US20140127240A1 - Novel Binder-Drug Conjugates (ADCs) and Use of Same - Google Patents

Novel Binder-Drug Conjugates (ADCs) and Use of Same Download PDF

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Publication number
US20140127240A1
US20140127240A1 US14/113,070 US201214113070A US2014127240A1 US 20140127240 A1 US20140127240 A1 US 20140127240A1 US 201214113070 A US201214113070 A US 201214113070A US 2014127240 A1 US2014127240 A1 US 2014127240A1
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United States
Prior art keywords
group
hydrogen
denotes
linkage site
formula
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US14/113,070
Inventor
Hans-Georg Lerchen
Lars Linden
Sherif El Sheikh
Jörg Willuda
Charlotte C. Kopitz
Joachim Schuhmacher
Simone Greven
Christoph Mahlert
Beatrix Stelte-Ludwig
Sven Golfier
Rudolf Beier
Iring Heisler
Axel Harrenga
Karl-Heinz Thierauch
Sandra Bruder
Heike Petrul
Hannah Jörissen
Sandra Brokowski
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Bayer Pharma AG
Bayer Intellectual Property GmbH
Seagen Inc
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Bayer Pharma AG
Bayer Intellectual Property GmbH
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Application filed by Bayer Pharma AG, Bayer Intellectual Property GmbH filed Critical Bayer Pharma AG
Assigned to BAYER PHARMA AKTIENGESELLSCHAFT, BAYER INTELLECTUAL PROPERTY GMBH reassignment BAYER PHARMA AKTIENGESELLSCHAFT ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: EL SHEIKH, SHERIF, WILLUDA, JOERG, BORKOWSKI, SANDRA, GOLFIER, SVEN, PETRUL, HEIKE, BEIER, RUDOLF, KOPITZ, CHARLOTTE C., MAHLERT, CHRISTOPH, STELTE-LUDWIG, BEATRIX, BRUDER, SANDRA, GREVEN, SIMONE, HEISLER, IRING, JOERISSEN, HANNAH, LERCHEN, HANS-GEORG, LINDEN, LARS, SCHUHMACHER, JOACHIM, THIERAUCH, KARL-HEINZ, HARRENGA, AXEL
Assigned to SEATTLE GENETICS, INC. reassignment SEATTLE GENETICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BAYER INTELLECTUAL PROPERTY GMBH, BAYER PHARMA AKTIENGESELLSCHAFT
Publication of US20140127240A1 publication Critical patent/US20140127240A1/en
Abandoned legal-status Critical Current

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Abstract

The present patent application relates to novel binder-drug conjugates (ADCs) of N,N-dialkylauristatins directed against the target epidermal growth factor receptor (EGFR, gene ID 1956), effective metabolites of these ADCs, methods for producing these ADCs, use of these ADCs for treatment and or prevention of diseases as well as the use of these ADCs to produce pharmaceutical drugs for treatment and/or prevention of diseases, in particular hyperproliferative and/or angiogenic diseases such as cancer, for example. Such treatments may be administered as monotherapy or in combination with other pharmaceutical drugs or other therapeutic measures.

Description

  • The present application relates to novel binder-drug conjugates (antibody-drug conjugates, ADCs) of N,N-dialkylauristatins, in particular those directed against the target epidermal growth factor receptor (EGFR, gene ID 1956), active metabolites of these ADCs, methods of synthesis of these ADCs, use of these ADCs for treatment and/or prevention of diseases and use of these ADCs for production of drugs for treatment and/or prevention of diseases, in particular hyperproliferative and/or angiogenic diseases, such as the various forms of cancer, for example. Such treatments may be administered as monotherapy or in combination with other drugs or other therapeutic measures.
  • Cancer is the result of uncontrolled cell growth of a wide variety of tissues. In many cases, the cells grow into the existing tissue (invasive growth) or metastasize to remote organs. Cancer occurs in a wide variety of organs and the pathology often has a tissue-specific course. The term cancer is therefore a generic term that describes a large group of specific diseases of various organs, tissues and types of cells.
  • Early-stage tumors can in some cases be removed by surgical and radiotherapeutic measures. Metastatic tumors can usually be treated only palliatively by chemotherapeutic agents. The goal here is to find the optimum combination of improving the quality of life and prolonging life.
  • Most of the chemotherapeutic agents administered parenterally today are not distributed to the tumor tissue or tumor cells in a targeted manner but instead are nonspecifically distributed throughout the patient's body through systemic administration, i.e., at sites where exposure to the drug is often undesirable, such as in healthy cells, tissues and organs, for example. This may lead to adverse effects or even serious general toxic effects, which then often severely limit the therapeutically usable drug dosage range or necessitate complete cessation of the medication.
  • The improved and selective availability of these chemotherapeutic agents in the tumor cell or the immediate surrounding tissue and the associated increase in effect, on the one hand, and minimization of toxic side effects, on the other hand, have therefore for many years been the focus of work in developing new chemotherapeutic drugs. There have been numerous attempts so far to develop efficient methods for introducing drugs into the target cell. However, it is still a difficult task to optimize the association between the drug and the intracellular target and to minimize the intercellular distribution of the drug, e.g., to neighboring cells.
  • Monoclonal antibodies, for example, are suitable for targeted addressing of tumor tissue and tumor cells. The importance of such antibodies for clinical treatment of cancer has grown enormously in recent years based on the efficacy of such agents as trastuzumab (Herceptin), rituximab (Rituxan), cetuximab (Erbitux) and bevacizumab (Avastin) which have been approved in the meantime for treatment of individual specific tumor conditions (see, for example, G. P. Adams and L. M. Weiner, Nat. Biotechnol. 23, 1147-1157 (2005)). As a result, there has been a significant increase in interest in so-called immunoconjugates, such as the aforementioned ADCs, for example, in which an internalizing antibody directed against a tumor-associated antigen is bound covalently to a cytotoxic agent by a linking unit (“linker”). After introducing the ADCs into the tumor cell and then splitting off the conjugate, either the cytotoxic agent itself or another cytotoxic metabolite formed from it is then released inside the tumor cell, where it can manifest its effect directly and selectively. In this way, the damage to normal tissue can be kept within significantly narrower limits in comparison with conventional chemotherapy for cancer (see, for example, J. M. Lambert, Curr. Opin. Pharmacol. 5, 543-549 (2005); A. M. Wu and P. D. Senter, Nat. Biotechnol. 23, 1137-1146 (2005); P. D. Senter, Curr. Opin. Chem. Biol. 13, 235-244 (2009); L. Ducry and B. Stump, Bioconjugate Chem. 21, 5-13 (2010)).
  • Instead of antibodies, binders from the field of small drug molecules may be used as binders to selectively bind to a specific target, such as, for example, a receptor (see, e.g., E. Ruoslahti et al., Science, 279, 377-380 (1998); D. Karkan et al., PLoS ONE 3 (6), e2469 (Jun. 25, 2008)). Conjugates of a cytotoxic drug and an addressing ligand having a defined cleavage site between the ligand and the drug for release of the drug are also known. One such “intended breaking point” may consist of a peptide chain, for example, which can be cleaved selectively at a certain site by a specific enzyme at the site of action (see, for example, R. A. Firestone and L. A. Telan, US Patent Application US 2002/0147138).
  • Monoclonal antibodies are suitable in particular for targeted address of tumor tissues and tumor cells, especially those directed against the target EGFR. The “epidermal growth factor receptor” (EGFR, gene ID 1956) is a trans-membrane glycoprotein (170 kDa) belonging to the tyrosine kinase subfamily. Although the EGF receptor is expressed in many normal cells, it is overexpressed in many forms of human cancer, including cancer of the large and small intestine, carcinomas of the head and neck, pancreatic cancer and gliomas. The extent of this over-expression correlates with a poor prognosis (Galizia, G. et al., Ann. Surg. Oncol., June 2006, 13(6):823-35).
  • Binding of the ligand EGF to the EGF receptor leads to dimerization of the receptor and activation of the intracellular kinase domains. These kinase domains undergo autophosphorylation and thus activate pro-proliferative signal cascades (including those via mitogen-activated protein kinases (MAPKs) and Akt). These signal cascades regulate the transcription of genes involved in cell growth and cell survival, motility and proliferation.
  • Signal transduction by the EGF receptor also results in activation of the wild-type KRAS gene, but the presence of an activating somatic mutation in the KRAS gene within a cancer cell leads to dysregulation of the signal pathways and to resistance to EGFR inhibitory treatments (Allegra et al., J. Clin. Oncol., 20 Apr. 2009, 27(12):2091-6).
  • In an ADC approach, an additional antitumor effect can be achieved by the attached cytotoxic agent in addition to inhibiting the interaction between ligands and receptor.
  • The following publications describe the EGF receptor and anti-EGFR antibodies in general: WO 00069459 A1, WO 2010145796 A2, WO 02100348 A2, EP 00979246 B1, EP 00531472 B1, Mendelsohn, J., Baselga, J., Oncogene (2000) 19, 6550-6565; M. L. Janmaat and G. Giaccone, Drugs of Today, Vol. 39, Suppl. C, 2003, pp. 61-80; Normanno. N., et al., Gene, Jan. 17, 2006, 366(1):2-16, Epidermal growth factor receptor (EGFR) signaling in cancer.
  • Auristatin E (AE) and monomethyl auristatin E (MMAE) are synthetic analogs of the dolastatins, a special group of linear pseudopeptides, which were originally isolated from marine sources, and some of which have a very potent cytotoxic activity with respect to tumor cells (for an overview, see, for example, G. R. Pettit, Prog. Chem. Org. Nat. Prod. 70, 1-79 (1997); G. R. Pettit et al., Anti-Cancer Drug Design 10, 529-544 (1995); G. R. Pettit et al., Anti-Cancer Drug Design 13, 243-277 (1998)).
  • Figure US20140127240A1-20140508-C00001
  • However, MMAE has the disadvantage of a comparatively high systemic toxicity. To improve the tumor selectivity, MMAE is used for targeted tumor therapy in conjunction with enzymatically cleavable valine-citrulline linkers in the ADC setting in particular (WO 2005/081711 A2; S. O. Doronima et al., Bioconjugate Chem. 17, 114-124 (2006)). After proteolytic cleavage, MMAE is preferably released from the corresponding ADCs intracellularly.
  • However, when used in the form of antibody-drug conjugates (ADCs), MMAE is not compatible with linking units (linkers) between the antibody and the drug, which do not have any enzymatically cleavable intended breaking point (S. O. Doronina et al., Bioconjugate Chem. 17, 114-124 (2006)).
  • Monomethyl auristatin F (MMAF) is an auristatin derivative with a C-terminal phenylalanine unit having only a moderate antiproliferative effect in comparison with MMAE. This can very likely be attributed to the free carboxyl group, which has a negative effect on the cell viability of this compound because of its polarity and charge. In this context, the methyl ester of MMAF (MMAF-OMe) has been described as a prodrug derivative, which has a neutral charge and can pass through the cell membrane; it also has an increased in vitro cytotoxicity, which is greater by several orders of magnitude in comparison with MMAF with respect to various carcinoma cell lines (S. O. Doronina et al., Bioconjugate Chem. 17, 114-124 (2006)). It may be assumed that this effect is caused by the MMAF itself, which is rapidly released by intracellular ester hydrolysis after the prodrug has been incorporated into the cells.
  • Figure US20140127240A1-20140508-C00002
  • However, drug compounds based on simple ester derivatives are generally at risk of chemical instability due to a nonspecific ester hydrolysis, which is independent of the intended site of action, for example, due to esterases present in blood plasma. This can greatly restrict the usability of such compounds in treatment.
  • Monomethyl auristatin F (MMAF) as well as various esters and amide derivatives thereof were disclosed in WO 2005/081711 A2. Additional auristatin analogs having a C-terminal amide-substituted phenylalanine unit are described in WO 01/18032 A2. MMAF analogs involving side chain modifications of phenylalanine are claimed in WO 02/088172 A2 and WO 2007/008603 A1. WO 2007/008848 A2 describes those in which the carboxyl group of phenylalanine is modified. Auristatin conjugates linked via the C-terminus were recently described in WO 2009/117531 A1 (see also S. O. Doronina et al., Bioconjugate Chem. 19, 1960-1963 (2008)).
  • In addition, auristatin derivatives such as MMAE and MMAF are also substrates for transporter proteins, which are expressed by many tumor cells, which can lead to development of resistance to these drugs.
  • The object of the present invention was to provide novel binder-drug conjugates (ADCs) which, due to the combination of novel N,N-dialkylauristatin derivatives with suitable novel linkers and binders, have a very attractive profile of effects with regard to their specific tumor effect and/or the lower potential of the metabolites formed intracellularly as a substrate with respect to transporter proteins, for example, and are therefore suitable for treatment and/or prevention of hyperproliferative and/or angiogenic diseases, e.g., cancers.
  • The subject matter of the present invention is binder-drug conjugates of the general formula (Ia)
  • Figure US20140127240A1-20140508-C00003
  • in which
    • n stands for a number from 1 to 50,
    • AK stands for a binder, preferably a chimeric humanized or human antibody, especially preferably an anti-EGFR antibody,
      • the group §-G-L1-B-L2-§§ stands for a linker,
        • wherein
        • § denotes the linkage site to the group AK and
        • §§ denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00004
  • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen or methyl,
      • R2 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00005
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
    • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00006
      • wherein
      • #6 denotes the linkage site to the carbonyl group,
      • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen or methyl,
      • R4 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00007
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00008
        • wherein
        • #9 denotes the linkage site to —CHC(R26)-T2,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R26 stands for hydrogen or hydroxyl,
      • T2 stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
      • R35 stands for methyl or hydroxyl,
      • as well as their salts and solvates as well as the solvates of the salts.
  • Compounds according to the invention include the compounds of formula (I) and their salts and solvates as well as the solvates of the salts, the compounds of the formulas given below, covered by formula (I), and their salts and solvates as well as the solvates of the salts as well as the compounds covered by formula (I) and referred to below as exemplary embodiments as well as their salts and solvates as well as the solvates of the salts inasmuch as the compounds covered by formula (I) and listed below are not already the salts and solvates as well as the solvates of the salts.
  • The compounds according to the invention may exist in different stereoisomeric forms depending on their structure, i.e., in the form of configurational isomers or optionally also as conformational isomers (enantiomers and/or diastereomers, including those in atropisomers). The present invention therefore includes the enantiomers and diastereomers and their respective mixtures. The stereoisomerically uniform components can be isolated in a known way from such mixtures of enantiomers and/or diastereomers. Chromatographic methods, in particular HPLC chromatography on a chiral or achiral phase, are preferably used for this purpose.
  • If the compounds according to the invention can occur in tautomeric forms, then the present invention also includes all tautomeric forms.
  • The present invention also includes all suitable isotope variants of the compounds according to the invention. Isotope variants of a compound according to the invention are understood here to refer to a compound, in which at least one atom within the compound according to the invention is exchanged with another atom of the same ordinal number but with a different atomic mass than the atomic mass normally or mainly occurring in nature. Examples of isotopes that may be incorporated into a compound according to the invention include those of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine such as 2H (deuterium), 3H (tritium), 13C, 14C, 15N, 17O, 18O, 32F, 33F, 33S, 34S, 35S, 36S, 18F, 36Cl, 82Br, 123I, 124I, 129I and 131I. Certain isotope variants of a compound according to the invention, such as in particular those in which one or more radioactive isotopes are incorporated, may be beneficial for investigating the mechanism of action or the distribution of the drug in the body, for example. Compounds labeled with 3H or 14C isotopes are especially suitable for this purpose because of their comparative ease of synthesizing and detection. In addition, the implantation of isotopes, such as deuterium, for example, may lead to certain therapeutic advantages as a result of a greater metabolic stability of the compound, such as prolonging the half-life in the body, for example, or reducing the required active dose. Therefore, such modifications of the compounds according to the invention may optionally also be preferred embodiments of the present invention. Isotope variants of the compounds according to the invention can be synthesized by the methods known to those skilled in the art, for example, according to the methods described below and the procedures given in the exemplary embodiments by using the corresponding isotopic modifications of the respective reagents and/or starting compounds.
  • Within the scope of the present invention, the preferred salts are the physiologically safe salts of the compounds according to the invention. This also includes salts that are not suitable for pharmaceutical applications per se but may be used for isolating or purifying the compounds according to the invention, for example.
  • Physiologically safe salts of the compounds according to the invention include acid addition salts of mineral acids, carboxylic acids and sulfonic acids, for example, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methane sulfonic acid, ethane sulfonic acid, benzene sulfonic acid, toluene 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 safe salts of the compounds according to the invention also include the salts of conventional bases such as preferably and for example, alkali metal salts (e.g., sodium and potassium salts), alkaline earth salts (e.g., calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines with 1 to 16 carbon atoms, such as preferably and for example, ethylamine, diethylamine, triethylamine, ethyl diisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzoylamine, N-methylpiperidine, N-methylmorpholine, arginine, lysine and 1,2-ethylene-diamine.
  • Within the scope of the invention, the solvates refer to forms of the compounds according to the invention which form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a special form of solvates having coordinated molecules of water. Hydrates are the preferred solvates within the scope of the present invention.
  • Furthermore, the present invention also includes prodrugs of the compounds according to the invention. The term “prodrugs” here refers to compounds which may be biologically active or inactive themselves but are converted to the compounds according to the invention during their dwell time in the body (for example, metabolically or hydrolytically).
  • Within the scope of the present invention, the substituents have the following meanings, unless otherwise specified:
  • (C1-C4)-Alkyl within the scope of the invention stands for a linear or branched alkyl radical with 1 to 4 carbon atoms. The following can be mentioned, preferably and for example: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 1-methylpropyl and tert-butyl.
  • Alkanediyl within the scope of the invention stands for a linear α,ω-divalent alkyl radical having the number of carbon atoms indicated in each case. The following can be mentioned, preferably and for example: methylene, ethane-1,2-diyl (1,2-ethylene), propane-1,3-diyl (1,3-propylene), butane-1,4-diyl (1,4-butylene), pentane-1,5-diyl (1,5-pentylene), hexane-1,6-diyl (1,6-hexylene), heptane-1,7-diyl (1,7-hexylene), octane-1,8-diyl (1,8-octylene), nonane-1,9-diyl (1,9-nonylene), decane-1,10-diyl (1,10-decylene).
  • (C3-C7)-Cycloalkyl and/or three- to seven-membered carbocycle within the scope of the invention stands for a monocyclic saturated cycloalkyl group with 3 to 7 carbon atoms. The following can be mentioned preferably and for example: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
  • The side group of an α-amino acid in the R19 meaning includes both the side groups of the naturally occurring α-amino acids and the side groups of the homologs and isomers of these α-amino acids. The α-amino acid may be present in both the L- and D-configurations or as a mixture of these L- and D-forms. Examples of side groups that can be mentioned include: methyl (alanine), propan-2-yl (valine), propan-1-yl (norvaline), 2-methylpropan-1-yl (leucine), 1-methylpropan-1-yl (isoleucine), butan-1-yl (norleucine), tert-butyl (2-tert-butylglycine), phenyl (2-phenylglycine), benzyl (phenylalanine), p-hydroxybenzyl (tyrosine), indol-3-ylmethyl (tryptophan), imidazol-4-ylmethyl (histidine), hydroxymethyl (serine), 2-hydroxyethyl (homoserine), 1-hydroxyethyl (threonine), mercaptomethyl (cysteine), methylthiomethyl (S-methylcysteine), 2-mercaptoethyl (homocysteine), 2-methylthioethyl (methionine), carbamoylmethyl (asparagine), 2-carbamoylethyl (glutamine), carboxymethyl (aspartic acid), 2-carboxyethyl (glutamic acid), 4-aminobutan-1-yl (lysine), 4-amino-3-hydroxybutan-1-yl (hydroxylysine), 3-aminopropan-1-yl (ornithine), 2-aminoethyl (2,4-diaminobutyric acid), aminomethyl (2,3-diaminopropionic acid), 3-guanidinopropan-1-yl (arginine), 3-ureidopropan-1-yl (citrulline). Preferred α-amino acid side groups in the meaning of R19 include methyl (alanine), propan-2-yl (valine), 2-methylpropan-1-yl (leucine), benzyl (phenylalanine), imidazole-4-ylmethyl (histidine), hydroxymethyl (serine), 1-hydroxyethyl (threonine), 4-aminobutan-1-yl (lysine), 3-aminopropan-1-yl (ornithine), 2-aminoethyl (2,4-diaminobutyric acid), aminomethyl (2,3-diaminopropionic acid), 3-guanidinopropan-1-yl (arginine). The L configuration is preferred in each case.
  • A four- to seven-membered heterocycle within the scope of the invention stands for a monocyclic saturated heterocycle having a total of four to seven ring atoms that contain one or two ring heteroatoms from the series of N, O, S, SO and/or SO2 and are linked via a ring carbon atom or optionally a ring nitrogen atom. A five- to seven-membered heterocycle with one or two ring heteroatoms from the series N, O and/or S, especially preferably a five- or six-membered heterocycle with one or two ring heteroatoms from the series of N and/or O is preferred. Examples include: azetidinyl, oxetanyl, pyrrolidinyl, pyrazolidinyl, tetrahydrofuranyl, thiolanyl, piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, hexahydroazepinyl and hexahydro-1,4-diazepinyl. Preferred examples include pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl and morpholinyl.
  • In the formula for the group for which A, B, D, G, L1, L2, L4, R1, R2, R3, R4 and/or R5 may stand, the end point of the line at which the symbol #6, *, **, #3, #1, #2, ##1, ##2, ##3, ##4, ***, ****, #4, #5, #6, #7, #8 and/or #9 appears does not stand for a carbon atom or a CH2 group but instead is a component of the bond to the respective atom identified, to which A, B, D, G, L1, L2, L4, R1, R2, R3, R4 and/or R5 is bound.
  • Within the scope of the present invention, it is true that for all radicals that occur several times, their meanings are independent of one another. If radicals are substituted in the compounds according to the invention, then the radicals may be substituted one or more times, unless otherwise specified. Substitution with one or two substituents that are the same or different is preferred. Substitution with one substituent is especially preferred.
  • Within the scope of the present invention, the terms that are used have the following meanings, unless otherwise specified:
  • The term “linker” is understood in the broadest sense to be a chemical unit comprising a covalent bond or a row of atoms covalently linking a binder to a drug. The term “linker” is preferably understood to be a series of atoms in the sense of the present invention, which covalently link a binder to a drug. In addition, linkers may be divalent chemical units, such as alkyldiyls, aryldiyls, heteroaryldiyls, heterocyclyldiyls, dicarboxylic acid esters, dicarboxylic acid amides.
  • The term “binder” is understood in the broadest sense to be a molecule, which binds to a target molecule that is present on a certain target cell population to be addressed by the binder-drug conjugate. The term “binder” is to be understood in its broadest interpretation, which also includes, for example, lectins, proteins that can bind certain sugar chains or phospholipid binding proteins. Such binders include, for example, high-molecular proteins (binder proteins), polypeptides or peptides (binder peptides), nonpeptidic molecules (e.g., aptamers (U.S. Pat. No. 5,270,163; review article by Keefe, A. D. et al., Nat. Rev. Drug Discov. 2010; 9:537-550) or vitamins) and all other cell-binding molecules or substances. Binder proteins include, for example, antibodies and antibody fragments or antibody mimetics such as affibodies, adnectins, anticalins, DARPins, avimers, nanobodies (review article by Gebauer, M. et al., Curr. Opinion in Chem. Biol. 2009; 13:245-255; Nuttall, S. D. et al., Curr. Opinion in Pharmacology, 2008; 8:608-617). Binder peptides include, for example, ligands of a ligand-receptor pair, e.g., VEGF of the ligand receptor pair VEGF/KDR, such as transferrin of the ligand-receptor pair transferrin/transferrin receptor or a cytokine/cytokine receptor, such as TNFα of the ligand-receptor pair TNFα/TNFα receptor.
  • Preferred binders according to the invention include antibodies (in particular human or humanized monoclonal antibodies) or antigen binding antibody fragments that bind to EGFR. In the case of antibodies such as anti-EGFR antibodies, n (i.e., the number of toxophore molecules per antibody molecule) is preferably in the range of 1 to 10, especially preferably 2 to 8.
  • A “target molecule” is understood in the broadest sense to be a molecule, which is present in the target cell population and may be a protein (e.g., a receptor of a growth factor) or a non-peptidic molecule (e.g., a sugar or phospholipid). It is preferably a receptor or an antigen.
  • The term “extracellular” target molecule describes a target molecule, which is bound to the cell and is found on the outside of a cell or part of a target molecule, which is found on the outside of a cell, i.e., a binder may bind to an intact cell at its extracellular target molecule. An extracellular target molecule may be anchored in the cell membrane or may be part of the cell membrane. Those skilled in the art are familiar with methods for identifying extracellular target molecules. For proteins this may take place by determining the transmembrane domain(s) and though orientation of the protein in the membrane. These specifications are usually stored in the protein data banks (e.g., SwissProt).
  • The term “cancer target molecule” describes a target molecule, which is present on one or more types of cancer cells in comparison with noncancer cells of the same type of tissue. The cancer target molecule is preferably selectively present on one or more types of cancer cells in comparison with noncancer cells of the same tissue type, where the term “selective” describes an at least two-fold enrichment on cancer cells in comparison with noncancer cells of the same type of tissue (a “selective cancer target molecule”). Use of cancer target cells allows selective treatment of cancer cells with the conjugates according to the invention.
  • The binder may be linked to the linker via a bond. Various possibilities of covalent bonding (conjugation) of organic molecules to antibodies are known from the literature. The linkage of the binder may be accomplished by means of a heteroatom of the binder. Heteroatoms of the binder according to the invention that may be used for linkage include sulfur (by means of a sulfhydryl group of the binder in one embodiment), oxygen (by means of a carboxyl or hydroxyl group of the binder according to the invention) and nitrogen (by means of a primary or secondary amine group or amide group of the binder in one embodiment). Conjugation of the toxophores to the antibodies via one or more sulfur atoms of cysteine radicals of the antibody and/or via one or more NH groups of lysine radicals of the antibody is/are preferred according to the invention. These heteroatoms may be present in the natural binder or may be introduced through chemical or molecular biological methods. According to the present invention, the linkage of the binder to the toxophore only has a low influence on the binding activity of the binder to the target molecule. In a preferred embodiment, the linkage has no effect on the binding activity of the binder to the target molecule.
  • The term “antibody” is understood in its broadest sense according to the present invention and includes immunoglobulin molecules, for example, intact or modified monoclonal antibodies, polyclonal antibodies or multispecific antibodies (e.g., bispecific antibodies). An immunoglobulin molecule preferably comprises a molecule having four polypeptide chains, two heavy chains (H chains) and two light chains (L chains), which are typically linked by disulfide bridges. Each heavy chain comprises one variable domain of the heavy chain (abbreviated VH) and one constant domain of the heavy chain. The constant domain of the heavy chain may comprise, for example, three domains CH1, CH2 and CH3. Each light chain comprises one variable domain (abbreviated VL) and one constant domain The constant domain of the light chain comprises one domain (abbreviated CL). The VH and VL domains can be further subdivided into regions of hypervariability, also known as complementarity determining regions (abbreviated CDR), and regions of a lower sequence variability (“framework region,” abbreviated FR). Each VH and VL region is typically made up of three CDRs and up to four FRs, for example, from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An antibody can be obtained from any species suitable for this, e.g., rabbit, llama, camel, mouse or rat. In one embodiment, the antibody is of human or murine origin. An antibody may be human, humanized or chimeric, for example.
  • The term “monoclonal” antibody refers to antibodies obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies of the population are identical except for naturally occurring mutations which may occur in a small number. Monoclonal antibodies recognize a single antigenic binding site with a high specificity. The term monoclonal antibody is not based on a specific synthesis process.
  • The term “intact” antibody relates to antibodies comprising both an antigen binding domain and the constant domain of the light and heavy chains. The constant domain may be a naturally occurring domain or a variant thereof in which several amino acid positions have been altered.
  • The term “modified intact” antibody refers to intact antibodies that have been fused via their amino terminus or carboxy terminus to another polypeptide or protein that does not originate from an antibody by means of a covalent bond (for example, a peptide linkage). In addition, antibodies may also be modified by inserting reactive cysteines at defined sites to facilitate coupling to a toxophore (see Junutula et al., Nat. Biotechnol., August 2008; 26(8):925-32).
  • The term “human” antibody denotes antibodies that can be obtained from a human or are synthetic human antibodies. A “synthetic” human antibody is an antibody that can be obtained entirely or partially by in silico synthesis sequences based on analysis of human antibody sequences. A human antibody may be coded by a nucleic acid, for example, isolated from a library of antibody sequences of human origin. One example of such an antibody is given by Soderlind et al., Nature Biotech. 2000, 18:853-856.
  • The term “humanized” or “chimeric” antibody describes antibodies consisting of a human sequence component and a nonhuman sequence component. In these antibodies, a portion of the sequences of the human immunoglobulin (recipient) have been replaced by sequence components of a nonhuman immunoglobulin (donor). The donor is frequently a murine immunoglobulin. In humanized antibodies, amino acids of the CDR of the recipient are replaced by amino acids of the donor. In some cases amino acids of the framework are also replaced by corresponding amino acids of the donor. In many cases the humanized antibody contains amino acids not present in the recipient or donor but inserted during optimization of the antibody. In chimeric antibodies, variable domains of donor immunoglobulin are fused to constant regions of a human antibody.
  • The term complementarity determining region (CDR) as used here refers to the amino acids of a variable antibody domain, which are necessary for binding to the antigen. A variable region will typically have three CDR regions, which are identified as CDR1, CDR2 and CDR3. Each CDR region may comprise amino acids according to the definition by Kabat and/or amino acids of a hypervariable loop defined according to Chotia. The definition according to Kabat includes, for example, the region of approximately amino acid positions 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) of the variable light chain and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) of the variable heavy chain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The definition according to Chotia comprises, for example, approximately the region of amino acid positions 26-32 (CDR1), 50-52 (CDR2) and 91-96 (CDR3) of the variable light chain and 26-32 (CDR1), 53-55 (CDR2) and 96-101 (CDR3) of the variable heavy chain (Chotia and Lesk; J. Mol. Biol. 196:901-917 (1987)). In many cases, a CDR may comprise amino acids from a CDR region as defined by Kabat and Chiota.
  • Antibodies can be divided into several various classes, depending on the amino acid sequence of the constant domain of the heavy chain. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, several of which can be divided further into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The constant domain of the heavy chain corresponding to the different classes are identified as [alpha/α], [delta/δ], [epsilon/ε], [gamma/γ] and [mu/μ]. Both the three-dimensional structure and the subunit structure of antibodies are known.
  • The term “functional fragment” or “antigen binding antibody fragment” of an antibody/immunoglobulin is defined as a fragment of an antibody/immunoglobulin (e.g., the variable domains of an IgG), which still comprises the antigen binding domains of the antibody/immunoglobulin. The “antigen binding domain” of an antibody typically comprises one or more hypervariable regions of an antibody, e.g., the CDR, CDR2 and/or CDR3 regions. However, the “framework” region of an antibody may also play a role in binding the antibody to the antigen. The framework region forms the framework for the CDRs. The antigen binding domain preferably comprises at least amino acids 1 through 103 of the variable light chain and amino acids 5 through 109 of the variable heavy chain, more preferably amino acids 3 through 107 of the variable light chain and 4 through 111 of the variable heavy chain, with the complete variable light and heavy chains being especially preferred, i.e., amino acids 1 through 109 of the VL and 1 through 113 of the VH (numbering according to WO 97/08320).
  • “Functional fragments” or “antigen binding antibody fragments” of the invention comprise not conclusively Fab, Fab′, F(ab′)2 and Fv fragments, diabodies, single domain antibodies (DAbs), linear antibodies, single chain antibodies (single chain Fv, abbreviated scFv) and multispecific antibodies, for example, bi- and tri-specific antibodies formed from antibody fragments (C.A.K. Borrebaeck, editor (1995), Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann and S. Duebel, editors (2001), Antibody Engineering (Springer Laboratory Manual), Springer Verlag). Antibodies other than “multispecific” or “multi-functional” include those with identical binding sites. Multispecific antibodies may be specific for different epitopes of an antigen or specific for epitopes of 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. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; or Kostelny et al., 1992, J. Immunol. 148:1547-1553). A F(ab′)2 or Fab molecule may be constructed so that the number of intermolecular disulfide interactions taking place between the CH1 and CL domains can be reduced or completely prevented.
  • “Functional fragment” or “antigen binding antibody fragments” may be fused to an additional polypeptide or protein which does not originate from an antibody by way of their amino terminus or carboxy terminus by means of a covalent bond (e.g., a peptide linkage). In addition, antibodies and antigen binding fragments may be modified so that reactive cysteines are inserted at defined sites to facilitate coupling to a toxophore (see Junutula et al., Nat. Biotechnol., August 2008, 26(8):925-32).
  • Polyclonal antibodies can be synthesized by methods with which the average person skilled in the art is familiar. Monoclonal antibodies can be synthesized by methods with which those skilled in the art are familiar (Köhler and Milstein, Nature, 256:495-497, 1975). Human and/or humanized monoclonal antibodies can be synthesized by methods with which the average person skilled in the art is familiar (Olsson et al., Meth. Enzymol. 92:3-16 and/or Cabilly et al., U.S. Pat. No. 4,816,567 or Boss et al., U.S. Pat. No. 4,816,397).
  • The average person skilled in the art is familiar with various methods for synthesis of antibodies and their fragments such as, for example, by means of transgenic mice (N. Lonberg and D. Huszar, Int. Rev. Immunol. 1995; 13(1):65-93) or Phage Display Technologies (Clackson et al., Nature, Aug. 15, 1991, 352(6336):624-628). Antibodies according to the invention can be obtained from recombinant antibody library consisting of the amino acid sequences of a plurality of antibodies created from a large number of healthy volunteer subjects. Antibodies can also be synthesized by means of known recombinant DNA technologies. The nucleic acid sequence of an antibody can be obtained by routine sequencing or is available from publicly accessible data banks.
  • An “isolated” antibody or binder has been purified to remove other constituents of the cell. Contaminating ingredients of a cell which can interfere with a diagnostic or therapeutic use may be, for example, enzymes, hormones or other peptidic or nonpeptidic components of a cell. An antibody or binder that has been purified to more than 95% by weight, based on the antibody and/or binder (determined by the Lowry method, UV-Vis spectroscopy or SDS capillary gel electrophoresis, for example). Furthermore, an antibody that has been purified to the extent that at least 15 amino acids of the amino terminus or an internal amino acid sequence can be determined or which has been purified to the point of homogeneity, where homogeneity is determined by SDS-PAGE under reducing or nonreducing conditions (detection may be performed by Coomassie blue staining or preferably by silver staining) may also be used. However, an antibody is normally synthesized by at least one purification step.
  • The term “specific binding” or “binds specifically” refers to an antibody or binder that binds to a predetermined antigen/target molecule. Specific binding of an antibody or binder typically describes an antibody, i.e., binder having an affinity of at least 10−7 M (as the Kd value; i.e., preferably those with a Kd value of less than 10−7 M), where the antibody, i.e., the binder, has an affinity for the predetermined antigen/target molecule that is at least twice as high as that of a nonspecific antigen/target molecule (e.g., bovine serum albumin or casein) which is not the predetermined antigen/target molecule or a closely related antigen/target molecule.
  • Antibodies which are specific against a cancer cell antigen can be synthesized by the average person skilled in the art using methods with which he is familiar (such as recombinant expression) or may be acquired commercially (for example, from Merck KGaA, Germany). Examples of known commercially available antibodies in cancer therapy include Erbitux® (cetuximab, Merck KGaA), Avastin® (bevacizumab, Roche) and Herceptin® (trastuzumab, Genentech). Trastuzumab is a recombinant humanized monoclonal antibody of the IgG1κ type which binds the extracellular domains of human epidermal growth receptor with a high affinity in a cell-based assay (Kd=5 nM). The antibody is synthesized recombinantly in CHO cells.
  • The compounds of formula (I) constitute a subgroup of the compounds of formula (Ia).
  • The preferred subject matter of the invention is binder-drug conjugates of the general formula (Ia), wherein
    • n stands for a number from 1 to 50,
    • AK stands for AK1 or AK2
      • wherein
      • AK1 stands for a binder (preferably for a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody) which is bound to the group G by a sulfur atom of the binder,
      • AK2 stands for a binder (preferably for a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody) which is bound to the group G by a nitrogen atom of the binder,
    • G for the case when AK=AK1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00009
      • wherein
      • #1 denotes the linkage site to the sulfur atom of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C1-C10)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00010
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • L1A stands for linear (C2-C10)-alkanediyl,
      • B1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00011
        • wherein
        • ##5 denotes the linkage site to the group L1A,
        • ##6 denotes the linkage site to the group L1B,
        • L5 stands for a bond or (C2-C4)-alkanediyl,
        • L6 stands for a bond or a group with the formula
  • Figure US20140127240A1-20140508-C00012
          • wherein
          • ##7 denotes the linkage site to the carbonyl group,
          • ##8 denotes the linkage site to L1B,
          • R33 stands for hydrogen, (C1-C4)-alkylcarbonyl, tert-butyloxy-carbonyl or benzyloxycarbonyl,
          • R34 stands for hydrogen or methyl,
        • R29 stands for hydrogen or (C1-C4)-alkyl,
        • R30 stands for hydrogen or (C1-C4)-alkyl,
        • or
        • R29 and R30 together with the atoms to which they are bound form a five- or six-membered heterocycle,
        • R31 stands for hydrogen or (C1-C4)-alkyl,
        • R32 stands for hydrogen or (C1-C4)-alkyl,
        • or
        • R31 and R32 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • L1B stands for linear (C2-C10)-alkanediyl,
      • and
      • wherein (C1-C10)-alkanediyl may be substituted with one to four substituents selected independently from one another from the group, comprising methyl, hydroxyl and benzyl,
      • and
      • wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally situated between them, to form a (C3-C6)-cycloalkyl ring or a phenyl ring,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00013
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • P stands for O or NH,
      • L3 stands for a bond or (C2-C4)-alkanediyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00014
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for hydrogen or methyl,
        • R28 stands for hydrogen, (C1-C4)-alkylcarbonyl, tert-butyloxycarbonyl or benzyloxycarbonyl,
      • Q1 stands for a four- to seven-membered heterocycle,
      • Q2 stands for a three- to seven-membered carbocycle or a four- to seven-membered heterocycle,
      • R14 stands for hydrogen or (C1-C4)-alkyl,
      • R15 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R14 and R15 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • R16 stands for hydrogen or (C1-C4)-alkyl,
      • R17 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • R18 stands for hydrogen or (C1-C4)-alkyl,
      • R19 stands for hydrogen or the side group of α-amino acid or its homologs or isomers,
      • R20 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
      • R21 stands for hydrogen or (C1-C4)-alkyl,
      • R22 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a three- to seven-membered carbocycle,
      • R23 stands for (C1-C4)-alkyl,
      • R24 stands for hydrogen or (C1-C4)-alkyl,
      • R27 stands for hydrogen or (C1-C4)-alkyl,
      • R36 stands for hydrogen, (C1-C4)-alkylcarbonyl, tert-butyloxycarbonyl or benzyloxy-carbonyl,
      • R37 stands for hydrogen or methyl,
      • or
      • R36 and R37 together with the atoms to which they are bound form a pyrrolidine ring,
    • L2 stands for linear (C2-C10)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00015
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
      • wherein (C2-C10)-alkanediyl may be substituted with one to four substituents, selected independently of one another from the group comprising methyl, hydroxyl and benzyl,
      • and
      • wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally present between them, to form a (C3-C6) cycloalkyl ring or a phenyl ring,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00016
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen or methyl,
      • R2 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00017
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O grouping contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00018
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen or methyl,
      • R4 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00019
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00020
        • wherein
        • #9 denotes the linkage site to —CHC(R26)-T2,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R26 stands for hydrogen or hydroxyl,
      • T2 stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
    • R35 stands for methyl or hydroxyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • Binder-drug conjugates of the general formula (Ia), wherein
    • n stands for an integer from 1 to 50,
    • AK stands for a binder, preferably a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
    • the group §-G-L1-B-§§ for a linker
      • wherein
      • § denotes the linkage site to the group AK and
      • §§ denotes the linkage site to the nitrogen atom,
      • L2 stands for linear (C2-C10)-alkanediyl or a group of the formula
  • Figure US20140127240A1-20140508-C00021
        • wherein
        • p stands for a number from 2 to 6,
        • ##3 denotes the linkage site to the group B,
        • ##4 denotes the linkage site to the nitrogen atom,
        • wherein (C2-C10)-alkanediyl may be substituted with one to four substituents, independently of one another, selected from the group comprising methyl, hydroxyl and benzyl,
        • and
        • wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another to form a (C3-C6) cycloalkyl ring or a phenyl ring, including the carbon atoms optionally situated between them,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00022
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen or methyl,
      • R2 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00023
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained therein stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00024
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen or methyl,
      • R4 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00025
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted with methoxycarbonyl or carboxyl in the phenyl group,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00026
        • wherein
        • #9 denotes the linkage site to —CHC(R26)-T2,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
    • R26 stands for hydrogen or hydroxyl,
    • T2 stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
    • R35 stands for methyl or hydroxyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention relates to binder-drug conjugates of general formula (Ia) as given above, wherein
    • n stands for a number from 1 to 50,
    • AK stands for AK1 or AK2
      • wherein
      • AK1 stands for a binder (preferably for a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody), which is bound to the group G via a sulfur atom of the binder,
      • AK2 stands for a binder (preferably for a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody) that is bound to the group G via a nitrogen atom of the binder,
    • G for the case when AK=AK1, stands for a group of the formula
  • Figure US20140127240A1-20140508-C00027
      • wherein
      • #1 denotes the linkage site to the sulfur atom of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C1-C10)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00028
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • L1A stands for linear (C2-C10)-alkanediyl,
      • B1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00029
        • wherein
        • ##5 denotes the linkage site to the group L1A,
        • ##6 denotes the linkage site to the group L1B,
        • L5 stands for a bond or (C2-C4)-alkanediyl,
        • L6 stands for a bond or a group with the formula
  • Figure US20140127240A1-20140508-C00030
          • wherein
          • ##7 denotes the linkage site to the carbonyl group,
          • ##8 denotes the linkage site to L1B,
          • R33 stands for hydrogen, (C1-C4)-alkylcarbonyl, tert-butyloxy-carbonyl or benzyloxycarbonyl,
          • R34 stands for hydrogen or methyl,
        • R29 stands for hydrogen or (C1-C4)-alkyl,
        • R30 stands for hydrogen or (C1-C4)-alkyl,
        • or
        • R29 and R30 together with the atoms to which they are bound form a five- or six-membered heterocycle,
        • R31 stands for hydrogen or (C1-C4)-alkyl,
        • R32 stands for hydrogen or (C1-C4)-alkyl,
        • or
        • R31 and R32 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • L1B stands for linear (C2-C10)-alkanediyl,
      • and
      • wherein (C1-C10)-alkanediyl may be substituted with one to four substituents selected independently of one another from the group comprising methyl, hydroxyl and benzyl
      • and
      • wherein two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-relation to one another may be bridge to form a (C3-C6)-cycloalkyl ring or a phenyl ring by inclusion of the carbon atoms optionally occurring between them,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00031
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • P stands for O or NH,
      • L3 stands for a bond or (C2-C4)-alkanediyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00032
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for hydrogen or methyl,
        • R28 stands for hydrogen, (C1-C4)-alkylcarbonyl, tert-butyloxycarbonyl or benzyloxycarbonyl,
      • Q1 stands for a four- to seven-membered heterocycle,
      • Q2 stands for a three- to seven-membered carbocycle or a four- to seven-membered heterocycle,
      • R14 stands for hydrogen or (C1-C4)-alkyl,
      • R15 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R14 and R15 together with the atoms to which they are bound may form a five- or six-membered heterocycle,
      • R16 stands for hydrogen or (C1-C4)-alkyl,
      • R17 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • R18 stands for hydrogen or (C1-C4)-alkyl,
      • R19 stands for hydrogen or the side group of a naturally occurring α-amino acid or its homologs or isomers,
      • R20 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
      • R21 stands for hydrogen or (C1-C4)-alkyl,
      • R22 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a three- to seven-membered carbocycle,
      • R23 stands for (C1-C4)-alkyl,
      • R24 stands for hydrogen or (C1-C4)-alkyl,
      • R27 stands for hydrogen or (C1-C4)-alkyl,
      • R36 stands for hydrogen, (C1-C4)-alkylcarbonyl, tert-butyloxycarbonyl or benzyloxy-carbonyl,
      • R37 stands for hydrogen or methyl,
      • or
      • R36 and R37 together with the atoms to which they are bound form a pyrrolidine ring,
    • L2 stands for linear (C2-C10)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00033
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
      • wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 substituents selected independently of one another from the group comprising methyl, hydroxyl and benzyl,
      • and
      • wherein two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-relation to one another to form a (C3-C6) cycloalkyl ring or a phenyl ring, including the carbon atoms optionally situated between them,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00034
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen or methyl,
      • R2 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached may form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00035
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A together with the N—O group contained therein stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00036
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen or methyl,
      • R4 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached forms a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00037
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00038
        • wherein
        • #9 denotes the linkage site to —CHC(R26)-T2,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R26 stands for hydrogen or hydroxyl,
      • T2 stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
    • R35 stands for methyl or hydroxyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the invention relates to binder-drug conjugates of the general formula (Ia), wherein
      • n stands for a number from 1 to 20,
      • AK stands for AK1 or AK2
        • wherein
        • AK1 stands for an antibody or an antigen binding antibody fragment which binds to EGFR and is bound to the group G via the sulfur atom of a cysteine radical of the binder,
        • AK2 stands for an antibody or an antigen binding antibody fragment which binds to EGFR and is bound to the group G via the NH side group of a lysine radical of the binder,
    • G for the case when AK=AK1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00039
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C2-C6)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00040
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • L1A stands for linear (C2-C6)-alkanediyl,
      • B1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00041
        • wherein
        • ##5 denotes the linkage site to the group L1A,
        • ##6 denotes the linkage site to the group L1B,
        • L5 stands for a bond,
        • L6 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00042
          • wherein
          • ##7 denotes the linkage site to the carbonyl group,
          • ##8 denotes the linkage site to L1B,
          • R33 for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
          • R34 stands for hydrogen or methyl,
        • R29 stands for hydrogen,
        • R30 stands for hydrogen,
        • R31 stands for hydrogen or methyl,
        • R32 stands for hydrogen or methyl,
      • L1B stands for linear (C2-C6)-alkanediyl,
      • and
      • wherein (C2-C6)-alkanediyl may be substituted with one to two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00043
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00044
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for hydrogen or methyl,
        • R28 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • Q1 stands for a four- to seven-membered heterocycle,
      • R14 stands for hydrogen,
      • R15 stands for hydrogen,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
      • R18 stands for hydrogen,
      • R19 stands for hydrogen, methyl, propan-2-yl, 2-methylpropan-1-yl or 1-methyl-propan-1-yl,
      • R20 stands for hydrogen or methyl,
      • or
      • R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
      • R21 stands for hydrogen or methyl,
      • R22 stands for hydrogen or methyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a cyclopropyl ring,
      • R23 stands for methyl,
      • R24 stands for hydrogen or methyl,
      • R27 stands for hydrogen,
      • R36 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • R37 stands for hydrogen or methyl,
      • or
      • R36 and R37 together with the atoms to which they are bound form a pyrrolidine ring,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00045
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
      • wherein (C2-C10)-alkanediyl may be substituted with one or two methyl substituents.
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00046
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached may form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00047
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained therein stands for a monocyclic or
  • Figure US20140127240A1-20140508-C00048
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached may form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00049
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom which they are attached may form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted with methoxycarbonyl or carboxyl in the phenyl group,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00050
        • wherein
        • #9 denotes the linkage site to —CHC(R26)-T2,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R26 stands for hydrogen or hydroxyl,
      • T2 stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
    • R35 stands for methyl or hydroxyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention relates to binder-drug conjugates of the general formula (Ia) as indicated above, wherein
    • n stands for a number from 1 to 20,
    • AK stands for AK1 or AK2
      • wherein
      • AK1 stands for an antibody or an antigen binding antibody fragment which binds to EGFR and is bound to the group G by the sulfur atom of a cysteine radical of the binder,
      • AK2 stands for an antibody or an antigen binding antibody fragment which binds to EGFR and is bound to the group G by the NH side group of a lysine radical of the binder,
    • G for the case when AK=AK1 stands for the group of the formula
  • Figure US20140127240A1-20140508-C00051
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C2-C6)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00052
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • L1A stands for linear (C2-C6)-alkanediyl,
      • B1 stands for a group with the formula
  • Figure US20140127240A1-20140508-C00053
        • wherein
        • ##5 denotes the linkage site to the group L1A,
        • ##6 denotes the linkage site to the group L1B,
        • L5 stands for a bond,
        • L6 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00054
          • wherein
          • ##7 denotes the linkage site to the carbonyl group,
          • ##8 denotes the linkage site to L1B,
          • R33 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
          • R34 stands for hydrogen or methyl,
        • R29 stands for hydrogen,
        • R30 stands for hydrogen,
        • R31 stands for hydrogen or methyl,
        • R32 stands for hydrogen or methyl,
      • L1B stands for linear (C2-C6)-alkanediyl,
      • and
      • wherein (C2-C6)-alkanediyl may be substituted with one to two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00055
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00056
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for hydrogen or methyl,
        • R28 stands for hydrogen, methylcarbonyl tert-butyloxycarbonyl,
      • Q1 stands for a four- to seven-membered heterocycle,
      • R14 stands for hydrogen,
      • R15 stands for hydrogen,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
      • R18 stands for hydrogen,
      • R19 stands for hydrogen, methyl, propan-2-yl, 2-methylpropan-1-yl or 1-methyl-propan-1-yl,
      • R20 stands for hydrogen or methyl,
      • or
      • R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
      • R21 stands for hydrogen or methyl,
      • R22 stands for hydrogen or methyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a cyclopropyl ring,
      • R23 stands for methyl,
      • R24 stands for hydrogen or methyl,
      • R27 stands for hydrogen,
      • R36 stands for hydrogen, (C1-C4)-alkylcarbonyl, tert-butyloxycarbonyl or benzyloxycarbonyl,
      • R37 stands for hydrogen or methyl,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00057
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
      • wherein (C2-C10)-alkanediyl may be substituted with one or two methyl substituents,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00058
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00059
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A together with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00060
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached may form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00061
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are bound may form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted with methoxycarbonyl or carboxyl in the phenyl group,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00062
        • wherein
        • #9 denotes the linkage site to —CHC(R26)-T2,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R26 stands for hydrogen or hydroxyl,
      • T2 stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
    • R35 stands for methyl or hydroxyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the invention relates to binder-drug conjugates of the general formula (Ia), wherein
    • n stands for a number between 1 and 10,
    • AK stands for AK1 or AK2
      • wherein
      • AK1 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the sulfur atom of a cysteine radical of the binder,
      • AK2 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the NH side group of a lysine radical of the binder,
    • G for the case when AK=AK1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00063
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C2-C6)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00064
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00065
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00066
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for methyl,
        • R28 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • Q1 stands for piperidine-1,4-diyl,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
      • R21 stands for hydrogen or methyl,
      • R22 stands for hydrogen or methyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a cyclopropyl ring,
      • R23 stands for methyl,
      • R24 stands for hydrogen,
      • R36 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • R37 stands for hydrogen or methyl,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00067
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00068
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00069
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00070
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00071
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00072
        • wherein
        • #9 denotes the linkage site to —CHCH2-phenyl,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
    • R35 stands for methyl or hydroxyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention relates to binder-drug conjugates of the general formula (Ia) as indicated above, wherein
    • n stands for a number from 1 to 10,
    • AK stands for AK1 or AK2
      • wherein
      • AK1 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the sulfur atom of a cysteine radical of the binder,
      • AK2 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the NH side group of a lysine radical of the binder,
    • G for the case when AK=AK1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00073
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C2-C6)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00074
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00075
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00076
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for methyl,
        • R28 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • Q1 stands for piperidine-1,4-diyl,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
      • R21 stands for hydrogen or methyl,
      • R22 stands for hydrogen or methyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a cyclopropyl ring,
      • R23 stands for methyl,
      • R24 stands for hydrogen,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00077
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00078
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 1-hydroxyethyl, benzyl, 1-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00079
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00080
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for benzyl, 1-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00081
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00082
        • wherein
        • #9 denotes the linkage site to —CHCH2-phenyl,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
    • R35 stands for methyl or hydroxyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention relates to binder-drug conjugates of the general formula (Ia) as indicated above, wherein
    • n stands for a number from 1 to 10,
    • AK stands for AK2
      • wherein
      • AK2 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the NH side group of a lysine radical of the binder,
    • G stands for carbonyl,
    • L1 stands for a bond,
    • B stands for a bond,
    • L2 stands for linear (C3-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00083
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00084
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for benzyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00085
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00086
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
      • R3 stands for hydrogen,
      • R4 stands for benzyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00087
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7 or —C(═O)—NR8R9,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen,
        • R9 stands for hydrogen or benzyl,
    • R35 stands for methyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention relates to binder-drug conjugates of the general formula (Ia) as indicated above, wherein
    • n stands for a number from 1 to 10,
    • AK stands for AK2,
      • wherein
      • AK2 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the NH side group of a lysine radical of the binder,
    • G stands for carbonyl,
    • L1 stands for a bond,
    • B stands for a bond,
    • L2 stands for linear (C3-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00088
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00089
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for benzyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00090
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00091
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
      • R3 stands for hydrogen,
      • R4 stands for benzyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00092
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7 or —C(═O)—NR8R9,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen,
        • R9 stands for hydrogen or benzyl,
        • R35 stands for methyl,
          as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention relates to binder-drug conjugates of the general formula (Ia) as indicated above, wherein
    • n stands for a number from 1 to 10,
    • AK stands for AK1
      • wherein
      • AK1 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the sulfur atom of a cysteine radical of the binder,
    • G stands for a group of the formula
  • Figure US20140127240A1-20140508-C00093
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
    • L1 stands for a bond, linear (C3-C5)-alkanediyl or a group of the formula
  • Figure US20140127240A1-20140508-C00094
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C3-C5)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00095
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00096
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for methyl,
        • R28 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
    • L2 stands for linear (C3-C5)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00097
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00098
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for benzyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00099
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00100
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
      • R3 stands for hydrogen,
      • R4 stands for benzyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00101
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7 or —C(═O)—NR8R9,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen,
        • R9 stands for hydrogen or benzyl,
    • R35 stands for methyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention relates to binder-drug conjugates of the general formula (Ia) as indicated above, wherein
    • n stands for a number from 1 to 10,
    • AK stands for AK1,
      • wherein
      • AK1 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the sulfur atom of a cysteine radical of the binder,
    • G stands for a group of the formula
  • Figure US20140127240A1-20140508-C00102
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
    • L1 stands for a bond, linear (C3-C5)-alkanediyl or a group of the formula
  • Figure US20140127240A1-20140508-C00103
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C3-C5)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00104
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00105
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for methyl,
        • R28 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
    • L2 stands for linear (C3-C5)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00106
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00107
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for benzyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00108
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00109
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
      • R3 stands for hydrogen,
      • R4 stands for benzyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00110
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7 or —C(═O)—NR8R9,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen,
        • R9 stands for hydrogen or benzyl,
    • R35 stands for methyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • Another subject matter of the present invention relates to compounds of the formula (XXXa)
  • Figure US20140127240A1-20140508-C00111
  • in which
    • Cys stands for a cysteine radical which is bound to a carbon atom of the succinimide via the sulfur atom of the side chain,
    • L1 stands for a bond, linear (C1-C10)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00112
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • L1A stands for linear (C2-C10)-alkanediyl,
      • B1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00113
        • wherein
        • ##5 denotes the linkage site to the group L1A,
        • ##6 denotes the linkage site to the group L1B,
        • L5 stands for a bond or (C2-C4)-alkanediyl,
        • L6 stands for a bond,
        • R29 stands for hydrogen or (C1-C4)-alkyl,
        • R30 stands for hydrogen or (C1-C4)-alkyl,
        • or
        • R29 and R30 together with the atoms to which they are bound form a five- or six-membered heterocycle,
        • R31 stands for hydrogen or (C1-C4)-alkyl,
        • R32 stands for hydrogen or (C1-C4)-alkyl,
        • or
        • R31 and R32 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • L1B stands for linear (C2-C10)-alkanediyl,
      • and
      • wherein (C1-C10)-alkanediyl may be substituted with one to four substituents selected independently of one another from the group comprising methyl, hydroxyl and benzyl
      • and
      • wherein two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-relation to one another to form a (C3-C6)-cycloalkyl ring or a phenyl ring by including the carbon atoms optionally situated between them,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00114
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • P stands for O or NH,
      • L3 stands for a bond or (C2-C4)-alkanediyl,
      • L4 stands for a bond,
      • Q1 stands for a four- to seven-membered heterocycle,
      • Q2 stands for a three- to seven-membered carbocycle or a four- to seven-membered heterocycle,
      • R14 stands for hydrogen or (C1-C4)-alkyl,
      • R15 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R14 and R15 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • R16 stands for hydrogen or (C1-C4)-alkyl,
      • R17 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • R18 stands for hydrogen or (C1-C4)-alkyl,
      • R19 stands for hydrogen or the side group of a natural α-amino acid or its homologs or isomers,
      • R20 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
      • R21 stands for hydrogen or (C1-C4)-alkyl,
      • R22 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a three- to seven-membered carbocycle,
      • R23 stands for (C1-C4)-alkyl,
      • R24 stands for hydrogen or (C1-C4)-alkyl,
      • R27 stands for hydrogen or (C1-C4)-alkyl,
    • L2 stands for linear (C2-C10)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00115
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
      • wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 substituents selected independently of one another from the group comprised of methyl, hydroxyl and benzyl,
      • and
      • wherein two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-relation to one another to form a (C3-C6) cycloalkyl ring or a phenyl ring, including the carbon atoms optionally situated between them,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00116
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen or methyl,
      • R2 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00117
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00118
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen or methyl,
      • R4 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00119
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00120
        • wherein
        • #9 denotes the linkage site to —CHC(R26)-T2,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R26 stands for hydrogen or hydroxyl,
      • T2 stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
    • R35 stands for methyl or hydroxyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention is compounds of formula (XXXa) as indicated above, wherein
  • Figure US20140127240A1-20140508-C00121
    • Cys stands for a cysteine radical which is bound to a carbon atom of the succinimide via the sulfur atom of the side chain,
    • L1 stands for a bond, linear (C2-C6)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00122
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • L1A stands for linear (C2-C6)-alkanediyl,
      • B1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00123
        • wherein
        • ##5 denotes the linkage site to the group L1A,
        • ##6 denotes the linkage site to the group L1B,
        • L5 stands for a bond,
        • L6 stands for a bond,
        • R29 stands for hydrogen,
        • R30 stands for hydrogen,
        • R31 stands for hydrogen or methyl,
        • R32 stands for hydrogen or methyl,
      • L1B stands for linear (C2-C6)-alkanediyl,
      • and
      • wherein (C2-C6)-alkanediyl may be substituted with one to two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00124
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond,
      • R14 stands for hydrogen,
      • R15 stands for hydrogen,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
      • R23 stands for methyl,
      • R24 stands for hydrogen or methyl,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00125
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00126
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00127
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic,
  • Figure US20140127240A1-20140508-C00128
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00129
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00130
        • wherein
        • #9 denotes the linkage site to —CHCH2-phenyl,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R35 stands for methyl or hydroxyl,
      • as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention is compounds of formula (XXXa) as indicated above, wherein
    • Cys stands for a cysteine radical which is bound to a carbon atom of the succinimide via the sulfur atom of the side chain,
    • L1 stands for a bond or linear (C2-C6)-alkanediyl,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00131
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond,
      • L4 stands for a bond,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00132
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00133
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for benzyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00134
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00135
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
      • R3 stands for hydrogen,
      • R4 stands for benzyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00136
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7 or —C(═O)—NR8R9,
        • wherein
        • R7 stands for hydrogen,
        • R8 stands for hydrogen,
        • R9 stands for hydrogen,
      • R35 stands for methyl,
      • as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention is compounds of formula (XXXa) as indicated above, wherein
    • Cys stands for a cysteine radical which is bound to a carbon atom of the succinimide via the sulfur atom of the side chain,
    • L1 stands for a bond or linear (C2-C6)-alkanediyl,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00137
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond,
      • L4 stands for a bond,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00138
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00139
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for benzyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00140
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00141
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
      • R3 stands for hydrogen,
      • R4 stands for benzyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00142
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7 or —C(═O)—NR8R9,
        • wherein
        • R7 stands for hydrogen,
        • R8 stands for hydrogen,
        • R9 stands for hydrogen,
    • R35 stands for methyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • Another subject matter of the present invention relates to compounds of formula (XXXI)
  • Figure US20140127240A1-20140508-C00143
  • in which
    • L1 stands for a bond, linear (C1-C10)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00144
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • L1A stands for linear (C2-C10)-alkanediyl,
      • B1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00145
        • wherein
        • ##5 denotes the linkage site to the group L1A,
        • ##6 denotes the linkage site to the group L1B,
        • L5 stands for a bond or (C2-C4)-alkanediyl,
        • L6 stands for a bond,
        • R29 stands for hydrogen or (C1-C4)-alkyl,
        • R30 stands for hydrogen or (C1-C4)-alkyl,
        • or
        • R29 and R30 together with the atoms to which they are bound form a five- or six-membered heterocycle,
        • R31 stands for hydrogen or (C1-C4)-alkyl,
        • R32 stands for hydrogen or (C1-C4)-alkyl,
        • or
        • R31 and R32 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • L1B stands for linear (C2-C10)-alkanediyl,
      • and
      • wherein (C1-C10)-alkanediyl may be substituted with one to four substituents selected independently from one another from the group comprising methyl, hydroxyl and benzyl
      • and
      • wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally situated between them, to form a (C3-C6)-cycloalkyl ring or a phenyl ring,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00146
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • P stands for O or NH,
      • Q1 stands for a four- to seven-membered heterocycle,
      • Q2 stands for a three- to seven-membered carbocycle or a four- to seven-membered heterocycle,
        • R18 stands for hydrogen or (C1-C4)-alkyl,
      • R19 stands for hydrogen or the side group of a natural α-amino acid or its homologs or isomers,
      • R20 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
      • R21 stands for hydrogen or (C1-C4)-alkyl,
      • R22 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a three- to seven-membered carbocycle,
      • R27 stands for hydrogen or (C1-C4)-alkyl,
    • L2 stands for linear (C2-C10)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00147
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
      • Wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 substituents selected independently of one another from the group comprising methyl, hydroxyl and benzyl,
      • and
      • wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally present between them to form a (C3-C6) cycloalkyl ring or a phenyl ring,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00148
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen or methyl,
      • R2 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00149
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00150
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen or methyl,
      • R4 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00151
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00152
        • wherein
        • #9 denotes the linkage site to —CHC(R26)-T2,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R26 stands for hydrogen or hydroxyl,
      • T2 stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
      • R35 stands for methyl or hydroxyl,
      • as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention is compounds of formula (XXXI) as indicated above, wherein
    • L1 stands for a bond, linear (C2-C6)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00153
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00154
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • R18 stands for hydrogen,
      • R19 stands for hydrogen, methyl, propan-2-yl, 2-methylpropan-1-yl or 1-methyl-propan-1-yl,
      • R20 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
      • R21 stands for hydrogen or methyl,
      • R22 stands for hydrogen or methyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a cyclopropyl ring,
      • R27 stands for hydrogen or methyl,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00155
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
      • wherein (C2-C10)-alkanediyl may be substituted with one or two methyl substituents,
      • and
      • wherein two carbon atoms of the alkanediyl chain may be bridged in 1,4-relation to one another, including the carbon atoms optionally present between them to form a phenyl ring,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00156
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00157
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00158
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00159
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00160
        • wherein
        • #9 denotes the linkage site to —CHCH2-phenyl,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R35 stands for methyl or hydroxyl,
      • as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention is compounds of formula (XXXI) as indicated above, wherein
    • L1 stands for a bond,
    • B stands for a bond,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00161
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00162
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for benzyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00163
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00164
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for benzyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00165
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7 or —C(═O)—NR8R9,
        • wherein
        • R7 stands for hydrogen,
        • R8 stands for hydrogen,
        • R9 stands for hydrogen,
      • R35 stands for methyl,
      • as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention is compounds of formula (XXXI) as indicated above, wherein
    • L1 stands for a bond,
    • B stands for a bond,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00166
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00167
      • wherein
      • ∩3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for benzyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00168
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00169
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for benzyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a
  • Figure US20140127240A1-20140508-C00170
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7 or —C(═O)—NR8R9,
        • wherein
        • R7 stands for hydrogen,
        • R8 stands for hydrogen,
        • R9 stands for hydrogen,
    • R35 stands for methyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention relates to compounds of formula (XXXa) and (XXXI) selected from the group:
    • N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,
    • N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide,
    • N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate,
    • N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,
    • as well as their salts and solvates as well as the solvates of the salts.
  • Another preferred subject matter of the present invention relates to binder-drug conjugates of the general formula (I)
  • Figure US20140127240A1-20140508-C00171
  • in which
    • n stands for a number from 1 to 50,
    • AK stands for a binder, preferably a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
    • the group §-G-L1-B-L2-§§ stands for a linker,
      • wherein
      • § denotes the linkage site to the group AK and
      • §§ denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00172
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 1-hydroxyethyl, benzyl, 1-phenylethyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00173
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic,
  • Figure US20140127240A1-20140508-C00174
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for 1-hydroxyethyl, benzyl, 1-phenylethyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00175
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are attached form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00176
        • wherein
        • #9 denotes the linkage site to —CHC(R26)-T2,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R26 stands for hydrogen or hydroxyl,
      • T2 stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
        as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the invention relates to binder-drug conjugates of the general formula (I), wherein
    • n stands for a number from 1 to 50,
    • AK stands for AK1 or AK2
      • wherein
      • AK1 denotes a binder which is bound by a nitrogen atom of the binder to the group G, preferably for a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
      • AK2 stands for a binder bound to group G by a nitrogen atom of the binder, preferably standing for a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
    • G for the case when AK=AK1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00177
      • wherein
      • #1 denotes the linkage site to the sulfur atom of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C1-C10)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00178
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C1-C10)-alkanediyl may be substituted with 1 to 4 methyl substituents,
      • and
      • wherein two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-relation to one another to form a (C3-C6)-cycloalkyl ring or a phenyl ring, including the carbon atoms optionally situated between them,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00179
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • P stands for O or NH,
      • L3 for a bond or (C2-C4)-alkanediyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00180
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for hydrogen or methyl,
      • Q1 stands for a four- to seven-membered heterocycle,
      • Q2 stands for a three- to seven-membered carbocycle or a four- to seven-membered heterocycle,
      • R14 stands for hydrogen or (C1-C4)-alkyl,
      • R15 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R14 and R15 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • R16 stands for hydrogen or (C1-C4)-alkyl,
      • R17 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • R18 stands for hydrogen or (C1-C4)-alkyl,
      • R19 stands for hydrogen or the side group of a natural α-amino acid or its homologs or isomers,
      • R20 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
      • R21 stands for hydrogen or (C1-C4)-alkyl,
      • R22 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a three- to seven-membered carbocycle,
      • R23 stands for (C1-C4)-alkyl,
      • R24 stands for hydrogen or (C1-C4)-alkyl,
      • R27 stands for hydrogen or (C1-C4)-alkyl,
    • L2 stands for linear (C2-C10)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00181
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
      • wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 methyl substituents,
      • and
      • wherein two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-relation to one another to form a (C3-C6)-cycloalkyl ring or a phenyl ring, including the carbon atoms optionally situated between them,
    • D has the meanings given above,
      as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the invention is binder-drug conjugates of the general formula (I)
  • in which
    • n stands for a number from 1 to 50,
    • AK stands for AK1 or AK2
      • wherein
      • AK1 stands for an antibody or an antigen binding antibody fragment and is bound to the group G via a sulfur atom, preferably standing for a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
      • AK2 stands for an antibody or an antigen binding antibody fragment and is bound to the group G via a nitrogen atom, preferably standing for a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
    • G, L1, B, L2 and D have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention is binder-drug conjugates of general formula (I)
  • in which
    • n stands for a number from 1 to 20,
    • AK stands for AK1 or AK2
      • wherein
      • AK1 stands for an antibody or an antigen binding antibody fragment which binds to
  • EGFR and is bound to the group G via the sulfur atom of a cysteine radical of the binder,
      • AK2 stands for an antibody or an antigen binding antibody fragment which binds to EGFR and is bound to the group G via the NH side group of a lysine radical of the binder,
    • G for the case when AK=AK1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00182
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00183
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00184
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • P stands for O or NH,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00185
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for methyl,
        • Q2 stands for cyclopentyl or cyclohexyl,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
      • R18 stands for hydrogen,
      • R19 stands for hydrogen, methyl, propan-2-yl, 2-methylpropan-1-yl or 1-methyl-propan-1-yl,
      • R20 stands for hydrogen or methyl,
      • or
      • R19 and R20 together with the atoms to which they are attached form a pyrrolidinyl ring,
    • L2 stands for linear (C2-C6)-alkanediyl,
      • wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00186
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00187
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00188
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for 1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00189
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are attached form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00190
        • wherein
        • #9 denotes the linkage site to —CHC(R26)-T2,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R26 stands for hydrogen or hydroxyl,
      • T2 stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
        as well as their salts and solvates as well as the solvates of the salts.
  • Especially preferred as the subject matter of the present invention are binder-drug conjugates of general formula (I)
  • in which
    • n stands for a number from 1 to 10,
    • AK stands for AK1 or AK2
      • wherein
      • AK1 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the sulfur atom of a cysteine radical of the binder,
      • AK2 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the NH side group of a lysine radical of the binder,
    • G for the case when AK=AK1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00191
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C2-C6)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00192
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00193
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00194
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for methyl,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
    • L2 stands for linear (C2-C6)-alkanediyl,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00195
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00196
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00197
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00198
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen or a group of the formula
  • Figure US20140127240A1-20140508-C00199
        • wherein
        • #9 denotes the linkage site to —CHC(R26)phenyl,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
          as well as their salts and solvates as well as the solvates of the salts.
  • Another subject matter of the present invention relates to compounds of formula (XXX)
  • Figure US20140127240A1-20140508-C00200
  • in which
    • Cys stands for a cysteine radical which is bound to a carbon atom of the succinimide via the sulfur atom of the side chain,
    • L1 stands for a bond, linear (C1-C10)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00201
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C1-C10)-alkanediyl may be substituted with 1 to 4 methyl substituents,
      • and
      • wherein two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-relation to one another to form a (C3-C6)-cycloalkyl ring or a phenyl ring, including the carbon atoms optionally situated between them,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00202
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • P stands for O or NH,
      • L3 for a bond or (C2-C4)-alkanediyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00203
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for hydrogen or methyl,
      • Q1 stands for a three- to seven-membered carbocycle or a four- to seven-membered azaheterocycle,
      • Q2 stands for a three- to seven-membered carbocycle or a four- to seven-membered azaheterocycle,
      • R14 stands for hydrogen or (C1-C4)-alkyl,
      • R15 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R14 and R15 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • R16 stands for hydrogen or (C1-C4)-alkyl,
      • R17 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • R18 stands for hydrogen or (C1-C4)-alkyl,
      • R19 stands for hydrogen or the side group of a natural α-amino acid or its homologs or isomers,
      • R20 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
      • R21 stands for hydrogen or (C1-C4)-alkyl,
      • R22 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a three- to seven-membered carbocycle,
      • R23 stands for (C1-C4)-alkyl,
      • R24 stands for hydrogen or (C1-C4)-alkyl,
    • L2 stands for linear (C2-C10)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00204
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
      • wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 methyl substituents,
      • and
      • wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally present between them to form a (C3-C6) cycloalkyl ring or a phenyl ring,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00205
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00206
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00207
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for 1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00208
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00209
        • wherein
        • #9 denotes the linkage site to —CHC(R26)-T2,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R26 stands for hydrogen or hydroxyl,
      • T2 stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
        as well as their salts and solvates as well as the solvates of the salts.
  • In addition, compounds of formula (XXX) that are especially preferred within the scope of the present invention are those in which
    • Cys stands for a cysteine radical which is bound to a carbon atom of the succinimide via the sulfur atom of the side chain,
    • L1 stands for a bond, linear (C2-C6)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00210
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00211
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond,
      • R14 stands for hydrogen,
      • R15 stands for hydrogen,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
      • R23 stands for methyl,
      • R24 stands for hydrogen or methyl,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00212
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00213
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00214
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00215
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00216
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl ethyl, n-propyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen or a group of the formula
  • Figure US20140127240A1-20140508-C00217
        • wherein
        • #9 denotes the linkage site to —CHC(R26)phenyl,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
          as well as their salts and solvates as well as the solvates of the salts.
  • Within the scope of the present invention, compounds of formula (Ia) are also preferred, in which
  • n=1-20, especially preferably n=1-10 and most especially preferably n=2-8.
  • Within the scope of the present invention, compounds of formula (Ia) are preferred, in which
    • AK stands for AK1
      • wherein
      • AK1 stands for an antibody or an antigen binding antibody fragment which binds to EGFR and is bound to the group G by the sulfur atom of a cysteine radical of the binder,
    • G stands for a group of the formula
  • Figure US20140127240A1-20140508-C00218
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
    • and
    • n, L1, B, L2, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), wherein
    • AK stands for AK2
      • wherein
      • AK2 stands for an antibody or an antigen binding antibody fragment which binds to EGFR and is bound to the group G via the NH side group of a lysine radical of the binder,
    • G stands for carbonyl,
    • and
    • n, L1, B, L2, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), in which
    • AK stands for AK1
      • wherein
      • AK1 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the sulfur atom of a cysteine radical of the binder,
    • G stands for a group of the formula
  • Figure US20140127240A1-20140508-C00219
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
    • and
    • n, L1, B, L2, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), in which
    • AK stands for AK2
      • wherein
      • AK2 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the NH side group of a lysine radical of the binder,
    • G stands for carbonyl,
    • and
    • n, L1, B, L2, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of general formula (Ia), in which
    • AK stands for AK2
      • wherein
      • AK2 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the NH side group of a lysine radical of the binder,
    • G stands for carbonyl,
    • L1 stands for a bond,
    • B stands for a bond,
    • L2 stands for linear (C3-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00220
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • n, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of general formula (Ia), in which
    • AK stands for AK1
      • wherein
      • AK1 stands for cetuximab, pantitumumab or nimutuzumab, which is bound to the group G via the sulfur atom of a cysteine radical of the binder,
    • G stands for a group of the formula
  • Figure US20140127240A1-20140508-C00221
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
    • L1 stands for a bond, linear (C3-C5)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00222
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C3-C5)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00223
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00224
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for methyl,
        • R28 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
    • L2 stands for linear (C3-C5)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00225
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • and
    • n, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • LI stands for a bond,
    • B stands for a bond,
    • L2 stands for linear (C3-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00226
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • and
    • n, AK, Cys, G, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), in which
    • L1 stands for linear (C1-C10)-alkanediyl or a group of the formula
  • Figure US20140127240A1-20140508-C00227
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C1-C10)-alkanediyl may be substituted with 1 to 4 substituents selected independently of one another from the group methyl, hydroxyl and benzyl,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00228
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 for a bond or (C2-C4)-alkanediyl,
      • L4 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00229
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for hydrogen or methyl,
        • R28 stands for hydrogen, (C1-C4)-alkylcarbonyl, tert-butyloxycarbonyl or benzyloxycarbonyl,
      • Q1 stands for a four- to seven-membered heterocycle,
      • R16 stands for hydrogen or (C1-C4)-alkyl,
      • R17 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • R23 stands for (C1-C4)-alkyl,
      • R24 stands for hydrogen or (C1-C4)-alkyl,
      • R36 stands for hydrogen, (C1-C4)-alkylcarbonyl, tert-butyloxycarbonyl or benzyloxy-carbonyl,
      • R37 stands for hydrogen or methyl,
      • or
      • R36 and R37 together with the atoms to which they are bound form a pyrrolidine ring,
    • L2 stands for linear (C2-C10)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00230
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
      • wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 substituents selected independently of one another from the group comprising methyl, hydroxyl and benzyl,
    • and
    • n, AK, G, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), in which
    • L1 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00231
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00232
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00233
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for hydrogen or methyl,
        • R28 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
      • R36 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • R37 stands for hydrogen or methyl,
      • or
      • R36 and R37 together with the atoms to which they are bound form a pyrrolidine ring,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00234
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • and
    • n, AK, G, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia) and (XXXa), in which
    • G stands for a group of the formula
  • Figure US20140127240A1-20140508-C00235
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
    • L1 stands for linear (C3-C5)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00236
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C3-C5)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00237
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond,
    • L2 for linear (C3-C5)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00238
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • and
    • n, AK1, Cys, D, R16 and R17 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia) and (XXXa), in which
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00239
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond,
    • n, AK, Cys, G, L1, L2, D, R16, R17 and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • L1 stands for a bond, linear (C3-C5)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00240
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C3-C5)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00241
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond,
      • L4 stands for a bond
      • R16 stands for hydrogen,
      • R17 stands for hydrogen,
    • L2 stands for linear (C3-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00242
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • n, AK, Cys, G, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • L1 stands for a bond,
    • B stands for a bond,
    • L2 stands for linear (C3-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00243
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • n, AK, Cys, G, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • L1 stands for linear (C3-C5)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00244
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C3-C5)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a group of the formula
  • Figure US20140127240A1-20140508-C00245
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond,
      • L4 stands for a bond
      • R16 stands for hydrogen,
      • R17 stands for hydrogen,
    • L2 stands for linear (C3-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00246
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • n, AK, Cys, G, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), in which
    • n stands for a number from 2 to 8, preferably 2 to 5,
    • AK stands for AK1 or AK2
      • wherein
      • AK1 stands for an antibody or an antigen binding antibody fragment which binds to C4.4a and is bound to the group G via the sulfur atom of a cysteine radical of the binder,
      • AK2 stands for an antibody or an antigen binding antibody fragment which binds to C4.4a and is bound to the group G via the NH side group of a lysine radical of the binder,
    • G for the case when AK=AK1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00247
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C3-C5)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00248
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C3-C5)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00249
  • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond,
      • L4 stands for a bond,
      • R16 stands for hydrogen,
      • R17 stands for hydrogen,
    • L2 stands for linear (C3-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00250
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • and
    • D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), in which
    • n stands for a number from 2 to 8, preferably 2 to 5,
    • AK stands for AK1 or AK2
      • wherein
      • AK1 stands for an antibody or an antigen binding antibody fragment which binds to C4.4a and is bound to the group G via the sulfur atom of a cysteine radical of the binder,
      • AK2 stands for an antibody or an antigen binding antibody fragment which binds to C4.4a and is bound to the group G via the NH side group of a lysine radical of the binder,
    • G for the case when AK=AK1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00251
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond,
    • B stands for a bond,
    • L2 stands for linear (C3-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00252
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), in which
    • n stands for a number from 2 to 8, preferably 2 to 5,
    • AK stands for AK1,
      • wherein
      • AK1 stands for an antibody or an antigen binding antibody fragment which binds to C4.4a and is bound to the group G via the sulfur atom of a cysteine radical of the binder,
    • G stands for a group of the formula
  • Figure US20140127240A1-20140508-C00253
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
    • L1 stands for linear (C3-C5)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00254
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C3-C5)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a group of the formula
  • Figure US20140127240A1-20140508-C00255
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond,
      • L4 stands for a bond
      • R16 stands for hydrogen,
      • R17 stands for hydrogen,
    • L2 stands for linear (C3-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00256
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), in which
    • L1 stands for a bond, linear (C3-C5)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00257
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • L1A stands for linear (C2-C6)-alkanediyl,
      • B1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00258
        • wherein
        • ##5 denotes the linkage site to the group L1A,
        • ##6 denotes the linkage site to the group L1B,
        • L5 stands for a bond or ethane-1,2-diyl,
        • L6 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00259
          • wherein
          • ##7 denotes the linkage site to the carbonyl group,
          • ##8 denotes the linkage site to L1B,
          • R33 stands for hydrogen, (C1-C4)-alkylcarbonyl or tert-butyloxy-carbonyl,
          • R34 stands for hydrogen or methyl,
        • R29 stands for hydrogen or methyl,
        • R30 stands for hydrogen or methyl,
        • R31 stands for hydrogen or methyl,
        • R32 stands for hydrogen or methyl,
      • L1B stands for linear (C3-C6)-alkanediyl,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00260
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • P stands for O,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00261
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for hydrogen or methyl,
        • R28 stands for hydrogen, (C1-C4)-alkylcarbonyl or tert-butyloxycarbonyl,
      • Q1 stands for a four- to seven-membered heterocycle,
      • Q2 stands for a three- to seven-membered carbocycle or a four- to seven-membered heterocycle,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • R23 stands for (C1-C4)-alkyl,
      • R24 stands for hydrogen or (C1-C4)-alkyl,
      • R36 stands for hydrogen, (C1-C4)-alkylcarbonyl or tert-butyloxycarbonyl,
      • R37 stands for hydrogen or methyl,
      • or
      • R36 and R37 together with the atoms to which they are bound form a pyrrolidine ring,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00262
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
        as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), in which
    • L1 stands for linear (C3-C5)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00263
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
    • B stands for a group of the formula
  • Figure US20140127240A1-20140508-C00264
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00265
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for methyl,
        • R28 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • Q1 stands for piperidine-1,4-diyl,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • R23 stands for methyl,
      • R24 stands for hydrogen,
      • R36 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • R37 stands for hydrogen or methyl,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00266
      • wherein
      • p stands for a number of 2 or 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
        as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00267
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen or methyl,
      • R2 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00268
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00269
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen or methyl,
      • R4 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00270
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00271
        • wherein
        • #9 denotes the linkage site to —CHC(R26)-T2,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R26 stands for hydrogen,
      • T2 stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
    • and
    • n, AK, Cys, G, L1, B, L2, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00272
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • the ring A with the N—O group contained in it stands for a heterocycle of the formula
  • Figure US20140127240A1-20140508-C00273
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
      • R3 stands for hydrogen,
      • R4 stands for benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • T1 stands for a group of the formula —C(═O)—OR7 or —C(═O)—NR8R9,
        • wherein
        • R7 stands for hydrogen,
        • R8 stands for hydrogen,
        • R9 stands for hydrogen,
    • n, AK, Cys, G, L1, B, L2, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00274
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • the ring A with the N—O group contained in it stands for a heterocycle of the formula
  • Figure US20140127240A1-20140508-C00275
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
      • R3 stands for hydrogen,
      • R4 stands for benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • T1 stands for a group of the formula —C(═O)—NR8R9,
        • wherein
        • R8 stands for hydrogen,
        • R9 stands for hydrogen,
    • n, AK, Cys, G, L1, B, L2, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00276
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • the ring A with the N—O group contained in it stands for a heterocycle of the formula
  • Figure US20140127240A1-20140508-C00277
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
      • R3 stands for hydrogen,
      • R4 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • T1 stands for a group of the formula —C(═O)—NR8R9,
        • wherein
        • R8 stands for hydrogen,
        • R9 stands for hydrogen,
    • n, AK, Cys, G, L1, B, L2, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00278
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R3 stands for hydrogen,
      • R4 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • T1 stands for a group of the formula —C(═O)—OR7 or —C(═O)—NR8R9,
        • wherein
        • R7 stands for hydrogen,
        • R8 stands for hydrogen,
        • R9 stands for hydrogen,
    • n, AK, Cys, G, L1, B, L2, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00279
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R3 stands for hydrogen,
      • R4 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • T1 stands for a group of the formula —C(═O)—NR8R9,
        • wherein
        • R8 stands for hydrogen,
        • R9 stands for hydrogen,
    • n, AK, Cys, G, L1, B, L2, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00280
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen or methyl,
      • R2 stands for isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00281
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00282
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
    • and
    • n, AK, Cys, G, L1, B, L2 and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00283
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00284
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00285
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
    • and
    • n, AK, Cys, G, L1, B, L2 and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • R35 stands for hydroxyl,
    • and
    • n, AK, Cys, G, L1, B, L2, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (Ia), (XXXa) and (XXXI), in which
    • R35 stands for methyl,
    • and
    • n, AK, Cys, G, L1, B, L2, D and R35 have the meanings given above
      as well as their salts and solvates as well as the solvates of the salts.
  • Also especially preferred within the scope of the present invention are compounds of formula (XXXa), in which
    • Cys stands for a L-cysteine radical which is bound to a carbon atom of the succinimide via the sulfur atom of the side chain,
      as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are compounds of formula (I) and (XXX), in which
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00286
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00287
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00288
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • n, AK, Cys, G, L1, L2 and B have the meanings given above
        as well as their salts and solvates as well as the solvates of the salts.
  • Also especially preferred within the scope of the present invention are compounds of formula (Ia) and (XXXa), in which
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00289
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for benzyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00290
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a heterocycle of the formula
  • Figure US20140127240A1-20140508-C00291
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
      • n, AK, Cys, G, L1, L2 and B have the meanings given above
        as well as their salts and solvates as well as the solvates of the salts.
  • Another especially preferred subject matter of the present invention is compounds of formula (I), in which
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00292
    •  wherein
      • T1 stands for —C(═O)—OH or —C(═O)—NH2 and
    • n, AK, G, L1, B, L2, #3, R3 and R4 have the meanings given above.
  • Also preferred within the scope of the present invention are compounds of formula (I), in which
    • n=1-20, especially preferably n=1-10 and most especially preferably n=2-8.
  • Also preferred within the scope of the present invention are compounds of formula (Ia) and (XXX), in which
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00293
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond,
      • n, AK, Cys, G, L1, L2, D, R16 and R17 have the meanings given above
        as well as their salts and solvates as well as the solvates of the salts.
  • Especially preferred within the scope of the present invention are compounds of formula (I) and (XXX), in which
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00294
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 and L4 stands for a bond,
      • n, AK, Cys, G, L1, L2, D, R16 and R17 have the meanings given above
        as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are binder-drug conjugates of general formula (I), in which
    • AK stands for AK1
      • wherein
      • AK1 stands for a binder that is bound to the group G via a sulfur atom of the binder, preferably a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
    • G stands for a group of the formula
  • Figure US20140127240A1-20140508-C00295
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
    • L1 stands for a bond, linear (C1-C10)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00296
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C1-C10)-alkanediyl may be substituted with 1 to 4 methyl substituents,
      • and
      • wherein two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-relation to one another to form a (C3-C6)-cycloalkyl ring or a phenyl ring, including the carbon atoms optionally situated between them,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00297
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 for a bond or (C2-C4)-alkanediyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00298
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for hydrogen or methyl,
      • Q1 stands for a four- to seven-membered heterocycle,
      • R14 stands for hydrogen or (C1-C4)-alkyl,
      • R15 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R14 and R15 together with the atoms to which they are bound form a five- or six-membered heterocycle,
      • R16 stands for hydrogen or (C1-C4)-alkyl,
      • R17 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a five- or six-membered heterocycle,
    • L2 stands for linear (C2-C10)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00299
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
      • wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 methyl substituents,
    • and
      • wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally present between them to form a (C3-C6) cycloalkyl ring or a phenyl ring,
        as well as their salts and solvates as well as the solvates of the salts.
  • Also preferred within the scope of the present invention are binder-drug conjugates of general formula (I), in which
    • AK stands for AK2
      • wherein
      • AK2 stands for a binder that is bound to the group G via the NH side group of a lysine radical of the binder,
    • G stands for carbonyl,
    • L1 stands for a bond, linear (C1-C10)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00300
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C1-C10)-alkanediyl may be substituted with 1 to 4 methyl substituents,
      • and
      • wherein two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-relation to one another to form a (C3-C6)-cycloalkyl ring or a phenyl ring, including the carbon atoms optionally situated between them,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00301
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • P stands for O or NH,
      • Q2 stands for a three- to seven-membered carbocycle or a four- to seven-membered heterocycle,
      • R19 stands for hydrogen or (C1-C4)-alkyl,
      • R20 stands for hydrogen or the side group of a natural α-amino acid or its homologs or isomers,
      • R20 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
      • R21 stands for hydrogen or (C1-C4)-alkyl,
      • R22 stands for hydrogen or (C1-C4)-alkyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a three- to seven-membered carbocycle,
      • R23 stands for (C1-C4)-alkyl,
      • R24 stands for hydrogen or (C1-C4)-alkyl,
    • L2 stands for linear (C2-C10)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00302
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
      • wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 methyl substituents,
      • and
      • wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally present between them to form a (C3-C6) cycloalkyl ring or a phenyl ring,
        as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention are binder-drug conjugates of general formula (Ia) as indicated above, in which
    • n stands for a number from 1 to 20,
    • AK stands for AK1 or AK2
      • wherein
      • AK1 stands for a binder that is bound to the group G via a sulfur atom of the binder, preferably a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
      • AK2 stands for a binder that is bound to the group G via a nitrogen atom of the binder, preferably a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
    • G for the case when AK=AK1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00303
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C2-C6)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00304
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • L1A stands for linear (C2-C6)-alkanediyl,
      • B1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00305
        • wherein
        • ##5 denotes the linkage site to the group L1A,
        • ##6 denotes the linkage site to the group L1B,
        • L5 stands for a bond,
        • L6 stands for a bond or a group with the formula
  • Figure US20140127240A1-20140508-C00306
          • wherein
          • ##7 denotes the linkage site to the carbonyl group,
          • ##8 denotes the linkage site to L1B,
          • R33 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
          • R34 stands for hydrogen or methyl,
        • R29 stands for hydrogen,
        • R30 stands for hydrogen,
        • R31 stands for hydrogen or methyl,
        • R32 stands for hydrogen or methyl,
      • L1B stands for linear (C2-C6)-alkanediyl,
      • and
      • wherein (C2-C6)-alkanediyl may be substituted with one to two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00307
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00308
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for hydrogen or methyl,
        • R28 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • Q1 stands for a four- to seven-membered heterocycle,
      • R14 stands for hydrogen,
      • R15 stands for hydrogen,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
      • R18 stands for hydrogen,
      • R19 stands for hydrogen, methyl, propan-2-yl, 2-methylpropan-1-yl or 1-methyl-propan-1-yl,
      • R20 stands for hydrogen or methyl,
      • or
      • R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
      • R21 stands for hydrogen or methyl,
      • R22 stands for hydrogen or methyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a cyclopropyl ring,
      • R23 stands for methyl,
      • R24 stands for hydrogen or methyl,
      • R27 stands for hydrogen,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00309
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
      • wherein (C2-C10)-alkanediyl may be substituted with one or two methyl substituents,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00310
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00311
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00312
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00313
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • or
        • R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
        • R10 stands for benzoyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00314
        • wherein
        • #9 denotes the linkage site to —CHC(R26)-T2,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R26 stands for hydrogen or hydroxyl,
      • T2 stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
      • R35 stands for methyl or hydroxyl,
        as well as their salts and solvates as well as the solvates of the salts.
  • The preferred subject matter of the present invention is binder-drug conjugates of general formula (Ia) as indicated above, in which
    • n stands for a number from 1 to 10,
    • AK stands for AK1 or AK2
      • wherein
      • AK1 stands for a binder bound to the group G via a sulfur atom of the binder,
      • AK2 stands for a binder bound to the group G via a nitrogen atom of the binder,
    • G for the case when AK=AK1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00315
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C2-C6)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00316
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00317
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00318
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for methyl,
        • R28 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • Q1 stands for piperidine-1,4-diyl,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
      • R21 stands for hydrogen or methyl,
      • R22 stands for hydrogen or methyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a cyclopropyl ring,
      • R23 stands for methyl,
      • R24 stands for hydrogen,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00319
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00320
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 1-hydroxyethyl, benzyl, 1-hydroxybenzyl, 1-phenylethyl, or 1H-indol-3-ylmethyl,
      • or
      • R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00321
        • wherein
        • #4 denotes the linkage site to the vicinal nitrogen atom,
        • #5 denotes the linkage site to the carbonyl group,
      • the ring A with the N—O group contained in it stands for a monocyclic or bicyclic, optionally substituted heterocycle of the formula
  • Figure US20140127240A1-20140508-C00322
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
        • R6 stands for hydrogen, hydroxyl or benzyloxy,
      • R3 stands for hydrogen,
      • R4 stands for benzyl, 1-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
      • or
      • R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
  • Figure US20140127240A1-20140508-C00323
        • wherein
        • #7 denotes the linkage site to the vicinal nitrogen atom,
        • #8 denotes the linkage site to the group T1,
      • T1 stands for a group of the formula —C(═O)—OR7, —C(═O)—NR8R9 or —CH2—O—R11,
        • wherein
        • R7 stands for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl, ethyl, n-propyl or benzyl,
        • R11 stands for benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
      • R5 stands for hydrogen, methyl or a group of the formula
  • Figure US20140127240A1-20140508-C00324
        • wherein
        • #9 denotes the linkage site to —CHCH2-phenyl,
        • R12 stands for phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
        • R13 stands for phenyl, which may be substituted with methoxycarbonyl or carboxyl,
      • R35 stands for methyl or hydroxyl,
      • as well as their salts and solvates as well as the solvates of the salts.
  • Especially preferred within the scope of the present invention are binder-drug conjugates of formula (Ia), in which
    • n stands for a number from 2 to 8,
    • AK stands for AK1 or AK2,
      • wherein
      • AK1 stands for a binder that is bound to the group G via a sulfur atom of the cysteine radical of the binder, preferably for a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
      • AK2 stands for a binder that is bound to the group G via a nitrogen atom of the lysine radical of the binder, preferably a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
    • G for the case when AK=AK1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00325
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C2-C6)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00326
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00327
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00328
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for methyl,
        • R28 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • Q1 stands for piperidine-1,4-diyl,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
      • R21 stands for hydrogen or methyl,
      • R22 stands for hydrogen or methyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a cyclopropyl ring,
      • R23 stands for methyl,
      • R24 stands for hydrogen,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00329
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00330
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • the ring A with the N—O group contained therein stands for
  • Figure US20140127240A1-20140508-C00331
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
      • R3 stands for hydrogen,
      • R4 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • T1 stands for a group of the formula —C(═O)—NR8R9,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl or ethyl,
    • R35 stands for methyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • Especially preferred within the scope of the present invention are binder-drug conjugates of formula (Ia), in which
    • n stands for a number from 2 to 8, preferably 2 to 5,
    • AK stands for AK1,
      • wherein
      • AK1 stands for a binder that is bound to the group G via a sulfur atom of the cysteine radical of the binder, preferably for a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
    • G stands for a group of the formula
  • Figure US20140127240A1-20140508-C00332
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
    • L1 stands for pentane-1,5-diyl,
    • B stands for a group of the formula
  • Figure US20140127240A1-20140508-C00333
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond,
      • L4 stands for a bond,
      • R16 stands for hydrogen,
      • R17 stands for hydrogen,
    • L2 stands for propane-1,3-diyl,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00334
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • the ring A with the N—O group contained therein stands for
  • Figure US20140127240A1-20140508-C00335
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
      • R3 stands for hydrogen,
      • R4 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • T1 stands for a group of the formula —C(═O)—NR8R9,
        • R8 stands for hydrogen,
        • R9 stands for hydrogen,
    • R35 stands for methyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • Especially preferred within the scope of the present invention are binder-drug conjugates of formula (Ia), in which
    • n stands for a number from 2 to 8, preferably 2 to 5,
    • AK stands for AK1,
      • wherein
      • AK1 stands for a binder that is bound to the group G via a sulfur atom of the cysteine radical of the binder, preferably for a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
    • G stands for a group of the formula
  • Figure US20140127240A1-20140508-C00336
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
    • L1 stands for a bond,
    • B stands for a bond,
    • L2 stands for hexane-1,6-diyl,
    • and D has the meaning given above,
      as well as their salts and solvates as well as the solvates of the salts.
  • Especially preferred within the scope of the present invention are binder-drug conjugates of formula (Ia), in which
    • n stands for a number from 2 to 8, preferably from 2 to 5,
    • AK stands for AK2,
      • wherein
      • AK2 stands for a binder that is bound to the group G via a nitrogen atom of the binder, preferably a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
    • G stands for carbonyl,
    • L1 stands for a bond,
    • B stands for a bond,
    • L2 stands for pentane-1,5-diyl,
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00337
      • wherein
      • #3 denotes the linkage site to the nitrogen atom,
      • R1 stands for hydrogen,
      • R2 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • the ring A with the N—O group contained therein stands for
  • Figure US20140127240A1-20140508-C00338
        • wherein
        • #6 denotes the linkage site to the carbonyl group,
      • R3 stands for hydrogen,
      • R4 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • T1 stands for a group of the formula —C(═O)—NR8R9,
      • R8 stands for hydrogen,
      • R9 stands for hydrogen,
    • R35 stands for methyl,
      as well as their salts and solvates as well as the solvates of the salts.
  • Especially preferred within the scope of the present invention are binder-drug conjugates of formula (Ia), in which
    • n stands for a number from 2 to 8, preferably 2 to 5,
    • AK stands for AK2,
      • wherein
      • AK2 stands for a binder that is bound to the group G via a nitrogen atom of the lysine radical of the binder, preferably a chimeric, humanized or human antibody, especially preferably an anti-EGFR antibody,
    • G stands for carbonyl,
    • L1 stands for a bond,
    • B stands for a bond,
    • L2 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00339
      • wherein
      • p stands for the number 3,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
    • and D has the meaning given above,
      as well as their salts and solvates as well as the solvates of the salts.
  • According to the invention, the drug-binder conjugate preferably comprises the following compounds in particular, where n stands for a number from 2 to 8, preferably 2 to 8, and AK stands for a chimeric, human or humanized antibody or an antigen binding antibody fragment which binds to mesothelin, C4.4a or EGFR:
  • Figure US20140127240A1-20140508-C00340
    Figure US20140127240A1-20140508-C00341
  • In addition, according to the invention the drug-binder conjugate is especially preferably selected from the following compounds:
  • Figure US20140127240A1-20140508-C00342
    Figure US20140127240A1-20140508-C00343
  • in which
    • n stands for a number from 2 to 8, preferably 2 to 5,
    • and
    • AK1A, AK1B, AK2A, AK3 and AK4 stand for the antibodies indicated.
    • AK
      binder-drug conjugate of the following formula Ia
  • Figure US20140127240A1-20140508-C00344
  • wherein
    • n stands for a number from 2 to 8;
    • AK stands for AK1 or AK2
      • wherein
      • AK1 stands for a chimeric, human or humanized antibody or an antigen binding antibody fragment which is bound to mesothelin, EGFR or C4.4a and is bound to the group G via the sulfur atom of a cysteine radical of the binder,
      • AK2 stands for a chimeric, human or humanized antibody or an antigen binding antibody fragment which is bound to mesothelin, EGFR or C4.4a and is bound to the group G via the NH side group of a lysine radical of the binder,
    • R35 stands for methyl;
    • D stands for a group of the formula
  • Figure US20140127240A1-20140508-C00345
  • wherein
    • #3 denotes the linkage site to the nitrogen atom,
    • R1 stands for hydrogen,
    • R2 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      the ring A with the N—O group contained therein stands for
  • Figure US20140127240A1-20140508-C00346
      • wherein
      • #6 denotes the linkage site to the carbonyl group,
      • R3 stands for hydrogen,
      • R4 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
      • T1 stands for a group of the formula —C(═O)—NR8R9,
        • R8 stands for hydrogen or methyl,
        • R9 stands for hydrogen, methyl or ethyl,
      • the group §-G-L1-B-L2-§§ stands for a linker,
        • wherein
        • § denotes the linkage site to the group AK and
        • §§ denotes the linkage site to the nitrogen atom,
    • G for the case when AK=AK1 stands for a group of the formula
  • Figure US20140127240A1-20140508-C00347
      • wherein
      • #1 denotes the linkage site to the cysteine radical of the binder,
      • #2 denotes the linkage site to the group L1,
      • or
      • for the case when AK=AK2, G stands for carbonyl,
    • L1 stands for a bond, linear (C2-C6)-alkanediyl, a group of the formula
  • Figure US20140127240A1-20140508-C00348
      • wherein
      • m stands for a number of 2 or 3,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
    • B stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00349
      • wherein
      • * denotes the linkage site to L1,
      • ** denotes the linkage site to L2,
      • L3 stands for a bond or ethane-1,2-diyl,
      • L4 stands for a bond or a group of the formula
  • Figure US20140127240A1-20140508-C00350
        • wherein
        • *** denotes the linkage site to the carbonyl group,
        • **** denotes the linkage site to L2,
        • R25 stands for methyl,
        • R28 stands for hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
      • Q1 stands for piperidine-1,4-diyl,
      • R16 stands for hydrogen or methyl,
      • R17 stands for hydrogen or methyl,
      • or
      • R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
      • R21 stands for hydrogen or methyl,
      • R22 stands for hydrogen or methyl,
      • or
      • R21 and R22 together with the atoms to which they are bound form a cyclopropyl ring,
      • R23 stands for methyl,
      • R24 stands for hydrogen,
    • L2 stands for linear (C2-C6)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00351
      • wherein
      • p stands for a number from 2 to 6,
      • ##3 denotes the linkage site to the group B,
      • ##4 denotes the linkage site to the nitrogen atom,
        as well as their salts and solvates as well as the solvates of the salts.
  • Especially preferred are conjugates of the following formula,
  • Figure US20140127240A1-20140508-C00352
  • wherein
    • n stands for a number from 2 to 8, preferably 2 to 5;
    • AK stands for a human or humanized antibody or an antigen binding antibody fragment which is bound to mesothelin, EGFR or C4.4a and is bound to the group G via the sulfur atom of a cysteine radical of the binder,
    • X1 stands for NH2 or
  • Figure US20140127240A1-20140508-C00353
    • and
    • X2 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl.
  • When the toxophore is bound to a cysteine radical of the antibody, the linker
  • Figure US20140127240A1-20140508-C00354
  • may be replaced by the following linker, for example:
  • Figure US20140127240A1-20140508-C00355
  • When the toxophore is bound to an NH group of the lysine radical of the antibody, the linker may be replaced by the following:
  • Figure US20140127240A1-20140508-C00356
  • According to the invention the drug-binder conjugate is especially comprised of the following compounds, where n stands for a number from 2 to 8, preferably 2 to 5, and AK stands for a chimeric, human or humanized antibody or an antigen binding antibody fragment which binds to mesothelin, EGFR or C4.4a:
  • Figure US20140127240A1-20140508-C00357
    Figure US20140127240A1-20140508-C00358
  • In these formulas, AK1F, AK1B and AK2B may be replaced by other chimeric, human or humanized anti-C4.4a antibodies, anti-EGFR antibodies or anti-mesothelin antibodies.
  • The definitions of radicals given in the respective combinations and/or preferred combinations of radicals in detail can also be replaced by definitions of radicals of other combinations independently of the respective combinations of radicals given.
  • Combinations of two or more of the aforementioned preferred ranges are most especially preferred.
  • An additional subject matter of the present invention is a method for synthesis of the compounds of formula (Ia) according to the invention, which is characterized in that a solution of the binder in PBS buffer
    • [A] is mixed with a suitable reducing agent such as, for example, dithiothreitol or tris-(2-carboxyethyl)phosphine hydrochloride and then is reacted with a compound of formula (IIa)
  • Figure US20140127240A1-20140508-C00359
      • in which D, L1, B, L2 and R35 each have the meanings given above,
      • to form a compound of formula (I-A)
  • Figure US20140127240A1-20140508-C00360
      • in which n, AK1, D, L1, B, L2 and R35 each have the meanings given above,
    • or
    • [B] reacting it with a compound of formula (Ma)
  • Figure US20140127240A1-20140508-C00361
      • in which D, L1, B, L2 and R35 each have the meanings given above,
      • to form a compound of formula (Ia-B)
  • Figure US20140127240A1-20140508-C00362
      • in which n, AK2, D, L1, B, L2 and R35 each have the meanings given above.
  • An additional subject matter of the present invention is a method for synthesis of the compounds of formula (I) according to the invention, which is characterized in that a solution of the binder in PBS buffer
    • [A] is mixed with a suitable reducing agent such as, for example, dithiothreitol or tris-(2-carboxyethyl)phosphine hydrochloride and then is reacted with a compound of formula (II)
  • Figure US20140127240A1-20140508-C00363
      • in which D, L1, B and L2 each have the meanings given above,
      • to form a compound of formula (I-A)
  • Figure US20140127240A1-20140508-C00364
      • in which n, AK1, D, L1, B and L2 each have the meanings given above,
    • or
    • [B] reacting it with a compound of formula (III)
  • Figure US20140127240A1-20140508-C00365
      • in which D, L1, B and L2 each have the meanings given above,
      • to form a compound of formula (I-B)
  • Figure US20140127240A1-20140508-C00366
      • in which n, AK2, D, L1, B and L2 each have the meanings given above.
    Cysteine Coupling:
  • Partial reduction of the antibody and subsequent conjugation of the (partially) reduced antibody with a compound of formula (II) and/or (IIa) takes place according to methods with which those skilled in the art are familiar; see, for example, Ducry et. al., Bioconj. Chem. 2010, 21, 5 and references therein, Klussman et. al., Bioconj. Chem. 2004, 15(4), 765-773. The mild reduction of the antibody by adding 2-6 equivalence of TCEP to the antibody present in a suitable buffer solution, preferably phosphate buffer, and stirring for 30-180 minutes at temperatures between 15° C. and 40° C., preferably at room temperature. Next the conjugation is performed by adding a solution of a compound of formula (II) and/or (IIa) in DMSO, acetonitrile or DMF to the solution of the (partially) reduced antibody in PBS buffer and then reacting them at a temperature of 0° C. to +40° C., in particular from +10° C. to +30° C. for a period of 30 minutes to 6 hours, in particular one to two hours.
    • Lysine Coupling:
  • First the compounds of formula (III) and/or (IIa) or comparable activated carboxyl components are synthesized by traditional methods of peptide chemistry. These compounds are then dissolved in inert solvents such as DMSO or DMF and added to the antibody, which is preferably present in phosphate buffer at a neutral pH. The solution is stirred for 1-16 hours at a temperature between 15° C. and 40° C., preferably at RT.
  • The synthesis processes described above are then illustrated as an example on the basis of the following schemes (schemes 1 and 2):
  • Figure US20140127240A1-20140508-C00367
  • Figure US20140127240A1-20140508-C00368
  • The compounds of formula (II) in which L1 and B stand for a bond can be synthesized by reductive amination of a compound of formula (IV)
  • Figure US20140127240A1-20140508-C00369
  • in which D has the meaning given above
    in an inert solvent with a compound of formula (V)
  • Figure US20140127240A1-20140508-C00370
  • in which
    • L2A has the meaning of L2 as defined above but is shortened in the alkyl chain length by one carbon atom,
    • PG1 stands for an amino protective group such as, for example, (9H-fluorene-9-ylmethoxy) carbonyl, tert-butoxycarbonyl or benzyloxycarbonyl,
      to form a compound of formula (VI)
  • Figure US20140127240A1-20140508-C00371
  • in which D, L2 and PG1 have the meanings given above,
    splitting off the protective group PG1 from this compound by methods with which those skilled in the art are familiar and then reacting the deprotected compound in an inert solvent in the presence of a suitable base with methyl-2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate to form a compound of formula (II-A)
  • Figure US20140127240A1-20140508-C00372
  • in which D and L2 each have the meanings given above.
  • The compounds of formula (II) in which B stands for a group of the formula (B1)
  • Figure US20140127240A1-20140508-C00373
  • in which *, **, R14 and R15 each have the meanings given above,
    can be synthesized by splitting off the protective group PG1 from a compound of formula (VI) by methods with which those skilled in the art are familiar and then reacting the deprotected compound in an inert solvent in the presence of a suitable base with a compound of formula (VII)
  • Figure US20140127240A1-20140508-C00374
  • in which L1 has the meaning given above
    to form a compound of formula (II-B)
  • Figure US20140127240A1-20140508-C00375
  • in which D, L1 and L2 each have the meanings given above.
  • The compounds of formula (II) in which B stands for a group of the formula (B2)
  • Figure US20140127240A1-20140508-C00376
  • in which *, **, L3, R16 and R17 each have the meanings given above,
    can be synthesized by reductive amination of a compound of formula (IV) in an inert solvent with a compound of formula (VIII)
  • Figure US20140127240A1-20140508-C00377
  • in which
    • L2A has the meaning given above for L2 but the alkyl chain length has been shortened by one carbon atom,
      to form a compound of formula (IX)
  • Figure US20140127240A1-20140508-C00378
  • in which D and L2 have the meanings given above
    and this compound is reacted in an inert solvent in the presence of a suitable coupling reagent and a suitable base with a compound of formula (X)
  • Figure US20140127240A1-20140508-C00379
  • in which L1 and L3 each have the meanings given above,
    to form a compound of formula (II-C)
  • Figure US20140127240A1-20140508-C00380
  • in which D, L1, L2 and L3 each have the meanings given above.
  • A compound of formula (II), in which B stands for a group of the formula (B3)
  • Figure US20140127240A1-20140508-C00381
  • in which *, **, L3, R16 and R17 each have the meanings given above and
    • L4A stands for a group of the formula
  • Figure US20140127240A1-20140508-C00382
      • wherein
      • *** denotes the linkage site to the carbonyl group,
      • **** denotes the linkage site to L2,
      • R25 stands for hydrogen or methyl,
        can be synthesized by reacting a compound a compound of formula (IX) in an inert solvent in the presence of a suitable base and a suitable coupling reagent with a compound of formula (XI-A) or (XI-B)
  • Figure US20140127240A1-20140508-C00383
  • in which R25 and PG1 each have the meanings given above and
    • PG2 stands for a suitable carboxyl protective group, in particular benzyl,
      to form a compound (XII-A) and/or (XII-B)
  • Figure US20140127240A1-20140508-C00384
  • in which D, PG1, PG2 and L2 have the meanings given above,
    then splitting off the protective group PG2 from this compound using methods known to those skilled in the art and reacting the deprotected compound in an inert solvent in the presence of a suitable coupling reagent and a suitable base with a compound of formula (X), and then splitting off the protective group PG1 by methods with which those skilled in the art are familiar to form a compound of formula (II-D-A) and/or (II-D-B)
  • Figure US20140127240A1-20140508-C00385
  • in which D, L1, L2 and L3 have the meanings given above.
  • A compound of formula (II) in which B stands for a group of the formula (B4)
  • Figure US20140127240A1-20140508-C00386
  • in which *, ** each have the meanings given above and
    • Q1A stands for an N-linked four- to seven-membered heterocycle,
      can be synthesized by reacting a compound of formula (IX) in an inert solvent in the presence of a suitable base and a suitable coupling reagent with a compound of formula (XX1)
  • Figure US20140127240A1-20140508-C00387
  • in which PG1 and Q1A each have the meanings given above,
    to form a compound of formula (XXII)
  • Figure US20140127240A1-20140508-C00388
  • in which PG1, Q1A, D and L2 have the meanings given above
    then splitting off the protective group PG1 from this compound by methods with which those skilled in the art are familiar and then reacting the deprotected compound in an inert solvent in the presence of a suitable coupling reagent and a suitable base with a compound of formula (XXIII)
  • Figure US20140127240A1-20140508-C00389
  • in which L1 has the meaning given above
    to form a compound of formula (II-D)
  • Figure US20140127240A1-20140508-C00390
  • in which Q1A, D, L1 and L2 have the meanings given above.
  • The compounds of formula (III), in which L1 and B stand for a bond can be synthesized by reacting a compound of formula (IX) with N-hydroxysuccinimide in an inert solvent in the presence of a suitable coupling regent and a suitable base to form a compound of formula (III-A):
  • Figure US20140127240A1-20140508-C00391
  • in which D and L2 each have the meanings given above.
  • The compounds of formula (III), in which L1 stands for a bond and B stands for a group of the formula (B5A)
  • Figure US20140127240A1-20140508-C00392
  • in which *, ** and P each have the meanings given above and
    • Q2A stands for a three- to seven-membered carbocycle,
      can be synthesized by reacting a compound of formula (IX) in an inert solvent in the presence of a suitable coupling reagent and a suitable base with a compound of formula (XIII)
  • Figure US20140127240A1-20140508-C00393
  • in which P, Q2A and PG2 each have the meanings given above,
    to form a compound of formula (XIV)
  • Figure US20140127240A1-20140508-C00394
  • in which D, P, Q2A, L2 and PG2 each have the meanings given above,
    splitting off the protective group PG2 from this compound by methods with which those skilled in the art are familiar and then reacting the deprotected compound in an inert solvent in the presence of a suitable base with N-hydroxysuccinimide to form a compound of formula (III-B)
  • Figure US20140127240A1-20140508-C00395
  • in which D, P, Q2A and L2 each have the meanings given above.
  • The compounds of formula (III), in which L1 stands for a bond and B stands for a group of the formula (B6)
  • Figure US20140127240A1-20140508-C00396
  • in which *, **, R18, R19 and R20 each have the meanings given above,
    can be synthesized by reacting a compound of formula (IX) in an inert solvent in the presence of a suitable coupling reagent and a suitable base with a compound of formula (XV)
  • Figure US20140127240A1-20140508-C00397
  • in which R18, R19, R20 and PG2 each have the meanings given above,
    to form a compound of formula (XVI)
  • Figure US20140127240A1-20140508-C00398
  • in which D, R18, R19, R20, L2 and PG2 each have the meanings given above,
    then splitting off the protective group PG2 from this compound by methods with which those skilled in the art are familiar and then reacting the deprotected compound in an inert solvent in the presence of a suitable coupling reagent and a suitable base with N-hydroxysuccinimide to form a compound of formula (III-C)
  • Figure US20140127240A1-20140508-C00399
  • in which D, R18, R19, R20 and L2 each have the meanings given above.
  • The compounds of formula (III), in which L1 stands for a bond and B stands for a group of formula (B7)
  • Figure US20140127240A1-20140508-C00400
  • in which *, **, R21 and R22 each have the meanings given above,
    may be synthesized by splitting off the protective group PG1 from a compound of formula (VI) by methods with which those skilled in the art are familiar, and then reacting the resulting deprotected compound in an inert solvent in the presence of a suitable base with a compound of formula (XVII)
  • Figure US20140127240A1-20140508-C00401
  • in which R21 and R22 each have the meanings given above,
    to form a compound of formula (III-D)
  • Figure US20140127240A1-20140508-C00402
  • in which D, R21, R22 and L2 each have the meanings given above.
  • The compounds of formula (III) in which B stands for a group of the formula (B8)
  • Figure US20140127240A1-20140508-C00403
  • in which *, **, R23 and R24 each have the meanings given above,
    can by synthesized by reacting a compound of formula (IX) in an inert solvent in the presence of a suitable coupling reagent and a suitable base with a compound of formula (XVIII)
  • Figure US20140127240A1-20140508-C00404
  • in which R23, R24 and PG1 each have the meanings given above
    to form a compound of formula (XIX)
  • Figure US20140127240A1-20140508-C00405
  • in which D, R23, R24, L2 and PG1 each have the meanings given above,
    splitting off the protective group PG1 from this compound by methods with which those skilled in the art are familiar and then reacting the deprotected compound in an inert solvent in the presence of a suitable coupling reagent and a suitable base with a compound of formula (XX)
  • Figure US20140127240A1-20140508-C00406
  • in which
    • L1A stands for linear (C1-C10)-alkanediyl or for a group of the formula
  • Figure US20140127240A1-20140508-C00407
      • wherein
      • m stands for a number from 2 to 6,
      • ##1 denotes the linkage site to the group G,
      • ##2 denotes the linkage site to the group B,
      • wherein (C1-C10)-alkanediyl may be substituted with 1 to 4 methyl substituents,
      • and
      • wherein two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-relation to one another may be bridged to form a (C3-C6)-cycloalkyl ring or a phenyl ring including the carbon atoms optionally situated between them,
        to form a compound of formula (III-E)
  • Figure US20140127240A1-20140508-C00408
  • in which D, R23, R24, L1A and L2 each have the meanings given above.
  • The compounds of formula (III), in which B stands for a group of the formula (B5B)
  • Figure US20140127240A1-20140508-C00409
  • in which * and ** each have the meanings given above and
    • Q2B stands for a N-linked four- to seven-membered heterocycle,
      can be synthesized by reacting a compound of formula (IX) in an inert solvent in the presence of a suitable base and a suitable coupling reagent with a compound of formula (XXIV
  • Figure US20140127240A1-20140508-C00410
  • in which PG1 and Q2B each have the meanings given above,
    to form a compound of formula (XXV)
  • Figure US20140127240A1-20140508-C00411
  • in which PG1, Q2B, D and L2 have the meanings given above,
    splitting off the protective group PG1 from this compound by methods with which those skilled in the art are familiar
    and then reacting the deprotected compound in an inert solvent in the presence of a suitable base with a compound of formula (XX) to yield a compound of formula (III-F)
  • Figure US20140127240A1-20140508-C00412
  • in which Q2B, D, L1A and L2 have the meanings given above.
  • The reactions (IV)+(V)→(VI) and (IV)+(VIII)→(IX) take place in the usual solvents that are typically used for reductive amination and are inert under the reaction conditions, optionally in the presence of an acid and/or a water extracting agent as the catalyst. Such solvents include, for example, alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol or tert-butanol, ethers such as tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane or bis-(2-methoxyethyl)ether or other solvents such as dichloromethane, 1,2-dichloroethane, N,N-dimethyl formamide and water. It is also possible to use mixtures of these solvents. The preferred solvent is a 1,4-dioxane/water mixture that is used with the addition of acetic acid or dilute hydrochloric acid as the catalyst.
  • Complex borohydrides in particular are suitable reducing agents for this reaction, such as, for example, sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, tetra-n-butylammonium borohydride or borane-pyridine complex. Sodium cyanoborohydride or borane pyridine complex is preferably used.
  • The reactions (IV)+(V)→(VI) and (IV)+(VIII)→(IX) usually take place in a temperature range from 0° C. to +120° C., preferably at +50° C. to +100° C. The reactions may be performed at normal, elevated or reduced pressure (e.g., from 0.5 to 5 bar). It is customary to work under normal pressure.
  • The coupling reactions described above (IX)+(X)→(II-C), (XII-A) and/or (XII-B)+(X)→(II-D-A) and/or (II-D-B), (IX)+(XIII)→(XIV), (IX)+(XV)→(XVI) and (XXII)+(XXIII)→(II-D) (amide formed from the respective amine and carboxylic acid components) are performed according to the standard methods of peptide chemistry (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 [Amino Acids, Peptides, Proteins], Verlag Chemie, Weinheim, 1982).
  • Inert solvents for these coupling reactions include, for example, ethers like diethyl ether, diisopropyl ether, tert-butylmethyl ether, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane or bis-(2-methoxyethyl)ether, hydrocarbons such as benzene, toluene, xylene, pentane, hexane, heptane, cyclohexane or petroleum fractions, halohydrocarbons such as dichloromethane, trichloromethane, tetrachloromethane, 1,2-dichloroethane, trichloroethylene or chlorobenzene or dipolar aprotic solvents such as acetone, methyl ethyl ketone, acetonitrile, ethyl acetate, pyridine, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), N,N′-dimethylpropylene urea (DMPU) or N-methylpyrrolidinone (NMP). It is likewise possible to use mixtures of such solvents. N,N-Dimethylformamide is preferred.
  • Suitable activation/condensation agents for these coupling reactions include, for example, carbodiimides such as N,N′-diethyl, N,N′-dipropyl, N,N′-diisopropyl, N,N′-dicyclohexyl-carbodiimide (DCC) or N-(3-dimethylaminoisopropyl)-N′-ethylcarbodiimide hydrochloride (EC), phosgene derivatives such as N,N′-carbonyldiimidazole (CDI) or isobutyl chloroformate, 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium 3-sulfate or 2-tert-butyl-5-methylisoxazolium perchlorate, acylamino compounds such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, phosphorus compounds such as propane phosphonic acid anhydride, cyanophosphonic acid diethyl ester, bis-(2-oxo-3-oxazolidinyl)phosphoryl chloride, benzo-triazole-1-yloxy-tris-(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yloxytris-(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), or uronium compounds such as O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), O-(benzo-triazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), 2-(2-oxo-1-(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) or O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), optionally in combination with additional excipients such as 1-hydroxybenzotriazole (HOBt) or N-hydroxysuccinimide (HOSu) as well as bases such as alkali carbonates, e.g., sodium or potassium carbonate or tertiary amine bases such as triethylamine, N-methylmorpholine, N-methylpiperidine, N,N-diisopropylethylamine, pyridine or 4-N,N-dimethylaminopyridine.
  • Within the context of the present invention, the preferred activation/condensation agents for such coupling reactions include N-(3-dimethylaminoisopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) in combination with 1-hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine or O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) likewise in combination with N,N-diisopropylethylamine
  • The coupling reactions (IX)+(X)→(II-C), (XII-A) and/or (XII-B)+(X)→(II-D-A) and/or (II-D-B), (IX)+(XIII)→(XIV), (IX)+(XV)→(XVI) and (XXII)+(XXIII)→(II-D) are usually performed in a temperature range from −20° C. to +60° C., preferably at 0° C. to +40° C. The reactions may be performed under normal, elevated or reduced pressure (e.g., from 0.5 to 5 bar). It is customary to work under normal pressure.
  • The ester-forming reactions (IX)+(XVIII)→(XII) and (IX)+(XI-A) and/or (XI-B)→(XII-A) and/or (XII-B), (IX)+(XXIV)→(XXV) as well as (IX)+(XXI)→(XXII) take place like the amide coupling reactions described above. These reactions preferably take place in dichloromethane using N-(3-dimethylaminoisopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 4-dimethylaminopyridine at a temperature of +50° C. to 100° C. under normal pressure.
  • The functional groups optionally present in the compounds—such as amino, hydroxyl and carboxyl groups in particular—may also be present in a temporarily protected form in the process steps described above, if this is expedient or necessary. Such protective groups are introduced and removed according to conventional methods known in peptide chemistry (see, for example, T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, Wiley, New York, 1999; M. Bodanszky and A. Bodanszky, The Practice of Peptide Synthesis, Springer Verlag, Berlin, 1984). In the presence of multiple protected groups, their re-release may optionally be performed simultaneously in a one-pot reaction or also in separate reaction steps.
  • The preferred amino protective groups PG1 include tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Z) or (9H-fluorene-9-ylmethoxy)carbonyl (Fmoc); tert-butyl or benzyl is preferably used as the protective group PG2 for a hydroxyl or carboxyl function. A tert-butyl or tert-butoxycarbonyl group is usually split off by treatment with a strong acid such as hydrochloric acid, hydrobromic acid or trifluoroacetic acid in an inert solvent such as diethyl ether, 1,4-dioxane, dichloromethane or acetic acid. This reaction may optionally also take place without the addition of an inert solvent. In the case of benzyl or benzyloxycarbonyl as the protective group, such a protective group is preferably removed by hydrogenolysis in the presence of a suitable palladium catalyst such as, for example, palladium on activated carbon. The (9H-fluorene-9-ylmethoxy)carbonyl group is generally split off with the help of a secondary amine base such as diethylamine or piperidine.
  • The reaction (VI)→(II-A) takes place in a solvent that is inert under the reaction conditions, such as, for example, ethers, e.g., tetrahydrofuran, 1,4-dioxane, 1,2-dimetoxyethane or bis-(2-methoxyethyl)ether, alcohols such as methanol, ethanol, isopropanol, n-butanol or tert-butanol or dipolar aprotic solvents such as acetone, methyl ethyl ketone, acetonitrile, ethyl acetate, pyridine, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethyl-acetamide (DMA), N,N′-dimethylpropylene urea (DMPU) or N-methylpyrrolidinone (NMP) or water. It is likewise possible to use mixtures of such solvents. A mixture of 1,4-dioxane and water is preferably used.
  • Suitable bases for the reaction (VI)→(II-A) include, for example, alkali carbonates such as potassium carbonate, sodium carbonate or lithium carbonate, alkali bicarbonates such as sodium or potassium bicarbonate or alkali alcoholates such as sodium methanolate, sodium ethanolate or potassium tert-butylate. Sodium bicarbonate is preferred.
  • The reaction (VI)→(II-A) takes place in a temperature range from 0° C. to +50° C., preferably at +10° C. to +30° C. The reaction may be performed under normal, elevated or reduced pressure (e.g., from 0.5 to 5 bar). It is customary to work under normal pressure.
  • The reaction (VI)+(VII)→(II-B) takes place in a solvent that is inert under the reaction conditions such as, for example, ethers, e.g., tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane or bis-(2-methoxyethyl)ether, alcohols such as methanol, ethanol, isopropanol, n-butanol or tert-butanol or dipolar aprotic solvents like acetone, methyl ethyl ketone, acetonitrile, ethyl acetate, pyridine, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), N,N′-dimethylpropylene urea (DMPU) or N-methylpyrrolidinone (NMP) or water. It is also possible to use mixtures of such solvents. DMF is preferred.
  • Suitable bases for the reaction (VI)+(VII)→(II-B) include, for example, tertiary amine bases such as triethylamine, N-methylmorpholine, N-methylpiperidine, N,N-diisopropylethylamine, pyridine or 4-N,N-dimethylaminopyridine. N,N-Diisopropylethylamine is preferred.
  • The reaction (VI)+(VII)→(II-B) takes place in a temperature range from 0° C. to +50° C., preferably at +10° C. to +30° C. The reaction may take place under normal, elevated or reduced pressure (e.g., from 0.5 to 5 bar). It is customary to work under normal pressure.
  • The reactions (IX)→(III-A), (XIV)→(III-B) and (XVI)→(III-C) as well as (VI)+(XVII)→(III-D), (XIX)+(XX)→(III-E) and (XXV)+(XX)→(III-F) take place in a solvent that is inert under the reaction conditions. Suitable solvents include, for example, ethers such as diethyl ether, diisopropyl ether, tert-butylmethyl ether, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane or bis-(2-methoxyethyl)ether, hydrocarbons such as benzene, toluene, xylene, pentane, hexane, heptane, cyclohexane or petroleum fractions, halohydrocarbons such as dichloromethane, trichloromethane, tetrachloromethane, 1,2-dichloroethane, trichloroethylene or chlorobenzene or dipolar aprotic solvents such as acetone, methyl ethyl ketone, acetonitrile, ethyl acetate, pyridine, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), N,N′-dimethylpropylene urea (DMPU) or N-methylpyrrolidinone (NMP). It is likewise possible to use mixtures of such solvents. N,N-Dimethylformamide is preferred.
  • Suitable bases for these reactions include, for example, tertiary amines like triethylamine, N-methylmorpholine, N-methylpiperidine, N,N-diisopropylethylamine, pyridine or 4-N,N-di-methylaminopyridine. N,N-Diisopropylethylamine is preferred, optionally with the addition of 4-N,N-dimethylaminopyridine.
  • The reactions (IX)→(III-A), (XIV)→(III-B) and (XVI)→(III-C) as well as (VI)+(XVII)→(III-D) and (XIX)+(XX)→(III-E) take place in a temperature range from 0° C. to +50° C., preferably at +10° C. to +30° C. The reaction may be carried out under normal, elevated or reduced pressure (e.g., from 0.5 to 5 bar). It is customary to work under normal pressure.
  • The compounds of formulas (II), (III), (I-A) and/or (I-B) are subsets of the compounds of formulas (IIa), (IIIa), (Ia-A) and/or (Ia-B), where R35 stands for methyl. Compounds (IIa) and (IIIa) are synthesized as in the synthesis of the compound of formula (II) and (III) as described above.
  • The methods described above are illustrated by the following synthesis schemes (schemes 3 through 13, 18) as examples:
  • Figure US20140127240A1-20140508-C00413
  • Figure US20140127240A1-20140508-C00414
  • Figure US20140127240A1-20140508-C00415
  • Figure US20140127240A1-20140508-C00416
  • Figure US20140127240A1-20140508-C00417
  • Figure US20140127240A1-20140508-C00418
  • Figure US20140127240A1-20140508-C00419
  • Figure US20140127240A1-20140508-C00420
  • Figure US20140127240A1-20140508-C00421
  • Figure US20140127240A1-20140508-C00422
  • Figure US20140127240A1-20140508-C00423
  • Figure US20140127240A1-20140508-C00424
  • The compounds of formula (IV) may be synthesized from commercially available amino acid building blocks or those known from 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) as with the processes known from the literature, by using the standard methods of peptide chemistry and as described in the present experimental part. The following synthesis schemes (schemes 14 through 16) illustrate this synthesis process as an example.
  • Figure US20140127240A1-20140508-C00425
  • Figure US20140127240A1-20140508-C00426
  • Figure US20140127240A1-20140508-C00427
  • The compounds of formulas (XI), (XIII), (XV), (XVII) and (XXI), including where applicable chiral or diastereomeric forms thereof are commercially available or have been described as such in the literature or can be synthesized by methods like those published in the literature in a manner that would be self-evident to those skilled in the art. Several detailed publications and specifications in the literature regarding the synthesis of the starting materials can also be found in the experimental part in the section for synthesis of the starting compounds and intermediates.
  • The compounds of formulas (V), (VII), (VIII), (X), (XVIII), (XX) and (XXIII) including where appropriate chiral or diastereomeric forms thereof are known in the literature or they can be synthesized by methods like those described in the literature in a manner obvious to those skilled in the art. Numerous detailed specifications as well as references from the literature regarding the synthesis of the starting materials can be found in the experimental part in the section on synthesis of the starting compounds and intermediates.
  • Alternatively individually steps of the synthesis sequence may be performed in a different order. This procedure is illustrated in the following synthesis schemes (schemes 17, 19 and 20) as an example.
  • Figure US20140127240A1-20140508-C00428
    Figure US20140127240A1-20140508-C00429
  • Figure US20140127240A1-20140508-C00430
  • Figure US20140127240A1-20140508-C00431
    Figure US20140127240A1-20140508-C00432
  • In one embodiment, the binder is bound to a target molecule that is present on a cancer cell. In a preferred embodiment, the binder binds to a cancer target molecule.
  • In another preferred embodiment, the target molecule is a selected cancer target molecule.
  • In an especially 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 are known to those skilled in the art. Examples of these are given below.
  • Examples of cancer target molecules include:
  • (1) EGF receptor (NCBI reference sequence NP—005219.2)
    Sequence (1210 amino acids):
  • >gi|29725609|ref|NP_005219.2| epidermal growth 
    factor receptor isoform a precursor [Homo sapiens]
    MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLS
    LQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIP
    LENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRF
    SNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCW
    GAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLV
    CRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYV
    VTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLS
    INATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKE
    ITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGL
    RSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCK
    ATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFV
    ENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM
    GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGM
    VGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPN
    QALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREA
    TSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLD
    YVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQH
    VKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSY
    GVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKC
    WMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRA
    LMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACI
    DRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKR
    PAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNST
    FDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRV
    APQSSEFIGA
  • The extracellular domain is underlined for emphasis.
  • (2) Mesothelin (SwissProt reference Q13421-3)
    Sequence (622 amino acids):
  • >sp|Q13421-3|MSLN_HUMAN isoform 2 of mesothelin  
    OS = Homo sapiens GN = MSLN
    MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDG
    VLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQ
    LRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVD
    LLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAES
    AEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRG
    LLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKT
    ACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLD
    VLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLE
    VNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELS
    SVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFL
    GGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGL
    KAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGT
    PCLLGPGPVLTVLALLLASTLA

    where mesothelin is coded by amino acids 296-598. Amino acids 37-286 code for “megakaryocyte potentiating factor.” Mesothelin is anchored in the cell membrane by a GPI anchor and is localized extracellularly.
    (3) Carboanhydrase IX (SwissProt reference Q16790)
    Sequence (459 amino acids):
  • >sp|Q16790|CAH9_HUMAN Carbonic anhydrase 9 OS =
    Homo sapiens GN = CA9 PE = 1 SV = 2
    MAPLCPSPWLPLLIPAPAPGLTVQLLLSLLLLVPVHPQRLPRMQEDSPLG
    GGSSGEDDPLGEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKP
    KSEEEGSLKLEDLPTVEAPGDPQEPQNNAHRDKEGDDQSHWRYGGDPPWP
    RVSPACAGRFQSPVDIRPQLAAFCPALRPLELLGFQLPPLPELRLRNNGH
    SVQLTLPPGLEMALGPGREYRALQLHLHWGAAGRPGSEHTVEGHRFPAEI
    HVVHLSTAFARVDEALGRPGGLAVLAAFLEEGPEENSAYEQLLSRLEEIA
    EEGSETQVPGLDISALLPSDFSRYFQYEGSLTTPPCAQGVIWTVFNQTVM
    LSAKQLHTLSDTLWGPGDSRLQLNFRATQPLNGRVIEASFPAGVDSSPRA
    AEPVQLNSCLAAGDILALVFGLLFAVTSVAFLVQMRRQHRRGTKGGVSYR
    PAEVAETGA
  • The extracellular domain is underlined for emphasis.
  • (4) C4.4a (NCBI reference sequence NP—055215.2; synonym LYPD3)
    Sequence (346 amino acids):
  • >gi|93004088|ref|NP_055215.2| 1y6/PLAUR domain-
    containing protein 3 precursor [Homo sapiens]
    MDPARKAGAQAMIWTAGWLLLLLLRGGAQALECYSCVQKADDGCSPNKMK
    TVKCAPGVDVCTEAVGAVETIHGQFSLAVRGCGSGLPGKNDRGLDLHGLL
    AFIQLQQCAQDRCNAKLNLTSRALDPAGNESAYPPNGVECYSCVGLSREA
    CQGTSPPVVSCYNASDHVYKGCFDGNVTLTAANVTVSLPVRGCVQDEFCT
    RDGVTGPGFTLSGSCCQGSRCNSDLRNKTYFSPRIPPLVRLPPPEPTTVA
    STTSVTTSTSAPVRPTSTTKPMPAPTSQTPRQGVEHEASRDEEPRLTGGA
    AGHQDRSNSGQYPAKGGPQQPHNKGCVAPTAGLAALLLAVAAGVLL
  • The matured extracellular domain is underlined for emphasis (SEQ ID NO: 1).
  • (5) CD52 (NCBI reference sequence NP—001794.2)
  • >gi|68342030|ref|NP_001794.2| CAMPATH-1 antigen 
    precursor [Homo sapiens]
    MKRFLFLLLTISLLVMVQIQTGLSGQNDTSQTSSPSASSNISGGIFLFFV
    ANAIIHLFCFS

    (6) HER2 (NCBI reference sequence NP—004439.2)
  • >gi|54792096|ref|NP_004439.2| receptor tyrosine-
    protein kinase erbB-2 isoform a [Homo sapiens]
    MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLY
    QGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLR
    IVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILK
    GGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCK
    GSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHS
    DCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACP
    YNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHL
    REVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVF
    ETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGI
    SWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRP
    EDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGL
    PREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARC
    PSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASP
    LTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPL
    TPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPV
    AIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQL
    MPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARN
    VLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFT
    HQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTID
    VYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPL
    DSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSS
    STRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQS
    LPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPP
    SPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQ
    GGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLG
    LDVPV

    (7) CD20 (NCBI reference sequence NP—068769.2)
  • >gi|23110987|ref|NP_068769. 2| B-lymphocyte 
    antigen CD20 [Homo sapiens]
    MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESK
    TLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSL
    LAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKME
    SLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIF
    AFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLT
    ETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP

    (8) The lymphocyte activating antigen CD30 (SwissProt ID P28908)
  • >gi|68348711|ref|NP_001234.2| tumor necrosis 
    factor receptor superfamily member 8 isoform 1 
    precursor [Homo sapiens]
    MRVLLAALGLLFLGALRAFPQDRPFEDTCHGNPSHYYDKAVRRCCYRCPM
    GLFPTQQCPQRPTDCRKQCEPDYYLDEADRCTACVTCSRDDLVEKTPCAW
    NSSRVCECRPGMFCSTSAVNSCARCFFHSVCPAGMIVKFPGTAQKNTVCE
    PASPGVSPACASPENCKEPSSGTIPQAKPTPVSPATSSASTMPVRGGTRL
    AQEAASKLTRAPDSPSSVGRPSSDPGLSPTQPCPEGSGDCRKQCEPDYYL
    DEAGRCTACVSCSRDDLVEKTPCAWNSSRTCECRPGMICATSATNSRARC
    VPYPICAAETVTKPQDMAEKDTTFEAPPLGTQPDCNPTPENGEAPASTSP
    TQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVG
    SSAFLLCHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSG
    ASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDL
    PEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEE
    ELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK

    (9) The lymphocyte adhesion molecule CD22 (SwissProt ID P20273)
  • >gi|157168355|ref|NP_001762.2| B-cell receptor
    CD22 isoform 1 precursor [Homo sapiens]
    MHLLGPWLLLLVLEYLAFSDSSKWVFEHPETLYAWEGACVWIPCTYRALD
    GDLESFILFHNPEYNKNTSKFDGTRLYESTKDGKVPSEQKRVQFLGDKNK
    NCTLSIHPVHLNDSGQLGLRMESKTEKWMERIHLNVSERPFPPHIQLPPE
    IQESQEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQAAVTSTSLTIKSVFT
    RSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKHTPKLEIKVTP
    SDAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVT
    KDQSGKYCCQVSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPAVEGSQVE
    FLCMSLANPLPTNYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAEN
    ILGTGQRGPGAELDVQYPPKKVTTVIQNPMPIREGDTVTLSCNYNSSNPS
    VTRYEWKPHGAWEEPSLGVLKIQNVGWDNTTIACAACNSWCSWASPVALN
    VQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEVQFFWEKNGRLL
    GKESQLNFDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSM
    SPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSLPYHSQKLRLEPVK
    VQHSGAYWCQGTNSVGKGRSPLSTLTVYYSPETIGRRVAVGLGSCLAILI
    LAICGLKLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLG
    CYNPMMEDGISYTTLRFPEMNIPRTGDAESSEMQRPPPDCDDTVTYSALH
    KRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYVILKH

    (10) The myeloid cell surface antigen CD33 (SwissProt ID P20138)
  • >gi|130979981|ref|NP_001763.3| myeloid cell
    surface antigen CD33 isoform 1 precursor
    [Homo sapiens]
    MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYY
    DKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNN
    CSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIP
    GTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIIT
    PRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGK
    QETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTH
    PTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNP
    SKDTSTEYSEVRTQ

    (11) The transmembrane glycoprotein NMB (SwissProt ID Q14956)
  • >gi|52694752|ref|NP_001005340.1| transmembrane
    glycoprotein NMB isoform a precursor
    [Homo sapiens]
    MECLYYFLGFLLLAARLPLDAAKRFHDVLGNERPSAYMREHNQLNGWSSD
    ENDWNEKLYPVWKRGDMRWKNSWKGGRVQAVLTSDSPALVGSNITFAVNL
    IFPRCQKEDANGNIVYEKNCRNEAGLSADPYVYNWTAWSEDSDGENGTGQ
    SHHNVFPDGKPFPHHPGWRRWNFIYVFHTLGQYFQKLGRCSVRVSVNTAN
    VTLGPQLMEVTVYRRHGRAYVPIAQVKDVYVVTDQIPVFVTMFQKNDRNS
    SDETFLKDLPIMFDVLIHDPSHFLNYSTINYKWSFGDNTGLFVSTNHTVN
    HTYVLNGTFSLNLTVKAAAPGPCPPPPPPPRPSKPTPSLATTLKSYDSNT
    PGPAGDNPLELSRIPDENCQINRYGHFQATITIVEGILEVNIIQMTDVLM
    PVPWPESSLIDFVVTCQGSIPTEVCTIISDPTCEITQNTVCSPVDVDEMC
    LLTVRRTFNGSGTYCVNLTLGDDTSLALTSTLISVPDRDPASPLRMANSA
    LISVGCLAIFVTVISLLVYKKHKEYNPIENSPGNVVRSKGLSVFLNRAKA
    VFFPGNQEKDPLLKNQEFKGVS

    (12) The adhesion molecule CD56 (SwissProt ID P13591)
  • >gi|94420689|ref|NP_000606.3| neural cell
    adhesion molecule 1 isoform 1 [Homo sapiens]
    MLQTKDLIWTLFFLGTAVSLQVDIVPSQGEISVGESKFFLCQVAGDAKDK
    DISWFSPNGEKLTPNQQRISVVWNDDSSSTLTIYNANIDDAGIYKCVVTG
    EDGSESEATVNVKIFQKLMFKNAPTPQEFREGEDAVIVCDVVSSLPPTII
    WKHKGRDVILKKDVRFIVLSNNYLQIRGIKKTDEGTYRCEGRILARGEIN
    FKDIQVIVNVPPTIQARQNIVNATANLGQSVTLVCDAEGFPEPTMSWTKD
    GEQIEQEEDDEKYIFSDDSSQLTIKKVDKNDEAEYICIAENKAGEQDATI
    HLKVFAKPKITYVENQTAMELEEQVTLTCEASGDPIPSITWRTSTRNISS
    EEKTLDGHMVVRSHARVSSLTLKSIQYTDAGEYICTASNTIGQDSQSMYL
    EVQYAPKLQGPVAVYTWEGN
    QVNITCEVFAYPSATISWFRDGQLLPSSNYSNIKIYNTPSASYLEVTPDS
    ENDFGNYNCTAVNRIGQESLEFILVQADTPSSPSIDQVEPYSSTAQVQFD
    EPEATGGVPILKYKAEWRAVGEEVWHSKWYDAKEASMEGIVTIVGLKPET
    TYAVRLAALNGKGLGEISAASEFKTQPVQGEPSAPKLEGQMGEDGNSIKV
    NLIKQDDGGSPIRHYLVRYRALSSEWKPEIRLPSGSDHVMLKSLDWNAEY
    EVYVVAENQQGKSKAAHFVFRTSAQPTAIPANGSPTSGLSTGAIVGILIV
    IFVLLLVVVDITCYFLNKCGLFMCIAVNLCGKAGPGAKGKDMEEGKAAFS
    KDESKEPIVEVRTEEERTPNHDGGKHTEPNETTPLTEPEKGPVEAKPECQ
    ETETKPAPAEVKTVPNDATQTKENESKA

    (13) The surface molecule CD70 (SwissProt ID P32970)
  • >gi|4507605|ref|NP_001243.1| CD70 antigen
    [Homo sapiens]
    MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPL
    ESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHR
    DGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQG
    CTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP

    (14) The surface molecule CD74 (SwissProt ID P04233)
  • >gi|10835071|ref|NP_004346.1| HLA class II
    histocompatibility antigen gamma chain
    isoform b [Homo sapiens]
    MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALY
    TGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKP
    PKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNAD
    PLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQ
    KPTDAPPKESLELEDPSSGLGVTKQDLGPVPM

    (15) The B-lymphocyte antigen CD19 (SwissProt ID P15391)
  • >gi|296010921|ref|NP_001171569.1| B-lymphocyte
    antigen CD19 isoform 1 precursor [Homo sapiens]
    MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQL
    TWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPG
    PPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGK
    LMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSC
    GVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPR
    ATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYL
    IFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGN
    VLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVG
    PEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPE
    DEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLAGSQS
    YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGR
    MGTWSTR

    (16) The surface protein mucin 1 (SwissProt ID P15941)
  • >gi|65301117|ref|NP_002447.4|mucin-1 isoform
    1 precursor [Homo sapiens]
    MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTE
    KNALSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYK
    QGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY
    NLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALA
    VCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKV
    SAGNGGSSLSYTNPAVAATSANL

    (17) The surface protein CD138 (SwissProt ID P18827)
  • >gi|29568086|ref|NP_002988.3| syndecan-1
    precursor [Homo sapiens]
    MRRAALWLWLCALALSLQPALPQIVATNLPPEDQDGSGDDSDNFSGSGAG
    ALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAGEGPK
    EGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHQASTTTATTAQEPAT
    SHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQL
    PAAEGSGEQDFTFETSGENTAVVAVEPDRRNQSPVDQGATGASQGLLDRK
    EVLGGVIAGGLVGLIFAVCLVGFMLYRMKKKDEGSYSLEEPKQANGGAYQ
    KPTKQEEFYA

    (18) Integrin alphaV (GenBank Accession No. NP—002201.1)
  • >gi|4504763|ref|NP_002201.1| integrin
    alpha-V isoform 1 precursor [Homo sapiens]
    MAFPPRRRLRLGPRGLPLLLSGLLLPLCRAFNLDVDSPAEYSGPEGSYFG
    FAVDFFVPSASSRMFLLVGAPKANTTQPGIVEGGQVLKCDWSSTRRCQPI
    EFDATGNRDYAKDDPLEFKSHQWFGASVRSKQDKILACAPLYHWRTEMKQ
    EREPVGTCFLQDGTKTVEYAPCRSQDIDADGQGFCQGGFSIDFTKADRVL
    LGGPGSFYWQGQLISDQVAEIVSKYDPNVYSIKYNNQLATRTAQAIFDDS
    YLGYSVAVGDFNGDGIDDFVSGVPRAARTLGMVYIYDGKNMSSLYNFTGE
    QMAAYFGFSVAATDINGDDYADVFIGAPLFMDRGSDGKLQEVGQVSVSLQ
    RASGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGFNDIAIAAPYGGEDKK
    GIVYIFNGRSTGLNAVPSQILEGQWAARSMPPSFGYSMKGATDIDKNGYP
    DLIVGAFGVDRAILYRARPVITVNAGLEVYPSILNQDNKTCSLPGTALKV
    SCFNVRFCLKADGKGVLPRKLNFQVELLLDKLKQKGAIRRALFLYSRSPS
    HSKNMTISRGGLMQCEELIAYLRDESEFRDKLTPITIFMEYRLDYRTAAD
    TTGLQPILNQFTPANISRQAHILLDCGEDNVCKPKLEVSVDSDQKKIYIG
    DDNPLTLIVKAQNQGEGAYEAELIVSIPLQADFIGVVRNNEALARLSCAF
    KTENQTRQVVCDLGNPMKAGTQLLAGLRFSVHQQSEMDTSVKFDLQIQSS
    NLFDKVSPVVSHKVDLAVLAAVEIRGVSSPDHIFLPIPNWEHKENPETEE
    DVGPVVQHIYELRNNGPSSFSKAMLHLQWPYKYNNNTLLYILHYDIDGPM
    NCTSDMEINPLRIKISSLQTTEKNDTVAGQGERDHLITKRDLALSEGDIH
    TLGCGVAQCLKIVCQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSL
    KSSASFNVIEFPYKNLPIEDITNSTLVTTNVTWGIQPAPMPVPVWVIILA
    VLAGLLLLAVLVFVMYRMGFFKRVRPPQEEQEREQLQPHENGEGNSET

    (19) The teratocarcinoma-derived growth factor 1 protein TDGF1 (GenBank Accession No.: NP—003203.1)
  • >gi|4507425|ref|NP_003203.1| teratocarcinoma-
    derived growth factor 1 isoform 1 precursor
    [Homo sapiens]
    MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYLAFRDDS
    IWPQEEPAIRPRSSQRVPPMGIQHSKELNRTCCLNGGTCMLGSFCACPPS
    FYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQAFLPGCD
    GLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY

    (20) The prostate-specific membrane antigen PSMA (SwissProt ID: □04609)
  • >gi|4758398|ref|NP_004467.1| glutamate
    carboxypeptidase 2 isoform 1 [Homo sapiens]
    MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEAT
    NITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQW
    KEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPG
    YENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKI
    VIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPG
    GGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYY
    DAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTN
    EVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVR
    SFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYI
    NADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKK
    SPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYP
    LYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDY
    AVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERL
    QDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKY
    AGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA

    (21) Tyrosine protein kinase EPHA2 (SwissProt ID: P29317)
  • >gi|32967311|ref|NP_004422.2| ephrin type-A
    receptor 2 precursor [Homo sapiens]
    MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGK
    GWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKF
    TVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEI
    TVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKK
    CPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVD
    GEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPS
    PEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTP
    PQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVS
    DLEPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTT
    SLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPD
    TTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIGGVAVGVVLLLV
    LAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTYVDPHTYEDPNQA
    VLKFTTEIHPSCVTRQKVIGAGEFGEVYKGMLKTSSGKKEVPVAIKTLKA
    GYTEKQRVDFLGEAGIMGQFSHHNIIRLEGVISKYKPMMIITEYMENGAL
    DKFLREKDGEFSVLQLVGMLRGIAAGMKYLANMNYVHRDLAARNILVNSN
    LVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWTAPEAISYRKFTSASDV
    WSFGIVMWEVMTYGERPYWELSNHEVMKAINDGFRLPTPMDCPSAIYQLM
    MQCWQQERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSG
    SEGVPFRTVSEWLESIKMQQYTEHFMAAGYTAIEKVVQMTNDDIKRIGVR
    LPGHQKRIAYSLLGLKDQVNTVGIPI

    (22) The surface protein SLC44A4 (GenBank Accession No. NP—001171515)
  • >gi|295849282|ref|NP_001171515.1| choline
    transporter-like protein 4 isoform 2
    [Homo sapiens]
    MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIV
    VGIVAWLYGDPRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNI
    ISVAENGLQCPTPQTVITSLQQELCPSFLLPSAPALGRCFPWTNVTPPAL
    PGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVALVL
    SLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQ
    LGFTTNLSAYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLK
    EASKAVGQMMSTMFYPLVTFVLLLICIAYWAMTALYLATSGQPQYVLWAS
    NISSPGCEKVPINTSCNPTAHLVNSSCPGLMCVFQGYSSKGLIQRSVFNL
    QIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQDIPTFPLISAFI
    RTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCCFK
    CCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLD
    KVTDLLLFFGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIM
    TSILGAYVIASGFFSVFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLL
    KILGKKNEAPPDNKKRKK

    (23) The surface protein BMPR1B (SwissProt: O00238)
    (24) The transport protein SLC7A5 (SwissProt: Q01650)
    (25) The epithelial antigen of the prostate STEAP1 (SwissProt: Q9UHE8)
    (26) The ovarian carcinoma antigen MUC16 (SwissProt: Q8WXI7)
    (27) The transport protein SLC34A2 (SwissProt: O95436)
    (28) The surface protein SEMA5b (SwissProt: Q9P283)
    (29) The surface protein LYPD1 (SwissProt: Q8N2G4)
    (30) The endothelin receptor type B EDNRB (SwissProt: P24530)
    (31) The ring finger protein RNF43 (SwissProt: Q68DV7)
    (32) The prostate carcinoma associated protein STEAP2 (SwissProt: □8NFT2)
    (33) The cation channel TRPM4 (SwissProt: Q8TD43)
    (34) The complement receptor CD21 (SwissProt: P20023)
    (35) The B-cell antigen receptor complex associated protein CD79b (SwissProt: P40259)
    (36) The cell adhesion antigen CEACAM6 (SwissProt: P40199)
    (37) The dipeptidase DPEP1 (SwissProt: P16444)
    (38) The interleukin receptor IL20Ralpha (SwissProt: Q9UHF4)
    (39) The proteoglycan BCAN (SwissProt: Q96GW7)
    (40) The ephrine receptor EPHB2 (SwissProt: P29323)
    (41) The prostatic stem cell associated protein PSCA (GenBank Accession No. NP—005663.2)
    (42) The surface protein LHFPL3 (SwissProt: Q86UP9)
    (43) The receptor protein TNFRSF13C (SwissProt: Q96RJ3)
    (44) The B-cell antigen receptor complex associated protein CD79a (SwissProt: P11912)
    (45) The receptor protein CXCR5 (SwissProt: P32302)
    (46) The ion channel P2X5 (SwissProt: Q93086)
    (47) The lymphocyte antigen CD180 (SwissProt: Q99467)
    (48) The receptor protein FCRL1 (SwissProt: Q96LA6)
    (49) The receptor protein FCRL5 (SwissProt: Q96RD9)
    (50) The MHC class II molecule Ia antigen HLA-DOB (GenBank Accession No: NP—002111.1)
    (51) The T-cell protein VTCN1 (SwissProt: Q7Z7D3).
    (52) Single-pass type-I membrane protein “Programmed cell death 1 ligand 1”
    (synonyms: CD274, B7H1, PDCD1L1, PDCD1LG1, PDL1) (SwissProt: Q9NZQ7)—both are isoforms
  • >sp|Q9NZQ7|PD1L1_HUMAN Programmed cell death
    1 ligand 1 OS = Homo sapiens GN = CD274
    PE = 1 SV = 1
    MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDL
    AALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQ
    ITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSE
    HELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRIN
    TTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLC
    LGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET

    (53) Single-pass type I membrane protein “ICOSLG” (synonyms: B7H2, B7RP1, ICOSL, KIAA0653, CD275)—(SwissProt: 075144), both are isoforms
  • >sp|O75144|ICOSL_HUMAN ICOS ligand OS = Homo
    sapiens GN = ICOSLG PE = 1 SV = 2
    MRLGSPGLLFLLFSSLRADTQEKEVRAMVGSDVELSCACPEGSRFDLND
    VYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSL
    RLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPH
    SPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGL
    YDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITEN
    PVSTGEKNAATWSILAVLCLLVVVAVAIGWVCRDRCLQHSYAGAWAVSP
    ETELTGHV

    (54) Tyrosine kinase “Fibroblast growth factor receptor 3” (FGFR-3, EC=2.7.10.1, CD333, JTK4), (SwissProt: P22607)—four isoforms (alternative splicing)
  • >sp|P22607|FGFR3_HUMAN Fibroblast growth factor
    receptor 3 OS = Homo sapiens GN = FGFR3 PE = 1
    SV = 1
    MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQL
    VFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLN
    ASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVD
    TGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFR
    GEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDV
    LERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVN
    GSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGN
    SIGFSHHSAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAA
    VTLCRLRSPPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIA
    RLSSGEGPTLANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAE
    AIGIDKDRAAKPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNII
    NLLGACTQGGPLYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQ
    LTFKDLVSCAYQVARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFG
    LARDVHNLDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWE
    IFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAP
    SQRPTFKQLVEDLDRVLTVTSTDEYLDLSAPFEQYSPGGQDTPSSSSSG
    DDSVFAHDLLPPAPPSSGGSRT

    (55) Single-pass type-I membrane protein “TYRP1” (CAS2, TYRP, TYRRP, DHICA oxidase, 5,6-dihydroxyindole-2-carboxylic acid oxidase, catalase B, glycoprotein 75, melanoma antigen gp75, tyrosinase-related protein 1), (SwissProt: P17643)
  • >sp|P17643|TYRP1_HUMAN 5,6-dihydroxyindole-2-
    carboxylic acid oxidase OS = Homo sapiens GN =
    TYRP1 PE = 1 SV = 2
    MSAPKLLSLGCIFFPLLLFQQARAQFPRQCATVEALRSGMCCPDLSPVS
    GPGTDRCGSSSGRGRCEAVTADSRPHSPQYPHDGRDDREVWPLRFFNRT
    CHCNGNFSGHNCGTCRPGWRGAACDQRVLIVRRNLLDLSKEEKNHFVRA
    LDMAKRTTHPLFVIATRRSEEILGPDGNTPQFENISIYNYFVWTHYYSV
    KKTFLGVGQESFGEVDFSHEGPAFLTWHRYHLLRLEKDMQEMLQEPSFS
    LPYWNFATGKNVCDICTDDLMGSRSNFDSTLISPNSVFSQWRVVCDSLE
    DYDTLGTLCNSTEDGPIRRNPAGNVARPMVQRLPEPQDVAQCLEVGLFD
    TPPFYSNSTNSFRNTVEGYSDPTGKYDPAVRSLHNLAHLFLNGTGGQTH
    LSPNDPIFVLLHTFTDAVFDEWLRRYNADISTFPLENAPIGHNRQYNMV
    PFWPPVTNTEMFVTAPDNLGYTYEIQWPSREFSVPEIIAIAVVGALLLV
    ALIFGTASYLIRARRSMDEANQPLLTDQYQCYAEEYEKLQNPNQSVV

    (56) Cell membrane protein, cleaved into secreted glypican-3 (GPC3, OCI5, GTR2-2, intestinal protein OCI-5, MXR7), (SwissProt: P51654)
  • >sp|P51654|GPC3_HUMAN Glypican-3 OS = Homo sapiens
    GN = GPC3 PE = 1 SV = 1
    MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGL
    KWVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASM
    ELKFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLTPQAFEFVGE
    FFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINE
    CLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDH
    LKFSKDCGRMLTRMWYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDK
    YWREYILSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTT
    TIGKLCAHSQQRQYRSAYYPEDLFIDKKVLKVAHVEHEETLSSRRRELI
    QKLKSFISFYSALPGYICSHSPVAENDTLCWNGQELVERYSQKAARNGM
    KNQFNLHELKMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVLDKNLDE
    EGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNS
    QQATPKDNEISTFHNLGNVHSPLKLLTSMAISVVCFFFLVH
  • In a preferred subject matter of the invention, the cancer target molecule is selected from the group consisting of the cancer target molecules (1) though (56).
  • In another preferred subject matter of the invention, the binder binds to an extracellular cancer target molecule, which is selected from the group consisting of the cancer target molecules (1) through (56).
  • In another preferred subject matter of the invention, the binder binds specifically to an extracellular cancer target molecule, which is selected from the group consisting of the cancer target molecules (1) through (56).
  • In an especially preferred subject matter 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), carboanhydrase IX (CA IX; Q16790, NP—001207.2), HER2, glypican-3, TYRP1, fibroblast growth factor receptor 3, single-pass type I membrane protein ICOSLG and programmed cell death 1 ligand 1.
  • In another especially preferred subject matter of the invention, the binder binds to an extracellular cancer target molecule, which is selected from the group consisting of EGF receptor (NP—005219.2), mesothelin (Q13421-3), C4.4a (NP—055215.2), carboanhydrase IX (CA IX; Q16790, NP—001207.2), HER2, glypican-3, TYRP1, fibroblast growth factor receptor 3, single-pass type I membrane protein ICOSLG and programmed cell death 1 ligand 1.
  • In one preferred embodiment, the binder is internalized by the target cell after binding to its extracellular target molecule on the target cell by the binding. The result of this is that the binder-drug conjugate which may be an immunoconjugate or an ADC, is absorbed by the target cell.
  • In one embodiment, the binder is a binding protein. In a preferred embodiment, the binder is an antibody, an antigen-binding antibody fragment, a multispecific antibody or an antibody mimetic.
  • Preferred antibody mimetics include affibodies, adnectins, anticalins, DARPins, avimers or nanobodies. Preferred multispecific antibodies include bi-specific and tri-specific antibodies.
  • In a preferred embodiment, the binder is an antibody or an antigen-binding antibody fragment; more preferably it is an isolated antibody or an isolated antigen-binding antibody fragment.
  • Preferred antigen binding antibody fragments include Fab, Fab′, F(ab)2 and Fv fragments, diabodies, Dabs, linear antibodies and scFv, Fab, diabodies and scFv are especially preferred.
  • In an especially preferred embodiment, the binder is an antibody. Especially preferred are monoclonal antibodies or antigen-binding antibody fragments thereof. Additionally especially preferred are human, humanized or chimeric antibodies or antigen-binding antibody fragments thereof.
  • Antibodies or antigen-binding antibody fragments that bind cancer target molecules can be synthesized by the average person skilled in the art using known methods, for example, recombinant synthesis or recombinant expression. Binders for cancer target molecules can be purchased commercially or can be synthesized by an average person skilled in the art by using known methods, e.g., chemical synthesis or recombinant expression. Additional methods of synthesis of antibodies or antigen-binding antibody fragments are described in WO 2007070538 (see page 22 “Antibodies”). Those skilled in the art are familiar with methods such as the so-called phage display technique which creates libraries (e.g., Morphosys HuCAL Gold) and can be used to discover antibodies or antigen-binding antibody fragments (see WO 200707058, pages 24 ff., Example 1 on page 70 and Example 2 on page 72). Additional methods of synthesis of antibodies using DNA libraries from B cells are described on page 26 of WO 2007070538, for example. Methods of humanizing antibodies are described on pages 30-32 of WO 2007070538 and in detail in Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033, 1989 or in WO 90/0786. In addition, those skilled in the art are familiar with methods of recombinant expression of proteins in general and in specific by antibodies (see, e.g., in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, vol. 152, Academic Press, Inc.; Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, vol. 1-3; Current Protocols in Molecular Biology, F. M. Ausabel 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 familiar with the corresponding vectors, promoters and signal peptides, which are necessary for expression of a protein/antibody.
  • Conventional methods are also described on pages 41-45 of WO 2007070538. Methods of synthesis of an IgG1 antibody are described in WO 2007070538, e.g., on pages 74 ff. of Example 6; these methods, described on page 80 of WO 2007070538, for example, make it possible to internalize an antibody after binding it to its antigen. Similarly, those skilled in the art can utilize the methods described in WO 2007070538 for synthesis of carboanhydrase IX (Mn) antibodies to synthesize antibodies having other target molecule specificities.
  • Especially preferred binders according to the invention are antibodies in particular human or humanized antibodies. The antibodies preferably have an affinity of at least 10−7 M (as a Kd value; i.e., preferably those with smaller Kd values than 10−7 M), preferably of at least 10−8 M, especially preferably in the range of 10−9 M to 10−11 M. These Kd values can be determined, for example, by surface plasmon resonance spectroscopy.
  • The antibody-drug conjugates according to the invention also have affinities in these ranges. Through conjugation of the active ingredients, the affinity is preferably not influenced significantly (the affinity is usually reduced by less than one order of magnitude, e.g., max. from 10−8 M to 10−7 M).
  • The antibodies used according to the invention are also preferably characterized by a high selectivity. Selectivity is high when the antibody according to the invention has a better affinity for the target protein than for another independent antigen, e.g., human serum albumin, said affinity being better by a factor of 2, a factor of 5, a factor of 10 or in particular preferably a factor of 100 (the affinity can be determined, for example, by surface plasmon resonance spectroscopy).
  • Furthermore, the antibodies used according to the invention are preferably cross-reactive. To facilitate preclinical trials, e.g., toxicological studies or efficacy studies (e.g., in xenograft mice) and to be able to interpret them better, it is advantageous if the antibody to be used according to the invention not only binds the human target protein but also binds the species target protein in the species used for these studies. In one embodiment, the antibody used according to the invention, which is cross-reactive with the antibody used according to the invention but is also cross-reactive with the human target protein of at least one additional species. For toxicological studies and efficacy studies, species of rodent, dog and non-human primate families are especially preferred. Preferred rodent species include the mouse and the rat. Preferred non-human primates include Rhesus monkeys, chimpanzees and long-tailed macaques.
  • In one embodiment, the antibody used according to the invention is also cross-reactive with the target protein of at least one additional species in addition to being cross-reactive with the human target protein, said additional species being selected from the group of species consisting of the mouse, the rat and the long-tailed macaque (Macaca fascicularis). Antibodies that are used according to the invention and are cross-reactive at least with the mouse target protein in addition to being cross-reactive with the human target protein are preferred in particular. Cross-reactive antibodies whose affinity for the target protein of the additional non-human species does not differ from the affinity for the human target protein by more than a factor of 50, in particular not more than a factor of 10 are preferred.
    • EGFR Antibodies
  • Examples of antibodies that bind the cancer target molecule EGFR include 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 that is produced in SP2/0 mouse myeloma cells and is distributed by ImClone Systems Inc., Merck KGaA/Bristol Myers Squibb Co. Cetuximab is indicated for treatment of metastatic EGFR-expressing colorectal carcinoma with the wild-type K-Ras gene. It has an affinity of 10−10 M.
    • Sequence:
  • Cetuximab light chain (kappa):
    DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY
    ASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGA
    GTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
    DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
    LSSPVTKSFNRGEC
    Cetuximab heavy chain:
    QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV
    IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALT
    YYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
    YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
    ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
    DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
    TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
    YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
    DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
  • Panitumumab (INN No. 8499) (Drug Bank Accession No. DB01269) is a recombinant monoclonal human IgG2 antibody that binds specifically to human EGF receptor 1 and is distributed by Abgenix/Amgen. Panitumumab originates from the immunization of transgenic mice (XenoMouse). These mice are capable of producing human immunoglobulins (light and heavy chains). A special B-cell clone that produces antibodies to EGFR was selected and was immortalized with CHO cells (Chinese hamster ovary cells). These cells are now being used for the production of a 100% human antibody. Panitumumab is indicated for the treatment of EGFR-expressing, metastatic colorectal carcinoma, which is refractory to chemotherapeutic treatment with fluoropyrimidine, oxaliplatin and irinotecan. It has an affinity of 10−11 M.
    • Sequence:
  • Panitumumab light chain (kappa):
    DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD
    ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGG
    GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
    DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
    LSSPVTKSFNRGEC
    Panitumumab heavy chain:
    QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWI
    GHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRD
    RVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD
    YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTY
    TCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM
    ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
    VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP
    PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG
    SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
  • Nimotuzumab (INN No. 8545) (EP 00586002, EP 00712863) is a humanized monoclonal IgG1 antibody that binds specifically to the human EGF receptor 1 and is distributed by YM BioSciences Inc. (Mississauga, Canada). It is produced in non-secreting NSO cells (mammalian cell line). Nimotuzumab has been approved for treatment of head and neck tumors, highly malignant astrocytomas and glioblastoma multiforme (not in EU or US) and pancreatic cancer (orphan drug, EMA). It has an affinity of 10−8 M.
  • Additional embodiments of EGFR antibodies include:
      • Zalutumumab/2F8/HuMax-EGFr, Genmab Co. A/S (WO 02100348, WO 2004-056847, INN No. 8605)
      • Necitumumab/11F8, ImClone/IMC-11F8, ImClone Systems Inc. (Eli Lilly & Co.) (WO 2005090407, (EP 01735348-A1, US 20070264253-A1, U.S. Ser. No. 07/598,350, 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/monoclonal antibody 425, Merck KGaA/Takeda (WO 09215683, INN No. 8103 (matuzumab))
      • RG-7160/GA-201/GA201/R-7160/R7160/RG7160/RO-4858696/RO-5083945/RO4858696/RO5083945, Glycart Biotechnology AG (Roche Holding AG) (WO 2010-112413 A1, WO 2010115554)
      • GT-MAB 5.2-GEX/CetuGEX, Glycotope GmbH (WO 2008028686 A2, EP 01900750 A1, EP 01911766 A1, EP 02073842 A2, US 20100028947 A1)
      • ISU-101, Isu Abxis Inc. (ISU Chemical Co. Ltd.)/Scancell (patent: WO 2008-004834 A1)
      • ABT-806/mAb 806/ch-806/anti-EGFR monoclonal antibody 806, Ludwig Institute for Cancer Research/Abbott/Life Science Pharmaceuticals (WO02092771, WO2005-081854 and WO-2009023265)
      • SYM-004 (consists of two chimeric IgG1 antibodies (992 and 1024)), Symphogen A/S (WO 2010022736 A2)
      • MR1-1/MR1-1KDEL, IVAX Corp (Teva Pharmaceutical Industries Ltd.) (Duke University), (patent: WO 2001062931 A2)
      • Antibodies to the deletion mutant, EGFRvIII, Amgen/Abgenix (WO 2005010151, U.S. Ser. No. 07/628,986)
      • SC-100, Scancell Ltd. (WO-2001088138-A1)
      • MDX-447/EMD 82633/BAB-447/H 447/MAb, EGFR, Medarex/Merck KGaA, Bristol-Myers Squibb (US)/Merck KGaA (DE)/Takeda (JP), (WO 09105871, WO 09215683)
      • Anti-EGFR mAb, Xencor (WO 2005056606)
      • DXL-1218/anti-EGFR monoclonal antibody (cancer), InNexus, InNexus Biotechnology Inc., pharmaceutical projects PH048638
  • In a preferred embodiment, the anti-EGFR antibodies are selected from the group consisting of cetuximab, panitumumab, nimotuzumab, zalutumumab, necitumumab, matuzumab, RG-716, GT-MAB 5.2-GEX, ISU-101, ABT-806, SYM-004, MR1-1, SC-100, MDX-447 and DXL-1218.
  • In an especially preferred embodiment, the anti-EGFR antibodies are selected from the group consisting of cetuximab, panitumumab, nimotuzumab, zalutumumab, necitumumab and matuzumab.
  • Those skilled in the art will be familiar with methods with which additional antibodies having a similar or better affinity and/or specificity for the target molecule can be synthesized from the CDR regions of the aforementioned antibodies by sequence variations.
  • In another embodiment, the anti-EGFR antibodies or antigen-binding antibody fragments are selected from the group consisting of
  • antibodies or antigen-binding antibody fragments comprising the three CDR regions of the light chain and the three CDR regions of the heavy chain of one of the following antibodies: cetuximab, panitumumab, nimotuzumab, zalutumumab, necitumumab, 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 preferred embodiment, the anti-EGFR antibodies or antigen-binding antibody fragments are selected from the group consisting of
  • antibodies or antigen-binding antibody fragments comprising the three CDR regions of the light chain and the three CDR regions of the heavy chain of one of the following antibodies: cetuximab, panitumumab, nimotuzumab, zalutumumab, necitumumab, matuzumab.
    • Carboanhydrase IX Antibodies
  • Especially preferred binders according to the invention include anti-CALX antibodies, in particular human or humanized anti-CALX antibodies. The antibodies preferably have an affinity of at least 10−7 M (as the Kd value, i.e., preferably those with Kd values of less than 10−7 M), preferably of at least 10−8 M, especially preferably in the range of 10−9 M to 10−11 M. The Kd values can be determined, for example, by surface plasmon resonance spectroscopy.
  • The antibody-drug conjugates according to the invention also have affinities in these ranges. The affinity is preferably not influenced significantly by conjugation of the active ingredients (the affinity is usually reduced less than one order of magnitude, i.e., from max. 10−8 M to 10−7 M, for example).
  • The antibodies used according to the invention are also characterized preferably by a high selectivity. A high selectivity occurs when the antibodies according to the invention have a better affinity for the target protein by a factor of at least 2, a factor of 5, a factor of 10 or especially preferably a factor of 100 than the affinity for another independent antigen, e.g., human serum albumin (the affinity can be determined, for example, by surface plasmon resonance spectroscopy).
  • Furthermore, the antibodies used according to the invention are preferably cross-reactive. To facilitate preclinical trials, e.g., toxicological or efficacy studies (e.g., in xenograft mice), and to be better able to interpret them, it is advantageous if the antibodies used according to the invention bind not only the human target protein but also bind the species target protein in the species used for the studies. In one embodiment, the antibody used according to the invention is cross-reactive with the target protein of at least one species in addition to the human target protein. For toxicological studies and efficacy studies, the preferred species for use are those of the rodent, dog and non-human primate families Preferred rodent species include the mouse and the rate. Preferred non-human primates include Rhesus monkeys, chimpanzees and long-tailed macaques.
  • In one embodiment, the antibody used according to the invention is cross-reactive with the target protein of at least one additional species selected from the group of species consisting of mouse, rat and long-tailed macaque (Macaca fascicularis) in addition to being cross-reactive with the human target protein. Especially preferred are antibodies that can be used according to the invention and are at least cross-reactive with the mouse target protein in addition to being cross-reactive with the human target protein. The preferred cross-reactive antibodies are those whose affinity for the target protein of the additional non-human species does not differ from the affinity for the human target protein by a factor of more than 50, in particular no more than a factor of 10. Anti-CAIX antibodies include those described, for example, in WO 2007/070538 A2. These antibodies may be used according to the invention.
  • Examples of antibodies that bind the cancer target molecule carboanhydrase IX are described in WO 2007/070538 A2 (e.g., Claims 1-16).
  • In a preferred embodiment, the anti-carboanhydrase IX antibodies or antigen-binding antibody fragments are selected from the group consisting of anti-carboanhydrase IX antibodies or antigen-binding antibody fragments 3ee9 (Claim 4 (a) in WO 2007070538 A2), 3ef2 (Claim 4 (b) in WO 2007070538 A2), 1e4 (Claim 4 (c) in WO 2007070538 A2), 3a4 (Claim 4 (d) in WO 2007070538 A2), 3ab4 (Claim 4 (e) in WO 2007070538 A2), 3ah10 (Claim 4 (f) in WO 2007070538 A2), 3bb2 (Claim 4 (g) in WO 2007070538 A2), 1aa1 (Claim 4 (h) in WO 2007070538 A2), 5a6 (Claim 4 (i) in WO 2007070538 A2) and 5aa3 (Claim 4 (j) in WO 2007070538 A2).
  • In a preferred embodiment, the anti-carboanhydrase IX antibodies or antigen-binding antibody fragments are selected from the group consisting of:
  • anti-carboanhydrase IX antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody 3ee9 (from WO 2007070538 A2),
    anti-carboanhydrase IX antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody 3ef2 (from WO 2007070538 A2),
    anti-carboanhydrase IX antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody 1e4 (from WO 2007070538 A2),
    anti-carboanhydrase IX antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody 3a4 (from WO 2007070538 A2),
    anti-carboanhydrase IX antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody 3ab4 (from WO 2007070538 A2),
    anti-carboanhydrase IX antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody 3ah10 (from WO 2007070538 A2),
    anti-carboanhydrase IX antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody 3bb2 (from WO 2007070538 A2),
    anti-carboanhydrase IX antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody 1aa1 (from WO 2007070538 A2),
    anti-carboanhydrase IX antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody 5a6 (from WO 2007070538 A2) and
    anti-carboanhydrase IX antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody 5aa3 (from WO 2007070538 A2).
  • The given sequences of the CDR regions are shown in FIGS. 2a-2c, pages 128-130 in WO 2007070538 A2.
  • In a preferred embodiment, the anti-carboanhydrase IX antibodies or antigen-binding antibody fragments are selected from the group consisting of:
  • an antibody or antigen-binding fragment consisting of the amino acid sequence of the variable light and variable heavy chains of the antibody 3ee9, as defined in WO 2007070538 A2 in FIG. 4b on page 137,
    an antibody or antigen-binding fragment consisting of the amino acid sequence of the variable light and variable heavy chains of the antibody 3ef2, as defined in WO 2007070538 A2 in FIG. 4c on page 138 and/or in FIG. 4b on page 137,
    an antibody or antigen-binding fragment consisting of the amino acid sequence of the variable light and variable heavy chains of the antibody 1e4, as defined in WO 2007070538 A2 in FIG. 4a on page 136,
    an antibody or antigen-binding fragment consisting of the amino acid sequence of the variable light and variable heavy chains of the antibody 3a4, as defined in WO 2007070538 A2 in FIG. 4a on page 136,
    an antibody or antigen-binding fragment consisting of the amino acid sequence of the variable light and variable heavy chains of the antibody 3ab4, as defined in WO 2007070538 A2 in FIG. 4a on page 136,
    an antibody or antigen-binding fragment consisting of the amino acid sequence of the variable light and variable heavy chains of the antibody 3ah10, as defined in WO 2007070538 A2 in FIG. 4a on page 136,
    an antibody or antigen-binding fragment consisting of the amino acid sequence of the variable light and variable heavy chains of the antibody 3bb2, as defined in WO 2007070538 A2 in FIG. 4b on page 137,
    an antibody or antigen-binding fragment consisting of the amino acid sequence of the variable light and variable heavy chains of the antibody 1aa1, as defined in WO 2007070538 A2 in FIG. 4a on page 136,
    an antibody or antigen-binding fragment consisting of the amino acid sequence of the variable light and variable heavy chains of the antibody 5a6, as defined in WO 2007070538 A2 in FIG. 4b on page 137, and
    an antibody or antigen-binding fragment consisting of the amino acid sequence of the variable light and variable heavy chains of the antibody 5aa3, as defined in WO 2007070538 A2 in FIG. 4b on page 137.
  • In an especially preferred embodiment, the anti-carboanhydrase IX antibody is the antibody 3ee9 from WO 2007070538 A2.
  • In an especially preferred embodiment, the anti-carboanhydrase IX antibody or the antigen-binding antibody fragment comprises the amino acid sequences of the CDR regions of the variable heavy chain of the antibody 3ee9 (VH3-CDR1: GFTFSSYGMS; VH3-CDR2: GISSLGSTTYYADSVKG; VH3-CDR3: TGSPGTFMHGDH, see FIG. 2a, page 128 in WO 2007070538 A2) and the amino acid sequences of the CDR regions of the variable light chain of the antibody 3ee9 (VLkl-CDR1: RASQDINNYLS; VLkl-CDR2: YGASNLQS; VLk1-CDR3: QQYYGRPT, see FIG. 2b, page 129 in WO 2007070538 A2).
  • In an especially preferred embodiment, the anti-carboanhydrase IX antibody or the antigen-binding antibody fragment comprises the amino acid sequences of the variable heavy chain of the antibody 3ee9 (VH3:ELVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSGISSLGST TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTGSPGTFMHHGDHWGQ GTLVTVSS, see FIG. 4b, page 137 in WO 2007070538 A2) and the amino acid sequences of the variable light chain of the antibody 3ee9 (VLk1:DIQMTQSPSSLSASVGDRVTITCRaSQDINNYLSWYQQKPGKAPKLLIYGASNLQS GVPSRFSGSGSGTDFTLTISLQPEDFAVYYC QQYYGRPTTFGQGTKVEIKRT, see FIG. 4b, page 137 in WO 2007070538 A2).
  • In a preferred embodiment, the anti-carboanhydrase IX antibody 3ee9 is a IgG antibody.
  • In an especially preferred embodiment, the anti-carboanhydrase IX antibody 3ee9 is an IgG1 antibody (3ee9-IgG1),
  • wherein the amino acid sequence of the heavy chain comprises the following sequence:
  • QVELVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSG
    ISSLGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTG
    SPGTFMHGDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
    KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
    TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
    PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
    NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
    QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
    VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
    K

    and the amino acid sequence of the light chain comprises the following sequence:
  • DIQMTQSPSSLSASVGDRVTITCRASQDINNYLSWYQQKPGKAPKLLIYG
    ASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYYGRPTTFGQ
    GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
    DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
    LSSPVTKSFNRGEC

    anti-carboanhydrase IX antibody 3ee9-IgG1:
  • Another aspect of the present invention is supplying the anti-carboanhydrase IX antibody 3ee9-IgG1.
    • C4.4a Antibody:
  • Especially preferred binders according to the invention are anti-C4.4a antibodies, in particular human or humanized anti-C4.4a antibodies. These antibodies have an affinity of preferably at least 10−7 M (as Kd value, i.e., preferably those with Kd values of less than 10−7 M), especially at least 10−8 M, most especially preferably in the range of 10−9 M to 10−11 M. The Kd values can be determined by surface plasmon resonance spectroscopy, for example.
  • The antibody-drug conjugates according to the invention also have affinities in these ranges. Through conjugation of the active ingredients, the affinity is preferably not influenced significantly (the affinity is usually reduced by less than one order of magnitude, e.g., max. from 10−8 M to 10−7 M).
  • The antibodies used according to the invention are also preferably characterized by a high selectivity. A high selectivity occurs when the antibody according to the invention has a better affinity for the target protein than for another independent antigen, e.g., human serum albumin by a factor of at least 2, preferably by a factor of 5 or in particular preferably a factor of 10 (the affinity can be determined, for example, by surface plasmon resonance spectroscopy).
  • Furthermore, the antibodies to be used according to the invention are preferably cross-reactive. To facilitate preclinical trials, e.g., toxicological studies or efficacy studies (e.g., in xenograft mice) and to be able to interpret them better, it is advantageous if the antibody to be used according to the invention not only binds the human target protein but also binds the species target protein in the species used for the studies. In one embodiment, the antibody used according to the invention is additionally cross-reactive with the target protein of at least one other species in addition to the human target protein. For toxicological studies and efficacy studies, species of the rodent, dog and non-human primate families are preferably used. Preferred rodent species include the mouse and the rat. Preferred non-human primates include Rhesus monkeys, chimpanzees and long-tailed macaques.
  • In one embodiment, the antibody used according to the invention is also cross-reactive with the target protein of at least one other species in addition to being cross-reactive with the human target protein, said additional species being selected from the group of species consisting of the mouse, rat and long-tailed macaque (Macaca fascicularis). Especially preferred antibodies for use according to the invention include those that are cross-reactive with at least the mouse target protein in addition to being cross-reactive with the human target protein. The preferred cross-reactive antibodies are those whose affinity for the target protein of the additional non-human species does not differ from the affinity for the human target protein by a factor of more than 50, in particular more than 10.
  • Anti-C4.4a antibodies are described in WO 01/23553 or WO 2011070088, for example. These antibodies may be used according to the present invention.
  • Examples of C4.4a antibodies and antigen-binding fragments are described below. The sequences of the antibodies are given in Table 1, where each row shows the respective CDR amino acid sequences of the variable light chain and/or of the variable heavy chain of the antibody listed in column 1. This table also shows the amino acid sequences of the variable light chain and of the variable heavy chain and also the amino acid sequence of the respective antibody listed lists in column 1.
  • In one embodiment, the anti-C4.4a antibodies or the antigen-binding antibody fragments bind to the S1 domain S1 (amino acid positions 1-85 of SEQ ID NO: 1) of C4.4a.
  • In one embodiment, the anti-C4.4a antibodies or the antigen-binding antibody fragments have cross-reactivity with human C4.4a (SEQ ID NO: 1) and with murine C4.4a (SEQ ID NO: 2).
  • In one exemplary embodiment, the anti-C4.4a antibodies or the antigen-binding antibody fragments thereof are internalized by the cell after binding to a C4.4a-expressing cell.
  • In another embodiment, the anti-C4.4a antibodies or the antigen-binding antibody fragments compete with the antibody M31-B01 and/or with the antibody M20-D02-S-A for binding to C4.4a. Antibodies M31-B01 and M20-D02-S-A compete for binding to C4.4a. Antibodies B01-1 to B01-12 were synthesized by affinity maturation from M31-B01 and compete with M31-B01 for binding to C4.4a. The antibodies D02-1 through D02-13 were synthesized by affinity maturation from M20-D02-S-A and compete with M20-D02-S-A for binding to C4.4a.
  • In another embodiment, the anti-4.4a antibodies or the antigen-binding antibody fragments comprise at least one, two or three of the CDR amino acid sequences listed in Table 1 or Table 2.
  • In another embodiment, the anti-4.4a antibodies or the antigen-binding antibody fragments comprise at least one, two or three CDR amino acid sequences of an antibody listed in Table 1 or Table 2.
  • In another embodiment, the anti-4.4a antibodies or the antigen-binding antibody fragments comprise at least one, two or three CDR amino acid sequences of the variable light chain and at least one, two or three CDR amino acid sequences of the variable heavy chain of an antibody listed in Table 1 or Table 2.
  • In another embodiment, the anti-4.4a antibodies or the antigen-binding antibody fragments, which are at least 50%, 60%, 70%, 80%, 90% or 95% identical to the CDR amino acid sequences of the variable light chain and are identical with the CDR amino acid sequences of the variable heavy chain comprise an antibody as listed in Table 1 or Table 2.
  • In another embodiment, the CDR sequences of the anti-C4.4a antibodies or of the antigen-binding fragments comprise:
  • CDR sequences of the heavy chain, which conform to CDR sequences SEQ ID NO: 297 (CDR H1), SEQ ID NO: 298 (CDR H2) and SEQ ID NO: 299 (CDR H3), and CDR sequences of the light chain, which conform to CDR sequences SEQ ID NO: 300 (CDR L1), SEQ ID NO: 22 (CDR L2) and SEQ ID NO: 301 (CDR L3), or
    CDR sequences of the heavy chain, which conform to CDR sequences SEQ ID NO: 302 (CDR H1), SEQ ID NO: 303 (CDR H2) and SEQ ID NO: 304 (CDR H3) and CDR sequences of the light chain, which conform to CDR sequences SEQ ID NO: 305 (CDR L1), SEQ ID NO: 306 (CDR L2) and SEQ ID NO: 307 (CDR L3).
  • In another embodiment, the anti-C4.4a antibodies or antigen-binding antibody fragments which are at least 50%, 60%, 70%, 80%, 90% or 95% identical to the variable light chain and to the variable heavy chain comprise an antibody as listed in Table 1 or Table 2.
  • In another embodiment, the anti-C4.4a antibodies or antigen-binding antibody fragments comprise the three CDR amino acid sequences of the variable light chain and the three CDR amino acid sequences of the variable heavy chain as listed in Table 1 or Table 2.
  • In another embodiment, the anti-C4.4a antibodies or antigen-binding antibody fragments comprise a variable light chain and/or a variable heavy chain of an antibody as listed in Table 1 or Table 2.
  • In another embodiment, the anti-C4.4a antibodies or antigen-binding antibody fragments comprise the variable light chain and the variable heavy chain of an antibody as listed in Table 1 or Table 2.
  • In a preferred embodiment, the C4.4a antibodies and the antigen-binding antibody fragments are selected from the group consisting of
  • antibody comprising the CDR sequences of the variable heavy chain represented by SEQ ID NO: 75-77 and which reflects the CDR sequences of the variable light chain, as represented by sequence SEQ ID NOS: 78-80 (B01-10),
    antibody comprising the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NOS: 5, 9 and 13 and comprising the CDR sequences of the variable light chain represented by the sequences SEQ ID NOS: 17, 21 and 25 (M31-B01),
    antibody comprising the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NOS: 6, 10 and 14 and comprising the CDR sequences of the variable light chain represented by the sequences SEQ ID NOS: 18, 22 and 26 (M20-D02-S-A),
    antibody comprising the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NOS: 7, 11 and 15 and comprising the CDR sequences of the variable light chain represented by the sequences SEQ ID NOS: 19, 23 and 27 (M60-G03),
    antibody comprising the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NOS: 8, 12 and 16 and comprising the CDR sequences of the variable light chain represented by the sequences SEQ ID NOS: 20, 24 and 28 (36-H02),
    antibody comprising the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NOS: 45-47 and comprising the CDR sequences of the variable light chain represented by the sequences SEQ ID NOS: 48-50 (B01-3),
    antibody comprising the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NOS: 55-57 and comprising the CDR sequences of the variable light chain represented by the sequences SEQ ID NOS: 58-60 (B01-5),
    antibody comprising the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NOS: 65-67 and comprising the CDR sequences of the variable light chain represented by the sequences SEQ ID NOS: 68-70 (B01-7),
    antibody comprising the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NOS: 85-87 and comprising the CDR sequences of the variable light chain represented by the sequences SEQ ID NOS: 88-90 (B01-12),
    antibody comprising the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NOS: 95-97 and comprising the CDR sequences of the variable light chain represented by the sequences SEQ ID NOS: 98-100 (D02-4),
    antibody comprising the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NOS: 105-107 and comprising the CDR sequences of the variable light chain represented by the sequences SEQ ID NOS: 108-110 (D02-6),
    antibody comprising the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NOS: 115-117 and comprising the CDR sequences of the variable light chain represented by the sequences SEQ ID NOS: 118-120 (D02-7),
    antibody comprising the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NOS: 125-127 and comprising the CDR sequences of the variable light chain represented by the sequences SEQ ID NOS: 128-130 (D02-11) and
    antibody comprising the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NOS: 135-137 and comprising the CDR sequences of the variable light chain represented by the sequences SEQ ID NOS: 138-140 (D02-13).
  • In a preferred embodiment, the C4.4a antibodies and the antigen-binding antibody fragments are selected from the group consisting of antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NO: 81 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 82 (B01-7), antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NOS: 33 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 29 (M31-B01), antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NO: 34 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 30 (M20-D02 S-A), antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NO: 35 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 31 (M60-G03), antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NO: 36 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 32 (M36-H02), antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NO: 51 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 52 (B01-3), antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NO: 61 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 62 (B01-5), antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NO: 71 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 72 (B01-7), antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NO: 91 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 92 (B01-12), antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NO: 101 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 102 (D02-4), antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NO: 111 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 112 (D02-6), antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NO: 121 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 122 (D02-7), antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NO: 131 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 132 (D02-11) and antibodies comprising the amino acid sequence of the variable heavy chain represented by the sequence SEQ ID NO: 141 and comprising the amino acid sequence of the variable light chain represented by the sequence SEQ ID NO: 142 (D02-13).
  • In another embodiment, the anti-C4.4a antibodies comprise the light chain and the heavy chain of an antibody as listed in Table 2.
  • In a preferred embodiment, the anti-C4.4a antibodies comprise the light chain and the heavy chain of an antibody as listed in Table 2.
  • In an especially preferred embodiment, the C4.4a antibody is selected from the group consisting of:
  • an antibody comprising the amino acid sequence of the light chain represented by SEQ ID NO: 346 and comprising the amino acid sequence of the heavy chain represented by SEQ ID NO: 347 (M31-B01),
    an antibody comprising the amino acid sequence of the light chain represented by SEQ ID NO: 352 and comprising the amino acid sequence of the heavy chain represented by SEQ ID NO: 353 (B01-3),
    an antibody comprising the amino acid sequence of the light chain represented by SEQ ID NO: 364 and comprising the amino acid sequence of the heavy chain represented by SEQ ID NO: 365 (B01-10) and
    an antibody comprising the amino acid sequence of the light chain represented by SEQ ID NO: 382 and comprising the amino acid sequence of the heavy chain represented by SEQ ID NO: 383 (D02-6).
  • TABLE 1
    Sequences of the C4.4a antibodies
    SEQ ID SEQ ID SEQ ID SEQ ID
    SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO:
    NO: NO: NO: NO: NO: NO: VH VL VH VL
    Antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 protein protein nucleotide nucleotide
    M31-B01 5 9 13 17 21 25 33 29 41 37
    M20-D02 S-A 6 10 14 18 22 26 34 30 42 38
    M60-G03 7 11 15 19 23 27 35 31 43 39
    M36-H02 8 12 16 20 24 28 36 32 44 40
    B01-3 45 46 47 48 49 50 51 52 53 54
    B01-5 55 56 57 58 59 60 61 62 63 64
    B01-7 65 66 67 68 69 70 71 72 73 74
    B01-10 75 76 77 78 79 80 81 82 83 84
    B01-12 85 86 87 88 89 90 91 92 93 94
    D02-4 95 96 97 98 99 100 101 102 103 104
    D02-6 105 106 107 108 109 110 111 112 113 114
    D02-7 115 116 117 118 119 120 121 122 123 124
    D02-11 125 126 127 128 129 130 131 132 133 134
    D02-13 135 136 137 138 139 140 141 142 143 144
    B01-nn1 145 146 147 148 149 150 151 152 308 309
    B01-nn2 153 154 155 156 157 158 159 160 310 311
    B01-nn3 161 162 163 164 165 166 167 168 312 313
    B01-nn4 169 170 171 172 173 174 175 176 314 315
    B01-nn5 177 178 179 180 181 182 183 184 316 317
    B01-2 185 186 187 188 189 190 191 192 318 319
    B01-4 193 194 195 196 197 198 199 200 320 321
    B01-6 201 202 203 204 205 206 207 208 322 323
    B01-8 209 210 211 212 213 214 215 216 324 325
    B01-9 217 218 219 220 221 222 223 224 326 327
    B01-11 225 226 227 228 229 230 231 232 328 329
    B01-12 233 234 235 236 237 238 239 240 330 331
    D02-ogl 241 242 243 244 245 246 247 248 332 333
    D02-5 249 250 251 252 253 254 255 256 334 335
    D02-8 257 258 259 260 261 262 263 264 336 337
    D02-9 265 266 267 268 269 270 271 272 338 339
    D02-10 273 274 275 276 277 278 279 280 340 341
    D02-11 281 282 283 284 285 286 287 288 342 343
    D02-12 289 290 291 292 293 294 295 296 344 345
  • TABLE 2
    Sequences of the light and heavy chains of the C4.4a antibodies
    Light chain Heavy chain
    Antibody SEQ ID NO: SEQ ID NO:
    M31-B01 346 347
    B01-1  348 349
    B01-2  350 351
    B01-3  352 353
    B01-4  354 355
    B01-5  356 357
    B01-6  358 359
    B01-7  360 361
    B01-8  362 363
    B01-10 364 365
    B01-11 366 367
    B01-12 368 369
    M20-D02 S-A 370 371
    D02-1  372 373
    D02-2  374 375
    D02-3  376 377
    D02-4  378 379
    D02-5  380 381
    D02-6  382 383
    D02-7  384 385
    D02-8  386 387
    D02-9  388 389
    D02-10 390 391
    D02-11 392 393
    D02-12 394 395
    D02-13 396 397
    • Anti-C4.4a Antibody IgG:
  • Another aspect of the present invention is providing an anti-C4.4a IgG1 antibody comprising the amino acid sequence of the light chain and the heavy chain of an antibody as listed in Table 2.
  • One example of an antibody that binds the cancer target molecule HER2 is trastuzumab (Genentech). Trastuzumab is a humanized antibody used for treatment of breast cancer, among other things. An example of an antibody that binds the cancer target molecule CD20 is rituximab (Genentech). Rituximab (CAS No. 174722-31-7) is a chimeric antibody used for treating non-Hodgkin's lymphoma. One example of an antibody that binds the cancer target molecule CD52 is alemtuzumab (Genzyme). Alemtuzumab (CAS No. 216503-57-0) is a humanized antibody that is used for treatment of chronic lymphatic leukemia.
    • Mesothelin Antibody
  • According to the invention an especially preferred binders are anti-mesothelin antibodies, in particular human or humanized anti-mesothelin antibodies. The antibodies preferably have an affinity of at least 10−7 M (as Kd value, i.e., preferably those with Kd values less than 10−7 M), preferably of at least 10−8 M, especially preferably in the range from 10−9 M to 10−11 M. The Kd values can be determined by surface plasmon resonance spectroscopy.
  • The antibody-drug conjugates according to the invention also have affinities in these ranges. Through conjugation of the active ingredients, the affinity is preferably not influenced significantly (the affinity is usually reduced by less than one order of magnitude, e.g., max. from 10−8 M to 10−7 M).
  • The antibodies used according to the invention are also preferably characterized by a high selectivity. A high selectivity occurs when the antibody according to the invention has a better affinity for the target protein than for another independent antigen, e.g., human serum albumin by a factor of at least 2, preferably by a factor of 5 or in particular preferably a factor of 10 (the affinity can be determined, for example, by surface plasmon resonance spectroscopy).
  • Furthermore, the antibodies used according to the invention are preferably cross-reactive. To facilitate preclinical trials, e.g., toxicological studies or efficacy studies (e.g., in xenograft mice) and to be able to interpret them better, it is advantageous if the antibody to be used according to the invention not only binds the human target protein but also binds the species target protein in the species used for these studies. In one embodiment, the antibody used according to the invention which is cross-reactive with the antibody used according to the invention but is also cross-reactive with the human target protein of at least one additional species. For toxicological studies and efficacy studies, species of the rodent, dog and non-human primate families are especially preferred. Preferred rodent species include the mouse and the rat. Preferred non-human primates include Rhesus monkeys, chimpanzees and long-tailed macaques.
  • In one embodiment, the antibody used according to the invention is also cross-reactive with the target protein of at least one additional species in addition to being cross-reactive with the human target protein, said additional species being selected from the group of species consisting of the mouse, the rat and the long-tailed macaque (Macaca fascicularis). Preferred antibodies in particular are those that are used according to the invention and are cross-reactive with at least the mouse target protein in addition to being cross-reactive with the human target protein. The preferred cross-reactive antibodies are those whose affinity for the target protein of the additional non-human species does not differ from the affinity for the human target protein by more than a factor of 50, in particular not more than a factor of 10.
  • The antibodies used according to the invention are additionally preferably characterized by invariant binding to mesothelin. Invariant binding is characterized, for example, in that the antibody used according to the invention binds to an epitope of mesothelin, which cannot be masked by another extracellular protein. Such an additional extracellular protein is, for example, the ovarian cancer antigen 125 protein (CA125). The antibodies used are preferably characterized in that their binding to mesothelin is not blocked by CA125.
  • Anti-mesothelin antibodies are described, for example, in WO 2009/068204. These antibodies may be used according to the invention.
  • Another aspect of the present invention is providing a novel anti-mesothelin antibody (MF-Ta) whose amino acid sequence comprises the CDR sequences of the variable heavy chain represented by the sequences SEQ ID NO:398 (HCDR1), SEQ ID NO:399 (HCDR2) and SEQ ID NO:400 (HCDR3) and the CDR sequences of the variable light chain represented by the sequences SEQ ID NO:401 (LCDR1), SEQ ID NO:402 (LCDR2) and SEQ ID NO:403 (LCDR3).
  • In a preferred embodiment, the amino acid sequence of the anti-mesothelin antibody MF-Ta or the antigen-binding antibody fragment comprises a sequence of the variable heavy chain represented by the sequences SEQ ID NO: 404 and the sequence of the variable light chain represented by the sequence SEQ ID NO: 405. In a preferred embodiment, the amino acid sequence of the anti-mesothelin antibody MF-Ta or the antigen-binding antibody fragment comprises the sequence of the variable heavy chain, which is coded by the nucleic acid sequence SEQ ID NO: 406 and the sequence of the variable light chain, which is coded by the nucleic acid sequence SEQ ID NO: 407.
  • In an especially preferred embodiment, the amino acid sequence of the anti-mesothelin antibody MF-Ta comprises the sequence of the heavy chain represented by the sequences SEQ ID NO: 408 and the sequence of the light chain represented by the sequence SEQ ID NO: 409.
  • In an especially preferred embodiment, the amino acid sequence of the anti-mesothelin antibody MF-Ta comprises the sequence of the heavy chain, which is coded by the nucleic acid sequence SEQ ID NO: 410 and the sequence of the light chain, which is coded by the nucleic acid sequence SEQ ID NO: 411.
  • Additional examples of antibodies that bind the cancer target molecule mesothelin are familiar to those skilled in the art and are described in WO 2009/068204, for example, and can be used for the binder-drug conjugates according to the invention.
  • In one embodiment of the binder-drug conjugates, the binder is an anti-mesothelin antibody or an antigen-binding antibody fragment wherein the antibody binds to mesothelin and has invariant binding.
  • In one embodiment, the binder-drug conjugate comprises an anti-mesothelin antibody or an antigen-binding antibody fragment comprises the amino acid sequences of the three CDR regions of the light chain and the amino acid sequences of the three CDR regions of the heavy chain of an antibody as described in WO 2009/068204 A1 (Table 7, pages 61-63).
  • In a preferred embodiment, the mesothelin antibodies or the antigen-binding antibody fragments are selected from the group consisting of:
  • anti-mesothelin antibodies or antigen-binding antibody fragments which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody MF-Ta,
    anti-mesothelin antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody MF-J (WO 2009068204 A1; Table 7, page 61),
    anti-mesothelin antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody MOR06640 (WO 2009/068204 A1; Table 7, page 61),
    anti-mesothelin antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody MF-226 (WO 2009/068204 A1; Table 7, page 61), and
    anti-mesothelin antibodies or antigen-binding antigen fragments thereof, which comprise the sequences of the three CDR regions of the light chain and the sequences of the three CDR regions of the heavy chain of the antibody MOR06626 (WO 2009/068204-A1; Table 7, page 61).
  • In an especially preferred embodiment the mesothelin antibodies or antigen-binding antibody fragments are selected from the group
  • anti-mesothelin antibodies or antigen-binding antibody fragments thereof comprising the sequence of the variable light chain and the sequence of the variable heavy chain of the antibody MF-Ta,
    anti-mesothelin antibodies or antigen-binding antibody fragments thereof comprising the sequence of the variable light chain and the sequence of the variable heavy chain of the antibody MF-J (WO 2009/068204 A1; Table 7, page 61),
    anti-mesothelin antibodies or antigen-binding antibody fragments thereof comprising the sequence of the variable light chain and the sequence of the variable heavy chain of the antibody MOR06640 (WO 2009/068204 A1; Table 7, page 61),
    anti-mesothelin antibodies or antigen-binding antibody fragments thereof comprising the sequence of the variable light chain and the sequence of the variable heavy chain of the antibody MF-226 (WO 2009/068204 A1; Table 7, page 61), and
    anti-mesothelin antibodies or antigen-binding antibody fragments thereof comprising the sequence of the variable light chain and the sequence of the variable heavy chain of the antibody MOR06626 (WO 2009/068204 A1; Table 7, page 61).
    • Additional Antibodies:
  • Trastuzumab (Genentech) is an example of antibody that binds the cancer target molecule HER2. Trastuzumab is a humanized antibody used for treatment of breast cancer among other things. Rituximab (Genentech) is an example of an antibody that binds the cancer target molecule CD20. Rituximab (CAS No. 174722-31-7) is a chimeric antibody used for treatment of non-Hodgkin's lymphoma. Alemtuzumab (Genzyme) is an example of an antibody that binds the cancer target molecule CD52. Alemtuzumab (CAS No. 216503-57-0) is a humanized antibody that is used for treatment of chronic lymphatic leukemia.
  • Additional examples of antibodies that bind to HER2 in addition to trastuzumab (INN No. 7637, CAS No. 180288-69-1) and pertuzumab (CAS No.: 380610-27-5) also include antibodies such as those proposed in WO 2009123894 A2, WO2008140603 A2 or WO 2011044368 A2. One example of anti-HER2 conjugate is trastuzumab emtansine (INN No. 9295).
  • Examples of antibodies that bind the cancer target molecule CD30 and can be used for treatment of cancer, e.g., Hodgkin's lymphoma include brentuximab, iratumumab and antibodies as disclosed in WO 2008092117, WO 2008036688 or WO 2006089232. An example of an anti-CD30 conjugate is brentuximab vedotin (INN No. 9144).
  • Examples of antibodies that bind the cancer target molecule CD22 and can be used for treatment of cancer, e.g., lymphoma include inotuzumab or epratuzumab. Examples of anti-CD22 conjugates include inotuzumab ozagamycin (INN No. 8574) or anti-CD22-MMAE and anti-CD22-MC-MMAE (CAS No.: 139504-50-0 and/or 474645-27-7).
  • Examples of antibodies that bind the cancer target molecule CD33 and can be used for treatment of cancer, e.g., leukemia include gemtuzumab or lintuzumab (INN No. 7580). One example of an anti-CD33 conjugate is gemtuzumab ozagamycin.
  • One example of an antibody that binds the cancer target molecule NMB and can be used for treatment of cancer, e.g., melanoma or breast cancer is glembatumumab (INN No. 9199). One example of an anti-NMB conjugate is glembatumumab vedotin (CAS No.: 474645-27-7).
  • One example of an antibody that binds the cancer target molecule CD56 and can be used for treatment of cancer, e.g., multiple myeloma, small-cell lung carcinoma, MCC or ovarian carcinoma is lorvotuzumab. One example of an anti-CD56 conjugate is lorvotuzumab mertansine (CAS No.: 139504-50-0).
  • Examples of antibodies that bind the cancer target molecule CD70 and can be used for treatment of cancer, e.g., non-Hodgkin's lymphoma or renal cell cancer are disclosed in WO 2007038637 A2 or WO 2008070593 A2. One example of an anti-CD70 conjugate is SGN-75 (CD70 MMAF)
  • One example of an antibody that binds the cancer target molecule CD74 and can be used for treatment of cancer, e.g., multiple myeloma, is milatuzumab. One example of an anti-CD74 conjugate is milatuzumab doxorubicin (CAS No.: 23214-92-8).
  • One example of an antibody that binds the cancer target molecule CD19 and can be used for treatment of cancer, e.g., non-Hodgkin's lymphoma is disclosed in WO 2008031056 A2. Additional antibodies and examples of an anti-CD19 conjugate (SAR3419) are disclosed in WO 2008047242 A2.
  • Examples of antibodies that bind the cancer target molecule Mucin-1 and can be used for treatment of cancer, e.g., non-Hodgkin's lymphoma include clivatuzumab or the antibodies disclosed in WO 2003106495 A2, WO 2008028686 A2. Examples of anti-mucin conjugates are disclosed in WO 2005009369 A2.
  • Examples of antibodies that bind the cancer target molecule CD138 and conjugate thereof that can be used for treatment of cancer, e.g., multiple myeloma are disclosed in WO 2009080829 A1, WO 2009080830 A1.
  • Examples of antibodies that bind the cancer target molecule integrin alphaV and can be used for treatment of cancer, e.g., melanoma, sarcoma or carcinoma include intetumumab (CAS No.: 725735-28-4), abciximab (CAS No.: 143653-53-6), etaracizumab (CAS No.: 892553-42-3) or the antibodies disclosed in U.S. Pat. No. 7,465,449 B2, EP 719859 A1, WO 2002012501 A1 or WO 2006062779 A2. Examples of anti-integrin alphaV conjugates include intetumumab DM4 and additional ADCs disclosed in WO 2007024536 A2.
  • Examples of antibodies that bind the cancer target molecule TDGF1 and can be used for treatment of cancer include the antibodies disclosed in WO 2002077033 A1, U.S. Pat. No. 7,318,924, WO 2003083041 A2 and WO 2002088170 A2. Examples of anti-TDGF1 conjugates are disclosed in WO2002088170-A2.
  • Examples of antibodies that bind the cancer target molecule and can be used for treatment of cancer, e.g., prostatic carcinoma are the antibodies disclosed in WO 199735615 A1, WO 199947554 A1 and WO 2001009192 A1. Examples of anti-PSMA conjugates are disclosed in WO 2009026274 A1.
  • Examples of antibodies that bind the cancer target molecule EPHA2 and can be used for producing a conjugate and for treatment of cancer are disclosed in WO 2004091375 A2.
  • Examples of antibodies that bind the cancer target molecule SLC44A4 and can be used for producing a conjugate and for treatment of cancer, e.g., pancreatic or prostatic carcinoma, are disclosed in WO 2009033094 A2 and US 20090175796 A1.
  • One example of an antibody that binds the cancer target molecule HLA-DOB is the antibody Lym-1 (CAS No.: 301344-99-0), which can be used for treatment of cancer, e.g., non-Hodgkin's lymphoma. Examples of anti-HLA-DOB conjugates are disclosed, for example, in WO 2005081711 A2.
  • Examples of antibodies that bind the cancer target molecule VTCN1 and can be used for producing a conjugate and for treatment of cancer, e.g., ovarian cancer, pancreatic, lung cancer or breast cancer are disclosed in WO 2006074418 A2.
  • An example of an antibody that can be used to bind the cancer target molecule PDL1 is the antibody 3G10 from patent WO 2007005874 A2. The antibody 3G10 may be used, for example, in the human IgG1 format and as the anti-PDL1 used in the exemplary embodiments where the antibody comprises the following sequences;
  • Light chain:
    EIVLTQSPATLSLSPGERATLSCRASQSVSSYLVWYQQKPGQAPRLLIYD
    ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQ
    GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
    DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
    LSSPVTKSFNRGEC
    Heavy chain:
    QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGFSWVRQAPGQGLEWMGW
    ITAYNGNTNYAQKLQGRVTMTTDTSTSTVYMELRSLRSDDTAVYYCARDY
    FYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF
    PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
    NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT
    LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
    RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
    LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
    DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
  • An example of an antibody that binds the cancer target molecule ICOSLG is the antibody 16H (SEQ ID NOS: 70 and 45) from WO 2007011941 A2. Anti-ICOSLG antibody 16H may be used in the human IgG1 format and as the anti-ICOSLG used in the exemplary embodiments comprising the following sequences:
  • Light chain:
    DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPEKAPKSLIYA
    ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDSYPRTFGQ
    GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
    DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
    LSSPVTKSFNRGEC
    Heavy chain:
    EVQLVESGGGLVQPGGSLRLSCAGSGFTFSSYWMSWVRQAPGKGLEWVAY
    IKQDGNEKYYVDSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCAREG
    ILWFGDLPTFWGQGILVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
    KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
    TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
    PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
    NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
    QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
    VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
  • One example of an antibody that binds the target molecule FGFR3 is the antibody 15D8 (SEQ ID NO: 74 for the heavy chain and SEQ ID NO: 76 for the light chain) from WO 2010002862 A2. Anti-FGFR3 antibody 15D8 may be used in the human IgG1 format, for example, and as the anti-FGFR3 used in the exemplary embodiment, this antibody comprises the following sequences:
  • Light chain:
    DIQLTQSPSSLSASVGDRVTITCSASSSVSYMYWFQQKPGKAPKPLIYLT
    SYLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSYPLTFGGG
    TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
    NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
    SSPVTKSFNRGEC
    Heavy chain:
    EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYNMYWVRQMPGKGLEWMGY
    IDPYNGGTSYNQKFKGKATLTVDKSISTAYLQWSSLKASDTAMYYCAREG
    GNYEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
    DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
    YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
    KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
    STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
    VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
    LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
  • One example of an antibody that binds the target cancer molecule 5,6-dihydroxyindole-2-carboxylic acid oxidase (TYRP1) is the antibody 20D7S (SEQ ID NOS: 30 and 32) from patent WO 2009114585 A1. In the exemplary embodiments, anti-TYRP1 antibody 20D7S may be used in human IgG1 format, for example, and when used as the anti-TYRP1, the antibody comprises the following sequences:
  • Light chain
    EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
    ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWLMYTFG
    QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
    VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ
    GLSSPVTKSFNRGEC
    Heavy chain:
    QVQLVQSGSELKKPGASVKISCKASGYTFTSYAMNWVRQAPGQGLESMGW
    INTNTGNPTYAQGFTGRFVFSMDTSVSTAYLQISSLKAEDTAIYYCAPRY
    SSSWYLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
    YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
    ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
    DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
    TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
    YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
    DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
  • One example of an antibody that binds the cancer target molecule glypican-3 is the antibody that is known from the patent U.S. Ser. No. 07/776,329 B2 and comprises the amino acid sequences SEQ ID NOS: 84 and 92 (human mouse chimera). The anti-glypican-3 antibody described above may be used in human IgG1 format, for example, and as the anti-glypican-3 used in the exemplary embodiments, this antibody has the following sequences:
  • Light chain:
    DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQ
    LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP
    PTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
    VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
    VTHQGLSSPVTKSFNRGEC
    Heavy chain:
    QVQLVESGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGA
    LDPKTGDTAYSQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARFY
    SYTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
    PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
    NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM
    ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
    VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
    PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
    SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
  • The compounds according to the invention have valuable pharmacological properties and can be used to prevent and treat diseases in humans and animals.
  • The binder-drug conjugates (ADCs) of formula (Ia) according to the invention have a high and specific cytotoxic activity with respect to tumor cells which can be demonstrated on the basis of assays performed in the present experimental part (C-1 to C-7e). This high and specific cytotoxic activity of the binder-drug conjugates (ADCs) of formula (Ia) according to the invention is achieved by the suitable combination of the novel N,N-dialkylauristatin derivatives and binders with linkers which have both an enzymatic, hydrolytic or reductively cleavable intended breaking point for release of the toxophore as well as those not having any such intended breaking point. The effect on the tumor cell is delineated very specifically by using stable linkers in particular, which do not have any intended breaking points that can be cleaved enzymatically, hydrolytically or reductively to release the toxophore and which still remain entirely or partially intact after receiving the ADC into the tumor cell and after complete intracellular enzymatic degradation of the antibody. The compatibility of ADCs with stable linkers presupposes, among things, that the metabolites formed intracellularly will be formed with enough efficiency to reach their target and be able to manifest their antiproliferative effect on the target there in adequate potency without first being removed from the tumor cell by transporter proteins. The metabolites formed intracellularly after incorporation of the compounds of formula (Ia) according to the invention have a reduced potential as a substrate with respect to transporter proteins, so that a redistribution to the systemic circulation and thus the triggering of potential adverse effects are suppressed by the toxophore itself. In addition the basic character at the amino terminus of the monomethylauristatin peptide is preserved by the novel N-alkyl binding. In particular with the binder-drug conjugates (ADCs) of formula (Ia) according to the invention, the total charge of the antibody remains constant regardless of the number of toxophore-linker charges.
  • The compatibility of the ADCs with a stable linker chemistry and the respective target in conjunction with metabolites that represent a substrate for transporter proteins to a slight extent offers an enlarged therapeutic window.
  • Because of this profile of properties, the compounds according to the invention are therefore suitable to a particular extent for treatment of hyperproliferative diseases in humans and infants in general. These compounds may on the one hand block, inhibit, reduce or decrease cell proliferation and cell division on the one hand while on the other hand potentiating the apoptosis.
  • The hyperproliferative diseases for treatment of which the compounds according to the invention may be used include in particular the group of cancer and tumor diseases. These are understood to include in particular the following diseases within the scope of the present invention without being limited to these: breast cancer and breast tumors (ductile and lobular forms, also in situ), respiratory tract tumors (small cell and non-small-cell carcinomas, bronchial carcinoma), brain tumors (e.g., of the brain stem and the hypothalamus, astrocytoma, medulloblastoma, ependymoma and neuroectodermal and pineal tumors), tumors of the digestive tract (esophagus, stomach, gallbladder, small intestine, large intestine, rectum), liver tumors (including hepatocellular carcinoma, cholangiocarcinoma and mixed hepatocellular cholangiocarcinoma), tumors of the head and neck area (larynx, hypopharynx, nasopharynx, oropharynx, lips and oral cavity), skin tumors (squamous epithelial carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer and non-melanoma type skin cancer), tumors of the soft tissues (including soft tissue sarcomas, malignant fibrous histiocytoma, lymphosarcoma and rhabdomyosarcoma), tumors of the eyes (including intraocular melanoma and retinoblastoma), tumors of the endocrine and exocrine glands (e.g., thyroid and parathyroid glands, pancreatic gland and esophageal gland), tumors of the urinary tract (bladder, penis, kidney, renal pelvis and urethral tumors) as well as tumors of the reproductive organs (endometrium, cervical, ovarian, vaginal, vulval and uterine carcinomas in the woman and prostatic and testicular carcinomas in males). These also include proliferative blood diseases in solid form and as circulating blood cells such as lymphomas, leukemias and myeloproliferative diseases, e.g., acute myeloid leukemia, acute lymphoblastic, chronic lymphocytic leukemia, chronic myelogenous leukemia and hairy cell leukemia as well as AIDS-related lymphomas, Hodgkin's lymphomas, non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, Burkitt's lymphomas and lymphomas of the central nervous system.
    • Preferred Hyperproliferative Diseases for Anti-CA9 Binder-Drug Conjugates
  • Hyperproliferative diseases for the treatment of which the compounds according to the invention may preferably be used include CA9 overexpressing tumors, breast cancers and breast tumors (ductile and lobular forms, also in situ); respiratory tract tumors (small cell and non-small-cell carcinomas, bronchial carcinomas), of which preferably non-small-cell lung carcinoma; brain tumors (e.g., of the brain stem and of the hypothalamus, astrocytoma, medulloblastoma, ependymoma and/or neuroectodermal and pineal tumors); tumors of the digestive organs (esophagus, stomach, gallbladder, small intestine, large intestine, rectum), of which those that are especially preferred are stomach and intestinal tumors; liver tumors (including hepatocellular carcinoma, cholangiocarcinoma and mixed hepatocellular cholangiocarcinoma); tumors of the head and neck area (larynx, hypopharynx, nasopharynx, oropharynx, lips, oral cavity, tongue and esophagus); tumors of the urinary tract (bladder, penis, kidney, renal pelvis and urethral tumors), of which the tumors of the kidneys and bladder are especially preferred; and/or tumors of the reproductive organs (endometrial, cervical, ovarian, vaginal, vulval and uterine carcinomas of the woman and/or prostatic and testicular carcinomas in the man), of which cervical and uterine carcinomas are especially preferred.
    • Preferred Hyperproliferative Diseases for Anti-EGFR Binder-Drug Conjugates
  • Hyperproliferative diseases for the treatment of which the compounds according to the invention may preferably be used include EGFR overexpressing tumors, respiratory tract tumors (e.g., small cell and non-small-cell carcinoma, bronchial carcinoma), of which non-small-cell lung carcinoma is especially preferred; tumors of the digestive tract (e.g., esophagus, stomach, gallbladder, small intestine, large intestine, rectum), of which the intestinal tumors are especially preferred; tumors of the endocrine and exocrine glands (e.g., thyroid and parathyroid glands, pancreatic gland and salivary gland), of which the pancreas is preferred; tumors of the head and neck area (e.g., larynx, hypopharynx, nasopharynx, oropharynx, lips, oral cavity, tongue and esophagus); and/or gliomas.
    • Preferred Hyperproliferative Diseases for Anti-Mesothelin Binder-Drug Conjugates
  • Hyperproliferative diseases for treatment of which the compounds according to the invention are preferably used include mesothelin overexpressing tumors, tumors of the reproductive organs (endometrial, cervical, ovarian, vaginal, vulva and uterine carcinomas in the woman and/or prostatic and testicular carcinomas in the man) of which ovarian carcinomas are preferred; tumors of the endocrine and exocrine glands (e.g., thyroid and parathyroid glands, pancreatic gland and salivary gland), of which the pancreas is preferred; respiratory tract tumors (e.g., small cell and non-small-cell carcinomas, bronchial carcinoma), of which non-small lung cancer is preferred; and/or mesotheliomas.
    • Preferred Hyperproliferative Diseases for Anti-C4.4a Binder-Drug Conjugates
  • Hyperproliferative diseases for treatment of which the compounds according to the invention are preferably used include C4.4a hyperexpressing tumors, squamous epithelial cell carcinomas (e.g., of the cervix, vulva, vagina, the anal canal, endometrium, fallopian tube, penis, scrotum, esophagus, breast, bladder, bile duct, endometrium, uterus and ovaries); breast cancer and breast tumors (e.g., ductal and lobular forms, also in situ); respiratory tract tumors (e.g., small-cell and non-small-cell carcinomas, bronchial carcinoma), of which the non-small-cell lung cancer is preferred along with squamous epithelial cell and adenocarcinomas of the lungs; tumors of the head and neck area (e.g., larynx, hypopharynx, nasopharynx, oropharynx, lips, oral cavity, tongue and esophagus, squamous epithelial cell carcinomas of the head and neck area); tumors of the urinary tract (bladder, penis, kidney, renal pelvis and urethral tumors, squamous epithelial cell carcinomas of the bladder), of which tumors of the kidney and bladder are especially preferred; skin tumors (squamous epithelial cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer and non-melanoma-type skin cancer), of which melanomas are especially preferred; tumors of the endocrine and exocrine glands (e.g., thyroid and parathyroid glands, pancreatic gland and salivary gland), of which the pancreas is preferred; tumors of the digestive organs (e.g., esophagus, stomach, gallbladder, small intestine, large intestine, rectum), of which colorectal carcinomas are especially preferred; and/or tumors of the reproductive organs (endometrial, cervical, ovarian, vaginal, vulval and uterine carcinomas in the woman and/or prostatic and testicular carcinomas in the man), of which uterine carcinomas are especially preferred.
  • These human diseases that have been described extensively can also occur with a comparable etiology in other mammals and can be treated there using the compounds according to the present invention.
  • The terms “treatment” or “to treat” is used in the conventional sense within the scope of this invention and refers to the care, treatment and consultation of a patient with the goal of combatting, reducing, diminishing or ameliorating a disease or health deviation and improving the quality of life which is impaired by this disease such as, for example, in a cancer.
  • An additional subject matter of the present invention is thus the use of the compounds according to the invention for treatment and/or prevention of diseases, in particular the diseases cited above.
  • An additional subject matter of the present invention is the use of the compounds according to the invention for producing a pharmaceutical drug for treatment and/or prevention of diseases, in particular the diseases cited above.
  • An additional subject matter of the present invention is the use of the compounds according to the invention in a method for treatment and/or prevention of diseases, in particular the diseases cited above.
  • An additional subject matter of the present invention is a method for treatment and/or prevention of diseases, in particular the diseases cited above, using an effective amount of at least one of the compounds according to the invention.
  • The inventor-drug conjugate is preferably used for treating cancer in a patient, where the cancer cells of the patient to be treated express the target (preferably EGFR, CA9, mesothelin or C4.4a), preferably having a greater expression of this target than in non-tumorous tissue.
  • A method for identifying patients who will response advantageously to an anti-target binder-drug conjugate for treatment of cancer includes determining the target expression in the patient's cancer cells. In one embodiment, the target expression is determined by target gene expression analysis. Those skilled in the art are familiar with methods for gene expression analysis such as RNA detection, quantitative or qualitative polymerase chain reaction or fluorescence in situ hybridization (FISH). In another preferred embodiment, the target expression is determined by means of immunohistochemistry using an anti-target antibody. Immunohistochemistry is preferably performed on tissue fixed in formaldehyde. The antibody used for the immunohistochemistry is the same antibody also used in the conjugate. The antibody used for the immunohistochemistry is a second antibody that recognizes the target protein/target, preferably specifically.
  • The compounds according to the invention may be used alone or, if necessary, in combination with one or more other pharmacologically active substances as long as this combination does not lead to adverse and unacceptable effects. Another subject matter of the present invention therefore relates to pharmaceutical drugs containing at least one of the compounds according to the invention and one or more additional active ingredients, in particular for treating and/or preventing the diseases listed above.
  • For example, the compounds according to the invention may be combined with known anti-hyperproliferative, cytostatic or cytotoxic for treatment of cancer. Suitable combination active ingredients and drugs that can be listed as examples include:
  • aldesleukin, alendronic acid, alfaferone, alitretinoin, allopurinol, aloprim, aloxi, altretamine, amino glutethimide, amifostine, amrubicin, amsacrine, anastrozol, anzmet, aranesp, arglabin, arsentrioxide, aromasine, 5-azacytidine, azathioprine, BCG or tice-BCG, bestatin, betamethasone acetate, betamethasone sodium phosphate, bexarotene, bleomycin sulfate, broxuridine, bortezomib, busulfane, calcitonine, campath, capecitabine, carboplatin, casodex, cefesone, celmoleukin, cerubidine, chlorambucil, cisplatin, cladribine, clodronic acid, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunoxome, decadrone, decadrone phosphate, delestrogen, denileukin diftitox, depo medrol, desloreline, dexrazoxane, diethylstilbestrol, diflucan, docetaxel, doxifluridine, doxorubicin, dronabinol, DW-166HC, eligard, elitek, ellence, emend, epirubicin, epoetin alfa, epogen, eptaplatin, ergamisole, estrace, estradiol, estramustine sodium phosphate, ethinyl estradiol, ethyol, etidronic acid, etopophos, etoposide, fadrozole, farstone, filgrastim, finasteride, fligrastim, floxuridine, fluconazole, fludarabin, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU), fluoxymesterone, flutamide, formestane, fosteabine, fotemustine, fulvestrant, gammagard, gemcitabine, gemtuzumab, gleevec, gliadel, gosereline, granisetrone hydrochloride, histreline, hycamtine, hydrocortone, erythrohydroxynonyladenine, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, interferon-alpha, interferon-alpha-2, interferon-alpha-2α, interferon-alpha-2β, interferon-alpha-n1, interferon-alpha-n3, interferon-beta, interferon-gamma-1α, interleukin-2, intron A, iressa, irinotecan, kytril, lentinane sulfate, letrozole, leucovorine, leuprolide, leuprolide acetate, levamisole, levofolic acid calcium salt, levothroid, levoxyl, lomustine, lonidamine, marinol, mechlorethamine, mecobalamine, medroxyprogesterone acetate, megestrole acetate, melphalane, menest, 6-mercaptopurine, mesna, methotrexate, metvix, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, modrenal, myocet, nedaplatin, neulasta, neumega, neupogen, nilutamide, nolvadex, NSC-631570, OCT-43, octreotide, ondansetrone hydrochloride, orapred, oxaliplatin, paclitaxel, pediapred, pegaspargase, pegasys, pentostatin, picibanil, pilocarpine hydrochloride, pirarubicin, plicamycin, porfimer sodium, prednimustin, prednisolone, prednisone, premarin, procarbazine, procrit, raltitrexed, rebif, rhenium 186 etidronate, rituximab, roferone A, romurtide, salagen, sandostatin, sargramostim, semustine, sizofiran, sobuzoxane, solu-medrol, streptozocine, strontium-89 cehlorid, Synthroid, tamoxifen, tamsulosine, tasonermine, tastolactone, taxoter, teceleukin, temozolomide, teniposide, testosterone propionate, testred, thioguanine, thiotepa, thyrotropin, tiludronic acid, topotecan, toremifen, tositumomab, tastuzumab, teosulfane, tretinoin, trexall, trimethylmelamine, trimetrexate, triptoreline acetate, triptoreline pamoate, uft, uridine, valrubicin, vesnarinone, vinblastine, vincristine, vindesine, vinorelbine, virulizine, zinecard, zinostatin stimalamer, zofran; ABI-007, acolbifene, actimmune, affinitak, aminopterine, arzoxifene, asoprisnil, atamestane, atrasentane, avastin, BAY 43-9006 (sorafenib), CCI-779, CDC-501, celebrex, cetuximab, crisnatol, cyproterone acetate, decitabin, DN-101, doxorubicin MTC, dSLIM, dutasteride, edotecarin, eflornithine, exatecane, fenretinide, histamine dihydrochloride, histreline hydrogel implant, holmium-166-DOTMP, ibandronic acid, interferon-gamma, intron PEG, ixabepilone, keyhole limpet hemocyanine, L-651582, lanreotide, lasofoxifen, libra, lonafarnib, miproxifen, minodronate, MS-209, liposomales MTP-PE, MX-6, nafareline, nemorubicin, neovastat, nolatrexed, oblimersen, onko-TCS, osidem, paclitaxel polyglutamate, pamidronate disodium, PN-401, QS-21, quazepam, Rr-1549, raloxifene, ranpirnase, 13-cis-retinic acid, satraplatin, seocalcitol, T-138067, tarceva, taxoprexine, thymosine-alpha-1, tiazofurine, tipifarnib, tirapazamine, TLK-286, toremifene, transmid 107R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100, zoledronic acid as well as combinations thereof.
  • In a preferred embodiment, the compounds according to the present invention may be combined with antihyperproliferative agents, which may include the following, for example, although this list is not conclusive:
  • aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine, bleomycin, busulfan, carboplatin, carmustin, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, 2′,2′-difluorodeoxycytidine, docetaxel, doxorubicin (adriamycin), epirubicin, epothilone and seine derivate, erythrohydroxynonyladenine, ethinyl estradiol, etoposide, fludarabine phosphate, 5-fluorodeoxyuridine, 5-fluordeoxyuridine monophosphate, 5-fluoruracil, fluoxymesterone, flutamide, hexamethyl melamine, hydroxyurea, hydroxyprogesterone caproate, idarubicin, ifosfamide, interferon, irinotecan, leucovorin, lomustine, mechlorethamine, medroxyprogesterone acetate, megestrol acetate, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitotane, mitoxantrone, paclitaxel, pentostatin, n-phosphonoacetyl-L-aspartate (PALA), plicamycin, prednisolone, prednisone, procarbazine, raloxifene, semustine, streptozocine, tamoxifen, teniposide, testosterone propionate, thioguanine, thiotepa, topotecan, trimethylmelamine, uridine, vinblastine, vincristine, vindesine and vinorelbine.
  • In one very promising aspect, the compounds according to the invention can also be combined with biological therapeutic agents such as antibodies (e.g., Avastin, Rituxan, Erbitux, Herceptin). The compounds according to the invention may also achieve positive effects in combination with treatments directed against angiogenesis, for example, with Avastin, axitinib, recentin, regorafenib, sorafenib or sunitinib. Combinations with inhibitors of the proteasome and of mTOR as well as combinations with antihormones and steroidal metabolic enzyme inhibitors are also especially suitable because of their favorable profile of side effects.
  • In general, the following goals can be pursued with the combination of compounds of the present invention with other active cytostatic or cytotoxic agents:
      • an improved efficacy in retarding the growth of a tumor, in reducing its size or even completely eliminating it in comparison with a treatment with a single active ingredient;
      • the possibility of using the chemotherapeutic agents that are used in a lower dosage than in monotherapy;
      • the possibility of a tolerable therapy with few adverse effects in comparison with an individual administration;
      • the possibility of treatment of a broader spectrum of tumors;
      • achieving a higher response rate to the treatment;
      • a longer survival time for the patients in comparison with today's standard therapy.
  • In addition, the compounds according to the invention may also be used in combination with radiation therapy and/or a surgical intervention.
  • An additional subject matter of the present invention is pharmaceutical drugs that contain at least one compound according to the invention, usually together with one or more inert nontoxic pharmaceutically suitable excipients as well as their use for the aforementioned purposes.
  • The compounds according to the invention may act systemically and/or locally. For this purpose they are applied in a suitable way such as, for example, orally or parenterally. The compounds according to the invention may act systemically and/or locally. For this purpose they are applied in a suitable way such as, for example, parenterally, possibly by inhalation or as an implant and/or stent.
  • For these application methods, the compounds according to the invention may be administered in suitable dosage forms.
  • Parenteral administration may be performed in order to bypass a resorption step (e.g., intravenously, intra-arterially, intracardially, intraspinally or intralumbarly) or with the inclusion of resorption (e.g., intramuscularly, subcutaneously, intracutaneously, percutaneously or intraperitoneally). Suitable forms of administration for parenteral administration include infusion and injection preparations in the form of solutions, suspensions, emulsions or lyophilisates.
  • Parenteral administration in particular intravenous administration is preferred.
  • i.v. Solution:
  • The compounds according to the invention may be converted to the dosage forms indicated. This may be done in the known way by “mixing with” and/or “dissolving in” inert nontoxic pharmaceutically suitable excipients (e.g., buffer substances, stabilizers, solubilizer, preservatives). These may include, for example: amino acids (glycine, histidine, methionine, arginine, lysine, leucine, isoleucine, threonine, glutamic acid, phenylalanine and others), sugars and related substances (glucose, saccharose, mannitol, trehalose, sucrose, mannose, lactose, sorbitol), glycerol, sodium, potassium, ammonium and calcium salts (e.g., NaCl, KCl or Na2HPO4 and many more), acetate/acetic acid buffer systems, phosphate buffer systems, citric acid and citrate buffer systems, trometamol (TRIS and TRIS salts), polysorbates (e.g., polysorbate 80 and polysorbate 20), poloxamers (e.g., poloxamer 188 and poloxamer 171), macrogols (PEG derivatives, e.g., 3350), Triton X-100, EDTA salts, glutathione, albumins (e.g., human), urea, benzyl alcohol, phenol, chlorocresol, metacresol, benzalkonium chloride and many others.
  • It has proven advantageous in general to administer doses of approx. 0.001 to 1 mg/kg, preferably approx. 0.01 to 0.5 mg/kg body weight to achieve effective results in parenteral administration.
  • Nevertheless, it may be necessary under some circumstances to deviate from the stated amounts, namely depending on the body weight, the method of administration, the individual response to the active ingredient, the type of preparation and the point in time or interval at which the application occurs. Thus, in many cases, it may be sufficient to use less than the aforementioned minimum amount, whereas in other cases the aforementioned upper limit must be exceeded. In the case of administration of larger amounts, it may be advisable to distribute the larger amount among several individual doses throughout the day.
  • The following examples are presented to illustrate the invention, although the invention is not limited to these examples.
  • The percentage amounts specified in the following tests and examples are percent by weight (wt %), unless otherwise indicated; parts refer to parts by weight. Solvent ratios, dilution ratios and concentration specifications for liquid-liquid solutions are each based on volume.
    • A. EXAMPLES Abbreviations and Acronyms
    • A431NS human tumour cell line
    • A549 human tumour cell line
    • ABCB1 ATP-binding cassette sub-family B member 1 (synonym for P-gp and MDR1)
    • abs. absolute
    • ADC antibody-drug-conjugate
    • Ac acetyl
    • aq. aqueous, aqueous solution
    • ATP adenosine triphosphate
    • BCRP breast cancer resistance protein, an efflux transporter
    • Boc tert-butoxycarbonyl
    • br. broad (in NMR)
    • Ex. example
    • ca. circa, approximately
    • CAD (carboanhydrase IX
    • CI chemical ionization (in MS)
    • d doublet (in NMR)
    • d day(s)
    • TLC thin-layer chromatography
    • DCI direct chemical ionization (in MS)
    • dd doublet of a doublet (in NMR)
    • DMAP 4-N,N-dimethylaminopyridine
    • DME 1,2-dimethoxyethane
    • DMEM Dulbecco's modified eagle medium (standardized nutrient medium for the cell culture)
    • DMF N,N-dimethylformamide
    • DMSO dimethyl sulphoxide
    • DPBS, D-PBS, PBS Dulbecco's phosphate-buffered saline solution PBS=DPBS=D-PBS, pH 7.4, from Sigma, No. D8537
      • Composition:
      • 0.2 g KCl
      • 0.2 g KH2PO4 (anhydrous)
      • 8.0 g NaCl
      • 1.15 g Na2HPO4 (anhydrous)
      • make up to 1 l with H2O
    • dt doublet of a triplet (in NMR)
    • DTT DL-dithiothreitol
    • EDC N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride
    • EGFR epidermal growth factor receptor
    • EI electron impact ionization (in MS)
    • ELISA enzyme-linked immunosorbent assay
    • eq. equivalent(s)
    • ESI electrospray ionization (in MS)
    • ESI-MicroTofq ESI—MicroTofq (name of the mass spectrometer, with Tof=time of flight and q=quadrupole)
    • FCS foetal calf serum
    • Fmoc (9H-fluoren-9-ylmethoxy)carbonyl
    • sat. Saturated
    • GTP guanosine 5′-triphosphate
    • h hour(s)
    • HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate
    • HCT-116 human tumour cell line
    • HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid
    • HOAc acetic acid
    • HOBt 1-hydroxy-1H-benzotriazole hydrate
    • HOSu N-hydroxysuccinimide
    • HPLC high-pressure, high-performance liquid chromatography
    • HT29 human tumour cell line
    • IC50 half-maximum inhibitory concentration
    • i.m. intramuscular, administration into the muscle
    • i.v. intravenous, administration into the vein
    • conc. Concentrated
    • LC-MS liquid chromatography-coupled mass spectrometry
    • LLC-PK1 cells Lewis lung carcinoma pork kidney cell line
    • L-MDR human MDR1 transfected LLC-PK1 cells
    • m multiplet (in NMR)
    • MDR1 multidrug resistance protein 1
    • min minute(s)
    • MS mass spectrometry
    • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
    • NCI-H292 human tumour cell line
    • NCI-H520 human tumour cell line
    • NMM N-methylmorpholine
    • NMP N-methyl-2-pyrrolidinone
    • NMR nuclear magnetic resonance spectrometry
    • NMRI mouse strain, originating from Naval Medical Research Institute (NMRI)
    • Nude mice experimental animals
    • NSCLC non-small cell lung cancer (non-small cell bronchial carcinoma)
    • PBS phosphate-buffered saline solution
    • Pd/C palladium on activated carbon
    • P-gp P-glycoprotein, a transporter protein
    • PNGaseF enzyme for sugar elimination
    • quant. quantitative (for yield)
    • quart quartet (in NMR)
    • quint quintet (in NMR)
    • Rf retention index (for TLC)
    • RT room temperature
    • Rt retention time (for HPLC)
    • s singlet (in NMR)
    • s.c. subcutaneous, administration beneath the skin
    • SCC-4 human tumour cell line
    • SCC-9 human tumour cell line
    • SCID mice experimental mice with a severe combined immunodeficiency
    • t triplet (in NMR)
    • tert tertiary
    • TFA trifluoroacetic acid
    • THF tetrahydrofuran
    • UV ultraviolet spectrometry
    • v/v volume to volume ratio (of a solution)
    • Z benzyloxycarbonyl
    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×1 mm; eluent A: 1 l water+0.25 ml 99% strength formic acid, eluent B: 1 l acetonitrile+0.25 ml 99% strength 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×1 mm; eluent A: 1 l water+0.5 ml 50% strength formic acid, eluent B: 1 l acetonitrile+0.5 ml 50% strength formic acid; gradient: 0.0 min 90% A→0.1 min 90% A→1.5 min 10% A→2.2 min 10% A; flow rate: 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×4 mm; eluent A: 1 l water+0.5 ml 50% strength formic acid, eluent B: 1 l acetonitrile+0.5 ml 50% strength 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×3.00 mm; eluent A: 1 l water+0.5 ml 50% strength formic acid, eluent B: 1 l acetonitrile+0.5 ml 50% strength 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 Speed ROD RP-18e, 50 mm×4.6 mm; preliminary column: Merck Chromolith Guard Cartridge Kit RP-18e, 5 mm×4.6 mm; injection volume: 5 μl; eluent A: 70% HClO4 in water (4 ml/litre), eluent 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×4.6 mm; Ord. No.: 1.51450.0001, preliminary column: Merck Chromolith Guard Cartridge Kit RP-18e, 5 mm×4.6 mm; Ord. No.: 1.51470.0001, eluent A: 70% HClO4 in water (4 ml/litre), eluent 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×4 mm; eluent A: 1 l water+0.5 ml 50% strength formic acid, eluent B: 1 l acetonitrile+0.5 ml 50% strength 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×4 mm; eluent A: 1 l water+0.5 ml 50% strength formic acid, eluent B: 1 l acetonitrile+0.5 ml 50% strength 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×1 mm; eluent A: 1 l water+0.25 ml 99% strength formic acid, eluent B: 1 l acetonitrile+0.25 ml 99% strength 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×150 mm; preliminary column: Phenomenex KrudKatcher Disposable Pre-Column; injection volume: 5 μl; eluent A: 1 l water+0.01% trifluoroacetic acid; eluent 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 whose preparation is not explicitly described below, they were obtained commercially from generally available sources. For all other reactants or reacents whose preparation is likewise not described below, and which were not available commercially or were obtained from sources which are not generally available, a reference is given to the published literature in which their preparation is described.
    • Method 11 (LC-MS):
  • Instrument: Waters ACQUITY SQD UPLC System; column: Waters Acquity UPLC HSS T3 1.8 g 30×2 mm; eluent A: 1 l water+0.25 ml 99% strength formic acid, eluent B: 1 l acetonitrile+0.25 ml 99% strength 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):
  • Instrument: Agilent 1200 Series with column oven and DAD; column: Merck Chromolith SpeedROD RP-18e, 50 mm×4.6 mm; Ord. No.: 1.51450.0001; preliminary column: Merck Chromolith Guard Cartridge Kit RP-18e, 5 mm×4.6 mm; Ord. No.: 1.51470.0001; eluent A: 70% HClO4 in water (4 ml/litre), eluent 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 3μ 50×3 mm; eluent A: 1 l water+0.01 mol ammonium carbonate, eluent 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 (2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoic acid (Boc-dolaproine)
  • Figure US20140127240A1-20140508-C00433
  • The title compound can be prepared in various 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., Tetrahedron Lett. 1991, 32, 2395; Vidal et al., Tetrahedron 2004, 60, 9715; Poncet et al., Tetrahedron 1994, 50, 5345. It was prepared either as the free acid or as a 1:1 salt with dicyclohexylamine.
    • Starting Compound 2a tert-butyl (3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoate hydrochloride (dolaisoleucine-OtBu×HCl)
  • Figure US20140127240A1-20140508-C00434
  • The title compound can be prepared in various 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 tert-butyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoate (dolaisoleucine-OtBu)
  • Figure US20140127240A1-20140508-C00435
  • The compound was prepared in analogy to starting compound 2a, except that the hydrogenation was performed without addition of 1N hydrochloric acid.
    • Starting Compound 3 Nα-(tert-butoxycarbonyl)-N-hydroxy-L-phenylalaninamide
  • Figure US20140127240A1-20140508-C00436
  • The title compound was prepared by the literature method (A. Ritter et al., J. Org. Chem. 1994, 59, 4602).
  • Yield: 750 mg (75% of theory)
  • LC-MS (Method 3): Rt=1.67 min; MS (ESIpos): m/z=281 (M+H)+.
    • Starting Compound 4 1,2-oxazolidine hydrochloride
  • Figure US20140127240A1-20140508-C00437
  • The title compound can be prepared by literature methods (see, for example, H. King, J. Chem. Soc. 1942, 432); it is also commercially available.
    • Starting Compound 5 1,2-oxazinane hydrochloride
  • Figure US20140127240A1-20140508-C00438
  • The title compound can be 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
  • Figure US20140127240A1-20140508-C00439
  • The title compound can be prepared in Boc-protected form by the literature method (see, for example, C. Johnson et al., Tetrahedron Lett. 1998, 39, 2059); the deprotection was effected in a customary manner by treatment with trifluoroacetic acid and subsequent neutralization.
  • Yield: 149 mg (89% of theory)
    • Starting Compound 7 tert-butyl (1S,2R)-1-(hydroxycarbamoyl)-2-phenylcyclopropyl carbamate
  • Figure US20140127240A1-20140508-C00440
  • The title compound was prepared by a literature method (A. Ritter et al., J. Org. Chem. 1994, 59, 4602) proceeding from commercially available (1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid (C. Cativiela et al., Chirality 1999, 11, 583).
  • Yield: 339 mg (59% of theory)
  • LC-MS (Method 1): Rt=0.82 min; MS (ESIpos): m/z=293 (M+H)+.
    • Intermediate 1 tert-butyl-(3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-L-valyl}(methyl)amino]-3-methoxy-5-methyl heptanoate
  • Figure US20140127240A1-20140508-C00441
  • 10.65 g (41.058 mmol) of tert-butyl (3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)-heptanoate (starting compound 2b) were taken up in 250 ml of dichloromethane, and the solution was cooled to −10° C. Then, while stirring, 10.317 g (41.058 mmol) of N-Rbenzyloxy)carbonylF L-valine, 16.866 g (61.586 mmol) of 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP) and 28.6 ml of N,N-diisopropylethylamine were added, and the mixture was subsequently stirred at RT for 20 h. The reaction mixture was then diluted with dichloromethane and shaken twice with saturated sodium chloride solution, dried over sodium sulphate, filtered and concentrated. The residue was purified by flash chromatography on silica gel with 4:1 petroleum ether/ethyl acetate as the eluent. The corresponding fractions were concentrated, and the residue was dried under high vacuum overnight. 10.22 g (51% of theory) of the title compound were obtained as a yellowish oil.
  • HPLC (Method 5): Rt=2.3 min;
  • LC-MS (Method 2): Rt=1.59 min; MS (ESIpos): m/z=493 (M+H)+.
    • Intermediate 2 tert-butyl (3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(L-valyl)amino]heptanoate
  • Figure US20140127240A1-20140508-C00442
  • 500 mg (1 mmol) of tert-butyl (3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoate (intermediate 1) were dissolved in 50 ml of methanol and, after addition of 100 mg of 10% palladium on activated carbon, hydrogenated under standard hydrogen pressure at RT for 1 h. The catalyst was then filtered off, and the solvent was removed under reduced pressure. This gave 370 mg (quant.) of the title compound as a virtually colourless oil.
  • HPLC (Method 5): Rt=1.59 min;
  • LC-MS (Method 1): Rt=0.74 min; MS (ESIpos): m/z=359 (M+H)+.
    • Intermediate 3 N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-tert-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00443
  • 4.64 g (13.13 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valine were dissolved in 20 ml of DMF and admixed successively with 4.28 g (11.94 mmol) of tert-butyl (3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(L-valyl)amino]heptanoate (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-1H-benzotriazole hydrate. The mixture was stirred at RT overnight. The reaction mixture was then poured into a mixture of semisaturated aqueous ammonium chloride solution and ethyl acetate. The organic phase was removed, washed successively with saturated sodium hydrogencarbonate solution and saturated sodium chloride solution, dried over magnesium sulphate, filtered and concentrated. The residue was used directly in the next stage, without further purification.
  • Yield: 9.1 g (quant., 60% purity)
  • HPLC (Method 5): Rt=2.7 min;
  • LC-MS (Method 2): Rt=1.99 min; MS (ESIpos): m/z=694 (M+H)+.
    • Intermediate 4 N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00444
  • 9.1 g of the crude product N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-tert-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 3) were taken up in 56.6 ml of dichloromethane, 56.6 ml of trifluoroacetic acid were added, and the mixture was stirred at RT for 2 h. Subsequently, the reaction mixture was concentrated in vacuo and the remaining residue was purified by flash chromatography, using dichloromethane, 3:1 dichloromethane/ethyl acetate and 15:5:0.5 dichloromethane/ethyl acetate/methanol as eluent. After purification of the corresponding fractions and concentration, 5.8 g (86% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=2.2 min;
  • LC-MS (Method 1): Rt=1.3 min; MS (ESIpos): m/z=638 (M+H)+.
    • Intermediate 5 tert-butyl (2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl carbamate
  • Figure US20140127240A1-20140508-C00445
  • 500 mg (1.9 mmol) of N-(tert-butoxycarbonyl)-L-phenylalanine were dissolved in 10 ml of DMF and admixed successively with 466 mg (3.8 mmol) of 1,2-oxazinane hydrochloride (Starting Compound 5), 433 mg (2.3 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 382 mg (2.8 mmol) of 1-hydroxy-1H-benzotriazole hydrate and 731 mg (5.7 mmol) of N,N-diisopropylethylamine. The mixture was stirred at RT overnight. The reaction mixture was then poured into a mixture of semisaturated aqueous ammonium chloride solution and ethyl acetate. The organic phase was removed, washed successively with saturated sodium hydrogencarbonate solution and saturated sodium chloride solution, dried over magnesium sulphate, filtered and concentrated. 620 mg (98% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.8 min;
  • LC-MS (Method 2): Rt=1.62 min; MS (ESIpos): m/z=235 (M-C4H8—CO2+H)+.
    • Intermediate 6 (2S)-2-amino-1-(1,2-oxazinan-2-yl)-3-phenylpropan-1-one trifluoroacetate
  • Figure US20140127240A1-20140508-C00446
  • 620 mg (1.85 mmol) of tert-butyl (2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl carbamate (Intermediate 5) were taken up in 5 ml of dichloromethane, 10 ml of trifluoroacetic acid were added and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated in vacuo, and the remaining residue was lyophilized from water/acetonitrile. In this way, 779 mg (91% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=0.45 min;
  • LC-MS (Method 3): Rt=1.09 min; MS (ESIpos): m/z=235 (M+H)+.
    • Intermediate 7 (2R,3R)-3-methoxy-2-methyl-N-[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]-3-[(2S)-pyrrolidin-2-yl]propanamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00447
  • 360 mg (1.25 mmol) of (2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoic acid (Starting Compound 1) were taken up in 10 ml of DMF and admixed successively with 579.2 mg (1.25 mmol) of (2S)-2-amino-1-(1,2-oxazinan-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′-tetramethyluronium hexafluorophosphate (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 up in ethyl acetate and extracted by shaking first with 5% aqueous citric acid solution, then with 5% aqueous sodium hydrogencarbonate solution and subsequently with saturated sodium chloride solution. The organic phase was concentrated, and the residue was purified by flash chromatography on silica gel with 16:4 dichloromethane/methanol as the eluent. The corresponding fractions were combined and the solvent was removed under in vacuo. After the residue had been dried under high vacuum, 503.5 mg (74% of theory) of the Boc-protected intermediate tert-butyl (2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidine-1-carboxylate were obtained.
  • HPLC (Method 12): Rt=2.0 min;
  • LC-MS (Method 1): Rt=1.12 min; MS (ESIpos): m/z=504 (M+H)+.
  • 503 mg (1 mmol) of this intermediate were taken up in 20 ml of dichloromethane, 10 ml of trifluoroacetic acid were added, and the mixture was stirred at RT for 30 min Subsequently, the reaction mixture was concentrated in vacuo and redistilled with dichloromethane. The remaining residue was precipitated from ethyl acetate with n-pentane, and the solvent was decanted off. The residue thus obtained was dissolved in water and extracted by shaking with ethyl acetate, and the aqueous phase was subsequently lyophilized. In this way, 462 mg (89% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 12): Rt=1.53 min;
  • LC-MS (Method 11): Rt=0.57 min; MS (ESIpos): m/z=404 (M+H)+.
    • Intermediate 8 N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00448
  • 51 mg (0.08 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 4) were dissolved in 10 ml of DMF, and 0.5 ml of piperidine was added. After stirring at RT for 10 min, the reaction mixture was concentrated in vacuo, and the residue was stirred with diethyl ether. The insoluble constituents were filtered off and washed repeatedly with diethyl ether. Then the filter residue was taken up in 5 ml of dioxane/water, and the solution was adjusted to pH 11 with 1 N sodium hydroxide solution. Under ultrasound treatment, a total of 349 mg (1.6 mmol) of di-tert-butyl dicarbonate were added in several portions, in the course of which the pH of the solution was kept at 11. After the reaction had ended, the dioxane was evaporated off and the aqueous solution was adjusted to a pH of 2-3 with citric acid. The mixture was extracted twice, with 50 ml each time of ethyl acetate. The organic phases were combined, dried over magnesium sulphate and concentrated under reduced pressure. The residue was taken up in diethyl ether and the of the title compound was precipitated with pentane. The solvent was removed by decantation. The residue was digested several times more with pentane and finally dried under high vacuum. 40 mg (97% of theory) of the title compound were thus obtained.
  • HPLC (Method 6): Rt=2.2 min;
  • LC-MS (Method 2): Rt=1.32 min; MS (ESIpos): m/z=516 (M+H)+.
    • Intermediate 9 tert-butyl-(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidine-1-carboxylate
  • Figure US20140127240A1-20140508-C00449
  • The title compound was prepared in analogy to the synthesis of Intermediates 5, 6 and 7 over three stages by coupling of commercially available (1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid with 1,2-oxazinane hydrochloride (Starting Compound 5), subsequent deprotection with trifluoroacetic acid and coupling with Starting Compound 1. The end product was purified by preparative HPLC.
  • HPLC (Method 5): Rt=2.12 min;
  • LC-MS (Method 2): Rt=1.25 min; MS (ESIpos): m/z=516 (M+H)+.
    • Intermediate 10 N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00450
  • 315 mg (0.494 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 4) were dissolved in 12 ml of DMF and admixed with 104 mg (0.543 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 83 mg (0.543 mmol) of 1-hydroxy-1H-benzotriazole hydrate, and the mixture was stirred at RT for 90 min. Subsequently, 112 μl of N,N-diisopropylethylamine and 149 mg (0.494 mmol) of (2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoic acid trifluoroacetate, which had been prepared beforehand from Starting Compound 1 by elimination of the Boc protecting group by means of trifluoroacetic acid, were added. The mixture was stirred at RT for 2 h and then concentrated under high vacuum. The remaining residue was purified twice by preparative HPLC. 140 mg (35% of theory) of the title compound were obtained in the form of a colourless foam.
  • HPLC (Method 5): Rt=2.40 min;
  • LC-MS (Method 1): Rt=1.38 min; MS (ESIpos): m/z=807 (M+H)+.
    • Intermediate 11 N-[(benzyloxy)carbonyl]-N-methyl-L-threonyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00451
  • First, N-[(benzyloxy)carbonyl]-N-methyl-L-threonine was released from 237 mg (0.887 mmol) of its dicyclohexylamine salt thereof by taking it up in ethyl acetate and extractive shaking with 5% aqueous sulphuric acid. The organic phase was dried over magnesium sulphate, filtered and concentrated. The residue was taken up in 16 ml of DMF and admixed successively with 365 mg (1 mmol) of tert-butyl (3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(L-valyl)amino]heptanoate (Intermediate 2), 185 mg (0.967 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 148 mg (0.967 mmol) of 1-hydroxy-1H-benzotriazole hydrate. The mixture was stirred at RT for 2 h. The reaction mixture was then poured into a mixture of semisaturated aqueous ammonium chloride solution and ethyl acetate. The organic phase was removed, washed successively with saturated sodium hydrogencarbonate solution and saturated sodium chloride solution, dried over magnesium sulphate, filtered and concentrated. The residue was purified by preparative HPLC. 283 mg (53% of theory) of the tert-butyl ester intermediate N-[(benzyloxy)carbonyl]-N-methyl-L-threonyl-N-[(3R,4S,5S)-1-tert-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were thus obtained.
  • HPLC (Method 5): Rt=2.17 min
  • 283 mg (0.466 mmol) of this intermediate were taken up in 5 ml of dichloromethane, 5 ml of anhydrous trifluoroacetic acid were added, and the mixture was stirred at RT for 2 h. Subsequently, the reaction mixture was concentrated under high vacuum and the remaining residue was purified by means of preparative HPLC. This gave 156 mg (61% of theory) of the title compound as a colourless foam.
  • HPLC (Method 5): Rt=1.50 min;
  • LC-MS (Method 2): Rt=1.09 min; MS (ESIpos): m/z=552 (M+H)+.
    • Intermediate 12 Benzyl-N-{(2R,3R)-3-methoxy-2-methyl-3[(2S)-pyrrolidin-2-yl}propanoyl]-L-phenylalaninate trifluoroacetate
  • Figure US20140127240A1-20140508-C00452
  • In the first step, Starting Compound 1 was released from 600 mg (1.28 mmol) of the corresponding dicyclohexylammonium salt by dissolving the salt in 100 ml of ethyl acetate and extractive shaking, first with 50 ml of 0.5% sulphuric acid and then with saturated sodium chloride solution. Then the organic phase was dried over magnesium sulphate, filtered, concentrated and reacted immediately with benzyl L-phenylalaninate in analogy to the synthesis of Intermediate 7, and then deprotected.
  • Yield: 650 mg (94% over 2 stages)
  • HPLC (Method 6): Rt=1.76 min;
  • LC-MS (Method 2): Rt=1.68 min; MS (ESIpos): m/z=425 (M+H)+.
    • Intermediate 13 Benzyl-(βS)—N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-β-methyl-L-phenylalaninate trifluoroacetate
  • Figure US20140127240A1-20140508-C00453
  • First, (2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoic acid was released from 351 mg (0.75 mmol) of the dicyclohexylamine salt (Starting Compound 1) by taking it up in ethyl acetate and extractive shaking with aqueous 5% potassium hydrogensulphate solution. The organic phase was dried over magnesium sulphate, filtered and concentrated. The residue was taken up in 10 ml of DMF and admixed successively with 373 mg (0.75 mmol) of benzyl-(βS)-β-methyl-L-phenylalaninate trifluoroacetate [prepared from commercially available (βS)-N-(tert-butoxycarbonyl)-β-methyl-L-phenylalanine by EDC/DMAP-mediated esterification with benzyl alcohol and subsequent cleaving of the Boc protecting group with trifluoroacetic acid], 428 mg (1.125 mmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and 392 μl of N,N-diisopropylethylamine. The mixture was stirred at RT for 20 h. The reaction mixture was then poured onto a mixture of semisaturated aqueous ammonium chloride solution and ethyl acetate. The organic phase was removed, washed successively with saturated sodium hydrogencarbonate solution and saturated sodium chloride solution, and subsequently concentrated. The residue was purified by means of preparative HPLC. This gave 230 mg (57% of theory) of the Boc-protected intermediate benzyl-(βS)-N-{(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-β-methyl-L-phenylalaninate.
  • HPLC (Method 6): Rt=2.3 min;
  • LC-MS (Method 1): Rt=1.36 min; MS (ESIpos): m/z=539 (M+H)+.
  • 230 mg (0.42 mmol) of this intermediate were taken up in 5 ml of dichloromethane, 5 ml of trifluoroacetic acid were added, and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated in vacuo. The remaining residue was the reaction mixture dried further in vacuo and then lyophilized from acetonitrile/water. In this way, 230 mg (quant.) of the title compound were obtained.
  • HPLC (Method 6): Rt=1.6 min
    • Intermediate 14 N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoro acetate
  • Figure US20140127240A1-20140508-C00454
  • 143 mg (0.223 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 4) were taken up in 15 ml of DMF and admixed successively with 141 mg (0.22 mmol) of (2R,3R)-3-methoxy-2-methyl-N-[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-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′-tetramethyluronium hexafluorophosphate (HATU) and 128 μl (0.74 mmol) of N,N-diisopropylethylamine The mixture was stirred at RT for 3 h. The reaction mixture was then poured into a mixture of semisaturated aqueous ammonium chloride solution and ethyl acetate. The organic phase was removed, washed successively with saturated sodium hydrogencarbonate solution and saturated sodium chloride solution, dried over magnesium sulphate, filtered and concentrated. This gave 275 mg (quant.) of the Fmoc-protected intermediate N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • HPLC (Method 5): Rt=2.73 min;
  • LC-MS (Method 4): Rt=3.19 min; MS (ESIpos): m/z=1023 (M+H)+.
  • 46 mg (0.045 mmol) of this intermediate were dissolved in 4 ml of DMF. After 1 ml of piperidine had been added, the reaction mixture was stirred at RT for 1 h. Subsequently, the reaction mixture was concentrated in vacuo and the residue was purified by means of preparative HPLC (eluent: acetonitrile+0.01% TFA/water+0.01% TFA). 22 mg (54% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.68 min;
  • LC-MS (Method 2): Rt=1.03 min; MS (ESIpos): m/z=801 (M+H)+
  • 1H NMR (600 MHz, DMSO-d6): 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) [further signals hidden under H2O peak].
    • Intermediate 15 N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00455
  • 126 mg (0.198 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 4) were taken up in 10 ml of DMF and admixed successively with 105 mg (0.198 mmol) of (2R,3R)-3-methoxy-2-methyl-N-[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-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-1H-benzotriazole hydrate and 79 μl (0.454 mmol) of N,N-diisopropylethylamine. The mixture was stirred at RT overnight. The reaction mixture was then poured into a mixture of semisaturated aqueous ammonium chloride solution and ethyl acetate. The organic phase was removed, washed successively with saturated sodium hydrogencarbonate solution and saturated sodium chloride solution, dried over magnesium sulphate, filtered and concentrated. This gave 220 mg (quant.) of the Fmoc-protected intermediate N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • HPLC (Method 5): Rt=2.77 min;
  • LC-MS (Method 1): Rt=1.5 min; MS (ESIpos): m/z=1037 (M+H)+.
  • 220 mg (0.212 mmol) of this intermediate were dissolved in 5 ml of DMF. After 1 ml of piperidine had been added, 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 means of preparative HPLC (eluent: acetonitrile+0.01% TFA/water+0.01% TFA). 91 mg (46% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.71 min;
  • LC-MS (Method 1): Rt=0.9 min; MS (ESIpos): m/z=815 (M+H)+
  • 1H NMR (600 MHz, DMSO-d6): 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) [further signals hidden under H2O peak].
    • Intermediate 16 N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00456
  • 617 mg (1.2 mmol) of tert-butyl (2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidine-1-carboxylate (Intermediate 24) were taken up in 44 ml of dichloromethane, 4.4 ml of trifluoroacetic acid were added, and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated in vacuo and the remaining residue was lyophilized from dioxane/water. 702 mg (quant.) of the deprotected compound (2R,3R)-3-methoxy-2-methyl-N-[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]-3-[(2S)-pyrrolidin-2-yl]propanamide trifluoroacetate were obtained as a crude product, which was used in the following stage without further purification.
  • 470 mg (0.74 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 4) were taken up in 57 ml of DMF and admixed successively with 390 mg (approx. 0.74 mmol) of the above-obtained (2R,3R)-3-methoxy-2-methyl-N-[(1S,2R)-1-(1,2-oxazinan-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′-tetramethyluronium hexafluorophosphate (HATU) and 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 semisaturated aqueous ammonium chloride solution and ethyl acetate. The organic phase was removed, washed successively with saturated sodium hydrogencarbonate solution and saturated sodium chloride solution, dried over sodium sulphate, filtered and concentrated. The residue was purified by preparative HPLC. This gave 453 mg (59% of theory) of the Fmoc-protected intermediate N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • HPLC (Method 5): Rt=2.58 min;
  • LC-MS (Method 1): Rt=3.10 min; MS (ESIpos): m/z=1035 (M+H)+.
  • 453 mg (0.438 mmol) of this intermediate were dissolved in 24 ml of DMF. After 2.4 ml of piperidine had been added, 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 (eluent: acetonitrile/0.1% TFA in water). 260 mg (64% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.64 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=813 (M+H)+
  • 1H NMR (400 MHz, DMSO-d6): 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) [further signals hidden under H2O peak].
    • Intermediate 17 N-benzyl-N-methyl-L-phenylalaninamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00457
  • 1000 mg (3.77 mmol) of N-(tert-butoxycarbonyl)-L-phenylalanine were dissolved in 10 ml of DMF and admixed with 457 mg (3.77 mmol) of N-methylbenzylamine, 2150 mg (5.65 mmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 657 μl of N,N-diisopropylethylamine. The reaction mixture was stirred at RT for 30 min and then concentrated in vacuo. The residue was taken up in dichloromethane and extracted by shaking three times with water. The organic phase was dried over magnesium sulphate and concentrated. The residue was purified by flash chromatography on silica gel with 3:1 petroleum ether/ethyl acetate as the eluent. The product fractions were concentrated, and the residue was dried under high vacuum. This gave 1110 mg (75% of theory) of the Boc-protected intermediate N-benzyl-Nα-(tert-butoxycarbonyl)-N-methyl-L-phenylalaninamide
  • HPLC (Method 6): Rt=2.1 min;
  • LC-MS (Method 1): Rt=1.14 min; MS (ESIpos): m/z=369 (M+H)+.
  • 1108 mg (3.007 mmol) of this intermediate were taken up in 30 ml of dichloromethane, 10 ml of trifluoroacetic acid were added, and the mixture was stirred at RT for 30 min Subsequently, the reaction mixture was concentrated in vacuo, the remaining residue was stirred with dichloromethane, and the solvent was distilled off. The residue was stirred twice more with pentane, the solvent was decanted off again each time and the of the title compound was finally dried under high vacuum. 1075 mg (93% of theory) of the title compound were thus obtained as a resin.
  • HPLC (Method 6): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.6 min; MS (ESIpos): m/z=269 (M+H)+.
    • Intermediate 18 N-benzyl-Nα-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-N-methyl-L-phenylalaninamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00458
  • First, (2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoic acid (Starting Compound 1) was released from 141 mg (0.491 mmol) of its dicyclohexylamine salt by taking it up in ethyl acetate and extractive shaking with 5% aqueous sulphuric acid. The organic phase was dried over magnesium sulphate, 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-phenylalaninamide trifluoroacetate (Intermediate 9), 190.3 mg (1.47 mmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and 256 μl of N,N-diisopropylethylamine were added. The mixture was stirred at RT for 1 h. The reaction mixture was then concentrated, the residue was taken up in ethyl acetate, and the solution was subsequently extracted by shaking successively with saturated ammonium chloride solution, saturated sodium hydrogencarbonate solution and water. The organic phase was dried over magnesium sulphate and concentrated. The residue was purified by flash chromatography on silica gel with 30:1 acetonitrile/water as the eluent. The product fractions were concentrated and the residue was dried under high vacuum. This gave 168 mg (64% of theory) of the Boc-protected intermediate tert-butyl (2S)-2-[(1R,2R)-3-({(2S)-1-[benzyl(methyl)amino]-1-oxo-3-phenylpropan-2-yl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1-carboxylate.
  • HPLC (Method 6): Rt=2.2 min;
  • LC-MS (Method 2): Rt=1.22 min; MS (ESIpos): m/z=538 (M+H)+.
  • 168 mg (0.312 mmol) of this intermediate were taken up in 15 ml of dichloromethane, 3 ml of trifluoroacetic acid were added, and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated in vacuo. The remaining residue was stirred first with dichloromethane, then with diethyl ether, and the solvent was distilled off again each time. After drying under high vacuum, 170 mg (99% of theory) of the title compound were obtained as a resin.
  • HPLC (Method 6): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.73 min; MS (ESIpos): m/z=438 (M+H)+.
    • Intermediate 19 Methyl-N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-phenylalaninate trifluoroacetate
  • Figure US20140127240A1-20140508-C00459
  • The title compound was prepared in analogy to the synthesis of Intermediate 18, proceeding from (2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoic acid (Starting Compound 1), which was released from the dicyclohexylamine salt, and methyl L-phenylalaninate hydrochloride.
  • HPLC (Method 5): Rt=0.6 min;
  • LC-MS (Method 3): Rt=1.17 min; MS (ESIpos): m/z=349 (M+H)+.
    • Intermediate 20 Benzyl-N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl}propanoyl]-L-tryptophanate trifluoroacetate
  • Figure US20140127240A1-20140508-C00460
  • The title compound was prepared in analogy to the synthesis of Intermediate 18, proceeding from (2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoic acid (Starting Compound 1), which was released from the dicyclohexylamine salt, and benzyl L-tryptophanate.
  • HPLC (Method 6): Rt=2.0 min;
  • LC-MS (Method 1): Rt=0.8 min; MS (ESIpos): m/z=464 (M+H)+.
    • Intermediate 21 Benzyl-(1S,2R)-1-({(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}amino)-2-phenylcyclopropanecarboxylate trifluoroacetate
  • Figure US20140127240A1-20140508-C00461
  • The title compound was prepared in analogy to the synthesis of Intermediate 18, proceeding from (2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoic acid (Starting Compound 1), which was released from the dicyclohexylamine salt, and benzyl (1S,2R)-1-amino-2-phenylcyclopropanecarboxylate. Benzyl-(1S,2R)-1-amino-2-phenylcyclopropane-carboxylate had been prepared beforehand by standard methods, by esterifying commercially available (1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid with benzyl alcohol and subsequent Boc cleaving with trifluoroacetic acid.
  • HPLC (Method 5): Rt=1.5 min;
  • LC-MS (Method 2): Rt=0.93 min; MS (ESIpos): m/z=437 (M+H)+.
    • Intermediate 22 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N′-methylhexanehydrazide trifluoroacetate
  • Figure US20140127240A1-20140508-C00462
  • 100 mg (473 μmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid were dissolved in 71 μl of DMF and then admixed with 139 mg (947 μmol) of tert-butyl-1-methylhydrazinecarboxylate, 182 mg (947 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 145 mg (947 μmol) of 1-hydroxy-1H-benzotriazole hydrate. The mixture was stirred at RT overnight and then concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization from dioxane/water, 129 mg (80% of theory) of the protected intermediate were obtained as a colourless foam.
  • Subsequently, the 129 mg (380 μmol) were deblocked with 2 ml of trifluoroacetic acid in 8 ml of dichloromethane. After stirring at RT for 1 h, the reaction mixture was concentrated in vacuo. The residue was lyophilized from acetonitrile/water, which left 125 mg (83% of theory) of the title compound as a colourless foam.
  • LC-MS (Method 1): Rt=0.38 min; MS (ESIpos): m/z=240 (M+H)+
    • Intermediate 23 N-(2-aminoethyl)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylbutanamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00463
  • First, 35 mg (164 μmol) of tert-butyl-[2-(methylamino)ethyl]carbamate-hydrochloride-trifluoroacetate, 30 mg (164 μmol) of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid, 75 mg (197 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorophosphate and 57 μl of N,N-diisopropylethylamine were combined in 5 ml of DMF and stirred at RT overnight. Subsequently, the solvent was removed in vacuo, and the remaining residue was purified by means of preparative HPLC. The corresponding fractions were concentrated and, by lyophilization from dioxane/water, 35 mg (63% of theory) of the protected intermediate were obtained.
  • HPLC (Method 12): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.71 min; MS (ESIpos): m/z=340 (M+H)+.
  • Subsequently, the entire amount of the protected intermediate was deblocked with 1 ml of trifluoroacetic acid in 5 ml of dichloromethane to obtain 28 mg (77% of theory) of the title compound.
  • LC-MS (Method 3): Rt=0.75 min; MS (ESIpos): m/z=240 (M+H)+.
    • Intermediate 24 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[2-(methylamino)ethyl]butanamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00464
  • First, 35 mg (164 μmol) of tert-butyl-(2-aminoethyl)methylcarbamate hydrochloride trifluoroacetate, 30 mg (164 μmol) of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid, 75 mg (197 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 57 μl of N,N-diisopropylethylamine were combined in 5 ml of DMF and stirred at RT for 30 min. Subsequently, the solvent was removed in vacuo, and the remaining residue was purified by means of preparative HPLC. The corresponding fractions were concentrated and, by lyophilization from dioxane/water, 51 mg (91% of theory) of the protected intermediate were obtained.
  • HPLC (Method 12): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.77 min; MS (ESIpos): m/z=340 (M+H)+.
  • Subsequently, the entire amount was deprotected with 1 ml of trifluoroacetic acid in 5 ml of dichloromethane to obtain 45 mg (69% of theory) of the title compound.
  • LC-MS (Method 1): Rt=0.19 min; MS (ESIpos): m/z=240 (M+H)+.
    • Intermediate 25 Benzyl-(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoate trifluoroacetate
  • Figure US20140127240A1-20140508-C00465
  • First, (2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoic acid was released from 1.82 g (3.88 mmol) of its dicyclohexylamine salt by taking it up in 150 ml of ethyl acetate and extractive shaking with 100 ml of 0.5% sulphuric acid. The organic phase was dried over magnesium sulphate, filtered and concentrated. The residue was taken up in 10 ml of dioxane and 10 ml of water, 1517 mg (4.66 mmol) of caesium carbonate were added, and the mixture was treated in an ultrasound bath for 5 min, then concentrated under in vacuo and redistilled once with DMF. The residue was then taken up in 15 ml of dichloromethane, and 1990 mg (11.64 mmol) of benzyl bromide were added to this. The mixture was treated in an ultrasound bath for 15 min and then concentrated in vacuo. The residue was partitioned between ethyl acetate and water, the organic phase was removed and extracted by shaking with saturated sodium chloride solution and then concentrated. The residue was then purified by preparative HPLC. This gave 1170 mg (80% of theory) of the Boc-protected intermediate.
  • Subsequently, these 1170 mg were deprotected immediately with 5 ml of trifluoroacetic acid in 15 ml of dichloromethane. After stirring at RT for 15 min, the reaction mixture was concentrated in vacuo. The residue was lyophilized from dioxane. After drying under high vacuum, there remained 1333 mg (84% of theory) of the title compound as a yellow oil.
  • HPLC (Method 6): Rt=1.5 min;
  • LC-MS (Method 1): Rt=0.59 min; MS (ESIpos): m/z=278 (M+H)+.
    • Intermediate 26 N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00466
  • 1200 mg (2.33 mmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 5) were combined with 910.8 mg (2.33 mmol) of benzyl (2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoate trifluoroacetate (Intermediate 14), 1327 mg (3.49 mmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 2027 μl of N,N-diisopropylethylamine in 50 ml of DMF, and the mixture was stirred at RT for 5 min. Thereafter, the solvent was removed in vacuo. The remaining residue was taken up in ethyl acetate and extracted by shaking it successively with 5% aqueous citric acid solution and saturated sodium hydrogencarbonate solution. The organic phase was removed and concentrated. The residue was purified by means of 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-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-(benzyloxy)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide as a resin.
  • LC-MS (Method 1): Rt=1.56 min; MS (ESIpos): m/z=775 (M+H)+.
  • The entire amount of this intermediate obtained was taken up in 25 ml of a mixture of methanol and dichloromethane (20:1), and the benzyl ester group was removed by hydrogenation under standard hydrogen pressure with 10% palladium on activated carbon as a catalyst. After stirring at RT for 30 min, the catalyst was filtered off and the filtrate was concentrated in vacuo. This gave 803 mg (91% of theory) of the title compound as a white solid.
  • HPLC (Method 6): Rt=2.1 min;
  • LC-MS (Method 1): Rt=1.24 min; MS (ESIpos): m/z=685 (M+H)+.
    • Intermediate 27 (1S,2R)-1-amino-2-phenyl-N-propylcyclopropanecarboxamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00467
  • The title compound was prepared by coupling commercially available (1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid with n-propylamine in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and subsequent Boc cleaving with trifluoroacetic acid (yield: 85% of theory over both stages).
  • HPLC (Method 6): Rt=1.2 min;
  • LC-MS (Method 1): Rt=0.52 min; MS (ESIpos): m/z=219 (M+H)+.
    • Intermediate 28 Ethyl-(1S,2R)-1-amino-2-phenylcyclopropanecarboxylate trifluoroacetate
  • Figure US20140127240A1-20140508-C00468
  • The title compound was prepared according to standard methods by esterifying commercially available (1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid with ethanol and subsequent Boc cleaving with trifluoroacetic acid.
  • LC-MS (Method 1): Rt=0.50 min; MS (ESIpos): m/z=206 (M+H)+.
    • Intermediate 29 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanoic acid
  • Figure US20140127240A1-20140508-C00469
  • To a solution of 1.39 g (8.95 mmol) of N-methoxycarbonylmaleimide in 44 ml of saturated sodium hydrogencarbonate solution were added, at 0° C., 1.5 g (8.95 mmol) of 4-amino-2,2-dimethylbutyric acid, and the mixture was stirred for 40 min. Subsequently, the cooling bath was removed, and the reaction mixture was stirred for 1 h more. While cooling with ice, the reaction mixture was then adjusted to pH 3 by adding sulphuric acid, then extracted with ethyl acetate. The combined organic phases were dried over magnesium sulphate and concentrated. 1.17 g (79% purity, 49% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.64 min; m/z=212 (M+H)+.
    • Intermediate 30 tert-butyl 2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanoyl]hydrazinecarboxylate
  • Figure US20140127240A1-20140508-C00470
  • To a solution of 50 mg (237 μmol) of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanoic acid in 2 ml of THF were added, at 0° C., first 26 μl (237 μmol) of 4-methylmorpholine and then 31 μl (237 μmol) of isobutyl chloroformate. After removing the cooling bath and stirring at RT for a further 15 min, 31.3 mg (237 μmol) of tert-butyloxycarbonyl hydrazide were added. The reaction mixture was stirred overnight and then concentrated. The residue was purified by preparative HPLC. 50.8 mg (66% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.71 min; m/z=324 (M−H)−.
    • Intermediate 31 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanehydrazide trifluoroacetate
  • Figure US20140127240A1-20140508-C00471
  • 50 mg (154 mmol) of tert-butyl 2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanoyl]hydrazinecarboxylate were 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. 55.2 mg (93% purity, 99% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.36 min; m/z=226 (M+H)+.
    • Intermediate 32 Adamantan-1-ylmethyl N-(tert-butoxycarbonyl)-L-phenylalaninate
  • Figure US20140127240A1-20140508-C00472
  • To a solution of 500 mg (1.89 mmol) of N-Boc-L-phenylalanine in 25 ml of dichloromethane were added, at RT, 1192 mg (6.2 mmol) of EDC, 578 μl (4.1 mmol) of triethylamine, 345 mg (2.8 mmol) of DMAP and 345 mg (2.1 mmol) of 1-adamantylmethanol. The reaction mixture was stirred overnight, then diluted with 50 ml of dichloromethane, and was successively washed with 10% aqueous citric acid solution, water and saturated sodium chloride solution. The organic phase was dried over magnesium sulphate, then concentrated, and the residue was purified by preparative HPLC. 769 mg (90% of theory) of the title compound were obtained.
  • LC-MS (Method 2): Rt=1.84 min; m/z=414 (M+H)+.
    • Intermediate 33 Adamantan-1-ylmethyl L-phenylalaninate hydrochloride
  • Figure US20140127240A1-20140508-C00473
  • 769 mg (1.86 mmol) of adamantan-1-ylmethyl N-(tert-butoxycarbonyl)-L-phenylalaninate (Intermediate 13) were dissolved in 25 ml of a 4 N solution of hydrogen chloride in dioxane and stirred at RT for 1 h. Subsequently, the reaction mixture was concentrated, and the residue was dried in vacuo. 619 mg (95% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.82 min; m/z=314 (M+H)+.
    • Intermediate 34 N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide
  • Figure US20140127240A1-20140508-C00474
  • To a solution of 20 mg (29 μmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide in 1 ml of DMF were added, at RT, 15.3 μl (88 μmol) of N,N-diisopropylethylamine, 6.7 mg (44 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 adamantan-1-yl L-phenylalaninate hydrochloride were added. After stirring overnight, the reaction mixture was separated directly into its components via preparative HPLC. 27.5 mg (93% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=1.70 min; m/z=980 (M+H)+.
    • Intermediate 35 N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00475
  • 27.5 mg (28 μmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide were dissolved in 1.8 ml of dichloromethane, and 361 μl of TFA were added. The reaction mixture was stirred 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 were obtained.
  • LC-MS (Method 1): Rt=1.14 min; m/z=880 (M+H)+.
    • Intermediate 36 tert-butyl (2S)-1-(benzyloxy)-3-phenylpropan-2-yl carbamate
  • Figure US20140127240A1-20140508-C00476
  • Under an argon atmosphere, 500 mg (1.99 mmol) of N-Boc-L-phenylalaninol were dissolved in 5 ml of DMF and cooled to 0° C. Subsequently, 159 mg (3.98 mmol) of a 60% suspension of sodium hydride in paraffin oil were added. The reaction mixture was stirred until the evolution of gas had ended, and then 260 μl (2.19 mmol) of benzyl bromide were added. The cooling bath was removed, and the reaction mixture was stirred at RT for 2 h. Thereafter, the reaction mixture was concentrated, the residue was taken up in ice water, and the mixture was extracted with dichloromethane. The organic phase was washed with saturated sodium chloride solution, dried over magnesium sulphate and concentrated. The residue was purified by means of preparative HPLC. 226 mg (33% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=1.28 min; m/z=342 (M+H)+.
    • Intermediate 37 (2S)-1-(benzyloxy)-3-phenylpropan-2-amine hydrochloride
  • Figure US20140127240A1-20140508-C00477
  • 220 mg (644 μmol) of tert-butyl (2S)-1-(benzyloxy)-3-phenylpropan-2-yl carbamate were dissolved in 11 ml of a 4 N solution of hydrogen chloride in dioxane and stirred at RT for 1 h. Then the reaction mixture was concentrated, and the residue was dried in vacuo. 138 mg (77% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.65 min; m/z=242 (M+H)+.
    • Intermediate 38 N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide
  • Figure US20140127240A1-20140508-C00478
  • To a solution of 20 mg (29 μmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide in 1 ml of DMF were added, at RT, 15.3 μl (88 μmol) of N,N-diisopropylethylamine, 6.7 mg (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 were added. After stirring overnight, the reaction mixture was separated directly into its components via preparative HPLC. 26 mg (98% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=1.51 min; m/z=909 (M+H)+.
    • Intermediate 39 N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00479
  • 26 mg (29 μmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide were dissolved in 1.8 ml of dichloromethane, and 370 μl of TFA were added. The reaction mixture was stirred at RT for 30 min and then concentrated. The residue was taken up in water and lyophilized. 26.4 mg (quant.) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.97 min; m/z=809 (M+H)+.
    • Intermediate 40 N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00480
  • 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 were admixed with 42 mg (0.11 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 25 μl of N,N-diisopropylethylamine, and the reaction mixture was stirred at RT for 5 min. This was followed by concentration and purification of the residue by means of preparative HPLC. After combining the corresponding fractions, concentrating and drying under high vacuum, 49 mg (81%) of the protected intermediate were obtained. Subsequently, the Boc group was cleaved using known conditions with trifluoroacetic acid in dichloromethane. Concentration was followed by the purification of the title compound via preparative HPLC, and 26 mg (52%) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.65 min;
  • LC-MS (Method 1): Rt=0.77 min; MS (ESIpos): m/z=718 (M+H)+.
    • Intermediate 41 3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoic acid trifluoroacetate
  • Figure US20140127240A1-20140508-C00481
  • 150 mg (541 μmol) of tert-butyl 3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoate were dissolved in 3 ml of dichloromethane, 1.5 ml of trifluoroacetic acid were added, and the reaction mixture was stirred at RT for 1 h, then concentrated.
  • 181 mg (100% of theory) of the title compound were obtained.
  • MS (EI): m/z 222 (M+H)+
    • Intermediate 42 3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)propanoic acid
  • Figure US20140127240A1-20140508-C00482
  • 186 mg (555 μmol) of 3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoic acid trifluoroacetate were dissolved in 2.6 ml of saturated sodium hydrogencarbonate solution and admixed at 0° C. with 86 mg (555 μmol) of N-methoxycarbonylmaleimide. The reaction mixture was stirred at 0° C. for 40 min and at RT for 1 h, then cooled again to 0° C., adjusted to pH 3 with sulphuric acid and extracted 3× with 25 ml of ethyl acetate. The combined organic phases were dried over magnesium sulphate and concentrated.
  • 126 mg (75% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.53 min; m/z=302 (M+H)+.
    • Intermediate 43 tert-butyl-15-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-oxo-7,10,13-trioxa-2,3-diazapentadecan-1-oate
  • Figure US20140127240A1-20140508-C00483
  • 125 mg (417 μmol) of 3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)propanoic acid were dissolved at 0° C. in 2.1 ml of THF and admixed 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, at 0° C., 55 mg (417 μmol) of tert-butyloxycarbonyl hydrazide were added. The reaction mixture was warmed to RT overnight, concentrated and purified via preparative HPLC.
  • 60 mg (33% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.66 min; m/z=416 (M+H)+.
    • Intermediate 44 3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)propanehydrazide trifluoroacetate
  • Figure US20140127240A1-20140508-C00484
  • 60 mg (145 μmol) of tert-butyl-15-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-oxo-7,10,13-trioxa-2,3-diazapentadecan-1-oate were 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 were obtained.
  • LC-MS (Method 1): Rt=0.35 min; m/z=316 (M+H)+.
    • Intermediate 45 Benzyl-(1S,2R)-1-amino-2-phenylcyclopropanecarboxylate trifluoroacetate
  • Figure US20140127240A1-20140508-C00485
  • The title compound was prepared according to standard methods by esterifying commercially available (1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid with benzyl alcohol and subsequent Boc cleaving with trifluoroacetic acid.
  • LC-MS (Method 1): Rt=0.72 min; MS (ESIpos): m/z=268 (M+H)+.
    • Intermediate 46 N-(tert-butoxycarbonyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00486
  • 383 mg (0.743 mmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 8) were combined with 485 mg (0.743 mmol) of benzyl-N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-phenylalaninate trifluoroacetate (Intermediate 12), 424 mg (1.114 mmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 388 μl of N,N-diisopropylethylamine in 15 ml of DMF, and the mixture was stirred at RT for 10 min Subsequently, the solvent was removed in vacuo. The remaining residue was taken up in ethyl acetate and extracted by shaking successively with 5% aqueous citric acid solution and saturated sodium hydrogencarbonate solution. The organic phase was removed and concentrated, and the residue was purified by means of preparative HPLC. The product fractions were combined and concentrated, and the residue was dried under high vacuum. 335 mg (48% of theory) of the benzyl ester intermediate were obtained as a foam.
  • LC-MS (Method 1): Rt=1.49 min; MS (ESIpos): m/z=922 (M+H)+.
  • 100 mg (0.11 mmol) of this intermediate were taken up in 15 ml of methanol and the benzyl ester group was removed by hydrogenation under standard hydrogen pressure with 10% palladium on activated carbon as a catalyst. After stirring at RT for 1 h, the catalyst was filtered off and the filtrate was concentrated in vacuo. After lyophilization from dioxane, 85 mg (94% of theory) of the title compound were obtained as a solid.
  • HPLC (Method 12): Rt=2.4 min;
  • LC-MS (Method 1): Rt=1.24 min; MS (ESIpos): m/z=832 (M+H)+.
    • Intermediate 47 N-benzyl-L-tryptophanamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00487
  • 202 mg (0.5 mmol) of 2,5-dioxopyrrolidin-1-yl N-(tert-butoxycarbonyl)-L-tryptophanate and 45 mg (0.42 mmol) of benzylamine were dissolved in 10 ml of DMF, and 110 μl (630 μmol) of N,N-diisopropylethylamine were added. The reaction mixture was stirred at RT for 3 h. Subsequently, the reaction mixture was concentrated in vacuo and the residue was purified by flash chromatography on silica gel (eluent: 20:0.5:0.05 dichloromethane/methanol/17% aq. ammonia). The corresponding fractions were combined and concentrated. The resulting residue was digested 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 were added. After stirring at RT for 45 minutes, the mixture was concentrated, and the residue was purified via preparative HPLC. After drying in vacuo, 117 mg (57% of theory over both stages) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.66 min; MS (ESIpos): m/z=294 (M+H)+.
    • Intermediate 48 (1S,2R)-1-amino-2-phenylcyclopropanecarboxamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00488
  • 50 mg (180 μmol) of commercially available (1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid were dissolved in 5 ml of DMF, 94 μl (541 μmol) of N,N-diisopropylethylamine, 31 mg (270 μmol) of N-hydroxysuccinimide and 41.5 mg (216 μmol) of EDC were added, and then the mixture was stirred at RT overnight. The reaction mixture was then concentrated, the residue was taken up in dioxane, 71 mg (901 μmol) of ammonium hydrogencarbonate were added, and the reaction mixture was then left to stand at RT for 3 days. The reaction mixture was then diluted with a 1:1 mixture of ethyl acetate and water. The organic phase was removed, dried over magnesium sulphate and concentrated. The resulting residue was subsequently taken up in 3 ml of dichloromethane, and 3 ml of anhydrous trifluoroacetic acid were added. Stirring at RT for 1 h was followed by concentration. The residue was stirred with pentane, suctioned off and lyophilized from dioxane. In this way, 32 mg (62% of theory over both stages) of the title compound were obtained.
  • HPLC (Method 6): Rt=0.38 min;
  • LC-MS (Method 1): Rt=0.20 min; MS (ESIpos): m/z=177 (M+H)+.
    • Intermediate 49 Nα-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-tryptophanamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00489
  • The title compound was prepared in analogy to the synthesis of Intermediate 13 from Starting Compound 1 and L-tryptophanamide hydrochloride.
  • HPLC (Method 5): Rt=1.4 min;
  • LC-MS (Method 1): Rt=0.92 min; MS (ESIpos): m/z=473 (M+H)+.
    • Intermediate 50 4-nitrophenyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl carbamate
  • Figure US20140127240A1-20140508-C00490
  • 813 mg (3.1 mmol) of triphenylphosphine were dissolved in 25 ml of THF and cooled to −70° C. under argon. After the dropwise addition of 627 mg (3.1 mmol) of diisopropyl azodicarboxylate, the mixture was stirred for 5 min. Subsequently, 500 mg (3.1 mmol) of tert-butyl-(2-aminoethyl) carbamate dissolved in 5 ml of THF were added dropwise, and the reaction mixture was stirred at −70° C. for another 5 min. Then 136.6 mg (1.55 mmol) of 2,2-dimethyl-1-propanol dissolved in 1 ml of THF and 301 mg (3.1 mmol) of maleimide were added, the reaction mixture was stirred at −70° C. for another 10 min, and then the mixture was warmed to RT. After stirring at RT for another 16 h, the solvent was removed in vacuo, and the residue was purified by means of preparative HPLC. This gave 463 mg (62%) of the protected intermediate.
  • After removing the Boc protecting group under standard conditions, 652 mg of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione were obtained as trifluoroacetate.
  • 112.9 mg (543 μmol) of nitrophenyl chloroformate were dissolved in 30 ml of THF and, after the addition of 100 mg (271.6 μmol) of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione trifluoroacetate, the mixture was stirred at RT for 30 min. The mixture was filtered, and the filtrate was concentrated to dryness and then slurried with diethyl ether. After suctioning off and drying, 60 mg (95% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=0.65 min;
  • LC-MS (Method 1): Rt=0.74 min; MS (ESIpos): m/z=306 (M+H)+.
    • Intermediate 51 (1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethanamine trifluoroacetate
  • Figure US20140127240A1-20140508-C00491
  • 200 mg (0.75 mmol) of N-(tert-butoxycarbonyl)-L-phenylalanine were initially provided at 0° C. in 5.5 ml of dichloromethane, and 128 mg (0.79 mmol) of 1,1′-carbonyldiimidazole were added to this. After 30 min, 103 mg (0.75 mmol) of benzoyl hydrazide were added. After a further 45 min at 0° C., 500 mg (1.5 mmol) of carbon tetrabromide and 395 mg (1.5 mmol) of triphenylphosphine were finally added. The reaction mixture was stirred first at 0° C. for 2 h and then at RT overnight. The mixture was subsequently concentrated on a rotary evaporator, and the residue was dried under high vacuum. The crude product thus obtained was purified by means of preparative HPLC. 217 mg (78% of theory) of the Boc-protected intermediate tert-butyl-[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl carbamate were obtained.
  • LC-MS (Method 12): Rt=1.15 min; MS (ESIpos): m/z=366 (M+H)+
  • 217 mg (0.59 mmol) of this intermediate were taken up in 3 ml of dichloromethane, 0.6 ml of trifluoroacetic acid were added, and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated in vacuo. The remaining residue was the reaction mixture and was dried further in vacuo, then lyophilized from dioxane. In this way, 214 mg (90% of theory) of the title compound were obtained.
  • LC-MS (Method 11): Rt=0.62 min; MS (ESIpos): m/z=266 (M+H)+
    • Intermediate 52 (1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethanamine trifluoroacetate
  • Figure US20140127240A1-20140508-C00492
  • 200 mg (0.75 mmol) of N-(tert-butoxycarbonyl)-D-phenylalanine were initially provided at 0° C. in 5.5 ml of dichloromethane, and 128.3 mg (0.79 mmol) of 1,1′-carbonyldiimidazole were added to this. After 30 min, 103 mg (0.75 mmol) of benzoyl hydrazide were 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 finally added. The reaction mixture was stirred first at 0° C. for 2 h and then at RT overnight. The mixture was subsequently concentrated on a rotary evaporator, and the residue was dried under high vacuum. The crude product thus obtained was purified by means of preparative HPLC. 219 mg (80% of theory) of the Boc-protected intermediate tert-butyl (1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl carbamate were obtained.
  • LC-MS (Method 2): Rt=1.36 min; MS (ESIpos): m/z=366 (M+H)+
  • 219 mg (0.6 mmol) of this intermediate were taken up in 3 ml of dichloromethane, 0.6 ml of trifluoroacetic acid were added, and the mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated in vacuo. The remaining residue was the reaction mixture and was dried further in vacuo, then lyophilized from dioxane. In this way, 196 mg (86% of theory) of the title compound were obtained as a solid.
  • HPLC (Method 10): Rt=2.41 min
    • Intermediate 53 (2S)-1-(benzylsulphonyl)-3-phenylpropan-2-amine
  • Figure US20140127240A1-20140508-C00493
  • 200 mg (1.13 mmol) of (4S)-4-benzyl-1,3-oxazolidin-2-one were initially provided in 3 ml of tert-butanol, and 280 mg (2.26 mmol) of benzyl mercaptan were added to this. The mixture was subsequently heated under reflux for 2 days. Thereafter, the reaction mixture was concentrated on a rotary evaporator, and the resulting intermediate (2S)-1-(benzylsulphanyl)-3-phenylpropan-2-amine was directly converted further, without workup.
  • HPLC (Method 10): Rt=2.63 min
  • LC-MS (Method 1): Rt=0.67 min; MS (ESIpos): m/z=258 (M+H)
  • The crude intermediate obtained above was dissolved in a solution of 2 ml of 30% hydrogen peroxide and 5 ml of formic acid, and the mixture was stirred at RT for 12 h. Then the reaction mixture was added to saturated sodium sulphate solution and extracted three times with ethyl acetate. The organic phase was dried over magnesium sulphate and concentrated in vacuo. The obtained crude product was purified by means of preparative HPLC. 343 mg (61% of theory) of the title compound were thus obtained.
  • HPLC (Method 10): Rt=2.40 min;
  • LC-MS (Method 12): Rt=0.65 min; MS (ESIpos): m/z=290 (M+H)
    • Intermediate 54 (2S,3E)-1,4-diphenylbut-3-en-2-amine
  • Figure US20140127240A1-20140508-C00494
  • 552.7 mg (9.85 mmol) of potassium hydroxide were dissolved in methanol, adsorbed onto 1.1 g of neutral aluminium oxide and then dried under high vacuum. To a solution of 240 mg (0.82 mmol) of (2S)-1-(benzylsulphonyl)-3-phenylpropan-2-amine and 1.56 g of the potassium hydroxide on aluminium oxide thus prepared in 6.2 ml of n-butanol were added dropwise, at 5-10° C., 307 μl (3.3 mmol) of dibromodifluoromethane. The reaction mixture was stirred at RT for 2 h, then filtered through Celite, and the residue was washed thoroughly with dichloromethane afterwards. The filtrate was concentrated, and the resulting residue was dried in vacuo. The crude product thus obtained was purified by means of preparative HPLC. 98 mg (35% of theory) of the title compound were obtained with an E/Z diastereomer ratio of 4:1.
  • HPLC (Method 10): Rt=2.46 min;
  • LC-MS (Method 12): Rt=0.75 min; MS (ESIpos): m/z=224 (M+H)+
  • The E/Z diastereomer mixture obtained above was dissolved in 2 ml of ethanol and 0.2 ml of N,N-diisopropylethylamine and separated by means of HPLC on chiral phase [column: Daicel Chiralpak AD-H, 5 μm, 250 mm×20 mm, eluent: 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 was dried in vacuo. 45 mg of the title compound were obtained.
  • 1H NMR (400 MHz, DMSO-d6) δ [ppm]=2.62-2.83 (m, 2H) 3.52-3.71 (m, 1H) 6.18-6.30 (m, 1H) 6.34-6.46 (m, 1H) 6.98-7.57 (m, 10H) [further signals hidden under solvent peaks].
    • Intermediate 55 N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00495
  • 20 mg (29 μmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 1 ml of DMF, 13.3 mg (35 μmol) of HATU and 15.3 μl (88 μmol) of N,N-diisopropylethylamine were added, and the mixture was stirred at RT for 30 min. Subsequently, 12.2 mg (32 μmol) of (1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethanamine trifluoroacetate were added. The reaction mixture was stirred at RT overnight and then separated by means of preparative HPLC. This gave 22 mg (81% of theory) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • LC-MS (Method 12): Rt=1.45 min; MS (ESIpos): m/z=933 (M+H)+
  • By subsequently cleaving the BOC protecting group with trifluoroacetic acid, 22.4 mg (98% of theory) of the title compound were then obtained.
  • LC-MS (Method 11): Rt=0.85 min; MS (ESIpos): m/z=833 (M+H)+
    • Intermediate 56 N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00496
  • N-(tert-Butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was prepared in analogy to the synthesis of Intermediate 55, by reacting 20 mg (29 μmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide with 12.2 mg (32 μmol) of (1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethanamine trifluoroacetate.
  • Yield: 17 mg (64% of theory)
  • HPLC (Method 10): Rt=3.74 min;
  • LC-MS (Method 1): Rt=1.45 min; MS (ESIpos): m/z=933 (M+H)+
  • By subsequently cleaving the BOC protecting group with trifluoroacetic acid, 17.1 mg (99% of theory) of the title compound were thus obtained.
  • HPLC (Method 10): Rt=2.55 min;
  • LC-MS (Method 11): Rt=0.85 min; MS (ESIpos): m/z=833 (M+H)
    • Intermediate 57 N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylsulphonyl)-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00497
  • N-(tert-Butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylsulphonyl)-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-valinamide was prepared in analogy to the synthesis of Intermediate 55, by reacting 20 mg (29 μmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide with 9.3 mg (20 μmol) of (2S)-1-(benzylsulphonyl)-3-phenylpropan-2-amine
  • Yield: 19.2 mg (68% of theory)
  • HPLC (Method 10): Rt=3.5 min;
  • LC-MS (Method 12): Rt=1.41 min; MS (ESIpos): m/z=957 (M+H)+
  • By subsequently cleaving the BOC protecting group with trifluoroacetic acid, 19.3 mg (99% of theory) of the title compound were thus obtained.
  • HPLC (Method 10): Rt=2.52 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=857 (M+H)+
    • Intermediate 58 N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3E)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00498
  • N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3E)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was prepared in analogy to the synthesis of Intermediate 55, by reacting 20 mg (29 μmol) N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide with 7.1 mg (32 μmol) of (2S,3E)-1,4-diphenylbut-3-en-2-amine
  • Yield: 15.1 mg (58% of theory)
  • HPLC (Method 10): Rt=4.2 min;
  • LC-MS (Method 12): Rt=1.51 min; MS (ESIpos): m/z=891 (M+H)
  • By subsequently cleaving the BOC protecting group with trifluoroacetic acid, 15.7 mg (99% of theory) of the title compound were then obtained.
  • HPLC (Method 10): Rt=2.62 min;
  • LC-MS (Method 12): Rt=0.97 min; MS (ESIpos): m/z=791 (M+H)+
    • Intermediate 61 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00499
  • 50 mg (0.054 mmol) of N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate (Intermediate 16) were dissolved in 8 ml of dioxane/water, and 70 ml (0.108 mmol) of a 15% solution of 4-oxobutanoic acid in water were added. The reaction mixture was subsequently stirred at 100° C. for 1 h. After cooling to RT, 3.7 mg (0.059 mmol) of sodium cyanoborohydride were added, and the mixture was adjusted to a pH of 3 by adding about 300 μl of 0.1 N hydrochloric acid. The reaction mixture was then stirred at 100° C. for another 2 h. After cooling, another 70 ml (0.108 mmol) of the 15% 4-oxobutanoic acid solution were added, and the reaction mixture was stirred again at 100° C. for 1 h. Then a further 3.7 mg (0.059 mmol) of sodium cyanoborohydride were added, and about 300 μl of 0.1 N hydrochloric acid were subsequently used to readjust the pH to 3. The reaction mixture was then stirred at 100° C. for another 2 h. For a conversion that was still incomplete, this procedure was repeated for a third time. The reaction mixture was finally concentrated, and the residue was purified by means of preparative HPLC. In this way, 32 mg (65% of theory) of the title compound were obtained in the form of a colourless foam.
  • HPLC (Method 5): Rt=1.64 min;
  • LC-MS (Method 9): Rt=4.76 min; MS (ESIpos): m/z=899 (M+H)+
  • 1H NMR (500 MHz, DMSO-d6): δ=8.95 and 8.8 (2m, 1H), 8.88 and 8.65 (2s, 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) [further signals hidden under H2O and DMSO peaks].
    • Intermediate 62 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00500
  • The title compound was prepared in analogy to the synthesis of Intermediate 61, by reacting 50 mg of N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate (Intermediate 14) with 4-oxobutanoic acid.
  • Yield: 34 mg (70% of theory)
  • HPLC (Method 5): Rt=1.64 min;
  • LC-MS (Method 9): Rt=4.77 min; MS (ESIpos): m/z=887 (M+H)+.
    • Intermediate 63 N-(4-carboxybenzyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00501
  • The title compound was prepared in analogy to the synthesis of Intermediate 61 by reacting 15 mg of N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate (Intermediate 16) with 4-formylbenzoic acid.
  • Yield: 7.5 mg (48% of theory)
  • HPLC (Method 5): Rt=1.75 min;
  • LC-MS (Method 1): Rt=0.97 min; MS (ESIpos): m/z=947 (M+H)+.
    • Intermediate 64 N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00502
  • 10 mg (0.011 mmol) of N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate (Intermediate 16) were dissolved in 2 ml of dioxane/water, and 2.8 mg (0.022 mmol) of 6-oxohexanoic acid were added. The reaction mixture was subsequently stirred at 100° C. for 1 h. After cooling to RT, 0.75 mg (0.012 mmol) of sodium cyanoborohydride were added, and the mixture was adjusted to a pH of 3 by adding 0.1 N hydrochloric acid. The reaction mixture was then stirred at 100° C. for another hour. After cooling, another 2.8 mg (0.022 mmol) of 6-oxohexanoic acid were added, and the reaction mixture was again stirred at 100° C. for 1 h. A further 0.75 mg (0.012 mmol) of sodium cyanoborohydride were added, and 0.1 N hydrochloric acid was subsequently used to readjust the pH to 3. The reaction mixture was then stirred at 100° C. for another 1 h. This procedure was then repeated for a third time. The reaction mixture was finally concentrated, and the crude product was purified by means of preparative HPLC. This gave 6.4 mg (64% of theory) of the title compound in the form of a colourless foam.
  • HPLC (Method 5): Rt=1.68 min;
  • LC-MS (Method 9): Rt=4.86 min; MS (ESIpos): m/z=927 (M+H)+.
    • Intermediate 65 N-(2-aminoethyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide bistrifluoroacetate
  • Figure US20140127240A1-20140508-C00503
  • The title compound was prepared by reacting 68 mg of N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate (Intermediate 14) with tert-butyl-(2-oxoethyl)carbamate and subsequent cleaving of the Boc protecting group with trifluoroacetic acid.
  • Yield: 49 mg (62% of theory over two stages)
  • HPLC (Method 5): Rt=1.58 min;
  • LC-MS (Method 2): Rt=1.05 min; MS (ESIpos): m/z=844 (M+H)+
  • 1H NMR (600 MHz, DMSO-d6): 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) [further signals hidden under H2O peak].
    • Intermediate 66 N-(3-aminopropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00504
  • The title compound was prepared in analogy to the synthesis of Intermediate 65 by reacting 25 mg (0.027 mmol) of N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate (Intermediate 16) with benzyl-(3-oxopropyl)carbamate and subsequent hydrogenolytic cleaving of the Z protecting group (with 10% palladium on charcoal as a catalyst, in ethanol as a solvent).
  • Yield: 11 mg (41% of theory over two stages)
  • HPLC (Method 5): Rt=1.53 min;
  • LC-MS (Method 1): Rt=0.72 min; MS (ESIpos): m/z=870 (M+H)+.
    • Intermediate 67 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide
  • Figure US20140127240A1-20140508-C00505
  • 26 mg (26 μmol) of N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide trifluoroacetate and 33.9 μl of a 15% aqueous succinaldehydic acid solution (53 μmol) were dissolved in 957 μl of a 1:1-dioxane/water mixture and heated to 100° C. for 1 h. After brief cooling, 1.81 mg (29 μmol) of sodium cyanoborohydride were added. The reaction mixture was adjusted to pH 3 by adding 0.1 N hydrochloric acid, and the mixture was heated to 100° C. for another 2 h. After again adding the same amounts of succinaldehydic acid solution, sodium cyanoborohydride and hydrochloric acid, the mixture was heated once again to 100° C. for 2 h. The reaction mixture was then directly separated into its components by means of preparative HPLC. 18.5 mg (73% of theory) of the title compound were thus obtained.
  • LC-MS (Method 1): Rt=1.17 min; m/z=967 (M+H)+.
    • Intermediate 68 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide
  • Figure US20140127240A1-20140508-C00506
  • 24 mg (26 μmol) of N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide trifluoroacetate and 33.7 μl of a 15% aqueous succinaldehydic acid solution (52 μmol) were dissolved in 953 μl of a 1:1-dioxane/water mixture and heated to 100° C. for 1 h. After brief cooling, 1.80 mg (29 μmol) of sodium cyanoborohydride were added. The reaction mixture was adjusted to pH 3 by adding 0.1 N hydrochloric acid and the mixture was heated to 100° C. for another 2 h. After adding the same amounts of succinaldehydic acid solution, sodium cyanoborohydride and hydrochloric acid again, the mixture was heated once again to 100° C. for 2 h. The reaction mixture was then directly separated into its components by means of preparative HPLC. 15.2 mg (65% of theory) of the title compound were thus obtained.
  • LC-MS (Method 1): Rt=1.01 min; m/z=895 (M+H)+
    • Intermediate 69 N-(3-carboxypropyl)-N-methyl-L-valyl-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-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00507
  • 53 mg (84 μmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 4) and 45 mg (84 μmol) of benzyl N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-phenylalaninate trifluoroacetate (Intermediate 12) were taken up in 2 ml of DMF, 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 overnight. Subsequently, the reaction mixture was concentrated and the residue was purified by means of preparative HPLC. This gave 59 mg (68% of theory) of the Fmoc-protected intermediate N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-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-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • LC-MS (Method 1): Rt=1.55 min; m/z=1044 (M+H)+.
  • 57 mg (0.055 mmol) of this intermediate were treated with 1.2 ml of piperidine in 5 ml of DMF to cleave the Fmoc protecting group. After concentration and purification by means of preparative HPLC, 39 mg (76% of theory) of the free amine intermediate N-methyl-L-valyl-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-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were obtained as the trifluoroacetate.
  • HPLC (Method 5): Rt=1.9 min;
  • LC-MS (Method 1): Rt=1.01 min; m/z=822 (M+H)+.
  • 37 mg (0.045 mmol) of this intermediate were dissolved in 5 ml of dioxane/water and, in analogy to the preparation of the compound in Intermediate 66, reacted with a 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride. 16 mg (39% of theory) of the title compound were obtained in the form of a colourless foam.
  • HPLC (Method 6): Rt=2.1 min;
  • LC-MS (Method 1): Rt=1.01 min; MS (ESIpos): m/z=908 (M+H)+.
    • Intermediate 70 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3S)-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-valinamide
  • Figure US20140127240A1-20140508-C00508
  • First, in analogy to the synthesis described in Intermediate 14, proceeding from Intermediates 4 and 26, the amine compound N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3S)-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-valinamide was prepared.
  • 30 mg (0.032 mmol) of this compound were dissolved in 6 ml of dioxane/water, and 41 μl (0.063 mmol) of a 15% aqueous solution of 4-oxobutanoic acid were added. The reaction mixture was subsequently stirred at 100° C. for 1 h. After cooling to RT, 2.2 mg (0.035 mmol) of sodium cyanoborohydride were added, and the mixture was adjusted to a pH of 3 by adding about 300 μl of 0.1 N hydrochloric acid. The reaction mixture was then stirred at 100° C. for another 2 h. After cooling, another 41 μl (0.063 mmol) of the 15% 4-oxobutanoic acid solution were added, and the reaction mixture was again stirred at 100° C. for 1 h. Then a further 2.2 mg (0.035 mmol) of sodium cyanoborohydride were added, and about 300 μl of 0.1 N hydrochloric acid were subsequently used to adjust the pH back to 3. The reaction mixture was then stirred at 100° C. for another 2 h. In the event of the conversion still being incomplete, this procedure was repeated for a third time. The reaction mixture was finally concentrated and the crude product was purified by means of preparative HPLC. This gave 24 mg (82% of theory) of the title compound in the form of a colourless foam.
  • HPLC (Method 5): Rt=1.9 min;
  • LC-MS (Method 9): Rt=5.15 min; MS (ESIpos): m/z=922 (M+H)+.
    • Intermediate 71 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-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-valinamide
  • Figure US20140127240A1-20140508-C00509
  • First, in analogy to the synthesis described in Intermediate 14, proceeding from Intermediates 4 and 39, the amine compound N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-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-valinamide was prepared. 7 mg (0.009 mmol) of this compound were then used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 2 mg (22% of theory) of the title compound in the form of a colourless foam.
  • HPLC (Method 6): Rt=1.9 min;
  • LC-MS (Method 2): Rt=1.06 min; MS (ESIpos): m/z=832 (M+H)+.
    • Intermediate 72 N-(3-carboxypropyl)-N-methyl-L-valyl-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]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00510
  • 212 mg (411 μmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 8) and 237 mg (411 μmol) of benzyl-N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-tryptophanate trifluoroacetate (Intermediate 20) were taken up in 30 ml of DMF, and 188 mg (493 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 215 μl N,N-diisopropylethylamine were added. The reaction mixture was stirred at RT for 20 h, then concentrated in vacuo, and the residue was purified by means of preparative HPLC. The product fractions were combined and concentrated, and the residue was dried under high vacuum. This gave 315 mg (80% of theory) of the Boc-protected intermediate N-(tert-butoxycarbonyl)-N-methyl-L-valyl-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]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide as a colourless foam.
  • LC-MS (Method 1): Rt=1.45 min; m/z=961 (M+H)+.
  • 50 mg (52 μmol) of this intermediate were treated with 1 ml of trifluoroacetic acid in 9 ml of dichloromethane to cleave the Boc protecting group. After concentration and purification by means of preparative HPLC, 29 mg (57% of theory) of the free amine intermediate N-methyl-L-valyl-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]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were obtained as trifluoroacetate.
  • LC-MS (Method 1): Rt=0.99 min; m/z=861 (M+H)+.
  • 29 mg (0.03 mmol) of this intermediate were dissolved in 6 ml of dioxane/water, and 39 μl (0.059 mmol) of a 15% aqueous solution of 4-oxobutanoic acid were added. The reaction mixture was subsequently stirred at 100° C. for 1 h. After cooling to RT, 2 mg (0.033 mmol) of sodium cyanoborohydride were added, and the mixture was adjusted to a pH of 3 by adding about 300 μl of 0.1 N hydrochloric acid. The reaction mixture was then stirred at 100° C. for a further 2 h. After cooling, another 39 μl (0.059 mmol) of the 15% 4-oxobutanoic acid solution were added, and the reaction mixture was again stirred at 100° C. for 1 h. Then a further 2 mg (0.033 mmol) of sodium cyanoborohydride were added, and about 300 μl of 0.1 N hydrochloric acid were subsequently used to adjust the pH back to 3. The mixture was then stirred at 100° C. for another 2 h. Thereafter, the reaction mixture was poured onto a 1:1 mixture of semisaturated aqueous ammonium chloride solution and ethyl acetate. The organic phase was removed, washed with saturated sodium chloride solution, dried over sodium sulphate and concentrated. The residue was freeze-dried from water/acetonitrile. This gave 27 mg (94% of theory) of the title compound in the form of a colourless foam.
  • HPLC (Method 5): Rt=2.2 min;
  • LC-MS (Method 9): Rt=5.04 min; MS (ESIpos): m/z=947 (M+H)+.
    • Intermediate 73 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(2S)-1-[benzyl(methyl)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-valinamide
  • Figure US20140127240A1-20140508-C00511
  • First, in analogy to the synthesis described in Intermediate 14, proceeding from Intermediates 4 and 38, the amine compound N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(2S)-1-[benzyl(methyl)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-valinamide was prepared. 25 mg (0.026 mmol) of this compound were then used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 13 mg (54% of theory) of the title compound in the form of a colourless foam.
  • HPLC (Method 12): Rt=2.2 min;
  • LC-MS (Method 9): Rt=5.01 min; MS (ESIpos): m/z=921 (M+H)+.
    • Intermediate 74 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(1S,2R)-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-valinamide
  • Figure US20140127240A1-20140508-C00512
  • 50 mg (73 μmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) and 28 mg (73 μmol) of benzyl (1S,2R)-1-amino-2-phenylcyclopropanecarboxylate trifluoroacetate (Intermediate 45) were taken up in 5 ml of DMF, and 42 mg (110 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 38 μl of N,N-diisopropylethylamine were added. The reaction mixture was stirred at RT for 5 h, then concentrated in vacuo, and the residue was purified by means of preparative HPLC. The product fractions were combined and concentrated. After lyophilization from dioxane/water, 35 mg (51% of theory) of the Boc-protected intermediate N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(1S,2R)-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-valinamide were obtained as a colourless foam.
  • LC-MS (Method 1): Rt=1.52 min; m/z=934 (M+H)+.
  • 35 mg of this intermediate were treated with 1 ml of trifluoroacetic acid in 5 ml of dichloromethane to cleave the Boc protecting group. After concentration and lyophilization from dioxane/water, 34 mg (97% of theory) of the free amine intermediate N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(1S,2R)-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-valinamide were obtained as the trifluoroacetate.
  • LC-MS (Method 1): Rt=0.91 min; m/z=834 (M+H)+.
  • 11 mg (0.011 mmol) of this intermediate were then used, in analogy to the preparation of Intermediate 66, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 2.5 mg (24% of theory) of the title compound in the form of a colourless foam.
  • HPLC (Method 12): Rt=2.2 min;
  • LC-MS (Method 9): Rt=5.1 min; MS (ESIpos): m/z=920 (M+H)+.
    • Intermediate 75 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S,2R)-2-phenyl-1-(propylcarbamoyl)cyclopropyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00513
  • First, in analogy to the synthesis described in Intermediate 74, by coupling of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) and (1S,2R)-1-amino-2-phenyl-N-propylcyclopropanecarboxamide trifluoroacetate (Intermediate 27) in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent cleaving of the Boc protecting group by means of trifluoroacetic acid, the amine compound N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S,2R)-2-phenyl-1-(propylcarbamoyl)cyclopropyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was prepared as trifluoroacetate. 14 mg (0.016 mmol) of this compound were then used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 11.3 mg (83% of theory) of the title compound.
  • HPLC (Method 6): Rt=1.9 min;
  • LC-MS (Method 2): Rt=1.27 min; MS (ESIpos): m/z=871 (M+H)+.
    • Intermediate 76 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-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-valinamide
  • Figure US20140127240A1-20140508-C00514
  • First, by coupling of Intermediate 46 (N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide) with Intermediate 48 (ethyl (1S,2R)-1-amino-2-phenylcyclopropanecarboxylate trifluoroacetate) in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent Boc cleaving, the starting compound N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-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-valinamide trifluoroacetate was prepared. 70 mg (0.079 mmol) of this starting material were then used, by reaction with 4-oxobutanoic acid, in analogy to Intermediate 61, to obtain 46 mg (68% of theory) of the title compound.
  • HPLC (Method 6): Rt=1.9 min;
  • LC-MS (Method 2): Rt=1.28 min; MS (ESIpos): m/z=858 (M+H)+
    • Intermediate 77 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-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-valinamide
  • Figure US20140127240A1-20140508-C00515
  • First, in analogy to the synthesis described in Intermediate 75, by coupling of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) and L-phenylalaninamide hydrochloride in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent cleaving of the Boc protecting group by means of trifluoroacetic acid, the amine compound N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-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-valinamide was prepared as the trifluoroacetate. 47 mg (0.049 mmol) of this compound were then used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 39 mg (96% of theory) of the title compound.
  • HPLC (Method 6): Rt=1.7 min;
  • LC-MS (Method 9): Rt=4.44 min; MS (ESIpos): m/z=817 (M+H)+
  • 1H NMR (500 MHz, DMSO-d6): 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) [further signals hidden under solvent peaks].
    • Intermediate 78 N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00516
  • This compound was prepared in analogy to Intermediate 66 over 2 stages, proceeding from 20 mg (16 μmol) of the compound from Intermediate 14 and benzyl-(6-oxohexyl)carbamate, and the hydrogenation was performed in methanol as the solvent.
  • Yield: 7.6 mg (55% of theory over 2 stages)
  • HPLC (Method 6): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.7 min; MS (ESIpos): m/z=901 (M+H)+.
    • Intermediate 79 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide
  • Figure US20140127240A1-20140508-C00517
  • 36 mg (43 μmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-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-valinamide (Intermediate 46) and 4.6 mg (43 μmol) of benzylamine were taken up in 5 ml of DMF, 7.5 μl (88 μmol) of N,N-diisopropylethylamine, 10 mg (65 μmol) of HOBt and 10 mg (52 μmol) of EDC were added, and then the mixture was stirred at RT overnight. Subsequently, the reaction mixture was concentrated and the residue was purified by means of preparative HPLC. 29 mg (73% of theory) of the Boc-protected intermediate N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide were obtained.
  • LC-MS (Method 1): Rt=1.43 min; m/z=921 (M+H)+.
  • 29 mg of this intermediate were treated with 1 ml of trifluoroacetic acid in 6 ml of dichloromethane to cleave the Boc protecting group. After concentration and lyophilization from dioxane/water, 30 mg (quant.) of the free amine intermediate N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide were obtained as the trifluoroacetate.
  • LC-MS (Method 1): Rt=0.95 min; m/z=821 (M+H)+.
  • 17 mg (0.018 mmol) of this intermediate were then used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 13 mg (80% of theory) of the title compound in the form of a colourless foam.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 9): Rt=4.97 min; MS (ESIpos): m/z=907 (M+H)+.
    • Intermediate 80 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-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-valinamide
  • Figure US20140127240A1-20140508-C00518
  • First, in analogy to the synthesis described in Intermediate 74, by coupling of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) and N-benzyl-L-tryptophanamide trifluoroacetate (Intermediate 47) in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent cleaving of the Boc protecting group by means of trifluoroacetic acid, the amine compound N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-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-valinamide was prepared as the trifluoroacetate. 10 mg (0.01 mmol) of this compound were then used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 2.5 mg (26% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 2): Rt=1.13 min; MS (ESIpos): m/z=946 (M+H)+.
    • Intermediate 81 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-carbamoyl-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00519
  • First, in analogy to the synthesis described in Intermediate 74, by coupling of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) and (1S,2R)-1-amino-2-phenylcyclopropanecarboxamide trifluoroacetate (Intermediate 48) in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent cleaving of the Boc protecting group by means of trifluoroacetic acid, the amine compound N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-carbamoyl-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was prepared as the trifluoroacetate. 14 mg (0.0163 mmol) of this compound were then used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 8 mg (57% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 9): Rt=4.64 min; MS (ESIpos): m/z=829 (M+H)+.
    • Intermediate 82 N-(3-carboxypropyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00520
  • First, in analogy to the synthesis described in Intermediate 69, by coupling of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 4) and Nα-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-tryptophanamide trifluoroacetate (Intermediate 49) in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent cleaving of the Fmoc protecting group by means of piperidine, the amine compound N-methyl-L-valyl-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-valinamide was prepared as the trifluoroacetate. 78 mg (0.088 mmol) of this compound were then used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 68 mg (90% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.8 min;
  • LC-MS (Method 9): Rt=4.49 min; MS (ESIpos): m/z=856 (M+H)+.
    • Intermediate 83 N-(5-carboxypentyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00521
  • This compound was prepared in analogy to the compound in Intermediate 82, proceeding from 20 mg (26 μmol) of N-methyl-L-valyl-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-valinamide trifluoroacetate, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride.
  • Yield: 5 mg (25% of theory)
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 11): Rt=0.72 min; MS (ESIpos): m/z=884 (M+H)+.
    • Intermediate 84 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(morpholin-4-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00522
  • First, in analogy to the synthesis described in Intermediate 79, by coupling of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-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-valinamide (Intermediate 46) and morpholine in the presence of EDC and HOBT and subsequent cleaving of the Boc protecting group by means of trifluoroacetic acid, the amine compound N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(morpholin-4-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was prepared as the trifluoroacetate. 30 mg (0.033 mmol) of this compound were then used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 22 mg (76% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 9): Rt=4.58 min; MS (ESIpos): m/z=887 (M+H)+.
    • Intermediate 85 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3R)-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-valinamide
  • Figure US20140127240A1-20140508-C00523
  • First, in analogy to the synthesis described in Intermediate 79, by coupling of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-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-valinamide (Intermediate 46) and N-benzyl-L-threoninamide trifluoroacetate in the presence of HATU and subsequent cleaving of the Boc protecting group by means of trifluoroacetic acid, the amine compound N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3R)-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-valinamide was prepared as the trifluoroacetate. 21 mg (0.024 mmol) of this compound were then used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 20 mg (97% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.54 min;
  • LC-MS (Method 9): Rt=4.49 min; MS (ESIpos): m/z=861 (M+H)+.
    • Intermediate 86 N-(3-carboxypropyl)-N-methyl-L-valyl-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-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00524
  • First, in analogy to the synthesis described in Intermediate 74, by coupling of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) and tert-butyl-L-phenylalaninate hydrochloride in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent cleaving of the Boc protecting group by means of trifluoroacetic acid to obtain the tert-butyl ester (stirring with trifluoroacetic acid in dichloromethane for 40 minutes), the amine compound N-methyl-L-valyl-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-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was prepared as the trifluoroacetate. 22 mg (0.02 mmol) of this compound were then used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 16 mg (94% of theory) of the title compound.
  • HPLC (Method 5): Rt=2.0 min;
  • LC-MS (Method 9): Rt=5.05 min; MS (ESIpos): m/z=874 (M+H)+.
    • Intermediate 87 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-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-valinamide
  • Figure US20140127240A1-20140508-C00525
  • This compound was prepared in analogy to the synthesis described in Intermediate 86, proceeding from 230 mg (336 μmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) and tert-butyl-L-tryptophanate hydrochloride over 3 stages.
  • Yield: 95 mg (31% of theory over 3 stages)
  • HPLC (Method 5): Rt=2.0 min;
  • LC-MS (Method 9): Rt=5.05 min; MS (ESIpos): m/z=913 (M+H)+.
    • Intermediate 88 N-(6-aminohexyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00526
  • First, in analogy to the syntheses described in Intermediate 69, by coupling of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 4) and Nα-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-tryptophanamide trifluoroacetate (Intermediate 49) in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent cleaving of the Fmoc protecting group by means of piperidine, the amine compound N-methyl-L-valyl-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-valinamide was prepared as the trifluoroacetate. 30 mg (0.03 mmol) of this compound were then used, in analogy to the preparation of the compound of Intermediate 61, by reaction with benzyl-(6-oxohexyl)carbamate, which had been obtained beforehand by oxidation of benzyl-(6-hydroxyhexyl)carbamate, in the presence of sodium cyanoborohydride, to obtain 17 mg (45% of theory) of the Z-protected compound. Subsequently, hydrogenolysis in methanol over 10% palladium/activated carbon yielded the title compound.
  • Yield: 14 mg (95% of theory)
  • HPLC (Method 5): Rt=1.5 min;
  • LC-MS (Method 1): Rt=0.73 min; MS (ESIpos): m/z=869 (M+H)+.
    • Intermediate 89 N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-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-valinamide
  • Figure US20140127240A1-20140508-C00527
  • First, in analogy to the synthesis described in Intermediate 86, by coupling of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) and tert-butyl-L-tryptophanate hydrochloride in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent cleaving of the Boc protecting group by means of trifluoroacetic acid to obtain the tert-butyl ester (stirring with 1:10 trifluoroacetic acid/dichloromethane for 30 min), the amine compound N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-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-valinamide was prepared as the trifluoroacetate. 71 mg (0.075 mmol) of this compound were then used, in analogy to the preparation of the compound of Intermediate 61, by reaction with benzyl-(6-oxohexyl)carbamate, which had been obtained beforehand by oxidation of benzyl-(6-hydroxyhexyl)carbamate, in the presence of sodium cyanoborohydride, to obtain 35 mg (44% of theory) of the Z-protected compound. Subsequently, hydrogenolysis in methanol over 10% palladium/activated carbon yielded 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 (ESIpos): m/z=926 (M+H)+.
    • Intermediate 90 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(1H-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-valinamide
  • Figure US20140127240A1-20140508-C00528
  • First, in analogy to the synthesis described in Intermediate 74, by coupling of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) and 2-(1H-indol-3-yl)ethanamine in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent cleaving of the Boc protecting group by means of trifluoroacetic acid, the amine compound N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(1H-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-valinamide was prepared as the trifluoroacetate. 100 mg (0.119 mmol) of this compound were then used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 50 mg (49% of theory) of the title compound. The title compound was purified here by flash chromatography on silica gel with dichloromethane/methanol/17% ammonia as the eluent, in the course of which the mixing ratio was switched from initially 15/2/02 to 15/4/0.5.
  • HPLC (Method 6): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=813 (M+H)+.
    • Intermediate 91 N-(3-carboxypropyl)-N-methyl-L-valyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00529
  • First, in analogy to the synthesis described in Intermediate 74, by coupling of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) and phenylethylamine in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent cleaving of the Boc protecting group by means of trifluoroacetic acid, the amine compound N-methyl-L-valyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide was prepared as the trifluoroacetate. 57 mg (0.071 mmol) of this compound were then used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 44 mg (80% of theory) of the title compound. The title compound can also be purified here by flash chromatography on silica gel with dichloromethane/methanol/17% ammonia as the eluent (15/2/02->15/4/0.5).
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 9): Rt=4.64 min; MS (ESIpos): m/z=774 (M+H)+.
    • Intermediate 92 N-(3-carboxypropyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00530
  • 100 mg (0.139 mmol) of N-methyl-L-valyl-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-valinamide (Intermediate 40) were used, in analogy to the preparation of Intermediate 61, by reaction with 4-oxobutanoic acid in the presence of sodium cyanoborohydride, to obtain 94 mg (84% of theory) of the title compound. The title compound was purified by preparative HPLC.
  • HPLC (Method 5): Rt=1.5 min;
  • LC-MS (Method 9): Rt=4.46 min; MS (ESIpos): m/z=804 (M+H)+.
    • Intermediate 93 N-(3-carboxypropyl)-N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00531
  • 22.4 mg (24 μmol) of N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate were dissolved in 1.4 ml of dioxane/water and, analogously to the preparation of Intermediate 61, reacted with 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride. After lyophilization from dioxane, 8.2 mg (38% of theory) of the title compound were obtained in the form of a white solid.
  • HPLC (Method 10): Rt=2.54 min
  • LC-MS (Method 12): Rt=0.94 min; MS (ESIpos): m/z=919 (M+H)+
    • Intermediate 94 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00532
  • 17.1 mg (18 μmol) of N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate were dissolved in 1.1 ml of dioxane/water and, analogously to the preparation of Intermediate 61, reacted with 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride. After lyophilization from dioxane, 14.8 mg (89% of theory) of the title compound were obtained in the form of a white solid.
  • HPLC (Method 10): Rt=2.54 min;
  • LC-MS (Method 12): Rt=0.92 min; MS (ESIpos): m/z=919 (M+H)+
    • Intermediate 95 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylsulphonyl)-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-valinamide
  • Figure US20140127240A1-20140508-C00533
  • 19.3 mg (20 μmol) N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylsulphonyl)-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-valinamide trifluoroacetate were dissolved in 1.2 ml of dioxane/water and, analogously to the preparation of Intermediate 61, reacted with 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride. After lyophilization from dioxane, 8.6 mg (45% of theory) of the title compound were obtained in the form of a solid.
  • LC-MS (Method 11): Rt=0.85 min; MS (ESIpos): m/z=943 (M+H)+
    • Intermediate 96 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3E)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00534
  • 15.5 mg (10 μmol) of N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3E)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate were dissolved in 1.0 ml of dioxane/water and, analogously to the preparation of Intermediate 61, reacted with 15% aqueous solution of 4-oxobutanoic acid in the presence of sodium cyanoborohydride. After lyophilization from dioxane, 10.3 mg (68% of theory) of the title compound were obtained in the form of a white solid.
  • HPLC (Method 10): Rt=2.59 min;
  • LC-MS (Method 11): Rt=0.94 min; MS (ESIpos): m/z=877 (M+H)+
    • Intermediate 97 N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00535
  • The title compound was prepared in analogy to the synthesis of Intermediate 66, by reaction of 200 mg (0.108 mmol) of N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate (Intermediate 16) with benzyl-(6-oxohexyl)carbamate and subsequent hydrogenolytic cleaving of the Z protecting group (with 5% palladium on charcoal as a catalyst, in methanol as a solvent).
  • Yield: 69 mg (65% of theory over two stages)
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.76 min; MS (ESIpos): m/z=912 (M+H)+.
    • Intermediate 98 N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-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-valinamide
  • Figure US20140127240A1-20140508-C00536
  • This compound was prepared in analogy to the synthesis described in Intermediate 80. The purification was effected by preparative HPLC.
  • Yield: 40 mg (29% of theory over 3 stages)
  • HPLC (Method 5): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.92 min; MS (ESIpos): m/z=974 (M+H)+.
    • Intermediate 99 (2S)-2-amino-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)propan-1-one trifluoroacetate
  • Figure US20140127240A1-20140508-C00537
  • 324 mg (0.81 mmol) of 2,5-dioxopyrrolidin-1-yl N-(tert-butoxycarbonyl)-L-tryptophanate were dissolved in 20 ml of DMF, and 200 mg (1.62 mmol) of 1,2-oxazinane hydrochloride (Starting Compound 5) and 850 μl of N,N-diisopropylethylamine were added. The reaction mixture was stirred at 50° C. overnight and then concentrated under in vacuo. The residue was taken up in dichloromethane and extracted with water. The organic phase was dried over magnesium sulphate and concentrated. The residue was purified by flash chromatography on silica gel with 4:1 dichloromethane/ethyl acetate as the eluent. The product fractions were concentrated, and the residue was dried under high vacuum. This gave 147.5 mg (48% of theory) of the Boc-protected intermediate.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=1.03 min; MS (ESIpos): m/z=374 (M+H)+.
  • Using 166 mg (444.5 μmol) of this intermediate, under standard conditions with 3 ml of trifluoroacetic acid in 20 ml of dichloromethane, the Boc protecting group was cleaved and, after HPLC purification, 155 mg (86% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.43 min;
  • LC-MS (Method 11): Rt=0.56 min; MS (ESIpos): m/z=274 (M+H)+.
    • Intermediate 100 N-(6-{[(benzyloxy)carbonyl]amino}hexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00538
  • 177 mg (260 μmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) and 100 mg (260 μmol) of (2S)-2-amino-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)propan-1-one trifluoroacetate (Intermediate 99) were taken up in 15 ml of DMF, and 118 mg (310 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 140 μl of N,N-diisopropylethylamine were added. The reaction mixture was stirred at RT for 30 min, then concentrated in vacuo, and the residue was purified by means of preparative HPLC. The product fractions were combined and concentrated. After lyophilization from dioxane, 170 mg (68% of theory) of the Boc-protected intermediate were obtained.
  • LC-MS (Method 1): Rt=1.36 min; m/z=940 (M+H)+.
  • 170 mg of this intermediate were treated with 3 ml of trifluoroacetic acid in 30 ml of dichloromethane for 30 min for cleaving the Boc protecting group. Then the reaction mixture was concentrated in vacuo, and the residue was purified by means of preparative HPLC to obtain 155 mg (86% of theory) of the deprotected N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide intermediate.
  • HPLC (Method 12): Rt=1.85 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=840 (M+H)+.
  • 50 mg (0.052 mmol) of this intermediate were then used, in analogy to the preparation of Intermediate 97, with benzyl-(6-oxohexyl)carbamate in the presence of sodium cyanoborohydride and subsequent hydrogenolytic cleaving of the Z protecting group (with 5% palladium on charcoal as a catalyst, in methanol as a solvent), to prepare the title compound.
  • Yield: 21 mg (37% of theory)
  • HPLC (Method 12): Rt=2.1 min;
  • LC-MS (Method 1): Rt=1.02 min; MS (ESIpos): m/z=1073 (M+H)+.
    • Intermediate 101 N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00539
  • 26.7 mg (24.87 μmol) of Intermediate 100 were dissolved in 10 ml of methanol and hydrogenated over palladium/activated carbon (5%) under standard hydrogen pressure for 30 min. The catalyst was filtered off and the solvent was evaporated off in vacuo. After the residue had been dried under high vacuum, 22.5 mg (96% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.76 min; MS (ESIpos): m/z=939 (M+H)+.
    • Intermediate 102 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(morpholin-4-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00540
  • This compound was prepared in analogy to the synthesis described in Intermediate 157 from N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(morpholin-4-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide and commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
  • Yield: 8 mg (71% of theory)
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=1094 (M+H)+.
    • Intermediate 103 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3R)-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-valinamide
  • Figure US20140127240A1-20140508-C00541
  • This compound was prepared in analogy to the synthesis described in Intermediate 157 from N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3R)-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-valinamide and commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
  • Yield: 3 mg (22% of theory)
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.78 min; MS (ESIpos): m/z=1069 (M+H)+.
    • Intermediate 104 N-{4-[(trans-4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}cyclohexyl)amino]-4-oxobutyl}-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00542
  • First, benzyl trans-4-aminocyclohexanecarboxylate trifluoroacetate was prepared from trans-4-aminocyclohexanecarboxylic acid by introducing the Boc protecting group, then introducing the benzyl ester protecting group and subsequently cleaving the Boc protecting group by conventional peptide chemistry methods.
  • 15 mg (18 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-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-valinamide were then dissolved in 5 ml of dimethylformamide and subsequently admixed with 13 mg (35 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 9 μl of N,N-diisopropylethylamine and with 15 mg (44 μmol) of benzyl trans-4-aminocyclohexanecarboxylate trifluoroacetate. The mixture was stirred at RT for 1 h and then concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. The corresponding fractions were combined and the solvent was evaporated off in vacuo. After the residue had been dried under high vacuum, 14.7 mg (78% of theory) of the protected intermediate were obtained as a colourless foam.
  • HPLC (Method 6): Rt=2.0 min;
  • LC-MS (Method 1): Rt=0.95 min; MS (ESIpos): m/z=1072 (M+H)+.
  • From this protected intermediate, the benzyl ester was first removed by hydrogenolytic means, and the free carboxyl component was obtained in quantitative yield. 14 mg (14 μmol; 1 equiv.) of the deprotected compound were taken up in 5 ml of DMF and admixed with 3.3 mg (29 μmol; 2.1 equiv.) of N-hydroxysuccinimide in the presence of 4.1 mg (21 μmol; 1.5 equiv.) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 7.5 μl (44 μmol; 3.1 equiv.) of N,N-diisopropylethylamine and 0.9 mg (7 μmol; 0.5 equiv.) of 4-dimethylaminopyridine, and the mixture was stirred at RT overnight. Then another 10 equiv. of N-hydroxysuccinimide, 5 equiv. of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 5 equiv. of N,N-diisopropylethylamine and 0.5 equiv. of 4-dimethylaminopyridine were added, and the reaction mixture was treated in an ultrasound bath for 5 h. Subsequently, the solvent was evaporated off, the residue was purified by means of preparative HPLC and the corresponding fractions were combined and concentrated. After lyophilization of the residue from dioxane, 9.7 mg (62% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 6): Rt=1.8 min;
  • LC-MS (Method 11): Rt=0.77 min; MS (ESIpos): m/z=1078 (M+H)+.
    • Intermediate 105 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00543
  • This compound was prepared in analogy to the synthesis described in Intermediate 157, proceeding from 4-{[(2S)-1-{[(2S)-1-{[(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-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methylbutan-2-yl]amino}-3-methyl-1-oxobutan-2-yl](methyl)amino}butanoic acid and commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The ester intermediate was obtained in 42% yield. In a second step, 6 mg (6 μmol) of this intermediate were cleaved with trifluoroacetic acid the tert-butyl ester. After HPLC purification, 3.4 mg (48% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.66 min;
  • LC-MS (Method 2): Rt=1.04 min; MS (ESIpos): m/z=1025 (M+H)+.
    • Intermediate 106 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00544
  • 14 mg (16 μmol) of N-(6-aminohexyl)-N-methyl-L-valyl-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-valinamide (Intermediate 88) were taken up in 750 μl of dioxane and admixed with 1.5 ml of saturated sodium hydrogencarbonate solution and then with 3.2 mg (21 μmol) of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate. The reaction mixture was stirred at RT for 1 h and then concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization, 5.5 mg (36% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.84 min; MS (ESIpos): m/z=949 (M+H)+.
    • Intermediate 107 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(1H-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-valinamide
  • Figure US20140127240A1-20140508-C00545
  • 38 mg (47 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(1H-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-valinamide were dissolved in 37 ml of DMF and then admixed with 71 mg (187 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 33 μl of N,N-diisopropylethylamine and with 37 mg (140 μmol) of commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The mixture was stirred at RT for 1 h. This was followed by concentration under high vacuum and purification of the remaining residue by means of preparative HPLC. Thus, 12.2 mg (26% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.85 min; MS (ESIpos): m/z=1020 (M+H)+.
    • Intermediate 108 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00546
  • The compound was prepared in analogy to Intermediate 107.
  • Yield: 2.5 mg (30% of theory)
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.9 min; MS (ESIpos): m/z=981 (M+H)+.
    • Intermediate 109 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00547
  • The compound was prepared in analogy to Intermediate 107 from the compound in Intermediate 92.
  • Yield: 35 mg (65% of theory)
  • HPLC (Method 5): Rt=1.9 min;
  • LC-MS (Method 11): Rt=0.76 min; MS (ESIpos): m/z=1011 (M+H)+.
    • Intermediate 110 N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00548
  • This compound was prepared in analogy to Intermediate 147 from the compound in Intermediate 83.
  • Yield: 2.4 mg (24% of theory)
  • HPLC (Method 6): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=981 (M+H)+.
    • Intermediate 111 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-1-methylhydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00549
  • This compound was prepared in analogy to Intermediate 140 from Intermediate 82 and Intermediate 22.
  • Yield: 6.5 mg (51% of theory)
  • HPLC (Method 6): Rt=1.8 min;
  • LC-MS (Method 1): Rt=4.71 min; MS (ESIpos): m/z=1077 (M+H)+.
    • Intermediate 112 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-carbamoyl-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00550
  • This compound was prepared in analogy to Intermediate 157 from the compound in Intermediate 81.
  • Yield: 5.7 mg (57% of theory)
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=1036 (M+H)+.
    • Intermediate 113 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00551
  • 95 mg (104 μmol) of 4-{[(2S)-1-{[(2S)-1-{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-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](methyl)amino}-3-methylbutan-2-yl]amino}-3-methyl-1-oxobutan-2-yl](methyl)amino}butanoic acid were dissolved in DMF and then admixed with 79.5 mg (209 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 73 μl of N,N-diisopropylethylamine and with 68 mg (261 μmol) of commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The mixture was stirred at RT for 2 h. This was followed by concentration under high vacuum and purification of the remaining residue by means of preparative HPLC. Thus, 104 mg (89% of theory) of the tert-butyl ester of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=2.0 min;
  • LC-MS (Method 1): Rt=0.93 min; MS (ESIpos): m/z=1121 (M+H)+.
  • The intermediate was taken up in 33.4 ml of dichloromethane, 17 ml of trifluoroacetic acid were added, 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 the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=1064 (M+H)+.
    • Intermediate 114 N-[6-({[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}amino)hexyl]-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00552
  • 5 mg (5 μmol) of N-(6-aminohexyl)-N-methyl-L-valyl-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-valinamide were taken up in 885 μl of DMF and admixed with 5.3 mg (8 μmol) of 4-nitrophenyl-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamate and 2.8 μl of N,N-diisopropylethylamine. The reaction mixture was stirred at RT for 2 h and then concentrated to dryness. The residue was purified by means of preparative HPLC.
  • Yield: 0.58 mg (11% of theory) of a colourless foam
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.83 min; MS (ESIpos): m/z=1035 (M+H)+.
    • Intermediate 115 N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00553
  • This compound was prepared in analogy to the compound in Intermediate 147, starting from 8 mg (9 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide. After concentration, the activated ester was purified by means of preparative HPLC and, after removal of the solvent in vacuo, reacted immediately with the antibody.
  • Yield: 3 mg (27% of theory) (hydrolysis-sensitive)
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=996 (M+H)+.
    • Intermediate 116 N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00554
  • This compound was prepared in analogy to the compound in Intermediate 147, starting from 5 mg (6 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide After concentration, the activated ester was purified by means of preparative HPLC and, after removal of the solvent in vacuo, reacted immediately with the antibody.
  • Yield: 3.2 mg (43% of theory) (hydrolysis-sensitive)
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.92 min; MS (ESIpos): m/z=984 (M+H)+.
    • Intermediate 117 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00555
  • This compound was prepared in analogy to Intermediate 157 from the compound in Intermediate 86.
  • Yield: 7 mg (42% of theory)
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.94 min; MS (ESIpos): m/z=1081 (M+H)+.
    • Intermediate 118 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2R)-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-valinamide
  • Figure US20140127240A1-20140508-C00556
  • The target compound was prepared in analogy to Intermediate 157 from 7 mg (7.8 μmol) of the compound in Intermediate 68. Yield: 6.3 mg (53% of theory)
  • LC-MS (Method 1): Rt=1.00 min; MS (ESIpos): m/z=1102 (M+H)+.
    • Intermediate 119 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00557
  • 7.4 mg (8.1 mmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide and 6.3 mg (24.2 mmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide hydrochloride were coupled and worked up in analogy to Intermediate 157. 1.6 mg (13% of theory) of the title compound were obtained as a solid.
  • LC-MS (Method 11): Rt=0.89 min; MS (ESIpos): m/z=1126 (M+H)+
    • Intermediate 120 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00558
  • 12.8 mg (13.9 mmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide and 10.9 mg (41.8 mmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide hydrochloride were coupled and worked up in analogy to Intermediate 157. 10.8 mg (59% of theory) of the title compound were obtained as a solid.
  • LC-MS (Method 11): Rt=0.90 min; MS (ESIpos): m/z=1126 (M+H)+
    • Intermediate 121 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylsulphonyl)-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-valinamide
  • Figure US20140127240A1-20140508-C00559
  • 7.4 mg (7.9 mmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylsulphonyl)-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-valinamide and 6.2 mg (23.5 mmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide hydrochloride were coupled and worked up in analogy to Intermediate 157. 6.9 mg (74% of theory) of the title compound were obtained as a solid.
  • LC-MS (Method 11): Rt=0.87 min; MS (ESIpos): m/z=1150 (M+H)+
    • Intermediate 122 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3E)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00560
  • 8 mg (9.1 mmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3E)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide and 7.2 mg (27.4 mmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide hydrochloride were coupled and worked up in analogy to Intermediate 157. 8.2 mg (82% of theory) of the title compound were obtained as a white solid.
  • LC-MS (Method 11): Rt=0.95 min; MS (ESIpos): m/z=1083 (M+H)+
    • Intermediate 123 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-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-valinamide
  • Figure US20140127240A1-20140508-C00561
  • 30 mg (30 μmol) of Intermediate 89 were taken up in 2 ml of 1,4-dioxane and admixed with 4 ml of saturated sodium hydrogencarbonate solution and then with 7.5 mg (50 μmol) of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate. The reaction mixture was stirred at RT for 1 h and then concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization, 24 mg (74% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=2.2 min;
  • LC-MS (Method 1): Rt=1.01 min; MS (ESIpos): m/z=1006 (M+H)+.
    • Intermediate 124 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00562
  • 22 mg (20 μmol) of Intermediate 123 were reacted with 4 ml of trifluoroacetic acid in 8 ml of dichloromethane at RT for 1 h. Thereafter, the reaction mixture was concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization, 11 mg (54% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.8 min;
  • LC-MS (Method 11): Rt=0.85 min; MS (ESIpos): m/z=950 (M+H)+.
    • Intermediate 125 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00563
  • 22.5 mg (20 μmol) of Intermediate 101 were taken up in 2 ml of 1:1 dioxane/water and then admixed with 5.6 mg (40 μmol) of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate and with 0.25 ml of saturated sodium hydrogencarbonate solution. The reaction mixture was stirred at RT for 30 min. Then another 0.25 ml of the saturated sodium hydrogencarbonate solution were added, and the reaction mixture was stirred at RT for another 15 min and then concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization, 12.8 mg (50% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.95 min; MS (ESIpos): m/z=1019 (M+H)+.
    • Intermediate 126 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00564
  • 64 mg (70 μmol) of N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 97) were taken up in 3 ml of 1:1 dioxane/water, then adjusted to pH 9 with 4 ml of saturated sodium hydrogencarbonate solution and subsequently admixed with 16.3 mg (110 μmol) of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate. The reaction mixture was stirred at RT for 1 h and then concentrated in vacuo. Then another 8 mg (55 μmol) of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate were added, and the reaction mixture was adjusted again to pH 9 and stirred at RT for another hour. This was followed by concentration and purification of the remaining residue by means of preparative HPLC. At first, 31 mg of an as yet uncyclized intermediate were obtained. 27 mg of this intermediate were taken up again in 2 ml of 1:1 dioxane/water and then admixed with 250 μl of saturated sodium hydrogencarbonate solution. After stirring at RT for 2 hours, the reaction mixture was concentrated, and the residue was purified by means of preparative HPLC. After lyophilization, 20 mg (29% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.96 min;
  • LC-MS (Method 1): Rt=0.97 min; MS (ESIpos): m/z=992 (M+H)+.
    • Intermediate 127 N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-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-valinamide
  • Figure US20140127240A1-20140508-C00565
  • 17 mg (18 μmol) of N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-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-valinamide (Intermediate 98) were dissolved in 2.8 ml of dichloromethane and admixed with 20 mg (174 mmol) of 1-hydroxypyrrolidine-2,5-dione and then admixed 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 concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization, 8.2 mg (43% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=2.0 min;
  • LC-MS (Method 1): Rt=0.98 min; MS (ESIpos): m/z=1071 (M+H)+.
    • Intermediate 128 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00566
  • 5 mg (5.6 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 845 μl of DMF and then admixed with 3.2 mg (17 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2.6 mg (17 μmol) of 1-hydroxy-1H-benzotriazole hydrate, 1.96 μl of N,N-diisopropylethylamine and with 5.9 mg (22.5 μmol) of commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. Thus, 2.2 mg (36% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.88 min; MS (ESIpos): m/z=1094 (M+H)+.
    • Intermediate 129 N-(6-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-6-oxohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00567
  • 4 mg (4.3 μmol) of N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 646 μl of DMF and then admixed with 2.5 mg (13 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2.0 mg (13 μmol) of 1-hydroxy-1H-benzotriazole hydrate, 2.25 μl of N,N-diisopropylethylamine and with 4.5 mg (17 μmol) of commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The mixture was stirred at RT for 3 h and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. Thus, 1.9 mg (39% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 9): Rt=4.9 min; MS (ESIpos): m/z=1134 (M+H)+.
    • Intermediate 130 N-(4-{[(2R)-1-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)propan-2-yl]oxy}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00568
  • 10.5 mg (11.7 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 3.7 ml of dichloromethane and then admixed with 6.7 mg (35 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.7 mg (5.8 μmol) of 4-dimethylaminopyridine and with 8.2 mg (47 μmol) of commercially available tert-butyl-[(2R)-2-hydroxypropyl]carbamate. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. Thus, 7.5 mg (61% of theory) of the Boc-protected intermediate were obtained as a colourless foam.
  • HPLC (Method 5): Rt=2.0 min;
  • LC-MS (Method 1): Rt=1.03 min; MS (ESIpos): m/z=1056 (M+H)+.
  • Subsequently, the Boc protecting group was cleaved with trifluoroacetic acid. 4.9 mg (0.005 mmol) of the deprotected crude product were then, without further purification, taken up in 1.8 ml of dichloromethane and admixed with 3.7 mg (0.011 mmol) of 1,1′-[(1,5-dioxopentane-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) of 4-dimethylaminopyridine. The mixture was stirred at RT for 2 h and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. Thus, 0.77 mg (15% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.93 min; MS (ESIpos): m/z=1167 (M+H)+.
    • Intermediate 131 N-{4-[(1-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}piperidin-4-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00569
  • 10 mg (11 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 2 ml of dichloromethane and then admixed with 4.3 mg (22 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.88 mg (6 μmol) of 4-dimethylaminopyridine and with 5.2 mg (22 μmol) of commercially available benzyl 4-hydroxypiperidine-1-carboxylate. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. Thus, 5 mg (40% of theory) of the Z-protected intermediate were obtained as a colourless foam.
  • HPLC (Method 5): Rt=2.1 min;
  • LC-MS (Method 1): Rt=1.04 min; MS (ESIpos): m/z=1116 (M+H)+.
  • Subsequently, the Z protecting group was cleaved by hydrogenolytic means in ethanol over palladium/activated carbon. 4.6 mg (0.005 mmol) of the deprotected crude product were then, without further purification, taken up in 1.8 ml of dichloromethane and admixed with 3.8 mg (0.012 mmol) of 1,1′-[(1,5-dioxopentane-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. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. Thus, 0.96 mg (16% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.94 min; MS (ESIpos): m/z=1193 (M+H)+.
    • Intermediate 132 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazinyl}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00570
  • 15 mg (16.7 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 2500 μl of DMF and then admixed with 9.6 mg (50 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 7.6 mg (50 μmol) of 1-hydroxy-1H-benzotriazole hydrate, 5.8 μl of N,N-diisopropylethylamine and with 17.4 mg (67 μmol) of commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. Thus, 11.2 mg (52% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 2): Rt=1.09 min; MS (ESIpos): m/z=1106 (M+H)+.
    • Intermediate 133 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazinyl}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3S)-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-valinamide
  • Figure US20140127240A1-20140508-C00571
  • 5.8 mg (6.3 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3S)-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-valinamide were dissolved in 943 μl of DMF and then admixed with 3.6 mg (19 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2.9 mg (19 μmol) of 1-hydroxy-1H-benzotriazole hydrate, 2.2 μl of N,N-diisopropylethylamine and with 6.6 mg (25 μmol) of commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. Thus, 4.5 mg (64% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=2.0 min;
  • LC-MS (Method 1): Rt=1.03 min; MS (ESIpos): m/z=1129 (M+H)+.
    • Intermediate 134 N-[3-({[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}amino)propyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00572
  • First, 4-nitrophenyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl carbamate was prepared under standard conditions, starting from commercially available 1-(2-aminoethyl)-1H-pyrrole-2,5-dione trifluoroacetate and 4-nitrophenyl chlorocarbonate.
  • 5 mg (6 μmol) of N-(3-aminopropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 1000 μl of DMF and then admixed with 2 μl of N,N-diisopropylethylamine and with 2.2 mg (9 μmol) of 4-nitrophenyl-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamate. The mixture was stirred at RT for 1 h and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. Thus, 1.6 mg (23% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 2): Rt=1.09 min; MS (ESIpos): m/z=1036 (M+H)+.
    • Intermediate 135 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00573
  • 10 mg (11 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-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-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 4000 μl of DMF and then admixed with 6.3 mg (33 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4.5 mg (33 μmol) of 1-hydroxy-1H-benzotriazole hydrate, 5.7 μl of N,N-diisopropylethylamine and with 11.5 mg (44 μmol) of commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. Thus, 2.6 mg (14% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 6): Rt=2.1 min;
  • LC-MS (Method 1): Rt=1.01 min; MS (ESIpos): m/z=1115 (M+H)+.
    • Intermediate 136 N-(4-{4-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]piperazin-1-yl}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00574
  • First, 1-[4-oxo-4-(piperazin-1-yl)butyl]-1H-pyrrole-2,5-dione trifluoroacetate was prepared under standard conditions, starting from tert-butyl piperazine-1-carboxylate and 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid over 2 stages.
  • 5 mg (5.6 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 1000 μl of DMF and then admixed with 2.1 mg (11 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1.7 mg (11 μmol) of 1-hydroxy-1H-benzotriazole hydrate, 2 μl of N,N-diisopropylethylamine and with 3.5 mg (5.6 μmol) of 1-[4-oxo-4-(piperazin-1-yl)butyl]-1H-pyrrole-2,5-dione trifluoroacetate. The mixture was stirred at RT overnight. Then 2.1 mg (5.6 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate were added, and the reaction mixture was stirred at RT for another 3 h. Subsequently, the solvent was removed in vacuo, and the remaining residue was purified by means of preparative HPLC. The corresponding fractions were concentrated and, by lyophilization from water, 0.6 mg (10% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 6): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.9 min; MS (ESIpos): m/z=1132 (M+H)+.
    • Intermediate 137 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-1-methylhydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00575
  • First, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N′-methylhexanehydrazide trifluoroacetate was prepared under standard conditions, starting from commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid and tert-butyl 1-methylhydrazinecarboxylate over 2 stages.
  • 6.9 mg (8 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 2540 μl of DMF and then admixed with 3.6 mg (9 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 3 μl of N,N-diisopropylethylamine and with 4.1 mg (12 μmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N′-methylhexanehydrazide trifluoroacetate. The mixture was stirred at RT overnight. Subsequently, the solvent was removed in vacuo, and the remaining residue was purified by means of preparative HPLC. Thus, 3.9 mg (45% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.93 min; MS (ESIpos): m/z=1108 (M+H)+.
    • Intermediate 138 N-{4-[(2-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl](methyl)amino}ethyl)(methyl)amino]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00576
  • Starting from tert-butylmethyl[2-(methylamino)ethyl]carbamate and 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid, over 2 stages, 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methyl-N-[2-(methylamino)ethyl]butanamide trifluoroacetate was prepared first.
  • 6.6 mg (7.3 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 2000 μl of DMF and then admixed with 5.6 mg (14.7 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 2.6 μl of N,N-diisopropylethylamine and with 4.1 mg (9 μmol) of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methyl-N-[2-(methylamino)ethyl]butanamide trifluoroacetate. After stirring at RT for 3 h, the same amounts of HATU and N,N-diisopropylethylamine were added once more, and the reaction mixture was then stirred at RT overnight. Subsequently, the solvent was removed in vacuo, and the remaining residue was purified by means of preparative HPLC. Thus, 4 mg (44% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 6): Rt=2.0 min;
  • LC-MS (Method 1): Rt=0.91 min; MS (ESIpos): m/z=1134 (M+H)+.
    • Intermediate 139 (2R,3S)-3-amino-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutan-2-yl (3R,4S,7S,10S)-4-[(2S)-butan-2-yl]-7,10-diisopropyl-3-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazapentadecan-15-oate
  • Figure US20140127240A1-20140508-C00577
  • 13 mg (14.7 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 10 ml of dichloromethane and then admixed with 8.4 mg (44 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 5.4 mg (44 μmol) of 4-dimethylaminopyridine and with 9 mg (29.3 μmol) of commercially available benzyl N-(tert-butoxycarbonyl)-L-threoninate. The mixture was stirred at RT for 5 h. Subsequently, the reaction mixture was extracted twice by shaking with water, and the organic phase was dried over sodium sulphate and concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization from dioxane/water, 14 mg (81% of theory) of the protected intermediate were obtained as a colourless foam.
  • HPLC (Method 12): Rt=2.3 min;
  • LC-MS (Method 1): Rt=1.13 min; MS (ESIpos): m/z=1178 (M+H)+.
  • Subsequently, the Z protecting group was cleaved by hydrogenolytic means in methanol over 10% palladium/activated carbon. 9.5 mg (0.0087 mmol) of the deprotected crude product were then, without further purification, taken up in 5 ml of DMF and admixed with 5 mg (26.2 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4 mg (26.2 μmol) of 1-hydroxy-1H-benzotriazole hydrate, 54.6 μl of N,N-diisopropylethylamine and with 9.1 mg (34.9 μmol) of commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The mixture was stirred at RT for 1 h and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. After lyophilization from dioxane, 9.5 mg (84% of theory) of the Boc-protected intermediate were obtained.
  • HPLC (Method 12): Rt=2.1 min;
  • LC-MS (Method 1): Rt=0.97 min; MS (ESIpos): m/z=1295 (M+H)+.
  • Subsequently, 9.5 mg (7.3 μmol) were deprotected with 0.5 ml of trifluoroacetic acid in 2 ml of dichloromethane of the Boc-protected intermediate and, after lyophilization from dioxane, 9 mg (82% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 12): Rt=2.1 min;
  • LC-MS (Method 1): Rt=0.84 min; MS (ESIpos): m/z=1195 (M+H)+.
    • Intermediate 140 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-1-methylhydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00578
  • 4.1 mg (12 μmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N′-methylhexanehydrazide trifluoroacetate (Intermediate 22) were dissolved together with 6.9 mg (8 μmol) of the compound from Intermediate 61 in 2.5 ml of DMF and then admixed with 3.5 mg (9 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 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 means of preparative HPLC. After lyophilization from dioxane, 2.6 mg (30% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.90 and 0.91 min; MS (ESIpos): m/z=1120 (M+H)+.
    • Intermediate 141 N-[4-({1-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]piperidin-4-yl}oxy)-4-oxobutyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00579
  • 44 mg (49 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 2 ml of dichloromethane and then admixed with 18.8 mg (98 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 3.8 mg (24 μmol) of 4-dimethylaminopyridine and with 23 mg (98 μmol) of commercially available benzyl 4-hydroxypiperidine-1-carboxylate. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. Thus, 22 mg (40% of theory) of the Z-protected intermediate were obtained as a colourless foam.
  • HPLC (Method 5): Rt=2.1 min;
  • LC-MS (Method 1): Rt=1.04 min; MS (ESIpos): m/z=1116 (M+H)+.
  • Subsequently, the Z protecting group was cleaved by hydrogenolytic means in ethanol over palladium/activated carbon.
  • 19 mg (19 μmol) of the deprotected crude product were then, without further purification, taken up in 4 ml of DMF and admixed with 7 mg (39 μmol) of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid, 11 mg (29 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 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 means of preparative HPLC. After lyophilization from dioxane, 7.5 mg (34% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.94 min; MS (ESIpos): m/z=1147 (M+H)+.
    • Intermediate 142 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00580
  • 9 mg (9.5 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-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]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 72) were dissolved in 1000 μl of DMF and then admixed with 10 mg (38 μmol) of commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide, 7.2 mg (19 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 8 μl of N,N-diisopropylethylamine, and the reaction mixture was stirred at RT for 1 h. Subsequently, the solvent was removed in vacuo and the remaining residue was purified by means of preparative HPLC. The corresponding fractions were concentrated and, by lyophilization, 6.4 mg (58% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.99 min; MS (ESIpos): m/z=1154 (M+H)+.
    • Intermediate 143 N-(4-{2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00581
  • 6 mg (6.7 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 61) were reacted with 3 mg (8.7 μmol) of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanehydrazide trifluoroacetate in analogy to Intermediate 142 to yield 2 mg (27% of theory) of the title compound.
  • HPLC (Method 12): Rt=2.1 min;
  • LC-MS (Method 3): Rt=1.92 min; MS (ESIpos): m/z=1106 (M+H)+.
    • Intermediate 144 N-(4-{2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00582
  • To a solution of 5 mg (5.6 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide in 1 ml of DMF were added 7.65 mg (22.5 μmol) of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanehydrazide 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, a further 0.95 mg (2.8 μmol) of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanehydrazide trifluoroacetate were added. After stirring overnight, the reaction mixture was concentrated and purified by means of preparative HPLC. 3.5 mg (85% purity, 48% of theory) of the title compound were obtained.
  • LC-MS (Method 3): Rt=1.86 min; m/z=1094 (M+H)+.
    • Intermediate 145 N-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00583
  • 12 mg (14 μmol) of N-(3-aminopropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 66) were taken up in 750 μl of dioxane and admixed with 1.5 ml of saturated sodium hydrogencarbonate solution and then with 3.2 mg (21 μmol) of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate. The reaction mixture was stirred at RT for 1 h and then concentrated under reduced pressure. The remaining residue was purified by means of preparative HPLC. After lyophilization, 4.2 mg (32% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.94 min; MS (ESIpos): m/z=950 (M+H)+.
    • Intermediate 146 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(2S)-1-[benzyl(methyl)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-valinamide
  • Figure US20140127240A1-20140508-C00584
  • 9 mg (9.8 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(2S)-1-[benzyl(methyl)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-valinamide (Intermediate 73) were reacted in analogy to Intermediate 133 with 10 mg (39 μmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide to yield 1.8 mg (15% of theory) of the title compound.
  • HPLC (Method 12): Rt=2.2 min;
  • LC-MS (Method 9): Rt=5.11 min; MS (ESIpos): m/z=1128 (M+H)+.
    • Intermediate 147 N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3S)-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-valinamide
  • Figure US20140127240A1-20140508-C00585
  • 16 mg (17 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3S)-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-valinamide (Intermediate 70) were dissolved in 2 ml of dichloromethane and admixed 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. After stirring at RT for 2 h, the same amounts of 1-hydroxypyrrolidine-2,5-dione and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added once again. Then stirring at RT overnight, the reaction mixture was concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization, 10 mg (56% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=2.0 min;
    • Intermediate 148 N-{4-[(2-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl](methyl)amino}ethyl)amino]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00586
  • 6 mg (7 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 61) were combined with 2.8 mg (8 μmol) of N-(2-aminoethyl)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylbutanamide trifluoroacetate, 10.1 mg (27 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 5 μl of N,N-diisopropylethylamine in 2 ml of DMF and stirred at RT overnight. Then another 5 mg (23.5 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 3 μl of N,N-diisopropylethylamine were added. After stirring at RT for a further 5 h, the solvent was removed in vacuo, and the remaining residue was purified by means of preparative HPLC. The corresponding fractions were concentrated and, by lyophilization from dioxane, 1.3 mg (15% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=2.1 min;
  • LC-MS (Method 2): Rt=1.21 min; MS (ESIpos): m/z=1120 (M+H)+.
    • Intermediate 149 N-{4-[(2-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]amino}ethyl)(methyl)amino]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00587
  • 6 mg (7 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 61) were combined with 3.1 mg (9 μmol) of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[2-(methylamino)ethyl]butanamide trifluoroacetate, 10.1 mg (27 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and 5 μl of N,N-diisopropylethylamine in 2 ml of DMF, and the mixture was stirred at RT for 4 h. Then the solvent was removed in vacuo, and the remaining residue was purified by means of preparative HPLC. The corresponding fractions were concentrated and, by lyophilization from dioxane, 1 mg (13.4% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=2.1 min;
  • LC-MS (Method 1): Rt=0.89 min; MS (ESIpos): m/z=1121 (M+H)+.
    • Intermediate 150 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S,2R)-2-phenyl-1-(propylcarbamoyl)cyclopropyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00588
  • 7.9 mg (9 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S,2R)-2-phenyl-1-(propylcarbamoyl)cyclopropyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 3 ml of DMF and then admixed with 10.4 mg (54 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 8.3 mg (54 μmol) of 1-hydroxy-1H-benzotriazole hydrate, 9 μl of N,N-diisopropylethylamine and with 9.5 mg (36 μmol) of commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. Thus, 4.3 mg (22% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 6): Rt=1.9 min;
  • LC-MS (Method 9): Rt=4.93 min; MS (ESIpos): m/z=1078 (M+H)+.
    • Intermediate 151 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-carbamoyl-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00589
  • The compound was prepared in analogy to Intermediate 150 starting from the compound in Intermediate 81.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=1036 (M+H)+.
    • Intermediate 152 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-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-valinamide
  • Figure US20140127240A1-20140508-C00590
  • 10 mg (12 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-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-valinamide were dissolved in 3 ml of DMF and then admixed with 8.9 mg (23 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 10 μl of N,N-diisopropylethylamine and with 12 mg (47 μmol) of commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The mixture was stirred at RT for 1 h. This was followed by concentration under high vacuum and purification of the remaining residue by means of preparative HPLC. Thus, 5.8 mg (37% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 6): Rt=2.0 min;
  • LC-MS (Method 9): Rt=4.99 min; MS (ESIpos): m/z=1066 (M+H)+.
    • Intermediate 153 N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,14-diazaoctadecan-8-yl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00591
  • To a solution of 5 mg (5.6 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide in 1 ml of DMF were added 9.7 mg (22.5 μmol) of 3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)propanehydrazide trifluoroacetate, 3.2 mg (16.9 μmol) of 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, another 1.2 mg (2.8 μmol) of 3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)propanehydrazide trifluoroacetate were added. The reaction mixture was stirred at RT overnight and then purified by means of preparative HPLC.
  • 3.6 mg (51% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.90 min; m/z=1185 (M+H)+.
    • Intermediate 154 (2R,3S)-3-amino-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutan-2-yl-(3R,4S,7S,10S)-4-[(2S)-butan-2-yl]-7,10-diisopropyl-3-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazapentadecan-15-oate
  • Figure US20140127240A1-20140508-C00592
  • 15 mg (17 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were dissolved in 10 ml of dichloromethane and then admixed with 12.8 mg (67 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 10 mg (83 μmol) of 4-dimethylaminopyridine and with 10.3 mg (33 μmol) of commercially available benzyl N-(tert-butoxycarbonyl)-L-threoninate. The mixture was heated to reflux for 4 h. Then the same amounts of coupling reagent and 4-dimethylaminopyridine were added again, and the reaction mixture was heated overnight with reflux. Subsequently, the reaction mixture was diluted with dichloromethane and extracted by shaking once with water, the organic phase was removed and concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. Thus, 7.7 mg (37% of theory) of the protected intermediate were obtained as a colourless foam.
  • HPLC (Method 12): Rt=2.5 min;
  • LC-MS (Method 1): Rt=1.13 min; MS (ESIpos): m/z=1190 (M+H)+.
  • Subsequently, the benzyl ester protecting group was removed by hydrogenation under standard hydrogen pressure in methanol over 10% palladium/activated carbon, and the acid thus obtained, as described in Intermediate 151, was coupled to 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. In a last step, the Boc protecting group was detached with trifluoroacetic acid. The remaining residue was purified by means of preparative HPLC. Thus, 0.22 mg (2.5% of theory over 3 stages) of the title compound was obtained as a colourless foam.
  • HPLC (Method 12): Rt=2.0 min;
  • LC-MS (Method 1): Rt=0.81 min; MS (ESIpos): m/z=1207 (M+H)+.
    • Intermediate 155 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-{(2S)-2-[(1R,2R)-3-{[(2S)-1-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-valinamide
  • Figure US20140127240A1-20140508-C00593
  • This compound was prepared in analogy to the synthesis described in Intermediate 152 from N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-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-valinamide and commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.82 min; MS (ESIpos): m/z=1024 (M+H)+.
    • Intermediate 156 N-(3-{[(1-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}cyclopropyl)carbonyl]amino}propyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00594
  • This compound was prepared in analogy to the synthesis described in the last stage of Intermediate 131 from N-(3-aminopropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide and 1,1′-[cyclopropane-1,1-diylbis(carbonyloxy)]dipyrrolidine-2,5-dione, which had been obtained from the corresponding dicarboxylic acid beforehand.
  • HPLC (Method 12): Rt=2.0 min;
  • LC-MS (Method 1): Rt=0.92 min; MS (ESIpos): m/z=1080 (M+H)+.
    • Intermediate 157 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00595
  • 15 mg (18 μmol) of (N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5)-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-valinamide were dissolved in 3.8 ml of DMF and then admixed with 27 mg (70 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 12 μl of N,N-diisopropylethylamine and with 14 mg (53 μmol) of commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The reaction mixture was stirred at RT for 1 h. This was followed by concentration under high vacuum and purification of the remaining residue by means of preparative HPLC. Thus, 6.2 mg (33% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.85 min; MS (ESIpos): m/z=1063 (M+H)+.
  • 1H-NMR (500 MHz, DMSO-d6, characteristic signals): 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-({(2R)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-methyl-1-oxopentan-2-yl}amino)-4-oxobutyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide
  • Figure US20140127240A1-20140508-C00596
  • 13 mg (14.7 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide were dissolved in 4 ml of dimethylformamide and then admixed with 9.4 mg (25 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 6 μl of N,N-diisopropylethylamine and with 7 mg (31 μmol) of commercially available tert-butyl-D-leucinate hydrochloride. The mixture was stirred at RT for 5 h and then concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization from dioxane/water, 6.5 mg (49% of theory) of the protected intermediate were obtained as a colourless foam.
  • HPLC (Method 5): Rt=2.2 min;
  • LC-MS (Method 1): Rt=1.21 min; MS (ESIpos): m/z=1076 (M+H)+.
  • Trifluoroacetic acid in dichloromethane was first used to cleave the Boc protecting group from this protected intermediate, yielding 6.2 mg (99% of theory) of the deprotected compound. 5.2 mg (5 μmol) of this intermediate were taken up in 1.5 ml of dichloromethane and reacted with 0.8 mg (7 μmol) of N-hydroxysuccinimide, in the presence of 1.2 mg (6 μmol) of 1-(3-dimethylaminopropyl)-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 by means of preparative HPLC. 1.3 mg of the title compound were obtained, some of which was hydrolysed into an educt.
    • Intermediate 159 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide
  • Figure US20140127240A1-20140508-C00597
  • This compound was prepared in analogy to the synthesis described in Intermediate 157 from N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide and commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
  • Yield: 6 mg (53% of theory)
  • HPLC (Method 5): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.94 min; MS (ESIpos): m/z=1114 (M+H)+.
    • Intermediate 160 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-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-valinamide
  • Figure US20140127240A1-20140508-C00598
  • This compound was prepared in analogy to the synthesis described in Intermediate 157 from 20 mg (21 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-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-valinamide and commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
  • Yield: 13 mg (52% of theory)
  • HPLC (Method 5): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.92 min; MS (ESIpos): m/z=1153 (M+H)+.
    • Intermediate 161 N-(6-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-6-oxohexyl)-N-methyl-L-valyl-N-[(3R,4S,5)-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-valinamide
  • Figure US20140127240A1-20140508-C00599
  • This compound was prepared in analogy to the synthesis described in Intermediate 157 from N-(5-carboxypentyl)-N-methyl-L-valyl-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-valinamide and commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
  • Yield: 0.8 mg (16% of theory)
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.78 min; MS (ESIpos): m/z=1092 (M+H)+.
    • Intermediate 162 N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00600
  • 18 mg (20 μmol) of N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 64) were dissolved in 3.2 ml of dichloromethane and admixed with 22 mg (190 mmol) of 1-hydroxypyrrolidine-2,5-dione and then with 11 mg (60 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.24 mg (0.17 μmol) of DMAP. After stirring at RT for 2 h, another 22 mg (190 mmol) of 1-hydroxypyrrolidine-2,5-dione, 11 mg (60 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.24 mg (0.17 μmol) of DMAP were added, and the reaction mixture was stirred at RT for another hour. This was followed by concentration in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization, 8.2 mg (41% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=2.0 min;
  • LC-MS (Method 11): Rt=0.9 min; MS (ESIpos): m/z=1024 (M+H)+.
    • Intermediate 163 [(1S,2R)-1-amino-2-phenylcyclopropyl](1,4-dihydro-3H-2,3-benzoxazin-3-yl)methanone trifluoroacetate
  • Figure US20140127240A1-20140508-C00601
  • First, starting with 265 mg (0.82 mmol) of tert-butyl (1S,2R)-1-(hydroxycarbamoyl)-2-phenylcyclopropyl carbamate (Starting Compound 7), and by reaction with 1,2-bis(bromomethyl)benzene analogously to a literature method (see H. King, J. Chem. Soc. 1942, 432), the Boc-protected intermediate tert-butyl-[(1S,2R)-1-(1,4-dihydro-3H-2,3-benzoxazin-3-ylcarbonyl)-2-phenylcyclopropyl]carbamate was prepared.
  • Yield: 108 mg (34% of theory)
  • LC-MS (Method 2): Rt=1.3 min; MS (ESIpos): m/z=395 (M+H)+.
  • 108 mg (0.27 mmol) of this intermediate were taken up in 3.7 ml of dichloromethane, 1.8 ml of trifluoroacetic acid were added, and the mixture was stirred at RT for 15 min. This was followed by concentration in vacuo and lyophilization of the remaining residue from dioxane. 112 mg of the title compound were obtained in quantitative yield as a colourless foam.
  • LC-MS (Method 1): Rt=0.7 min; MS (ESIpos): m/z=295 (M+H)+.
    • Intermediate 164 N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-(1,4-dihydro-3H-2,3-benzoxazin-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00602
  • 166 mg (0.196 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 10) were taken up in 40 ml of DMF and admixed successively with 80 mg (0.196 mmol) of [(1S,2R)-1-amino-2-phenylcyclopropyl](1,4-dihydro-3H-2,3-benzoxazin-3-yl)methanone trifluoroacetate (Intermediate 163), 112 mg (0.294 mmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and 682 μl (3.9 mmol) of N,N-diisopropylethylamine. The mixture was subsequently stirred at RT overnight. The reaction mixture was then concentrated in vacuo, the residue was taken up in ethyl acetate, and the solution was washed with saturated aqueous sodium chloride solution. The organic phase was dried over magnesium sulphate, filtered and concentrated. The residue was finally purified by means of preparative HPLC. In this way, 19 mg (9% of theory) of the Fmoc-protected intermediate N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-(1,4-dihydro-3H-2,3-benzoxazin-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-valinamide were obtained.
  • HPLC (Method 5): Rt=1.68 min;
  • LC-MS (Method 1): Rt=1.51 min; MS (ESIpos): m/z=1083 (M+H)+.
  • 19 mg (0.015 mmol) of this intermediate were dissolved in 4 ml of DMF. After adding 817 μl of piperidine, the reaction mixture was stirred at RT for 5 min. This was followed by concentration in vacuo, and the residue was first digested with diethyl ether and then purified by means of preparative HPLC (eluent: acetonitrile+0.1% TFA/0.1% aq. TFA). The corresponding fractions were combined, the solvent was removed in vacuo, and then the residue was lyophilized from dioxane/water. 12 mg (92% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 6): Rt=2.0 min;
  • LC-MS (Method 1): Rt=0.94 min; MS (ESIpos): m/z=861 (M+H)+.
    • Intermediate 165 N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-(1,4-dihydro-3H-2,3-benzoxazin-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-valinamide
  • Figure US20140127240A1-20140508-C00603
  • 20 mg (0.021 mmol) of Intermediate 164 were used, in analogy to the preparation of Intermediate 97, together with benzyl-(6-oxohexyl)carbamate in the presence of sodium cyanoborohydride and with subsequent hydrogenolytic cleaving of the Z protecting group (using 5% palladium on carbon as catalyst, in methanol as a solvent), to prepare the title compound.
  • Yield: 4.5 mg (23% of theory over 2 stages)
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.9 min; MS (ESIpos): m/z=960 (M+H)+.
    • Intermediate 166 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-(1,4-dihydro-3H-2,3-benzoxazin-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-valinamide
  • Figure US20140127240A1-20140508-C00604
  • 4.4 mg (4.5 μmol) of Intermediate 165 were taken up in 1 ml of 1:1 dioxane/water and then admixed with 1 mg (6.8 μmol) of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate and with 50 μl of saturated aqueous sodium hydrogencarbonate solution. The reaction mixture was stirred at RT for 30 min. Then another 50 μl of the saturated aqueous sodium hydrogencarbonate solution were added, and the reaction mixture was stirred at RT for a further 15 min and then concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization, 1 mg (21% of theory) of the title compound were obtained as a colourless foam.
  • HPLC (Method 12): Rt=2.1 min;
  • LC-MS (Method 1): Rt=1.08 min; MS (ESIpos): m/z=1040 (M+H)+.
    • Intermediate 167 Benzyl 3-{2-[2-(2-oxoethoxy)ethoxy]ethoxy}propanoate
  • Figure US20140127240A1-20140508-C00605
  • The title compound was prepared from 6 g (21.55 mmol) of commercially available 3-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}propanoic acid under standard conditions, first by esterification with benzyl chloride and caesium carbonate and subsequent oxidation with sulphur trioxide-pyridine complex.
  • Yield: 611 mg (10% of theory over 2 stages)
  • LC-MS (Method 2): Rt=1.69 min; MS (ESIpos): m/z=311 (M+H)+.
    • Intermediate 168 N-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00606
  • First, in analogy to the synthesis described in Intermediate 69, by coupling of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 4) and Nα-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-tryptophanamide trifluoroacetate (Intermediate 49) in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent cleaving of the Fmoc protecting group by means of piperidine, the amine compound N-methyl-L-valyl-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-valinamide was prepared as the trifluoroacetate.
  • 25 mg (0.028 mmol) of this compound and 17.5 mg (0.06 mmol) of Intermediate 167 were combined in 2 ml of methanol and admixed 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 overnight. Then, the same amounts of borane-pyridine complex and acetic acid were added once more, and the reaction mixture was stirred at RT for another 24 h. This was followed by concentration in vacuo, and the residue was purified by means of preparative HPLC. After concentration of the corresponding fractions and lyophilization from 1:1 dioxane/water, 26.5 mg (88% of theory) of the Z-protected title compound were obtained.
  • HPLC (Method 12): Rt=2.04 min;
  • LC-MS (Method 1): Rt=0.97 min; MS (ESIpos): m/z=1064 (M+H)+.
  • 25 mg (0.024 mmol) of this intermediate were taken up in 10 ml of methanol and hydrogenated over 10% palladium on activated carbon under standard hydrogen pressure at RT for 45 min. The catalyst was then filtered off, and the solvent was removed in vacuo. After lyophilization from dioxane, 19.7 mg (85% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.83 min; MS (ESIpos): m/z=974 (M+H)+.
    • Intermediate 169 N-{2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00607
  • 10 mg (10 μmol) of Intermediate 168 were dissolved in 3 ml of DMF and admixed with 3.5 mg (30 mmol) of 1-hydroxypyrrolidine-2,5-dione and then with 2.4 mg (10 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 5 μl of N,N-diisopropylethylamine. After stirring at RT for 20 h, 8 mg (0.02 mmol) of HATU were added, and the reaction mixture was stirred once again at RT overnight and then concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization from dioxane, 8.6 mg (64% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 11): Rt=0.81 min; MS (ESIpos): m/z=1071 (M+H)+.
    • Intermediate 170 N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00608
  • This compound was prepared in analogy to Intermediate 101 over 2 stages, starting from 26 mg (0.028 mmol) of Intermediate 15.
  • Yield: 16.7 mg (63% of theory over 2 stages)
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.81 min; MS (ESIpos): m/z=914 (M+H)+.
    • Intermediate 171 N-(6-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]amino}hexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00609
  • 6.7 mg (7.3 μmol) of the compound from Intermediate 170 and 3 mg (14.7 μmol) of commercially available 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid were taken up in 2 ml of DMF and admixed with 5.6 mg (14.7 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and 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 by means of preparative HPLC. The corresponding fractions were combined, the solvent was removed in vacuo, and then the residue was lyophilized from dioxane. Thus, 4.5 mg (56% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=2.0 min;
  • LC-MS (Method 1): Rt=1.12 min; MS (ESIpos): m/z=1079 (M+H)+.
    • Intermediate 172 Benzyl 2-{2-[2-(2-oxoethoxy)ethoxy]ethoxy}ethyl)carbamate
  • Figure US20140127240A1-20140508-C00610
  • The title compound was prepared from commercially available 2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethanol under standard conditions by first introducing the Z protecting group and then oxidizing with sulphur trioxide-pyridine complex.
  • HPLC (Method 12): Rt=1.4 min;
  • LC-MS (Method 11): Rt=0.65 min; MS (ESIpos): m/z=326 (M+H)+.
    • Intermediate 173 Benzyl {2-[2-(2-oxoethoxy)ethoxy]ethyl}carbamate
  • Figure US20140127240A1-20140508-C00611
  • The title compound was prepared in analogy to Intermediate 172 from commercially available 2-[2-(2-aminoethoxy)ethoxy]ethanol under standard conditions by first introducing the Z protecting group and then oxidizing with sulphur trioxide-pyridine complex.
  • HPLC (Method 12): Rt=1.3 min;
  • LC-MS (Method 11): Rt=0.68 min; MS (ESIpos): m/z=282 (M+H)+.
    • Intermediate 174 N-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenyl cyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00612
  • 47 mg (0.05 mmol) of Intermediate 16 were reductively aminated in analogy to the preparation of Intermediate 167 with benzyl-(2-{2-[2-(2-oxoethoxy)ethoxy]ethoxy}ethyl)carbamate in the presence of borane-pyridine complex. Subsequently, the Z protecting group was removed by hydrogenolytic means with 5% palladium on carbon as a catalyst and in methanol as a solvent, and 38 mg (66% of theory over 2 stages) of the title compound were prepared.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.8 min; MS (ESIpos): m/z=988 (M+H)+.
    • Intermediate 175 N-[2-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)ethyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00613
  • The preparation was done in analogy zu Intermediate 166 starting from 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): Rt=0.97 min; MS (ESIpos): m/z=1068 (M+H)+.
    • Intermediate 176 N-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00614
  • The preparation was done in analogy to Intermediates 174 and 175 starting with the reductive amination of Intermediate 16 with Intermediate 173, subsequent deprotection and formation of the maleimide.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 11): Rt=0.8 min; MS (ESIpos): m/z=981 (M+H)+.
    • Intermediate 177 N-[2-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)ethyl]-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00615
  • The preparation was done in analogy to Intermediates 174 and 175 starting with the reductive amination of Intermediate 16 with Intermediate 172, subsequent deprotection and formation of the maleimide.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=1025 (M+H)+.
    • Intermediate 178 N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00616
  • The preparation was done in analogy to Intermediate 162 starting from 6 mg of Intermediate 82.
  • LC-MS (Method 1): Rt=0.82 min; MS (ESIpos): m/z=953 (M+H)+.
    • Intermediate 179 4-[(1E,3S)-3-amino-4-phenylbut-1-en-1-yl]benzenesulphonic acid trifluoroacetate
  • Figure US20140127240A1-20140508-C00617
  • A mixture of 13.6 mg (0.06 mmol) of palladium(II) acetate, 469 mg (1.46 mmol) of potassium 4-iodobenzenesulphonate, 300 mg (1.21 mmol) of (S)-tert-butyl-1-phenylbut-3-en-2-yl-carbamate, 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 for 15 min to 160° C. The crude product was subsequently purified directly by means of preparative HPLC. This yielded 312 mg of a mixture of 31% of the BOC-protected compound and 69% of the free amine.
  • This mixture was subsequently taken up in 30 ml of dichloromethane, admixed with 1 ml of trifluoroacetic acid and stirred at RT for 20 h. After concentrating in vacuo, the residue was stirred in with diethyl ether, and the precipitate that formed was suctioned off and washed with diethyl ether. This yielded 200 mg (62% of theory) of the title compound.
  • LC-MS (Method 11): Rt=0.44 min; MS (ESIpos): m/z=304 (M+H)+.
    • Intermediate 180 4-[(3R)-3-amino-4-phenylbutyl]benzenesulphonic acid
  • Figure US20140127240A1-20140508-C00618
  • 100 mg (0.25 mmol) of 4-[(1E,3S)-3-amino-4-phenylbut-1-en-1-yl]benzenesulphonic acid trifluoroacetate were suspended in 10 ml of acetic acid and a few drops of DMF and water, admixed with 70 mg (0.07 mmol) of palladium on carbon (10%) and hydrogenated at hydrogen pressure 2.2 bar for 24 h. The solution was filtered and the filtrate purified by means of preparative HPLC.
  • 29 mg (76% purity, 21% of theory) of product were obtained.
  • LC-MS (Method 1): Rt=0.46 min; MS (ESIpos): m/z=306 (M+H)+.
    • Intermediate 181 N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00619
  • To a solution of 90 mg (0.13 mmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide in 4 ml of DMF were added 60 mg (0.16 mmol) of HATU and 69 μl of (0.39 mmol) Hünig's base. The reaction mixture was stirred at RT for 30 min and then admixed with 60 mg (0.15 mmol) 60.3 mg (0.13 mmol) of 4-[(1E,3S)-3-amino-4-phenylbut-1-en-1-yl]benzenesulphonic acid trifluoroacetate. After stirring overnight, the reaction mixture was purified by means of preparative HPLC. This yielded 127 mg of a 44:56 mixture of the title compound and of the already deprotected amine.
  • LC-MS (Method 1): Rt=1.21 min; MS (ESIpos): m/z=971 (M+H)+; Rt=0.84 min; MS (ESIpos): m/z=871 (M+H)+ for the deprotected compound.
    • Intermediate 182 N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00620
  • 90 mg of Intermediate 180 were dissolved in 4.6 ml of dichloromethane, and 0.92 ml of trifluoroacetic acid were added. The reaction mixture was stirred at RT for 30 min and then concentrated. The obtained crude product was purified by means of preparative HPLC.
  • 91 mg (98% of theory) of the target compound were obtained.
  • LC-MS (Method 1): Rt=0.85 min; MS (ESIpos): m/z=871 (M+H)+
    • Intermediate 183 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00621
  • 16.7 μl (0.03 mmol) of a 15% aqueous succinaldehyde solution were initially provided in 943 μl of methanol and admixed with 17 mg (0.02 mmol) of N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate (Intermediate 181) and 1.1 μl (0.02 mmol) of acetic acid. The reaction mixture was stirred for 5 min at RT, and then 2.9 μl (0.02 mmol) of borane-pyridine complex were added. After 1 h, a further 2 equivalents 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 means of preparative HPLC.
  • This yielded 20 mg (83% purity, 80% of theory) of the title compound.
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=957 (M+H)+
    • Intermediate 184 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00622
  • 8 mg (7.5 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide, 2.8 mg (8.2 μmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide trifluoroacetate, 3.4 mg (9 μmol) of HATU 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 means of preparative HPLC.
  • 3 mg (31% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.90 min; MS (ESIpos): m/z=1164 (M+H)+
    • Intermediate 185 N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00623
  • To a solution of 8 mg (7.5 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide in 2 ml of DMF were added 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. The reaction mixture was stirred at RT for 20 h. Subsequently, 1.3 μl (7.5 μmol) of Hünig's base were added, and the mixture was stirred for another 1 h. The reaction mixture was then purified by means of preparative HPLC. 2.6 mg (72% purity, 21% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.89 min; MS (ESIpos): m/z=1054 (M+H)+
    • Intermediate 186 N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00624
  • To a solution of 43 mg (0.06 mmol) of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide in 1.9 ml of DMF were added 29 mg (0.07 mmol) of HATU and 33 μl (0.19 mmol) of Hünig's base. The reaction mixture was stirred at RT for 30 min and then admixed with 29 mg (0.07 mmol) of 4-[(3R)-3-amino-4-phenylbutyl]benzenesulphonic acid trifluoroacetate. After stirring overnight, the reaction mixture was purified by means of preparative HPLC. This yielded 58 mg of a 45:55 mixture of the title compound and of the already deprotected amine.
  • LC-MS (Method 1): Rt=1.09 min; MS (ESIpos): m/z=973 (M+H)+; Rt=0.87 min; MS (ESIpos): m/z=873 (M+H)+ for the deprotected compound.
    • Intermediate 187 N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00625
  • 58 mg of Intermediate 186 were dissolved in 4.1 ml of dichloromethane, 0.41 ml of trifluoroacetic acid were added, and the mixture was stirred at RT for 30 min. After concentration in vacuo, the crude product was purified by means of preparative HPLC.
  • 50 mg (90% purity, 85% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=873 (M+H)+
    • Intermediate 188 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00626
  • 1711 (0.26 mmol) of a 15% aqueous succinaldehyde solution were initially provided in 2.5 ml of methanol and admixed with 50 mg (0.05 mmol) of N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate and 11.6 μl (0.2 mmol) of acetic acid. The reaction mixture was stirred for 5 min at RT, and then 30 al (0.24 mmol) of borane-pyridine complex were added. After stirring for 24 hours, another equivalent of borane-pyridine complex was added, and the mixture was stirred for another 2 h. The reaction mixture was then purified by means of preparative HPLC.
  • 40 mg (90% purity, 66% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.91 min; MS (ESIpos): m/z=959 (M+H)+
    • Intermediate 189 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00627
  • 10 mg (9.3 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide, 3.5 mg (10.3 μmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide trifluoroacetate, 4.3 mg (11.2 μmol) of HATU and 4.9 μl (28 μmol) of Hünig's base were stirred in 1 ml of DMF at RT for 20 h. Subsequently, the reaction mixture was purified by means of preparative HPLC.
  • 4.2 mg (92% purity, 33% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.91 min; MS (ESIpos): m/z=1166 (M+H)+
    • Intermediate 190 N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00628
  • To a solution of 10 mg (9.3 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide in 2.5 ml of DMF were added 10.7 mg (93 μmol) of N-hydroxysuccinimide, 10.6 mg (28 μmol) of EDCI and 0.12 mg (0.9 μmol) of DMAP. The reaction mixture was stirred at RT for 20 h and then purified by means of preparative HPLC.
  • 3.8 mg (72% purity, 25% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.90 min; MS (ESIpos): m/z=1055 (M+H)+
    • Intermediate 191 (2R,3R)—N-[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00629
  • The title compound was prepared in analogy to the synthesis of Intermediate 7 over two stages from Starting Compound 1 and (2S)-2-amino-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)propan-1-one trifluoroacetate (Intermediate 99).
  • Yield over 2 stages: 62 mg (67% of theory)
  • HPLC (Method 6): Rt=1.65 min;
  • LC-MS (Method 1): Rt=0.7 min; MS (ESIpos): m/z=443 (M+H)+.
    • Intermediate 192 N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00630
  • 1015 mg (1.59 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 4) were taken up in 50 ml of DMF, admixed with 654 mg (2.39 mmol) of 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP) and 2.8 ml of N,N-diisopropylethylamine, then stirred at RT for 10 min. Then 1083 mg (1.75 mmol) of (2R,3R)—N-[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanamide trifluoroacetate (Intermediate 191) were added, and then the mixture was treated in an ultrasound bath at RT for 30 min. The reaction mixture was then concentrated in vacuo, and the residue was taken up in 300 ml of ethyl acetate. The organic phase was washed successively with 5% aqueous citric acid solution and 5% aqueous sodium hydrogencarbonate solution, dried over magnesium sulphate, filtered and concentrated. The crude product thus obtained (1684 mg) was, without further purification, taken up in 20 ml of acetonitrile, 2 ml of piperidine were added to this, and the reaction mixture was then stirred at RT for 10 min. Then the mixture was concentrated in vacuo, and the residue was admixed with diethyl ether. The solvent was again concentrated by evaporation, and the residue was purified by flash chromatography on silica gel (eluent: 15:1:0.1->15:2:0.2 dichloromethane/methanol/17% aqueous ammonia solution). The corresponding fractions were combined, the solvent was removed in vacuo, and the residue was lyophilized from acetonitrile/water. Thus, 895 mg (67% over 2 stages) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.84 min; MS (ESIpos): m/z=840 (M+H)+.
  • 1H NMR (500 MHz, DMSO-d6): 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) [further signals hidden under H2O peak].
    • Intermediate 193 N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00631
  • 50 mg (0.052 mmol) of N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide (Intermediate 192) and 204 μl einer of a 15% aqueous solution of 4-oxobutanoic acid were combined in 2 ml of methanol and admixed with 23.4 mg (0.252 mmol) of borane-pyridine complex and 6 μl of acetic acid. The reaction mixture was stirred at RT overnight. This was followed by concentration in vacuo, and the residue was purified by means of preparative HPLC. After concentration of the corresponding fractions, 38 mg (78% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 9): Rt=4.7 min; MS (ESIpos): m/z=926 (M+H)+.
    • Intermediate 194 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00632
  • This compound was prepared in analogy to the synthesis described in Intermediate 157 from 10 mg (11 μmol) of N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide and commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
  • Yield: 4.4 mg (35% of theory)
  • HPLC (Method 5): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.90 min; MS (ESIpos): m/z=1133 (M+H)+.
    • Intermediate 195 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00633
  • This compound was prepared in analogy to Intermediate 166 starting from 9 mg (0.010 mmol) of Intermediate 170.
  • Yield: 1.1 mg (10% of theory)
  • HPLC (Method 12): Rt=2.0 min;
  • LC-MS (Method 1): Rt=0.99 min; MS (ESIpos): m/z=994 (M+H)+.
    • Intermediate 196 (2S)-2-amino-1-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-3-phenylpropan-1-one trifluoroacetate
  • Figure US20140127240A1-20140508-C00634
  • 41 mg (0.37 mmol) of 2,5-dioxopyrrolidin-1-yl N-(tert-butoxycarbonyl)-L-phenylalaninate were taken up in 10 ml of DMF and admixed with 149 mg (0.41 mmol) of 2-oxa-3-azabicyclo[2.2.2]oct-5-ene (Starting Compound 6) and 72 μl (0.41 mmol) of N,N-diisopropylethylamine. The mixture was stirred at RT for 1 h. The solvent was then removed in vacuo, and the residue was taken up in ethyl acetate and extracted by shaking with 5% aqueous citric acid solution and then with 5% aqueous sodium hydrogencarbonate solution. The organic phase was concentrated, and the residue was purified by flash chromatography on silica gel with 10:1 toluene/ethanol as the eluent. The corresponding fractions were combined, and the solvent was removed in vacuo. After the residue had been dried under high vacuum, 69 mg (47% of theory) of the Boc-protected intermediate tert-butyl-[(2S)-1-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-1-oxo-3-phenylpropan-2-yl]carbamate were thus obtained as a diastereomer mixture.
  • LC-MS (Method 1): Rt=1.1 min; MS (ESIpos): m/z=359 (M+H)+.
  • 64 mg (0.18 mmol) of this intermediate were taken up in 10 ml of dichloromethane, 1 ml of trifluoroacetic acid was added, and the mixture was stirred at RT for 30 min. This was followed by concentration in vacuo and lyophilization of the remaining residue from water/dioxane. In this way, 66 mg (quant.) of the title compound were obtained as a foam.
  • HPLC (Method 6): Rt=1.45 min;
  • LC-MS (Method 3): Rt=1.12 min; MS (ESIpos): m/z=259 (M+H)+.
    • Intermediate 197 (2R,3R)-3-methoxy-2-methyl-N-[(2S)-1-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-1-oxo-3-phenylpropan-2-yl]-3-[(2S)-pyrrolidin-2-yl]propanamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00635
  • First, (2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoic acid (Starting Compound 1) was released from 83 mg (0.18 mmol) of its dicyclohexylamine salt by taking it up in ethyl acetate and extractive shaking with 5% aqueous potassium hydrogensulphate solution. The organic phase was dried over magnesium sulphate, filtered and concentrated. The residue was taken up in 10 ml of DMF and admixed successively with 66 mg (0.18 mmol) of (2S)-2-amino-1-(2-oxa-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′-tetramethyluronium hexafluorophosphate (HATU) and 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 means of preparative HPLC. This yielded 52 mg (56% of theory) of the Boc-protected intermediate tert-butyl-(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidine-1-carboxylate.
  • HPLC (Method 6): Rt=2.13 min;
  • LC-MS (Method 1): Rt=1.13 min; MS (ESIpos): m/z=528 (M+H)+.
  • 52 mg (0.1 mmol) of this intermediate were taken up in 10 ml of dichloromethane, 1 ml of trifluoroacetic acid was added, and the mixture was stirred at RT for 20 min. This was followed by concentration in vacuo and stirring of the remaining residue with 20 ml of diethyl ether. After 10 min, the mixture was filtered, and the filter residue was dried under high vacuum. In this way, 39 mg (72% of theory) of the title compound were obtained.
  • HPLC (Method 6): Rt=1.62 min;
  • LC-MS (Method 1): Rt=0.68 min; MS (ESIpos): m/z=428 (M+H)+.
    • Intermediate 198 N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(2-oxa-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00636
  • 44.5 mg (0.071 mmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 4) were taken up in 10 ml of DMF and admixed successively with 38.6 mg (0.071 mmol) of (2R,3R)-3-methoxy-2-methyl-N-[(2S)-1-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-1-oxo-3-phenylpropan-2-yl]-3-[(2S)-pyrrolidin-2-yl]propanamide trifluoroacetate (Intermediate 197), 32.5 mg (0.086 mmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and 41 μl (0.235 mmol) of N,N-diisopropylethylamine. The mixture was stirred at RT for 1 h. The reaction mixture was then concentrated in vacuo, and the residue was taken up in ethyl acetate. The organic phase was washed successively with 5% aqueous citric acid solution and 5% aqueous sodium hydrogencarbonate solution, dried over magnesium sulphate, filtered and concentrated. This yielded 73 mg (98% of theory) of the Fmoc-protected intermediate N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(2-oxa-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-valinamide.
  • HPLC (Method 6): Rt=2.78 min;
  • LC-MS (Method 3): Rt=2.96 min; MS (ESIpos): m/z=1047 (M+H)+.
  • 73 mg (0.071 mmol) of this intermediate were dissolved in 5 ml of DMF. After adding 0.5 ml of piperidine, the reaction mixture was stirred at RT for 10 min. This was followed by concentration in vacuo, and the residue was digested repeatedly with diethyl ether. After the diethyl ether had been decanted off, the residue was purified by means of preparative HPLC (eluent: acetonitrile/0.1% aq. TFA). 16 mg (26% of theory) of the title compound were obtained as a foam.
  • HPLC (Method 6): Rt=1.94 min;
  • LC-MS (Method 3): Rt=1.71 min; MS (ESIpos): m/z=825 (M+H)+1H NMR (400 MHz, DMSO-d6): 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) [further signals hidden under H2O peak].
    • Intermediate 199 N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(2-oxa-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-valinamide
  • Figure US20140127240A1-20140508-C00637
  • The title compound was prepared in analogy to Intermediates 193 and 194 starting from 23 mg (24 μmol) of N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(2-oxa-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-valinamide trifluoroacetate (Intermediate 198).
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 2): Rt=2.1 min; MS (ESIpos): m/z=1118 (M+H)+.
    • Intermediate 200 N-[2-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)ethyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00638
  • The preparation was done in analogy to Intermediates 174 and 175 starting with the reductive alkylation of Intermediate 192 with Intermediate 172, subsequent deprotection and formation of the maleimide.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=1025 (M+H)+.
    • Intermediate 201 N-{6-[(bromoacetyl)amino]hexyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00639
  • 22 mg (0.023 mmol) of N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide (Intermediate 101) were dissolved in 9.5 ml of THF and admixed at 0° C. with 4.2 μl of triethylamine. A solution of bromoacetyl chloride in THF was added dropwise, and the reaction mixture was stirred at 0° C. for 30 min. The reaction mixture was concentrated and the residue was purified by means of preparative HPLC. Thus, 6.9 mg (26% of theory) of the title compound were obtained as a foam.
  • HPLC (Method 5): Rt=1.8 min;
  • LC-MS (Method 11): Rt=0.9 min; MS (ESIpos): m/z=1059 and 1061 (M+H)+.
    • Intermediate 202 N-{2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00640
  • The preparation was at first done in analogy to Intermediate 168 starting with the reductive alkylation of Intermediate 192 with Intermediate 167 and subsequent hydrogenolytic cleavage of the benzyl ester of N-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide.
  • 13 mg (10 μmol) of this intermediate were dissolved in 5 ml of DMF and admixed with 2.1 mg (20 mmol) of 1-hydroxypyrrolidine-2,5-dione, 6.5 μl of N,N-diisopropylethylamine and 7.1 mg (0.02 mmol) of HATU. The reaction mixture was stirred at RT overnight and then concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization from acetonitrile/water, 9.2 mg (62% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=2.0 min;
  • LC-MS (Method 2): Rt=2.1 min; MS (ESIpos): m/z=1141 (M+H)+.
    • Intermediate 203 tert-butyl-(6-hydrazino-6-oxohexyl)carbamate
  • Figure US20140127240A1-20140508-C00641
  • This compound was prepared by standard peptide chemistry methods by coupling of 6-[(tert-butoxycarbonyl)amino]hexanoic acid with benzyl hydrazinecarboxylate in the presence of EDCI and HOBT and subsequent hydrogenolytic cleavage of the benzyloxycarbonyl protecting group.
  • LC-MS (Method 11): Rt=0.59 min; MS (ESIpos): m/z=246 (M+H)+.
    • Intermediate 204 N-{4-[2-(6-aminohexanoyl)hydrazino]-4-oxobutyl}-N-methyl-L-valyl-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00642
  • 146 mg (50 μmol) of (N-(3-carboxypropyl)-N-methyl-L-valyl-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-valinamide were dissolved in 5 ml of DMF and then admixed with 30.6 mg (80 μmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 19 μl of N,N-diisopropylethylamine and with 22.4 mg (60 μmol) of tert-butyl-(6-hydrazino-6-oxohexyl)carbamate. The reaction mixture was stirred at RT for 1.5 h. This was followed by concentration under high vacuum and purification of the remaining residue by means of preparative HPLC. Thus, 43 mg (68% of theory) of the protected intermediate were obtained, which were 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 stirred in with dichloromethane, and the solvent was removed again in vacuo. Thus, 45 mg (68% of theory over 2 stages) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.6 min;
  • LC-MS (Method 11): Rt=0.66 min; MS (ESIpos): m/z=983 (M+H)+.
    • Intermediate 205 N-(4-{2-[6-({[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}amino)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00643
  • This compound was prepared in analogy to Intermediate 114 starting from Intermediates 50 and 204.
  • Yield: 4 mg (78% of theory)
  • HPLC (Method 12): Rt=1.7 min;
  • LC-MS (Method 11): Rt=0.73 min; MS (ESIpos): m/z=1149 (M+H)+.
    • Intermediate 206 N-(6-{[3-({3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl}disulphanyl)propanoyl]amino}hexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00644
  • 8 mg (10 μmol) of Intermediate 101 were dissolved in 2 ml of DMF and admixed with 8.6 mg (20 μmol) of 1,1′-{disulphanediylbis[(1-oxopropane-3,1-diyl)oxy]}dipyrrolidine-2,5-dione and 3.7 μl of N,N-diisopropylethylamine. The reaction mixture was stirred at RT for 2 h, and then the solvent was evaporated off in vacuo, and the residue was purified by means of preparative HPLC. 7.2 mg (68% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.9 min;
  • LC-MS (Method 11): Rt=0.94 min; MS (ESIpos): m/z=615 [½ (M+2H+]
    • Intermediate 207 (1S,2R)-1-amino-2-phenylcyclopropanecarboxylic acid trifluoroacetate
  • Figure US20140127240A1-20140508-C00645
  • The title compound was obtained in quantitative yield by deprotecting 210 mg (0.76 mmol) of commercially available (1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic acid with trifluoroacetic acid.
  • LC-MS (Method 1): Rt=0.23 min; MS (ESIpos): m/z=178 (M+H)+.
    • Intermediate 208 9H-fluoren-9-ylmethyl-(6-oxohexyl)carbamate
  • Figure US20140127240A1-20140508-C00646
  • The title compound was prepared from 1 g (2.95 mmol) of commercially available 9H-fluoren-9-ylmethyl-(6-hydroxyhexyl)carbamate under standard conditions, by oxidation with sulphur trioxide-pyridine complex. 840 mg (85% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=2.0 min;
  • LC-MS (Method 1): Rt=1.1 min; MS (ESIpos): m/z=338 (M+H)+.
    • Intermediate 209 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-carboxy-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00647
  • First prepared was, in analogy to the synthesis described in Intermediate 75, by coupling of N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) and (1S,2R)-1-amino-2-phenylcyclopropanecarboxylic acid trifluoroacetate (Intermediate 207) in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and the subsequent cleaving of the Boc protecting group by means of trifluoroacetic acid, the amine compound N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-carboxy-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide as the trifluoroacetate.
  • To 22 mg (0.026 mmol) of this compound in 10 ml of methanol were then added 17 mg (0.05 mmol) of 9H-fluoren-9-ylmethyl-(6-oxohexyl)carbamate (Intermediate 208) and 2.3 mg of acetic acid, and also 11.4 mg (0.12 mmol) of borane-pyridine complex. The reaction mixture was stirred at RT overnight. Then the same amounts of borane-pyridine complex and acetic acid, and also 8 mg of fluoren-9-ylmethyl-(6-oxohexyl)carbamate were added once again, and the reaction mixture was stirred at RT for a further 24 h. This was followed by concentration in vacuo, and the residue was purified by means of preparative HPLC. After concentration of the corresponding fractions, the product was used immediately in the next stage.
  • 33 mg of the still contaminated intermediate were taken up 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 the obtained residue was purified by means of preparative HPLC. Thus, 11 mg (55% of theory over 2 stages) of the aminocarboxylic acid intermediate were obtained.
  • HPLC (Method 12): Rt=1.7 min;
  • LC-MS (Method 11): Rt=0.7 min; MS (ESIpos): m/z=843 (M+H)+.
  • 6 mg (7.12 μmol) of this intermediate were taken up in 1 ml of dioxane and then admixed with 6.6 mg (42.7 μmol) of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate and with 5 al of saturated aqueous sodium hydrogencarbonate solution. The reaction mixture was stirred at RT for 1 h. Then another 3 portions each of 50 al of the saturated aqueous sodium hydrogencarbonate solution were added, and the reaction mixture was stirred at RT for another 30 min. Then the reaction mixture was acidified to pH 2 with trifluoroacetic acid and subsequently concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization from acetonitrile/water, 4 mg (60% of theory) of the title compound were obtained as a foam.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 11): Rt=0.88 min; MS (ESIpos): m/z=923 (M+H)+.
    • Intermediate 210 N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00648
  • 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-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide (Intermediate 192) and 65.4 mg (0.5 mmol) of 6-oxohexanoic acid were combined in 9 ml of methanol and admixed with 10 μl of acetic acid and 37.4 mg (0.4 mmol) of borane-pyridine complex. The reaction mixture was stirred at RT overnight. This was followed by concentration in vacuo, and the residue was taken up 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 means of preparative HPLC. After concentration of the corresponding fractions, 70 mg (86% of theory) of N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide were obtained as the trifluoroacetate.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=955 (M+H)+.
  • 1H NMR (500 MHz, DMSO-d6 characteristic signals): 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 were dissolved in 1.8 ml of dichloromethane and admixed 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, and the reaction mixture was stirred at RT for 2 h. Subsequently, the reaction mixture was concentrated in vacuo and the remaining residue was purified by means of preparative HPLC. After lyophilization from acetonitrile/water, 21.3 mg (88% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.94 min; MS (ESIpos): m/z=1052 (M+H)+.
    • Intermediate 211 N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00649
  • 15 mg (20 μmol) of N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate (Intermediate 15) were reductively alkylated with 6-oxohexanoic acid, in analogy to Intermediate 210.
  • Yield: 9.2 mg (61% of theory)
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=929 (M+H)+.
  • 9 mg (10 μmol) of this intermediate were dissolved in 3 ml of DMF and admixed with 5.6 mg (48 μmol) of 1-hydroxypyrrolidine-2,5-dione, 5 μl of N,N-diisopropylethylamine and 5.5 mg (0.015 mmol) of HATU, and the reaction mixture was treated in an ultrasound bath for 6 h. In the course of this, 5.5 mg of HATU were re-added every hour. Subsequently, the reaction mixture was concentrated in vacuo, and the residue was taken up in acetonitrile/water and adjusted to pH 2 with trifluoroacetic acid. After concentrating again in vacuo, the remaining residue was purified by means of preparative HPLC. After lyophilization from acetonitrile/water, 5.8 mg (57% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=2.0 min;
  • LC-MS (Method 1): Rt=0.95 min; MS (ESIpos): m/z=1027 (M+H)+.
    • Intermediate 212 N-{2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00650
  • The preparation was at first done in analogy to Intermediate 168 starting with the reductive alkylation of Intermediate 15 with Intermediate 167 and subsequent hydrogenolytic cleavage of the benzyl ester of N-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide.
  • 8.4 mg (8 μmol) of this intermediate were dissolved in 3 ml of DMF and admixed with 9.5 mg (80 μmol) of 1-hydroxypyrrolidine-2,5-dione, 10 μl of N,N-diisopropylethylamine and 9.4 mg (25 μmol) of HATU, and the reaction mixture was stirred at RT overnight and then concentrated in vacuo. Subsequently, the reaction mixture was concentrated in vacuo, and the residue was taken up in acetonitrile/water and adjusted to pH 2 with trifluoroacetic acid. After concentrating again in vacuo, the remaining residue was purified by means of preparative HPLC. After lyophilization from acetonitrile/water, 4 mg (32% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=2.0 min;
  • LC-MS (Method 1): Rt=0.96 min; MS (ESIpos): m/z=1117 (M+H)+.
    • Intermediate 213 N-{6-[(trans-4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}cyclohexyl)amino]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00651
  • This compound was prepared in analogy to Intermediate 104 starting from N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide, the synthesis of which was described under Intermediate 210. 9.3 mg of the title compound (37% of theory over 3 stages) were obtained.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.9 min; MS (ESIpos): m/z=1177 (M+H)+.
    • Intermediate 214 N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00652
  • This compound was prepared in analogy to Intermediate 210 by conversion of Intermediate 92 to the active ester.
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 11): Rt=0.82 min; MS (ESIpos): m/z=901 (M+H)+.
    • Intermediate 215 N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00653
  • First, from Intermediate 40, in analogy to Intermediate 183 with borane-pyridine complex, was prepared N-(5-carboxypentyl)-N-methyl-L-valyl-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-valinamide. From this compound, in analogy to Intermediate 210, the active ester was then generated. 34 mg (36% of theory over 2 stages) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.85 min; MS (ESIpos): m/z=930 (M+H)+.
    • Intermediate 216 N-(4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}benzyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00654
  • First, in analogy to the preparation of Intermediate 183, Intermediate 192 was reacted with 4-formylbenzoic acid with borane-pyridine complex to yield N-(4-carboxybenzyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-[1-(1,2-oxazinan-2-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-valinamide. This compound was then used, in analogy to Intermediate 210, to generate 11 mg (68% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.8 min;
  • LC-MS (Method 1): Rt=1.13 min; MS (ESIpos): m/z=1072 (M+H).
    • Intermediate 217 N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5)-{(2S)-2-[(1R,2R)-3-{[(2S)-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-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00655
  • 53 mg (84 μmol) of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 4) and 45 mg (84 μmol) of benzyl-N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-phenylalaninate trifluoroacetate (Intermediate 12) were taken up in 2 ml of DMF, 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 overnight. Subsequently, the reaction mixture was concentrated and the residue was purified by means of preparative HPLC. This yielded 59 mg (68% of theory) of the Fmoc-protected intermediate N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-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-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide.
  • LC-MS (Method 1): Rt=1.55 min; m/z=1044 (M+H)+.
  • 57 mg (0.055 mmol) of this intermediate were treated with 1.2 ml of piperidine in 5 ml of DMF to cleave the Fmoc protecting group. After concentration and purification by means of preparative HPLC, 39 mg (76% of theory) of the free amine intermediate N-methyl-L-valyl-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-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were obtained as the trifluoroacetate.
  • HPLC (Method 5): Rt=1.9 min;
  • LC-MS (Method 1): Rt=1.01 min; m/z=822 (M+H)+.
  • 60 mg (0.06 mmol) of this intermediate were reacted, in analogy to Intermediate 210, with 6-oxohexanoic acid in the presence of borane-pyridine complex. 45 mg (75% of theory) of the title compound were obtained as a foam.
  • HPLC (Method 5): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.97 min; MS (ESIpos): m/z=9936 (M+H)+.
    • Intermediate 218 N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-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-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00656
  • This compound was prepared by conversion of 42 mg (0.05 mmol) of Intermediate 217 to the active ester.
  • Yield: 26 mg (54%)
  • HPLC (Method 5): Rt=2.1 min;
  • LC-MS (Method 1): Rt=1.01 min; MS (ESIpos): m/z=1034 (M+H)+.
    • Intermediate 219 N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00657
  • 20 mg (0.02 mol) of the compound from Intermediate 218 were taken up in 2.4 ml of methanol and hydrogenated over 5% palladium on activated carbon under standard hydrogen pressure at RT for 30 min. The catalyst was then filtered off, and the solvent was removed in vacuo. The residue was lyophilized from 1:1 acetonitrile/water. This yielded 14 mg (92% of theory) of the title compound as a colourless foam.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): 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-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00658
  • 0.5 g (1.01 mmol) of Intermediate 1 were admixed in 10 ml of dichloromethane with 1 ml of trifluoroacetic acid. After treatment in an ultrasound bath for 30 min, the batch was concentrated and redistilled first with DCM and then with diethyl ether, then dried under high vacuum. The oily residue was used without further purification in the next stage.
  • 500 mg of this intermediate were dissolved in 20 ml of DMF and admixed with 466 mg (3.8 mmol) of Intermediate 191, 382 mg (1.01 mmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and 440 al (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 dichloromethane and extracted by shaking first twice with 5% aqueous citric acid solution and then with saturated aqueous sodium hydrogencarbonate solution. The organic phase was concentrated, and the residue was purified by flash chromatography on silica gel with 95:5 dichloromethane/methanol as the eluent. The corresponding fractions were combined, and the solvent was removed in vacuo. After the residue had been dried under high vacuum, 562 mg (65% of theory over both stages) of the Z-protected intermediate were obtained.
  • 562 mg (0.57 mmol) of this intermediate were taken up in 50 ml of methanol and hydrogenated with 155 mg of 10% palladium on activated carbon under standard hydrogen pressure at RT for 20 min. The catalyst was then filtered off, and the solvent was removed in vacuo. The residue was purified by means of preparative HPLC. The corresponding fractions were combined, the solvent was evaporated in vacuo, and the residue was lyophilized from dioxane. This yielded 361 mg (87% of theory) of the title compound as a foam.
  • HPLC (Method 5): double peak with 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 (ESIpos): m/z=944 (M+H)+.
    • Intermediate 221 N-{(2S)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropyl}-N-methyl-L-valine
  • Figure US20140127240A1-20140508-C00659
  • 100 mg (0.76 mmol) of commercially available N-methyl-L-valine and 285 mg (1.14 mmol) of commercially available tert-butyl (2S)-1-oxo-3-phenylpropan-2-yl carbamate were combined in 22 ml of methanol and admixed with 340 mg (3.66 mmol) of borane-pyridine complex and 701 of acetic acid. The reaction mixture was stirred at RT overnight. This was followed by concentration in vacuo, and the residue was purified by flash chromatography on silica gel with dichloromethane/methanol/17% aqueous ammonia solution as the eluent. After concentration of the corresponding fractions and lyophilization from 1:1 dioxane/water, 259 mg (93% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.6 min;
  • LC-MS (Method 11): Rt=0.76 min; MS (ESIpos): m/z=365 (M+H)+.
    • Intermediate 222 N-[(2S)-2-amino-3-phenylpropyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00660
  • 40 mg (0.11 mmol) of N-{(2S)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropyl}-N-methyl-L-valine (Intermediate 221) were dissolved in 5 ml of DMF and admixed 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-oxazinan-2-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-valinamide (Intermediate 220), 50 mg (0.13 mmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and 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 washed first with 5% aqueous citric acid solution and then with water. The organic phase was concentrated, and the residue was purified by means of preparative HPLC. The corresponding fractions were combined, and the solvent was removed in vacuo. After lyophilization from dioxane, 60 mg (50% of theory) of the protected intermediate were obtained.
  • HPLC (Method 12): Rt=2.2 min;
  • LC-MS (Method 1): Rt=1.17 min; MS (ESIpos): m/z=1073 (M+H)+.
  • 60 mg (0.05 mmol) of this intermediate were taken up in 10 ml of dichloromethane, 2 ml of trifluoroacetic acid were added, and the reaction mixture was stirred at RT for 1.5 h. Subsequently, the reaction mixture was concentrated in vacuo, and the remaining residue was purified by means of preparative HPLC. The corresponding fractions were combined, the solvent was removed in vacuo, and the residue was lyophilized from dioxane/water. In this way, 25 mg (42% of theory) of the title compound were obtained as a foam.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.95 min; MS (ESIpos): m/z=974 (M+H)+.
    • Intermediate 223 N-[(2S)-2-({[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}amino)-3-phenylpropyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00661
  • The preparation was done in analogy to Intermediate 134 starting from 5 mg (4.6 μmol) of Intermediate 222. 3.4 mg (65% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=2.0 min;
  • LC-MS (Method 1): Rt=0.99 min; MS (ESIpos): m/z=1140 (M+H)+.
    • Intermediate 224 N-[(2S)-2-({[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}amino)propyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00662
  • The preparation was done in analogy to the synthesis of Intermediate 223.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.92 min; MS (ESIpos): m/z=1064 (M+H)+.
    • Intermediate 225 N-(2-aminoethyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00663
  • 100 mg (0.76 mmol) of commercially available N-methyl-L-valine and 182 mg (1.14 mmol) of commercially available tert-butyl 2-oxoethyl carbamate were combined in 20 ml of methanol and admixed with 340 mg (3.66 mmol) of borane-pyridine complex and 65 μl of acetic acid. The reaction mixture was stirred at RT overnight. This was followed by concentration under reduced pressure, and the residue was purified by flash chromatography on silica gel with dichloromethane/methanol/17% aqueous ammonia solution (15/4/0.5) as the eluent. After concentration of the corresponding fractions and lyophilization from 1:1 dioxane/water, 190 mg in 39% purity (35% of theory) of the intermediate were obtained, which were converted further without further purification.
  • 50 mg (0.07 mmol) of this intermediate were dissolved in 10 ml of DMF and admixed 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-oxazinan-2-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-valinamide (Intermediate 220), 32 mg (0.09 mmol) of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and 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 by shaking first with 5% aqueous citric acid solution and then with water. The organic phase was concentrated and the residue was purified by means of preparative HPLC. The corresponding fractions were combined, and the solvent was removed in vacuo. After lyophilization from dioxane, 53 mg (76% of theory) of the protected intermediate were obtained.
  • HPLC (Method 12): Rt=2.0 min;
  • LC-MS (Method 1): Rt=1.02 min; MS (ESIpos): m/z=984 (M+H)+.
  • 53 mg (0.05 mmol) of this intermediate were taken up in 10 ml of dichloromethane, 2 ml of trifluoroacetic acid were added, and the reaction mixture was stirred at RT for 30 min. Subsequently, the reaction mixture was concentrated in vacuo and the remaining residue was purified by means of preparative HPLC. The corresponding fractions were combined, the solvent was removed in vacuo, and the residue was lyophilized from dioxane/water. In this way, 21 mg (40% of theory) of the title compound were obtained with 65% purity.
  • HPLC (Method 12): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=884 (M+H)+.
    • Intermediate 226 N-[2-({[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}amino)ethyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00664
  • The preparation was done starting from Intermediate 225 in analogy to the synthesis of Intermediate 134. 11.6 mg (59% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.90 min; MS (ESIpos): m/z=1050 (M+H)+.
    • Intermediate 227 N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-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]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00665
  • This compound was prepared analogously to Intermediate 218 by conversion to the active ester.
  • Yield: 18 mg (51% of theory)
  • HPLC (Method 5): Rt=2.1 min;
  • LC-MS (Method 1): Rt=0.98 min; MS (ESIpos): m/z=1073 (M+H)+.
    • Intermediate 228 (2R,3S)-3-[(tert-butoxycarbonyl)amino]-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutan-2-yl(3R,4S,7S,10S)-4-[(2S)-butan-2-yl]-7,10-diisopropyl-3-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazapentadecan-15-oate
  • Figure US20140127240A1-20140508-C00666
  • The title compound was prepared by coupling the Boc-protected intermediate obtained from the synthesis of Intermediate 154 with commercially available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
  • HPLC (Method 12): Rt=2.1 min;
  • LC-MS (Method 1): Rt=0.97 min; MS (ESIpos): m/z=1308 (M+H)+.
    • Intermediate 229 (2R,3S)-3-acetamido-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutan-2-yl (3R,4S,7S,10S)-4-[(2S)-butan-2-yl]-7,10-diisopropyl-3-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazapentadecan-15-oate
  • Figure US20140127240A1-20140508-C00667
  • The title compound was prepared from 7.5 mg (2.5 μmol) of Intermediate 154 by acetylation with 2.3 μl of acetic anhydride in 1 ml of DMF in the presence of 0.4 μl of N,N-diisopropylethylamine.
  • Yield: 1.4 mg (40% of theory)
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=1250 (M+H)+.
    • Intermediate 230 (2R,3S)-3-[(tert-butoxycarbonyl)amino]-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutan-2-yl (3R,4S,7S,10S)-4-[(2S)-butan-2-yl]-3-(2-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazapentadecan-15-oate
  • Figure US20140127240A1-20140508-C00668
  • This compound was prepared in analogy to Intermediate 228 starting from Intermediate 193. 16 mg (30% of theory over 3 stages) of the title compound were obtained.
  • HPLC (Method 12): Rt=2.0 min;
  • LC-MS (Method 1): Rt=1.02 min; MS (ESIpos): m/z=1335 (M+H)+.
    • Intermediate 231 (2R,3S)-3-acetamido-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxobutan-2-yl (3R,4S,7S,10S)-4-[(2S)-butan-2-yl]-3-(2-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazapentadecan-15-oate
  • Figure US20140127240A1-20140508-C00669
  • This compound was prepared from 8 mg (6 μmol) of Intermediate 230, first by deprotection with trifluoroacetic acid and subsequent acetylation with acetic anhydride in DMF in the presence of N,N-diisopropylethylamine. 2 mg (37% of theory over 2 stages) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.88 min; MS (ESIpos): m/z=1277 (M+H)+.
    • Intermediate 232 Benzyl-N-[(4-nitrophenoxy)carbonyl]-beta-alaninate
  • Figure US20140127240A1-20140508-C00670
  • 200 mg (0.57 mmol) of commercially available 4-methylbenzenesulphonic acid benzyl beta-alaninate and 229 mg (1.14 mmol) of 4-nitrophenyl chlorocarbonate were taken up in 15 ml of tetrahydrofuran, and the reaction mixture was then heated to reflux for 30 min. Subsequently, the reaction mixture was concentrated in vacuo, and the residue was purified by means of preparative HPLC. After concentration of the corresponding fractions and drying of the residue under high vacuum, 86 mg (44% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=1.07 min; MS (ESIpos): m/z=345 (M+H)+.
    • Intermediate 233 N-{2-[({3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl}carbamoyl)amino]ethyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00671
  • 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 were added. The mixture was stirred at RT overnight and then concentrated under high vacuum. The remaining residue was purified by means of preparative HPLC. After concentration of the corresponding fractions and drying of the residue under high vacuum, 5.4 mg (38% of theory) of the protected intermediate were obtained.
  • HPLC (Method 5): Rt=2.1 min;
  • LC-MS (Method 1): Rt=0.6 in; MS (ESIpos): m/z=1089 (M+H)+.
  • 5.4 mg (5 μmol) of this intermediate were dissolved in 5 ml of methanol and, after adding 2 mg of 10% palladium on activated carbon, hydrogenated under standard hydrogen pressure at RT for 20 min. The catalyst was then filtered off, and the solvent was removed in vacuo. After drying of the residue under high vacuum, 5 mg (quant.) of the acid intermediate were obtained.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.84 min; MS (ESIpos): m/z=999 (M+H)+.
  • 5 mg (10 μmol) of this intermediate were dissolved in 1 ml of DMF and admixed with 5.8 mg (50 mmol) of 1-hydroxypyrrolidine-2,5-dione and then with 2.6 μl of N,N-diisopropylethylamine and 3.8 mg (10 μmol) of HATU. After stirring at RT for 20 h, the reaction mixture was concentrated in vacuo. The remaining residue was purified by means of preparative HPLC. After lyophilization from 1:1 dioxane/water, 1.1 mg (20% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=1096 (M+H)+.
    • Intermediate 234 N-(6-{[(benzyloxy)carbonyl]amino}hexyl)-N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00672
  • 25 mg (30 μmol) of N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 55) and 45 mg (180 μmol) of benzyl-(6-oxohexyl)carbamate were taken up in 3 ml of methanol and acidified with acetic acid. At room temperature, 15 μl (144 μmol; 9.4M) of borane-pyridine complex were subsequently added. The batch was subsequently stirred at RT for 24 h, and acetic acid and 15 μl (144 μmol; 9.4M) of borane-pyridine complex were added again after 8 h. The reaction mixture was subsequently adjusted to pH 2 with TFA and purified by means of 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): Rt=1.03 min; m/z=1066 (M+H)+.
    • Intermediate 235 N-(6-aminohexyl)-N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00673
  • 15 mg (14 μmol) of N-(6-{[(benzyloxy)carbonyl]amino}hexyl)-N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 234) were taken up in 3 ml of methanol, and 1.8 mg of palladium on carbon (5%) were added. The reaction mixture was subsequently hydrogenated under standard hydrogen pressure at RT for 2 h. The catalyst was then filtered off, and the solvent was removed in vacuo. The residue was lyophilized from 1:1 acetonitrile/water. 11 mg (86% of theory) of the title compound were obtained as a foam.
  • LC-MS (Method 1): Rt=0.81 min; m/z=932 (M+H)+.
    • Intermediate 236 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00674
  • 11 mg (12 μmol) of N-(6-aminohexyl)-N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 235) were taken up in 500 μl of 1:1 dioxane/water and admixed with 253 μl of 1M aqueous sodium hydrogencarbonate solution and then with 2.8 mg (18 μmol) of methyl 2,5-dioxo-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 means of preparative HPLC. After lyophilization, 0.8 mg (7% of theory) of the title compound was obtained.
  • LC-MS (Method 1): Rt=1.01 min; m/z=1012 (M+H)+.
    • Intermediate 237 N-(5-carboxypentyl)-N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00675
  • 25 mg (30 μmol) of N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 55) and 23 mg (180 μmol) of 6-oxohexanoic acid were taken up in 3 ml of methanol and acidified with acetic acid. At room temperature, 15 μl (144 μmol; 9.4M) of borane-pyridine complex were subsequently added. The reaction mixture was subsequently stirred at RT for 20 h, and acetic acid and 15 μl (144 μmol; 9.4M) of borane-pyridine complex were added again after 8 h. The reaction mixture was subsequently adjusted to pH 2 with trifluoroacetic acid and purified by means of preparative HPLC. The product fractions were combined and concentrated, and the residue was lyophilized. 21 mg (74% of theory) of the title compound were thus obtained as a foam.
  • LC-MS (Method 1): Rt=0.91 min; m/z=947 (M+H)+.
    • Intermediate 238 N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-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-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00676
  • 21 mg (22 μmol) of Intermediate 237 were dissolved in 1 ml of DMF and admixed 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′-tetramethyluronium hexafluorophosphate (HATU) and 19 μl of N,N-diisopropylethylamine. After stirring at RT for 2 h, the reaction mixture was purified by means of preparative HPLC. After lyophilization from dioxane, 22 mg (96% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=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-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00677
  • First, N-[(benzyloxy)carbonyl]-N-methyl-L-threonine was released from 237 mg (0.887 mmol) of its dicyclohexylamine salt by taking it up in ethyl acetate and extractive shaking with 5% aqueous sulphuric acid. The organic phase was dried over magnesium sulphate, filtered and concentrated. 14.7 mg (0.055 mmol) of N-[(benzyloxy)carbonyl]-N-methyl-L-threonine were taken up in 3 ml of DMF and admixed successively 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-1H-benzotriazole hydrate. The mixture was subsequently stirred at RT for 2 h. The solvent was then removed in vacuo, and the residue purified by means of preparative HPLC. 29 mg (54% of theory) of the Z-protected intermediate were thus obtained.
  • LC-MS (Method 1): Rt=1.15 min; MS (ESIpos): m/z=976 (M+H)+.
  • 29 mg (0.003 mmol) of this intermediate were dissolved in 5 ml of methanol and hydrogenated over 5 mg of 5% palladium/carbon at RT and standard pressure for 1 h. The catalyst was subsequently filtered off and the solvent evaporated. The remaining residue was purified by means of preparative HPLC. 17 mg (54% of theory) of the title compound were obtained.
  • LC-MS (Method 1): Rt=0.77 min; MS (ESIpos): m/z=842 (M+H)+.
    • Intermediate 240 N-{6-[(2,5-dioxopyrrolidin-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-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00678
  • This compound was prepared in analogy to Intermediate 210 from 15.6 mg (0.016 mmol) of Intermediate 239. 10.8 mg (67% of theory over 2 stages) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.85 min; MS (ESIpos): m/z=1053 (M+H)+.
    • Intermediate 241 N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-hydroxyphenyl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00679
  • First, in analogy to Intermediate 5, trifluoroacetic acid-(2S)-2-amino-3-(4-hydroxyphenyl)-1-(1,2-oxazinan-2-yl)propan-1-one (1:1) was prepared. This component was then used to obtain the title compound, in analogy to the synthesis described in Intermediate 75, by coupling with N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent cleaving of the Boc protecting group by means of trifluoroacetic acid.
  • HPLC (Method 12): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.85 min; MS (ESIpos): m/z=817 (M+H)+.
    • Intermediate 242 N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-hydroxyphenyl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00680
  • 50 mg (0.05 mmol) of Intermediate 241 were reacted, in analogy to Intermediate 210, with 6-oxohexanoic acid in the presence of borane-pyridine complex. Subsequently, 22.5 mg (0.02 mmol) of the obtained acid were converted to the activated ester. 13.5 mg (36% of theory over 2 stages) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=1028 (M+H)+.
    • Intermediate 243 N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-hydroxyphenyl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00681
  • The preparation was done, in analogy to Intermediate 78, by reductive alkylation of Intermediate 241 with benzyl-(6-oxohexyl)carbamate and borane-pyridine complex and subsequent hydrogenation in methanol as the solvent.
  • Yield: 17.5 mg (34% of theory over 2 stages)
  • HPLC (Method 12): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.63 min; MS (ESIpos): m/z=916 (M+H)+.
    • Intermediate 244 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-hydroxyphenyl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00682
  • The preparation was done in analogy to Intermediate 166 starting from Intermediate 243.
  • Yield: 1.3 mg (12% of theory)
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.89 min; MS (ESIpos): m/z=996 (M+H)+.
    • Intermediate 245 2,5-dioxopyrrolidin-1-yl O-[(3R,4S,7S,10S)-4-[(2S)-butan-2-yl]-3-(2-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,15-trioxo-2-oxa-5,8,11-triazapentadecan-15-yl]-N-(tert-butoxycarbonyl)-L-threonyl-beta-alaninate
  • Figure US20140127240A1-20140508-C00683
  • First, Intermediate 193, as described for Intermediate 154, was reacted with benzyl N-(tert-butoxycarbonyl)-L-threoninate, and then the benzyl ester was removed by hydrogenolytic means. 30 mg (0.027 mmol) of the thus obtained N-[4-({(1S,2R)-1-[(tert-butoxycarbonyl)amino]-1-carboxypropan-2-yl}oxy)-4-oxobutyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide were then coupled with 4-methylbenzenesulphonic acid benzyl-beta-alaninate in the presence of HATU, and the benzyl ester was removed again by hydrogenolysis (yield: 24 mg (71% of theory over 2 stages)). Finally, 10 mg (0.008 mmol) of the obtained acid were converted to the activated ester. After HPLC purification, 2.7 mg (23% of theory) of the title compound were obtained.
  • HPLC (Method 5): Rt=1.9 min;
  • LC-MS (Method 1): Rt=1.01 min; MS (ESIpos): m/z=1295 (M+H)+
    • Intermediate 246a (2S)-2-amino-1-(4-hydroxy-1,2-oxazolidin-2-yl)-3-(1H-indol-3-yl)propan-1-one trifluoroacetate (Diastereomer 1)
  • Figure US20140127240A1-20140508-C00684
  • 1.6 g (3.982 mmol) of 2,5-dioxopyrrolidin-1-yl N-(tert-butoxycarbonyl)-L-tryptophanate were dissolved in 15 ml of DMF and admixed with 500 mg (3.982 mmol) of 1,2-oxazolidin-4-ol and 100 μl of N,N-diisopropylethylamine. The reaction mixture was stirred at RT overnight. Then another 100 μl of N,N-diisopropylethylamine were added, and the mixture was first treated in an ultrasound bath for 5 h, then stirred at RT overnight and subsequently concentrated in vacuo. The remaining residue was taken up in ethyl acetate and extracted first twice with 5% aqueous citric acid solution, then with saturated aqueous sodium hydrogencarbonate solution and finally with water. The organic phase was concentrated and the residue separated into the diastereomers by means of flash chromatography on silica gel with 95:5 dichloromethane/methanol as the eluent. The corresponding fractions of both diastereomers were combined and the solvent was removed in vacuo. After drying of the residues under high vacuum, 272 mg (18% of theory) of Diastereomer 1 (Rf=0.18 (95:5 dichloromethane/methanol) and 236 mg (16% of theory) of Diastereomer 2 (Rf=0.13 (95:5 dichloromethane/methanol) as wells as 333 mg (22% of theory) of a mixed fraction of the Boc-protected intermediates were obtained.
  • 5 ml of trifluoroacetic acid in 20 ml of dichloromethane were used under standard conditions for cleaving the Boc protecting group from 272 mg (725 μmol) of Diastereomer 1 of this intermediate and, after lyophilization from dioxane/water, 290 mg (quant) of the title compound were obtained in 75% purity and used without further purification in the next stage.
  • HPLC (Method 12): Rt=1.1 min;
  • LC-MS (Method 13): Rt=1.80 min; MS (ESIpos): m/z=276 (M+H)+
    • Intermediate 246b (2S)-2-amino-1-(4-hydroxy-1,2-oxazolidin-2-yl)-3-(1H-indol-3-yl)propan-1-one trifluoroacetate (Diastereomer 2)
  • Figure US20140127240A1-20140508-C00685
  • 5 ml of trifluoroacetic acid in 20 ml of dichloromethane were used under standard conditions for cleaving the Boc protecting group from 236 mg (630 μmol) of Diastereomer 2 of the intermediate described in 246a and, after concentration, stirring with diethyl ether and drying of the residue under high vacuum, 214 mg (76%) of the title compound were obtained.
  • LC-MS (Method 13): Rt=1.84 min; MS (ESIpos): m/z=276 (M+H)+
    • Intermediate 247a N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(4-hydroxy-1,2-oxazolidin-2-yl)-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-valinamide (Diastereomer 1)
  • Figure US20140127240A1-20140508-C00686
  • To synthesize this compound, the coupling of Intermediates 26 and 246a with subsequent cleaving of the Boc protecting group was first performed as described for Intermediate 74. Subsequently, the alkylation with 6-oxohexanoic acid in the presence of borane-pyridine complex and subsequent conversion of the acid to the active ester were performed, as described for Intermediate 210. The title compound was purified by means of preparative HPLC.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=1053 (M+H)+
    • Intermediate 247b N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(4-hydroxy-1,2-oxazolidin-2-yl)-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-valinamide (Diastereomer 2)
  • Figure US20140127240A1-20140508-C00687
  • To synthesize this compound, the coupling of Intermediates 26 and 246b with subsequent cleaving of the Boc protecting group was first performed as described for Intermediate 74. Subsequently, the alkylation with 6-oxohexanoic acid in the presence of borane-pyridine complex and subsequent conversion of the acid to the active ester were performed, as described for Intermediate 210. The title compound was purified by means of preparative HPLC.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=1053 (M+H)+
    • Intermediate 248 N-(5-carboxypentyl)-N-methyl-L-valyl-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-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00688
  • First, in analogy to the synthesis described in Intermediate 86, the amine compound tert-butyl N-[(2R,3R)-3-methoxy-3-{(2S)-1-[(3R,4S,5S)-3-methoxy-5-methyl-4-(methyl{(2S)-3-methyl-2-[(N-methyl-L-valyl)amino]butyl}amino)heptanoyl]pyrrolidin-2-yl}-2-methylpropanoyl]-L-tyrosinate was prepared as the trifluoroacetate by coupling N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 26) and tert-butyl-L-tyrosinate in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and subsequent cleaving of the Boc protecting group by means of trifluoroacetic acid to obtain the tert-butyl ester (stirring with trifluoroacetic acid in dichloromethane for 40 min). 38 mg (0.04 mmol) of this compound were then used to obtain 31 mg (99% of theory) of the title compound, in analogy to the preparation of Intermediate 210, by reaction with 6-oxohexanoic acid in the presence of borane-pyridine complex.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.88 min; MS (ESIpos): m/z=918 (M+H)+.
    • Exemplary Embodiments Anti-EGFR1 Antibodies Used Cetuximab (INN No. 7906)
  • Additional names: IMC-225, C225, EMR-62202, BMS-564717, Fab C225
  • Cetuximab (Drug Bank Accession No. DB00002) is a chimeric anti-EGFR1-antibody that is produced in SP2/0 mouse myeloma cells and is distributed by ImClone Systems Inc./Merck KGaA/Bristol-Myers Squibb Co.
  • Cetuximab is indicated for treatment of metastatic EGFR-expressing colorectal carcinoma with the wild-type K-Ras gene. It has an affinity of 10−10 M.
    • Sequence:
  • Light chain (kappa):
    DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY
    ASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGA
    GTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
    DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
    LSSPVTKSFNRGEC
    Heavy chain:
    QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV
    IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALT
    YYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
    YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
    ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
    DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
    TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
    YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
    DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
    • Panitumumab (INN No. 8499)
  • Panitumumab (additional names: ABX-EGF, E7.6.3) (Drug Bank Accession No. DB01269) is a recombinant monoclonal human IgG2 antibody that binds specifically to the human EGF receptor 1 and is distributed by Abgenix/Amgen.
  • Panitumumab originates from the immunization of transgenic mice (XenoMouse). These mice are capable of producing human immunoglobulins (light chains and heavy chains). A special B cell clone was selected which produces antibodies to EGFR and it was immortalized with CHO cells (Chinese hamster ovary cells). These cells are now used for the production of a 100% human antibody.
  • Panitumumab is indicated for treatment of an EGFR-expressing metastatic colorectal carcinoma that is refractory to a chemotherapeutic treatment with fluoropyrimidine, oxaliplatin and irinotecan. It has an affinity of 10−11 M.
    • Sequence:
  • Light chain (kappa):
    DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD
    ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGG
    GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
    DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
    LSSPVTKSFNRGEC
    Heavy chain:
    QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWI
    GHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRD
    RVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD
    YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTY
    TCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM
    ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
    VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP
    PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG
    SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
    • Nimotuzumab (INN No. 8545)
  • Nimotuzumab (additional names: TheraCIM-h-R3; h-R3; Theraloc; BioMAb; BIOMAb-EGFR; Vecthix; KI-0501) (patents EP 00586002, EP 00712863) is a humanized monoclonal IgG1 antibody that binds specifically to human EGF receptor 1 and is produced by YM BioSciences Inc. (Mississauga, Canada). It is produced in non-secreting NSO cells (mammalian line).
  • Nimotuzumab is approved for treatment of head and neck tumors, highly malignant astrocytomas and glioblastoma multiform (not in the EU and US) and pancreatic cancer (orphan drug, EMA). It has an affinity of 10−8 M.
    • B. PRODUCTION OF ADCs
  • The intermediates described above can be linked to the anti-EGF receptor antibodies cetuximab, nimotuzumab or panitumumab, for example, as well as additional antibodies listed below, and such linkages may optionally be via cysteine or lysine side chains of the antibody protein according to the methods described below.
    • B-1.1 Workup of the EGFR Antibodies Before Conjugation
  • Erbitux commercial product (Erbitux® 5 mg/mL infusion solution 100 mL, PZN 0493540, N1, 500 mg, Merck), Vectibix commercial product (Vectibix® 20 mg/mL concentrate for preparing an infusion solution, one puncturable vial (N1) 100 mg, 20 mL, PZN 6078606, Amgen) or CIMAher commercial product (CIMAher® 50 mg AMP 4×10 mL, imported from Cuba, YM BioSciences Inc. (Mississauga, Canada) were obtained commercially from a pharmacy.
  • To remove the polysorbate 80 contained in the formulation, it was bound to protein A (MabSelectSure) and rinsed with 15% isopropanol. After elution with acidic acetate buffer, the mixture was rebuffered after gel filtration on D-PBS, and the resulting material was coupled to the respective toxophores.
    • B-1.2 General Procedure for Expression of Antibodies in Mammalian Cells
  • The antibodies, e.g., anti-PDL1 or other antibodies to the various targets are produced in mammalian cell culture by transfecting HEK293 6E cells transiently with a suitable CMV promoter-based expression plasmid. The light and heavy chains of the antibodies were cloned either together in a single-vector system or separately in a two-vector system. The cell culture standard was up to 1.5 L in an agitated flask or 10 L in the “Wave Bag.” The expression occurred at 37° C. for 5-6 days in F17 medium (Invitrogen) supplemented with tryptone TN1 (Organo-technie) with 1% “FCS ultra-low IgG” (Invitrogen) and 0.5 mM valproic acid. The expression yields were between 7 and 310 mg/L.
  • B-1.3 General Method for Purifying Antibodies from Cell Supernatants
  • The antibodies, e.g., PDL1 or other antibodies to the various targets were obtained from the cell culture supernatants. The cell culture supernatants were clarified by centrifugation of cells. Then the supernatant was purified by affinity chromatography on a MabSelectSure (GE Healthcare) chromatography column. The column was therefore equilibrated in DPBS, pH 7.4 (Sigma/Aldrich), the cell supernatant was applied and the column was washed with approx. 10 column volumes of DPBS, pH 7.4, +500 mM sodium chloride. The antibodies were eluted in 50 mM sodium acetate, pH 3.5, +500 mM sodium chloride and then purified further by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS, pH 7.4.
    • B-1.4 General Method for Coupling to Cysteine Side Chains
  • The following antibodies were used in the coupling reaction:
  • Anti-EGFR1 antibodies:
    • cetuximab
    • nimotuzumab
    • panitumumab
      Other antibodies:
    • anti-PDL1
    • anti-ICOSLG
    • anti-FGFR3
    • herceptin
    • anti-TYRP1
    • anti-glypican-3
  • To a solution of the corresponding antibody in PBS buffer in the concentration range between 1 mg/mL and 10 mg/mL, 3 eq. of tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) dissolved in PBS buffer were added and stirred for one hour at RT. Then, depending on the desired load, between two and ten equivalents of the maleimide precursor compound to be coupled or the halide precursor compound (intermediates 102, 103, 105-109, 111-114, 117-126, 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) were added as a solution in DMSO. The amount of DMSO should not exceed 10% of the total volume. The batch was stirred for 60-120 minutes at RT and then applied to PD10 columns (Sephadex® G-25, GE Healthcare) equilibrated in PBS and then eluted with PBS buffer. If necessary, the concentration was increased further by ultracentrifugation.
  • Unless otherwise indicated, 5 mg of the corresponding antibody was generally used in PBS buffer for reduction and the following coupling. After purification on the PD10 column, the solutions of the corresponding ADC in 3.5 mL PBS buffer were each obtained. The protein concentration indicated in each case was then determined for these solutions. In addition, the load of the antibody (drug/mAb ratio) was determined by the methods described below.
  • According to this method, the immunoconjugates synthesized in Examples 1-34, 36-37, 39-41, 43-44, 52-53, 55, 338-339, 341-344, 349, 351-352, 354, 356-358 and 374 were prepared.
  • In the structural formulas presented, AK1A-AK1J have the following meanings:
    • AK1A=cetuximab (partially reduced)-S§1
    • AK1B=nimotuzumab (partially reduced)-S§1
    • AK1C=panitumumab (partially reduced)-S§1
    • AK1D=anti-PDL1 (partially reduced)-S§1
    • AK1E=anti-ICOSLG (partially reduced)-S§1
    • AK1F=anti-FGFR3 (partially reduced)-S§1
    • AK1G=herceptin (partially reduced)-S§1
    • AK1H=anti-TYRP1 (partially reduced)-S§1
    • AK1J=anti-glypican-3 (partially reduced)-S§1
      wherein
    • §1 denotes the linkage to the succinimide group
    • and
    • S stands for the sulfur atom of a cysteine radical of the partially reduced antibody.
    B-1.5 General Method for Coupling to Lysine Side Chains
  • The following antibodies were used in the coupling reactions:
  • Anti-EGFR1 antibodies:
    • cetuximab
    • nimotuzumab
    • panitumumab
      Other antibodies:
    • anti-PDL1
    • anti-ICOSLG
    • anti-FGFR3
    • herceptin
    • anti-TYRP1 hIgG1-kapp
    • anti-glypican-3
  • Between 2 and 5 eq. of the precursor compound to be coupled from the intermediates 104, 110, 115, 116, 127, 130, 131, 147, 156, 158, 162, 169, 178, 185, 190, 202, 206, 210-216, 218, 219, 227, 233, 238, 240, 242, 245, 247a and 247b were added as a solution in DMSO to a solution of the corresponding antibody in PBS buffer in the concentration range between 1 mg/mL and 10 mg/mL, depending on the desired load. After stirring for 30 minutes at RT, the same amount of precursor compound in DMSO was again added. The amount of DMSO should not exceed 10% of the total volume. After stirring for 30 minutes more at RT the batch was poured over PD10 columns (Sephadex® G-25) and then eluted with PBS buffer. Another concentration step was optionally performed by ultrafiltration. If necessary, for better separation of low-molecular components, the concentration step by ultrafiltration was repeated after diluting again with PBS buffer.
  • Unless otherwise indicated, 5 mg of the corresponding antibody in PBS buffer was generally used for coupling. After purification on the PD10 column, solutions of the corresponding ADCs in 3.5 mL PBS buffer were obtained. For these solutions, the respective protein concentrations then given were determined, and the antibody load (drug/mAb ratio) was determined according to the methods described below.
  • According to this method, the immunoconjugates described in Examples 35, 38, 42, 54, 337, 340, 345-348, 350, 353, 355, 359, 363, 375 and 376 were prepared.
  • In the structural formulas shown here, AK2A, AK2B, AK2C, AK2D, AK2E, AK2F, AK2G, AK2H and AK2J have the following meanings:
    • AK2A=cetuximab-NH§2
    • AK2B=nimotuzumab-NH§2
    • AK2C=panitumumab-NH§2
    • AK2D=anti-PDL1-NH§2
    • AK1E=anti-ICOSLG-NH§2
    • AK2F=anti-FGFR3-NH§2
    • AK2G=herceptin-NH§2
    • AK2H=anti-TYRP1-NH§2
    • AK2J=anti-glypican-3-NH§2
      wherein
    • §2 denotes the linkage to the carbonyl group
    • and
    • NH stands for the side chain amino group of a lysine radical of the antibody.
    B-1.6A General Method for Preparing Cysteine Adducts
  • 10 μmol of the maleimide precursor compounds described above was placed in 3 mL DMF and mixed with 2.1 mg (20 μmol) L-cysteine. The batch was stirred for 2 hours at RT, then concentrated in vacuo and next purified by preparative HPLC.
  • In the structural formulas shown here, Cys has the following meaning:
  • Figure US20140127240A1-20140508-C00689
  • wherein
    • §3 denotes the linkage to the linker toxophore unit.
    B-1.6b General Method for Preparing Lysine Adducts:
  • 10 μmol of the active ester precursor compounds described above was placed in 5 mL DMF and mixed with α-amino-protected L-lysine in the presence of 30 μmol N,N-diisopropylethylamine. The reaction mixture was stirred for 2 hours at RT and then concentrated in vacuo and next was purified by preparative HPLC. Then the protective group was removed by known methods.
    • Further Purification and Characterization of the Conjugates According to the Invention
  • After the reaction was successful, the reaction mixture was concentrated in some cases by ultracentrifugation, for example, and then was desalinated and purified by chromatography using a Sephadex® G-25 column, for example. Elution was performed using phosphate-buffered saline solution (PBS), for example. Then the solution was sterile filtered and frozen. Alternatively, the conjugate may be lyophilized.
    • B-1.7 Determining the Toxophore Load
  • The toxophore load was determined as follows on the resulting solutions of the conjugates in PBS buffer as described in the exemplary embodiments:
  • The toxophore load of lysine-linked ADCs was determined by mass spectrometric determination of the molecular weights of the individual conjugated species. The antibody conjugates were first deglycosylated by PNGaseF, the sample was acidified and next, after HPLC separation, the sample was analyzed by mass spectrometry using ESI MicroTofQ (Bruker Daltonik). All the spectra over the signal in the TIC (total ion chromatogram) were added up and the molecular weights of the various conjugate species were calculated on the basis of MaxEnt deconvolution. After signal integration of the various species, the DAR (drug/antibody ratio) was calculated.
  • For protein identification, after deglycosylation and/or denaturing, in addition to determination of the molecular weight, tryptic digestion was performed, confirming the identity of the protein on the basis of the tryptic peptides identified after denaturing, reduction and derivatization.
  • The toxophore load of cysteine-linked conjugates was determined by reversed-phase chromatography of the reduced and dentured ADCs. Guanidinium hydrochloride (GuHCl, 28.6 mg) and a solution of DL-dithiothreitol (DTT, 500 mM, 3 μL) were added to the ADC solution (1 mg/mL, 50 μL). The mixture was incubated for one hour at 55° C. and then analyzed by HPLC.
  • HPLC analysis was performed on an adjuvant 1260 HPLC system with detection at 220 nm, using a Polymer Laboratories PLRP-S polymeric reversed-phase column (catalog number PL1912-3802) (2.1×150 mm, 8 μm particle size, 1000 Å) at a flow rate of 1 mL/min with the following gradient: 0 min, 25% B; 3 min, 25% B; 28 min, 50% B. Eluent A consisted of 0.05% trifluoroacetic acid (TFA) in water, and eluent B consisted of 0.05% trifluoroacetic acid in acetonitrile.
  • The peaks detected were assigned based on a comparison of the retention times with the light chain (LO) and the heavy chain (HO) of the unconjugated antibody. Peaks detected exclusively in the conjugated sample were assigned to the light chain with one toxophore (L1) and to the heavy chains with one, two and three toxophores (H1, H2, H3).
  • The average toxophore load of the antibody was calculated from the peak areas determined by integration as the sum of the integration results of all peaks times 2, weighted by the number of toxophores, divided by the total of the integration results of all peaks with simple weighting. In isolated cases, it may happen that the toxophore load cannot be determined accurately due to co-elution of some peaks.
    • B-1.8 Testing the Antigen Binding of the ADC
  • The ability of the binder to bind to the target molecule was tested after successful coupling. Those skilled in the art are familiar with a variety of methods for doing so; for example, the affinity of the conjugate can be tested by means of ELISA technology or surface plasmon resonance analysis (BIAcore™ measurements). The skilled person can measure the conjugate concentration using conventional methods, e.g., protein assay for antibody conjugates (see also Doronina et al., Nature Biotechnol. 2003; 21:778-784 and Polson et al., Blood 2007, 1102:616-623).
    • B2 Producing Antibody-Drug Conjugates (ADCs)
  • The intermediates described above were linked to the anti-mesothelin antibody MF-Ta, for example, with the linkage optionally taking place via the cysteine or lysine side chains of the antibody protein using the methods described below. The anti-mesothelin antibody MF-Ta was produced by methods like those described in WO 2009/068204 A1. The antibody MF-Ta was expressed in eukaryotic CHO cells (stable cell line) and purified by protein A and gel filtration before being subjected to conjugation in DPBS buffer.
    • B-2.1 General Working Procedure 1 (Coupling Via Cysteine):
  • To a solution of the corresponding antibody in PBS buffer in the concentration range between 1 mg/mL and 10 mg/mL, 3 eq. of tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) dissolved in PBS buffer were added and stirred for one hour at RT. Next, depending on the desired load, between 2 and 10 eq. of the maleimide precursor compound or the halide precursor compound to be coupled (intermediates 128, 129, 132-146, 148-155, 157, 159-161, 171, 175-177, 184, 189, 194-195, 199-201, 205, 209, 223-224, 226, 228-231, 236 and 244) were added as a solution in DMSO. The amount of DMSO should not exceed 10% of the total volume. The reaction mixture was stirred for 60-120 minutes at RT and then applied to PD10 columns (Sephadex® G-25) and eluted with PBS buffer. The solution was then optionally concentrated by ultrafiltration. Concentration by ultrafiltration was repeated, if necessary, after diluting again with PBS buffer to achieve a better separation of low-molecular components.
  • Unless otherwise indicated, 5 mg of the corresponding antibody in PBS buffer was generally used for reduction and the subsequent coupling. After purification over the PD10 column, solutions of the corresponding ADCs in 3.5 mL PBS buffer were thus obtained. Then the respective protein concentration was determined for each of these solutions. In addition, the antibody load (drug/mAb ratio) was determined by the methods described in B-4.
  • The immunoconjugates synthesized in Examples 56, 57, 60-74, 76-83, 85, 86, 88-92, 94-101, 103, 106-112, 114, 115, 126, 128-131, 133-135, 137-139, 141-142, 151, 153-154, 366 and 377 were prepared by this method.
  • In the structural formulas given, AK3 has the meaning
      • AK3=MF-Ta (partially reduced)-S§1,
        wherein
    • §1 denotes the linkage to the succinimide group,
    • MF-Ta (partially reduced) stands for the partially reduced MF-Ta antibody (heavy chain SEQ ID NO: 408 and light chain SEQ ID NO: 409),
    • and
    • S stands for the sulfur atom of a cysteine radical of the partially reduced antibody.
    B-2.2 General Working Procedure 2 (Coupling Via Lysine Side Chains):
  • Between 2 and 5 eq. of the precursor compound to be coupled (intermediates 104, 110, 115, 116, 127, 130, 131, 147, 156, 158, 162, 169, 178, 185, 190, 202, 206, 210-216, 218, 219, 227, 233, 238, 240, 242, 245, 247a and 247b) were added as a solution in DMSO to a solution of the corresponding antibody in PBS buffer in the concentration range between 1 mg/mL and 10 mg/mL, depending on the desired load. After stirring for 30 minutes at RT, the same amount of precursor compound in DMSO was added. The amount of DMSO should not exceed 10% of the total volume. After stirring for 30 minutes more at RT, the batch was poured over PD10 columns (Sephadex® G-25) and eluted with PBS buffer. Further concentration by ultrafiltration was optionally also performed. Concentration was repeated by ultrafiltration, if necessary, after diluting again with PBS buffer to improve the separation of low-molecular components.
  • Unless otherwise indicated, 5 mg of the corresponding antibody in PBS buffer was generally used for coupling. After purification over the PD10 column, solutions of the corresponding ADC in 3.5 mL PBS buffer were obtained. Then the protein concentration indicated was determined for these solutions and the load of the antibody (drug/mAb ratio) was determined by the methods described under B-4.
  • Following this method, the immunoconjugates synthesized in Examples 58, 59, 75, 84, 87, 93, 102, 104, 105, 113, 116, 127, 132, 136, 140, 143-150, 152, 367-369 and 378-380 were prepared.
  • In the structural formulas given, AK4 has the meaning
      • AK4=MF-Ta-NH§2,
        wherein
    • §2 denotes the linkage to the carbonyl group
    • MF-Ta stands for the unreduced MF-Ta antibody (heavy chain SEQ ID NO: 408 and light chain SEQ ID NO: 409)
    • and
    • NH stands for the side chain amino group of a lysine radical of the antibody.
    B-2.3A General Method for Synthesis of Cysteine Adducts:
  • 10 μmol of the maleimide precursor compounds described above was dissolved in 3 mL DMF and mixed with 2.1 mg (10 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and purified by preparative HPLC.
  • Cys in the structural formulas given has the meaning
  • Figure US20140127240A1-20140508-C00690
  • wherein
    • §3 denotes the linkage to the linker toxophore unit.
    B-2.3b General Method for Preparing Lysine Adducts:
  • 10 μmol of the active ester precursor compounds described above was placed in 5 mL DMF and mixed with α-amino-protected L-lysine in the presence of 30 μmol N,N-diisopropylethylamine. The reaction mixture was stirred for 2 hours at RT and then concentrated in vacuo and next was purified by preparative HPLC. Then the protective group was removed by known methods.
    • Further Purification and Characterization of the Conjugates According to the Invention
  • After a successful reaction, the reaction mixture was concentrated by ultracentrifugation, for example, in some cases and then was desalinated and purified by chromatography using a Sephadex® G-25 column, for example. Elution was performed using phosphate-buffered saline solution (PBS), for example. Then the solution was sterile filtered and frozen. Alternatively, the conjugate may be lyophilized.
    • B-2.4 Determining the Toxophore Load
  • The toxophore load was determined as follows on the resulting solutions of the conjugates in PBS buffer as described in the exemplary embodiments:
  • The toxophore load of lysine-linked ADCs was determined by mass spectrometric determination of the molecular weights of the individual conjugated species. First, the antibody conjugates were deglycosylated by PNGaseF, then the sample was acidified; next, after HPLC separation, the sample was analyzed by mass spectrometry using ESI MicroTofQ (Bruker Daltonik). All the spectra over the signal in the TIC (total ion chromatogram) were added up and the molecular weights of the various conjugate species were calculated based on MaxEnt deconvolution. The DAR (drug/antibody ratio) was calculated after signal integration of the various species.
  • For protein identification, after deglycosylation and/or denaturing, tryptic digestion was performed, in addition to determination of the molecular weight, the identity of the protein being confirmed on the basis of the tryptic peptides identified after denaturing, reduction and derivatization.
  • The toxophore load of cysteine-linked conjugates was determined by reversed-phase chromatography of the reduced and dentured ADCs. Guanidinium hydrochloride (GuHCl, 28.6 mg) and a solution of DL-dithiothreitol (DTT, 500 mM, 3 μL) were added to the ADC solution (1 mg/mL, 50 μL). The mixture was then incubated for one hour at 55° C. and analyzed by HPLC.
  • The HPLC analysis was performed on an adjuvant 1260 HPLC system with detection at 220 nm, using a Polymer Laboratories PLRP-S polymeric reversed-phase column (catalog no. PL1912-3802) (2.1×150 mm, 8 μm particle size, 1000 Å) at a flow rate of 1 mL/min with the following gradient: 0 min, 25% B; 3 min, 25% B; 28 min, 50% B. Eluent A was 0.05% trifluoroacetic acid (TFA) in water, and eluent B was 0.05% trifluoroacetic acid in acetonitrile.
  • The peaks detected were assigned based on a comparison of the retention times with the light chain (L0) and the heavy chain (H0) of the unconjugated antibody. Peaks detected exclusively in the conjugated sample were assigned to the light chain with one toxophore (L1) and to the heavy chains with one, two and three toxophores (H1, H2, H3).
  • The average toxophore load of the antibody was calculated from the peak areas determined by integration as two times the sum of the integration results of all peaks, weighted by the number of toxophores, divided by the total of the integration results of all peaks with simple weighting. In isolated cases, it may happen that the toxophore load cannot be determined accurately due to co-elution of some peaks.
    • B-2.5 Testing the Antigen Binding of the ADC
  • The ability of the binder to bind to the target molecule was tested after successful coupling. Those skilled in the art are familiar with a variety of methods for doing so; for example, the affinity of the conjugate can be tested by ELISA technology or surface plasmon resonance analysis (BIAcore™ measurements). The skilled person can measure the conjugate concentration using conventional methods, e.g., by protein determination for antibody conjugates (see also Doronina et al., Nature Biotechnol. 2003; 21:778-784 and Polson et al., Blood 2007, 1102:616-623).
    • B3 Synthesis of Antibody-Drug Conjugates (ADCs) B-3.1 General Method for Generating Anti-C4.4a Antibodies
  • The anti-C4.4a antibodies described by the sequences according to Table 1 and Table 2 were generated by screening of a phage display library for recombinant human C4.4a SEQ ID NO: 1 and murine C4.4a SEQ ID NO: 2 and for C4.4a-expressing cells. The antibodies thereby obtained were reformatted into the human IgGl format and used for the exemplary embodiments described here.
    • B-3.2 General Method for Expression of Anti-C4.4a Antibodies in Mammalian Cells
  • The antibodies, e.g., M31-B01 (light chain SEQ ID NO: 346 and heavy chain SEQ ID NO: 347) or other antibodies according to Table 2, were produced in mammalian cell culture. To do so, HEK293 6E cells were transfected transiently using a suitable CMV promoter-based expression plasmid. The heavy and light chains of the antibodies were cloned either together in a single-vector system or separately in a two-vector system. This cell culture standard was up to 1.5 L in a shaken flask or 10 L in a Wave Bag. The cells were expressed for 5-6 days at 37° C. in F17 medium (Invitrogen) supplemented with tryptone TN1 (Organotechnie) with 1% FCS ultralow IgG (Invitrogen) and 0.5 mM valproic acid. Expression yields were between 100 mg/L and 600 mg/L.
  • B-3.3 General Method for Purification of Antibodies from Cell Supernatants
  • The antibodies, e.g., M31-B01 (light chain SEQ ID NO: 346 and heavy chain SEQ ID NO: 347) or additional antibodies according to Table 2 were obtained from the cell culture supernatants. The cell supernatants were clarified of cells by centrifugation. Then the cell supernatant was purified by affinity chromatography on a MabSelectSure (GE Healthcare) chromatography column. To do so, the column was equilibrated in DPBS, pH 7.4 (Sigma/Aldrich), the cell supernatant was applied and the column was washed with approx. 10 column volumes of DPBS, pH 7.4, +500 mM NaCl. The antibodies were eluted in 50 mM sodium acetate, pH 3.5, +500 mM NaCl and then purified further by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS, pH 7.4.
    • B-3.4 General Method for Coupling to Cysteine Side Chains
  • The following antibodies were used in the coupling reactions:
    • anti-C4.4a M31-B01
    • anti-C4.4a B01-3
    • anti-C4.4a B01-10
    • anti-C4.4a B01-7
    • anti-C4.4a D02-4
    • anti-C4.4a D02-6
    • anti-C4.4a D02-7
  • Three equivalents of tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) dissolved in PBS buffer were added to a solution of the corresponding antibody in PBS buffer in the concentration range between 1 mg/mL and 10 mg/mL and stirred for one hour at RT. Next, between 2 and 10 eq. of the maleimide precursor compound from intermediates 128, 129, 132-146, 148-155, 157, 159-161, 166, 171, 175-177, 184, 188, 190, 194-195, 199-201, 205, 209, 223-224, 226, 228-231, 236 and 244 to be coupled, depending on the desired load, were added as a solution in DMSO. The amount of DMSO should not exceed 10% of the total volume. The batch was stirred for 60-120 minutes at RT and then applied to PD10 columns (Sephadex® G-25, GE Healthcare) equilibrated in PBS and eluted with PBS buffer. If necessary, further concentration was performed by ultracentrifugation.
  • Unless otherwise indicated, 5 mg of the corresponding antibody in PBS buffer was generally used for reduction and for the subsequent coupling. After purification on the PD10 column, solutions of the corresponding ADC in 3.5 mL PBS buffer were obtained. The protein concentration indicated was then determined for each of these solutions. In addition, the load of the antibody (drug/mAb ratio) was determined according to the methods described below.
  • The immunoconjugates prepared in Examples 163-165, 167-192, 194-198, 200-221, 223-228, 230-232, 242, 244-247, 249, 250, 254-257, 259-260, 269, 271-275, 371 and 385 were produced by this method.
  • In the structural formulas shown, AK5A through AK5G have the meanings given below:
    • AK5A=anti-C4.4a antibody M31-B01 (partially reduced)-S§1
    • AK5B=anti-C4.4a antibody B01-3 (partially reduced)-S§1
    • AK5C=anti-C4.4a antibody B01-10 (partially reduced)-S§1
    • AK5D=anti-C4.4a antibody B01-7 (partially reduced)-S§1
    • AK5E=anti-C4.4a antibody D02-4 (partially reduced)-S§1
    • AK5F=anti-C4.4a antibody D02-6 (partially reduced)-S§1
    • AK5G=anti-C4.4a antibody D02-7 (partially reduced)-S§1
      wherein
    • §1 denotes the linkage to the succinimide group
    • and
    • S stands for the sulfur atom of a cysteine radical of the partially reduced antibody.
    B-3.5 General Method for Coupling to Lysine Side Chains:
  • The following antibodies were used in the coupling reactions:
    • anti-C4.4a antibody M31-B01
    • anti-C4.4a antibody B01-3
  • To a solution of the corresponding antibody in PBS buffer in the concentration range between 1 mg/mL and 10 mg/mL, between 2 and 5 eq. of the precursor compound to be coupled, depending on the desired load, from the intermediates 104, 110, 115, 116, 127, 130, 131, 147, 156, 158, 162, 169, 178, 185, 190, 202, 206, 210-216, 218-219, 227, 233, 238, 240, 242, 245, 247a and 247b were added as a solution in DMSO. After stirring for 30 minutes at RT, the same amount of precursor compound in DMSO was added again. In doing so, the amount of DMSO should not exceed 10% of the total volume. After stirring for 30 minutes more at RT, the batch was applied to PD10 columns (Sephadex® G-25) and eluted with PBS buffer. The batch was optionally concentrated further by ultrafiltration. For better separation of low-molecular components, the ultrafiltration concentration step was repeated after diluting with PBS buffer again, if necessary.
  • Usually, unless otherwise indicated, 5 mg of the corresponding antibody in PBS buffer was used for coupling. After purification on the PD10 column, solutions of the corresponding ADC in 3.5 mL PBS buffer were thus obtained. Then the specific protein concentration indicated was determined for these solutions, and the antibody load (drug/mAb ratio) was determined according to the methods described below.
  • The immunoconjugates synthesized in Examples 166, 193, 199, 222, 229, 243, 248, 251-253, 258, 261-268, 270, 276, 370, 372-373 and 386-388 were prepared by this method.
  • In the structural formulas given, AK6A and AK6B have the following meanings
    • AK6A=anti-C4.4a antibody M31-B01-NH§2
    • AK6B=anti-C4.4a antibody B01-3-NH§2
      wherein
    • §2 denotes the linkage to the carbonyl group
    • and
    • NH stands for the side chain amino group of a lysine radical of the antibody.
    B-3.6 General Method for Synthesis of Cysteine Adducts:
  • 10 μmol of the maleimide precursor compounds described above was placed in 3 mL DMF and mixed with 2.1 mg (10 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and purified by preparative HPLC.
  • Cys in the structural formulas given has the meaning
  • Figure US20140127240A1-20140508-C00691
  • wherein
    • §3 denotes the linkage to the linker toxophore unit.
    B-3.6 General Method 2.3a for Synthesis of Cysteine Adducts:
  • 10 μmol of the maleimide precursor compounds described above was placed in 3 mL DMF and mixed with 2.1 mg (10 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and purified by preparative HPLC.
  • Cys in the structural formulas given has the meaning
  • Figure US20140127240A1-20140508-C00692
  • wherein
    • §3 denotes the linkage to the linker toxophore unit.
    Further Purification and Characterization of the Conjugates According to the Invention
  • After successful reaction, the reaction mixture was concentrated further in some cases by ultracentrifugation, for example, and then was desalinated and purified by chromatography, for example, using a Sephadex® G-25 column. Then elution was performed using phosphate-buffered saline solution (PBS), for example. Then the solution was sterile filtered and frozen. Alternatively, the conjugate may be lyophilized.
    • B-3.7 Determining the Toxophore Load
  • The toxophore load was determined as follows on the resulting solutions of the conjugates in PBS buffer, as described in the exemplary embodiments:
  • The toxophore load of lysine-linked ADCs was determined by mass spectrometric determination of the molecular weights of the individual conjugated species. The antibody conjugates were first deglycosylated by PNGaseF, the sample was acidified and then, after HPLC separation, the sample was analyzed by mass spectrometry using ESI-MicroTofQ (Bruker Daltonik). All the spectra over the signal in the TIC (total ion chromatogram) were added up and the molecular weights of the various conjugate species were calculated based on MaxEnt deconvolution. The DAR (drug/antibody ratio) was then calculated after signal integration of the various species.
  • For protein identification, after deglycosylation and/or denaturing, in addition to determination of the molecular weight, tryptic digestion was performed, confirming the identity of the protein on the basis of the tryptic peptides identified after denaturing, reduction and derivatization.
  • The toxophore load of cysteine-linked conjugates was determined by reversed-phase chromatography of the reduced and dentured ADCs. Guanidinium hydrochloride (GuHCl, 28.6 mg) and a solution of DL-dithiothreitol (DTT, 500 mM, 3 μL) were added to the ADC solution (1 mg/mL, 50 μL). The mixture was then incubated for one hour at 55° C. and then analyzed by HPLC.
  • The HPLC analysis was performed on an adjuvant 1260 HPLC system with detection at 220 nm, using a Polymer Laboratories PLRP-S polymeric reversed-phase column (catalog number PL1912-3802) (2.1×150 mm, 8 μm particle size, 1000 Å) at a flow rate of 1 mL/min with the following gradient: 0 min, 25% B; 3 min, 25% B; 28 min, 50% B. Eluent A consisted of 0.05% trifluoroacetic acid (TFA) in water; eluent B consisted of 0.05% trifluoroacetic acid in acetonitrile.
  • The peaks detected were assigned on the basis of a comparison of the retention times with the light chain (L0) and the heavy chain (H0) of the unconjugated antibody. Peaks detected exclusively in the conjugated sample were assigned to the light chain with one toxophore (L1) and to the heavy chains with one, two and three toxophores (H1, H2, H3). The average toxophore load of the antibody was calculated from the peak areas determined by integration as two times the sum of the integration results of all peaks, weighted by the number of toxophores, dividing by the total of the integration results of all peaks with simple weighting. In isolated cases, it may happen that the toxophore load cannot be determined accurately due to co-elution of some peaks.
    • B-3.8 Testing the Antigen Binding of the ADC
  • The ability of the binder to bind to the target molecule was tested after successful coupling. Those skilled in the art are familiar with a variety of methods for doing so. For example, the affinity of the conjugate can be tested by means of ELISA technology or surface plasmon resonance analysis (BIAcore™ measurements). The skilled person can measure the conjugate concentration using conventional methods, e.g., by protein determination for antibody conjugates (see also Doronina et al., Nature Biotechnol. 2003; 21:778-784 and Polson et al., Blood 2007, 1102:616-623).
    • B4 Producing Antibody-Drug Conjugates (ADCs)
  • The intermediates described above were linked to the anti-CA9 antibody (3ee9), for example, with the linkage taking place optionally by way of the cysteine or lysine side chains of the antibody protein using the methods described below.
    • B-4.1 General Method for Generating Anti-CA9 Antibodies
  • The antibodies to CA9, e.g., the antibody 3ee9, were obtained by panning the HuCAL GOLD phage display library for recombinant antigen. The Fab antibody fragments thereby isolated were recloned to the IgG format (WO 2007/070538 A2).
    • B-4.2 General Method for Expression of Anti-CA9 Antibodies in Mammalian Cells and for Purification
  • The anti-CA9 IgG antibodies, e.g., 3ee9, were expressed by transient transfection of HEK 293 and purified from their cell supernatants by methods familiar to those skilled in the art. These methods are described in WO 2007/070538 A2.
    • B-4.3 General Method for Coupling to Cysteine Side Chains
  • To a solution of the corresponding anti-CA9 antibody, e.g., 3ee9, which may be present PBS buffer or in Tris buffer in the concentration range between 1 mg/mL and 10 mg/mL, for example, three equivalents of tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) dissolved in PBS buffer were added and stirred for one hour at RT. Then, depending on the desired load, between 2 and 10 eq. of the maleimide precursor compound or the halide precursor compound from intermediates 102, 103, 105-109, 111-114, 117-126, 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 to be coupled were added as a solution in DMSO. The amount of DMSO should not exceed 10% of the total volume. The batch was stirred for 60-120 minutes at RT, then applied to PD10 columns (Sephadex® G-25, GE Healthcare) and eluted with PBS buffer. A further concentration was optionally performed by ultracentrifugation. If necessary, for better separation of low-molecular components, the concentration step by ultrafiltration was repeated after diluting again with PBS buffer.
  • Unless otherwise indicated, 5 mg of the corresponding antibody was generally used in PBS buffer for reduction and subsequent coupling. After purification on the PD10 column, solutions of the corresponding ADC in 3.5 mL PBS buffer were obtained. Then the respective protein concentration given was determined for each of these solutions. In addition, the antibody load (drug/mAb ratio) was determined by the methods described below.
  • According to this method the immunoconjugates synthesized in Examples 280-289, 291-302, 304-305, 313, 315-318, 320-321, 324-325, 327-328 and 330-331 were prepared.
  • In the structural formulas shown, AK1 has the meaning
      • AK7=3ee9 (partially reduced)-S§1,
        wherein
    • §1 denotes the linkage to the succinimide group
    • 3ee9 (partially reduced) stands for the partially reduced anti-CA9 3ee9 antibody,
    • and
    • S stands for the sulfur atom of a cysteine radical of the partially reduced antibody.
    B-4.4 General Method for Coupling to Lysine Side Chains
  • To a solution of the corresponding anti-CA antibody 3ee9 in PBS buffer in the concentration range between 1 mg/mL and 10 mg/mL, between two and five equivalents, depending on the desired load, of the precursor compound of the intermediates 104, 110, 115, 116, 127, 130, 131, 147, 156, 158, 162, 169, 178, 185, 190, 202, 206, 210-216, 218, 219, 227, 233, 238, 240, 242, 245, 247a and 247b to be coupled were added as a solution in DMSO. After stirring for 30 minutes at RT, the same amount of precursor compound in DMSO was added again. The amount of DMSO should not exceed 10% of the total volume. After stirring for 30 minutes more at RT, the batch was applied to PD10 columns (Sephadex® G-25) and eluted with PBS buffer. A further concentration step by ultrafiltration was optionally performed. Concentration by ultrafiltration was repeated, if necessary, after diluting with PBS buffer again for better separation of the low-molecular components.
  • Unless otherwise indicated, 5 mg of the corresponding antibody in PBS buffer was generally used for coupling. After purification on the PD10 column, solutions of the corresponding ADC in 3.5 mL PBS buffer were obtained. Then the respective protein concentration indicated was determined for each of these solutions, and the antibody load (drug/mAb ratio) by the methods described below.
  • According to this method the immunoconjugates synthesized in Examples 290, 303, 306, 314, 319, 322-323, 326, 329, 332-333 and 384 were prepared.
  • In the structural formulas given, AK2 has the meaning
      • AK8=anti-CA9-NH§2
        wherein
    • §2 denotes the linkage to the carbonyl group
    • anti-CA9 stands for the unreduced CA9 antibody 3ee9,
    • and
    • NH stands for the side chain amino group of a lysine radical of the antibody.
    B-4.5a General Method for Synthesis of Cysteine Adducts
  • 10 μmol of the maleimide precursor compounds described above was placed in 3 mL DMF and mixed with 2.1 mg (20 μmol) L-cysteine. The batch was stirred for 2 hours at RT, then concentrated in vacuo and purified by preparative HPLC.
  • Cys in the structural formulas given has the following meaning:
  • Figure US20140127240A1-20140508-C00693
  • wherein
    • §3 denotes the linkage to the linker toxophore unit.
    B-4.5b General Method for Synthesis of Lysine Adducts:
  • 10 μmol of the active ester precursor compounds described above was placed in 5 mL DMF and mixed with α-amino-protected L-lysine in the presence of 30 μmol N,N-diisopropylethylamine. The reaction mixture was stirred for 2 hours at RT and then concentrated in vacuo and next was purified by preparative HPLC. Then the protective group was removed by known methods.
    • Further Purification and Characterization of the Conjugates According to the Invention
  • After successful reaction, in some cases the reaction mixture was concentrated by ultracentrifugation, for example, and then was desalinated and purified by chromatography for example, using a Sephadex® G-25 column. Elution was performed using phosphate-buffered saline solution (PBS), for example. Then the solution was sterile filtered and frozen. Alternatively, the conjugate may be lyophilized.
    • B-4.6 Determining the Toxophore Load
  • The toxophore load was determined as follows on the resulting solutions of the conjugates in PBS buffer as described in the exemplary embodiments:
  • The toxophore load of lysine-linked ADCs was determined by mass spectrometric determination of the molecular weights of the individual conjugated species. The antibody conjugates were first deglycosylated by PNGaseF, the sample was acidified and then, after HPLC separation, the sample was analyzed by mass spectrometry using ESI MicroTofQ (Bruker Daltonik). All the spectra over the signal in the TIC (total ion chromatogram) were added up and the molecular weights of the various conjugate species were calculated on the basis of MaxEnt deconvolution. After signal integration of the various species, the DAR (drug/antibody ratio) was calculated.
  • For protein identification, after deglycosylation and/or denaturing, tryptic digestion was performed in addition to determination of the molecular weight, with the identity of the protein being confirmed on the basis of the tryptic peptides identified after denaturing, reduction and derivatization.
  • The toxophore load of cysteine-linked conjugates was determined by reversed-phase chromatography of the reduced and dentured ADCs. Guanidinium hydrochloride (GuHCl, 28.6 mg) and a solution of DL-dithiothreitol (DTT, 500 mM, 3 μL) were added to the ADC solution (1 mg/mL, 50 μL). The mixture was then incubated for one hour at 55° C. and then analyzed by HPLC.
  • HPLC analysis was performed on an adjuvant 1260 HPLC system with detection at 220 nm, using a Polymer Laboratories PLRP-S polymeric reversed-phase column (catalog number PL1912-3802) (2.1×150 mm, 8 μm particle size, 1000 Å) at a flow rate of 1 mL/min with the following gradient: 0 min, 25% B; 3 min, 25% B; 28 min, 50% B. Eluent A was 0.05% trifluoroacetic acid (TFA) in water, and eluent B was 0.05% trifluoroacetic acid in acetonitrile.
  • The peaks detected were assigned based on a comparison of the retention times with the light chain (L0) and the heavy chain (H0) of the unconjugated antibody. Peaks detected exclusively in the conjugated sample were assigned to the light chain with one toxophore (L1) and to the heavy chains with one, two and three toxophores (H1, H2, H3).
  • The average toxophore load of the antibody was calculated from the peak areas determined by integration as two times the sum of the integration results of all peaks, weighted by the number of toxophores, divided by the total of the integration results of all peaks with simple weighting. In isolated cases it may occur that the toxophore load cannot be determined accurately due to co-elution of some peaks.
    • B-4.7 Testing the Antigen Binding of the ADC
  • The ability of the binder to bind to the target molecule was tested after successful coupling. Those skilled in the art are familiar with a variety of methods for doing so; for example, the affinity of the conjugate can be tested by means of ELISA technology or surface plasmon resonance analysis (BIAcore™ measurements). The skilled person can measure the conjugate concentration using conventional methods, for example, by protein determination for antibody conjugates (see also Doronina et al., Nature Biotechnol. 2003; 21:778-784 and Polson et al., Blood 2007, 1102:616-623).
    • EMBODIMENTS Example 1
  • Figure US20140127240A1-20140508-C00694
  • Protein concentration: 1.27 mg/ml
  • Drug/mAb ratio: 1.5
    • Example 2
  • Figure US20140127240A1-20140508-C00695
  • Protein concentration: 0.64 mg/ml
  • Drug/mAb ratio: 3.3
    • Example 3
  • Figure US20140127240A1-20140508-C00696
  • Protein concentration: 0.61 mg/ml
  • Drug/mAb ratio: 5.1
    • Example 4
  • Figure US20140127240A1-20140508-C00697
  • Protein concentration: 0.61 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 5
  • Figure US20140127240A1-20140508-C00698
  • Protein concentration: 1.37 mg/ml
  • Drug/mAb ratio: 1.9
    • Example 6
  • Figure US20140127240A1-20140508-C00699
  • Protein concentration: 1.12 mg/ml
  • Drug/mAb ratio: 1.3
    • Example 7
  • Figure US20140127240A1-20140508-C00700
  • 270 mg cetuximab in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation
  • Protein concentration: 10.46 mg/ml
  • Drug/mAb ratio: 2.8
    • Example 8
  • Figure US20140127240A1-20140508-C00701
  • Protein concentration: 1.86 mg/ml
  • Drug/mAb ratio: 2.4
    • Example 9
  • Figure US20140127240A1-20140508-C00702
  • Protein concentration: 1.39 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 10
  • Figure US20140127240A1-20140508-C00703
  • Protein concentration: 0.66 mg/ml
  • Drug/mAb ratio: 2.4
    • Example 11
  • Figure US20140127240A1-20140508-C00704
  • Protein concentration: 0.66 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 12
  • Figure US20140127240A1-20140508-C00705
  • Protein concentration: 0.9 mg/ml
  • Drug/mAb ratio: 1.1
    • Example 13
  • Figure US20140127240A1-20140508-C00706
  • Protein concentration: 1.52 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 14
  • Figure US20140127240A1-20140508-C00707
  • Protein concentration: 1.44 mg/ml
  • Drug/mAb ratio: 3.2
    • Example 15
  • Figure US20140127240A1-20140508-C00708
  • Protein concentration: 1.23 mg/ml
  • Drug/mAb ratio: 2.7
    • Example 16
  • Figure US20140127240A1-20140508-C00709
  • Protein concentration: 1.27 mg/ml
  • Drug/mAb ratio: 1.3
    • Example 17
  • Figure US20140127240A1-20140508-C00710
  • Protein concentration: 1.61 mg/ml
  • Drug/mAb ratio: 4.7
    • Example 18
  • Figure US20140127240A1-20140508-C00711
  • Protein concentration: 1.24 mg/ml
  • Drug/mAb ratio: 2.4
    • Example 19
  • Figure US20140127240A1-20140508-C00712
  • Protein concentration: 1.49 mg/ml
  • Drug/mAb ratio: 1.9
    • Example 20
  • Figure US20140127240A1-20140508-C00713
  • Protein concentration: 1.49 mg/ml
  • Drug/mAb ratio: 2.0
    • Example 21
  • Figure US20140127240A1-20140508-C00714
  • Protein concentration: 1.46 mg/ml
  • Drug/mAb ratio: >0.9
    • Example 22
  • Figure US20140127240A1-20140508-C00715
  • Protein concentration: 1.28 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 23
  • Figure US20140127240A1-20140508-C00716
  • Protein concentration: 1.33 mg/ml
  • Drug/mAb ratio: 1.8
    • Example 24
  • Figure US20140127240A1-20140508-C00717
  • Protein concentration: 1.39 mg/ml
  • Drug/mAb ratio: >0.8
    • Example 25
  • Figure US20140127240A1-20140508-C00718
  • Protein concentration: 1.26 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 26
  • Figure US20140127240A1-20140508-C00719
  • Protein concentration: 1.51 mg/ml
  • Drug/mAb ratio: 1.8
    • Example 27
  • Figure US20140127240A1-20140508-C00720
  • Protein concentration: 1.6 mg/ml
  • Drug/mAb ratio: 1.8
    • Example 28
  • Figure US20140127240A1-20140508-C00721
  • Protein concentration: 1.21 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 29
  • Figure US20140127240A1-20140508-C00722
  • Protein concentration: 1.53 mg/ml
  • Drug/mAb ratio: 2.7
    • Example 30
  • Figure US20140127240A1-20140508-C00723
  • Protein concentration: 1.4 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 31
  • Figure US20140127240A1-20140508-C00724
  • Protein concentration: 1.66 mg/ml
  • Drug/mAb ratio: 2.2
    • Example 32
  • Figure US20140127240A1-20140508-C00725
  • Protein concentration: 1.21 mg/ml
  • Drug/mAb ratio: 2.2
    • Example 33
  • Figure US20140127240A1-20140508-C00726
  • Protein concentration: 1.46 mg/ml
  • Drug/mAb ratio: 2
    • Example 34
  • Figure US20140127240A1-20140508-C00727
  • Protein concentration: 1.2 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 35
  • Figure US20140127240A1-20140508-C00728
  • Protein concentration: 1.66 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 36
  • Figure US20140127240A1-20140508-C00729
  • Protein concentration: 1.48 mg/ml
  • Drug/mAb ratio: 2.2
    • Example 37
  • Figure US20140127240A1-20140508-C00730
  • Protein concentration: 1.45 mg/ml
  • Drug/mAb ratio: 2.7
    • Example 38
  • Figure US20140127240A1-20140508-C00731
  • Protein concentration: 1.5 mg/ml
  • Drug/mAb ratio: 0.15
    • Example 39
  • Figure US20140127240A1-20140508-C00732
  • Protein concentration: 1.5 mg/ml
  • Drug/mAb ratio: 2.1
    • Example 40
  • Figure US20140127240A1-20140508-C00733
  • Protein concentration: 1.54 mg/ml
  • Drug/mAb ratio: >0.9
    • Example 41
  • Figure US20140127240A1-20140508-C00734
  • Protein concentration: 1.39 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 42
  • Figure US20140127240A1-20140508-C00735
  • Protein concentration: 1.52 mg/ml
  • Drug/mAb ratio: 1.5
    • Example 43
  • Figure US20140127240A1-20140508-C00736
  • Protein concentration: 1.44 mg/ml
  • Drug/mAb ratio: 2.6
    • Example 44
  • Figure US20140127240A1-20140508-C00737
  • Protein concentration: 1.45 mg/ml
  • Drug/mAb ratio: 1.9
    • Examples for Cysteine Adducts Example 45 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00738
  • 10 mg (10 μmol) of Intermediate 113 were taken up in 5.2 ml DMF and mixed with 2.28 mg (20 μmol) L-cysteine. The batch was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 6 mg (54% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.5 min;
  • LC-MS (Method 1): Rt=0.77 min; MS (ESIpos): m/z=1185 (M+H)+.
    • Example 46 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00739
  • 9 mg (8.3 μmol) of Intermediate 132 were taken up in 4 ml DMF and mixed with 3 mg (24.4 μmol) L-cysteine. The batch was stirred overnight at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 6.8 mg (68% of theory) of the title compound.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.78 min; MS (ESIpos): m/z=1227 (M+H)+.
    • Example 47 N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00740
  • 10 mg (10 μmol) of Intermediate 106 were taken up in 5.8 ml DMF and mixed with 2.5 mg (20 μmol) L-cysteine. The batch was stirred for 2 hours at RT, then concentrated in vacuo and next
  • HPLC (Method 5) R=1.5 min;
  • LC-MS (Method 11): Rt=0.71 min; MS (ESIpos): m/z=1070 (M+H)+.
    • Example 48 N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00741
  • 10 mg (10 μmol) of Intermediate 124 were taken up in 4 ml DMF and mixed with 2.5 mg (20 μmol) L-cysteine. The batch was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 7.2 mg (64% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.8 min; MS (ESIpos): m/z=1071 (M+H)+.
    • Example 49 N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00742
  • 10 mg (10 μmol) of Intermediate 125 were taken up in 4 ml DMF and mixed with 2.4 mg (20 μmol) L-cysteine. The batch was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 7.7 mg (69% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 2): Rt=1.91 min; MS (ESIpos): m/z=1140 (M+H)+.
    • Example 50 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-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-valinamide
  • Figure US20140127240A1-20140508-C00743
  • 10 mg (10 μmol) of Intermediate 160 were taken up in 3 ml DMF and mixed with 2.1 mg (20 μmol) L-cysteine. The batch was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 8.1 mg (73% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=1274 (M+H)+.
    • Example 51 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)—{(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-valinamide
  • Figure US20140127240A1-20140508-C00744
  • 10 mg (10 μmol) of Intermediate 157 were taken up in 5.2 ml DMF and mixed with 2.28 mg (20 μmol) L-cysteine. The batch was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 5.8 mg (48% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.45 min;
  • LC-MS (Method 1): Rt=0.74 min; MS (ESIpos): m/z=1184 (M+H)+.
    • Example 52
  • Figure US20140127240A1-20140508-C00745
  • 5 mg cetuximab in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.73 mg/ml
  • Drug/mAb ratio: 2.8
    • Example 53
  • Figure US20140127240A1-20140508-C00746
  • 5 mg cetuximab in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 0.86 mg/ml
  • Drug/mAb ratio: 4.9
    • Example 54
  • Figure US20140127240A1-20140508-C00747
  • 5 mg cetuximab in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.64 mg/ml
  • Drug/mAb ratio: 0.7
    • Example 55
  • Figure US20140127240A1-20140508-C00748
  • 5 mg cetuximab in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.43 mg/ml
  • Drug/mAb ratio: 3.2
    • Example 56
  • Figure US20140127240A1-20140508-C00749
  • Protein concentration: 0.96 mg/ml
  • Drug/mAb ratio: 3.1
    • Example 57
  • Figure US20140127240A1-20140508-C00750
  • Protein concentration: 0.44 mg/ml
  • Drug/mAb ratio: 4.6
    • Example 58
  • Figure US20140127240A1-20140508-C00751
  • Protein concentration: 1.09 mg/ml
  • Drug/mAb ratio: 2.1
    • Example 59
  • Figure US20140127240A1-20140508-C00752
  • Protein concentration: 0.87 mg/ml
  • Drug/mAb ratio: 3.8
    • Example 60
  • Figure US20140127240A1-20140508-C00753
  • Protein concentration: 0.45 mg/ml
  • Drug/mAb ratio: 6.5
    • Example 61
  • Figure US20140127240A1-20140508-C00754
  • Protein concentration: 0.15 mg/ml
  • Drug/mAb ratio: 3.1
    • Example 62
  • Figure US20140127240A1-20140508-C00755
  • Protein concentration: 0.94 mg/ml
  • Drug/mAb ratio: 2.8
    • Example 63
  • Figure US20140127240A1-20140508-C00756
  • Protein concentration: 0.45 mg/ml
  • Drug/mAb ratio: 0.9
    • Example 64
  • Figure US20140127240A1-20140508-C00757
  • Protein concentration: 0.51 mg/ml
  • Drug/mAb ratio: 6.6
    • Example 65
  • Figure US20140127240A1-20140508-C00758
  • Protein concentration: 0.47 mg/ml
  • Drug/mAb ratio: 4.2
    • Example 66
  • Figure US20140127240A1-20140508-C00759
  • Protein concentration: 0.45 mg/ml
  • Drug/mAb ratio: 5.9
    • Example 67
  • Figure US20140127240A1-20140508-C00760
  • Protein concentration: 0.47 mg/ml
  • Drug/mAb ratio: 3.3
    • Example 68
  • Figure US20140127240A1-20140508-C00761
  • Protein concentration: 0.53 mg/ml
  • Drug/mAb ratio: 2.8
    • Example 69
  • Figure US20140127240A1-20140508-C00762
  • Protein concentration: 0.92 mg/ml
  • Drug/mAb ratio: 3.5
    • Example 70
  • Figure US20140127240A1-20140508-C00763
  • Protein concentration: 0.09 mg/ml
  • Drug/mAb ratio: nd
    • Example 71
  • Figure US20140127240A1-20140508-C00764
  • Protein concentration: 0.62 mg/ml
  • Drug/mAb ratio: 1.8
    • Example 72
  • Figure US20140127240A1-20140508-C00765
  • Protein concentration: 0.55 mg/ml
  • Drug/mAb ratio: 3.8
    • Example 73
  • Figure US20140127240A1-20140508-C00766
  • Protein concentration: 0.54 mg/ml
  • Drug/mAb ratio: 4.4
    • Example 74
  • Figure US20140127240A1-20140508-C00767
  • Protein concentration: 0.56 mg/ml
  • Drug/mAb ratio: 4.0
    • Example 75
  • Figure US20140127240A1-20140508-C00768
  • Protein concentration: 1.1 mg/ml
  • Drug/mAb ratio: 0.3
    • Example 76
  • Figure US20140127240A1-20140508-C00769
  • Protein concentration: 0.61 mg/ml
  • Drug/mAb ratio: 0.9
    • Example 77
  • Figure US20140127240A1-20140508-C00770
  • Protein concentration: 0.57 mg/ml
  • Drug/mAb ratio: 1.2
    • Example 78
  • Figure US20140127240A1-20140508-C00771
  • 100 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation.
  • Protein concentration: 11.2 mg/ml
  • Drug/mAb ratio: 3.4
    • Example 79
  • Figure US20140127240A1-20140508-C00772
  • Protein concentration: 1.56 mg/ml
  • Drug/mAb ratio: 2.8
    • Example 80
  • Figure US20140127240A1-20140508-C00773
  • Protein concentration: 0.60 mg/ml
  • Drug/mAb ratio: 2.4
    • Example 81
  • Figure US20140127240A1-20140508-C00774
  • Protein concentration: 0.584 mg/ml
  • Drug/mAb ratio: 2.6
    • Example 82
  • Figure US20140127240A1-20140508-C00775
  • Protein concentration: 0.39 mg/ml
  • Drug/mAb ratio: 0.8
    • Example 83
  • Figure US20140127240A1-20140508-C00776
  • 100 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation.
  • Protein concentration: 13.2 mg/ml
  • Drug/mAb ratio: 4.6
    • Example 84
  • Figure US20140127240A1-20140508-C00777
  • Protein concentration: 0.98 ml
  • Drug/mAb ratio: 1.1
    • Example 85
  • Figure US20140127240A1-20140508-C00778
  • Protein concentration: 0.55 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 86
  • Figure US20140127240A1-20140508-C00779
  • 40 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation.
  • Protein concentration: 10.6 mg/ml
  • Drug/mAb ratio: 4.1
    • Example 87
  • Figure US20140127240A1-20140508-C00780
  • Protein concentration: 0.96 mg/ml
  • Drug/mAb ratio: 0.4
    • Example 88
  • Figure US20140127240A1-20140508-C00781
  • 70 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation.
  • Protein concentration: 12.7 mg/ml
  • Drug/mAb ratio: 3.6
    • Example 89
  • Figure US20140127240A1-20140508-C00782
  • Protein concentration: 1.1 mg/ml
  • Drug/mAb ratio: 2.7
    • Example 90
  • Figure US20140127240A1-20140508-C00783
  • Protein concentration: 1.24 mg/ml
  • Drug/mAb ratio: 2.6
    • Example 91
  • Figure US20140127240A1-20140508-C00784
  • Protein concentration: 0.99 mg/ml
  • Drug/mAb ratio: 2.3
    • Example 92
  • Figure US20140127240A1-20140508-C00785
  • Protein concentration: 1.22 mg/ml
  • Drug/mAb ratio: 3.3
    • Example 93
  • Figure US20140127240A1-20140508-C00786
  • Protein concentration: 1.34 mg/ml
  • Drug/mAb ratio: 1.2
    • Example 94
  • Figure US20140127240A1-20140508-C00787
  • Protein concentration: 1.28 mg/ml
  • Drug/mAb ratio: 3.2
    • Example 95
  • Figure US20140127240A1-20140508-C00788
  • 70 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation.
  • Protein concentration: 10.9 mg/ml
  • Drug/mAb ratio: 5.1
    • Example 96
  • Figure US20140127240A1-20140508-C00789
  • 100 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation.
  • Protein concentration: 10.3 mg/ml
  • Drug/mAb ratio: 4.3
    • Example 97
  • Figure US20140127240A1-20140508-C00790
  • Protein concentration: 1.08 mg/ml
  • Drug/mAb ratio: 2.8
    • Example 98
  • Figure US20140127240A1-20140508-C00791
  • Protein concentration: 1.24 mg/ml
  • Drug/mAb ratio: 2.8
    • Example 99
  • Figure US20140127240A1-20140508-C00792
  • Protein concentration: 1.28 mg/ml
  • Drug/mAb ratio: 3.8
    • Example 100
  • Figure US20140127240A1-20140508-C00793
  • Protein concentration: 1.07 mg/ml
  • Drug/mAb ratio: 3.0
    • Example 101
  • Figure US20140127240A1-20140508-C00794
  • Protein concentration: 1.35 mg/ml
  • Drug/mAb ratio: 4.0
    • Example 102
  • Figure US20140127240A1-20140508-C00795
  • 100 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation.
  • Protein concentration: 12.2 mg/ml
  • Drug/mAb ratio: 5.6
    • Example 103
  • Figure US20140127240A1-20140508-C00796
  • Protein concentration: 1.32 mg/ml
  • Drug/mAb ratio: 3.2
    • Example 104
  • Figure US20140127240A1-20140508-C00797
  • Protein concentration: 1.01 mg/ml
  • Drug/mAb ratio: 0.9
    • Example 105
  • Figure US20140127240A1-20140508-C00798
  • Protein concentration: 1.03 mg/ml
  • Drug/mAb ratio: 0.3
    • Example 106
  • Figure US20140127240A1-20140508-C00799
  • Protein concentration: 0.62 mg/ml
  • Drug/mAb ratio: 3.1
  • This ADC was concentrated by Vivaspin centrifugation and diluted again, followed by another concentration and dilution.
    • Example 107
  • Figure US20140127240A1-20140508-C00800
  • Protein concentration: 1.26 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 108
  • Figure US20140127240A1-20140508-C00801
  • Protein concentration: 1.55 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 109
  • Figure US20140127240A1-20140508-C00802
  • Protein concentration: 1.23 mg/ml
  • Drug/mAb ratio: 3.5
    • Example 110
  • Figure US20140127240A1-20140508-C00803
  • Protein concentration: 1.44 mg/ml
  • Drug/mAb ratio: 4.1
    • Example 111
  • Figure US20140127240A1-20140508-C00804
  • Protein concentration: 0.92 mg/ml
  • Drug/mAb ratio: 3.5
    • Example 112
  • Figure US20140127240A1-20140508-C00805
  • 5 mg MF-T in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 0.77 mg/ml
  • Drug/mAb ratio: >1.5 (not exactly detectable)
    • Example 113
  • Figure US20140127240A1-20140508-C00806
  • Protein concentration: 1.3 mg/ml
  • Drug/mAb ratio: 2.0
    • Example 114
  • Figure US20140127240A1-20140508-C00807
  • 500 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation.
  • Protein concentration: 11.2 mg/ml
  • Drug/mAb ratio: 3.7
    • Example 115
  • Figure US20140127240A1-20140508-C00808
  • 100 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation.
  • Protein concentration: 11.4 mg/ml
  • Drug/mAb ratio: 3.9
    • Example 116
  • Figure US20140127240A1-20140508-C00809
  • 60 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation.
  • Protein concentration: 10.5 mg/ml
  • Drug/mAb ratio: 4.4
    • Example 117 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00810
  • 10 mg (10 μmol) of Intermediate 157 were taken up in 5.2 ml DMF and mixed with 2.28 mg (20 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 5.8 mg (48% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.45 min;
  • LC-MS (Method 1): Rt=0.74 min; MS (ESIpos): m/z=1184 (M+H)+.
    • Example 118 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00811
  • 10 mg (10 μmol) of Intermediate 113 were taken up in 5.2 ml DMF and mixed with 2.28 mg (20 mmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 6 mg (54% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.5 min;
  • LC-MS (Method 1): Rt=0.77 min; MS (ESIpos): m/z=1185 (M+H)+.
    • Example 119 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00812
  • 9 mg (8.3 μmol) of Intermediate 132 were taken up in 4 ml DMF and mixed with 3 mg (24.4 μmol) L-cysteine. The reaction mixture was stirred overnight at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 6.8 mg (68% of theory) of the title compound.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.78 min; MS (ESIpos): m/z=1227 (M+H)+.
    • Example 120 N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-{(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-valinamide
  • Figure US20140127240A1-20140508-C00813
  • 10 mg (10 μmol) of Intermediate 106 were taken up in 5.8 ml DMF and mixed with 2.5 mg (20 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 5.2 mg (46% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.5 min;
  • LC-MS (Method 11): Rt=0.71 min; MS (ESIpos): m/z=1070 (M+H)+.
    • Example 121 N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00814
  • 10 mg (10 μmol) of Intermediate 124 were taken up in 4 ml DMF and mixed with 2.5 mg (20 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 7.2 mg (64% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.8 min; MS (ESIpos): m/z=1071 (M+H)+.
    • Example 122 N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00815
  • 10 mg (10 μmol) of Intermediate 125 were taken up in 4 ml DMF and mixed with 2.4 mg (20 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 7.7 mg (69% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 2): Rt=1.91 min; MS (ESIpos): m/z=1140 (M+H)+.
    • Example 123 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-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-valinamide
  • Figure US20140127240A1-20140508-C00816
  • 10 mg (10 μmol) of Intermediate 160 were taken up in 3 ml DMF and mixed with 2.1 mg (20 mmol) L-cysteinet. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 8.1 mg (73% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=1274 (M+H)+.
    • Example 124 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide
  • Figure US20140127240A1-20140508-C00817
  • 3.5 mg (3 μmol) of Intermediate 159 were taken up in 1 ml DMF and mixed with 0.76 mg (6 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 2.6 mg (65% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.75 min;
  • LC-MS (Method 1): Rt=0.85 min; MS (ESIpos): m/z=1235 (M+H)+.
    • Example 125 N-(6-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-6-oxohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00818
  • 3.6 mg (3 μmol) of Intermediate 129 were taken up in 1 ml DMF and mixed with 0.77 mg (6 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 1.55 mg (39% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=1255 (M+H).
    • Example 126
  • Figure US20140127240A1-20140508-C00819
  • 5 mg MF-T[a] in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 0.9 mg/ml
  • Drug/mAb ratio: 1
    • Example 127
  • Figure US20140127240A1-20140508-C00820
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.86 mg/ml
  • Drug/mAb ratio: 2.9
    • Example 128
  • Figure US20140127240A1-20140508-C00821
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.05 mg/ml
  • Drug/mAb ratio: 4.4
    • Example 129
  • Figure US20140127240A1-20140508-C00822
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.13 mg/ml
  • Drug/mAb ratio: 2.8
    • Example 130
  • Figure US20140127240A1-20140508-C00823
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.41 mg/ml
  • Drug/mAb ratio: 3.9
    • Example 131
  • Figure US20140127240A1-20140508-C00824
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.38 mg/ml
  • Drug/mAb ratio: 4.3
    • Example 132
  • Figure US20140127240A1-20140508-C00825
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.32 mg/ml
  • Drug/mAb ratio: 1
    • Example 133
  • Figure US20140127240A1-20140508-C00826
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.14 mg/ml
  • Drug/mAb ratio: 5.3
    • Example 134
  • Figure US20140127240A1-20140508-C00827
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.25 mg/ml
  • Drug/mAb ratio: 4.8
    • Example 135
  • Figure US20140127240A1-20140508-C00828
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.12 mg/ml
  • Drug/mAb ratio: 1.7
    • Example 136
  • Figure US20140127240A1-20140508-C00829
  • 150 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation, diluted again with PBS and concentrated again.
  • Protein concentration: 12.2 mg/ml
  • Drug/mAb ratio: 4.1
    • Example 137
  • Figure US20140127240A1-20140508-C00830
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 0.86 mg/ml
  • Drug/mAb ratio: 3.4
    • Example 138
  • Figure US20140127240A1-20140508-C00831
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.43 mg/ml
  • Drug/mAb ratio: 3.7
    • Example 139
  • Figure US20140127240A1-20140508-C00832
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 0.8 mg/ml
  • Drug/mAb ratio: 0.7
    • Example 140
  • Figure US20140127240A1-20140508-C00833
  • 50 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation, diluted again with PBS and reconcentrated.
  • Protein concentration: 9.5 mg/ml
  • Drug/mAb ratio: 2.9
    • Example 141
  • Figure US20140127240A1-20140508-C00834
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.52 mg/ml
  • Drug/mAb ratio: 3.2
    • Example 142
  • Figure US20140127240A1-20140508-C00835
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.25 mg/ml
  • Drug/mAb ratio: 4.6
    • Example 143
  • Figure US20140127240A1-20140508-C00836
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.47 mg/ml
  • Drug/mAb ratio: 1.6
    • Example 144
  • Figure US20140127240A1-20140508-C00837
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 0.99 mg/ml
  • Drug/mAb ratio: 5.5
    • Example 145
  • Figure US20140127240A1-20140508-C00838
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.02 mg/ml
  • Drug/mAb ratio: 4.0
    • Example 146
  • Figure US20140127240A1-20140508-C00839
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.63 mg/ml
  • Drug/mAb ratio: 3.8
    • Example 147
  • Figure US20140127240A1-20140508-C00840
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.27 mg/ml
  • Drug/mAb ratio: 3.0
    • Example 148
  • Figure US20140127240A1-20140508-C00841
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.58 mg/ml
  • Drug/mAb ratio: 0.6
    • Example 149
  • Figure US20140127240A1-20140508-C00842
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.31 mg/ml
  • Drug/mAb ratio: 6.6
    • Example 150
  • Figure US20140127240A1-20140508-C00843
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.75 mg/ml
  • Drug/mAb ratio: 1.8
    • Example 151
  • Figure US20140127240A1-20140508-C00844
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.44 mg/ml
  • Drug/mAb ratio: 2.5
    • Example 152
  • Figure US20140127240A1-20140508-C00845
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.96 mg/ml
  • Drug/mAb ratio: 5.6
    • Example 153
  • Figure US20140127240A1-20140508-C00846
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.58 mg/ml
  • Drug/mAb ratio: 4.2
    • Example 154
  • Figure US20140127240A1-20140508-C00847
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.48 mg/ml
  • Drug/mAb ratio: 4.6
    • Example 155
  • Figure US20140127240A1-20140508-C00848
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.5 mg/ml
  • Drug/mAb ratio: 3.1
    • Example 156
  • Figure US20140127240A1-20140508-C00849
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: mg/ml
  • Drug/mAb ratio:
    • Example 157
  • Figure US20140127240A1-20140508-C00850
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.62 mg/ml
  • Drug/mAb ratio: 2.2
    • Example 158
  • Figure US20140127240A1-20140508-C00851
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.37 mg/ml
  • Drug/mAb ratio: 2.8
    • Example 159
  • Figure US20140127240A1-20140508-C00852
  • 5 mg MF-Ta in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.43 mg/ml
  • Drug/mAb ratio: 4.0
    • Example 160** N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00853
  • 15.5 mg (15 μmol) of Intermediate 210 were taken up in 5 ml DMF and mixed with 4.4 mg (18 μmol) N2-(tert.-butoxycarbonyl)-L-lysine and 7.7 μL (44 μmol) N,N-diisopropylethylamine. The reaction mixture was stirred overnight at RT, then concentrated in vacuo. The residue was purified by means of preparative HPLC. The yield was 14 mg (81% of theory) of the protected intermediate of the title compound, which were then taken up in 1 ml dichloromethane and deprotected with 1 ml trifluoroascetic acid. The reaction mixture was concentrated and, after lyophilization of the residue from acetonitrile/wasser 1:1, 15 mg (97% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.79 min; MS (ESIpos): m/z=1083 (M+H)+.
    • Example 161 N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00854
  • 40 mg (40 μmol) of Intermediate 226 were taken up in 5 ml DMF and mixed with 11.5 mg (40 μmol) N2-[(benzyloxy)carbonyl]-L-lysine and 13 μl (80 μmol) N,N-diisopropylethylamine. The reaction mixture was stirred overnight at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 32.5 mg (70% of theory) of the protected intermediate of the title compound.
  • These 32.5 mg of the intermediate were dissolved in 10 ml methanol and, after adding 2 mg 10% palladium on activated carbon, hydrogenated for 30 min at RT under standard hydrogen pressure. The catalyst was then filtered off and the solvent removed in vacuo. Following lyophilization of the residue from dioxane/water 1:1, 26 mg (99% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.76 min; MS (ESIpos): m/z=1014 (M+H)+.
    • Example 162 N-[(18S)-18-amino-18-carboxy-12-oxo-3,6,9-trioxa-13-azaoctadec-1-yl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00855
  • 3.5 mg (3 μmol) of Intermediate 202 were taken up in 2 ml DMF and mixed with 0.8 mg (3 μmol) N2-(tert-butoxycarbonyl)-L-lysine and 1.6 μl (10 μmol) N,N-diisopropylethylamine. The reaction mixture was stirred overnight at RT and then concentrated in vacuo. The residue was taken up in acetonitrile/water 1:1, adjusted to pH 2 with trifluoroascetic acid and then purified by means of preparative HPLC. The yield was 1 mg (25% of theory) of the protected intermediate of the title compound that was then taken up in 500 μl dichloromethane and deprotected with 500 μl trifluoroascetic acid. The reaction mixture was concentrated and, following lyophilization of the residue from acetonitrile/water 1:1, 1 mg (89% of theory) of the title compound was obtained.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.82 min; MS (ESIpos): m/z=1173 (M+H)+.
    • Example 163
  • Figure US20140127240A1-20140508-C00856
  • 70 mg anti-C4.4a M31-B01 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 12.2 mg/ml
  • Drug/mAb ratio: 1.5
    • Example 164
  • Figure US20140127240A1-20140508-C00857
  • Protein concentration: 0.87 mg/ml
  • Drug/mAb ratio: 5.8
    • Example 165
  • Figure US20140127240A1-20140508-C00858
  • Protein concentration: 1.16 mg/ml
  • Drug/mAb ratio: 3.1
    • Example 166
  • Figure US20140127240A1-20140508-C00859
  • Protein concentration: 1.24 mg/ml
  • Drug/mAb ratio: 1.6
    • Example 167
  • Figure US20140127240A1-20140508-C00860
  • Protein concentration: 0.88 mg/ml
  • Drug/mAb ratio: 6.9
    • Example 168
  • Figure US20140127240A1-20140508-C00861
  • Protein concentration: 1.2 mg/ml
  • Drug/mAb ratio: 2.8
    • Example 169
  • Figure US20140127240A1-20140508-C00862
  • Protein concentration: 0.9 mg/ml
  • Drug/mAb ratio: 3.9
    • Example 170
  • Figure US20140127240A1-20140508-C00863
  • Protein concentration: 0.52 mg/ml
  • Drug/mAb ratio: 1.6
    • Example 171
  • Figure US20140127240A1-20140508-C00864
  • Protein concentration: 0.47 mg/ml
  • Drug/mAb ratio: 6.6
    • Example 172
  • Figure US20140127240A1-20140508-C00865
  • Protein concentration: 0.77 mg/ml
  • Drug/mAb ratio: 6.9
    • Example 173
  • Figure US20140127240A1-20140508-C00866
  • Protein concentration: 0.47 mg/ml
  • Drug/mAb ratio: 4.0
    • Example 174
  • Figure US20140127240A1-20140508-C00867
  • Protein concentration: 1.46 mg/ml
  • Drug/mAb ratio: 2.5
    • Example 175
  • Figure US20140127240A1-20140508-C00868
  • Protein concentration: 0.45 mg/ml
  • Drug/mAb ratio: 3.3
    • Example 176
  • Figure US20140127240A1-20140508-C00869
  • Protein concentration: 0.98 mg/ml
  • Drug/mAb ratio: 3.6
    • Example 177
  • Figure US20140127240A1-20140508-C00870
  • 70 mg anti-C4.4a M31-B01 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 9.42 mg/ml
  • Drug/mAb ratio: 4.1
    • Example 178
  • Figure US20140127240A1-20140508-C00871
  • Protein concentration: 0.65 mg/ml
  • Drug/mAb ratio: 1.8
    • Example 179
  • Figure US20140127240A1-20140508-C00872
  • Protein concentration: 1.07 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 180
  • Figure US20140127240A1-20140508-C00873
  • Protein concentration: 0.47 mg/ml
  • Drug/mAb ratio: 4.4
    • Example 181
  • Figure US20140127240A1-20140508-C00874
  • Protein concentration: 0.43 mg/ml
  • Drug/mAb ratio: 4.8
    • Example 182
  • Figure US20140127240A1-20140508-C00875
  • Protein concentration: 1.01 mg/ml
  • Drug/mAb ratio: 2.6
    • Example 183
  • Figure US20140127240A1-20140508-C00876
  • Protein concentration: 0.53 mg/ml
  • Drug/mAb ratio: 0.6
    • Example 184
  • Figure US20140127240A1-20140508-C00877
  • Protein concentration: 0.55 mg/ml
  • Drug/mAb ratio: 1.3
    • Example 185
  • Figure US20140127240A1-20140508-C00878
  • Protein concentration: 0.65 mg/ml
  • Drug/mAb ratio: 1.1
    • Example 186
  • Figure US20140127240A1-20140508-C00879
  • Protein concentration: 1.04
  • Drug/mAb ratio: 3.5
    • Example 187
  • Figure US20140127240A1-20140508-C00880
  • Protein concentration: 0.62 mg/ml
  • Drug/mAb ratio: 2.4
    • Example 188
  • Figure US20140127240A1-20140508-C00881
  • 90 mg anti-C4.4a M31-B01 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 11.2 mg/ml
  • Drug/mAb-Ratio: 2.3
    • Example 189
  • Figure US20140127240A1-20140508-C00882
  • Protein concentration: 1.11 mg/ml
  • Drug/mAb-Ratio: 2.4
    • Example 190
  • Figure US20140127240A1-20140508-C00883
  • 70 mg anti-C4.4a M31-B01 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 10.7 mg/ml
  • Drug/mAb ratio: 2.2
    • Example 191
  • Figure US20140127240A1-20140508-C00884
  • Protein concentration: 0.87 mg/ml
  • Drug/mAb ratio: 1.8
    • Example 192
  • Figure US20140127240A1-20140508-C00885
  • Protein concentration: 1.3 mg/ml
  • Drug/mAb ratio: 2.1
    • Example 193
  • Figure US20140127240A1-20140508-C00886
  • Protein concentration: 1.3 mg/ml
  • Drug/mAb ratio: 0.3
    • Example 194
  • Figure US20140127240A1-20140508-C00887
  • 70 mg anti-C4.4a M31-B01 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 12.0 mg/ml
  • Drug/mAb ratio: 3.2
    • Example 195
  • Figure US20140127240A1-20140508-C00888
  • 90 mg anti-C4.4a M31-B01 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 10.2 mg/ml
  • Drug/mAb ratio: 4.3
    • Example 196
  • Figure US20140127240A1-20140508-C00889
  • Protein concentration: 1.37 mg/ml
  • Drug/mAb ratio: 2.6
    • Example 197
  • Figure US20140127240A1-20140508-C00890
  • Protein concentration: 1.14 mg/ml
  • Drug/mAb ratio: 2.0
    • Example 198
  • Figure US20140127240A1-20140508-C00891
  • Protein concentration: 1.07 mg/ml
  • Drug/mAb ratio: 3.5
    • Example 199
  • Figure US20140127240A1-20140508-C00892
  • Protein concentration: 1.14 mg/ml
  • Drug/mAb ratio: 1.9
    • Example 200
  • Figure US20140127240A1-20140508-C00893
  • Protein concentration: 1.22 mg/ml
  • Drug/mAb ratio: 3.3
    • Example 201
  • Figure US20140127240A1-20140508-C00894
  • Protein concentration: 1.3 mg/ml
  • Drug/mAb ratio: 3.2
    • Example 202
  • Figure US20140127240A1-20140508-C00895
  • Protein concentration: 1.23 mg/ml
  • Drug/mAb ratio: 3.3
    • Example 203
  • Figure US20140127240A1-20140508-C00896
  • Protein concentration: 1.64 mg/ml
  • Drug/mAb ratio: 1.8
    • Example 204
  • Figure US20140127240A1-20140508-C00897
  • Protein concentration: 1.07 mg/ml
  • Drug/mAb ratio: 3.1
    • Example 205
  • Figure US20140127240A1-20140508-C00898
  • Protein concentration: 1.14 mg/ml
  • Drug/mAb ratio: 2.3
    • Example 206
  • Figure US20140127240A1-20140508-C00899
  • Protein concentration: 1.23 mg/ml
  • Drug/mAb ratio: 3.4
    • Example 207
  • Figure US20140127240A1-20140508-C00900
  • Protein concentration: 1.22 mg/ml
  • Drug/mAb ratio: 2.5
    • Example 208
  • Figure US20140127240A1-20140508-C00901
  • Protein concentration: 1.22 mg/ml
  • Drug/mAb ratio: 2.4
    • Example 209
  • Figure US20140127240A1-20140508-C00902
  • Protein concentration: 1.32 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 210
  • Figure US20140127240A1-20140508-C00903
  • Protein concentration: 1.44 mg/ml
  • Drug/mAb ratio: 2.3
    • Example 211
  • Figure US20140127240A1-20140508-C00904
  • 250 mg anti-C4.4a B01-10 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 12.8 mg/ml
  • Drug/mAb ratio: 5.2
    • Example 212
  • Figure US20140127240A1-20140508-C00905
  • Protein concentration: 0.9 mg/ml
  • Drug/mAb ratio: 2
    • Example 213
  • Figure US20140127240A1-20140508-C00906
  • 250 mg anti-C4.4a B01-3 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 8.0 mg/ml
  • Drug/mAb ratio: 4.5
    • Example 214
  • Figure US20140127240A1-20140508-C00907
  • 250 mg anti-C4.4a B01-10 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 12.3 mg/ml
  • Drug/mAb ratio: 5.2
    • Example 215
  • Figure US20140127240A1-20140508-C00908
  • 250 mg anti-C4.4a B01-10 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 10.2 mg/ml
  • Drug/mAb ratio: 4.4
    • Example 216
  • Figure US20140127240A1-20140508-C00909
  • 50 mg anti-C4.4a B01-3 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 11.5 mg/ml
  • Drug/mAb ratio: 5.2
    • Example 217
  • Figure US20140127240A1-20140508-C00910
  • 250 mg anti-C4.4a D02-6 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 13 mg/ml
  • Drug/mAb ratio: 5.2
    • Example 218
  • Figure US20140127240A1-20140508-C00911
  • 250 mg anti-C4.4a B01-3 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 10.3 mg/ml
  • Drug/mAb ratio: 4.9
    • Example 219
  • Figure US20140127240A1-20140508-C00912
  • Protein concentration: 0.88 mg/ml
  • Drug/mAb ratio: 3.2
    • Example 220
  • Figure US20140127240A1-20140508-C00913
  • Protein concentration: 1.18 mg/ml
  • Drug/mAb ratio: 3.4
    • Example 221
  • Figure US20140127240A1-20140508-C00914
  • Protein concentration: 1.23 mg/ml
  • Drug/mAb ratio: 3.0
    • Example 222
  • Figure US20140127240A1-20140508-C00915
  • Protein concentration: 1.3 mg/ml
  • Drug/mAb ratio: 3.3
    • Example 223
  • Figure US20140127240A1-20140508-C00916
  • Protein concentration: 1.11 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 224
  • Figure US20140127240A1-20140508-C00917
  • Protein concentration: 1.25 mg/ml
  • Drug/mAb ratio: 2.4
    • Example 225
  • Figure US20140127240A1-20140508-C00918
  • Protein concentration: 0.88 mg/ml
  • Drug/mAb ratio: 5.0
    • Example 226
  • Figure US20140127240A1-20140508-C00919
  • Protein concentration: 1.23 mg/ml
  • Drug/mAb ratio: 3.3
    • Example 227
  • Figure US20140127240A1-20140508-C00920
  • Protein concentration: 0.93 mg/ml
  • Drug/mAb ratio: 1.8
    • Example 228
  • Figure US20140127240A1-20140508-C00921
  • Protein concentration: 0.85 mg/ml
  • Drug/mAb ratio: 5.3
    • Example 229
  • Figure US20140127240A1-20140508-C00922
  • Protein concentration: 1.51 mg/ml
  • Drug/mAb ratio: 1.4
    • Example 230
  • Figure US20140127240A1-20140508-C00923
  • 150 mg anti-C4.4a B01-3 in DPBS pH 7.4 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 11.0 mg/ml
  • Drug/mAb ratio: 4.5
    • Example 231
  • Figure US20140127240A1-20140508-C00924
  • Protein concentration: 1.2 mg/ml
  • Drug/mAb ratio: 3.3
    • Example 232
  • Figure US20140127240A1-20140508-C00925
  • Protein concentration: 1.25 mg/ml
  • Drug/mAb ratio: 3.1
    • Example 233 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00926
  • 10 mg (10 μmol) of Intermediate 157 were taken up in 5.2 ml DMF and mixed with 2.28 mg (20 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 5.8 mg (48% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.45 min;
  • LC-MS (Method 1): Rt=0.74 min; MS (ESIpos): m/z=1184 (M+H)+.
    • Example 234 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00927
  • 10 mg (10 μmol) of Intermediate 113 were taken up in 5.2 ml DMF and mixed with 2.28 mg (20 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 6 mg (54% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.5 min;
  • LC-MS (Method 1): Rt=0.77 min; MS (ESIpos): m/z=1185 (M+H)+.
    • Example 235 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00928
  • 9 mg (8.3 μmol) of Intermediate 132 were taken up in 4 ml DMF and mixed with 3 mg (24.4 μmol) L-cysteine. The reaction mixture was stirred overnight at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 6.8 mg (68% of theory) of the title compound.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.78 min; MS (ESIpos): m/z=1227 (M+H)+.
    • Example 236 N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C00929
  • 10 mg (10 μmol) of Intermediate 106 were taken up in 5.8 ml DMF and mixed with 2.5 mg (20 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 5.2 mg (46% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.5 min;
  • LC-MS (Method 11): Rt=0.71 min; MS (ESIpos): m/z=1070 (M+H)+.
    • Example 237 N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00930
  • 10 mg (10 μmol) of Intermediate 124 was taken up in 4 ml DMF and mixed with 2.5 mg (20 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 7.2 mg (64% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.8 min; MS (ESIpos): m/z=1071 (M+H)+.
    • Example 238 N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C00931
  • 10 mg (10 μmol) of Intermediate 125 were taken up in 4 ml DMF and mixed with 2.4 mg (20 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 7.7 mg (69% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 2): Rt=1.91 min; MS (ESIpos): m/z=1140 (M+H)+.
    • Example 239 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-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-valinamide
  • Figure US20140127240A1-20140508-C00932
  • 10 mg (10 μmol) of intermediate 160 were taken up in 3 ml DMF and mixed with 2.1 mg (20 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 8.1 mg (73% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=1274 (M+H)+.
    • Example 240 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-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-valinamide
  • Figure US20140127240A1-20140508-C00933
  • 3.5 mg (3 μmol) of Intermediate 159 were taken up in 1 ml DMF and mixed with 0.76 mg (6 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 2.6 mg (65% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.75 min;
  • LC-MS (Method 1): Rt=0.85 min; MS (ESIpos): m/z=1235 (M+H)+.
    • Example 241 N-(6-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-6-oxohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00934
  • 3.6 mg (3 μmol) of Intermediate 129 were taken up in 1 ml DMF and mixed with 0.77 mg (6 μmol) L-cysteine. The reaction mixture was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 1.55 mg (39% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.87 min; MS (ESIpos): m/z=1255 (M+H)+.
    • Example 242
  • Figure US20140127240A1-20140508-C00935
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 0.83 mg/ml
  • Drug/mAb ratio: 1.6
    • Example 243
  • Figure US20140127240A1-20140508-C00936
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.59 mg/ml
  • Drug/mAb ratio: 3.1
  • Drug/mAb ratio: 2.9
    • Example 244
  • Figure US20140127240A1-20140508-C00937
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.25 mg/ml
  • Drug/mAb ratio: 4.0
    • Example 245
  • Figure US20140127240A1-20140508-C00938
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.27 mg/ml
  • Drug/mAb ratio: 3.6
    • Example 246
  • Figure US20140127240A1-20140508-C00939
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.54 mg/ml
  • Drug/mAb ratio: 4.7
    • Example 247
  • Figure US20140127240A1-20140508-C00940
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.73 mg/ml
  • Drug/mAb ratio: 4.7
    • Example 248
  • Figure US20140127240A1-20140508-C00941
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the reaction mixture was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.66 mg/ml
  • Drug/mAb ratio: 1.3
    • Example 249
  • Figure US20140127240A1-20140508-C00942
  • Protein concentration: 2.11 mg/ml
  • Drug/mAb ratio: 5.5
    • Example 250
  • Figure US20140127240A1-20140508-C00943
  • Protein concentration: 1.53 mg/ml
  • Drug/mAb ratio: 3.4
    • Example 251
  • Figure US20140127240A1-20140508-C00944
  • Protein concentration: 1.5 mg/ml
  • Drug/mAb ratio: 0.2
    • Example 252
  • Figure US20140127240A1-20140508-C00945
  • Protein concentration: 1.32 mg/ml
  • Drug/mAb ratio: 0.1
    • Example 253
  • Figure US20140127240A1-20140508-C00946
  • 80 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation, diluted again with PBS and reconcentrated.
  • Protein concentration: 10.3 mg/ml
  • Drug/mAb ratio: 3.1
    • Example 254
  • Figure US20140127240A1-20140508-C00947
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.09 mg/ml
  • Drug/mAb ratio: 1.8
    • Example 255
  • Figure US20140127240A1-20140508-C00948
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.52 mg/ml
  • Drug/mAb ratio: 4.2
    • Example 256
  • Figure US20140127240A1-20140508-C00949
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.1 mg/ml
  • Drug/mAb ratio: 3.3
    • Example 257
  • Figure US20140127240A1-20140508-C00950
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.43 mg/ml
  • Drug/mAb ratio: 4.8
    • Example 258
  • Figure US20140127240A1-20140508-C00951
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation diluted again with PBS and reconcentrated.
  • Protein concentration: 1.36 mg/ml
  • Drug/mAb ratio: 4.6
    • Example 259
  • Figure US20140127240A1-20140508-C00952
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.33 mg/ml
  • Drug/mAb ratio: 4.0
    • Example 260
  • Figure US20140127240A1-20140508-C00953
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.33 mg/ml
  • Drug/mAb ratio: 4.6
    • Example 261
  • Figure US20140127240A1-20140508-C00954
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.47 mg/ml
  • Drug/mAb ratio: 1.6
    • Example 262
  • Figure US20140127240A1-20140508-C00955
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.49 mg/ml
  • Drug/mAb ratio: 4.5
    • Example 263
  • Figure US20140127240A1-20140508-C00956
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.29 mg/ml
  • Drug/mAb ratio: 3.3
    • Example 264
  • Figure US20140127240A1-20140508-C00957
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.74 mg/ml
  • Drug/mAb ratio: 3.5
    • Example 265
  • Figure US20140127240A1-20140508-C00958
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.09 mg/ml
  • Drug/mAb ratio: 3.2
    • Example 266
  • Figure US20140127240A1-20140508-C00959
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.63 mg/ml
  • Drug/mAb ratio: 0.2
    • Example 267
  • Figure US20140127240A1-20140508-C00960
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.41 mg/ml
  • Drug/mAb ratio: 7.6
    • Example 268
  • Figure US20140127240A1-20140508-C00961
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 2.0 mg/ml
  • Drug/mAb ratio: 1.6
    • Example 269
  • Figure US20140127240A1-20140508-C00962
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.67 mg/ml
  • Drug/mAb ratio: 2.8
    • Example 270
  • Figure US20140127240A1-20140508-C00963
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.91 mg/ml
  • Drug/mAb ratio: 5.3
    • Example 271
  • Figure US20140127240A1-20140508-C00964
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.82 mg/ml
  • Drug/mAb ratio: 4.6
    • Example 272
  • Figure US20140127240A1-20140508-C00965
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.9 mg/ml
  • Drug/mAb ratio: 4.2
    • Example 273
  • Figure US20140127240A1-20140508-C00966
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.89 mg/ml
  • Drug/mAb-Ratio: 2.7
    • Example 274
  • Figure US20140127240A1-20140508-C00967
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.73 mg/ml
  • Drug/mAb-Ratio: 2.3
    • Example 275
  • Figure US20140127240A1-20140508-C00968
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.71 mg/ml
  • Drug/mAb-Ratio: 3.3
    • Example 276
  • Figure US20140127240A1-20140508-C00969
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.47 mg/ml
  • Drug/mAb ratio: 3.9
    • Example 277 N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00970
  • 15.5 mg (15 μmol) of Intermediate 210 were taken up in 5 ml DMF and mixed with 4.4 mg (18 μmol) N2-(tert.-butoxycarbonyl)-L-lysine as well as 7.7 μL (44 μmol) N,N-diisopropylethylamine. The reaction mixture was stirred overnight at RT and then concentrated in vacuo. Next, the residue was purified by means of preparative HPLC. The yield was 14 mg (81% of theory) of the protected intermediate of the title compound that was subsequently taken up in 1 ml dichloromethane and deprotected with 1 ml trifluoroascetic acid. The batch was concentrated and, following lyophilization of the residue from acetonitrile/water (1:1), 15 mg (97% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.79 min; MS (ESIpos): m/z=1083 (M+H)+.
    • Example 278 N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C00971
  • 40 mg (40 μmol) of Intermediate 227 were taken up in 5 ml DMF and mixed with 11.5 mg (40 μmol) N2-[(benzyloxy)carbonyl]-L-lysine as well as 13 μl (80 μmol) N,N-diisopropylethylamine. The reaction mixture was stirred overnight at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 32.5 mg (70% of theory) of the protected intermediate of the title compound.
  • These 32.5 mg of the intermediate were dissolved in 10 ml methanol and, after adding 2 mg 10% palladium on activated carbon, hydrogenated for 30 min at RT under standard hydrogen pressure. The catalyst was then filtered off and the solvent removed in vacuo. Following lyophilization of the residue from dioxane/water 1:1, 26 mg (99% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.76 min; MS (ESIpos): m/z=1014 (M+H)+.
    • Example 279 N-[(18S)-18-amino-18-carboxy-12-oxo-3,6,9-trioxa-[3-azaoctadec-1-yl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C00972
  • 3.5 mg (3 μmol) of Intermediate 202 were taken up in 2 ml DMF and mixed with 0.8 mg (3 μmol) N2-(tert. butoxycarbonyl)-L-lysine as well as 1.6 μl (10 μmol) N,N-diisopropylethylamine. The reaction mixture was stirred overnight at RT and then concentrated in vacuo. Next, the residue was taken up in acetonitrile/water (1:1), adjusted to pH 2 with trifluoroascetic acid, and purified by means of preparative HPLC. The yield was 1 mg (25% of theory) of the protected intermediate of the title compound that was subsequently taken up in 500 μl dichloromethane and deprotected with 500 μl trifluoroascetic acid. The batch was concentrated and, following lyophilization of the residue from acetonitrile/water (1:1), 1 mg (89% of theory) of the title compound was obtained.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.82 min; MS (ESIpos): m/z=1173 (M+H)+.
    • Example 280
  • Figure US20140127240A1-20140508-C00973
  • Protein concentration: 0.9 mg/ml
  • Drug/mAb ratio: 2.8
    • Example 281
  • Figure US20140127240A1-20140508-C00974
  • Protein concentration: 1.08 mg/ml
  • Drug/mAb ratio: 1.1
    • Example 282
  • Figure US20140127240A1-20140508-C00975
  • Protein concentration: 0.98 mg/ml
  • Drug/mAb ratio: 2.4
    • Example 283
  • Figure US20140127240A1-20140508-C00976
  • Protein concentration: 1.23 mg/ml
  • Drug/mAb ratio: 4.6
    • Example 284
  • Figure US20140127240A1-20140508-C00977
  • 100 mg anti-CA9 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation. The solution was then diluted again, reconcentrated, and the process was repeated one more time.
  • Protein concentration: 9.2 mg/ml
  • Drug/mAb ratio: 3.2
    • Example 285
  • Figure US20140127240A1-20140508-C00978
  • Protein concentration: 1.21 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 286
  • Figure US20140127240A1-20140508-C00979
  • Protein concentration: 1.26 mg/ml
  • Drug/mAb ratio: 4.2
    • Example 287
  • Figure US20140127240A1-20140508-C00980
  • Protein concentration: 1.01 mg/ml
  • Drug/mAb ratio: 3.0
    • Example 288
  • Figure US20140127240A1-20140508-C00981
  • Protein concentration: 1.28 mg/ml
  • Drug/mAb ratio: 2.3
    • Example 289
  • Figure US20140127240A1-20140508-C00982
  • Protein concentration: 1.12 mg/ml
  • Drug/mAb ratio: 2.6
    • Example 290
  • Figure US20140127240A1-20140508-C00983
  • Protein concentration: 1.4 mg/ml
  • Drug/mAb ratio: 3.3
    • Example 291
  • Figure US20140127240A1-20140508-C00984
  • Protein concentration: 1.3 mg/ml
  • Drug/mAb ratio: 2.5
    • Example 292
  • Figure US20140127240A1-20140508-C00985
  • Protein concentration: 1.27 mg/ml
  • Drug/mAb ratio: 2.6
    • Example 293
  • Figure US20140127240A1-20140508-C00986
  • Protein concentration: 1.55 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 294
  • Figure US20140127240A1-20140508-C00987
  • 100 mg anti-CA9 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 11.8 mg/ml
  • Drug/mAb ratio: 4.4
    • Example 295
  • Figure US20140127240A1-20140508-C00988
  • 100 mg anti-CA9 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 12.29 mg/ml
  • Drug/mAb ratio: 3.8
    • Example 296
  • Figure US20140127240A1-20140508-C00989
  • Protein concentration: 1.1 mg/ml
  • Drug/mAb ratio: 1.6
    • Example 297
  • Figure US20140127240A1-20140508-C00990
  • Protein concentration: 1.00 mg/ml
  • Drug/mAb ratio: 1.9
    • Example 298
  • Figure US20140127240A1-20140508-C00991
  • 100 mg anti-CA9 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 11.5 mg/ml
  • Drug/mAb ratio: 4.9
    • Example 299
  • Figure US20140127240A1-20140508-C00992
  • Protein concentration: 0.98 mg/ml
  • Drug/mAb ratio: 2.5
    • Example 300
  • Figure US20140127240A1-20140508-C00993
  • Protein concentration: 0.99 mg/ml
  • Drug/mAb ratio: 2.0
    • Example 301
  • Figure US20140127240A1-20140508-C00994
  • Protein concentration: 0.87 mg/ml
  • Drug/mAb ratio: 2.1
    • Example 302
  • Figure US20140127240A1-20140508-C00995
  • 100 mg anti-CA9 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 12.2 mg/ml
  • Drug/mAb ratio: 4.6
    • Example 303
  • Figure US20140127240A1-20140508-C00996
  • Protein concentration: 1.58 mg/ml
  • Drug/mAb ratio: 1.9
    • Example 304
  • Figure US20140127240A1-20140508-C00997
  • 70 mg anti-CA9 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 11.5 mg/ml
  • Drug/mAb ratio: 3.9
    • Example 305
  • Figure US20140127240A1-20140508-C00998
  • 60 mg anti-CA9 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 11.6 mg/ml
  • Drug/mAb ratio: 3.9
    • Example 306
  • Figure US20140127240A1-20140508-C00999
  • 60 mg anti-CA9 were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation.
  • Protein concentration: 10 mg/ml
    • Example 307 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C01000
  • 10 mg (10 μmol) of Intermediate 113 were taken up in 5.2 ml DMF and mixed with 2.28 mg (20 μmol) L-cysteine. The batch was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 6 mg (54% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.5 min;
  • LC-MS (Method 1): Rt=0.77 min; MS (ESIpos): m/z=1185 (M+H)+.
    • Example 308 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C01001
  • 9 mg (8.3 μmol) of Intermediate 132 were taken up in 4 ml DMF and mixed with 3 mg (24.4 μmol) L-cysteine. The batch was stirred overnight at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 6.8 mg (68% of theory) of the title compound.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.78 min; MS (ESIpos): m/z=1227 (M+H)+.
    • Example 309 N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-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-valinamide
  • Figure US20140127240A1-20140508-C01002
  • 10 mg (10 μmol) of Intermediate 106 were taken up in 5.8 ml DMF and mixed with 2.5 mg (20 μmol) L-cysteine. The batch was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 5.2 mg (46% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.5 min;
  • LC-MS (Method 11): Rt=0.71 min; MS (ESIpos): m/z=1070 (M+H)+.
    • Example 310 N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C01003
  • 10 mg (10 μmol) of Intermediate 124 were taken up in 4 ml DMF and mixed with 2.5 mg (20 μmol) L-cysteine. The batch was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 7.2 mg (64% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.6 min;
  • LC-MS (Method 1): Rt=0.8 min; MS (ESIpos): m/z=1071 (M+H)+.
    • Example 311 N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide
  • Figure US20140127240A1-20140508-C01004
  • 10 mg (10 μmol) of Intermediate 125 were taken up in 4 ml DMF and mixed with 2.4 mg (20 μmol) L-cysteine. The batch was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 7.7 mg (69% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 2): Rt=1.91 min; MS (ESIpos): m/z=1140 (M+H)+.
    • Example 312 N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-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-valinamide
  • Figure US20140127240A1-20140508-C01005
  • 10 mg (10 μmol) of Intermediate 160 was taken up in 3 ml DMF and mixed with 2.1 mg (20 μmol) L-cystein. The batch was stirred for 2 hours at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 8.1 mg (73% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=1274 (M+H)+.
    • Example 313
  • Figure US20140127240A1-20140508-C01006
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.23 mg/ml
  • Drug/mAb ratio: ˜1-1.5
    • Example 314
  • Figure US20140127240A1-20140508-C01007
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.98 mg/ml
  • Drug/mAb ratio: 2.8
    • Example 315
  • Figure US20140127240A1-20140508-C01008
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.0 mg/ml
  • Drug/mAb ratio: 3
    • Example 316
  • Figure US20140127240A1-20140508-C01009
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.59 mg/ml
  • Drug/mAb ratio: 3.1
    • Example 317
  • Figure US20140127240A1-20140508-C01010
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.75 mg/ml
  • Drug/mAb ratio: 3.3
    • Example 318
  • Figure US20140127240A1-20140508-C01011
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.54 mg/ml
  • Drug/mAb ratio: 3.5
    • Example 319
  • Figure US20140127240A1-20140508-C01012
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 2 mg/ml
  • Drug/mAb ratio: 1.1
    • Example 320
  • Figure US20140127240A1-20140508-C01013
  • Protein concentration: 1.66 mg/ml
  • Drug/mAb ratio: 4.9
    • Example 321
  • Figure US20140127240A1-20140508-C01014
  • Protein concentration: 1.7 mg/ml
  • Drug/mAb ratio: 3.0
    • Example 322
  • Figure US20140127240A1-20140508-C01015
  • Protein concentration: 1.08 mg/ml
  • Drug/mAb ratio: 1.9
    • Example 323
  • Figure US20140127240A1-20140508-C01016
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.57 mg/ml
  • Drug/mAb ratio: 2.9
    • Example 324
  • Figure US20140127240A1-20140508-C01017
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.7 mg/ml
  • Drug/mAb ratio: 1.4
    • Example 325
  • Figure US20140127240A1-20140508-C01018
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.53 mg/ml
  • Drug/mAb ratio: 3.6
    • Example 326
  • Figure US20140127240A1-20140508-C01019
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.77 mg/ml
  • Drug/mAb ratio: 6.1
    • Example 327
  • Figure US20140127240A1-20140508-C01020
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.14 mg/ml
  • Drug/mAb ratio: 2.5
    • Example 328
  • Figure US20140127240A1-20140508-C01021
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.57 mg/ml
  • Drug/mAb ratio: 3.8
    • Example 329
  • Figure US20140127240A1-20140508-C01022
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.72 mg/ml
  • Drug/mAb ratio: 3.9
    • Example 330
  • Figure US20140127240A1-20140508-C01023
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.56 mg/ml
  • Drug/mAb ratio: 2.9
    • Example 331
  • Figure US20140127240A1-20140508-C01024
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.81 mg/ml
  • Drug/mAb ratio: 3.5
    • Example 332
  • Figure US20140127240A1-20140508-C01025
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.54 mg/ml
  • Drug/mAb ratio: 1.3
    • Example 333
  • Figure US20140127240A1-20140508-C01026
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.72 mg/ml
  • Drug/mAb ratio: 4.0
    • Example 334 N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C01027
  • 15.5 mg (15 μmol) of Intermediate 210 were taken up in 5 ml DMF and mixed with 4.4 mg (18 μmol) N2-(tert.-butoxycarbonyl)-L-lysine as well as 7.7 μl (44 μmol) N,N-diisopropylethylamine. The reaction mixture was stirred overnight at RT and then concentrated in vacuo. Next, the residue was purified by means of preparative HPLC. 14 mg (81% of theory) of the protected intermediate of the title compound were obtained, which were then taken up in 1 ml dichloromethane and deprotected with 1 ml trifluoroascetic acid. The reaction mixture was concentrated and, after lyophilization of the residue from acetonitrile/water (1:1), 15 mg (97% of theory) of the title compound were obtained.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.79 min; MS (ESIpos): m/z=1083 (M+H)+.
    • Example 335 N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C01028
  • 40 mg (40 μmol) of Intermediate 227 were taken up in 5 ml DMF and mixed with 11.5 mg (40 μmol) N2-[(benzyloxy)carbonyl]-L-lysine as well as 13 μl (80 μmol) N,N-diisopropylethylamine. The reaction mixture was stirred overnight at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 32.5 mg (70% of theory) of the protected intermediate of the title compound.
  • 32.5 mg of this intermediate was dissolved in 10 ml methanol and, after adding 2 mg 10% palladium on activated carbon, hydrogenated for 30 min at RT under standard hydrogen pressure. The catalyst was then filtered off and the solvent removed in vacuo. After lyophilization of the residue from dioxane/water (1:1), the yield was 26 mg (99% of theory) of the title compound.
  • HPLC (Method 12): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.76 min; MS (ESIpos): m/z=1014 (M+H)+.
    • Example 336 N-[(18S)-18-amino-18-carboxy-12-oxo-3,6,9-trioxa-[3-azaoctadec-1-yl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C01029
  • 3.5 mg (3 μmol) of Intermediate 202 were taken up in 2 ml DMF and mixed with 0.8 mg (3 μmol) N2-(tert.-butoxycarbonyl)-L-lysine as well as 1.6 μl (10 μmol) N,N-diisopropylethylamine. The reaction mixture was stirred overnight at RT and then concentrated in vacuo. Next, the residue was taken up in acetonitrile/water (1:1), adjusted to pH 2 with trifluoroascetic acid and then purified by means of preparative HPLC. 1 mg (25% of theory) of the protected intermediate of the title compound was obtained, which was subsequently taken up in 500 μl dichloromethane and deprotected with 500 μl trifluoroascetic acid. The reaction mixture was concentrated and, after lyophilization of the residue from acetonitrile/water (1:1), the yield was 1 mg (89% of theory) of the title compound.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.82 min; MS (ESIpos): m/z=1173 (M+H)+.
    • Example 337
  • Figure US20140127240A1-20140508-C01030
  • 5 mg cetuximab in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.57 mg/ml
  • Drug/mAb ratio: 4.6
    • Example 338
  • Figure US20140127240A1-20140508-C01031
  • 5 mg cetuximab were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.48 mg/ml
  • Drug/mAb ratio: 3.4
    • Example 339
  • Figure US20140127240A1-20140508-C01032
  • 5 mg cetuximab in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.21 mg/ml
  • Drug/mAb ratio: 2.4
    • Example 340
  • Figure US20140127240A1-20140508-C01033
  • 5 mg cetuximab in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.75 mg/ml
  • Drug/mAb ratio: 3.4
    • Example 341
  • Figure US20140127240A1-20140508-C01034
  • 5 mg cetuximab in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.69 mg/ml
  • Drug/mAb ratio: 2.9
    • Example 342
  • Figure US20140127240A1-20140508-C01035
  • 5 mg cetuximab were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.27 mg/ml
  • Drug/mAb ratio: 2.9
    • Example 343
  • Figure US20140127240A1-20140508-C01036
  • 5 mg panitumuab in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.27 mg/ml
  • Drug/mAb ratio: not detectable
    • Example 344
  • Figure US20140127240A1-20140508-C01037
  • 5 mg cetuximab were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.55 mg/ml
  • Drug/mAb ratio: 3.1
    • Example 345
  • Figure US20140127240A1-20140508-C01038
  • 5 mg cetuximab in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.67 mg/ml
  • Drug/mAb ratio: 3.5
    • Example 346
  • Figure US20140127240A1-20140508-C01039
  • 5 mg cetuximab in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.44 mg/ml
  • Drug/mAb ratio: 2.5
    • Example 347
  • Figure US20140127240A1-20140508-C01040
  • 5 mg cetuximab in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.73 mg/ml
  • Drug/mAb ratio: 1.2
    • Example 348
  • Figure US20140127240A1-20140508-C01041
  • 2 mg anti-PDL1 in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.04 mg/ml
  • Drug/mAb ratio: 4.8
    • Example 349
  • Figure US20140127240A1-20140508-C01042
  • 3 mg anti-PDL1 in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.7 mg/ml
  • Drug/mAb ratio: 2.3
    • Example 350
  • Figure US20140127240A1-20140508-C01043
  • 2 mg anti-ICOSLG in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.77 mg/ml
  • Drug/mAb ratio: 3.7
    • Example 351
  • Figure US20140127240A1-20140508-C01044
  • 4 mg anti-FGFR3 in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.41 mg/ml
  • Drug/mAb ratio: 1.8
    • Example 352
  • Figure US20140127240A1-20140508-C01045
  • 3 mg herceptin in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.49 mg/ml
  • Drug/mAb ratio: 2.3
    • Example 353
  • Figure US20140127240A1-20140508-C01046
  • 5 mg herceptin in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.62 mg/ml
  • Drug/mAb ratio: 5.0
    • Example 354
  • Figure US20140127240A1-20140508-C01047
  • 5 mg herceptin in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.63 mg/ml
  • Drug/mAb ratio: 2.4
    • Example 355
  • Figure US20140127240A1-20140508-C01048
  • 5 mg herceptin in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.65 mg/ml
  • Drug/mAb ratio: 3.9
    • Example 356
  • Figure US20140127240A1-20140508-C01049
  • 5 mg herceptin in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.78 mg/ml
  • Drug/mAb ratio: 3.1
    • Example 357
  • Figure US20140127240A1-20140508-C01050
  • 5 mg herceptin in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.72 mg/ml
  • Drug/mAb ratio: 3.2
    • Example 358
  • Figure US20140127240A1-20140508-C01051
  • 5 mg herceptin in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.95 mg/ml
  • Drug/mAb-Ratio: ˜3.7
    • Example 359
  • Figure US20140127240A1-20140508-C01052
  • 5 mg herceptin in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.79 mg/ml
  • Drug/mAb ratio: 9.1
    • Example 360 N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C01053
  • 15.5 mg (15 μmol) of Intermediate 210 were taken up in 5 ml DMF and mixed with 4.4 mg (18 μmol) N2-(tert.-butoxycarbonyl)-L-lysine as well as 7.7 μl (44 μmol) N,N-diisopropylethylamine. The reaction mixture was stirred overnight at RT, then concentrated in vacuo. The residue was purified by means of preparative HPLC. 14 mg (81% of theory) of the protected intermediate of the title compound were obtained, which were then taken up in 1 ml dichloromethane and deprotected with 1 ml trifluoroascetic acid. The batch was concentrated and, after lyophilization of the residue from acetonitrile/water (1:1), the yield was 15 mg (97% of theory) of the title compound.
  • HPLC (Method 12): Rt=1.8 min;
  • LC-MS (Method 1): Rt=0.79 min; MS (ESIpos): m/z=1083 (M+H)+.
    • Example 361 N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
  • Figure US20140127240A1-20140508-C01054
  • 40 mg (40 μmol) of Intermediate 227 were taken up in 5 ml DMF and mixed with 11.5 mg (40 μmol) N2-[(benzyloxy)carbonyl]-L-lysine as well as 13 μl (80 μmol) N,N-diisopropylethylamine.
  • The reaction mixture was stirred overnight at RT, then concentrated in vacuo and next purified by means of preparative HPLC. The yield was 32.5 mg (70% of theory) of the protected intermediate of the title compound.
  • 32.5 mg of this intermediate were taken up in 10 ml methanol and, after adding 2 mg 10% palladium on activated carbon, hydrogenated at RT under hydrogen normal pressure. The cataly-st was then filtered off and the solvent removed in vacuo. After lyophilization of the residue from dioxane/water (1:1), the yield was 26 mg (99% of theory) of the title compound.
  • HPLC (Method 12): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.76 min; MS (ESIpos): m/z=1014 (M+H)+.
    • Example 362 N-[(18S)-18-amino-18-carboxy-12-oxo-3,6,9-trioxa-13-azaoctadec-1-yl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C01055
  • 3.5 mg (3 μmol) of Intermediate 202 were taken up in 2 ml DMF and mixed with 0.8 mg (3 μmol) N2-(tert.-butoxycarbonyl)-L-lysine as well as 1.6 μl (10 μmol) N,N-diisopropylethylamine. The reaction mixture was stirred overnight at RT, then concentrated in vacuo. The residue was taken up in acetonitrile/water (1:1), adjusted to pH 2 with trifluoroascetic acid, then purified by means of preparative HPLC. The yield was 1 mg (25% of theory) of the protected intermediate of the title compound that was subsequently taken up in 500 μl dichloromethane and deprotected with 500 μl trifluoroascetic acid. The batch was concentrated and, after lyophilization of the residue from acetonitrile/water (1:1), the yield was 1 mg (89% of theory) of the title compound.
  • HPLC (Method 12): Rt=1.9 min;
  • LC-MS (Method 1): Rt=0.82 min; MS (ESIpos): m/z=1173 (M+H)+.
    • Example 363
  • Figure US20140127240A1-20140508-C01056
  • 2.2 mg anti-TYRP1 in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.14 mg/ml
  • Drug/mAb ratio: 4.1
    • Example 364
  • Figure US20140127240A1-20140508-C01057
  • 3 mg anti-glypican-3 were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.17 mg/ml
  • Drug/mAb ratio: 3.0
    • Example 365
  • Figure US20140127240A1-20140508-C01058
  • 3 mg anti-glypican-3 in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.25 mg/ml
  • Drug/mAb ratio: 2.9
    • Example 366
  • Figure US20140127240A1-20140508-C01059
  • 5 mg MF-Ta in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 0.81 mg/ml
  • Drug/mAb ratio: 2.5
    • Example 367
  • Figure US20140127240A1-20140508-C01060
  • 5 mg MF-Ta in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.06 mg/ml
  • Drug/mAb ratio: 1.8
    • Example 368
  • Figure US20140127240A1-20140508-C01061
  • 5 mg MF-Ta in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.36 mg/ml
  • Drug/mAb ratio: 7.2
    • Example 369
  • Figure US20140127240A1-20140508-C01062
  • 5 mg MF-Ta in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.57 mg/ml
  • Drug/mAb ratio: 2.9
    • Example 370
  • Figure US20140127240A1-20140508-C01063
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 0.89 mg/ml
  • Drug/mAb ratio: 1.8
    • Example 371
  • Figure US20140127240A1-20140508-C01064
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 0.57 mg/ml
  • Drug/mAb ratio: 1.5
    • Example 372
  • Figure US20140127240A1-20140508-C01065
  • 5 mg anti-C4.4a B01-3 were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.39 mg/ml
  • Drug/mAb ratio: 7.1
    • Example 373
  • Figure US20140127240A1-20140508-C01066
  • 5 mg anti-C4.4a B01-3 were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.54 mg/ml
  • Drug/mAb ratio: 2.4
    • Example 374
  • Figure US20140127240A1-20140508-C01067
  • 5 mg cetuximab in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.55 mg/ml
  • Drug/mAb-Ratio: 1.8
    • Example 375
  • Figure US20140127240A1-20140508-C01068
  • 5 mg cetuximab were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.36 mg/ml
  • Drug/mAb ratio: 1.9
    • Example 376
  • Figure US20140127240A1-20140508-C01069
  • 5 mg cetuximab in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.73 mg/ml
  • Drug/mAb-Ratio: 3.7
    • Example 377
  • Figure US20140127240A1-20140508-C01070
  • 5 mg MF-Ta in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.44 mg/ml
  • Drug/mAb ratio: 2.5
    • Example 378
  • Figure US20140127240A1-20140508-C01071
  • 5 mg MF-Ta in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.74 mg/ml
  • Drug/mAb-Ratio: 3.6
    • Example 379 Diastereomer 1
  • Figure US20140127240A1-20140508-C01072
  • Intermediate 247a and 5 mg MF-Ta in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.57 mg/ml
  • Drug/mAb ratio: 4.2
    • Example 380 Diastereomer 2
  • Figure US20140127240A1-20140508-C01073
  • Intermediate 247a and 5 mg MF-Ta in PBS were presently used for the coupling and, following purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.42 mg/ml
  • Drug/mAb ratio: 4.0
    • Example 381 N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-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-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C01074
  • 8.6 mg (8 μmol) of Intermediate 240 were taken up in 5 ml DMF and mixed with 4.0 mg (16 μmol) N2-(tert.-butoxycarbonyl)-L-lysine as well as 2 μl (16 μmol) N,N-diisopropylethylamine. The reaction mixture was stirred for 4 hours at RT, then mixed again with the same quantities of N2-(tert.-butoxycarbonyl)-L-lysine and N,N-diisopropylethylamine and stirred overnight at RT. The batch was then concentrated in vacuo. Next, the residue was purified by means of preparative HPLC. The yield was 7 mg (72% of theory) of the protected intermediate of the title compound that was then taken up in 1 ml of dichloromethane and deprotected with 0.5 ml trifluoroascetic acid. The batch was concentrated and the residue purified by means of preparative HPLC. After drying under high vacuum, the yield was 3.3 mg (47% of theory) of the title compound.
  • HPLC (Method 5): Rt=1.5 min;
  • LC-MS (Method 1): Rt=0.8 min; MS (ESIpos): m/z=1084 (M+H)+.
    • Example 382 N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-hydroxyphenyl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C01075
  • 8 mg (8 μmol) of Intermediate 242 were taken up in 3 ml DMF and mixed with 2.9 mg (12 μmol) N2-(tert.-butoxycarbonyl)-L-lysine as well as 2.7 μl (16 μmol) N,N-diisopropylethylamine. The reaction mixture was stirred overnight at RT, then mixed again with the same quantities of N2-(tert.-butoxycarbonyl)-L-lysine and N,N-diisopropylethylamine, then stirred for another 4 hours at RT. The batch was then concentrated in vacuo. Next, the residue was purified by means of preparative HPLC. Following lyophilization from acetonitrile/water, the yield was 6.5 mg (72% of theory) of the protected intermediate of the title compound that was then taken up in 5 ml dichloromethane and deprotected with 0.75 ml trifluoroascetic acid. The batch was concentrated and, following lyophilization of the residue from dioxane/water, the yield was 5 mg (76% of theory) of the title compound.
  • HPLC (Method 12): Rt=1.7 min;
  • LC-MS (Method 1): Rt=0.69 min; MS (ESIpos): m/z=1059 (M+H)+.
    • Example 383 N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-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-valinamide trifluoroacetate
  • Figure US20140127240A1-20140508-C01076
  • 38 mg (41 μmol) of Intermediate 248 were first converted to the N-hydroxysuccinimide ester. 72 mg of the obtained raw product were taken up in 5 ml DMF and mixed with 24 mg (100 μmol) N2-(tert.-butoxycarbonyl)-L-lysine as well as 23 μl N,N-diisopropylethylamine. The reaction mixture was stirred overnight at RT, then mixed again with 16 mg N2-(tert.-butoxycarbonyl)-L-lysine and 12 μl N,N-diisopropylethylamine and finally treated for another 2 hours in an ultrasonic bath. The batch was then concentrated in vacuo and the residue purified by means of preparative HPLC. After lyophilization from acetonitrile/water, the yield was 20 mg (50% of theory) of the protected intermediate of the title compound.
  • 15 mg (12 μmol) of this intermediate were then taken up in 3 ml dichloromethane and mixed with 1 ml trifluoroascetic acid. After stirring for 40 min at RT, further 1.5 ml trifluoroascetic acid were added, and the batch was treated for 1 h in an ultrasonic bath. The batch was then concentrated and, after lyophilization of the residue from dioxane/water, the yield was 13 mg (90% of theory) of the title compound.
  • HPLC (Method 12): Rt=1.5 min;
  • LC-MS (Method 1): Rt=0.68 min; MS (ESIpos): m/z=990 (M+H)+.
    • Example 384
  • Figure US20140127240A1-20140508-C01077
  • 5 mg anti-CA9 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.4 mg/ml
  • Drug/mAb ratio: 3.0
    • Example 385
  • Figure US20140127240A1-20140508-C01078
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.48 mg/ml
  • Drug/mAb ratio: 2.4
    • Example 386
  • Figure US20140127240A1-20140508-C01079
  • 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again.
  • Protein concentration: 1.43 mg/ml
  • Drug/mAb-Ratio: 3.6
    • Example 387 Diastereomer 1
  • Figure US20140127240A1-20140508-C01080
  • Intermediate 247a and 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.45 mg/ml
  • Drug/mAb ratio: 3.8
    • Example 388 Diastereomer 2
  • Figure US20140127240A1-20140508-C01081
  • Intermediate 247a and 5 mg anti-C4.4a B01-3 in PBS were presently used for the coupling and, after purification on a Sephadex column, the batch was concentrated by ultracentrifugation and diluted again with PBS.
  • Protein concentration: 1.42 mg/ml
  • Drug/mAb ratio: 4.0
    • C. EVALUATION OF BIOLOGICAL EFFICACY
  • The biological activity of the compound according to the invention can be demonstrated by in vitro and in vivo tests, such as those with which those skilled in the art are familiar.
  • The biological effect of the compounds according to the invention was revealed in the assays described below:
    • C-1.1 In Vitro Cell Proliferation Test
  • Human EGFR-expressing tumors cells are used to test the efficacy of anti-EGFR ADCs. The cells may be, for example, NCI-H292 or A431 with a high expression. Cells with a low EGFR expression, such as HT29 or cells with practically no EGFR expression such as NCI-H520 are used as the controls for the EGFR-dependent cytotoxicity.
    • Description of the Experiment
  • Day 1: The cells are plated out in the medium in 100 μL/well in a 96-well plate (Perkin Elmer, white, catalog 6005680). Cells for determination of the time zero are plated out in a parallel plate. All plates are incubated overnight at 37° C.
  • Day 2: A three-fold dilution series of the test substances in medium is prepared and 100 μL of the three-fold dilutions is pipetted into each well in the plates. The plates are incubated for 96 hours at 37° C. in an incubator. The time zero plate is measured: 100 μL/well CTG solution (Promega Cell Titer Glo solution (catalog nos. G755B and G756B)) are pipetted into the corresponding wells and incubated for 2 min on a shaker for +10 minutes in the dark. Next the luminescence is measured using a VICTOR V instrument (Perkin Elmer).
  • Day 6: Measurement of all other batches: 100 μL/well CTG solution (Promega Cell Titer Glo solution (catalog nos. G755B and G756B)) is pipetted into the corresponding wells and incubated for 2 min on a shaker for +10 minutes in the dark. Then the luminescence is measured using a VICTOR V instrument (Perkin Elmer).
  • The luminescence is used as a marker for the number of viable cells.
  • The measured value of the time zero plate is equated with zero, and the measured value of the cells incubated only in medium without active ingredient is equated with 100%. The result is a sigmoidal dose-effect curve from which the IC50 can be determined (GraphPad Prism software).
  • A431: 2500 cells/well, medium: DMEM Hams, Biochrom, #FG4815+10% FCS
  • NCI-H292: 2500 cells/well, medium: RPMI1640; Biochrom, #FG1215+10% FCS
  • HT29L 2500 cells/well, medium: DMEM Hams; Biochrom, #FG4815+10% FCS
  • Substances that inhibit cell proliferation at <1×10−7 M are classified as effective.
  • Substances that inhibit cell proliferation at <1×10−9 M are classified as especially effective.
  • Table 3 below lists the IC50 values1) of representative exemplary embodiments from this assay.
  • TABLE 3
    IC50 (nM) IC50 (nM) IC50 (nM)
    Example A431 NCI-H292 HT29
    1 0.03 <0.03 <0.03
    2 1.50 1.47
    3 0.28 0.12
    4 0.19 0.17
    5 <0.03 <0.03 <0.03
    6 2.79 0.79 6.22
    7 0.91 0.06 10.40
    10 0.08 <0.03 2.46
    11 2.91 0.36 46.70
    12 0.39 <0.03 0.27
    13 1.59 0.49 2.94
    38 0.60 0.24 1.06
    1) The efficacy data given are based on the ADC batches described in concrete terms here and may deviate in other batches having a different drug/antibody ratio.

    C-1.2 Protocol of Proliferation Assay with Short Substance Incubation (Pulse Assay)
  • The protocol is performed as described above but the substance is removed by suction after 4 hours of incubation with the test substances and is replaced by fresh medium. The analysis is performed as described above after a total of 96 hours.
  • Table 4 below lists the IC50 values1,2) of representative exemplary embodiments from this assay.
  • TABLE 4
    Example Antibody A431 NCI H292
    7 Cetuximab 0.99 0.01
    8 Nimotuzumab >200 34.4
    9 Panitumumab 90.5 26.4
    10 Cetuximab 0.0526 0.0884
    14 Cetuximab 0.258 0.248
    15 Cetuximab 13.4 3.5
    16 Cetuximab 0.943 0.689
    17 Cetuximab 0.285 0.133
    18 Cetuximab 17.4 3.63
    19 Cetuximab 0.89
    20 Cetuximab 31.9 42.3
    21 Nimotuzumab 54.6 120
    22 Panitumumab 44.3 51.8
    23 Cetuximab 88.1 124
    24 Nimotuzumab 111 >200
    25 Panitumumab 53 77
    26 Cetuximab 10.6 3.02
    27 Nimotuzumab 11.4 20.4
    28 Panitumumab 5.78 8.6
    29 Cetuximab 0.37 0.03
    30 Panitumumab 23.2 3.34
    31 Nimotuzumab 13.71 4.24
    32 Cetuximab 50.8 20.4
    33 Nimotuzumab 49 42
    34 Panitumumab 51.1 32.2
    35 Cetuximab 0.716 0.125
    36 Cetuximab 0.357 0.0589
    37 Cetuximab 0.52 0.17
    38 Cetuximab 7.21
    39 Cetuximab 1.49 0.03
    40 Nimotuzumab 36 2.38
    41 Panitumumab 110 6.91
    42 Cetuximab 2.84 0.0949
    43 Cetuximab 2.44 0.587
    44 Cetuximab 0.456 0.571
    52 Cetuximab >200 0.961
    53 Cetuximab 0.253 0.133
    54 Cetuximab 166 1.83
    55 Cetuximab 1.47 0.11
    337 Cetuximab 0.175 0.0746
    338 Cetuximab 1.79 0.152
    339 Cetuximab 2.57 0.194
    340 Cetuximab 0.225 0.101
    341 Cetuximab 2.25 0.232
    342 Cetuximab 24.5 0.267
    343 Panitumumab >200 20.3
    1) The efficacy data given are based on the ADC batches described in concrete terms here and may deviate in other batches having a different drug/antibody ratio.
    2) This shows the averages of two experiments (A431) or three (NCI-H292) experiments.
    • C-1.3 Determination of the Influence on Tubulin Polymerization
  • Cancer cells are degenerate cells which often lead to development of a tumor due to an increased cell division. Microtubules form the spindle fibers of the spindle apparatus and are an essential component of the cell cycle. Regulated synthesis and degradation of microtubules permits an accurate classification of chromosomes on daughter cells and constitutes a continuous dynamic process. A disturbance in the dynamics leads to a faulty cell division and ultimately leads to cell death. However, the increased cell division of cancer cells also makes them especially sensitive to spindle fiber toxins which are a fixed component of chemotherapy. Spindle fiber toxins such as paclitaxel or epothilone lead to a greatly increased polymerization rate of the microtubules, whereas the vinca alkaloids or monomethyl auristatin E (MMAE) lead to a greatly reduced polymerization rate of the microtubules. Those cases involve a sensitive disturbance in the essential dynamics of the cell cycle. The compounds tested within the scope of the present invention result in a reduced polymerization rate of the microtubules.
  • The “Fluorescence-Based Microtubule Polymerization Assay Kit” from Cytoskeleton (Denver, Colo., USA, order no. BK011) was used to investigate tubulin polymerization. In this assay, GTP is added to unpolymerized tubulin, so that polymerization can take place spontaneously. The assay is based on binding of the fluorophore 4′,6′-diamidino-2-phenylindole (DAPI) to tubulin. Free and bond DAPI can be differentiated on the basis of their different emission spectra. Tubulin polymerization can be tracked according to the increase in fluorescence of bound DAPI fluorophores, because DAPI has a much higher affinity for polymerized tubulin in comparison with unpolymerized tubulin.
  • To perform this assay, the test substances dissolved in DMSO were diluted from their initial concentration of 10 mM to 1 μM in water. In addition to the buffer controls, polymerization-increasing paclitaxel was also included as an assay control, and on the other hand, polymerization-inhibiting vinblastine was also included. For the measurement 96-well plates with a half bottom area were used, tracking the kinetics of tubulin polymerization for one hour at 37° C. in a fluorimeter. The excitation wavelength was 355 nm, and the emission was tracked at 460 nm. For the range of the linear increase within the first 10 minutes, the change in fluorescence per minute (ΔF/min) which represents the polymerization rate of the microtubules was calculated. The potency of the test substances was quantified on the basis of the respective reduction in polymerization rate.
  • The value of the inhibition of MMAF at a concentration of 1 μM is set at 100%.
  • Table 5 shows the data for inhibition of tubulin polymerization of representative exemplary embodiments.
  • TABLE 5
    Toxophore/ Concentration Percentage inhibition of
    Example (μM) tubulin polymerization
    MMAF 1 100
    MMAF 10 34
    MMAF 100 0
    360 1 45
    360 10 1
    361 1 80
    361 10 14
    362 1 60
    362 10 0
    233 1 88
    233 10 25
    234 1 109
    234 10 27
    235 1 120
    236 1 117
    236 10 64
    237 1 107
    237 10 25
    238 1 121
    238 10 35
    239 1 111
    239 10 45
    240 1 110
    381 1 102
    381 10 31
    382 1 88
    382 10 21
    383 1 90
    383 10 17
  • The toxophore MMAF and the exemplary embodiments inhibit tubulin polymerization in a concentration-dependent. Tubulin polymerization is completely inhibited at 100 μM MMAF. Example 115 inhibits the tubulin polymerization rate at 1 μM to 45% of the value measured at 1 μM MMAF.
    • C-1.4 Efficacy Test In Vivo
  • The efficacy of the conjugates according to the invention was tested in vivo, e.g., by means of xenograft models. Those skilled in the art are familiar with state-of-the-art methods for testing the efficacy of a conjugate according to the invention (see, for example, WO 2005081711; Polson et al., Cancer Res. Mar. 15, 2009, 69(6):2358-64). For example, a tumor cell line that expresses the target molecule of the binder would be implanted in rodents (e.g., mice). Then a conjugate according to the invention or a control antibody or an isotonic saline solution would be administered to the implant animals. The substance would be administered either one or more times. After an incubation time of several days, the tumor size would be determined in comparison with animals treated with conjugate and with the control group.
    • C-1.4a Growth Inhibition/Regression of Experimental Tumors in the Mouse
  • Human tumor cells that express the antigen for ADC are injected subcutaneously into the flanks of immunosuppressed mice for inoculation, for example, nude mice or SCID mice. Of the cell culture, 1-10 million cells are isolated, centrifuged and resuspended with Medium or Medium/Matrigel. The cell suspension is then injected subcutaneously into the mice.
  • A tumor will then grow within a few days. The treatment begins after establishment of the tumor but at a tumor size of 20 mm2. To investigate the effect of larger tumors, the treatment may be started only when the tumor size reaches 50-100 mm2.
  • Treatment with ADCs is performed via the intravenous route into the caudal vein of the mouse. The ADC is administered in a volume of 5 mL/kg.
  • The treatment regime depends on the pharmacokinetics of the antibody. The standard treatment consists of treatment three times every fourth day. However, the treatment may also be continued further or a second cycle with three days of treatment may also follow at a later point in time.
  • Eight animals are used per treatment group as the standard. This number may be increased if especially great fluctuations in tumor growth or according to treatment are to be expected. In addition to the groups receiving the active substances, one group as the control group is treated only with the buffer according to the same scheme.
  • In the case of this experiment, the area of the tumor is measured regularly using a caliper in two dimensions (length/width). The area of the tumor is determined by length×width.
  • At the end of the experiment, the tumors are excised and weighed. The comparison of the average tumor weights of the treatment group with the control group is reported as T/C.
    • C-1.4b Efficacy in the BxPC3 Pancreatic Carcinoma Tumor Model
  • Two million BxPC3 cells are injected subcutaneously into the flanks of female NMRI nude mice for inoculation.
  • In a tumor cell of 40 mm2 on day 15, the treatment is initiated with an intravenous dose of 10 mg/kg (days 15, 19, 22). Following the treatment, the tumor growth is tracked until day 77. The animals in the control group had to be sacrificed on day 50 for veterinary reasons because of the large tumors.
  • A naked anti-EGFR antibody shows a delayed tumor growth at approx. 14 days.
  • Animals treated with the anti-EGFR ADCs (Example 7 and Example 10) did not exhibit any further tumor growth until the end of the experiment on day 77.
    • C-1.4c Efficacy in the NCI-H292 NSCLC Tumor Model
  • Five million NCI-H292 cells were injected subcutaneously into the flanks of female NMRI nude mice for inoculation.
  • At a tumor size of 100 mm2 on day 15, the treatment was initiated with an intravenous dose of 3 mg/kg (days 15, 19, 23). The tumor growth was tracked until day 27 after the treatment. At the end of the experiment, the tumors were excised and weighed. The experiment was terminated on day 27 because the animals in the control group had to be euthanized due to the large tumors. The animals treated with the naked anti-EGFR antibody exhibited inhibited tumor growth. The animals treated with the anti-EGFR ADCs show regression of the tumor. After an incubation time of several days, the tumor size was determined in comparison of conjugate-treated animals with the control group (T/C). The animals treated with the conjugate had tumors of a smaller size.
  • The T/C ratio for the ADCs is between 0.05 (Example 10) and 0.1 (Example 7), whereas that with the naked anti-EGFR antibody is 0.3.
    • C-2.1 In Vitro Cell Proliferation Tests
  • The cytotoxic effect of the conjugates according to the invention was tested in an in vitro cell proliferation test by incubating a mammal cell that expresses the target molecule of the binder either endogenously or recombinantly with the conjugate according to the invention. After an incubation time of several hours to several days, cell proliferation was determined on the basis of the cell count in comparison with controls to which no conjugated was added. The unconjugated toxophore alone may be added as additional controls and/or cells that do not express the target molecule of the binder may be used. The cell count was determined by methods with which those skilled in the art are familiar, for example, by counting or by using a test kit which allows a determination of the cell count based on a measurement of ATP (e.g., ATPlite™, Perkin Elmer).
  • The IC50 value of the conjugates according to the invention was determined in this way. The selectivity of the conjugate could be determined by comparing the IC50 value of the conjugate in measurements on cells carrying the target molecule of the binder and cells not carrying that molecule.
    • C-2.2 Determination of the Antiproliferative Effect of Anti-Mesothelin ADC on the Human Colon Carcinoma Cell Line HT29
  • A defined cell count of the human colon carcinoma cell line HT29 wt (2500 c/well, wild type) was sown in a 96-well MTP in whole medium (10% FCS RPMI) and incubated overnight at 37° C., 5% carbon dioxide. In parallel with that transfected HT29 cells with stable mesothelin expression were plated out in a 96-well MTP in whole medium and incubated overnight (2500 c/well, 37° C., 5% carbon dioxide).
  • After 18 hours, the inoculation medium was replaced by fresh medium with 10% FCS. The treatment was initiated with the addition of the compounds according to the invention. The transfected cells and the HT29 wt cells were treated identically here.
  • Of the substances to be tested, dose-effect curves were determined in a concentration range from 10−5 M to 1014 M (1:10 dilution series).
  • Incubation times of 48-96 hours were selected.
  • Proliferation was determined with the help of the MTT assay (ATCC, Manassas, Va., USA, catalog no. 30-1010K). After the selected time had elapsed, the HT29 wt cells were incubated for 4 hours with MTT before lysis of the cells was performed overnight by adding detergent.
  • The dye thus formed was detected at 570 nm.
  • The 100% value was defined as the proliferation not with test substance but otherwise identically treated cells. The data obtained from this test represents triple determinations and at least two independent experiments were conducted.
  • Table 6 below lists the IC50 values1) of representative exemplary embodiments of this assay:
  • TABLE 6
    HT29 meso+ HT29 wt HT29 meso+ HT29 wt
    Example IC50 (nM) IC50 (nM) Example IC50 (nM) IC50 (nM)
    56 0.17 5.3 101 3 500
    57 0.4 162 102 7.2 294
    58 5 22 103 10 32
    59 1 13 104 1.8 0.9
    60 0.06 8 105 0.6 1.4
    60 0.16 5.4 106 0.8 15
    61 0.019 0.27 107 3 2
    62 20 >300 108 0.74 9.2
    63 0.03 1 109 0.09 1.1
    64 0.11 1.4 110 0.002 0.28
    65 0.14 3.3 111 0.7 162
    66 1.5 8.6 112 0.018 4
    67 0.015 11 113 0.4 2.5
    68 2 9 114 0.6 4.1
    69 0.07 20 115 0.06 10
    70 0.02 2 129 5.3 57
    71 0.6 11 130 0.13 672
    72 0.05 8 131 0.09 769
    73 25 221 132 3.3 >1000
    74 9 >50 133 2.1 219
    75 0.2 0.29 134 0.03 199
    76 0.35 3 135 0.25 317
    77 0.8 3.6 136 1.6 3000
    78 1.1 6 137 0.04 10
    79 0.5 5 138 0.3 >1000
    80 4.4 14 139 70 >1000
    81 0.02 1.4 140 6.8 >1000
    82 24 >1000 141 43 >1000
    83 0.3 31 142 0.27 554
    84 6 14 143 0.3 54
    85 0.2 2 144 2.7 74
    86 0.45 >500 145 7.4 135
    87 0.9 3 146 13 >1000
    88 0.01 0.2 147 5.6 18
    89 0.06 3.1 148 425 >1000
    90 2.6 60 149 3.1 33
    91 0.35 1.1 150 <0.1 0.15
    92 38 167 151 >1000 >1000
    93 52 89 152 4.1 >1000
    94 0.6 >500 153 30 >1000
    95 0.013 4.9 154 2.2 >1000
    96 25 >500 155 0.5 >500
    97 0.2 0.2 156 4.6 >500
    98 1.7 6.3 157 0.09 839
    99 0.27 1.6 158 0.18 602
    100 2 44 159 177 >500
    366 1.3 16 367 0.06 10
    368 3.1 >500 369 0.02 14
    377 1.6 >500 378 0.04 >500
    379 7 >500 380 15 >500
    1) Efficacy data listed here is based on the exemplary embodiments described in the present experimental section with the drug/mAb ratios indicated. The values may deviate with different drug/mAb ratios.

    C-2.3 Pharmacokinetics in the HT29 Tumor Model with Mesothelin-Transfected HT29 Cells and Non-Transfected Cells
  • After i.v. administration of 16 mg/kg from Example 60, the plasma and tumor concentrations of Example 60 were measured along with potentially occurring metabolites such as those of Example 119, for example. The area under the curve (AUC) of the compound from Example 119 in the plasma of animals with mesothelin-transfected tumors was approx. 0.50 mg·h/L; in the tumor, the exposure of the compound from Example 119 was approx. 400 times higher (AUC=203 mg·h/L). In the animals with non-transfected tumors, the exposure in the plasma in Example 119 was identical to the exposure in the plasma of animals with transfected tumors. However, the AUC in the tumor of the non-transfected animals was approx. eight times lower than in the transfected animals. This is indicative of a definite targeting effect in the tumor in the presence of the antigen.
  • Analysis for Quantitation of the Compound from Example 119
  • The measurement of the compound from Example 119 in the plasma and the tumor was performed after precipitation of the proteins with methanol by high pressure liquid chromatography (HPLC) coupled to a tandem mass spectrometer (MS).
  • For workup of 100 μL plasma, it was mixed with 400 μL methanol and 10 μL internal standard (ISTD, 50 ng/mL methanol) and agitated for 10 seconds. After centrifuging for 5 minutes at 16,000 g, 250 μL supernatant was transferred to an autosampler vial, topped off with 250 μL ammonium acetate buffer (AAC, 10 mM, pH 6.8) and agitated again.
  • In workup of a tumor, it was mixed with a four-fold amount of methanol. The sample was pulverized for 6 minutes at 30 beats per minute in the Tissuelyser II (Quiagen) and then centrifuged for 5 minutes at 16,000 g; 50 μL of the supernatant was transferred to an autosampler vial and topped off with 50 μL ammonium acetate buffer (10 mM, pH 6.8) and 5 μL ISTD. After agitating again, the tumor sample was ready for measurement.
  • Finally, the measurement of both matrix samples was performed on the HPLC coupled atmospheric pressure ionization/tandem mass spectrometer by means of turbo ion spray interface (TISP) on an API 4000 instrument from the company SCIEX. The following m/z transitions were measured:
    • Example 119 (quantifier) 614.652→570.9
    • Example 119 (qualifier 1) 614.652→555.0
    • Example 119 (qualifier 2) 614.652→500.4
    • Internal standard (ISTD) 726.665→694.5
  • HPLC/LC MSMS (TISP) analysis was performed under the following gradient conditions on an HP1100 pump (Agilent) using the Gemini column (5 μL C18 110 A, 50×3 mm, Phenomenex); flow rate 0.4 mL/min; gradient: 0.0 min to 1.0 min 10% acetonitrile/90% AAC, 1.0 min to 3.0 min 10% acetonitrile/90% AAC→50% acetonitrile/50% AAC, 3.0 min to 5.5 min 50% acetonitrile/50% AAC, 5.5 min to 5.6 min 50% acetonitrile/50% AAC→10% acetonitrile/90% AAC, 5.6 min to 6.0 min 10% acetonitrile/90% AAC.
  • For the calibration, plasma samples with concentrations of 0.5-2000 μg/L were mixed. The limit of quantitation (LOQ) was 2 μg/L. The linear range was from 2 to 1000 μg/L.
  • For calibration of the tumor samples, the supernatants of untreated tumors with concentrations of 0.5 to 200 μg/L were mixed. The limit of quantitation was 5 μg/L. The linear range was from 5 to 200 μg/L.
  • Quality controls for validity testing contained 5 and 50 μg/L, with an additional 500 μg/L in the plasma. The concentrations determined on these samples deviated by as much as 20% from the ideal value (data not included).
    • C-2.4 Efficacy Test In Vivo
  • The efficacy of the conjugates according to the invention was tested in vivo by using xenograft models, for example. Those skilled in the art are familiar with the state-of-the-art methods for testing the efficacy of a conjugate according to the invention (see, for example, WO 2005081711; Polson et al., Cancer Res. Mar. 15, 2009, 69(6):2358-64). For example, a tumor cell line that expresses the target molecule of the binder would be implanted in rodents (e.g., mice). Then a conjugate according to the invention or a control antibody or an isotonic saline solution would be administered to the implant animals. The substance would be administered one or more times. After an incubation time of several days, the tumor size would be determined in comparison with animals treated with the conjugate and with the control group. The size of the tumors was smaller in the animals treated with the conjugate.
    • C-2.4a Testing Anti-Mesothelin ADCs in Experimental Tumors in the Mouse
  • Human mesothelin-expressing tumor cells are injected subcutaneously into the flanks of immunosuppressed mice, for example, nude or SCID mice; 1-10 million cells are isolated from the cell culture, centrifuged and resuspended in 100 μL Medium or 1:1 Medium/Matrigel. The cell suspension is then injected subcutaneously into the mice.
  • A tumor will then grow within a few days. The treatment begins after establishment of the tumor but at a tumor size of 20 mm2. To investigate the effect of larger tumors, the treatment may be started only when the tumor size reaches 50-100 mm2.
  • Treatment with ADCs is performed via the intravenous route into the caudal vein of the mouse. The ADC is dissolved in PBS and administered in a volume of 5 mL/kg.
  • The treatment regime depends on the pharmacokinetics of the antibody. The standard treatment consists of treatment three times every fourth day. However, the treatment may also be continued further or a second cycle with three days of treatment may also follow at a later point in time.
  • Eight animals are used per treatment group as the standard. This number may be increased if especially great fluctuations in tumor growth or according to treatment are to be expected. In addition to the groups receiving the active substances, one group as the control group is treated only with the buffer according to the same scheme.
  • In this experiment, the area of the tumor is measured regularly in two dimensions (length/width) using a caliper. The area of the tumor is determined by length×width.
  • At the end of the experiment, the tumors are excised and weighed. The comparison of the average tumor weights of the treatment group (T) with the control group (C) is reported as T/C.
  • C-2.4b Efficacy in the HT29 Colon Carcinoma Tumor Model with Mesothelin-Transfected HT29 Cells
  • One million HT29 cells (stable transfection with mesothelin) were injected subcutaneously into the flanks of NMRI nude mice for inoculation. At a tumor size of 20-30 mm2 on day 6, intravenous treatment begins (days 6, 10, 14). Following the treatment, the tumor growth was tracked until day 48.
    • C-2.4c Efficacy in the Ovcar3 Ovarian Carcinoma Tumor Model
  • Seven million Ovcar3 cells were injected subcutaneously into the flanks of NMRI nude mice for inoculation.
  • At a tumor size of 25-30 mm2 on day 31, the intravenous treatment was initiated in the dosage range of 5-30 mg/kg (days 31, 35, 39). Following the treatment, the tumor growth was tracked until day 94. At the end of the experiment, the tumors were excised and weighed.
    • C-3.1 Analysis of the Cytotoxic Effect of the ADCs Directed Against C4.4a
  • The cytotoxic effect of the anti-C4.4a ADCs was analyzed on various cell lines:
      • A549 (CCL-185, ATCC) transfected with the sequence for the complete C4.4a receptor
      • A549, mock transfected
      • A549 wild type (DSMZ, lot 11)
      • NCI-H292, endogenous C4.4a-expressing lung cancer cell line CRL-1848, ATCC)
      • SCC-4 endogenous C4.4a-expressing squamous epithelial cell carcinoma cell line (CRL-1624, ATCC)
      • SCC-9 endogenous C4.4a-expressing squamous epithelial cell carcinoma cell line (CRL-1629, ATCC)
      • HCT-116 endogenous C4.4a-expressing colon carcinoma cell line (CCL-247, ATCC)
      • HCT-116/VM46, HCT-116 transfected with VM46
      • A431NS(CRL-2592, ATCC)
  • The cells were cultured according to the standard method as stipulated in the American Tissue Type Collection (ATCC) for the respective cell lines. For this procedure, the cells were isolated with a solution of trypsin (0.05%) and EDTA (0.02%) in PBS (Biochrom AG #L2143), pelletized, resuspended in culture medium, counted and sown in a 94-hole culture plate with a white bottom (Costar #3610) (2500 cells in 100 μL/well) and then incubated at 37° C. and 5% carbon dioxide in an incubator. After 24 hours, the antibody-drug conjugates were added to the cells in 100 μL culture medium in concentrations of 10−7 M to 10−11 M (double values) and incubated at 37° C. and 5% carbon dioxide in an incubator. After 72 hours, the cell viability was determined by the Cell Titer Glow Luminescent Cell Viability Assay (Promega #G7573 and #G7571). To do so, 100 μL of substrate was added per cell batch, then the plates were covered with aluminum foil, agitated for 2 minutes with the plate agitator at 180 rpm, left to stand for 8 minutes on the laboratory bench and then measured using the Victor X2 instrument (Perkin Elmer). The substrate detects the ATP content in the viable cells, thus generating a luminescence signal, the height of which is directly proportional to the vitality of the cells. The IC50 is calculated from the measured data using the Graph Pad Prism laboratory software.
  • Table 7 below shows the IC50 value1) of representative exemplary embodiments of this assay:
  • IC50 (nM) IC50 (nM)
    A549: A549
    Example C4.4a Mock
    163 0.081 11.15
    164 0.7 50
    165 0.47 4.75
    166 0.6 100
    167 0.4 20
    0.2 26
    0.1 17
    168 0.53 4.50
    169 0.39 32
    170 0.01 0.15
    171 0.43 10
    172 0.01 25
    173 4 >100
    174 0.58 6.36
    175 0.7 14.9
    176 0.1 65.5
    177 0.030 9.53
    178 3.8 21
    179 0.62 4.19
    180 0.4 >100
    181 1.2 66.1
    182 0.46 4.20
    183 4.5 12.7
    184 5 16
    185 0.4 0.7
    186 0.3 23
    187 5.4 53
    188 0.052 11.27
    189 0.65 6.70
    190 0.062 >100
    191 0.02 2.5
    192 0.1 71
    193 0.32 9
    194 0.035 6.19
    195 0.037 ~30
    196
    197 0.3 20
    198 0.08 >100
    199 0.1 kH
    200 0.03 50
    201 0.04 1.5
    202 0.6 50
    203 0.4 2
    204 0.1 14
    205 0.062 6.33
    206 0.044 6.93
    207 0.058 4.01
    208 0.062 7.74
    209 0.066 9.11
    210 0.061 6.78
    211 0.076 100
    212 0.02 0.02
    213 0.044 44
    214 0.04 45
    215 0.046 26
    216 0.074 >100
    217 0.053 >100
    218 0.037 60
    219 0.3 1
    220 0.04 >100
    221 0.1 >100
    222 0.04 >100
    223 0.44 6.8
    224 0.09 50
    225 0.1 0.4
    226 0.04 0.52
    227 0.03 0.04
    228 0.03 0.04
    229 0.08 26
    230 0.02 >100
    231 0.17 0.27
    212 0.06 7
    242 3.0 >100
    243 0.045 >100
    244 0.06 >100
    245 0.27 >100
    246 0.13 >100
    247 0.14 >100
    248 0.17 >100
    249 0.28 >100
    250 1.1 >100
    251 1.3 >100
    252 >100 >100
    253 0.15 kH
    254 0.29 >100
    255 0.04 >100
    256 0.035 100
    257 0.036 >100
    258 0.018 >100
    259 0.062 >100
    260 0.06 >100
    261 0.l 80
    262 0.1 kH
    263 0.3 kH
    264 0.1 kH
    265 0.2 30
    266 3 kH
    267 0.03 50
    268 0.05 20
    269 >100 kH
    270 0.03 >100
    271 1 >100
    272 0.2 kH
    373 0.03 7
    385 0.04 >100
    386 0.02 >100
    387 0.04 >100
    388 0.04 >100
    1) Efficacy data listed here is based on the exemplary embodiments described in the present experimental section with the drug/mAb ratios indicated. The values may deviate with different drug/mAb ratios.
    • C-3.3 In Vitro Tests for Determining Cell Permeability
  • The cell permeability of a substance can be investigated by means of in vitro testing in a flux assay using Caco2 cells (M. D. Troutman and D. R. Thakker, Pharm. Res. 20(8):1210-1224 (2003)). To do so, the cells are cultured on 24-hole filter plates for 15-16 days. To determine the permeation, the respective test substance in a HEPES buffer was applied to the cells either apically (A) or basally (B) and incubated for 2 hours. After 0 h and 2 h, samples were taken from the cis and trans compartments. The samples were separated by HPLC (Agilent 1200, Biblingen, Germany) using reverse-phase columns. The HPLC system was linked to a triple quadrupole mass spectrometer API 4000 (Applied Biosystems Applera, Darmstadt, Germany) via a turbo ion spray interface. Permeability was evaluated on the basis of a Papp value which was calculated by means of the formula published by Schwab et al. (D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)). A substance was classified as having active transport when the ratio of Papp(B−A) to Papp(A−B) was >2 or <0.5.
  • The permeability of B to A (Papp(B−A)) is of crucial importance for toxophores but are released intracellularly: the lower this permeability, the longer is the dwell time of the substance in the cell after intracellular release and thus also the time available for interaction with the biochemical target (here tubulin).
  • The following table shows permeability data of representative exemplary embodiments of this assay:
  • TABLE 8
    Exemplary Papp (B-A)
    embodiment (nm/s)
    233 2
    234 1.6
    235 2.5
    236 5
    237 1
    239 7
    277 2
    278 1
  • In comparison with this monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) have a Papp(B−A) value of 73 nm/s in this test.
    • C-3.4 In Vitro Tests for Determining the Substrate Properties for P-Glycoprotein (P-gp)
  • Many tumor cells express transporter proteins for drugs, which is often associated with the development of a resistance to cytostatics. Substances that are not substrates of such transporter proteins such as P-glycoprotein (P-gp) or BCRP, for example, might thus have an improved profile of effect.
  • The substrate properties of a substance for P-gp (ABCB1) are determined by means of flux assay using LLC-PK1 cells that overexpress P-gp (L-MDR1 cells) (A. H. Schinkel et al., J. Clin. Invest. 96, 1698-1705 (1995)). To do so, LLC-PK1 cells or L-MDR1 cells were cultured on 96-hole filter plates for 3-4 days. To determine the permeation, the respective test substance was applied to the cells and incubated for 2 hours, either alone or in the presence of an inhibitor (e.g., ivermectin or verapamil) in a HEPES buffer either apically (A) or basally (B). After 0 h and 2 h, samples were taken from the cis and trans compartments. The samples were separated by HPLC using reverse-phase columns. The HPLC system was coupled to a triple quadrupole mass spectrometer API 3000 (Applied Biosystems Applera, Darmstadt, Germany) by way of a turbo ion spray interface. The permeability was evaluated on the basis of a Papp value which was calculated by using the formula published by Schwab et al. (D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)). A substance was classified as being a P-gp substrate if the efflux ratio Papp(B−A) to Papp(A−B) was >2.
  • The efflux ratios in L-MDR1 and LLC-PK1 cells or the efflux ratio in the presence or absence of an inhibitor can be compared as additional criteria for evaluating the P-gp substrate properties. The respective substance is considered to be a P-gp substrate if these values differ by more than a factor of 2.
  • TABLE 9
    Exemplary Papp (B-A)
    embodiment (nm/s)
    233 3
    234 3.6
    235 2.1
    236 3.6
    237 4
    239 2
    277 6
    278 4
  • Again in this assay the permeability of the examples of B to A cited here is low, i.e., the dwell time of the toxophores in the cells is long.
    • C-3.5 Efficacy Test In Vivo
  • The efficacy of the conjugates according to the invention was tested in vivo, for example, by means of xenograft models. Those skilled in the art are familiar with the state-of-the-art methods for testing the efficacy of a conjugate according to the invention (see, for example, WO 2005081711; Polson et al., Cancer Res. Mar. 15, 2009, 69(6):2358-64). For example, a tumor cell line that expresses the target molecule of the binder would be implanted in rodents (e.g., mice). Then a conjugate according to the invention or a control antibody or an isotonic saline solution would be administered to the implant animals. The substance would be administered either one or more times. The tumor growth was determined using a caliper twice a week. After an incubation time of several days, the tumor size would be determined in comparison with animals treated with conjugate and with the control group.
    • C-3.5a Testing of ADCs in Experimental Tumors in the Mouse
  • Human tumor cells that express the antigen for ADC are injected subcutaneously into the flanks of immunosuppressed mice for inoculation, for example, nude mice or SCID mice. Of the cell culture, 1-10 million cells are isolated, centrifuged and resuspended with 100 μL medium or 50% medium/50% Matrigel. The cell suspension is then injected subcutaneously into the mice.
  • A tumor will then grow within a few days. The treatment begins after establishment of the tumor but at a tumor size of 25 mm2.
  • Treatment with ADCs is performed via the intravenous route into the caudal vein of the mouse.
  • The ADC is dissolved in PBS and administered in a volume of 10 mL/kg.
  • The treatment regime depends on the pharmacokinetics of the antibody. The standard treatment consists of treatment three times every fourth day. However, the treatment may also be continued further or a second cycle with three days of treatment may also follow at a later point in time.
  • Eight animals are used per treatment group as the standard. This number may be increased if especially great fluctuations in tumor growth or according to treatment are to be expected. In addition to the groups receiving the active substances, one group as the control group is treated only with the buffer according to the same scheme.
  • In the case of this experiment, the area of the tumor is measured regularly using a caliper in two dimensions (length/width). The area of the tumor is determined by length×width.
  • At the end of the experiment, the tumors are excised and weighed. The quotient of the average tumor weights of the treatment group (T) and the control group (C) is given as T/C. If the control group and treatment group are ended at different points in time, the T/C value is calculated on the basis of the tumor areas of the last joint measurement of all treatment groups and control groups.
  • One million SCC-4 cells are injected subcutaneously into the flanks of female NMRI nude mice for inoculation.
  • The intravenous treatment with the ADCs is started when the average tumor size has reached 30-35 mm2. When the control groups have reached the maximum allowed size, the test is terminated and the tumors are excised and weighed. All the C-4.4a targeting ADCs tested were found to have inhibited tumor growth in a dose dependent ratio. In a dose of 30 mg/kg all the tested ADCs achieved a T/C ratio of <0.1 (Example 216, Example 211, Example 215, Example 213). A significant antitumor effect was achieved in comparison with the controls for all the tested ADCs down to a dose of 15 mg/kg.
  • One million NCI-H292 cells are injected subcutaneously into the flanks of female NMRI nude mice for inoculation.
  • The intravenous treatment with the ADCs was initiated at an average tumor size of 30-35 mm2. The control groups and treatment groups were terminated whenever the maximum allowed tumor size was reached. Therefore, differences in the subsequent growth of tumors after the end of treatment can contribute toward a further characterization of the ADCs. The tumor areas at the last joint measurement time were therefore used to determine the antitumor effect in comparison with the controls (T/C). In the NCI-H292 mouse model used in this experiment, it is demonstrated that all the tested ADCs were able to reduce tumor growth in a dose dependent manner in comparison with the control. For Example 216, a significant antitumor effect was achieved down to a dose of 1.9 mg/kg; for Example 211, this was achieved down to a dose of 3.75 mg/kg. The minimal T/C values achieved in this model include a T/C of 0.16 at 30 mg/kg for Example 216; a T/C of 0.17 at 30 mg/kg for Example 211; a T/C of 0.16 at 30 mg/kg for Example 215, and a T/C of 0.17 at 15 mg/kg for Example 213.
  • C-4. Pharmacokinetics in the A549 Tumor Model with C4.4a-Transfected and Non-Transfected A549 Cells
  • After i.v. administration of 7-30 mg/kg of various ADCs, the plasma and tumor concentrations of ADC and any potential metabolites that might occur were measured and the pharmacokinetic parameters such as the clearance (CL), area under the curve (AUC) and half-life (t1/2) were calculated.
    • Analysis for Quantitation of the Metabolites Occurring Potentially
  • After precipitation of the proteins with methanol, the compounds in the plasma and tumor were measured by high pressure liquid chromatography (HPLC) coupled to a tandem mass spectrometer (MS).
  • For workup of 100 μL plasma, it was mixed with 400 μL methanol and 10 μL internal standard (ISTD, 50 ng/mL in methanol) and shaken for 10 seconds. After centrifuging for 5 minutes at 16,000 g, 250 μL supernatant was transferred to an autosampler vial, topped off with 250 μL ammonium acetate buffer (AAC, 10 mM, pH 6.8) and shaken again.
  • In workup of a tumor, it was mixed with a four-fold amount of methanol. The sample was pulverized for 6 minutes at 30 beats per minute in the Tissuelyser II (Quiagen) and then centrifuged for 5 minutes at 16,000 g; 50 μL of the supernatant was transferred to an autosampler vial and topped off with 50 μL ammonium acetate buffer (10 mM, pH 6.8) and 5 μL ISTD. The tumor sample was ready for measurement.
  • Finally, the two matrix samples were measured on the atmospheric pressure ionization/tandem mass spectrometer by means of turbo ion spray interface (TISP) on an API4000 instrument from the company SCIEX with the mass spectrometer linked to an HPLC.
  • HPLC/LC-MSMS (TISP) analysis was performed on an HP1100 pump (Agilent) with a Gemini column (5 μm C18 110 A, 50×3 mm, Phenomenex).
  • For calibration, plasma samples with concentrations of 0.5-2000 μg/L were mixed. The limit of quantitation (LOQ) was 2 μg/L. The linear range extended from 2 to 1000 μg/L.
  • For calibration of the tumor samples, the supernatant of untreated tumors with concentrations of 0.5-200 μg/L was mixed. The limit of quantitation was 5 μg/L. The linear range extended from 5 to 200 μg/L.
  • Quality controls for validity testing contained 5 and 50 μg/L plus 500 μg/L in the plasma. The concentrations determined on these samples deviated as much as 20% from the ideal value (data not included).
    • C-4.1 In Vitro Cell Proliferation Tests
  • The cytotoxic effect of the conjugates according to the invention was tested in an in vitro cell proliferation test by incubating a mammalian cell that expresses the target molecule of the binder either endogenously or recombinantly with the conjugate according to the invention. After an incubation time of several hours to several days, cell proliferation was determined on the basis of the cell count in comparison with controls to which no conjugated was added. The unconjugated toxophore alone may be added as additional controls and/or cells that do not express the target molecule of the binder may be used. The cell count was determined by methods with which those skilled in the art are familiar, for example, by counting or by using a test kit which allows a determination of the cell count based on a measurement of ATP (e.g., ATPlite™, Perkin Elmer). The IC50 value of the conjugates according to the invention was determined in this way. The selectivity of the conjugate was determined by comparing the IC50 value of the conjugate in measurements on cells carrying the target molecule of the binder and cells not carrying that molecule.
    • C-4.2 In Vitro Cytotoxicity on the Cell Lines HT29, DLD-1 and SNU-5
  • For testing the CA9 selective, cytotoxic effect on tumor cells that are endogenously CAIX-positive or CAIX-negative, the human colon carcinoma cell lines HT29 (CA9-positive) and DLD-1 (CA9-negative) as well as the gastric carcinoma cell line SNU-5 (CA9-positive) were used. A defined cell count of the cell line HT29 (5000 c/well) was sown in a 96-well MTP for luminescent in whole medium (DMEM/HAM's F12, 10% FCS heat inactivated) and incubated overnight at 37° C., 5% CO2. A similar procedure was followed with the SNU-5 cell line, but the medium here was ISCOVE's+10% FCS (heat inactivated). The antigen-negative cell line DLD-1 was plated out in parallel in a 96-well MTP for luminescence in whole medium (RPMI 1640, 10% FCS, heat inactivated) and incubated overnight (5000 c/well, 37° C., 5% CO2).
  • After 24 hours, the substances to be tested were concentrated three times in RPMI/5% FCS and prepared. The treatment began with the addition of the substances to be tested and/or the ADC to the cells. The HT29, SNU-5 and DLD-1 cells were treated identically.
  • Of the substances to be tested, dose-effect curves were determined in a concentration range of 3×10−7 M to 10−12 M.
  • Incubation times of 2 to 96 hours were selected.
  • Detection of proliferation was performed with the help of the Cell Titer Glo Luminescent Cell Viability Assay (PROMEGA catalog no. #G7571). After the selected incubation time had elapsed, the Cell Titer Glo reagent was incubated with the cells for 20 minutes and then the measurement of the luminescence was performed with the luminescence reader VICTOR Light (Perkin Elmer).
  • The proliferation not with test substance but with otherwise identically treated cells was defined as the 100% value. The data obtained from this test represents triple determinations and at least two independent experiments were performed.
  • The following table lists the IC50 values of representative exemplary embodiments from this assay:
  • TABLE 10
    EC50 (nM) EC50 (nM) EC50 (nM)
    Example HT29 DLD-1 SNU-5
    280 2.9 58 n.d.
    281 1.7 23 n.d.
    282 <0.01 <0.01 n.d.
    283 0.3 20 n.d.
    284 0.7 39 3
    284 1.6 5.8 2.2
    285 0.5 11.1 n.d.
    286 0.03 6.6 n.d.
    287 >30 >68 n.d.
    288 39 72 n.d.
    289 49 >300 n.d.
    290 115 7938 84
    291 27 107 n.d.
    292 223 74 n.d.
    293 0.1 0.6 n.d.
    294 13.6 5.5 21.4
    295 >100 >100 63
    296 0.4 16.4 n.d.
    297 <0.01 <0.01 n.d.
    298 2.5 31 7.8
    299 4.1 32.3 n.d.
    300 5 41 n.d.
    301 >30 >300 n.d.
    302 >30 >300 n.d.
    303 2.2 18.3 3.3
    304 0.05 8 0.6
    305 0.9 23 2.2
    306 66 >100 33
    • C-4.3 Determining the Antiproliferative Effect of Anti-CAIX ADC on the Human Pancreatic Carcinoma Cell Line MIAPaCa 2 and Colon Carcinoma Cell Line HT29
  • A defined cell count of the human pancreatic carcinoma cell line MIAPaCa 2 (2500 c/well, wild type) was sown in a 96-well MTP in whole medium (DMEM, 10% FCS, 2.5% equine serum) and incubated overnight at 37° C., 5% CO2. Transfected MIAPaCa 2 cells (MIAPaCaMSL) with stable CAIX expression were plated out in a 96-well MTP in whole medium and incubated overnight (2500 c/well, 37° C., 5% CO2).
  • To test the cytotoxic effect on cells that endogenously express CAIX, the colon carcinoma cell line HT29 was used. The cells (2500 c/well, wild type) were also sown in a 96-well MTP and incubated overnight in whole medium (RPMI, 10% FCS).
  • After 18 hours, the inoculation medium was replaced by fresh medium with serum. Treatment was initiated by adding the substances to be tested and/or the ADC. The transfected cells and MIAPaCa2 cells as well as the HT29 cells were treated identically.
  • Of the substances to be tested, dose-effect curves in a concentration range of 10−5M to 10−14 M (1:10 dilution series) were determined.
  • Incubation times of 48-96 hours were selected.
  • Proliferation was detected with the help of the MTT assay (ATCC, Manassas, Va., USA, catalog no. 30-1010K). After the end of the selected incubation time, the MTT reagent was incubated for 4 hours with the cells before lysis of the cells was performed overnight by adding the detergent.
  • The dye that was formed was detected at 570 nm.
  • Proliferation not with test substance but otherwise identically treated cells was defined as the 100% value. Data obtained from this test represents triple determinations and at least two independent experiments were conducted in each case.
  • The following table shows the IC50 values of representative exemplary embodiments from this assay:
  • TABLE 11
    MIAPaCa MIAPaCa MIAPaCa MIAPaCa
    Ex- 2/CA9+ 2 wt Ex- 2/CA9+ 2 wt
    ample IC50 (nM) IC50 (nM) ample IC50 (nM) IC50 (nM)
    280 0.4 8 296 0.6 0.9
    281 1.2 4.1 297 0.02 0.4
    282 0.05 0.5 298 1.1 9
    283 0.4 2.1 299 0.8 3.3
    284 0.9 5 300 0.3 1.1
    285 0.6 1.8 301 22 34
    286 0.8 2.7 302 3.9 39
    287 69 >1000 303 0.009 0.015
    288 12 42 304 0.019 0.03
    289 78 >1000 305 2.6 3.7
    290 1.9 357 306 21 25
    291 22 n.d. 313 3.8 44
    292 35 1686 314 150 >1000
    293 <0.1 <0.1 315 3.2 23
    294 1 7 320 19 78
    295 25 1400 321 220 1142
    322 172 >1000 325 137 >1000
    323 238 >500 326 2 >500
    324 11 15 327 19 26
    • C-4.4 Efficacy Test In Vivo
  • The efficacy of the conjugates according to the invention was tested in vivo, for example, by means of xenograft models. Those skilled in the art are familiar with the state-of-the-art methods for testing the efficacy of a conjugate according to the invention (see, for example, WO 2005081711; Polson et al., Cancer Res. Mar. 15, 2009, 69(6):2358-64). For example, a tumor cell line that expresses the target molecule of the binder would be implanted in rodents (e.g., mice). Then a conjugate according to the invention or a control antibody or an isotonic saline solution would be administered to the implant animals. The substance would be administered either one or more times. After an incubation time of several days, the tumor size would be determined in comparison with animals treated with conjugate and with the control group.
    • C-4.4a Testing the Efficacy of Anti-CA9 ADCs in Experimental Tumors in the Mouse
  • Human CA9-expressing tumor cells are injected s.c. into the flanks of immunosuppressed mice, for example, nude or SCID mice; 1-10 million cells are isolated from the cell culture, centrifuged and resuspended with 100 μL Medium or Medium/Matrigel. The cell suspension is then injected subcutaneously into the mice.
  • A tumor will then grow within a few days. The treatment begins after establishment of the tumor but at a tumor size of 20 mm2. To investigate the effect of larger tumors, the treatment may be started only when the tumor size reaches 50-100 mm2.
  • Treatment with ADCs is performed via the intravenous route into the caudal vein of the mouse. The ADC is dissolved in PBS and administered in a volume of 5 mL/kg.
  • The treatment regime depends on the pharmacokinetics of the antibody. The standard treatment consists of treatment three times every fourth day. However, the treatment may also be continued further or a second cycle with three days of treatment may also follow at a later point in time.
  • Eight animals are used per treatment group as the standard. This number may be increased if especially great fluctuations in tumor growth or according to treatment are to be expected. In addition to the groups receiving the active substances, one group as the control group is treated only with the buffer according to the same scheme.
  • In the case of this experiment, the area of the tumor is measured regularly using a caliper in two dimensions (length/width).
  • At the end of the experiment, the tumors are excised and weighed. The quotient of the average tumor weights of the treatment group (T) and the control group (C) is given as T/C. If the control group and treatment group are terminated on different days, the tumor area at the last measurement point at which all the groups were still in the experiment was used to calculate the T/C.
    • C-4.4b Efficacy in SNU-5 Gastric Carcinoma Tumor Model
  • Three million SNU-5 cells were injected subcutaneously into the flanks of nodSCID mice for inoculation.
  • At a tumor size of 30-40 mm2 on day 15, the treatment is initiated by intravenous injection of doses in the range between 5 and 30 mg/kg (days 15, 19, 23). Following the treatment the tumor growth of all groups was tracked. The control groups were ended when the tumors had reached the maximum allowed tumor size. Either all the groups were terminated together at this point in time, the tumors were excised and weighed and the T/C value was formed based on the tumor weight, or the treatment groups were observed with regard to further growth of the tumors. In the latter case, the T/C value was calculated based on the tumor area at the last joint measurement time. The animals treated with the anti-CA9 ADCs showed inhibited tumor growth.
    • C-4.4c HT29 Model
  • Female athymic nude mice carrying the nu/nu gene were used for the human xenograft study of mice. These inbred mice (NMRI background) were obtained from Taconic, Denmark with a body weight between 18 and 21 g. Human HT29 colon carcinoma cells were cultured in the recommended medium with 10% fetal calf serum (FCS) in accordance with the ATCC protocol. The cells were harvested for transplantation in the subconfluent state (80% confluence). On day 0, tumors were initiated by subcutaneous (s.c.) injection of 1×106 HT29 cells in 50% Matrigel/50% culture medium (without FCS) in the mice. The transplantation volume was 100 μL, the transplantation site was the left flank. Tumor growth was determined by measuring the area of the tumor (calculation: longest diameter×length of the perpendicular to that diameter), measured by caliper. According to the statement of object, the tumors were used up to a predefined size of 20-30 or 40-50 mm2. At this point in time, the animals were randomized and assigned to the individual test groups—control groups and treatment groups. The treatment with ADCs was administered every fourth day for a total of three times (Q4D×3). The form of the treatment was an intravenous (i.v.) injection into the caudal vein. The treatment of each animal was based on the individual body weight and the treatment volumes were 5-10 mL/kg body weight. The area of the tumor and the animal's weight were determined twice a week, monitoring the body weight as a measure of the treatment-associated toxicity. Animals were euthanized when signs of toxicity developed or when the tumor reached the size of 150 mm2 or when tumors became necrotic. At the time of termination of a group, the animals were euthanized, the tumors were excised and the respective tumor wet weight was determined. The response to treatment was calculated as the ratio of treatment to controls based on the tumor area or the final tumor weight where appropriate.
    • D. EXEMPLARY EMBODIMENTS FOR PHARMACEUTICAL COMPOSITIONS
  • The compounds according to the invention can be converted to pharmaceutical preparations by the following procedure:
  • i.v. Solution:
  • The compound according to the invention is dissolved in a concentration below the saturation solubility in a physiologically safe solvent (e.g., isotonic saline solution, D-PBS or a formulation with glycine and sodium chloride in citrate puffer with the addition of polysorbate 80). The solution is sterile-filtered and bottled in sterile and pyrogen-free injection containers.
  • i.v. Solution
  • The compounds according to the invention can be converted to the dosage forms indicated. This may be done in a known way by “mixing with” or “dissolving in” inert nontoxic pharmaceutical suitable excipients (e.g., buffer substances, stabilizers, solubilizers, preservatives). The following may be included for example: amino acids (glycine, histidine, methionine, arginine, lysine, leucine, isoleucine, threonine, glutamic acid, phenylalanine and others), sugars and related substances (glucose, saccharose, mannitol, trehalose, sucrose, mannose, lactose, sorbitol), glycerol, sodium, potassium, ammonium and calcium salts (e.g., sodium chloride, potassium chloride or disodium hydrogen phosphate and many more) acetate/acetic acid buffer systems, phosphate buffer systems, citric acid and citrate buffer systems trometamol (TRIS and TRIS salts), polysorbates (e.g., polysorbate 80 and polysorbate 20), poloxamers (e.g., poloxamer 188 and poloxamer 171), macrogols (PEG-derivatives, e.g., 3350), Triton X-100, EDTA salts, glutathione, albumins (e.g., human), urea, benzyl alcohol, phenol, chlorocresol, metacresol, benzalkonium chloride and many others.
  • Lyophilisate for Later Conversion to an i.v., s.c. or i.m. Solution:
  • Alternatively, the compounds according to the invention may be converted to a stable lyophilisate (possibly with the help of the excipients listed above) and reconstituted with a suitable solvent (e.g., water for injection, isotonic, saline solution) before administration and then administered.

Claims (33)

1. Binder-drug ingredient conjugates of general formula (Ia)
Figure US20140127240A1-20140508-C01082
in which
n is a number from 1 to 50,
AK is a binder,
the group §-G-L1-B-L2-§§ is a linker,
wherein
§ denotes the linkage site to the group AK and
§§denotes the linkage site to the nitrogen atom,
D is a group of the formula
Figure US20140127240A1-20140508-C01083
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen or methyl,
R2 is isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01084
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O group contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01085
#6 denotes the linkage site to the carbonyl group,
R6 is hydrogen, hydroxyl or benzyloxy,
R3 is hydrogen or methyl,
R4 is isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01086
wherein
#7 denotes the linkage site with the vicinal nitrogen atom,
#8 denotes the linkage site to the group T1,
T1 is a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
wherein
R7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
R8 is hydrogen or methyl,
R9 is hydrogen, methyl, ethyl, n-propyl or benzyl,
or
R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
R10 is benzoyl,
R11 is benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
R5 is hydrogen, methyl or a group of the formula
Figure US20140127240A1-20140508-C01087
wherein
#9 denotes the linkage site to —CHC(R26)-T2,
R12 is phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
R13 is phenyl, which may be substituted with methoxycarbonyl or carboxyl,
R26 is hydrogen or hydroxyl,
T2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl, and
R35 is methyl or hydroxyl,
as well as their salts, solvates, and solvates of the salts.
2. Binder-drug conjugates of the general formula (Ia) according to claim 1, wherein
n is a number from 1 to 50,
AK is AK1 or AK2
wherein
AK1 is a binder which is bound to the group G by a sulfur atom of the binder,
AK2 is a binder which is bound to the group G by a nitrogen atom of the binder,
G for the case when AK=AK1, is a group of the formula
Figure US20140127240A1-20140508-C01088
wherein
#1 denotes the linkage site to the sulfur atom of the binder,
#2 denotes the linkage site to the group L1,
or
for the case when AK=AK2, G is carbonyl,
L1 is a bond, linear (C1-C10)-alkanediyl, or a group of the formula
Figure US20140127240A1-20140508-C01089
wherein
m is a number from 2 to 6,
##1 denotes the linkage site to the group G,
##2 denotes the linkage site to the group B,
L1A is linear (C2-C10)-alkanediyl,
B1 is a group of the formula
Figure US20140127240A1-20140508-C01090
wherein
##5 denotes the linkage site to the group L1A,
##6 denotes the linkage site to the group L1B,
L5 is a bond or (C2-C4)-alkanediyl,
L6 is a bond or a group with the formula
Figure US20140127240A1-20140508-C01091
wherein
##7 denotes the linkage site to the carbonyl group,
##8 denotes the linkage site to L1B,
R33 is hydrogen, (C1-C4)-alkylcarbonyl, tert-butyloxycarbonyl or benzyloxycarbonyl,
R34 is hydrogen or methyl,
R29 is hydrogen or (C1-C4)-alkyl,
R30 is hydrogen or (C1-C4)-alkyl,
or
R29 and R30 together with the atoms to which they are bound form a five- or six-membered heterocycle,
R31 is hydrogen or (C1-C4)-alkyl,
R32 is hydrogen or (C1-C4)-alkyl,
or
R31 and R32 together with the atoms to which they are bound form a five- or six-membered heterocycle,
L1B is linear (C2-C10)-alkanediyl,
and
wherein (C1-C10)-alkanediyl may be substituted with one to four substituents selected independently of one another from the group consisting of methyl, hydroxyl and benzyl
and
wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally situated between them, to form a (C3-C6)-cycloalkyl ring or a phenyl ring,
B is a bond or a group of the formula
Figure US20140127240A1-20140508-C01092
wherein
* denotes the linkage site to L1,
** denotes the linkage site to L2,
P is O or NH,
L3 is a bond or (C2-C4)-alkanediyl,
L4 is a bond or a group of the formula
Figure US20140127240A1-20140508-C01093
wherein
*** denotes the linkage site to the carbonyl group,
**** denotes the linkage site to L2,
R25 is hydrogen or methyl,
R28 is hydrogen, (C1-C4)-alkylcarbonyl, tert-butyloxycarbonyl or benzyloxycarbonyl,
Q1 is a four- to seven-membered heterocycle,
Q2 is a three- to seven-membered carbocycle or a four- to seven-membered heterocycle,
R14 is hydrogen or (C1-C4)-alkyl,
R15 is hydrogen or (C1-C4)-alkyl,
or
R14 and R15 together with the atoms to which they are bound form a five- or six-membered heterocycle,
R16 is hydrogen or (C1-C4)-alkyl,
R17 is hydrogen or (C1-C4)-alkyl,
or
R16 and R17 together with the atoms to which they are bound form a five- or six-membered heterocycle,
R18 is hydrogen or (C1-C4)-alkyl,
R19 is hydrogen or the side group of a natural α-amino acid or its homologs or isomers,
R20 is hydrogen or (C1-C4)-alkyl,
or
R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
R21 is hydrogen or (C1-C4)-alkyl,
R22 is hydrogen or (C1-C4)-alkyl,
or
R21 and R22 together with the atoms to which they are bound form a three- to seven-membered carbocycle,
R23 is (C1-C4)-alkyl,
R24 is hydrogen or (C1-C4)-alkyl,
R27 is hydrogen or (C1-C4)-alkyl,
R36 is hydrogen, (C1-C4)-alkylcarbonyl, tert-butyloxycarbonyl or benzyloxycarbonyl,
R37 is hydrogen or methyl,
or
R36 and R37 together with the atoms to which they are bound form a pyrrolidine ring,
L2 is linear (C2-C10)-alkanediyl or for a group of the formula
Figure US20140127240A1-20140508-C01094
wherein
p is a number from 2 to 6,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 substituents selected independently of one another from the group consisting of methyl, hydroxyl and benzyl,
and
wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally present between them to form a (C3-C6) cycloalkyl ring or a phenyl ring,
D is a group of the formula
Figure US20140127240A1-20140508-C01095
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen or methyl,
R2 is isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01096
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O grouping contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01097
wherein
#6 denotes the linkage site to the carbonyl group,
R6 is hydrogen, hydroxyl or benzyloxy,
R3 is hydrogen or methyl,
R4 is isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, di-phenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01098
wherein
#7 denotes the linkage site to the vicinal nitrogen atom,
#8 denotes the linkage site to the group T1,
T1 is a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
wherein
R7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
R8 is hydrogen or methyl,
R9 is hydrogen, methyl, ethyl, n-propyl or benzyl,
or
R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
R10 is benzoyl,
R11 is benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
R5 is hydrogen, methyl or a group of the formula
Figure US20140127240A1-20140508-C01099
wherein
#9 denotes the linkage site to —CHC(R26)-T2,
R12 is phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
R13 is phenyl, which may be substituted with methoxycarbonyl or carboxyl,
R26 is hydrogen or hydroxyl,
T2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl, and
R35 is methyl or hydroxyl,
as well as their salts, solvates, and solvates of the salts.
3-6. (canceled)
7. Compounds of formula (XXXa)
Figure US20140127240A1-20140508-C01100
in which
Cys is a cysteine radical which is bound to a carbon atom of the succinimide via the sulfur atom of the side chain,
L1 is a bond, linear (C1-C10)-alkanediyl, or a group of the formula
Figure US20140127240A1-20140508-C01101
wherein
m is a number from 2 to 6,
##1 denotes the linkage site to the group G,
##2 denotes the linkage site to the group B,
L1A is linear (C2-C10)-alkanediyl,
B1 is a group of the formula
Figure US20140127240A1-20140508-C01102
wherein
##5 denotes the linkage site to the group L1A,
##6 denotes the linkage site to the group L1B,
L5 is a bond or (C2-C4)-alkanediyl,
L6 is a bond,
R29 is hydrogen or (C1-C4)-alkyl,
R30 is hydrogen or (C1-C4)-alkyl,
or
R29 and R30 together with the atoms to which they are bound form a five- or six-membered heterocycle,
R31 is hydrogen or (C1-C4)-alkyl,
R32 is hydrogen or (C1-C4)-alkyl,
or
R31 and R32 together with the atoms to which they are bound form a five- or six-membered heterocycle,
L1B is linear (C2-C10)-alkanediyl,
and
wherein (C1-C10)-alkanediyl may be substituted with one to four substituents selected independently of one another from the group consisting of methyl, hydroxyl and benzyl
and
wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally situated between them, to form a (C3-C6)-cycloalkyl ring or a phenyl ring,
B is a bond or a group of the formula
Figure US20140127240A1-20140508-C01103
wherein
* denotes the linkage site to L1,
** denotes the linkage site to L2,
P is O or NH,
L3 is a bond or (C2-C4)-alkanediyl,
L4 is a bond,
Q1 is a four- to seven-membered heterocycle,
R14 is hydrogen or (C1-C4)-alkyl,
R15 is hydrogen or (C1-C4)-alkyl,
or
R14 and R15 together with the atoms to which they are bound form a five- or six-membered heterocycle,
R16 is hydrogen or (C1-C4)-alkyl,
R17 is hydrogen or (C1-C4)-alkyl,
or
R16 and R17 together with the atoms to which they are bound form a five- or six-membered heterocycle,
R27 is hydrogen or (C1-C4)-alkyl,
L2 is linear (C2-C10)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01104
wherein
p is a number from 2 to 6,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 substituents selected independently of one another from the group consisting of methyl, hydroxyl and benzyl,
and
wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally present between them to form a (C3-C6) cycloalkyl ring or a phenyl ring,
D is a group of the formula
Figure US20140127240A1-20140508-C01105
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen or methyl,
R2 is isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01106
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O group contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01107
wherein
#6 denotes the linkage site to the carbonyl group,
R6 is hydrogen, hydroxyl or benzyloxy,
R3 is hydrogen or methyl,
R4 is isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01108
wherein
#7 denotes the linkage site to the vicinal nitrogen atom,
#8 denotes the linkage site to the group T1,
T1 is a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
wherein
R7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
R8 is hydrogen or methyl,
R9 is hydrogen, methyl, ethyl, n-propyl or benzyl,
or
R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
R10 is benzoyl,
R11 is benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
R5 is hydrogen, methyl or a group of the formula
Figure US20140127240A1-20140508-C01109
wherein
#9 denotes the linkage site to —CHC(R26)-T2,
R12 is phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
R13 is phenyl, which may be substituted with methoxycarbonyl or carboxyl,
R26 is hydrogen or hydroxyl,
T2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl, and
R35 is methyl or hydroxyl,
as well as their salts, solvates, and solvates of the salts.
8. Compounds of the formula (XXXa) according to claim 7, wherein
Cys is a cysteine radical which is bound to a carbon atom of the succinimide via the sulfur atom of the side chain,
L1 is a bond, linear (C2-C6)-alkanediyl, or a group of formula
Figure US20140127240A1-20140508-C01110
wherein
m is a number from 2 to 3,
##1 denotes the linkage site to the group G,
##2 denotes the linkage site to the group B,
L1A is linear (C2-C6)-alkanediyl,
B1 is a group of the formula
Figure US20140127240A1-20140508-C01111
wherein
##5 denotes the linkage site to the group L1A,
##6 denotes the linkage site to the group L1B,
L5 is a bond,
L6 is a bond,
R29 is hydrogen,
R30 is hydrogen,
R31 is hydrogen or methyl,
R32 is hydrogen or methyl,
L1B is linear (C2-C6)-alkanediyl,
and
wherein (C2-C6)-alkanediyl may be substituted with one to two methyl substituents,
B is a bond or a group of the formula
Figure US20140127240A1-20140508-C01112
wherein
* denotes the linkage site to L1,
** denotes the linkage site to L2,
L3 is a bond or ethane-1,2-diyl,
L4 is a bond,
R14 is hydrogen,
R15 is hydrogen,
R16 is hydrogen or methyl,
R17 is hydrogen or methyl,
or
R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
R23 is methyl,
R24 is hydrogen or methyl,
L2 is linear (C2-C6)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01113
wherein
p is a number from 2 to 3,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
D is a group of the formula
Figure US20140127240A1-20140508-C01114
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-yl-methyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01115
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O group contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01116
wherein
#6 denotes the linkage site to the carbonyl group,
R6 is hydrogen, hydroxyl or benzyloxy,
R3 is hydrogen,
R4 is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-yl-methyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01117
wherein
#7 denotes the linkage site to the vicinal nitrogen atom,
#8 denotes the linkage site to the group T1,
T1 is a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
wherein
R7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
R8 is hydrogen or methyl,
R9 is hydrogen, methyl, ethyl, n-propyl or benzyl,
or
R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
R10 is benzoyl,
R11 is benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
R5 is hydrogen, methyl or a group of the formula
Figure US20140127240A1-20140508-C01118
wherein
#9 denotes the linkage site to —CHCH2phenyl,
R12 is phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
R13 is phenyl, which may be substituted with methoxycarbonyl or carboxyl, and
R35 is methyl or hydroxyl,
as well as their salts, solvates, and solvates of the salts.
9. Compounds of the formula (XXXa) according to claim 7, wherein
Cys is a cysteine radical which is bound to a carbon atom of the succinimide via the sulfur atom of the side chain,
L1 is a bond or linear (C2-C6)-alkanediyl,
B is a bond or a group of the formula
Figure US20140127240A1-20140508-C01119
wherein
* denotes the linkage site to L1,
** denotes the linkage site to L2,
L3 is a bond,
L4 is a bond,
R16 is hydrogen or methyl,
R17 is hydrogen or methyl,
L2 is linear (C2-C6)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01120
wherein
p is a number from 2 to 3,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
D is a group of the formula
Figure US20140127240A1-20140508-C01121
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is benzyl or 1H-indol-3-ylmethyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01122
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O group contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01123
wherein
#6 denotes the linkage site to the carbonyl group,
R3 is hydrogen,
R4 is benzyl or 1H-indol-3-ylmethyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01124
wherein
#7 denotes the linkage site to the vicinal nitrogen atom,
#8 denotes the linkage site to the group T1,
T1 is a group of the formula —C(═O)—OR7 or —C(═O)—NR8R9,
wherein
R7 is hydrogen,
R8 is hydrogen,
R9 is hydrogen, and
R35 is methyl,
as well as their salts, solvates, and solvates of the salts.
10. Compounds of formula (XXXI)
Figure US20140127240A1-20140508-C01125
in which
L1 is a bond, linear (C1-C10)-alkanediyl, or a group of the formula
Figure US20140127240A1-20140508-C01126
wherein
m is a number from 2 to 6,
##1 denotes the linkage site to the group G,
##2 denotes the linkage site to the group B,
L1A is linear (C2-C10)-alkanediyl,
B1 is a group of the formula
Figure US20140127240A1-20140508-C01127
wherein
##5 denotes the linkage site to the group L1A,
##6 denotes the linkage site to the group L1B,
L5 is a bond or (C2-C4)-alkanediyl,
L6 is a bond,
R29 is hydrogen or (C1-C4)-alkyl,
R30 is hydrogen or (C1-C4)-alkyl,
or
R29 and R30 together with the atoms to which they are bound form a five- or six-membered heterocycle,
R31 is hydrogen or (C1-C4)-alkyl,
R32 is hydrogen or (C1-C4)-alkyl,
or
R31 and R32 together with the atoms to which they are bound form a five- or six-membered heterocycle,
L1B is linear (C2-C10)-alkanediyl,
and
wherein (C1-C10)-alkanediyl may be substituted with one to four substituents selected independently of one another from the group consisting of methyl, hydroxyl and benzyl
and
wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally situated between them, to form a (C3-C6)-cycloalkyl ring or a phenyl ring,
B is a bond or a group of the formula
Figure US20140127240A1-20140508-C01128
wherein
* denotes the linkage site to L1,
** denotes the linkage site to L2,
P is O or NH,
Q1 is a four- to seven-membered heterocycle,
Q2 is a three- to seven-membered carbocycle or a four- to seven-membered heterocycle,
R18 is hydrogen or (C1-C4)-alkyl,
R19 is hydrogen or the side group of a natural α-amino acid or its homologs or isomers,
R20 is hydrogen or (C1-C4)-alkyl,
or
R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
R21 is hydrogen or (C1-C4)-alkyl,
R22 is hydrogen or (C1-C4)-alkyl,
or
R21 and R22 together with the atoms to which they are bound form a three- to seven-membered carbocycle,
R27 is hydrogen or (C1-C4)-alkyl,
L2 is linear (C2-C10)-alkanediyl or for a group of the formula
Figure US20140127240A1-20140508-C01129
wherein
p is a number from 2 to 6,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 substituents selected independently of one another from the group consisting of methyl, hydroxyl and benzyl,
and
wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally present between them to form a (C3-C6) cycloalkyl ring or a phenyl ring,
D is a group of the formula
Figure US20140127240A1-20140508-C01130
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen or methyl,
R2 is isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01131
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O group contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01132
wherein
#6 denotes the linkage site to the carbonyl group,
R6 is hydrogen, hydroxyl or benzyloxy,
R3 is hydrogen or methyl,
R4 is isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01133
wherein
#7 denotes the linkage site to the vicinal nitrogen atom,
#8 denotes the linkage site to the group T1,
T1 is a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
wherein
R7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
R8 is hydrogen or methyl,
R9 is hydrogen, methyl, ethyl, n-propyl or benzyl,
or
R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
R10 is benzoyl,
R11 is benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
R5 is hydrogen, methyl or a group of the formula
Figure US20140127240A1-20140508-C01134
wherein
#9 denotes the linkage site to —CHC(R26)-T2,
R12 is phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
R13 is phenyl, which may be substituted with methoxycarbonyl or carboxyl,
R26 is hydrogen or hydroxyl,
T2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl, and
R35 is methyl or hydroxyl,
as well as their salts, solvates, and solvates of the salts.
11. Compounds of the formula (XXXI) according to claim 10, wherein
L1 is a bond, linear (C2-C6)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01135
wherein
m is a number from 2 to 3,
##1 denotes the linkage site to the group G,
##2 denotes the linkage site to the group B,
wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
B is a bond or a group of the formula
Figure US20140127240A1-20140508-C01136
wherein
* denotes the linkage site to L1,
** denotes the linkage site to L2,
R18 is hydrogen,
R19 is hydrogen, methyl, propan-2-yl, 2-methylpropan-1-yl or 1-methylpropan-1-yl,
R20 is hydrogen or (C1-C4)-alkyl,
or
R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
R21 is hydrogen or methyl,
R22 is hydrogen or methyl,
or
R21 and R22 together with the atoms to which they are bound form a cyclopropyl ring,
R27 is hydrogen or methyl,
L2 is linear (C2-C6)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01137
wherein
p is a number from 2 to 3,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
wherein (C2-C10)-alkanediyl may be substituted with one or two methyl substituents,
and
wherein two carbon atoms of the alkanediyl chain may be bridged in 1,4-relation to one another, including the carbon atoms optionally present between them to form a phenyl ring,
D is a group of the formula
Figure US20140127240A1-20140508-C01138
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-yl-methyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01139
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O group contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01140
wherein
#6 denotes the linkage site to the carbonyl group,
R6 is hydrogen, hydroxyl or benzyloxy,
R3 is hydrogen,
R4 is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-yl-methyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01141
wherein
#7 denotes the linkage site to the vicinal nitrogen atom,
#8 denotes the linkage site to the group T1,
T1 is a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
wherein
R7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
R8 is hydrogen or methyl,
R9 is hydrogen, methyl, ethyl, n-propyl or benzyl,
or
R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
R10 is benzoyl,
R11 is benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
R5 is hydrogen, methyl or a group of the formula
Figure US20140127240A1-20140508-C01142
wherein
#9 denotes the linkage site to —CHCH2phenyl,
R12 is phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
R13 is phenyl, which may be substituted with methoxycarbonyl or carboxyl, and
R35 is methyl or hydroxyl,
as well as their salts, solvates, and solvates of the salts.
12. Compounds of the formula (XXXI) according to claim 10, wherein
L1 is a bond,
B is a bond,
L2 is linear (C2-C6)-alkanediyl or for a group of the formula
Figure US20140127240A1-20140508-C01143
wherein
p is a number from 2 to 3,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
D is a group of the formula
Figure US20140127240A1-20140508-C01144
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is benzyl or 1H-indol-3-ylmethyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01145
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O group contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01146
wherein
#6 denotes the linkage site to the carbonyl group,
R3 is hydrogen,
R4 is benzyl or 1H-indol-3-ylmethyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01147
wherein
#7 denotes the linkage site to the vicinal nitrogen atom,
#8 denotes the linkage site to the group T1,
T1 is a group of the formula —C(═O)—OR7 or —C(═O)—NR8R9,
wherein
R7 is hydrogen,
R8 is hydrogen,
R9 is hydrogen, and
R35 is methyl,
as well as their salts, solvates, and solvates of the salts.
13. A compound of formula (XXXa) or (XXXI) selected from the group consisting of:
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide,
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-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-valinamide trifluoroacetate, and
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-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-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,
as well as their salts, solvates, and solvates of the salts.
14. Binder-drug conjugates of the general formula (Ia) according to claim 1, wherein R35 is methyl, thereby providing a binder-drug conjugate of general formula (I)
Figure US20140127240A1-20140508-C01148
in which
n is a number from 1 to 50,
AK is a binder,
the group §-G-L1-B-L2-§§ is a linker,
wherein
§ denotes the linkage site to the group AK and
§§ denotes the linkage site to the nitrogen atom,
D is a group of the formula
Figure US20140127240A1-20140508-C01149
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01150
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O group contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01151
wherein
#6 denotes the linkage site to the carbonyl group,
R6 is hydrogen, hydroxyl or benzyloxy,
R3 is hydrogen,
R4 is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01152
wherein
#7 denotes the linkage site to the vicinal nitrogen atom,
#8 denotes the linkage site to the group T1,
T1 is a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
wherein
R7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
R8 is hydrogen or methyl,
R9 is hydrogen, methyl, ethyl, n-propyl or benzyl,
or
R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
R10 is benzoyl,
R11 is benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
R5 is hydrogen, methyl or a group of the formula
Figure US20140127240A1-20140508-C01153
wherein
#9 denotes the linkage site to —CHC(R26)-T2,
R12 is phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
R13 is phenyl, which may be substituted with methoxycarbonyl or carboxyl,
R26 is hydrogen or hydroxyl, and
T2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
as well as their salts, solvates, and solvates of the salts.
15. Binder-drug conjugates according to claim 14 of the general formula (I),
in which
n is a number from 1 to 50,
AK is AK1 or AK2
wherein
AK1 is a binder bound to the group G via a sulfur atom of the binder,
AK2 is a binder bound to the group G via a nitrogen atom of the binder,
G for the case when AK=AK1, is a group of the formula
Figure US20140127240A1-20140508-C01154
wherein
#1 denotes the linkage site to the sulfur atom of the binder,
#2 denotes the linkage site to the group L1,
or
for the case when AK=AK2, G is carbonyl,
L1 is a bond, linear (C1-C10)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01155
wherein
m is a number from 2 to 6,
##1 denotes the linkage site to the group G,
##2 denotes the linkage site to the group B,
wherein (C1-C10)-alkanediyl may be substituted with 1 to 4 methyl substituents,
and
wherein two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-relation to one another may be bridged to form a (C3-C6)-cycloalkyl ring or a phenyl ring including the carbon atoms optionally situated between them,
B is a bond or a group of the formula
Figure US20140127240A1-20140508-C01156
wherein
* denotes the linkage site to L1,
** denotes the linkage site to L2,
P is O or NH,
L3 is a bond or (C2-C4)-alkanediyl,
L4 is a bond or a group of the formula
Figure US20140127240A1-20140508-C01157
wherein
*** denotes the linkage site to the carbonyl group,
**** denotes the linkage site to L2,
R25 is hydrogen or methyl,
Q1 is a four- to seven-membered heterocycle,
Q2 is a three- to seven-membered carbocycle or a four- to seven-membered heterocycle,
R14 is hydrogen or (C1-C4)-alkyl,
R15 is hydrogen or (C1-C4)-alkyl,
or
R14 and R15 together with the atoms to which they are bound form a five- or six-membered heterocycle,
R16 is hydrogen or (C1-C4)-alkyl,
R17 is hydrogen or (C1-C4)-alkyl,
or
R16 and R17 together with the atoms to which they are bound form a five- or six-membered heterocycle,
R18 is hydrogen or (C1-C4)-alkyl,
R19 is hydrogen or the side group of a natural α-amino acid or its homologs or isomers,
R20 is hydrogen or (C1-C4)-alkyl,
or
R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
R21 is hydrogen or (C1-C4)-alkyl,
R22 is hydrogen or (C1-C4)-alkyl,
or
R21 and R22 together with the atoms to which they are bound form a three- to seven-membered carbocycle,
R23 is (C1-C4)-alkyl,
R24 is hydrogen or (C1-C4)-alkyl,
R27 is hydrogen or (C1-C4)-alkyl,
L2 is linear (C2-C10)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01158
wherein
p is a number from 2 to 6,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 methyl substituents,
and
wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally present between them to form a (C3-C6) cycloalkyl ring or a phenyl ring, and
D has the meaning as described in claim 14,
as well as their salts, solvates, and solvates of the salts.
16. Binder-drug conjugates according to claim 14 of the general formula (I),
in which
n is a number from 1 to 50,
AK is AK1 or AK2
wherein
AK1 is an antibody or an antigen-binding antibody fragment and is bound to the group G via a sulfur atom,
AK2 is an antibody or an antigen-binding antibody fragment and is bound to the group G via a nitrogen atom, and
n, G, L1, B, L2, and D have the meanings given in claim 14,
as well as their salts, solvates, and solvates of the salts.
17-18. (canceled)
19. Method for production of compounds of formula (I) as defined in claim 14, the method comprising
[A] mixing a solution of binder in a buffer with a reducing agent selected from the group consisting of dithiothreitol and tris-(2-carboxyethyl)phosphine hydrochloride, and then reacting the resulting solution with a compound of formula (II)
Figure US20140127240A1-20140508-C01159
in which D, L1, B and L2 each have the meanings given in claim 14,
to form a compound of formula (I-A)
Figure US20140127240A1-20140508-C01160
in which n, AK1, D, L1, B and L2 each have the meanings given in claim 14,
or
[B] mixing a solution of binder in a buffer with a reducing agent selected from the group consisting of dithiothreitol and tris-(2-carboxyethyl)phosphine hydrochloride, and then reacting the resulting solution with a compound of formula (III)
Figure US20140127240A1-20140508-C01161
in which D, L1, B and L2 each have the meanings given in claim 14,
to form a compound of formula (I-B)
Figure US20140127240A1-20140508-C01162
in which n, AK2, D, L1, B and L2 each have the meanings given in claim 14.
20. (canceled)
21. Compounds of the general formula (XXXa) according to claim 7, wherein R35 is methyl, thereby providing a compound of the general formula (XXX)
Figure US20140127240A1-20140508-C01163
in which
Cys is a cysteine radical which is bound to a carbon atom of the succinimide via the sulfur atom of the side chain,
L1 is a bond, linear (C1-C10)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01164
wherein
m is a number from 2 to 6,
##1 denotes the linkage site to the group G,
##2 denotes the linkage site to the group B,
wherein (C1-C10)-alkanediyl may be substituted with 1 to 4 methyl substituents,
and
wherein two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-relation to one another may be bridged to form a (C3-C6)-cycloalkyl ring or a phenyl ring including the carbon atoms optionally situated between them,
B is a bond or a group of the formula
Figure US20140127240A1-20140508-C01165
wherein
* denotes the linkage site to L1,
** denotes the linkage site to L2,
P is O or NH,
L3 is a bond or (C2-C4)-alkanediyl,
L4 is a bond or a group of the formula
Figure US20140127240A1-20140508-C01166
wherein
*** denotes the linkage site to the carbonyl group,
**** denotes the linkage site to L2,
R25 is hydrogen or methyl,
Q1 is a three- to seven-membered carbocycle or a four- to seven-membered azaheterocycle,
Q2 is a three- to seven-membered carbocycle or a four- to seven-membered azaheterocycle,
R14 is hydrogen or (C1-C4)-alkyl,
R15 is hydrogen or (C1-C4)-alkyl,
or
R14 and R15 together with the atoms to which they are bound form a five- or six-membered heterocycle,
R16 is hydrogen or (C1-C4)-alkyl,
R17 is hydrogen or (C1-C4)-alkyl,
or
R16 and R17 together with the atoms to which they are bound form a five- or six-membered heterocycle,
R18 is hydrogen or (C1-C4)-alkyl,
R19 is hydrogen or the side group of a natural α-amino acid or its homologs or isomers,
R20 is hydrogen or (C1-C4)-alkyl,
or
R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
R21 is hydrogen or (C1-C4)-alkyl,
R22 is hydrogen or (C1-C4)-alkyl,
or
R21 and R22 together with the atoms to which they are bound form a three- to seven-membered carbocycle,
R23 is (C1-C4)-alkyl,
R24 is hydrogen or (C1-C4)-alkyl,
R27 is hydrogen or (C1-C4)-alkyl,
L2 is linear (C2-C10)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01167
wherein
p is a number from 2 to 6,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 methyl substituents,
and
wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally present between them to form a (C3-C6) cycloalkyl ring or a phenyl ring,
D is a group of the formula
Figure US20140127240A1-20140508-C01168
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01169
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O group contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01170
wherein
#6 denotes the linkage site to the carbonyl group,
R6 is hydrogen, hydroxyl or benzyloxy,
R3 is hydrogen,
R4 is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01171
wherein
#7 denotes the linkage site to the vicinal nitrogen atom,
#8 denotes the linkage site to the group T1,
T1 is a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH-Ro or —CH2—O—R11,
wherein
R7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
R8 is hydrogen or methyl,
R9 is hydrogen, methyl, ethyl, n-propyl or benzyl,
or
R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
R10 is benzoyl,
R11 is benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
R5 is hydrogen, methyl or a group of the formula
Figure US20140127240A1-20140508-C01172
wherein
#9 denotes the linkage site to —CHC(R26)-T2,
R12 is phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
R13 is phenyl, which may be substituted with methoxycarbonyl or carboxyl,
R26 is hydrogen or hydroxyl, and
T2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
as well as their salts, solvates, and solvates of the salts.
22. Compounds of the formula (XXX) according to claim 21, wherein
Cys is a cysteine radical which is bound to a carbon atom of the succinimide via the sulfur atom of the side chain,
L1 is a bond, linear (C2-C6)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01173
wherein
m is a number from 2 to 6,
##1 denotes the linkage site to the group G,
##2 denotes the linkage site to the group B,
wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
B is a bond or a group of the formula
Figure US20140127240A1-20140508-C01174
wherein
* denotes the linkage site to L1,
** denotes the linkage site to L2,
L3 is a bond or ethane-1,2-diyl,
L4 is a bond,
R14 is hydrogen,
R15 is hydrogen,
R16 is hydrogen or methyl,
R17 is hydrogen or methyl,
or
R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
R23 is methyl,
R24 is hydrogen or methyl,
L2 is linear (C2-C6)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01175
wherein
p is a number from 2 to 3,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
D is a group of the formula
Figure US20140127240A1-20140508-C01176
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is 1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01177
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O group contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01178
wherein
#6 denotes the linkage site to the carbonyl group,
R6 is hydrogen, hydroxyl or benzyloxy,
R3 is hydrogen,
R4 is benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01179
wherein
#7 denotes the linkage site to the vicinal nitrogen atom,
#8 denotes the linkage site to the group T,
T1 is a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
wherein
R7 is hydrogen, methyl, ethyl, n-propyl, benzyl or adamantylmethyl,
R8 is hydrogen or methyl,
R9 is hydrogen, methyl, ethyl, n-propyl or benzyl,
R10 is benzoyl,
R11 is benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
R5 is hydrogen or a group of the formula
Figure US20140127240A1-20140508-C01180
wherein
#9 denotes the linkage site to —CHC(R26)phenyl,
R12 is phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH, and
R13 is phenyl, which may be substituted with methoxycarbonyl or carboxyl,
as well as their salts, solvates, and solvates of the salts.
23-44. (canceled)
45. Pharmaceutical drug containing a binder-drug conjugate according to claim 1, in combination with an inert nontoxic pharmaceutically suitable excipient.
46. Pharmaceutical drug containing a binder-drug conjugate according to claim 1, in combination with one or more antihyperproliferative, cytostatic or cytotoxic substances.
47. (canceled)
48. Method for treatment or prevention of hyperproliferative or angiogenic diseases in humans and animals, the method comprising administering an effective amount of at least one binder-drug conjugate according to claim 1.
49. Binder-drug conjugates of the general formula (Ia) according to claim 1, wherein
n is a number from 1 to 20,
AK is AK1 or AK2
wherein
AK1 is a binder bound to the group G via a sulfur atom of the binder,
AK2 is a binder bound to the group G via a nitrogen atom of the binder,
G for the case when AK=AK1, is a group of the formula
Figure US20140127240A1-20140508-C01181
wherein
#1 denotes the linkage site to the cysteine radical of the binder,
#2 denotes the linkage site to the group L1,
or
for the case when AK=AK2, G is carbonyl,
L1 is a bond, linear (C2-C6)-alkanediyl, or a group of the formula
Figure US20140127240A1-20140508-C01182
wherein
m is a number from 2 to 6,
##1 denotes the linkage site to the group G,
##2 denotes the linkage site to the group B,
L1A is linear (C2-C6)-alkanediyl,
B1 is a group of the formula
Figure US20140127240A1-20140508-C01183
wherein
##5 denotes the linkage site to the group L1A,
##6 denotes the linkage site to the group L1B,
L5 is a bond,
L6 is a bond or a group with the formula
Figure US20140127240A1-20140508-C01184
wherein
##7 denotes the linkage site to the carbonyl group,
##8 denotes the linkage site to L1B,
R33 is hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
R34 is hydrogen or methyl,
R29 is hydrogen,
R30 is hydrogen,
R31 is hydrogen or methyl,
R32 is hydrogen or methyl,
L1B is linear (C2-C10)-alkanediyl,
and
wherein (C2-C6)-alkanediyl may be substituted with one to two methyl substituents,
B is a bond or a group of the formula
Figure US20140127240A1-20140508-C01185
wherein
* denotes the linkage site to L1,
** denotes the linkage site to L2,
L3 is a bond or ethane-1,2-diyl,
L4 is a bond or a group of the formula
Figure US20140127240A1-20140508-C01186
wherein
*** denotes the linkage site to the carbonyl group,
**** denotes the linkage site to L2,
R25 is hydrogen or methyl,
R28 is hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
Q1 is a four- to seven-membered heterocycle,
R14 is hydrogen,
R15 is hydrogen,
R16 is hydrogen or methyl,
R17 is hydrogen or methyl,
or
R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
R18 is hydrogen,
R19 is hydrogen, methyl, propan-2-yl, 2-methylpropan-1-yl or 1-methylpropan-1-yl,
R20 is hydrogen or methyl,
or
R19 and R20 together with the atoms to which they are bound form a pyrrolidinyl ring,
R21 is hydrogen or methyl,
R22 is hydrogen or methyl,
or
R21 and R22 together with the atoms to which they are bound form a cyclopropyl ring,
R23 is methyl,
R24 is hydrogen or methyl,
R27 is hydrogen,
L2 is linear (C2-C6)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01187
wherein
p is a number from 2 to 6,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
wherein (C2-C10)-alkanediyl may be substituted with one or two methyl substituents,
D is a group of the formula
Figure US20140127240A1-20140508-C01188
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-yl-methyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01189
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O group contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01190
wherein
#6 denotes the linkage site to the carbonyl group,
R6 is hydrogen, hydroxyl or benzyloxy,
R3 is hydrogen,
R4 is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-yl-methyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01191
wherein
#7 denotes the linkage site to the vicinal nitrogen atom,
#8 denotes the linkage site to the group T1,
T1 is a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
wherein
R7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
R8 is hydrogen or methyl,
R9 is hydrogen, methyl, ethyl, n-propyl or benzyl,
or
R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
R10 is benzoyl,
R11 is benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
R5 is hydrogen, methyl or a group of the formula
Figure US20140127240A1-20140508-C01192
wherein
#9 denotes the linkage site to —CHC(R26)-T2,
R12 is phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
R13 is phenyl, which may be substituted with methoxycarbonyl or carboxyl,
R26 is hydrogen or hydroxyl,
T2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl, and
R35 is methyl or hydroxyl,
as well as their salts, solvates, and solvates of the salts.
50. Binder-drug conjugates of the general formula (Ia) according to claim 1, wherein
n is a number from 1 to 10,
AK is AK1 or AK2
wherein
AK1 is a binder bound to the group G via a sulfur atom of the binder,
AK2 is a binder bound to the group G via a nitrogen atom of the binder,
G for the case when AK=AK1, is a group of the formula
Figure US20140127240A1-20140508-C01193
wherein
#1 denotes the linkage site to the cysteine radical of the binder,
#2 denotes the linkage site to the group L1,
or
for the case when AK=AK2, G is carbonyl,
L1 is a bond, linear (C2-C6)-alkanediyl, or a group of the formula
Figure US20140127240A1-20140508-C01194
wherein
m is a number from 2 to 3,
##1 denotes the linkage site to the group G,
##2 denotes the linkage site to the group B,
wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
B is a bond or a group of the formula
Figure US20140127240A1-20140508-C01195
wherein
* denotes the linkage site to L1,
** denotes the linkage site to L2,
L3 is a bond or ethane-1,2-diyl,
L4 is a bond or a group of the formula
Figure US20140127240A1-20140508-C01196
wherein
*** denotes the linkage site to the carbonyl group,
**** denotes the linkage site to L2,
R25 is methyl,
R28 is hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
Q1 is piperidine-1,4-diyl,
R16 is hydrogen or methyl,
R17 is hydrogen or methyl,
or
R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
R21 is hydrogen or methyl,
R22 is hydrogen or methyl,
or
R21 and R22 together with the atoms to which they are bound form a cyclopropyl ring,
R23 is methyl,
R24 is hydrogen,
L2 is linear (C2-C6)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01197
wherein
p is a number from 2 to 6,
##3 is linkage site to the group B,
##4 is linkage site to the nitrogen atom,
D is a group of the formula
Figure US20140127240A1-20140508-C01198
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is 1-hydroxyethyl, benzyl, 1-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-yl-methyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01199
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O group contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01200
wherein
#6 denotes the linkage site to the carbonyl group,
R6 is hydrogen, hydroxyl or benzyloxy,
R3 is hydrogen,
R4 is benzyl, 1-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01201
wherein
#7 denotes the linkage site to the vicinal nitrogen atom,
#8 denotes the linkage site to the group T1,
T1 is a group of the formula —C(═O)—OR7, —C(═O)—NR8R9 or —CH2—O—R11,
wherein
R7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
R8 is hydrogen or methyl,
R9 is hydrogen, methyl, ethyl, n-propyl or benzyl,
R11 is benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
R5 is hydrogen, methyl or a group of the formula
Figure US20140127240A1-20140508-C01202
wherein
#9 denotes the linkage site to —CHCH2phenyl,
R12 is phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
R13 is phenyl, which may be substituted with methoxycarbonyl or carboxyl, and
R35 is methyl or hydroxyl,
as well as their salts, solvates, and solvates of the salts.
51. Method for producing compounds of the general formula (Ia) as defined in claim 1, the method comprising
[A] mixing a solution of binder in a buffer with a reducing agent selected from the group consisting of dithiothreitol and tris-(2-carboxyethyl)phosphine hydrochloride and then reacting the resulting solution with a compound of formula (IIa)
Figure US20140127240A1-20140508-C01203
in which D, L1, B, L2 and R35 each have the meanings given in claim 1,
to form a compound of formula (Ia-A)
Figure US20140127240A1-20140508-C01204
in which n, AK1, D, L1, B, L2 and R35 each have the meanings given in claim 1,
or
[B] mixing a solution of binder in a buffer with a reducing agent selected from the group consisting of dithiothreitol and tris-(2-carboxyethyl)phosphine hydrochloride and then reacting the resulting solution with a compound of formula (IIIa)
Figure US20140127240A1-20140508-C01205
in which D, L1, B, L2 and R35 each have the meanings given in claim 1,
to form a compound of formula (Ia-B)
Figure US20140127240A1-20140508-C01206
in which n, AK2, D, L1, B, L2 and R35 each have the meanings given in claim 1.
52. (canceled)
53. Binder-drug conjugates of the general formula (Ia) according to claim 1, wherein
n is a number from 1 to 50,
AK is a bond,
the group §-G-L1-B-§§ is a linker,
wherein
§ denotes the linkage site to the group AK and
§§ denotes the linkage site to the nitrogen atom,
L2 is linear (C2-C10)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01207
wherein
p is a number from 2 to 6,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
wherein (C2-C10)-alkanediyl may be substituted with 1 to 4 substituents selected independently of one another from the group consisting of methyl, hydroxyl and benzyl,
and
wherein two carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-relation to one another, including the carbon atoms optionally present between them to form a (C3-C6) cycloalkyl ring or a phenyl ring,
D is a group of the formula
Figure US20140127240A1-20140508-C01208
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen or methyl,
R2 is isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
or
R1 and R2 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01209
wherein
#4 denotes the linkage site to the vicinal nitrogen atom,
#5 denotes the linkage site to the carbonyl group,
the ring A with the N—O group contained in it is a mono- or bicyclic, optionally substituted heterocycle of the formula
Figure US20140127240A1-20140508-C01210
wherein
#6 denotes the linkage site to the carbonyl group,
R6 is hydrogen, hydroxyl or benzyloxy,
R3 is hydrogen or methyl,
R4 is isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
or
R3 and R4 together with the carbon atom to which they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
Figure US20140127240A1-20140508-C01211
wherein
#7 denotes the linkage site to the vicinal nitrogen atom,
#8 denotes the linkage site to the group T1,
T1 is a group of the formula —C(═O)—OR7, —C(═O)—NR8R9, —C(═O)—NH—NH—R10 or —CH2—O—R11,
wherein
R7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
R8 is hydrogen or methyl,
R9 is hydrogen, methyl, ethyl, n-propyl or benzyl,
or
R8 and R9 together with the nitrogen atom to which they are bound form a four- to seven-membered heterocycle,
R10 is benzoyl,
R11 is benzyl, which may be substituted in the phenyl group with methoxycarbonyl or carboxyl,
R5 is hydrogen, methyl or a group of the formula
Figure US20140127240A1-20140508-C01212
wherein
#9 denotes the linkage site to —CHC(R26)-T2,
R12 is phenyl, which may be substituted with methoxycarbonyl, carboxyl or a group of the formula —S(O)2OH,
R13 is phenyl, which may be substituted with methoxycarbonyl or carboxyl,
R26 is hydrogen or hydroxyl,
T2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl, and
R35 is methyl or hydroxyl,
as well as their salts, solvates, and solvates of the salts.
54. Binder-drug conjugates of the general formula (Ia) according to claim 1, wherein
n is a number from 2 to 8,
AK is AK1 or AK2,
wherein
AK1 is a binder that is bound to the group G via a sulfur atom of the cysteine radical of the binder,
AK2 is a binder that is bound to the group G via a nitrogen atom of the lysine radical of the binder,
G for the case when AK=AK1, is a group of the formula
Figure US20140127240A1-20140508-C01213
wherein
#1 denotes the linkage site to the cysteine radical of the binder,
#2 denotes the linkage site to the group L1,
or
for the case when AK=AK2, G is carbonyl,
L1 is a bond, linear (C2-C6)-alkanediyl, or a group of the formula
Figure US20140127240A1-20140508-C01214
wherein
m is a number from 2 to 3,
##1 denotes the linkage site to the group G,
##2 denotes the linkage site to the group B,
wherein (C2-C6)-alkanediyl may be substituted with one or two methyl substituents,
B is a bond or a group of the formula
Figure US20140127240A1-20140508-C01215
wherein
* denotes the linkage site to L1,
** denotes the linkage site to L2,
L3 is a bond or ethane-1,2-diyl,
L4 is a bond or a group of the formula
Figure US20140127240A1-20140508-C01216
wherein
*** denotes the linkage site to the carbonyl group,
**** denotes the linkage site to L2,
R25 is methyl,
R28 is hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
Q1 is piperidine-1,4-diyl,
R16 is hydrogen or methyl,
R17 is hydrogen or methyl,
or
R16 and R17 together with the atoms to which they are bound form a piperazinyl ring,
R21 is hydrogen or methyl,
R22 is hydrogen or methyl,
or
R21 and R22 together with the atoms to which they are bound form a cyclopropyl ring,
R23 is methyl,
R24 is hydrogen,
L2 is linear (C2-C6)-alkanediyl or a group of the formula
Figure US20140127240A1-20140508-C01217
wherein
p is a number from 2 to 6,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
D is a group of the formula
Figure US20140127240A1-20140508-C01218
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
the ring A with the N—O group contained therein is
Figure US20140127240A1-20140508-C01219
wherein
#6 denotes the linkage site to the carbonyl group,
R3 is hydrogen,
R4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
T1 is a group of the formula —C(═O)—NR8R9,
wherein
R8 is hydrogen or methyl,
R9 is hydrogen, methyl or ethyl, and
R35 is methyl,
as well as their salts, solvates, and solvates of the salts.
55. Binder-drug conjugates of the general formula (Ia) according to claim 1, wherein
n is a number from 2 to 8,
AK is AK1,
wherein
AK1 is binder that is bound to the group G via a sulfur atom of the cysteine radical of the binder,
G is a group of the formula
Figure US20140127240A1-20140508-C01220
wherein
#1 denotes the linkage site to the cysteine radical of the binder,
#2 denotes the linkage site to the group L1,
L1 is pentane-1,5-diyl,
B is a group of the formula
Figure US20140127240A1-20140508-C01221
wherein
* denotes the linkage site to L1,
** denotes the linkage site to L2,
L3 is a bond,
L4 is a bond,
R16 is hydrogen,
R17 is hydrogen,
L2 is propane-1,3-diyl,
D is a group of the formula
Figure US20140127240A1-20140508-C01222
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
the ring A with the N—O group contained therein is
Figure US20140127240A1-20140508-C01223
wherein
#6 denotes the linkage site to the carbonyl group,
R3 is hydrogen,
R4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
T1 is a group of the formula —C(═O)—NR8R9,
wherein
R8 is hydrogen,
R9 is hydrogen, and
R35 is methyl,
as well as their salts, solvates, and solvates of the salts.
56. Binder-drug conjugates of the general formula (Ia) according to claim 1, wherein
n is a number from 2 to 8,
AK is AK1,
wherein
AK1 is a binder that is bound to the group G via a sulfur atom of the cysteine radical of the binder,
G is a group of the formula
Figure US20140127240A1-20140508-C01224
wherein
#1 denotes the linkage site to the cysteine radical of the binder,
#2 denotes the linkage site to the group L1,
L1 is a bond,
B is a bond,
L2 is hexane-1,6-diyl,
D is a group of the formula
Figure US20140127240A1-20140508-C01225
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
the ring A with the N—O group contained therein is
Figure US20140127240A1-20140508-C01226
wherein
#6 denotes the linkage site to the carbonyl group,
R3 is hydrogen,
R4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
T1 is a group of the formula —C(═O)—NR8R9,
wherein
R8 is hydrogen, and
R9 is hydrogen,
as well as their salts, solvates, and solvates of the salts.
57. Binder-drug conjugates of the general formula (Ia) according to claim 1, wherein
n is a number from 2 to 8,
AK is AK2,
wherein
AK2 is a binder that is bound to the group G via a nitrogen atom of the binder,
G is carbonyl,
L1 is a bond,
B is a bond,
L2 is pentane-1,5-diyl,
D is a group of the formula
Figure US20140127240A1-20140508-C01227
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
the ring A with the N—O group contained therein is
Figure US20140127240A1-20140508-C01228
wherein
#6 denotes the linkage site to the carbonyl group,
R3 is hydrogen,
R4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
T1 is a group of the formula —C(═O)—NR8R9,
wherein
R8 is hydrogen,
R9 is hydrogen, and
R35 is methyl,
as well as their salts, solvates, and solvates of the salts.
58. Binder-drug conjugates of the general formula (Ia) according to claim 1, wherein
n is a number from 2 to 8,
AK is AK2,
wherein
AK2 is a binder that is bound to the group G via a nitrogen atom of the lysine radical of the binder,
G is carbonyl,
L1 is a bond,
B is a bond,
L2 is a group of the formula
Figure US20140127240A1-20140508-C01229
wherein
p is the number 3,
##3 denotes the linkage site to the group B,
##4 denotes the linkage site to the nitrogen atom,
D is a group of the formula
Figure US20140127240A1-20140508-C01230
wherein
#3 denotes the linkage site to the nitrogen atom,
R1 is hydrogen,
R2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
the ring A with the N—O group contained therein is
Figure US20140127240A1-20140508-C01231
wherein
#6 denotes the linkage site to the carbonyl group,
R3 is hydrogen,
R4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
T1 is a group of the formula —C(═O)—NR8R9,
wherein
R8 is hydrogen, and
R9 is hydrogen,
as well as their salts, solvates, and solvates of the salts.
US14/113,070 2011-04-21 2012-04-20 Novel Binder-Drug Conjugates (ADCs) and Use of Same Abandoned US20140127240A1 (en)

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