WO2018140275A2

WO2018140275A2 - Novel auristatin derivatives and related antibody-drug conjugates (adcs) and methods of preparation thereof

Info

Publication number: WO2018140275A2
Application number: PCT/US2018/014167
Authority: WO
Inventors: Joachim Krüger
Original assignee: Seattle Genetics, Inc.
Priority date: 2017-01-26
Filing date: 2018-01-18
Publication date: 2018-08-02
Also published as: WO2018140275A3

Abstract

A compound according to the general Formula (8): wherein R₁ is hydrogen or C₁-C₄ alkyl; R₂ is C₁-C₄ alkyl, benzyl, or 1H-indol-3-ylmethyl; T is selected from the group consisting of -CH(OR3)-R4, -C(=O)-OR₃, or -C(=O)-NR₃ R₅, wherein R₃ is hydrogen or C₁-C₄ alkyl; R₄ is phenyl; R₅ is hydrogen or C₁-C₄ alkyl; L¹ is R⁶-C(=O)-R⁷; where R⁶ is C₁-C₆ alkyl; R⁷ is a bond or NH-NH; L² is a bond,, or; where n is 1-4; and Ab is an antibody or antigen-binding antibody fragment, and a process for preparing the same. In certain embodiments, Ab is an antibody or antigen-binding antibody fragment which binds to C4.4a.

Description

Novel auristatin derivatives and related antibody-drug conjugates (ADCs) and methods of preparation thereof

BACKGROUND OF THE INVENTION:

The present application relates to novel compounds, including auristatin derivatives and binder-drug conjugates, for example antibody-drug conjugates (ADCs) of N,N-dialkylauristatins that are directed against the target C4.4a; to active metabolites of these compounds; and to processes for preparing these compounds.

Cancer diseases are the consequence of uncontrolled cell growth in a wide variety of tissues. In many cases the new cells penetrate existing tissue (invasive growth), or they metastase into remote organs. Cancer diseases occur in a wide variety of organs, and the illnesses often progress in a tissue-specific manner. The designation "cancer disease" as a generic term therefore describes a large group of defined diseases of different organs, tissues, and cell types.

Early-stage tumors may be able to be removed by surgical and radiotherapeutic measures. Metastasized tumors can frequently only be given palliative therapy by means of chemotherapeutic agents. The objective in that case is to achieve the optimum combination of improving quality of life and prolonging remaining lifetime.

The majority of the chemotherapeutic agents which are presently administered parenterally are often not target-directed at the tumor tissue or the tumor cells, but instead, as a result of their systemic administration, are distributed non-specifically within the body, hence including at locations at which exposure to the drug is

undesirable, such as in healthy cells, tissues, and organs, for example. This may lead to unwanted side-effects and even to serious effects of general toxicity, which then often greatly limit the therapeutically useful dose range of the drug, or necessitate complete cessation of medication.

The improved and selective availability of these chemotherapeutic agents in the tumor cell or the immediately surrounding tissue, and the associated boost in effect, on the one hand, and minimization of toxic side-effects, on the other hand, have therefore been a focal point for a number of years in the development of new chemotherapeutic agents. Many attempts have been made to date to develop efficient methods of introducing the drug into the target cell. Optimizing the association between drug and intracellular target and minimizing the intercellular distribution of drug, to adjacent cells, for example, nevertheless continue to constitute a difficult problem.

Monoclonal antibodies, for example, are suitable for the target-directed addressing of tumor tissue and tumor cells. The significance of such antibodies for the clinical treatment of cancer diseases has seen a considerable general increase in recent years, based on the activity of such agents as trastuzumab (Herceptin), rituximab

(Rituxan), cetuximab (Erbitux) and bevacizumab (Avastin), which have since been approved for the therapy of individual, specific tumor diseases [see e.g. G. P. Adams and L. M. Weiner, Nat. Biotechnol. 23, 1147-1157 (2005)]. Consequently there has also been a marked increase in interest in so-called immunoconjugates such as, for example, the aforementioned ADCs, in which an internalizing antibody directed against a tumor- associated antigen is joined covalently via a linking unit ("linker") to a cytotoxic agent. Following introduction of the ADC into the tumor cell and subsequent cleavage of the conjugate, either the cytotoxic agent itself or another metabolite with cytotoxic activity, formed from the cytotoxic agent, is released within the tumor cell, where it is able to develop its effect directly and selectively. In this way it would be possible to keep the damage to normal tissue within significantly closer limits in comparison to a

conventional chemotherapy of the cancer disease [see e.g. 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, it is also possible for binders from the small-molecule drug sphere to be used as binders which bind selectively to a specific target location ("target"), such as to a receptor, for example [see e.g. E. Ruoslahti et al., Science 279, 377-380 (1998); D. Karkan et al, PLoS ONE 3 (6), e2469 (June 25, 2008)]. Also known are conjugates of cytotoxic drug and addressing ligand that exhibit a defined cleavage point between ligand and drug for the release of the drug. A "predetermined break point" of this kind may exist, for example, within a peptide chain which can be cleaved selectively at a particular site by a specific enzyme at the location of action [see e.g. R. A. Firestone and L. A. Telan, US Patent Application US 2002/0147138].

One example of binders suitable for the target-directed addressing of tumor tissue and tumor cells are monoclonal antibodies directed against the antigen C4.4a. C4.4a (gene: LYPD3) was first described as a metastasis-associated, cell surface protein in rat pancreas tumor cells (Rosel M. et al., Oncogene 1998, 17(15): 1989-2002). Human C4.4a was isolated from a placental cDNA library (Wiirfel, J. et. al. Gene 2001,262:35-41). C4.4a exhibits structural homology with the uPA receptor and contains two LY6 domains, which exhibit the typical three-finger folding pattern and are linked via 9 disulphide bridges (Jacobsen B. & Ploug M., Current Medicinal Chemistry 2008, 15:2559-2573). C4.4a is anchored in the cell via glycophosphatidylinositol (GPI). The protein is highly glycosylated and contains numerous N- and O-glycosylation sites. C4.4a exhibits strong expression in tumor cells of lung cancer, large bowel cancer, breast cancer, ovarian cancer, pancreatic cancer, kidney cancer, head-and-neck tumors, and melanomas. RNA analyses have shown C4.4a expression in ~ 50% of primary pulmonary tumors and ~ 75% of lung cancer metastases, although expression in healthy lung tissue was not detectable (Wiirfel J. et. al., Gene 2001, 262:35-41). C4.4a can be used as a prognostic marker in non-small-cell lung cancer - a high level of C4.4a expression correlates with a poor prognosis (Hansen L. et al., Lung Cancer 2007, 58:260-266). The same is true for large bowel cancer. C4.4a is cleaved off from the surface of the tumor cell and can be used as a prognostic serum marker (K. Konishi et al., Cancer Science 2010). A detailed expression analysis of melanomas has shown that C4.4a is expressed in ~ 60% of primary malignant melanomas and in 100% of lymph-node and skin metastases (Seiter S. et al., J Invest Dermatol. 2001, 116(2):344-347). Upregulation of C4.4a gene expression is observed in breast cancer tissue as compared with adjacent normal tissues (Fletcher G.C., Br. J. Cancer 2003, 88(4):579-585). C4.4a is an ideal target protein for a tumor therapy, since C4.4a expression in healthy tissues is confined to skin keratinocytes and esophageal endothelial cells, and also to placenta cells (Wiirfel J. et. al., Gene 2001, 262:35-41). WO01/23553 describes the use of a C4.4a inhibitor (e.g., an anti-C4.4a antibody) which in a cancer therapy is able to inhibit C4.4a expression or activity.

The precise function of C4.4a is unknown. In the course of wound healing, it is upregulated in migrating keratinocytes (Hansen L. et al., Biochem J. 2004, 380:845-857). It is thought that C4.4a plays a part in tumor cell invasion, presumably through interaction with the extracellular matrix (Rosel M. et al., Oncogene 1998, 17(15): 1989- 2002; Paret C. et al., British Journal of Cancer 2007, 97: 1146-1156). Potential ligands are laminin 1 and 5, and also galectin 3 (Paret C, Int. J. Cancer 2005, 115:724-733). Auristatin E (AE) and monomethylauristatin E (MMAE) are synthetic analogues of the dolastatins, a specific group of linear pseudopeptides which were originally isolated from marine sources and which have in some cases very potent cytotoxic activity with respect to tumor cells [for a review see e.g. 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);

-Cancer Drug Design 13, 243-277 (1998)].

Auristatin E (AE): R = CH₃

Monomethylauristatin E (MMAE): R = H

MMAE, however, possesses the disadvantage of a comparatively high systemic toxicity. For improving tumor selectivity, MMAE is used more particularly in conjunction with enzymatically cleavable valine-citrulline linkers in the ADC setting for more targeted tumor therapy [WO 2005/081711-A2; S. O. Doronina et al., Bioconjugate Chem. V7, 114-124 (2006)]. Following proteolytic cleavage, MMAE is released preferably intracellularly from corresponding ADCs.

When employed in the form of antibody-drug conjugates (ADCs), however, MMAE is not compatible with linking units (linkers) between antibody and drug that do not have an enzymatically cleavable predetermined break point [S. O. Doronina et al., Bioconjugate Chem. 17, 114-124 (2006)].

Monomethylauristatin F (MMAF) is an auristatin derivative having a C-terminal phenylalanine unit which exhibits only moderate antiproliferative activity in comparison to MMAE. This fact is very probably attributable to the free carboxyl group, whose polarity and charge adversely affect the capacity of this compound to access cells. In this connection, the methyl ester of MMAF (MMAF-OMe) has been described, as a neutral- charged prodrug derivative with cell access capability, which, in comparison to MMAF, has an in vitro cytotoxicity for various carcinoma cell lines that is increased by a number of orders of magnitude [S. O. Doronina et al., Bioconjugate Chem. 7, 114-124 (2006)]. It can be assumed that this effect is brought about by MMAF itself, which, following uptake of the prodrug into the cells, is rapidly released by intracellular ester hydrolysis.

Monomethylauristatin F (MMAF): R = H

Monomethylauristatin F methyl ester (MMAF-OMe): R = CH. However, drug compounds based on simple ester derivatives are generally subject to the risk of chemical instability on account of non-specific ester hydrolysis,

independent of the intended site of action, by means, for example, of esterases that are present in the blood plasma; this non-specific hydrolysis may significantly restrict the usefulness of such compounds in therapy.

Monomethylauristatin F (MMAF) and also various ester derivatives and amide derivatives thereof have been disclosed in WO 2005/081711-A2. Further auristatin analogues with a C-terminal, amidically substituted phenylalanine unit are described in WO 01/18032-A2. WO 02/088172-A2 and WO 2007/008603-A1 claim MMAF analogues which relate to side-chain modifications of the phenylalanine, while

WO 2007/008848-A2 claims those in which the carboxyl group of the phenylalanine has been modified. Auristatin conjugates linked via the C-terminus have been recently described in WO 2009/117531-Al [see also S. O. Doronina et al., Bioconjugate Chem. 19, 1960-1963 (2008)].

Additional auristatin derivatives and ADCs thereof have also been described in WO 2011/154359, WO 2012/041805, WO 2012/123423, WO 2012/143499, WO

2012/143497, WO 2012/143496, WO 2012/143495, and WO 2013/087716, national- stage applications thereof, and related counterpart applications. These publications describe new auristatin compounds, as well as ADCs that include novel linkers. These publications further disclose methods of production of those various compounds and numerous intermediate compounds useful in those production processes. There remains a need in the art for improved processes for producing auristatin derivatives and related ADCs, including a need for improved intermediate compounds useful for producing auristatin derivatives and related ADCs. The present application discloses novel intermediates useful for the production of auristatin derivatives and related ADCs, as well as novel production processes that employ those novel intermediates to produce auristatin derivatives and related ADCs.

SUMMARY OF THE INVENTION:

In one embodiment, the present application provides binder-drug conjugates of the general formula (8):

in which

R¹ is hydrogen or C₁-C₄ alkyl;

R² is C₁-C₄ alkyl, benzyl, or lH-indol-3-ylmethyl;

T is selected from the group consisting of -CH(OR₃)-R4, -C(=0)-OR₃, or - C(=0)-NR³ R⁵,

wherein

R³ is hydrogen or C₁-C₄ alkyl;

R⁴ is phenyl;

R⁵ is hydrogen or C₁-C₄ alkyl;

L¹ is R⁶-C(=0)-R⁷;

where

R⁶ is Ci-C₆ alkyl

R⁷ is a bond or NH-NH

L² is a bond or a group of the formula

where n is 1-4;

wherein Ab is an antibody or antigen-binding antibody fragment which which is bonded via the sulphur atom of a cysteine residue to the compound of formula (4).

In certain other embodiments, Ab is an intact antibody, while in other

embodiments Ab is an antigen-binding antibody fragment. In further embodiments, Ab is a polycolonal antibody or an antigen-binding fragment thereof, while in other embodiments Ab is a monoclonal antibody or an antigen-binding fragment thereof. In additional embodiments, Ab is a human antibody, while in other embodiments Ab is a chimeric or humanized antibody. In still further embodiments, the antibody or antigen- binding antibody fragment binds specifically to its target molecule, for example C4.4a. In particular embodiments, Ab is an antibody or antigen-binding abntibody fragment which binds C4.4a. In other particular embodiments, Ab is a monoclonal antibody or an antigen-binding fragment thereof which binds to C4.4a.

In other embodiments, the present application provides compounds of the general formula (4):

wherein

Ri is hydrogen or C₁-C₄ alkyl;

R-2 is C1-C4 alkyl, benzyl, or lH-indol-3-ylmethyl;

T is selected from the group consisting of -CH(OR₃)-R4, -C(=0)-OR₃, or - C(=0)- R₃ R₅, wherein

R-3 is hydrogen or C₁-C₄ alkyl;

R₄ is phenyl;

R₅ is hydrogen or C₁-C₄ alkyl;

L¹ is R⁶-C(=0)-R⁷;

where

R⁶ is Ci-C₆ alkyl

R⁷ is a bond or H- H

L² is a bond or a group of the formula

where n is 1-4.

In further embodiments, the present application provides compounds used in the production of compounds of the formula (4) or binder-drug conjugates of the formula (8), including:

compounds of the formula (1):

compounds of the formula (2):

In other embodiments, the present application provides a process for preparing the mpounds of the general formula (4):

wherein

Ri is hydrogen or C₁-C₄ alkyl;

R₂ is C₁-C₄ alkyl, benzyl, or lH-indol-3-ylmethyl;

T is selected from the group consisting of -CH(OR₃)-R4, -C(=0)-OR₃, or - C(=0)- R₃ R₅,

wherein

R₃ is hydrogen or C₁-C₄ alkyl;

R₄ is phenyl;

R₅ is hydrogen or C₁-C4 alkyl;

L¹ is R⁶-C(=0)-R⁷;

where

R⁶ is Ci-C₆ alkyl

R⁷ is a bond or H- H

L² is a bond or a group of the formula

where n is 1-4;

comprising the steps of:

(a) reacting a compound of formula (5):

ith a group of the formula LG²-L²-OH to give a compound of formula (1):

where LG² is hydrogen or hydroxyl and L² has the meaning discussed above; (b) further reacting the compound of formula (1) with a compound of formula (6):

where LG¹ is hydrogen or hydroxyl and L¹ has the meaning discussed above, wherein if LG² is hydrogen then LG¹ is hydroxyl and if LG² is hydroxyl then LG¹ is hydrogen;

to give a compound of formula (2):

(c) removing the -tBu protecting group from formula (2) to form a compound of f rmula (3):

(d) reacting the compound of formula (3) with a compound having the general

formula 7):

wherein Ri, R₂, and T are set forth as defined above, to form the compound according to formula (4).

In certain embodiments, Ri is hydrogen; R₂ is lH-indol-3-ylmethyl; T is -C(=0)- R₃ R₅, wherein R₃ and R₅ re hydrogen; L¹ is R⁶-C(=0)-R⁷, wherein R⁶ is a C₅ alkyl

and R⁷ is NH- H; and L² is

wherein n=3.

In other embodiments, Ri is hydrogen; R₂ is a Ci alkyl; T is -CH(OR₃)-R4, wherein R₃ is hydrogen and R₄ is phenyl; L¹ is R⁶-C(=0)-R⁷, wherein R⁶ is a C₅ alkyl and

R⁷ is a bond; and L²

In other embodiments, Ri is hydrogen; R₂ is benzyl; T is -C(=0)-OR₃, wherein R₃ is hydrogen; L¹ is R⁶-C(=0)-R⁷, wherein R⁶ is a C₅ alkyl and R⁷ is a bond; and L² is a bond.

In additional embodiments, the present application provides a process wherein the compound of the formula (4) is conjugated with a monoclonal antibody that binds C4.4a to form a compound according to the general formula (8):

wherein

Ri is hydrogen or C₁-C₄ alkyl;

R₂ is C₁-C₄ alkyl, benzyl, or lH-indol-3-ylmethyl;

T is selected from the group consisting of -CH(OR₃)-R₄, -C(=0)-OR₃, or - C(=0)- R₃ R₅,

wherein

R₃ is hydrogen or C₁-C₄ alkyl;

R₄ is phenyl;

R₅ is hydrogen or C₁-C4 alkyl;

L¹ is R⁶-C(=0)-R⁷;

where

R⁶ is Ci-C₆ alkyl

R⁷ is a bond or H- H L² is a bond or a group of the formula

where n is 1-4; and

Ab is an antibody or antigen-binding antibody fragment which which is bonded via the sulphur atom of a cysteine residue to the compound of formula (4).

In certain other embodiments, Ab is an intact antibody, while in other embodiments Ab is an antigen-binding antibody fragment. In further embodiments, Ab is a polycolonal antibody or an antigen-binding fragment thereof, while in other embodiments Ab is a monoclonal antibody or an antigen-binding fragment thereof. In additional embodiments, Ab is a human antibody, while in other embodiments Ab is a chimeric or humanized antibody. In still further embodiments, the antibody or antigen- binding antibody fragment binds specifically to its target molecule, for example C4.4a. In particular embodiments, Ab is an antibody or antigen-binding abntibody fragment which binds C4.4a. In other particular embodiments, Ab is a monoclonal antibody or an antigen-binding fragment thereof which binds to C4.4a.

In certain embodiments, Ri is hydrogen; R₂ is lH-indol-3-ylmethyl; T is -C(=0)- R₃ R₅, wherein R₃ and R₅ wherein R⁶ is a C₅ alkyl

and R⁷ is NH- H; and L² is

In other embodiments, Ri is hydrogen; R₂ is a Ci alkyl; T is -CH(OR₃)-R4, wherein R₃ is , wherein R⁶ is a C₅ alkyl and

R is a bond; and L is

DETAILED DESCRIPTION OF THE INVENTION:

In one aspect, the present application provides novel compounds useful for producing aunstatin derivatives and ADCs derived from the same. In other aspects, the present application provides methods of producing auristatin derivatives and ADCs derived therefrom using certain intermediate compounds.

in which

Ri is hydrogen or C₁-C₄ alkyl;

R₂ is C₁-C₄ alkyl, benzyl, or lH-indol-3-ylmethyl;

T is selected from the group consisting of -CH(OR₃)-R₄, -C(=0)-OR₃, or - C(=0)-NR₃ R₅,

wherein

R₃ is hydrogen or C₁-C₄ alkyl;

R₄ is phenyl; R₅ is hydrogen or C₁-C₄ alkyl;

L¹ is R⁶-C(=0)-R⁷;

where R⁶ is Ci-C₆ alkyl

R⁷ is a bond or H- H

L² is a bond or a group of the formula

where n is 1-4;

In another embodiment, the present application provides compounds of the general formula (4):

wherein

Ri is hydrogen or C₁-C₄ alkyl;

R₂ is C₁-C₄ alkyl, benzyl, or lH-indol-3-ylmethyl;

wherein

R₃ is hydrogen or C₁-C₄ alkyl;

R₄ is phenyl;

R₅ is hydrogen or C₁-C4 alkyl;

L¹ is R⁶-C(=0)-R⁷;

where R⁶ is Ci-C₆ alkyl;

R⁷ is a bond or H- H;

L² is a bond oer a group of the formula

where n is 1-4.

In further embodiments, the present application provides compounds used production of compounds of the formula (4) or (8), including:

compounds of the formula (1):

compounds of the formula (2):

and compounds of the formula (3):

In additional embodiments, the present application provides a process for preparing the compounds of the general formula (4):

wherein

Ri is hydrogen or C₁-C₄ alkyl;

R₂ is C₁-C₄ alkyl, benzyl, or lH-indol-3-ylmethyl;

wherein

R₃ is hydrogen or C₁-C₄ alkyl; R₄ is phenyl;

R-5 is hydrogen or Ci-C₄ alkyl;

L¹ is R⁶-C(=0)-R⁷;

where R⁶ is Ci-C₆ alkyl;

R⁷ is a bond or H- H;

L² is a bond or a group of the formula

where n is 1-4;

comprising the steps of:

(a) reacting a compound of formula (5):

with LG²-L²-OH to ive a compound of formula (1):

where LG² is hydrogen or hydroxyl

(b) f rther reacting the compound of formula (1) with a compound of formula (6):

where LG¹ is hydrogen or hydroxyl, wherein, if LG² is hydrogen then LG¹ is hydroxyl and is LG² is hydroxyl then LG¹ is hydrogen; to give a compound of formula (2):

(d) reacting the compound of formula (3) with a compound having the general

formula 7):

and R⁷ is NH- H; and L² is

R⁷ is a bond; and L²

In another embodiment, the present application provides a process wherein the compound of the formula (4) is conjugated with a monoclonal antibody that binds C4.4a to form a compound according to the general formula (8):

wherein

Ri is hydrogen or C₁-C₄ alkyl;

R₂ is C₁-C₄ alkyl, benzyl, or lH-indol-3-ylmethyl;

wherein

R₃ is hydrogen or C₁-C₄ alkyl;

R₄ is phenyl;

R₅ is hydrogen or C₁-C₄ alkyl;

L¹ is R⁶-C(=0)-R⁷;

where R⁶ is Ci-C₆ alkyl;

R⁷ is a bond or H- H;

L² is a bond or a group of the formula

where n is 1-4;

Ab is an antibody or antigen-binding antibody fragment which is bonded via the sulphur atom of a cysteine residue to the compound of formula (4).

In certain other embodiments, Ab is an intact antibody, while in other embodiments Ab is an antigen-binding antibody fragment. In further embodiments, Ab is a polycolonal antibody or an antigen-binding fragment thereof, while in other embodiments Ab is a monoclonal antibody or an antigen-binding fragment thereof. In additional embodiments, Ab is a human antibody, while in other embodiments Ab is a chimeric or humanized antibody. In still further embodiments, the antibody or antigen- binding antibody fragment binds specifically to its target molecule, for example C4.4a. In particular embodiments, Ab is an antibody or antigen-binding antibody fragment which binds C4.4a. In other particular embodiments, Ab is a monoclonal antibody or an antigen-binding fragment thereof which binds to C4.4a.

and R⁷ is NH- H; and L² is

, wherein n=3.

In other embodiments, Ri is hydrogen; R₂ is a Ci alkyl; T is -CH(OR₃)-R₄, wherein R₃ is , wherein R⁶ is a C5 alkyl and

R⁷ is a bond; and L² is

In other embodiments, Ri is hydrogen; R₂ is benzyl; T is -C(=0)-OR₃, wherein R₃ is hydrogen; L¹ is R⁶-C(=0)-R⁷, wherein R⁶ is a C5 alkyl and R⁷ is a bond; and L² is a bond.

The present application further relates to ADCs selected from the following compounds:



Depending on their structure, the compounds of the invention may exist in different stereoisomeric forms, i.e., in the form of configurational isomers or else where appropriate as conformational isomers (enantiomers and/or diastereoisomers, including those in the case of atropisomers). The present invention therefore encompasses the enantiomers and diastereomers and their respective mixtures. The stereoisomerically homogeneous constituents can be isolated from such mixtures of enantiomers and/or diastereomers in a known way; for this purpose it is preferred to use chromatographic processes, more particularly HPLC chromatography on an achiral or chiral phase.

Where the compounds of the invention can occur in tautomeric forms, the present invention encompasses all of the tautomeric forms.

The present invention also encompasses all suitable isotopic variants of the compounds of the invention. An isotopic variant of a compound of the invention is understood here to mean a compound in which at least one atom within the compound of the invention has been exchanged for another atom of the same atomic number but with a different atomic mass from the atomic mass which occurs commonly or predominantly in nature. Examples of isotopes which can be incorporated into an inventive compound are those of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine, chlorine, bromine and iodine such as ²H (deuterium), ³H (tritium), ¹³C, ¹⁴C, ¹⁵N, ¹⁷0, ¹⁸0, ³²P, ³³P, ³³S, ³⁴S, ³⁵S, ³⁶S, ¹⁸F, ³⁶C1, ⁸²Br, ¹²³I, ¹²⁴I, ¹²⁹I and ¹³¹I. Particular isotope variants of a compound of the invention, such as more particularly those in which one or more radioactive isotopes are incorporated, may be of benefit, for example, for investigating the mechanism of action or the distribution of drug in the body; owing to the comparative ease of preparation and detectability, compounds labelled with ³H or ¹⁴C isotopes are especially suitable for these purposes. Furthermore, the incorporation of isotopes, such as of deuterium, for example, may lead to certain therapeutic advantages as a consequence of greater metabolic stability of the compound, such as an extension to the half-life in the body or a reduction in the active dose required, for example; such modifications of the compounds of the invention may therefore, where appropriate, also constitute a preferred embodiment of the present invention. Isotopic variants of the compounds of the invention can be prepared by the processes known to the skilled person, as for example in accordance with the methods described later on below and the procedures reproduced in the working examples, by using corresponding isotopic modifications of the respective reagents and/or starting compounds.

The present application further encompasses salts and solvates of the compounds disclosed herein. Preferred salts in the context of the present invention are

physiologically acceptable salts of the compounds of the invention. Also encompassed are salts which although themselves not suitable for pharmaceutical applications may nevertheless be used, for example, for isolating or purifying the compounds of the invention.

Physiologically acceptable salts of the compounds of the invention encompass acid addition salts of mineral acids, carboxylic acids and sulphonic acids, examples being salts of hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, benzenesulphonic acid, toluenesulphonic acid, naphthalenedisulphonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

Physiologically acceptable salts of the compounds of the invention also encompass salts of customary bases, such as, by way of example and preferably, alkali metal salts (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium and magnesium salts) and ammonium salts, derived from ammonia or organic amines having 1 to 16 C atoms, such as, by way of example and preferably, ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine,

triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N- methylpiperidine, N-methylmorpholine, arginine, lysine and 1,2-ethylenediamine.

Solvates in the context of the invention are those forms of the compounds of the invention that form a complex in the solid or liquid state through coordination with solvent molecules. Hydrates are one specific form of solvates, in which the coordination takes place with water. Preferred solvates in the context of the present invention are hydrates.

Furthermore, the present invention also encompasses prodrugs of the compounds of the invention. The term "prodrugs" here identifies compounds which may themselves be biologically active or inactive but are converted during their residence in the body into compounds of the invention (by metabolism or hydrolysis, for example).

Definitions:

In the context of the present invention the terms used, unless otherwise specified, have the following definitions:

(Ci-C4)-Alkyl in the context of the invention is a linear or branched alkyl radical having 1 to 4 carbon atoms. By way of example and with preference, the following may be mentioned: methyl, ethyl, ^-propyl, isopropyl, «-butyl, isobutyl, 1-methylpropyl and tert-butyl.

An aryl in the context of the invention is any functional group or substituent derived from an aromatic ring. More specifically, heteroaryls are cyclic compounds having atoms of at least two different elements as members of its ring(s). Preference is given to heteroaryls having one or two ring heteroatoms from the series N, O and/or S, more preferably heteroaryls having one or two ring heteroatoms from the series N and/or O. By way of example, the following may be mentioned: furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, phenyl, pyridinyl, pyridazinyl, pyrimidinyl, or pyrazinyl; fused heteroaryl groups include indolyl, isoindolyl, benzofuryl, benzothienyl, indazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzothiadiazolyl, benzotriazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, and quinazolinyl.

A four- to seven-membered "heterocycle" within the scope of the invention stands for a mono-cyclic 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 S0₂ 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, thiolanyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, hexahydroazepinyl and hexahydro-l,4-diazepinyl. Preferred examples include pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl and morpholinyl.

The term "binder" is understood in the broadest sense as a molecule which binds to a target molecule which is present on a particular target cell population to be addressed with the binder-drug conjugate. The term "binder" should be understood in its broadest interpretation and encompasses, for example, lectins, proteins which are able to bind particular sugar chains, or phospholipid-binding proteins. Such binders comprise, for example, high molecular mass proteins (binding proteins), polypeptides or peptides (binding peptides), non-peptidic (e.g. aptamers (US5,270,163) (review article by Keefe AD., et al., Nat. Rev. Drug Discov. 2010; 9:537-550), or vitamins) and all other cell- binding molecules or substances. Binding proteins are, for example, antibodies and antibody fragments or antibody mimetics such as, for example, affibodies, adnectins, anticalins, DARPins, avimers, nanobodies (review articles 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). Binding peptides are, for example, ligands of a ligand- receptor pair, such as VEGF in the ligand-receptor pair VEGF/KDR, such as transferrin of the ligand-receptor pair transferrin/transferrin receptor, or cytokines/cytokine receptor, such as T F alpha in the ligand receptor pair T Falpha/T F alpha receptor.

Preferred binders in accordance with the invention are (more particularly human, monoclonal) antibodies or antigen-binding antibody fragments which bind to C4.4a. In the case of anti-C4.4a antibodies, the number of toxophore molecules per antibody molecule, is preferably in the range from 1 to 10, more 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). Preferably it is a receptor or an antigen.

The term "extracellular" target molecule describes a target molecule which is attached to the cell and which is located on the outside of a cell or the part of a target molecule which is located 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. The skilled person knows of methods for identifying extracellular target molecules. For proteins this may be done via determination of the transmembrane domain(s) and the orientation of the protein in the membrane. This data is generally recorded in protein databases (e.g. SwissProt).

The term "cancer target molecule" describes a target molecule which is multiply present on one or more cancer cell types in comparison to non-cancer cells of the same tissue type. The cancer target molecule is preferably present selectively on one or more cancer cell types in comparison to non-cancer cells of the same tissue type, with

"selectively" describing an at least twofold accumulation on cancer cells in comparison to non-cancer cells of the same tissue type (a "selective cancer target molecule"). The use of cancer target molecules allows selective therapy of cancer cells with the conjugates of the invention.

The binder may be linked via a bond to the linker. Known from the literature are various possibilities of covalent coupling (conjugation) of organic molecules to antibody. The linking of the binder may take place by means of a heteroatom of the binder.

Particular heteroatoms of the binder that may be used for linking are sulphur (in one embodiment via a sulphhydryl group of the binder), oxygen (in accordance with the invention by means of a carboxyl or hydroxy group of the binder), and nitrogen (in one embodiment via a primary or secondary amine group or amide group of the binder). In certain embodiments, the conjugation of the toxophores to the antibody is via one or more sulphur atoms of cysteine residues of the antibody and/or via one or more NH groups of lysine residues of the antibody. These heteroatoms may be present in the natural binder or may be introduced by means of methods of chemistry or molecular biology. In accordance with the invention, the linking of the binder to the toxophore has little influence over the binding activity of the binder to the target molecule. In a preferred embodiment the linking has no influence on the binding activity of the binder to the target molecule.

The term "antibody" is understood in accordance with the present invention in its broadest sense and encompasses immunoglobulin molecules, examples being 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 linked typically by disulphide bridges. Each heavy chain comprises a variable domain of the heavy chain (abbreviated to VH) and a constant domain of the heavy chain. The constant domain of the heavy chain may encompass, for example, three domains CHI, CH2 and CH3. Each light chain comprises a variable domain (abbreviated to VL) and a constant domain. The constant domain of the light chain comprises one domain (abbreviated to CL). The VH and VL domains may be further subdivided into regions having hypervariability, also called complementarity- determining regions (abbreviated to CDR), and regions having a low sequence variability ("framework region", abbreviated to FR). Each VH and VL region is typically composed of three CDRs and up to four FRs. For example, in the following order from the amino terminus to the carboxy terminus: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. An antibody may be obtained from any species suitable for the antibody, such as, for example, rabbit, lama, camel, mouse or rat. In one embodiment the antibody is of human or murine origin. An antibody may for example be human, humanized or chimeric.

The term "monoclonal" antibody identifies antibodies which have been 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 small numbers.

Monoclonal antibodies recognize a single antigenic binding site with a high specificity. The term "monoclonal antibody" does not refer to a particular production method.

The term "intact" antibody refers to antibodies which comprise not only an antigen- binding domain but also the constant domain of the light and heavy chain. The constant domain may be a naturally occurring domain, or a variant thereof in which one or more amino acid positions have been altered.

The term "modified intact" antibody refers to intact antibodies which have been fused with another polypeptide or protein, not originating from an antibody, via the amino terminus or carboxyl terminus thereof, by means of a covalent bond (e.g. a peptide linkage). Furthermore, antibodies may be modified by introducing reactive cysteines at defined locations, in order to facilitate coupling to a toxophore (see Junutula et al. Nat Biotechnol. 2008 Aug;26(8):925-32).

The term "human" antibody identifies antibodies which can be obtained from a human being or are synthetic human antibodies. A "synthetic" human antibody is an antibody which in parts or as a whole is obtainable from synthetic sequences in silico which are based on the analysis of human antibody sequences. A human antibody may be encoded, for example, by a nucleic acid which has been isolated from a library of antibody sequences which are of human origin. One example of such antibodies can be found in Soderlind et al., Nature Biotech. 2000, 18: 853-856.

The term "humanized" or "chimeric" antibody describes antibodies which consist of a non-human and of a human sequence component. In these antibodies, part of the sequences of the human immunoglobulin (recipient) is replaced by sequence components of a non-human immunoglobulin (donor). In many cases the donor is a murine immunoglobulin. With humanized antibodies, amino acids of the CDR in the recipient are replaced by amino acids of the donor. In some cases, amino acids of the framework as well are replaced by corresponding amino acids of the donor. In some cases the humanized antibody contains amino acids which were present neither in the recipient nor in the donor and which were inserted during the optimization of the antibody. In the case of chimeric antibodies, for example, the variable domains of the donor immunoglobulin, or else the entire Fab fraction, in other words VL-CL and VH + CHI, are fused with the constant regions of a human antibody.

The term "complementarity-determining region" (CDR) as used here refers to those amino acids in a variable antibody domain that are necessary for binding to the antigen. Every variable region typically has three CDR regions, identified as CDR1, CDR2 and CDR3. Each CDR region may comprise amino acids according to the definition of Kabat and/or amino acids of a hypervariable loop, defined according to Chotia. The definition according to Kabat encompasses, for example, the region of approximately amino acid position 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 Immulological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. ( 1991)). The definition according to Chotia encompasses, for example, the region of approximately amino acid position 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 Chothia and Lesk; J Mol Biol 196: 901-917 (1987)). In some cases a CDR may comprise amino acids from one CDR region as defined by Kabat and Chotia.

Depending on the amino acid sequence of the constant domain of the heavy chain, antibodies may be divided into different classes. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and a number of them may be broken down into further subclasses (isotypes), e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The constant domains of the heavy chain that correspond to the different classes are identified as [alpha/a], [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 fragments" of a antibody/immunoglobulin is defined as a fragment of an antibody/immunoglobulin (e.g. the variable domains of an IgG) which further encompasses the antigen binding domains of the antibody/immunoglobulin. The "antigen binding domain" of an antibody typically encompasses one or more hypervariable regions of an antibody, e.g. the CDR1, CDR2 and/or CDR3 region. However, the "framework" or "scaffold" region of an antibody may also play a part with regard to the binding of the antibody to the antigen. The framework region forms the scaffold for the CDRs. The antigen-binding domain preferably encompasses at least amino acids 4 to 103 of the variable light chain and amino acid 5 to 109 of the variable heavy chain, more preferably amino acid 3 to 107 of the variable light chain and 4 to 111 of the variable heavy chain, particular preference being given to the complete variable light and heavy chains, i.e. amino acid 1 - 109 of the VL and 1 to 113 of the VH (numbering according to WO97/08320).

"Functional fragments" or "antigen-binding antibody fragments" of the invention encompass, non -conclusively, Fab, Fab', F(ab')₂ and Fv fragments, diabodies, Single Domain Antibodies (DAbs), linear antibodies, individual chains of antibodies (single-chain Fv, abbreviated to ScFv); and multispecific antibodies, such as bi and tri -specific antibodies, for example, formed from antibody fragments C. A. K Borrebaeck, editor (1995) Antibody

Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, editors (2001) Antibody Engineering (Springer Laboratory Manual), Springer

Verlag). Antibodies other than "multispecific" or "multifunctional" antibodies are those having identical binding sites. Multispecific antibodies may be specific for different epitopes of an antigen or may be specific for epitopes of more than one antigen (see, for example

W093/17715; 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; 4,7 14,68 1; 4,925,648; 5,573,920; 5,601,8 19; or Kostelny et al., 1992, J. Immunol. 148: 1547 1553). An F(ab')₂ or Fab molecule may be constructed such that the number of intermolecular disulphide interactions occurring between the Chi and the CL domains can be reduced or else completely prevented.

"Functional fragments" or "antigen-binding antibody fragments" may be fused with another polypeptide or protein, not originating from an antibody, via the amino terminus or carboxyl terminus thereof, by means of a covalent bond (e.g. a peptide linkage). Furthermore, antibodies and antigen-binding fragments may be modified by introducing reactive cysteines at defined locations, in order to facilitate coupling to a toxophore (see Junutula et al. Nat

Biotechnol. 2008 Aug;26(8):925-32).

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

A person of ordinary skill in the art is aware of diverse methods for preparing human antibodies and fragments thereof, such as, for example, by means of transgenic mice (N Lonberg and D Huszar, Int Rev Immunol. 1995; 13(l):65-93) or Phage Display Technologies (Clackson et al., Nature. 1991 Aug 15;352(6336):624-8). Antibodies of the invention may be obtained from recombinant antibody libraries consisting for example of the amino acid sequences of a multiplicity of antibodies compiled from a large number of healthy volunteers. Antibodies may also be produced by means of known recombinant DNA technologies. The nucleic acid sequence of an antibody can be obtained by routine sequencing or is available from publically accessible databases.

An "isolated" antibody or binder has been purified to remove other constituents of the cell. Contaminating constituents of a cell which may interfere with a diagnostic or therapeutic use are, for example, enzymes, hormones, or other peptidic or non-peptidic constituents of the cell. A preferred antibody or binder is one which has been purified to an extent of more than 95%, relative to the antibody or binder (determined for example by Lowry method, UV-Vis spectroscopy or by SDS capillary gel electrophoresis), the purification thereof being such that it is possible to determine at least 15 amino acids of the amino terminus or of an internal amino acid sequence, or which has been purified to homogeneity, the homogeneity being determined by SDS-PAGE under reducing or non-reducing conditions (detection may be determined by means of Coomassie Blau staining or preferably by silver coloration). However, an antibody is normally prepared by one or more purification steps. The term "specific binding" or "binds specifically" refers to an antibody or binder which binds to a predetermined antigen/target molecule. Specific binding of an antibody or binder typically describes an antibody or binder having an affinity of at least 10^"7 M (as Kd value; i.e. preferably those with smaller Kd values than 10^"7 M), with the antibody or binder having an at least two times higher affinity for the predetermined antigen/target molecule than for 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 prepared by a person of ordinary skill in the art by means of methods with which he or she is familiar (such as recombinant expression, for example) or may be acquired commercially (as for example from Merck KGaA, Germany). Examples of known commercially available antibodies in cancer therapy are Erbitux® (cetuximab, Merck KGaA), Avastin®

(bevacizumab, Roche) and Herceptin® (trastuzumab, Genentech). Trastuzumab is a recombinant humanized monoclonal antibody of the IgGl kappa type which in a cell- based assay (Kd = 5 nM) binds the extracellular domains of the human epidermal growth receptor with high affinity. The antibody is produced recombinantly in CHO cells.

Anti-C4.4a antibodies and antigen-binding fragments thereof:

In certain embodiments, 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 fascicular is). 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. Additional examples of C4.4a antibodies and antigen-binding fragments are described below. The sequences of the CDR regions and/or heavy and light chains of the antibodies are given in Tables 1 and 2, where each row provides the SEQ ID Nos of the respective amino acid sequences of the CDR regions and/or of the variable light chain and/or of the variable heavy chain of the antibody listed in column 1.

In one embodiment, the anti-C4.4a antibodies or the antigen-binding antibody fragments bind to the SI domain SI (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-BOl and/or with the antibody M20-D02-S-A for binding to C4.4a. Antibodies M31-BOl and M20-D02-S-A compete for binding to C4.4a. Antibodies BOl-1 to B01-12 were synthesized by affinity maturation from M31- B01 and compete with M31-BOl 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 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-C4.4a antibodies or the antigen-binding antibody fragments, are at least 50%, 60%, 70%, 80%, 90% or 95% identical to the CDR amino acid sequences and/or the variable heavy and light chain sequences 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 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 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 HI), 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 LI), 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 HI), 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 LI), SEQ ID NO: 306 (CDR L2) and SEQ ID NO: 307 (CDR L3).

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 (BOl-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 (BOl-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 (BOl-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 (BOl-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 one particular 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 (BOl-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).

A further aspect of the present invention is the provision of an anti-C4.4a IgGl antibody which comprises the amino acid sequence of the light chain and of the heavy chain of an antibody given in Table 2.

Table 1: Sequences of the CDR Regions of Select C4.4a antibodies

o O o re

Cd Cd Cd Cd Cd Cd Cd Cd Cd Cd Cd _Q o o o o o o o o o O o o o o > 3 C t t to to

o re to ON to S

ό to Cd o r> o o a- re to

oo

re era^' s

O ON ON ON ON a

ON to o 00 ON to O 00 ON to o 00 ON

re

B o

S re O

ON ON ON ON ON K

*-

S o s

¾ o S O"^' a re^'

Light chain Heavy chain

Antibody SEQ ID NO: SEQ ID NO:

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

Treatment methods:

The compounds of the invention possess valuable pharmacological properties and can be used for the prevention and treatment of diseases in humans and animals.

The binder-drug conjugates (ADCs) of the invention formed as a result of Step (e) in Scheme 1, exhibit a high and specific cytotoxic activity with regard to tumor cells. This high and specific cytotoxic activity on the part of the binder-drug conjugates (ADCs) of the invention formed as a result of Step (e) in Scheme 1, is achieved through the appropriate combination of the new Ν,Ν-dialkylauristatin derivative and binder with linkers which exhibit not only an enzymatically, hydrolytically or reductively cleavable predetermined break point, for the release of the toxophores, but also no such

predetermined break point. More particularly, through the use of stable linkers which have no enzymatically, hydrolytically or reductively cleavable predetermined break point for the release of the toxophores, and which, following uptake of the ADCs into the tumor cell and following complete intracellular, enzymatic breakdown of the antibody, still remain wholly or partly intact, the activity is confined very specifically to the tumor cell. Compatibility between ADCs and stable linkers presupposes, among other things, that the metabolites formed intracellularly can be formed with sufficient efficacy, are able to reach their target and are able there to develop their anti-proliferative activity on the target with sufficient potency, without being carried out of the tumor cell again beforehand by transporter proteins. The metabolites formed intracellularly after the compounds of the formula (8) of the invention have been taken up exhibit a reduced potential as a substrate with respect to transporter proteins, thereby suppressing their redistribution into the systemic circulation and hence the triggering of potential side effects by the toxophore itself.

The compatibility of the ADCs with a stabile linker chemistry and with the target in question, in conjunction with metabolites which represent a substrate for transporter proteins to a relatively low degree, offers an enlarged therapeutic window.

More particularly, the binder-drug conjugates of the invention formed as a result of Step (e) in Scheme 1 exhibit a high and specific cytotoxic activity with respect to tumor cells which express C4.4a. The activity with respect to tumor cells which do not express C4.4a is significantly weaker at the same time.

On the basis of this profile of properties, the compounds of the invention are therefore suitable to a particular degree for the treatment of hyperproliferative diseases in humans and in mammals generally. The compounds are able on the one hand to inhibit, block, reduce or lower cell proliferation and cell division, and on the other hand to increase apoptosis.

The hyperproliferative diseases for the treatment of which the compounds of the invention can be employed include in particular the group of cancer and tumor diseases. In the context of the present invention, these are understood as meaning, in particular, the following diseases, but without being limited to them: mammary carcinomas and mammary tumors (ductal and lobular forms, also in situ), tumors of the respiratory tract (parvicellular and non-parvicellular carcinoma, bronchial carcinoma), cerebral tumors (e.g. of the brain stem and of the hypothalamus, astrocytoma, medulloblastoma, ependymoma and neuro-ectodermal and pineal tumors), tumors of the digestive organs (oesophagus, stomach, gall bladder, small intestine, large intestine, rectum), liver tumors (including hepatocellular carcinoma, cholangiocellular carcinoma and mixed

hepatocellular and cholangiocellular carcinoma), tumors of the head and neck region (larynx, hypopharynx, nasopharynx, oropharynx, lips and oral cavity), skin tumors (squamous epithelial carcinoma, Kaposi sarcoma, malignant melanoma, Merkel cell skin cancer and non-melanomatous skin cancer), tumors of soft tissue (including soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytomas, lymphosarcomas and rhabdomyosarcomas), tumors of the eyes (including intraocular melanoma and retinoblastoma), tumors of the endocrine and exocrine glands (e.g. thyroid and parathyroid glands, pancreas and salivary gland), tumors of the urinary tract (tumors of the bladder, penis, kidney, renal pelvis and ureter) and tumors of the reproductive organs (carcinomas of the endometrium, cervix, ovary, vagina, vulva and uterus in women and carcinomas of the prostate and testicles in men). These also include proliferative blood diseases in solid form and as circulating blood cells, such as lymphomas, leukaemias and myeloproliferative diseases, e.g. acute myeloid, acute lymphoblastic, chronic

lymphocytic, chronic myelogenic and hair cell leukaemia, and also AIDS -correlated lymphomas, Hodgkin's lymphomas, non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, Burkitt's lymphomas and lymphomas in the central nervous system.

Hyperproliferative diseases for the treatment of which the compounds of the invention can be preferably employed are C4.4a-overexpressing tumors, squamous epithelial carcinomas (e.g. of the cervix, vulva, vagina, of the anal duct, endometrium, fallopian tube, penis, scrotum, of the oesophagus, breast, of the bladder, of the bile duct, endometrium, uterus and ovary); mammary carcinomas and mammary tumors (e.g. ductal and lobular forms, also in situ); tumors of the respiratory tract (e.g. parvicellular and non-parvicellular carcinoma, bronchial carcinoma), including preferably non-parvicellular carcinoma of the lung, squamous epithelial carcinoma and adenocarcinoma of the lung; tumors of the head and neck region (e.g. larynx, hypopharynx, nasopharynx, oropharynx, lips, oral cavity, tongue and oesophagus, squamous epithelial carcinomas of the head and neck region); tumors of the urinary tract (tumors of the bladder, penis, kidney, renal pelvis and ureter, squamous epithelial carcinomas of the bladder), including more preferably tumors of the kidneys and of the bladder; skin tumors (squamous epithelial carcinoma, Kaposi sarcoma, malignant melanoma, Merkel cell skin cancer and non- melanomatous skin cancer), including more preferably melanomas; tumors of the endocrine and exocrine glands (e.g. thyroid and parathyroid glands, pancreas and salivary gland), including preferably pancreas; tumors of the digestive organs (e.g. oesophagus, stomach, gall bladder, small intestine, large intestine, rectum), including especially colorectal carcinomas; and/or tumors of the reproductive organs (carcinomas of the endometrium, cervix, ovary, vagina, vulva and uterus in women and/or carcinomas of the prostate and testicles in men), including more preferably uterine carcinomas. These well-described diseases in humans can also occur with a comparable aetiology in other mammals and can be treated there with the compounds of the present invention.

In the context of this invention the term "treatment" or "treat" is used in the conventional sense and means attending to, caring for and nursing a patient with the aim of combating, reducing, attenuating or alleviating an illness or health abnormality and improving the living conditions impaired by this illness, such as, for example, with a cancer disease.

The anti-C4.4a binder-drug conjugate of the invention is used preferably for treating cancer in a patient, where the cancer cells of the patient that are to be treated have C4.4a expression. Treatment is administered more preferably to patients whose C4.4a expression in cancer cells is higher than in healthy cells.

One method of identifying patients who respond advantageously to an anti-C4.4a binder-drug conjugate for the treatment of cancer involves determining the C4.4a expression in cancer cells of the patient. In one embodiment the C4.4a expression is determined by C4.4a gene expression analysis. The skilled person knows of methods for gene expression analysis such as, for example, RNA detection, quantitative or qualitative polymerase chain reaction or fluorescence in situ hybridization (FISH). In another preferred embodiment the C4.4a expression is determined by means of

immunohistochemistry with an anti-C4.4a antibody. The immunohistochemistry is carried out preferably on formaldehyde-fixed tissue. The antibody for use in the immunohistochemistry is the same antibody which is also used in the conjugate. The antibody for use in the immunohistochemistry is a second antibody which - preferably specifically - recognizes the C4.4a target protein.

The compounds according to the invention can be employed by themselves or, if required, in combination with one or more other pharmacologically active substances, as long as this combination does not lead to undesirable and unacceptable side effects. The present invention furthermore therefore provides medicaments comprising at least one of the compounds of the invention and one or more further drugs, in particular for the treatment and/or prevention of the abovementioned diseases.

For example, the compounds of the present invention can be combined with known antihyperproliferative, cytostatic or cytotoxic substances for the treatment of cancer diseases. Suitable drugs in the combination which may be mentioned by way of example are as follows:

aldesleukin, alendronic acid, alfaferone, alitretinoin, allopurinol, aloprim, aloxi, altretamine, aminoglutethimide, amifostine, amrubicin, amsacrine, anastrozole, anzmet, aranesp, arglabin, arsenic trioxide, aromasin, 5-azacytidine, azathioprine, BCG or tice- BCG, bestatin, betamethasone acetate, betamethasone sodium phosphate, bexarotene, bleomycin sulphate, broxuridine, bortezomib, busulfan, calcitonin, campath,

capecitabine, carboplatin, casodex, cefesone, celmoleukin, cerubidin, chlorambucil, cisplatin, cladribin, clodronic acid, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunoxome, decadron, decadron phosphate, delestrogen, denileukin diftitox, depomedrol, deslorelin, dexrazoxane, diethylstilbestrol, diflucan, docetaxel, doxifluridine, doxorubicin, dronabinol, DW-166HC, eligard, elitek, ellence, emend, epirubicin, epoetin-alfa, epogen, eptaplatin, ergamisol, estrace, estradiol, estramustine sodium phosphate, ethinylestradiol, ethyol, etidronic acid, etopophos, etoposide, fadrozole, farstone, filgrastim, finasteride, filgrastim, floxuridine, fluconazole, fludarabin, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU),

fluoxymesterone, flutamide, formestane, fosteabine, fotemustine, fulvestrant, gammagard, gemcitabine, gemtuzumab, gleevec, gliadel, goserelin, granisetron hydrochloride, histrelin, hycamtin, hydrocortone, erythro-hydroxynonyladenine, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, interferon-alpha, interferon- alpha-2, interferon-alpha-2a, interferon-alpha-2p, interferon-alpha-nl, interferon-alpha- n3, interferon-beta, interferon-gamma-la, interleukin-2, intron A, iressa, irinotecan, kytril, lentinan sulphate, letrozole, leucovorin, leuprolide, leuprolide acetate, levamisole, levofolic acid calcium salt, levothroid, levoxyl, lomustine, lonidamine, marinol, mechlorethamine, mecobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, 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, ondansetron hydrochloride, orapred, oxaliplatin, paclitaxel, pediapred, pegaspargase, pegasys, pentostatin, picibanil, pilocarpine hydrochloride, pirarubicin, plicamycin, porfimer sodium, prednimustine, prednisolone, prednisone, premarin, procarbazine, procrit, raltitrexed, rebif, rhenium- 186 etidronate, rituximab, roferon-A, romurtide, salagen, sandostatin, sargramostim, semustine, sizofiran, sobuzoxane, solu-medrol, streptozocin, strontium-89 chloride, synthroid, tamoxifen, tamsulosin, tasonermin, tastolactone, taxoter, teceleukin, temozolomide, teniposide, testosterone propionate, testred, thioguanine, thiotepa, thyrotropin, tiludronic acid, topotecan, toremifen, tositumomab, tastuzumab, teosulfan, tretinoin, trexall, trimethylmelamine, trimetrexate, triptorelin acetate, triptorelin pamoate, UFT, uridine, valrubicin, vesnarinone, vinblastine, vincristine, vindesine, vinorelbine, virulizin, zinecard, zinostatin-stimalamer, zofiran; ABI-007, acolbifen, actimmune, affinitak, aminopterin, arzoxifen, asoprisnil, atamestane, atrasentan, avastin, BAY 43-9006 (sorafenib), CCI-779, CDC-501, celebrex, cetuximab, crisnatol, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dSLEVI, dutasteride, edotecarin, eflornithine, exatecan, fenretinide, histamine

dihydrochloride, histrelin 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, liposomal MTP-PE, MX-6, nafarelin, nemorubicin, neovastat, nolatrexed, oblimersen, onko-TCS, osidem, paclitaxel polyglutamate, pamidronate disodium, PN-401, QS-21, quazepam, R- 1549, raloxifen, ranpirnas, 13-cz^'s-retic acid, satraplatin, seocalcitol, T- 138067, tarceva, taxoprexin, thymosin-alpha-1, tiazofurin, tipifarnib, tirapazamine, TLK-286, toremifen, transMID-107R, valspodar, vapreotide, vatalanib, verteporfin, vinflunin, Z-100, zoledronic acid and combinations of these.

In a preferred embodiment, the compounds of the present invention can be combined with antihyperproliferative agents, which can be, by way of example - without this list being conclusive as follows:

aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine, bleomycin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, 2',2'- difluorodeoxycytidine, docetaxel, doxorubicin (adriamycin), epirubicin, epothilone and its derivatives, erythro-hydroxynonyladenin, ethinylestradiol, etoposide, fludarabin phosphate, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil, fluoxymesterone, flutamide, hexamethylmelamine, 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, raloxifen, semustine, streptozocin, tamoxifen, teniposide, testosterone propionate, thioguanine, thiotepa, topotecan, trimethylmelamine, uridine, vinblastine, vincristine, vindesine and vinorelbine.

The compounds of the invention can also be combined in a very promising manner with biological therapeutics such as antibodies (e.g. avastin, rituxan, erbitux, herceptin). The compounds of the invention can also achieve positive effects in combination with therapies directed against angiogenesis, such as, for example, with avastin, axitinib, recentin, regorafenib, sorafenib or sunitinib. Combinations with inhibitors of the proteasome and of mTOR and also with antihormones and steroidal metabolic enzyme inhibitors are likewise particularly suitable because of their favourable profile of side effects.

Generally, the following aims can be pursued with the combination of compounds of the present invention with other agents having a cytostatic or cytotoxic action:

• an improved activity in slowing down the growth of a tumor, in reducing its size or even in its complete elimination compared with treatment with an individual drug;

• the possibility of employing the chemotherapeutics used in a lower dosage than in monotherapy;

• the possibility of a more tolerable therapy with few side effects compared with

individual administration;

• the possibility of treatment of a broader spectrum of tumor diseases;

• the achievement of a higher rate of response to the therapy;

• a longer survival time of the patient compared with present-day standard therapy.

The compounds according to the invention can moreover also be employed in combination with radiotherapy and/or surgical intervention.

The present invention furthermore provides medicaments which comprise at least one compound of the invention, conventionally together with one or more inert, nontoxic, pharmaceutically suitable excipients, and the use thereof for the abovementioned purposes.

The compounds of the invention can act systemically and/or locally. They can be administered in a suitable manner for this purpose, such as for example orally, parenterally, pulmonally, nasally, sublingually, lingually, buccally, rectally, dermally, transdermally, conjunctivally, otically or as an implant or stent.

The compounds of the invention can be administered in suitable administration forms for these administration routes.

Administration forms which function according to the prior art, release the compounds of the invention rapidly and/or in a modified manner and contain the compounds of the invention in crystalline and/or amorphized and/or dissolved form are suitable for oral administration, such as e.g. tablets (non-coated or coated tablets, for example with coatings which are resistant to gastric juice or dissolve in a delayed manner or are insoluble and control the release of the compound of the invention), films/oblates or tablets, which disintegrate rapidly in the oral cavity, films/lyophilizates, capsules (for example hard or soft gelatine capsules), film-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.

Parenteral administration can be effected with bypassing of an absorption step (e.g. intravenously, intraarterially, intracardially, intraspinally or intralumbally) or with inclusion of an absorption (e.g. intramuscularly, subcutaneously, intracutaneously, percutaneously or intraperitoneally). Administration forms which are suitable for parenteral administration include injection and infusion formulations in the form of solutions, suspensions, emulsions, lyophilizates or sterile powders.

For the other administration routes e.g. inhalation medicament forms (including powder inhalers, nebulizers), nasal drops, solutions or sprays, tablets, films/oblates or capsules for lingual, sublingual or buccal administration, suppositories, ear or eye preparations, vaginal capsules, aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (e.g. patches), milk, pastes, foams, sprinkling powders, implants or stents are suitable.

Oral and parenteral administration are preferred, in particular oral and intravenous administration.

The compounds of the invention can be converted into the administration forms mentioned. This can be effected in a manner known per se by mixing with inert, non- toxic, pharmaceutically suitable excipients. These excipients include inter alia carrier substances (for example microcrystalline cellulose, lactose, mannitol), solvents (e.g. liquid polyethylene glycols), emulsifiers and dispersing or wetting agents (for example sodium dodecyl sulphate, polyoxysorbitan oleate), binders (for example

polyvinylpyrrolidone), synthetic and natural polymers (for example albumin), stabilizers (e.g. antioxidants, such as, for example, ascorbic acid), colorants (e.g. inorganic pigments, such as, for example, iron oxides) and taste and/or odour correctants.

In general, it has proved advantageous in the case of parenteral administration to administer amounts of from about 0.001 to 1 mg/kg, preferably about 0.01 to 0.5 mg/kg of body weight to achieve effective results. In the case of oral administration the dosage is about 0.01 to 100 mg/kg, preferably about 0.01 to 20 mg/kg and very particularly preferably 0.1 to 10 mg/kg of body weight.

Nevertheless it may be necessary to deviate from the amounts mentioned, and in particular depending on the body weight, administration route, individual behaviour towards the active compound, nature of the formulation and point of time or interval at which administration takes place. Thus in some cases it may be sufficient to manage with less than the abovementioned minimum amount, while in other cases the upper limit mentioned must be exceeded. In the case where relatively large amounts are administered, it may be advisable to distribute these into several individual doses over the day.

EXAMPLES:

Synthesis procedures may be characterized using the following characterization techniques:

High Performance Liquid Chromatography (HPLC): Zorbax SB-Aq 150 x 3 mm, 3.5 mm column; T = 45°C, detection at 210 nm, flow 0.5 mL/min, gradient A: acetonitrile B: aqueous phosphate buffer pH 2.4; 0 min 95% B, 20 min 80% A.

Liquid Chromatography/Mass Spectroscopy (LC/MS) MHZ-QP-GOLD: Instrument: Micromass Quattro Premier with Waters UPLC Acquity; column: Thermo Hypersil GOLD 1.9 μ 50 x 1 mm; eluent A: 1 L water + 0.5 ml 50% aq. formic acid, eluent B: 1 I acetonitrile + 0.5 ml 50% aq. formic acid; gradient: 0.0 min 97% A→ 0.5 min 97% A→ 3.2 min 5% A→ 4.0 min 5% A oven: 50°C; flow: 0.3 mL/min; UV detection: 210 nm. MCW SQ-HSST3 : Instrument: Waters ACQUITY SQD UPLC System; column: Waters Acquity UPLC HSS T3 1.8 μ 50 x 1 mm; eluent A: 1 I Wasser + 0.25 ml 99% formic acid, eluent B: 1 I acetonitrile + 0.25 ml 99% formic acid; gradient: 0.0 min 90% A→ 1.2 min 5% A→ 2.0 min 5% A oven: 50°C; flow: 0.40 mL/min; UV-detection: 208 - 400 nm.

MCW_SQ-HSST3-Long: Instrument: Waters ACQUITY SQD UPIC System; column: Waters Acquity UPLC HSS T3 1.8 μ 50 x 1 mm; eluent A: 1 I water + 0.25 ml 99% formic acid, eluent B: 1 I acetonitrile + 0.25 ml 99% formic acid; gradient: 0.0 min 95% A→ 6.0 min 5% A→ 7.5 min 5% A oven: 50°C; flow: 0.35 mL/min; UV-detection: 210 - 400 nm.

EXAMPLE 1 :

Starting Compound (Formula (5)):

(3R,4S,5S)-tert-butyl 4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido) butanamido)-3-methoxy-5-methylheptanoate

Starting compound 1 can be prepared in various ways according to published methods; see, for example, International Publication No. WO/2007/008848 to Doronina et al.

Preparation of Intermediate 1 (Formula (1)):

Preparation of (6R,7S, 10S, 13S)-7-((S)-sec-butyl)-10, 13-diisopropyl-6-methoxy-2,2,8, 14- tetramethyl-4,9, 12-trioxo-3-oxa-8, 11, 14-triazaoctadecan-l 8-oic acid

Intermediate 1

To a solution of 30.2 g (64.1 mmol) of (3R,4S,5S)-tert-butyl 4-((S)-N,3 -dimethyl - 2-((S)-3-methyl-2-(methylamino)butanamido) butanamido)-3-methoxy-5- methylheptanoate (Formula (5)) in 300 ml isopropanol, a solution of 77.5 g (160.2 mmol) 4-oxobutanoic acid (20% in water) was added. Subsequently, 67.9 g (320.4 mmol) of sodium triacetoxyborhydride was added in 5 portions over a period of 1 hour and was stirred for 1 hour at 20°C. The solvent is partly distilled off in vacuum, resulting in a residue of 225 g of the crude reaction mixture. For work-up, 300 ml of

dichloromethane and 300 ml of a 10% aqeous citric acid was added. After stirring for 10 min, the organic layer was washed with 300 ml water, 300 ml of a 5% sodium

hydrogencarbonate solution and again 300 ml of water. The organic layer was dried over magnesium sulfate and evaporated to dryness in vacuum to give 34.9 g (62.6 mmol, 97.7%) of (6R,7S,10S,13S)-7-((S)-sec-butyl)-10,13-diisopropyl-6-methoxy-2,2,8,14- tetramethyl-4,9, 12-trioxo-3-oxa-8, l l,14-triazaoctadecan-18-oic acid as a colorless foam.

HPLC: R_t = 14.04 min; 96.0 area %. LC/MS (MHZ-QP-GOLD): R_t = 2.04 min, 100 area %, M/Z = 557.4

1H- MR (500 MHz, DMSO-i¾): δ [ppm] = 11.8 (br. s, 1H), 8.09 (d, 1H), 4.61 (s, 1H), 4.48 (t, 1H), 3.75 (m, 1H), 3.23 (s, 3H), 2.97 (s, 3H), 2.67 (d, 1H), 2.56 (m, 1H), 2.46 (m, 1H), 2.33 (m, 1H), 2.18 (m, 6H), 1.95-1.89 (m, 2H), 1.74 (m, 1H), 1.61(m, 2H), 1.41 (s, 8H), 1.27 (m, 2H), 0.90-0.86 (m, 13H), 0.73 (t, 3H), 0.69 (d, 3H). Preparation of Intermediate 2 (Formula (2)):

Preparation of (3R,4S,7S, 10S)-tert-butyl 4-((S)-sec-butyl)-23-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)-7, 10-diisopropyl-3-methoxy-5, l l-dimethyl-6,9,15, 18-tetraoxo- -pentaazatricosan- 1 -oate

Intermediate 2

Via EDC coupling:

To a solution of 34.9 g (62.6 mmol) of (6R,7S, 10S,13S)-7-((S)-sec-butyl)-10, 13- diisopropyl-6-methoxy-2,2,8, 14-tetramethyl-4,9, 12-trioxo-3-oxa-8, 11,14- triazaoctadecan-18-oic acid (Intermediate 1, Formula (1)) and 19.7 g (75.1 mmol) of 6- (2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanehydrazide in 700 ml of dichloromethane, 65.4 ml (375.4 mmol) of diisopropyl ethyl amine, 8.9 g (62.6 mmol) of ethyl

cyanoglyoxylate-2-oxime and 24.0 g (125.1 mmol) of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride was added. The combined mixture was stirred at 20°C for 3 hours. For work-up, the reaction mixture was washed with 700 ml of aqueous 0.1 M hydrochloric acid, 700 ml of aqueous 5% sodium hydrogencarbonate and 700 ml of water. The organic layer was dried over magnesium sulfate and the solvent was distilled off in vacuum. The residue was purified by flash chromatography using an eluent of dichloromethane/methanol 20: 1. (3R,4S,7S, 10S)-tert-butyl 4-((S)-sec-butyl)-23-(2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl)-7, 10-diisopropyl-3-methoxy-5, l l-dimethyl-6,9,15, 18- tetraoxo-5,8, l l,16, 17-pentaazatricosan-l-oate was obtained in a yield of 29.9 g (39.1 mmol, 63%) in a purity of 98.8 area % as measured by HPLC. The collected side fractions gave an additional 6.9 g (9.0 mmol, 14%) of Intermediate 2 in a purity of 93.2 area %.

Via acid chloride:

To a solution of 19.6 g (35.1 mmol) of Intermediate 1 in a mixture of 196 ml dichloromethane and 0.4 ml of DMF, 6.1 ml (70.3 mmol) of oxallyl chloride was added evenly over a time period of 15 minutes at 0°C. The combination was stirred for 3 hours at 20°C. The solvents were distilled off in vacuum and the residue was re-dissolved in dichloromethane and then the dichloromethane was again distilled off to dryness to yield 23.5 g of the intermediate acid chloride as a yellow foam. To a solution of 1 1.0 g (42.2 mmol) of 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanehydrazide in 150 ml dichloromethane and 14 ml pyridine, 43 mg (0.35 mmol) of 4-dimethylamino pyridine was added. The combination was cooled to 0-5°C and 23.5 g (max. 35.1 mmol) of the yellow foam intermediate acid chloride was added as a solution in 150 ml

dichloromethane over a time frame of 30 minutes. The final combination was stirred for 1 hour at 0-5°C and then quenched with 250 ml of a 0.1 M aqueous hydrochloric acid. The organic layer was washed with 250 ml of a 5% aqueous solution of sodium hydrogencarbonate followed by 250 ml of water. The organic layer was dried over magnesium sulfate and the solvent was distilled off in vacuum. The residue was purified by flash chromatography using an isocratic eluent of dichloromethane/methanol 20: 1.

Several fractions of (3R,4S,7S, 10S)-tert-butyl 4-((S)-sec-butyl)-23-(2,5-dioxo-

2,5-dihydro-lH-pyrrol-l-yl)-7, 10-diisopropyl-3-methoxy-5, l l-dimethyl-6,9, 15, 18- tetraoxo-5,8, 1 1, 16, 17-pentaazatricosan-l-oate were obtained: 14.6 g (54% yield, HPLC: 98.4 area %); 3.9 g (15% yield, HPLC: 96.3 area %) 1.6 g (6% yield, HPLC: 95.0 area %). A total yield of 20.1 g (75%) were obtained.

Mass Spectroscopy (ESI(+) Orbitrap): [M+H]⁺ = 765.51 (m/z calc. for C₃9H₆₉N₆09 = 765.51)

1H MR (500 MHz, OMSO-d₆) δ ppm = 9.61 (m, 2H), 8.08 (d, 1H), 7.00 (s, 2H), 4.61 (m, 1H), 4.48 (t, 1H), 3.76 (m, 1H), 3.37 (t, 2H), 3.24(s, 3H), 2.97(bs, 3H), 2.68 (d, 1H), 2.56 (d, 1H), 2.47 (s, 1H), 2.32 (m, 1H), 2.19 (s, 3H), 2.17 (m, 1H), 2.08 (t, 4H), 1.93 (m, 2H), 1.75 (m, 1H), 1.61 (m, 2H), 1.49 (m, 4H), 1.40 (s, 9H), 1.28 (m, 1H), 1.22 (m, 2H), 0.89 (m, 13H), 0.73 (t, 3H), 0.69 (d, 3H). Preparation of Intermediate 3 (Formula (3)):

Preparation of (3R,4S,7S, 10S)-4-((S)-sec-butyl)-23-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)-7, 10-dii sopropyl-3 -methoxy-5, 1 1 -dimethyl-6,9, 15, 18-tetraoxo-5, 8, 1 1, 16, 17- entaazatricosan-l-oic acid

6

Intermediate 3

To a solution of 10.0 g (13.1 mmol) of (3R,4S,7S, 10S)-tert-butyl 4-((S)-sec- butyl)-23-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-7, 10-diisopropyl-3-methoxy-5, l l- dimethyl-6,9, 15, 18-tetraoxo-5,8, l l, 16, 17-pentaazatricosan-l-oate (Intermediate 2) in 200 ml of dichloromethane, 67 ml of trifluoro acetic acid was added. The combination was stirred for 3 hours at 20°C and then the solvent was distilled off in vacuum. An additional 200 ml of dichloromethane was added and again distilled off in vacuum. 20.4 g (max. 13.1 mmol) of (3R,4S,7S, 10S)-4-((S)-sec-butyl)-23-(2,5-dioxo-2,5-dihydro-lH- pyrrol-l-yl)-7, 10-diisopropyl-3-methoxy-5, l l-dimethyl-6,9, 15, 18-tetraoxo-5,8, l l, 16, 17- pentaazatricosan-l-oic acid was formed.

HPLC: R_t = 1 1.7 min; 97.5 area %.

LC/MS (method MCW_SQ-HSST3-Long): Rt = 1.73 min, 100 area %, M/Z = 708 1H MR (500 MHz, OMSO-d₆) δ ppm = 10.87 (bs, 1H), 9.30 (bs, 1H), 8.29 (bs, 1H), 8.20 (d, 1H), 7.66 (d, 1H), 7.52 (bs, 1H), 7.32 (d, 1H), 7.20 (d, 1H), 7.07 (s, 1H), 7.05 (t, 1H), 6.97 (t, 1H), 4.55 (m, 1H), 3.42 (m, 1H), 3.13 (s, 3H), 3.08 (m, 2H), 2.93 (m, 3H), 2.48 (m, 1H), 1.71 (m, 1H), 1.52 (m, 2H), 1.44 (m, 1H), 1.04 (d, 3H). Preparation of Intermediate 4:

Intermediate 4 To a solution of 30.0 g (64.0 mmol) of ((2R,3R)-3-((S)-l-

(tertbutoxycarbonyl)pyrrolidin-2-yl)-3 -Methoxy-2-Methylpropanoic acid (N-Boc- dolaproine) in 600 mL of ethyl acetate was added 15.3 g (64.0 mmol) H-Trp- H₂*HC1, 33.4 mL (192.0 mmol) diisopropyl ethyl amine (DIPEA), and 30.8 g (96.0 mmol) N,N,N',N'-Tetramethyl-0-(benzotriazol-l-yl)uronium tetrafluorob orate (TBTU) and it was stirred at 20°C for 16 hours. Following this reaction, the product was washed twice each with 600 mL of a 0.1 M aqueous hydrochloric acid, with 600 mL of an aqueous 10% solution of potassium carbonate, and with 600 mL of water. The organic layer was dried over magnesium sulfate and the solvent was distilled off in vacuo to give 28.6 g of the crude product as a foam. The resulting product was dissolved it 57 mL of a 20: 1 mixture of dichloromethane/methanol and purified by flash chromatography using an isocratic 20: 1 mixture of dichloromethane/methanol as eluate. Intermediate 4 was obtained as a colorless foam (23.8 g, 50.4 mmol, 78.7% yield).

HPLC (method a): R_t = 14.3 min; 98.4 area%.

LC/MS (method MCW SQ-HSST3): Rt = 0.89 min, 100 area%, M/Z = 472.27

1H- MR (500 MHz, OMSO-d₆): δ [ppm] = 10.77 (s, 1H), 7.84 (m, 1H), 7.63 (m, 1H), 7.39 (d, 1H), 7.12 (m, 1H), 7.03 (m, 2H), 6.98 (7, 1H), 4.51 (m, 1H), 3.77-3.41 (m, 1H), 3.29 (m, 2H), 3.21 (s, 3H), 3.29 (m, 1H), 3.1 1-2.95 (m, 3H), 2.28 (m, 1H), 1.65 (m, 2H), 1.42-1-29 (m, 1 1H), 1.02 (d, 3H). Preparation of Intermediate 5:

Intermediate 5 To a solution of 46.3 g (98.0 mmol) of Intermediate 4 in 463 mL of acetonitrile were added seed crystals of product 9 and 112.5 mL (489.9 mmol) of a 4 M solution of HCl in dioxane was dosed over 20 min at 20°C. It was stirred for 1.5 hrs at 20°C and then filtered. The product was rinsed with 80 mL acetonitrile twice and then dried in vacuo at 40°C over a period of 16 hrs. The crude crystals (43.9 g) were resupended in 439 mL of acetonitrile then 4.4 mL of water were added and the solution was stirred for 16 hrs at 20°C. The final solution was filtered, washed with 80 mL of acetonitrile and dried in vacuo at 40°C over a period of 64 hrs to yield 34.5 g (77.5 mmol, 79.1 %) of Intermediate 5 as dihydrochloride salt. HPLC (method a): R_t = 6.8 min; 99.1 area%.

MS (ESI⁺): [M+H]⁺ = 373.0 (m/z calc. for C₂oH₂₉N₄0₃ = 373.0)

1H MR (500 MHz, DMSO-i¾) δ ppm = 10.87 (bs, 1H), 9.30 (bs, 1H) 8.29 (bs, 1H) 8.20 (d, 1H) 7.66 (d, 1H), 7.52 (bs, 1H), 7.32 (d, 1H), 7.20 (d, 1H), 7.07 (s, 1H), 7.05 (t, 1H), 6.97 (t, 1H), 4.55 (m, 1H), 3.42 (m, 1H), 3.13 (s, 3H), 3.08 (m, 2H), 2.93 (m, 3H), 2.48 (m, 1H), 1.71 (m, 1H), 1.52 (m, 2H), 1.44 (m, 1H), 1.04 (d, 3H).

Preparation of final toxophore:

Synthesis of (S)-N-((3R,4S,5S)-l-((S)-2-((lR,2R)-3-(((S)-l-amino-3-(lH-indol-

3 -yl)- 1 -oxopropan-2-yl)amino)- 1 -methoxy-2-methyl-3 -oxopropyl)pyrrolidin- 1 -yl)-3 - methoxy-5-methyl-l-oxoheptan-4-yl)-2-((S)-2-((4-(2-(6-(2,5-dioxo-2,5-dihydro-lH- pyrrol-l-yl)hexanoyl)hydrazinyl)-4-oxobutyl)(m ethyl) amino)-3-methylbutanamido)-N,3- dimethylbutanamide

known.' intermediate fS

In WQ%}11»434S5 3AV 1 Bseae. io

CAS: imm~37-9

To a solution of 8.1 g (max. 5.2 mmol) of (3R,4S,7S, 10S)-4-((S)-sec-butyl)-23- (2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-7,10-diisopropyl-3-methoxy-5,l l-dimethyl- 6,9,15, 18-tetraoxo-5, 8, 11,16, 17-pentaazatricosan-l-oic acid (Intermediate 3) and 2.8 g (6.3 mmol) of (2R,3R)-N-((S)-l-amino-3-(lH-indol-3-yl)-l-oxopropan-2-yl)-3-methoxy- 2-methyl-3-((S)-pyrrolidin-2-yl)propanamide in 81 ml of dichloromethane, 10.6 g (104.4 mmol) of N-methyl morpholine and 5.0 g (15.7 mmol) of Ν,Ν,Ν',Ν'-tetramethyl-O- (benzotriazol-l-yl)uronium tetrafluorob orate (TBTU) was added at 0°C. The

combination was stirred for 2 hours at 20°C.

For work-up, the reaction mixture was washed twice with each of 80 ml of a 0.1

M aqueous hydrochloric acid, followed by a rinse with 80 ml of water. The organic layer was dried over sodium sulfate and the solvent was distilled off in vacuum to give 5.9 g of crude (S)-N-((3R,4S, 5 S)- 1 -((S)-2-(( lR,2R)-3 -(((S)- 1 -amino-3 -( lH-indol-3 -yl)- 1 - oxopropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-3-methoxy-5- methyl-l-oxoheptan-4-yl)-2-((S)-2-((4-(2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanoyl)hydrazinyl)-4-oxobutyl)(methyl) amino)-3-methylbutanamido)-N,3- dimethylbutanamide.

The crude material was dissolved in a mixture of 118 ml acetonitrile/water (3 : 1) + 0.1% trifluoro acetic acid, and purified by preparative C18-HPLC using a gradient of acetonitrile/water containing 0.1% trifluoro acetic acid.

From the product fractions, acetonitrile was distilled off in vacuum and the pH was set to 6.1 using a saturated aqueous sodium hydrogencarbonale solution. The aqueous layer was extracted with 375 ml dichloromethane. The layers were separated and the solvent was distilled off in vacuum to give 4.2 g (3.9 mmol, 75% over two steps) of purified (S)-N-((3R,4S,5S)-l-((S)-2-((lR,2R)-3-(((S)-l-amino-3-(lH-indol-3-yl)-l- oxopropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-3-methoxy-5- methyl-l-oxoheptan-4-yl)-2-((S)-2-((4-(2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanoyl)hydrazinyl)-4-oxobutyl)(methyl) amino)-3-methylbutanamido)-N,3- dimethylbutanamide.

HPLC: R_t = 13.2 min, 99.5 area %.

MS (ESI⁺): [M + H]⁺ = 1063.7 (m/z calc. for C₅5H₈₇NioOii = 1063.

1H MR (500 MHz, DMSO-i¾, rotational isomers) δ ppm = 10.76-10.71 (s, 1H), 9.61 (m, 2H), 8.04-7.84 (m, 2H), 7.59 (m, 1H), 7.45-7.40 (bs, 1H), 7.28 (m, 1H), 7.16-6.90 (m, 4H), 6.98 (s, 2H), 4.73-4.43 (m, 3H), 3.95 (m, 1H), 3.80-2.88 (m, 6H), 3.36 (t, 2H), 3.22-3.19 (s, 3H), 3.16 (s, 3H), 3.03-2.98 (s, 3H), 2.68 (m, 1H), 2.55-2.13 (m, 5H), 2.20- 2.19 (s, 3H), 2.07 (t, 4H), 1.92 (m, 2H), 1.85-1.11 (m, 13H), 1.05; 1.00(d, 3H), 0.95- 0.83(14H), 0.78-0.65 (m, 6H).

Preparation of antibody-drug conjugate:

Synthesis of (S)-N-((3R,4S,5S)-l-((S)-2-((lR,2R)-3-(((S)-l-amino-3-(lH-indol-

3 -yl)- 1 -oxopropan-2-yl)amino)- 1 -methoxy-2-methyl-3 -oxopropyl)pyrrolidin- 1 -yl)-3 - methoxy-5-methyl-l-oxoheptan-4-yl)-2-((S)-2-((4-(2-(6-(2,5-dioxo-2,5-dihydro-lH- pyrrol-l-yl)hexanoyl)hydrazinyl)-4-oxobutyl)(m ethyl) amino)-3-methylbutanamido)-N,3- dimethylbutanamide conjugated to an anti-C4.4a monoclonal antibody

To a solution of a corresponding monoclonal 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 (S)-N-((3R,4S,5S)-l-((S)-2-((lR,2R)-3-(((S)-l-amino-3-(lH-indol-3-yl)- 1 -oxopropan-2-yl)amino)- 1 -methoxy-2-methyl-3 -oxopropyl)pyrrolidin- 1 -yl)-3 -methoxy- 5-methyl-l-oxoheptan-4-yl)-2-((S)-2-((4-(2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl) hexanoyl)hydrazinyl)-4-oxobutyl)(methyl) amino)-3-methylbutanamido)-N,3- dimethylbutanamide (compound formed from the synthesis of Example 1) 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 procedure of Example 1 can be summarized by the following scheme:

Sc

Step (c)

Example 2 - Synthesis of brentuximab vedotin with a monomethylauristatin E (MMAE) drug-linker. Synthesis of the compound of Example 2 can be made using similar processes as in Example 1, as shown in the following scheme: Sche

DCM, Oxyma, EDC, DIPEA water quench, aq. work-up flash chromatography (DCM/MeOH)

Step (c)

DCM, TFA

evaporation, crude to next step

aq. work-up

Step (e)

TCEP protocol

Example 3 - Synthesis of maleimidocaproyl-monomethylauristatin F (MMAF) drug- linker conjugated to an anti-C4.4a monoclonal antibody. Synthesis of the compound of Example 3 can be made using similar processes as in Example 1, as shown in the following scheme:

Scheme



Claims

Claims:

1 A process for preparing the compounds of the general Formula (4):

Formula (4) wherein

Ri is hydrogen or C₁-C₄ alkyl;

R₂ is C₁-C4 alkyl, benzyl, or lH-indol-3-ylmethyl;

wherein

R₃ is hydrogen or C₁-C4 alkyl;

R₄ is phenyl;

R₅ is hydrogen or C₁-C4 alkyl;

L¹ is R⁶-C(=0)-R⁷;

where

R⁶ is Ci-C₆ alkyl;

R⁷ is a bond or H- H;

L² is a bond or a group of the formula

where n is 1-4;

comprising the steps of:

(a) reacting a compound of Formula (5):

Formula (5) with a grou of the formula LG²-L²-OH to give a compound of Formula (1):

Formula (1) where LG² is hydrogen or hydroxyl;

(b) further reacting the compound of Formula (1) with a compound of Formula (6):

Formula (6) where LG¹ is hydrogen or hydroxyl, wherein, if LG² is hydrogen then LG¹ is hydroxyl and if LG² is hydroxyl then LG¹ is hydrogen;

to give mpound of Formula (2):

Formula (2) (c) removing the -tBu protecting group from Formula (2) to form a compound of Formul

Formula (3)

(d) reacting the compound of Formula (3) with a compound having the general Formula (7):

Formula (7)

2. The process of claim 1, wherein

R₁ is hydrogen;

R₂ is lH-indol-3-ylmethyl;

T is -C(=0)-NR₃ R₅, wherein R₃ and R₅ are hydrogen;

L¹ is R⁶- =0)-R⁷, wherein R⁶ is a C₅ alkyl and R⁷ is H- H; and

L² is

, wherein n=3.

The process of claim 1, wherein

Ri is hydrogen;

R₂ is a Ci alkyl; T is -CH(OR₃)-R₄, wherein R₃ is hydrogen and R₄ is phenyl;

L¹ is R⁶-C(=0)-R⁷, wherein R⁶ is a C₅ alkyl and R⁷ is a bond; and

L² is

4. The process of claim 1, wherein

Ri is hydrogen;

R₂ is benzyl;

T is -C(=0)-OR₃, wherein R₃ is hydrogen;

L¹ is R⁶-C(=0)-R⁷, wherein R⁶ is a C5 alkyl and R⁷ is a bond; and

L² is a bond.

5. The process of any one of claims 1-4, further comprising: the step of conjugating the compound of Formula (4) with a monoclonal antibody that binds C4.4a to form a mpound according to the general Formula (8):