₂ is an antibody or antigen-binding antibody fragment and is bonded to a G group via a nitrogen atom, and G, L ¹ , B, L ² and D have as claimed The definitions shown in paragraphs 14 or 15 of the patent, as well as the solvates of their salts, solvates and salts. A binding agent-drug conjugate of the general formula (I) according to any one of claims 14 to 16, wherein n is a number from 1 to 20, and AK is AK ₁ or AK ₂ wherein AK ₁ is a mesothelium And an antibody or antigen-binding antibody fragment that is bonded to the G group via a sulfur atom of a cysteic acid residue of the binding agent, and AK ₂ is an NH salt that binds to mesothelin and the amino acid residue via the binding agent. An antibody or antigen-binding antibody fragment having a side group bonded to a G group, and when AK=AK ₁ , G is a group of the following formula Wherein # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK=AK ₂ , G is a carbonyl group, and L ¹ is a bond. a linear (C ₂ -C ₆ )-alkylene group or a group of the formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₂ -C ₆ )-alkylene may pass through Or substituted with 2 methyl substituents, B being a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is methyl, and Q ¹ is 4- to 6-membered carbocyclic or piperidine-1. 4-diyl, R ¹⁴ is hydrogen, R ¹⁵ is hydrogen, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ¹⁸ is hydrogen, R ¹⁹ is hydrogen, methyl, prop-2-yl, 2-methylprop-1-yl or 1-methylprop-1-yl, and R ²⁰ is hydrogen or methyl. Or R ¹⁹ and R ^{20 together} with the atoms to which they are bonded form a pyrrolidinyl ring, R ²¹ is hydrogen or methyl, R ²² is hydrogen or methyl, or R ²¹ and R ²² are bonded together with the atoms to which they are bonded a cyclopropyl ring, R ²³ is a methyl group, R ²⁴ is hydrogen or methyl, and L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₆ )-alkylene may be 1 or Substituted by two methyl substituents, D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, or R ¹ And R ² and its bonded carbon atom together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Where # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H - Ind-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a a 4- to 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, methyl or the following formula Group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1 H -indol-3-yl group or a 1 H group - indol-3-ylmethyl, and solvates of the salts, solvates and salts thereof. A binding agent-drug conjugate of the general formula (I) according to any one of claims 14 to 17, wherein n is a number from 1 to 10, and AK is AK ₁ or AK ₂ wherein AK ₁ is an antibody, It comprises a CDR sequence of six MF-Ta antibodies, a variable light chain of MF-Ta antibody and a light chain and a heavy chain of a variable heavy chain or MF-Ta antibody, and is a cysteine residue via a binding agent. The sulfur atom is bonded to the G group, and AK ₂ is an antibody comprising the CDR sequence of the six MF-Ta antibodies, the variable light chain of the MF-Ta antibody, and the light chain of the variable heavy chain or MF-Ta antibody. And a heavy chain, and bonded to the G group via the NH pendant group of the amine acid residue of the binding agent, when AK=AK ₁ , G is a group of the following formula Wherein # ¹ indicates the position of attachment to the cysteine residue of the binding agent, # ² indicates the position of attachment to the L ¹ group, or when AK=AK ₂ , G is a carbonyl group, and L ¹ is a bond. Linear (C ₂ -C ₆ )-alkylene or a group of the formula Wherein m is the number 2 or 3, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₂ -C ₆ )-alkylene may be 1 or Substituted by two methyl substituents, B is a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond or B-1,2-diyl, and L ⁴ is a bond or a group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is methyl, Q ¹ is piperidine-1,4-diyl, and R ¹⁶ is hydrogen. Or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ²¹ is hydrogen or methyl, R ²² is hydrogen or methyl, and R ²¹ and R ^{22 together} with the atoms to which they are bonded form a cyclopropyl ring, R ²³ is a methyl group, and R ²⁴ is a hydrogen, L ² is a linear (C ₂ -C ₆ )-alkylene group, and D is a group of the following formula Where # ³ is the position to which the nitrogen atom is attached, and R ² is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, or R ¹ and R ^{2 are} related thereto. The bonded carbon atoms together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Where # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is benzyl, 1-phenylethyl or 1 H -indol-3-yl Methyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, benzyl or adamantylmethyl, R ⁸ is hydrogen or methyl, and R ⁹ is Hydrogen, methyl, ethyl, n-propyl or benzyl, R ¹¹ is benzyl, which may be substituted on the phenyl group via a methoxycarbonyl or carboxyl group, and R ⁵ is hydrogen or a group of the formula Wherein # ⁹ is a position to which a -CHC(R ²⁶ )phenyl group is bonded, and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, and R ¹³ is A phenyl group which may be substituted with a methoxycarbonyl group or a carboxyl group, and a solvate of a salt, a solvate thereof and a salt thereof. A process for the preparation of a compound of the formula (I) according to the invention in any one of claims 14 to 18, characterized in that a buffer solution [A] containing a binder is combined with a suitable reducing agent, such as disulfide Threitol or hydrazine (2-carboxyethyl)phosphine hydrochloride is mixed and subsequently reacted with a compound of formula (II) Wherein D, L ¹ , B and L ² each have the definitions as shown in the first to fifth aspects of the patent application, to obtain a compound of the formula (IA), Wherein n, AK ₁ , D, L ¹ , B and L ² each have a definition as shown in items 1 to 5 of the patent application, or [B] reacts with a compound of the formula (III), Wherein D, L ¹ , B and L ² each have a definition as shown in the first to fifth aspects of the patent application, to obtain a compound of the formula (IB), Wherein n, AK ₂ , D, L ¹ , B and L ² each have the definitions as shown in items 1 to 5 of the patent application scope. A compound prepared by the method of claim 19, wherein AK ₁ and AK ₂ are CDR sequences comprising six MF-Ta antibodies, variable light chains of MF-Ta antibodies, and variable heavy chains or MF An antibody to the light and heavy chains of the -Ta antibody, as well as solvates of the salts, solvates and salts thereof. a compound of formula (XXX), Wherein Cys is a cysteine residue bonded to a carbon atom of amber quinone via a sulfur atom of a pendant group, and L ¹ is a bond, a linear (C ₁ -C ₁₀ )-alkylene group or Group Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₁ -C ₁₀ )-alkylene can pass through Substituted to 4 methyl substituents, and two carbon atoms in which the intermediate alkylene chains are 1, 2, 1, 3 or 1, 4 in relation to each other (including any carbon atoms in between may be bridged) to form a carbon atom (C ₃ -C ₆ )-cycloalkyl ring or phenyl ring, B is a bond or a group of the formula Where * indicates the position of attachment to L ¹ , ** indicates the position of attachment to L ² , P is O or NH, L ³ is a bond or (C ₂ -C ₄ )-alkylene, and L ⁴ is or Group of the following formula Wherein *** indicates the position of attachment to the carbonyl group, **** indicates the position of attachment to L ² , R ²⁵ is hydrogen or methyl, Q ¹ is 3- to 7-membered carbon ring or 4- to a 7-membered nitrogen heterocycle, Q ² is a 3- to 7-membered carbocyclic ring or a 4- to 7-membered nitrogen heterocyclic ring, R ¹⁴ is hydrogen or (C ₁ -C ₄ )-alkyl, and R ¹⁵ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁴ and R ^{15 together} with the atoms to which they are bonded form a 5- or 6-membered heterocyclic ring, R ¹⁶ is hydrogen or (C ₁ -C ₄ )-alkyl , R ¹⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a 5- or 6-membered heterocyclic ring, R ¹⁸ is hydrogen or (C ₁ - C ₄ )-alkyl, R ¹⁹ is hydrogen or a side group of a natural α-amino acid or an analogue or isomer thereof, R ²⁰ is hydrogen or (C ₁ -C ₄ )-alkyl, or R ¹⁹ and R ²⁰ and its bonded atoms together form a pyrrolidinyl ring, R ²¹ is hydrogen or (C ₁ -C ₄ )-alkyl, R ²² is hydrogen or (C ₁ -C ₄ )-alkyl, or R ²¹ and R ^{22 together} with the atoms to which they are bonded form a 3- to 7-membered carbocyclic ring, R ²³ is (C ₁ -C ₄ )-alkyl, and R ²⁴ is hydrogen or (C ₁ -C ₄ )-alkane a group, R ²⁷ is hydrogen or (C ₁ -C ₄ )-alkyl, L ² is a linear (C ₂ -C ₁₀ )-alkylene group or a group of the formula Wherein p is a number from 2 to 6, ## ³ indicates the position of attachment to the B group, and ## ⁴ indicates the position of attachment to the nitrogen atom, wherein (C ₂ -C ₁₀ )-alkylene may be passed through 1 to Four methyl substituents substituted, and two carbon atoms in which the intermediate alkylene chain is 1, 2, 1, 3 or 1, 4 in relation to each other, including any carbon atoms in between, can be bridged to form one ( C ₃ -C ₆ )-cycloalkyl ring or phenyl ring, D is a group of the formula Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, or R ¹ And R ² and its bonded carbon atom together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Where # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indole Ind-3-ylmethyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl Or adamantylmethyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, or R ⁸ and R ^{9 together} with the nitrogen atom to which they are bonded form a a 4- to 7-membered heterocyclic ring, R ¹⁰ is a benzhydryl group, and R ¹¹ is a benzyl group which may be substituted with a methoxycarbonyl group or a carboxyl group on the phenyl group, and R ⁵ is hydrogen, methyl or the following formula Group Wherein # ⁹ is linked to the position of -CHC(R ²⁶ )-T ² , and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, R ¹³ Is a phenyl group which may be substituted by a methoxycarbonyl group or a carboxyl group, R ²⁶ is hydrogen or a hydroxyl group, and T ² is a phenyl group, a benzyl group, a 1H-indol-3-yl group or a 1H-indol-3-ylmethyl group. And solvates of salts, solvates and salts thereof. A compound of the formula (XXX) according to claim 21, wherein Cys is a cysteine residue bonded to a carbon atom of amber quinone via a sulfur atom of a pendant group, and L ¹ is a bond, a linear chain (C ₂ -C ₆ )-alkylene or a group of the formula Wherein m is a number from 2 to 6, ## ¹ indicates the position of attachment to the G group, and ## ² indicates the position of attachment to the B group, wherein (C ₂ -C ₆ )-alkylene may pass through Or substituted with 2 methyl substituents, B being a bond or a group of the formula Where * is the connection position with L ¹ , ** is the connection position with L ² , L ³ is a bond or B-1,2-diyl, L ⁴ is a bond, R ¹⁴ is hydrogen, R ¹⁵ Is hydrogen, R ¹⁶ is hydrogen or methyl, R ¹⁷ is hydrogen or methyl, or R ¹⁶ and R ^{17 together} with the atom to which they are bonded form a pipe a base ring, R ²³ is a methyl group, R ²⁴ is hydrogen or a methyl group, and L ² is a linear (C ₂ -C ₆ )-alkylene group or a group of the following formula Where p is the number 2 or 3, ## ³ indicates the position of attachment to the B group, ## ⁴ indicates the position of attachment to the nitrogen atom, and the group of D below Where # ³ is the position to which the nitrogen atom is attached, R ¹ is hydrogen, and R ² is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1 H -indol-3-ylmethyl, or R ¹ And R ² and its bonded carbon atom together form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁴ indicates the position of attachment to the adjacent nitrogen atom, # ⁵ indicates the position of attachment to the carbonyl group, and the A ring in which the NO group is present, which is a mono- or bicyclic ring of the following formula, as needed Substituted heterocycle Where # ⁶ is the position to which the carbonyl group is attached, R ⁶ is hydrogen, hydroxy or benzyloxy, R ³ is hydrogen, and R ⁴ is benzyl, 1-phenylethyl or 1 H -indol-3-yl Methyl, or R ³ and R ^{4 together} with the carbon atom to which they are bonded form a (1 S , 2 R )-2-phenylcyclopropane-1,1-diyl group of the formula Where # ⁷ is the position of the connection to the adjacent nitrogen atom, # ⁸ is the position of the connection with the T ¹ group, and T ¹ is the formula -C(=O)-OR ⁷ , -C(=O)-NR ^{a group of 8} R ⁹ , -C(=O)-NH-NH-R ¹⁰ or -CH ₂ -OR ¹¹ wherein R ⁷ is hydrogen, methyl, ethyl, n-propyl, benzyl or adamantyl Methyl, R ⁸ is hydrogen or methyl, R ⁹ is hydrogen, methyl, ethyl, n-propyl or benzyl, R ¹⁰ is benzinyl, R ¹¹ is benzyl, which may be on the phenyl group Substituted by methoxycarbonyl or carboxy, R ⁵ is hydrogen or a group of the formula Wherein # ⁹ is a position to which a -CHC(R ²⁶ )phenyl group is bonded, and R ¹² is a phenyl group which may be substituted with a methoxycarbonyl group, a carboxyl group or a group of the formula -S(O) ₂ OH, and R ¹³ is A phenyl group which may be substituted with a methoxycarbonyl group or a carboxyl group, and a solvate of a salt, a solvate thereof and a salt thereof. The binding agent-drug conjugate of any one of claims 1 to 6 or 14 to 18, wherein the conjugate is bound to a cancer target molecule. The binder-drug conjugate of any one of claims 1 to 6, 14 to 18 or 23, wherein the binder is bound to an extracellular target molecule. A binding agent-drug conjugate according to any one of claims 1 to 6, 14 to 18 or 23 to 24, wherein the conjugate is associated with an extracellular cancer target molecule. The binding agent-drug conjugate according to any one of claims 1 to 6, 14 to 18 or 23 to 25, wherein the target molecule, extracellular target molecule, cancer target molecule or The extracellular cancer target molecule is a protein. A binding agent-drug conjugate according to any one of claims 1 to 6, 14 to 18 or 23 to 26, wherein the conjugate is expressed after binding to an extracellular target molecule The cells of the molecule are internalized. The binder-drug conjugate of any one of claims 1 to 6, 14 to 18, or 23 to 27, wherein the binder is a binding protein. The binding agent-drug conjugate according to any one of claims 1, 2, 14, 15, 20 or 23 to 28, wherein the conjugate is an antibody or an antigen-binding antibody fragment or antibody mimetic thereof . A binding agent-drug conjugate according to any one of claims 1 to 6, 14 to 18 or 23 to 29, 10, wherein the antibody is a monoclonal antibody. A binding agent-drug conjugate according to any one of claims 1 to 6, 14 to 18 or 23 to 30, wherein the antibody is a human, humanized or chimeric antibody. The binding agent-drug conjugate of any one of claims 1 to 6, 14 to 18 or 23 to 31, wherein the antibody is a whole antibody or a modified whole antibody. The binding agent-drug conjugate of any one of claims 1 to 6, 14 to 18 or 23 to 32, wherein the antibody is an antibody of the IgG class. A binding agent-drug conjugate according to any one of claims 1 to 6, 14 to 18 or 23 to 33, wherein the conjugate is an extracellular cancer selected from the group consisting of Target molecule binding: EGF receptor (NP_005219.2), mesothelin (Q13421-3), C4.4a (NP_055215.2) and carbonic anhydrase IX (CA IX; NP_001207.2). A binder-drug conjugate according to any one of claims 1 to 6, 14 to 18 or 23 to 34, wherein the binder is specific to a cell selected from the group consisting of The external cancer target molecule binds: EGF receptor (NP_005219.2), mesothelin (Q13421-3), C4.4a (NP_055215.2), and carbonic anhydrase IX (CA IX; NP_001207.2). A binding agent-drug conjugate according to any one of claims 1 to 6, 14 to 18 or 23 to 35, wherein the anti-system binds to C4.4a. The binding agent-drug conjugate of any one of claims 1 to 6, 14 to 18 or 23 to 36, wherein the anti-system binds to mesothelin and has a constant binding. A binding agent-drug conjugate according to any one of claims 1 to 6, 14 to 18 or 23 to 37, wherein the antibody specificity binds to mesothelin. A binding agent-drug conjugate according to any one of claims 1 to 6, 14 to 18 or 23 to 38, wherein the conjugate is an anti-mesothelin antibody or an antigen-binding antibody fragment, The lines include the antibodies described in WO 2009/068204-A1 (Table 7; pages 61-63) The amino acid sequence of the light chain CDR regions and the amino acid sequence of the three heavy chain CDR regions. The binding agent-drug conjugate of any one of claims 1 to 3, 14 to 17 or 23 to 39, wherein the anti-mesothelin antibody or antigen-binding antibody fragment is composed of the following Selected from the group: an amino acid sequence comprising three light chain CDR regions of the MF-Ta antibody, and an anti-mesothelin antibody or antigen-binding antibody fragment thereof, including the MF- of the amino acid sequence of the three heavy chain CDR regions Three light chain CDR region sequences of the J antibody, and three heavy chain CDR region sequences of the anti-mesothelin antibody or antigen-binding antibody fragment thereof, including three light chain CDR region sequences of the MOR06640 antibody, and three heavy chain CDR sequences a region sequence anti-mesothelin antibody or antigen-binding antibody fragment thereof, comprising three light chain CDR region sequences of MF-226 antibody, and three heavy chain CDR region sequences of anti-mesothelin antibodies or antigen-binding antibody fragments thereof, And an anti-mesothelin antibody or antigen-binding antibody fragment thereof comprising three light chain CDR region sequences of the MOR06626 antibody and three heavy chain CDR region sequences. The binding agent-drug conjugate according to any one of claims 1 to 3, 14 to 17 or 23 to 40, wherein the anti-mesothelin antibody or antigen-binding antibody fragment is composed of the following Selected from the group: An anti-mesothelin antibody or antigen-binding antibody fragment thereof comprising a variable light chain sequence of a MF-Ta antibody and a variable heavy chain sequence, including a variable light chain sequence of the MF-J antibody and an anti-interval of the variable heavy chain sequence a dermal antibody or antigen-binding antibody fragment thereof, comprising a variable light chain sequence of a MOR06640 antibody and a variable heavy chain anti-mesothelin antibody or antigen-binding antibody fragment thereof, including a variable light chain sequence of the MF-226 antibody An anti-mesothelin antibody or antigen-binding antibody fragment thereof of variable heavy chain sequence, and an anti-mesothelin antibody or antigen-binding antibody fragment thereof comprising the variable light chain sequence of the MOR06626 antibody and the variable heavy chain sequence. An antibody or antigen-binding antibody fragment that binds to mesothelin, wherein the antibody sequence comprises an amino acid sequence of a variable heavy chain represented by the sequence of SEQ ID NO: 404, and is represented by the sequence of SEQ ID NO: 405 The amino acid sequence of the light chain. An antibody or antigen-binding antibody fragment that binds to mesothelin, wherein the antibody sequence comprises an amino acid sequence of a variable heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 406, and the nucleic acid of SEQ ID NO: 407 The amino acid sequence of the variable light chain encoded by the sequence. An antibody that binds to mesothelin, wherein the antibody sequence comprises an amino acid sequence of the heavy chain represented by the sequence of SEQ ID NO: 408, and an amino acid sequence of the light chain represented by the sequence of SEQ ID NO: 409. The antibody or antigen-binding antibody fragment of claim 42-44, which is specifically associated with mesothelin. The binding agent-drug conjugate of any one of claims 1 to 3, 14 to 17 or 23 to 38, wherein the anti-mesothelin anti-system comprises the sequence of SEQ ID NO: 408 The amino acid sequence of the heavy chain, and the amino acid sequence of the light chain represented by the sequence of SEQ ID NO: 409. A binder-drug conjugate or compound according to any one of claims 1 to 18, 21 to 41 or 46, for use in the treatment and/or prevention of a disease. A binding agent-drug conjugate or compound according to any one of claims 1 to 18, 21 to 41 or 46, for use in the treatment and/or prevention of hyperproliferative and/or angiogenic diseases. In the method. A binding agent-drug conjugate or compound according to any one of claims 1 to 18, 21 to 41 or 46, for use in the manufacture of a medicament for the treatment and/or prevention of hyperproliferation and/or An angiogenic disease. A pharmaceutical product comprising a binder-drug bond as claimed in any one of claims 1 to 18, 21 to 41 or 46 to 48 The compound or compound is combined with an inert, non-toxic pharmaceutically acceptable excipient. A pharmaceutical product comprising a binding agent-drug conjugate or compound and one or more anti-proliferative, cytostatic agents according to any one of claims 1 to 18, 21 to 41 or 46 to 48 Or a combination of cytotoxic substances. The pharmaceutical product of claim 50 or 51 is for the treatment and/or prevention of hyperproliferation and/or angiogenesis. A method for treating and/or preventing hyperproliferative and/or angiogenic diseases in humans and animals, using at least one of an effective amount as in any one of claims 1 to 18 or 21 to 41 Agent-drug conjugate or compound, or a pharmaceutical product according to claim 50 or 51. A process for the preparation of a compound of the invention of the formula (Ia) according to any one of claims 1 to 6 of the invention, characterized in that the solution [A] of the binding agent in a buffer is combined with a suitable reducing agent, for example two Mixing thiothreitol or hydrazine (2-carboxyethyl)phosphine hydrochloride, and subsequently reacting with a compound of formula (IIa), Wherein D, L ¹ , B, L ² and R ³⁵ each have the definitions as shown in the first to sixth aspects of the patent application, to obtain a compound of the formula (Ia-A), Wherein n, AK ₁ , D, L ¹ , B, L ² and R ³⁵ each have the definitions as defined in claims 1 to 6 or [B] reacts with the compound of formula (IIIa), Wherein D, L ¹ , B, L ² and R ³⁵ each have the definitions as shown in the first to sixth aspects of the patent application, to obtain a compound of the formula (Ia-B), Wherein n, AK ₂ , D, L ¹ , B, L ² and R ³⁵ each have the definitions as shown in the items 1 to 6 of the patent application. A binder-drug conjugate selected from the group consisting of: In each of the examples, n is a number from 2 to 8, preferably from 2 to 5, and AK ₁ is bonded to mesothelin and bonded to the G group via a sulfur atom of a cysteine residue of the binding agent. Human or humanized antibody or antigen-binding antibody fragment, AK ₂ is a human or humanized antibody or antigen that binds to mesothelin and binds to the G group via the NH side group of the amine acid residue of the binding agent. Antibody fragment.