Antibody-drug conjugates with cleavable linkers

Abstract

The present invention relates to prodrugs comprising a linker containing a five- or six-membered cyclic acetal and an adjacent specific cleavage site, as well as precursor compounds for the synthesis of said prodrugs. In one aspect, the present invention relates to antibody-targeted amatoxin conjugates comprising the linker, methods for their synthesis, and uses of said antibody-targeted amatoxin conjugates. In a further aspect, the present invention relates to pharmaceutical compositions comprising said conjugates and uses of said conjugates or compositions for therapeutic purposes, particularly tumor therapy and oncology.

Description

[Technical field]

[0001] The present invention relates to prodrugs comprising a linker comprising a 5- or 6-membered cyclic acetal and an adjacent specific cleavage site, as well as precursor compounds for the synthesis of said prodrugs.In one embodiment, the present invention relates to antibody-targeted amatoxin conjugates comprising said linkers, methods for their synthesis, and the use of said antibody-targeted amatoxin conjugates.In a further embodiment, the present invention relates to pharmaceutical compositions comprising said conjugates, and the use of said conjugates or compositions for therapeutic purposes, in particular for tumor therapy and oncology. [Background technology]

[0002] Prodrugs are pharmacologically inactive drugs that must be converted to their active form by biochemical or chemical reactions such as hydrolysis or phosphorylation. The idea of ​​prodrugs in cancer therapy is to reduce unintended side effects by designing compounds that interact with specific targets. These prodrugs contain a target-binding moiety that is linked to a therapeutic or toxic moiety, often via a linker moiety.

[0003] Prodrugs containing acid-labile moieties for the release of therapeutic or toxic moieties are often used in pharmaceutical research and applications. In the field of antibody-drug conjugates (ADCs), acid-labile linkers allow the release of toxic payloads after targeting of the construct to tumor cells by target-specific antibodies. The concept involves receptor-mediated endocytosis (uptake into cells) of the ADC and subsequent intracellular trafficking to lysosomes. When the ADC binds to tumor antigens on its surface, it is taken up into endosomes, where it subsequently matures and fuses with lysosomes. In the lysosomes, the drug is released via cleavage of the linker by specific proteases such as cathepsin B, or degradation of the ADC. Lysosomes are characterized by an acidic pH of 4–5 (Chalouni and Doll, 2018), which is considered to be sufficiently acidic for hydrolysis of acid-labile bonds. Acid-labile linkers such as acyl-hydrazones have been successfully used in ADCs that have received marketing approval (Chalouni and Doll, 2018).

[0004] Other acid-labile groups, such as acetals, have been considered as components of linkers for ADCs. Molecules containing acyclic acetals based on aliphatic aldehydes or ketones have been shown to be cleavable at pH 5, but cyclic acetals of aliphatic carbonyl compounds have not yet been studied in this regard; these structures may have too high stability and release by the acidic intracellular environment seems questionable.

[0005] WO2014 / 135282 discloses methods for the preparation of amatoxins containing cyclic carbonates and cyclic acetals with improved properties for use as ADCs, but does not teach compounds with a controllable release mechanism.

[0006] Gillies et al. (2004) described five- or six-membered cyclic acetals based on benzaldehyde derivatives included in low molecular weight prodrugs. Besides aliphatic acetals, the introduction of aromatic substituents at the acetal carbon atom allows tuning of the acidic stability by further substituents. Hammett's rule allows the prediction of the reactivity of benzyl acetals related to the substitution pattern of the aromatic ring. By following this principle, it was possible to synthesize cyclic acetals with half-lives in the range of 17.6 hours at pH 5.0 and several weeks at pH 7.4.

[0007] WO2015 / 152182 (NOF Corporation) describes hydrophilic polymer derivatives with cyclic benzylidene acetal linkers, whose hydrolysis rate at the pH of the weakly acidic environment in vivo can be controlled through the position and nature of the substituents on the benzyl structure; in particular, possible acid-labile linkers for polymer-drug conjugates based on 5- or 6-membered cyclic acetals derived from benzaldehyde derivatives are disclosed. This document does not disclose any specific examples of cytotoxic payloads and biological data. Furthermore, the teachings of WO2015 / 152182 require additional moieties for the covalent attachment of the cyclic acetal to the drug compound. These linker structures do not participate in acid cleavage and remain attached to the released payload, possibly negatively affecting its pharmacodynamic activity.

[0008] Because cleavage of the cyclic acetals disclosed in the prior art requires an acidic environment, it is essential that the constructs are taken up by cells and transported to lysosomes where only suitable acidic conditions are found. Thus, the cyclic acetals disclosed in the prior art are limited to targeting moieties that provide efficient uptake after receptor binding.

[0009] However, other potential target structures may remain on the cell surface upon binding of the target binding moiety and / or not be transported to late endosomes and lysosomes after uptake and therefore may not be amenable to this technique.

[0010] The combination of cleavable peptide structures and self-disintegrating spacers between the target-binding antibody and the drug of ADCs has been described as applicable to payloads bearing primary or secondary amines. Jeffrey et al. (2005) and WO2016 / 142049 disclose para-aminobenzyl ether linkers that can be applied to phenol moieties. Carboxylic acids of payloads can be addressed by analogous para-aminobenzyl amides (WO2017 / 149077).

[0011] Another type of related autoimmune moiety frequently used for small molecule prodrugs is disclosed in Tranoy-Opalinski et al. (2014), where the specific side chain contains a β-glucuronide, starting with a terminal phenol moiety and undergoing spontaneous fragmentation after enzymatic cleavage by β-glucuronidase. This type of linker is also used in the ADC field and is described, for example, in Jeffrey et al. (2006). Summary of the Invention [Problem to be solved by the invention]

[0012] Therefore, in view of the prior art, one objective of the present invention is to provide a highly efficient release mechanism of a drug from a prodrug under controlled conditions that allows for a non-pause release of the drug, thus increasing the therapeutic efficiency and avoiding loss of drug activity due to chemical residues remaining on the released therapeutic drug.

[0013] One object of the present invention is to provide a target-binding moiety amatoxin conjugate that contains an efficient pH-independent release mechanism for the pause-free release of highly active amatoxins.

[0014] Another object of the present invention is to provide modified amatoxins for the synthesis of said conjugates.

[0015] It is yet another object of the present invention to provide improved target-binding moiety amatoxin conjugates for therapeutic use, in particular for tumor therapy and oncology. [Means for solving the problem]

[0016] These objects are achieved according to the invention by the methods and means according to the independent claims. The dependent claims relate to preferred embodiments. [Brief description of the drawings]

[0017] [Figure 1] Schematic diagram of the drug release mechanism in the linker system of the present invention. The triangle represents an enzymatically cleavable structure, preferably a peptide structure. A is an electron donating group that can be selected from, but is not limited to, O, NH or S. [Diagram 2] Cytotoxicity test results on SKBR-3 cells in BrdU assay after 96 hours of incubation. [Diagram 3] Cytotoxicity test results on NCI-N87 cells in BrdU assay after 96 h incubation. [Figure 4] Cytotoxicity test results for JIMT-1 cells in BrdU assay after 96 h incubation. [Diagram 5] Results of efficacy studies of acetal linker conjugates in an in vivo SKOV-3 xenograft tumor mouse model. [Figure 6] Results of an in vivo tolerability study of conjugates T-D265C-30.2669 and T-D265C-30.2684 in NOD / SCID mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Further details, features, characteristics and advantages of the subject matter of the present invention are disclosed in the dependent claims as well as in the following description of the respective figures and examples which show preferred embodiments of the invention in an exemplary manner, however these figures should not be understood as limiting the scope of the invention in any way.

[0019] Detailed Description of the Invention Before describing the present invention in detail, it is to be understood that the present invention is not limited to the specific component parts of the described devices or process steps of the described methods, as the devices and methods may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include singular and / or plural referents unless the context clearly dictates otherwise. Furthermore, when a parameter range delimited by numerical values ​​is given, the range is also to be understood to be inclusive of these limits.

[0020] Furthermore, it should be understood that the embodiments disclosed herein are not meant to be understood as separate embodiments unrelated to each other. Features discussed in one embodiment are intended to be disclosed in relation to other embodiments shown herein. In one case, if a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, a person skilled in the art will understand that this does not necessarily mean that said feature is not intended to be disclosed in said other embodiment. A person skilled in the art will understand that it is the gist of the present application to disclose said features for other embodiments as well, but that this has not been done for the purpose of clarity and to keep the specification to a manageable volume.

[0021] Furthermore, the contents of the prior art documents referred to in this specification are incorporated by reference, in particular those prior art documents that disclose standard or conventional methods, in which case the incorporation by reference has the primary purpose of providing a full enabling disclosure and avoiding lengthy repetition.

[0022] The present invention provides a solution to the limitations discussed above with respect to controllable, pause-free release of drugs from prodrugs, particularly antibody-drug conjugates (ADCs).

[0023] The present invention relates to a specific drug release mechanism for the improvement of prodrug systems applied in targeted therapy such as antibody-drug conjugates or peptide-drug conjugates. The prodrug according to the present invention comprises a benzylic 5- or 6-membered ring acetal linked to an electron donating group and a side chain containing and / or displaying a specific cleavage site. Cleavage of the cleavage site triggers the assisted release of the acetal, resulting in the liberation of the uninhibited drug (Figure 1).

[0024] Various cytotoxic drugs, such as amatoxin, aphidicolin, apotolidin, calicheamicin, digitoxin, digoxin, etoposide, glucopyricidine A, hypothemycin, isatropolone A, lactacystin, muscotoxin A, pancratistatin, phalloidin, fenpanstatin, phytosphingosine, proscillaridin A, pseurotin A, rebeccamycin, sinefungin, G-strophanthin, swainsonine, turbostatin 1-4, consist of 1,2- and 1,3-diols that can be used to generate cyclic acetals, at least one of which is restored when the drug is released from the compounds of the invention.

[0025] The present invention relates to compounds containing benzylic cyclic acetals with enhanced hydrolysis following cleavage of specific side chains resulting in moieties at the 1-2 (ortho-) or 1-4 (para-) positions of the acetal carbons with strong positive mesomeric effect, and methods for their introduction. Enzymatic cleavage of side chains containing and / or representing specific cleavage sites (e.g., peptides) releases electron donating groups (e.g., 2- or 4-amino groups) with strong electron donating properties, which results in spontaneous fragmentation of the benzylidene linker and diol via an ortho- or para-quinone intermediate, liberating the free payload.

[0026] The enzymatic cleavage is carried out by specific proteases, such as cathepsins, preferably cathepsin B, elastases or matrix metalloproteases. In some embodiments, the proteases are tumor-specific enzymes in the extracellular matrix, such as matrix metalloproteases or β-glucuronidase.

[0027] Surprisingly, the inventors have found that the combination of a cleavable linker element and a self-immolating moiety can be successfully applied to cyclic acetals in prodrugs, particularly amatoxin containing ADCs. The compounds and methods of the present invention allow for efficient, controllable, pH-independent release of drugs from prodrugs, particularly amatoxin-containing ADCs, at the cyclic acetal, in a manner that does not include residual or structural elements remaining from spacers, linkers or target-binding moieties that may impose the risk of reducing drug activity and therapeutic efficiency of the released drug moiety.

[0028] According to one aspect, the present invention relates to a compound of formula I or II which comprises a cytotoxic drug moiety.

[0029] [ka]

[0030] During the ceremony D is a cytotoxic drug moiety that includes at least one moiety that upon cleavage from said compound results in a 1,2- or 1,3-diol; Z is CH 2 , C.H. 2 -CH 2 or CHR-CHR, where R is independently H or an alkyl group optionally substituted with a heteroatom; R1 is H or C 1 -C 6 is an alkyl group; A is an electron donating group; E is the cleavage site; R2 is independently H or an electron withdrawing or donating group.

[0031] Cytotoxic drugs according to the present invention may be selected from, for example, but are not limited to, amatoxin, aphidicolin, apotolidin, calicheamicin, digitoxin, digoxin, etoposide, glucopyricidine A, hypothemycin, isatropolone A, lactacystin, muscotoxin A, pancratistatin, phalloidin, fenpanstatin, phytosphingosine, proscillaridin A, pseurotin A, rebeccamycin, sinefungin, G-strophanthin, swainsonine, and turbostatin 1-4.

[0032] Amatoxins are cyclic peptides composed of eight amino acids. They can be isolated, for example, from Amanita phalloides mushrooms or prepared synthetically. Amatoxins specifically inhibit DNA-dependent RNA polymerase II in mammalian cells, thereby also inhibiting transcription and protein biosynthesis in affected cells. Inhibition of transcription in cells causes growth and proliferation arrest. Although not covalently bound, the complex between amanitin and RNA polymerase II is very tight (KD=3nM). Dissociation of amanitin from the enzyme is a very slow process, and therefore recovery of affected cells is unlikely. If transcription is inhibited for too long, the cell will undergo programmed cell death (apoptosis).

[0033] As used herein, the term "amatoxin" includes all cyclic peptides isolated from the genus Amanita and composed of eight amino acids as described in Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Bioc 1978 1978 Dec;5(185-260); all chemical derivatives thereof; all synthetic analogues thereof constructed from building blocks according to the primary structure of the natural compound (cyclic, eight amino acids), all synthetic or semi-synthetic analogues containing non-hydroxylated amino acids, and all synthetic or semi-synthetic analogues in which the thioether sulfoxide moiety is replaced by an atom different from sulfur, such as a sulfide, sulfone, or carbon atom, for example, as in the carba analogue of amanitin, in each case where such derivatives or analogues are functionally active by inhibiting mammalian RNA polymerase II.

[0034] As used herein, a "derivative" of a compound refers to a species that has a similar chemical structure to the compound, but contains at least one chemical group not present in the compound and / or lacks at least one chemical group present in the compound. The compound to which the derivative is compared is known as the "parent" compound. Typically, a "derivative" can be produced from the parent compound in one or more chemical reaction steps.

[0035] As used herein, an "analog" of a compound is structurally related to, but not identical to, the compound and exhibits at least one activity of the compound. The compound to which the analog is compared is known as the "parent" compound. Such activities include, but are not limited to, binding activity to another compound; inhibitory activity (e.g., enzyme inhibitory activity); toxic effects; and activating activity (e.g., enzyme activating activity). An analog need not exhibit such activity to the same extent as the parent compound. A compound is considered an analog within the context of this application if it exhibits an activity related to at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%) of the activity of the parent compound. Thus, "analog of amatoxin," as used herein, refers to a compound that is structurally related to any one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, and amanuric acid and exhibits at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%) of inhibitory activity against mammalian RNA polymerase II compared to at least one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, and amanuric acid. "Amatoxin analogs" suitable for use in the present invention may even exhibit greater inhibitory activity against mammalian RNA polymerase II than any one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, or amanuric acid. Inhibitory activity is measured as the concentration at which 50% inhibition occurs (IC 50 The inhibitory activity against mammalian RNA polymerase II can be indirectly measured by measuring the inhibitory activity against cell proliferation.

[0036] "Semi-synthetic analogues" refer to analogues obtained by chemical synthesis using compounds from natural sources (e.g., plant materials, bacterial cultures, fungal cultures or cell cultures) as starting materials. Typically, the "semi-synthetic analogues" of the present invention are synthesized starting from compounds isolated from mushrooms of the Amanitacea family. In contrast, "synthetic analogues" refer to analogues synthesized by so-called total synthesis from small (typically petrochemical) building blocks. Usually, this total synthesis is carried out without the aid of biological processes.

[0037] Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferred amatoxins have functional groups (e.g., carboxyl, amino, hydroxyl, thiol, or thiol capture groups) that can react with the linker molecules or target binding moieties defined above. Particularly suitable amatoxins for the complexes of the present invention are α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, and amanuric acid, as well as their salts, chemical derivatives, semisynthetic analogs, and synthetic analogs. Particularly preferred amatoxins for use in the present invention are α-amanitin, β-amanitin, and amaninamide.

[0038] According to one aspect of the invention, the amatoxin may be selected from the group consisting of α-amanitin, β-amanitin, amanin, amaninamide and analogues, derivatives and salts thereof.

[0039] As used herein, the term "electron donating group" refers to a substituent having a nitrogen, oxygen, sulfur, or lone pair of electrons that can be donated to an aromatic π-electron system such as amines (NH2, NHR, NR2), phenols (OH) and their conjugate bases O-, alkoxy groups (OR), phenyl esters (OCOR) or thiols, as well as alkyl groups that have a positive inductive effect.

[0040] The term "electron-withdrawing group" is intended to refer to substituents which exhibit a negative mesomeric effect on aromatic π-systems, such as halogen, nitro-, carbonyl-, cyano- and sulfonyl groups, or groups which have a negative inductive effect, such as trifluoromethyl or trialkylammonium groups.

[0041] According to one aspect of the invention, the electron donating group A is selected from O, NH and S.

[0042] As used herein, the term "cleavage site" refers to a moiety that is susceptible to specific cleavage at a defined location under particular conditions, such as a particular enzyme or reductive environment in a particular body or cellular compartment.

[0043] According to one embodiment of the invention, the cleavable site E is an enzymatically cleavable moiety comprising two or more amino acids. Preferably, said enzymatically cleavable moiety comprises a valine-alanine (Val-Ala), a valine-citrulline (Val-Cit), a valine-lysine (Val-Lys), a valine-arginine (Val-Arg) dipeptide, a phenylalanine-lysine-glycine-proline-leucine-glycine (Phe Lys Gly Pro Leu Gly) or an alanine-alanine-proline-valine (Ala Ala Pro Val) peptide, or a β-glucuronide or a β-galactoside.

[0044] According to one embodiment, the cleavage site is cleavable by at least one agent selected from the group consisting of cysteine ​​proteases, metalloproteases, serine proteases, threonine proteases, and aspartic proteases.

[0045] Cysteine ​​proteases, also known as thiol proteases, are proteases that share a common catalytic mechanism involving a nucleophilic cysteine ​​thiol in a catalytic triad or dyad.

[0046] Metalloproteases are proteases whose catalytic mechanism involves a metal. Most metalloproteases require zinc, but some use cobalt. The metal ion coordinates to the protein through three ligands. The ligands that coordinate the metal ion can vary by histidine, glutamic acid, aspartic acid, lysine, and arginine. The fourth coordination position is captured by a labile water molecule.

[0047] Serine proteases are enzymes that cleave peptide bonds in proteins, with serine acting as the nucleophilic amino acid at the active site of the enzyme. Serine proteases are broadly classified into two categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.

[0048] Threonine proteases are a family of proteolytic enzymes that contain a threonine (Thr) residue in their active site. The prototypic members of this class of enzymes are the catalytic subunits of the proteasome, but acyltransferases have convergently evolved the same active site geometry and mechanism. Aspartic proteases are catalytic type protease enzymes that use an activated water molecule bound to one or more aspartic acid residues for catalysis of their peptide substrates. In general, they have two highly conserved aspartates at the active site and are optimally active at acidic pH. Almost all known aspartyl proteases are inhibited by pepstatin.

[0049] In certain embodiments, the cleavable site is cleavable by at least one agent selected from the group consisting of cathepsin A or B, matrix metalloproteinase (MMP), elastase, β-glucuronidase, and β-galactosidase.

[0050] In another embodiment of the invention, cleavage site E is a disulfide bond and specific cleavage is effected by a reducing environment, for example an intracellular reducing environment.

[0051] In another embodiment, the present invention relates to a conjugate comprising a compound of the present invention and a TL moiety, wherein said TL moiety replaces at least one of the residues R2 in formula I or II, L is a linker, T is a target binding moiety.

[0052] As used herein, the term "linker" refers to a bifunctional group capable of covalently attaching a target-binding moiety to a compound of the invention, including a cytotoxic drug moiety.

[0053] The linker L according to the present invention comprises or consists of an alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, or heteroaralkylene group, containing 1 to 4 heteroatoms selected from N, O, and S, said linker being optionally substituted. The linker may also be only a covalent bond.

[0054] In one embodiment of the invention, the linker L comprises a moiety selected from at least one of the following moieties: disulfide, ether, thioether, amine, ester, carboxamide, urethane, and urea moieties.

[0055] A "linker" in the context of this application refers to a molecule that increases the distance between the two components, for example to reduce steric interference between the target binding moiety and the compound according to the invention (e.g., an amatoxin or an amatoxin derivative) (which would otherwise reduce the ability of the amatoxin to interact with RNA polymerase II). The linker may serve another purpose, since it may facilitate the release of the amatoxin specifically in the cells targeted by the target binding moiety. The linker, and preferably the bond between the linker and the compound of the invention, preferably an amatoxin, on one side, and the bond between the linker and the antibody on the other side, are stable under physiological conditions outside the cell, e.g., blood, but can be cleaved inside the cell, in particular inside the target cell, e.g., a cancer cell or an immune cell. To provide this selective stability, the linker may contain a functional group that is preferably pH-sensitive or protease-sensitive. Alternatively, the bond connecting the linker to the target binding moiety may provide the selective stability. Preferably, the linker has a length of at least 1, preferably 1 to 30 atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 atoms), where one side of the linker is reacted with the amatoxin and the other side is reacted with the target binding moiety. In the context of the present invention, the linker is preferably an optionally substituted C1-30-alkyl, C1-30-heteroalkyl, C2-30-alkenyl, C2-30-heteroalkenyl, C2-30-alkynyl, C2-30-heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl group. The linker may contain one or more structural elements such as amides, esters, ethers, thioethers, disulfides, carbohydrate moieties, etc. The linker may also contain a combination of two or more of these structural elements. Each of these structural elements may be present in the linker one or more times, for example, two, three, four, five, or six times. In some embodiments, the linker may contain a disulfide bond.It is understood that the linker must be attached to the amatoxin and the target binding moiety either in a single step or in two or more subsequent steps. For that purpose, the linker preferably has two groups at the proximal and distal ends that can either (i) form a covalent bond to a group, preferably an activated group on the amatoxin or the target binding peptide, or (ii) be activated to form a covalent bond with a group on the amatoxin. Thus, when a linker is present, it is preferred that chemical groups are at the distal and proximal ends of the linker, which are the result of coupling reactions such as, for example, esters, ethers, urethanes, peptide bonds, etc.

[0056] As used herein, the term "target binding moiety" refers to any molecule or portion of a molecule that can specifically bind to a target molecule or target epitope. Target binding moieties suitable for use in the present invention typically have a molecular weight of forty thousand Da (40 kDa) or greater.

[0057] As used herein, the terms "target molecule" and "target epitope" refer to the antigen and epitope of the antigen that are specifically bound by the target binding moiety, respectively.Preferably, the target molecule is a tumor-associated antigen, in particular an antigen or epitope that is present on the surface of one or more tumor cell types or tumor-associated cells at an increased concentration and / or different configuration compared to the surface of non-tumor cells.Preferably, said antigen or epitope is present on the surface of one or more tumor or tumor stromal cell types, but not on the surface of non-tumor cells.In another embodiment, said antigen or epitope is preferentially expressed on cells involved in autoimmune disease.In another embodiment, said antigen or epitope is preferentially expressed on cells involved in inflammatory disease.

[0058] In one aspect of the invention the target binding moiety T is selected from the group (i) an antibody or antigen-binding fragment thereof; (ii) an antibody-like protein, and (iii) Nucleic acid aptamers.

[0059] The term "antibody or antigen-binding fragment thereof" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules (i.e., molecules that contain an antigen-binding site that specifically binds to an antigen). Also included are immunoglobulin (Ig)-like proteins selected by techniques including, for example, phage display that specifically bind to a target molecule, such as the target protein Her-2 / neu or EpCAM. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule. Antibodies and antigen-binding fragments thereof suitable for use in the present invention include polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized (particularly CDR-grafted), deimmunized, or chimeric antibodies, single chain antibodies (e.g., scFv), Fab fragments, F(ab') 2 These include, but are not limited to, fragments, fragments produced by a Fab expression library, diabodies or tetrabodies (Poljak RJ, 1994), nanobodies, anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to an antibody of the invention), and epitope-binding fragments of any of the above.

[0060] In one aspect of the invention, the antibody or antigen-binding fragment thereof is selected from a diabody, a tetrabody, a nanobody, a chimeric antibody, a deimmunized antibody, a humanized antibody or a human antibody.

[0061] In one embodiment of the invention, the antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fd, Fv, single-chain Fv, disulfide-linked Fv (dsFv), and fragments comprising at least one VL and / or VH domain.

[0062] As used herein, the term "antibody-like protein" refers to a protein that is designed (e.g., by mutagenesis of Ig loops) to specifically bind to a target molecule. Typically, such antibody-like proteins contain at least one variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the antibody-like protein to a level comparable to that of an antibody. The length of the variable peptide loop typically consists of 10-20 amino acids. The scaffold protein can be any protein with good solubility properties. Preferably, the scaffold protein is a small globular protein. Antibody-like proteins include, but are not limited to, affibodies, anticalins, and designed ankyrin repeat proteins (Binz et al., 2005). Antibody-like proteins can be derived from large libraries of mutants, for example, by panning from large phage display libraries, and can be isolated similarly to conventional antibodies. Antibody-like binding proteins can also be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins.

[0063] As used herein, the term "nucleic acid aptamer" refers to a nucleic acid molecule engineered through repeated rounds of in vitro selection or SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to bind to a target molecule (Wang et al., 2019). Nucleic acid aptamers can be DNA or RNA molecules. Aptamers can include modifications, e.g., modified nucleotides such as 2'-fluorine substituted pyrimidines.

[0064] In one aspect, the invention relates to a conjugate of formula III or IV: [ka]

[0065] During the ceremony, X is S, SO, or SO 2 and; R1 is H or C 1 -C 6is an alkyl group; R2 is independently H or an electron withdrawing or donating group; R4 is H, OH, OC 1 -C 8 -Alkyl, NO 2 , N.H. 2 , F, Cl, Br, or SH; R5 is OH, NH 2 or NHOH; R6, R7 are the side chains of natural or unnatural amino acids; L is a linker, and T is a target binding moiety.

[0066] The conjugate of formula III or IV releases its payload (a cytotoxic drug) following peptide cleavage at the anilide site by a peptidase such as, but not limited to, a cathepsin, elastase, or matrix metalloprotease.

[0067] In another aspect, the invention relates to a conjugate of formula V: [ka]

[0068] During the ceremony, X is S, SO, or SO 2 and; Y is CH2 or CO; R1 is H or C 1 -C 6 is an alkyl group; R2 is independently H or an electron withdrawing or donating group; at least one R2 group is substituted on said TL moiety. R4 is H, OH, OC 1 -C 8 -Alkyl, NO 2 , N.H. 2 , F, Cl, Br, or SH; R5 is OH, NH 2 or NHOH; L is a linker, and T is a target binding moiety.

[0069] The conjugate of formula V releases its payload (the cytotoxic drug) following glycosidic cleavage by an enzyme such as, but not limited to, β-glucuronidase or β-galactosidase.

[0070] In another aspect, the invention relates to a conjugate of formula VI: [ka]

[0071] During the ceremony, X is S, SO, or SO 2 and; R1 is H or C 1 -C 6 is an alkyl group; R2 is independently H or an electron withdrawing or donating group, with the proviso that if R8 is not a TL moiety, then one R2 is substituted with said TL moiety; R4 is H, OH, OC 1 -C 8 -Alkyl, NO 2 , N.H. 2 , F, Cl, Br, or SH; R5 is OH, NH 2 or NHOH; R8 is a straight or branched chain alkyl group, or is a TL moiety if neither R2 is a TL moiety; L is a linker, and T is a target binding moiety.

[0072] The conjugate of formula VI releases its payload (the cytotoxic drug) following disulfide cleavage by a reducing environment, such as an intracellular reducing environment.

[0073] In another aspect, the invention relates to a conjugate of formula VII: [ka]

[0074] During the ceremony, X is S, SO, or SO 2 and; R1 is H or C 1 -C 6 is an alkyl group; R2 is independently H or an electron withdrawing or donating group, with the proviso that if R9 is not a TL moiety, then one R2 is substituted with said TL moiety; R4 is H, OH, OC 1 -C 8 -Alkyl, NO 2 , N.H. 2 , F, Cl, Br, or SH; R5 is OH, NH 2 or NHOH; R9 is H or a straight or branched chain alkyl group, or a TL moiety if neither R2 is a TL moiety; L is a linker, and T is a target binding moiety.

[0075] The conjugate of formula VII releases its payload (the cytotoxic drug) following enzymatic cleavage of the phosphate ester.

[0076] In another aspect, the invention relates to a conjugate of formula VIII. [ka]

[0077] In a further aspect, the invention relates to a conjugate of formula IX or X: [ka]

[0078] In another aspect, the present invention relates to a conjugate of the present invention for use as a medicament.

[0079] In another embodiment, the present invention relates to a method for synthesizing the conjugates of the present invention by reacting a 1,2- or 1,3-diol with dimethyl benzylidene acetal under acidic conditions in an aprotic solvent.

[0080] The present invention relates to a method for the synthesis of the conjugates of the invention by reacting amatoxins with dimethyl benzylidene acetal, preferably under acidic conditions in an aprotic solvent, said amatoxins being preferably selected from the group consisting of alpha-amanitin, beta-amanitin, amanine, amaninamide and their respective thioethers.

[0081] In a preferred embodiment, the process according to the invention relates to a process, wherein the aprotic solvent is DMF and / or the acid is trifluoroacetic acid.

[0082] As used herein, the term "under acidic conditions" is intended to refer to a pH of 5.0 or less. Particularly preferred methods are carried out at a pH of about 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, or 1.0.

[0083] Cyclic acetals can be formed by reacting aldehydes or ketones with diol compounds under acidic conditions. Typically, these reactions require elevated temperatures and removal of the water formed as a by-product by azeotropic distillation or water-binding agents such as calcium sulfate or molecular sieves. Such methods are often impractical when using sensitive and valuable starting materials. In such cases, it is advantageous to preactivate the less sensitive and less expensive components as acyl acetals. The formation of the cyclic acetal can then be achieved by a catalytic amount of acid, which is favored by the entropic effect in the formation of the cyclic from the non-cyclic compound.

[0084] For the cyclic acetal of the present invention, diols are usually more valuable and less stable compounds.Therefore, aromatic carbonyl groups are preferably converted to acyclic acetals with low boiling alcohols such as methanol, ethanol, propanol, butanol or trifluoroethanol in the presence of a water-binding agent.More preferably, the water-binding agent is an orthoester and the solvent is a low boiling alcohol.Preferred orthoesters are trimethyl orthoformate, triethyl orthoacetate, triethyl orthoacetate or tripropyl orthoformate.Most preferred is the use of trimethyl orthoformate.

[0085] The acid catalyst is preferably anhydrous acid such as concentrated sulfuric acid, hydrogen chloride in an organic solvent, e.g. HCl / EtOH, HCl / MeOH, HCl / 1,4-dioxane, 4-toluenesulfonic acid, trifluoroacetic acid, or a polymeric sulfonic acid such as a strong cation exchanger in the protonated form, e.g. Dowex® 50W X8, Amberlite® IR-120, etc. Most preferred is the use of hydrogen chloride in 1,4-dioxane and 4-toluenesulfonic acid.

[0086] The reaction of the acyclic acetal with the diol compound is preferably carried out in a high boiling aprotic solvent such as DMSO, DMF, DMA, NMP, 1,4-dioxane xylene or toluene. The use of DMF is preferred. The acid catalyst in this reaction can be selected from the list above. A low boiling acid such as HCl / 1,4-dioxane or trifluoroacetic acid is preferred. The use of trifluoroacetic acid is most preferred.

[0087] In another embodiment, the present invention relates to a conjugate of the present invention for use in the treatment of cancer in a patient, in particular the cancer is selected from the group consisting of breast cancer, pancreatic cancer, bile duct cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, gastric cancer, renal cancer, malignant melanoma, leukemia, and malignant lymphoma.

[0088] In yet another aspect, the present invention relates to a pharmaceutical composition comprising the conjugate of the present invention, optionally further comprising one or more pharma- ceutically acceptable diluents, carriers, excipients, fillers, binders, lubricants, disintegrants, adsorbents, and / or preservatives.

[0089] In certain embodiments, the pharmaceutical composition is used in the form of a systemically administered medicament. This includes parenteral agents, including injections and infusions, among others. Injections are formulated in the form of ampoules or as so-called ready-to-use injections, such as ready-to-use or disposable syringes, or alternatively as puncturable flasks for multiple withdrawals. The administration of injections can be in the form of subcutaneous (sc), intramuscular (im), intravenous (iv) or intradermal (ie) application. In particular, it is possible to prepare suitable injection formulations, respectively, as suspensions of crystals, solutions, nanoparticles or colloidal dispersions (e.g., hydrosols).

[0090] The particular combinations of elements and features in the detailed embodiments described above are merely illustrative, and interchangeability and substitution of these teachings with other teachings in the specification and patents / applications incorporated by reference are also expressly contemplated. As will be appreciated by those skilled in the art, variations, modifications, and other implementations of what is described herein may occur to those skilled in the art without departing from the spirit and scope of the invention as claimed. Thus, the foregoing description is merely illustrative and is not intended to be limiting. The scope of the invention is defined in the following claims and their equivalents. Moreover, reference signs used in the specification and claims do not limit the scope of the invention as described in the claims. EXAMPLES

[0091] While the present invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description are to be considered as illustrative or exemplary and not restrictive, and the present invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the techniques of the appended claims. In the claims, the word "comprises" does not exclude other elements or steps, and the indefinite articles "a" or "an" do not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be interpreted as limiting the scope.

[0092] Example 1: 4-Dimethoxymethyl-phenol (HDP 30.2628) [ka]

[0093] 4-Hydroxybenzaldehyde (1.221 g, 10 mmol) was dissolved in 30 ml of methanol, 10.9 ml (100 mmol) of trimethyl orthoformate and 250 μl (1 mmol) of hydrogen chloride in dioxane 4M were added and the solution was heated under reflux for 1 h. TLC-control (n-hexane / ethyl acetate 1:1) showed complete conversion of the aldehyde (Rf=0.36) to the dimethyl acetal (Rf=0.45). After rotary evaporation to 10 ml, the reaction was quenched with 50 ml of saturated sodium bicarbonate solution and the mixture was extracted with 50 ml of ethyl acetate. The organic layer was washed with 50 ml of brine and diluted with MgSO 4 After drying on a kettle and evaporation, 1.739 mg of 4-dimethoxymethyl-phenol was obtained as an oil. The product was homogenous by TLC and HPLC and was used in the next step without further purification.

[0094] Example 2: 3-Dimethoxymethyl-phenol (HDP 30.2629) [ka] The procedure of Example 1 was applied to 3-hydroxybenzaldehyde to give 3-dimethoxymethyl-phenol in quantitative yield.

[0095] Example 3: 4-Dimethoxymethyl-2-fluoro-phenol (HDP 30.2630) [ka] The procedure of Example 1 was applied to 1 g (7.14 mmol) of 3-fluoro-4-hydroxybenzaldehyde to give 4-dimethoxymethyl-2-fluoro-phenol in quantitative yield.

[0096] Example 4: 4-Bromo-3-dimethoxymethyl-phenol (HDP 30.2631) [ka] The procedure of Example 1 was applied to 2-bromo-3-hydroxybenzaldehyde to give 4-bromo-3-dimethoxymethyl-phenol in quantitative yield.

[0097] Example 5: 1-[11-Bromo-3,6,9-trioxaundecyloxy]-4-dimethoxy-methyl-benzene (HDP 30.2637) [ka]

[0098] The crude 4-dimethoxymethyl-phenol (1,739 g, max. 10 mmol) from Example 1 and 6.400 g (20 mmol) of 1,11-dibromo-3,6,6-trioxaundecane were dissolved in 40 ml of dry DMF, 3.910 g (12 mmol) of cesium carbonate were added and the mixture was heated at 50° C. for 1 h. The DMF was then evaporated and the residue was stirred in 50 ml of dichloromethane and then filtered. The filtrate was evaporated and purified on silica gel with a gradient of n-hexane to ethyl acetate to give 1.862 g (46%) of the product 1-[11-bromo-3,6,9-trioxaundecyloxy]-4-dimethoxy-methyl-benzene as an oil.

[0099] 1 H NMR (500 MHz, CDCl 3 ) d 7.39-7.32 (m, 2H), 6.94-6.87 (m, 2H), 5.35 (s, 1H), 4.17-4.11 (m, 2H), 3.91-3.84 (m, 2H), 3.80 (t, J = 6.3 Hz, 2H), 3.76-3.71 (m, 2H), 3.71-3.66 (m, 6H), 3.46 (t, J = 6.3 Hz, 2H), 3.31 (s, 6H). 13 C NMR (126 MHz, CDCl 3 ) d 158.85, 130.53, 127.88, 114.21, 102.99, 71.19, 70.84, 70.70, 70.64, 70.53, 69.73, 67.42, 52.59, 30.33. MS (ESI + ) Found: 429.17; Calculated [M+Na] + :429.09 (C 17 H 27 BrNaO 6 )

[0100] Example 6: 1-[11-Bromo-3,6,9-trioxaundecyloxy]-3-dimethoxy-methyl-benzene (HDP 30.2641) [ka]

[0101] The procedure of Example 5 was applied to the crude product of Example 2 to give 2.079 g (51%) of the title compound (HDP 30.2641) as an oil. 1 H NMR (500 MHz, CDCl 3 ) d 7.30-7.23 (m, 1H), 7.06-7.00 (m, 2H), 6.88 (ddd, J = 8.3, 2.5, 1.1 Hz, 1H), 5.35 (s, 1H), 4.17-4.12 (m, 2H), 3.88-3.84 (m, 2H), 3.81 (t, J = 6.3 Hz, 2H), 3.77-3.71 (m, 2H), 3.71-3.66 (m, 6H), 3.46 (t, J = 6.3 Hz, 2H), 3.32 (s, 6H). 13 C NMR (126 MHz, CDCl 3 ) d 158.92, 139.76, 129.32, 119.38, 115.01, 112.74, 103.06, 71.31, 70.95, 70.82, 70.76, 70.65, 69.87, 67.54, 52.84, 30.44. MS (ESI + ) Measured value: 375.17 / 377.17; Calculated value [MH-OMe] + :375.08 / 377.08 (C 16 H 24 Bro 5 )

[0102] Example 7: 1-(11-Bromo-3,6,6-trioxaundecyl)-4-dimethoxymethyl-2-fluoro-benzene (HDP 30.2642) [ka]

[0103] The procedure of Example 5 was applied to the crude product of Example 3 to give 2.032 g (67%) of the title compound (HDP 30.2642) as an oil. MS (ESI + ) Measured value: 393.17 / 395.17; Calculated value [MH-OMe] + :393.07 / 395.07 (C 16 H 23 BrFO 5 ) 1 H NMR (500 MHz, CDCl 3 ) d 7.18 (dd, J = 12.0, 2.1 Hz, 1H), 7.13 (ddt, J = 8.4, 1.9, 0.8 Hz, 1H), 6.97 (t, J = 8.4 Hz, 1H), 5.33 (s, 1H), 4.23-4.17 (m, 2H), 3.90-3.86 (m, 2H), 3.81 (t, J = 6.3 Hz, 2H), 3.76-3.72 (m, 2H), 3.68 (d, J = 9.6 Hz, 6H), 3.46 (t, J = 6.3 Hz, 2H), 3.30 (s, 6H). 13 C NMR (126 MHz, CDCl 3 ) d 152.60 (d, J = 246.1 Hz), 146.92 (d, J = 10.9 Hz), 131.90 (d, J = 5.7 Hz), 122.61 (d, J = 3.6 Hz), 114.93 (d, J = 4.0 Hz), 114.79, 102.20 (d, J = 1.6 Hz), 71.31, 71.06, 70.80, 70.74, 70.65, 69.71, 69.21, 52.67, 30.44.

[0104] Example 8: 1-Bromo-4-[11-bromo-3,6,9-trioxaundecyloxy]-2-dimethoxy-methyl-benzene (HDP 30.2643) [ka]

[0105] The procedure of Example 5 was applied to the crude product of Example 4 to give 2.757 g (57%) of the title compound (HDP 30.2643) as an oil. 1 H NMR (500 MHz, CDCl 3 ) d 7.42 (d, J = 8.7 Hz, 1H), 7.18 (d, J = 3.2 Hz, 1H), 6.79 (dd, J = 8.7, 3.1 Hz, 1H), 5.50 (s, 1H), 4.16-4.09 (m, 2H), 3.87-3.83 (m, 2H), 3.81 (t, J = 6.3 Hz, 2H), 3.74-3.71 (m, 2H), 3.70-3.66 (m, 6H), 3.47 (t, J = 6.3 Hz, 2H), 3.38 (s, 6H). 13 C NMR (126 MHz, CDCL3) d 158.22, 137.89, 133.57, 117.11, 114.31, 113.52, 102.99, 71.34, 70.99, 70.83, 70.77, 70.67, 69.77, 67.89, 54.08, 30.44. MS (ESI + ) Actual values: 502.00 / 504.00 / 506.00; Calculated value [M+NH 4 ] + :502.04 / 504.04 / 506.04 (C 17 H 30 Br 2 NO 6 ) Actual values: 453.08 / 455.08 / 457.08; Calculated value [MH-OMe] + : 452.99 / 454.99 / 456.99 (C 16 H 23 Br 2 O 5 )

[0106] Example 9: 1-[11-Fmoc-amino-3,6,9-trioxaundecyloxy]-4-dimethoxy-methyl-benzene (HDP 30.2640) [ka]

[0107] The product of Example 5, HDP 30.2637 (1.725 g, 4.24 mmol) was dissolved in 30 ml of dry DMF, 975 mg (12.7 mmol, 3 eq.) of sodium azide was added and the suspension was stirred overnight at ambient temperature. The solvent was then evaporated and the residue was taken up in 50 ml of tert-butyl methyl ether (MTBE) and washed with sodium bicarbonate and brine (50 ml each). The organic layer was diluted with MgSO 4 After drying at 40° C. and evaporation, a clear oil (1.435 g, 92% yield) containing the intermediate 1-[11-azido-3,6,9-trioxaundecyloxy]-4-dimethoxy-methyl-benzene was obtained. MS (ESI + ) Found: 392.25; Calculated [M+Na] + : 392.18(C 17 H 27 N 3 NaO 6 ) Measured value: 338.25; Calculated value [MH-OMe] + :338.17(C 16 H 24 N 3 O 5 )

[0108] The above azide intermediate was dissolved in THF and 2.038 mg (2 eq.) of triphenylphosphine was added followed by 2 ml of water. The mixture was stirred at room temperature overnight until gas evolution ceased. After 21 hours, the volatiles were evaporated and the remaining mixture of phosphine and intermediate 1-[11-amino-3,6,9-trioxaundecyloxy]-4-dimethoxy-methyl-benzene was dissolved in 20 ml of DMF.

[0109] 1.57 g (1.2 eq.) of Fmoc-N-hydroxysuccinimide ester and N-ethyldiisopropylamine (1320 μl; 2 eq.) were added. After stirring for 17 h at room temperature, a further 785 mg of Fmoc-OSu was added. After complete conversion of the intermediate amino, the solvent was evaporated and the residue was dissolved in 100 ml of dichloromethane, washed with water (2×100 ml) and brine (100 ml), dried (MgSO 4 ) and reduced to dryness. The crude product was purified on silica gel with a gradient of 0-100% MTBE in n-hexane to give 617 mg (28%) of the title compound as a sticky oil.

[0110] 1 H NMR (500 MHz, CDCl 3 ) d 7.75 (dt, J = 7.6, 0.9 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.39 (tt, J = 7.4, 0.9 Hz, 2H), 7.35-7.28 (m, 4H), 6.90-6.84 (m, 2H), 5.42 (t, J = 5.7 Hz, 1H), 5.33 (s, 1H), 4.40 (d, J = 6.9 Hz, 2H), 4.21 (t, J = 6.9 Hz, 1H), 4.08 (t, J = 4.9 Hz, 2H), 3.80 (t, J = 4.9 Hz, 2H), 3.72-3.58 (m, 8H), 3.55 (t, J = 5.1 Hz, 2H), 3.37 (q, J = 5.4 Hz, 2H), 3.29 (s, 6H). 13 C NMR (126 MHz, CDCl 3 ) d 158.77, 156.47, 143.97, 141.26, 127.84, 127.69, 127.60, 126.98, 125.02, 119.90, 114.14, 102.95, 70.79, 70.55, 70.30, 69.99, 69.85, 69.67, 67.32, 66.44, 52.55, 47.24, 40.90. MS (ESI + ) Found: 588.33; Calculated [M+Na] + :588.26 (C 32 H 39 NNaO 8 ) Measured value: 534.33; Calculated value [MH-OMe] + :534.25 (C 31 H 36 NO 7 )

[0111] Example 10: 1-[11-Fmoc-amino-3,6,9-trioxaundecyloxy]-3-dimethoxy-methyl-benzene (HDP 30.2650) [ka]

[0112] The procedure of example 9 was applied to 1.082 g of the crude product of example 6 to give 106 mg (7%) of the title compound (HDP 30.2650) as an oil. MS (ESI + ) Measured value: 588.33; Calculated value [M+Na] + :588.26 (C 32 H 39 NNaO 8 )

[0113] Example 11: 1-(11-Fmoc-amino-3,6,6-trioxaundecyl)-4-dimethoxymethyl-2-fluoro-benzene (HDP 30.2651) [ka]

[0114] The procedure of Example 9 was applied to 1.900 g of the crude product of Example 7 to give 112 mg (4%) of the title compound (HDP 30.2651) as an oil. MS (ESI + ) Found: 606.33; Calculated [M+Na]+ :606.25 (C 32 H 38 FNNaO 8 ) Measured value: 552.33; Calculated value [MH-OMe] + :552.24 (C 31 H 35 FNO 7 )

[0115] Example 12: 1-Bromo-4-[11-Fmoc-amino-3,6,9-trioxaundecyloxy]-2-dimethoxy-methyl-benzene (HDP 30.2652) [ka]

[0116] The procedure of example 9 was applied to 2.625 g of the crude product of example 8 to give 193 mg (6%) of the title compound (HDP 30.2652) as an oil. MS (ESI + ) Actual value: 666.17 / 668.17 Calculated value [M+Na] + :666.17 / 668.17 (C32H38BrNNaO8)

[0117] Example 13: Fmoc-Val-Ala-PAP-CHO (HDP 30.2623) [ka]

[0118] In a three-neck flask equipped with a thermometer, dropping funnel and rubber septum, 20 mL of dry dichloromethane was treated with (446 μL, 5.2 mmol) oxalyl chloride and the solution was cooled to −80° C. under an argon atmosphere. DMSO (739 μl, 10.4 mmol) was added dropwise via syringe through the rubber septum while maintaining the temperature below −70° C. After stirring for 15 min, Fmoc-Val-Ala-PAB-OH (HDP 30.1419, 2.062 g, 4.0 mmol) dissolved in 20 ml of dichloromethane was added dropwise via addition funnel over 30 min and the mixture was stirred for an additional 30 min.

[0119] Triethylamine (2.634 ml, 19.0 mmol) was then added via syringe and after 5 min the cooling bath was removed and the reaction was allowed to warm to room temperature. 25 ml of water and 25 ml of dichloromethane were then added and the layers were separated. The aqueous layer was extracted with 20 ml of dichloromethane and the combined organic layers were washed with 20 ml of 0.2 M citric acid, 3x20 ml of water and 20 ml of brine. Dry (MgSO 4 ), the solvent was evaporated and the crude product (965 mg) was purified on silica gel with a gradient of 0-20% ethyl acetate in dichloromethane to give 631 mg (31%) of the product as an amorphous solid.

[0120] 1 H NMR (500 MHz, D 6 -DMSO) d 10.36 (s, 1H), 9.89 (s, 1H), 8.22 (d, J = 6.8 Hz, 1H), 7.91-7.79 (m, 6H), 7.74 (t, J = 8.6 Hz, 2H), 7.45-7.29 (m, 5H), 4.47 (p, J = 7.0 Hz, 1H), 4.37-4.28 (m, 1H), 4.28-4.19 (m, 2H), 3.95 (dd, J = 8.9, 7.0 Hz, 1H), 2.02 (h, J = 6.7 Hz, 1H), 1.35 (d, J = 7.1 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H), 0.88 (d, J = 6.7 Hz, 3H). 13 C NMR (126 MHz, D 6-DMSO) d 191.35, 171.75, 171.03, 156.05, 144.40, 143.79, 143.69, 140.61, 131.30, 130.68, 127.52, 127.50, 126.93, 125.22, 119.96, 119.95, 118.76, 65.63, 59.86, 49.20, 46.64, 30.32, 19.06, 18.12, 17.70. MS (ESI + ) Found: 536.25 Calculated [M+Na] + :536.22 (C 30 H 31 N 3 NaO 5 )

[0121] Example 14: Fmoc-Val-Ala-PAP-CH(OMe) 2 (HDP 30.2677) [ka]

[0122] Step 13 product HDP 30.2623 (514 mg, 1.00 mmol) was dissolved in 20 ml of methanol. Trimethyl orthoformate (5.47 ml, 50 mmol) and 4-toluenesulfonic acid monohydrate (21 mg, 0.1 mmol) and 20 ml of dichloromethane were added and the mixture was refluxed under argon atmosphere. After 5 more hours, trimethyl orthoformate (5.47 ml) and 4-toluenesulfonic acid (210 mg) were added and stirring was continued for another 2 hours. The reaction mixture was cooled and poured into 50 ml of saturated sodium bicarbonate with vigorous stirring. After gas evolution had subsided. 100 ml of dichloromethane was added and the cloudy mixture was centrifuged at 4000 x g in 40 ml portions. The clear upper layer was removed and the organic layer was washed with 15 ml of water and centrifuged. The organic layers were combined and evaporated and the residue was distilled with 2 x 20 ml of methanol to remove traces of water. The residue was taken up in 100 ml of dichloromethane and the insoluble components were removed by filtration. The filtrate was evaporated and the residue was triturated with 50 ml of MTBE at 40°C for 1 hour, then cooled to room temperature and filtered under suction. The precipitate was washed with 20 ml of MTBE and dried in vacuum to give 445 mg (79%) of the title product as an amorphous solid.

[0123] Example 15: Fmoc-Val-Ala-OAB-OH (HDP 30.2761) [ka]

[0124] The dipeptide Fmoc-Val-Ala-OH (4.105 g, 10.0 mol) and 4-aminobenzyl alcohol (1.293 g, 1.05 eq.) were dissolved in 60 ml of anhydrous tetrahydrofuran (THF). 2-Ethoxy-N-(ethoxycarbonyl)-1,2-dihydroquinoline (EEDQ, 2.597 g, 1.05 eq.) was added and the mixture was stirred at room temperature protected from light.

[0125] After 3 days the reaction mixture which had formed a gelatinous material was diluted with 140 ml of MTBE and stirred until a fine precipitate formed which was suction filtered, washed with 50 ml of MTBE and dried to give 4.480 g (87%) of the title product HDP 30.2761 as a colorless solid. 1 H NMR (500 MHz, D 6 -DMSO) d 9.40 (s, 1H), 8.20 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.74 (t, J = 7.6 Hz, 2H), 7.53 (d, J = 8.0 Hz, 1H), 7.46-7.36 (m, 4H), 7.32 (tt, J = 7.5, 1.3 Hz, 2H), 7.23 (td, J = 7.6, 1.7 Hz, 1H), 7.15 (td, J = 7.4, 1.3 Hz, 1H), 5.24 (t, J = 5.6 Hz, 1H), 4.56-4.42 (m, 3H), 4.36-4.28 (m, 1H), 4.28-4.20 (m, 2H), 3.96 (dd, J = 9.1, 6.8 Hz, 1H), 2.04 (h, J = 6.8 Hz, 1H), 1.35 (d, J = 7.1 Hz, 3H), 0.90 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.8 Hz, 3H). 13 C NMR (126 MHz, D 6 -DMSO) d 171.09, 170.90, 156.09, 143.83, 143.72, 140.65, 140.63, 135.08, 134.74, 127.55, 127.54, 127.23, 126.97, 126.87, 125.27, 124.64, 123.66, 120.00, 119.98, 65.65, 60.03, 59.86, 48.90, 46.65, 40.01, 39.84, 39.67, 39.50, 39.34, 39.17, 39.00, 31.23, 30.33, 19.15, 18.04, 17.89. MS (ESI + ) Measured value: 538.25 Calculated value [M+Na] + :538.23 (C 30 H 33 N 3 NaO 5 )

[0126] Example 16: Fmoc-Val-Ala-OAP-CH(OMe)2 (HDP 30.2769) [ka]

[0127] The benzyl alcohol HDP 30.2761 (4.48 g, 8.69 mmol) from step 15 was dissolved in 18 ml of DMSO and 36 ml of anhydrous dichloromethane followed by 3.66 g, 1.0 equiv.) Dess-Martin periodinane and after stirring for 2 h the resulting brownish solution was diluted with 90 ml of chloroform and washed with 90 ml of water. The aqueous phase was back extracted with 2×40 ml of chloroform and the combined organics were washed by shaking with 90 ml of saturated sodium bicarbonate until a pH of 7-8 was reached. Then, 30 ml of 20% sodium thiosulfate solution was added and shaking was continued for 2 min. After separation, the organic layer was washed with a further 90 ml of water and dried (MgSO 4 The remaining 4.875 g was stirred with 150 ml MTBE, forming a fine precipitate which was filtered off with suction, washed with 50 ml MTBE and dried to give 4.307 g (97%) of the intermediate aldehyde as a colourless solid. MS (ESI + ) Measured value: 536.25 Calculated value [M+Na] + :536.22 (C 30 H 31 N 3 NaO 5 )

[0128] Then, 4.187 g (8.152 mmol) of the aldehyde Fmoc-Val-Ala-OAP-CHO were suspended in 80 ml of methanol, trimethyl orthoformate (45 ml, 50 equiv.) and 4-toluenesulfonic acid monobasic (1.551 g, 1 equiv.) were added and the mixture was refluxed under argon for 1 h. After cooling to room temperature, the reaction mixture was poured into 100 ml of saturated sodium bicarbonate with vigorous stirring. 100 ml of chloroform was added to dissolve the gelatinous material and the phases were separated. The aqueous phase was back-extracted with 2x50 ml of chloroform and the combined organic layers were washed with 50 ml of half-saturated brine and dried (MgSO 4 ) and evaporated. The crude product was purified on silica gel with a gradient of 0-20% ethyl acetate in dichloromethane to give 3.279 g (72%) of the title product HDP 30.2769 as an amorphous solid.

[0129] 1 H NMR (500 MHz, D 6 -DMSO) d 9.27 (s, 1H), 8.30 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.75 (t, J = 7.2 Hz, 2H), 7.70 (d, J = 8.1 Hz, 1H), 7.48-7.38 (m, 4H), 7.32 (ddd, J = 8.7, 6.7, 1.4 Hz, 3H), 7.18 (td, J = 7.6, 1.3 Hz, 1H), 5.45 (s, 1H), 4.46 (p, J = 7.0 Hz, 1H), 4.37-4.29 (m, 1H), 4.28-4.20 (m, 2H), 3.99 (dd, J = 9.2, 6.8 Hz, 1H), 3.26 (s, 3H), 3.26 (s, 3H), 2.05 (h, J = 6.7 Hz, 1H), 1.35 (d, J = 7.1 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H). 13 C NMR (126 MHz, D 6-DMSO) d 176.52, 176.05, 161.32, 149.06, 148.95, 145.89, 145.87, 140.68, 134.89, 133.97, 132.82, 132.80, 132.38, 132.23, 130.56, 130.55, 129.58, 129.11, 125.27, 106.40, 70.84, 65.01, 58.68, 58.37, 54.27, 51.85, 35.65, 24.41, 23.26, 22.69. MS (ESI + ) Measured value: 582.33 Calculated value [M+Na] + :582.26 (C 32 H 37 N 3 NaO 6 )

[0130] Example 17: BMP-Val-Ala-OAP-CH(OMe) 2 (HDP 30.2776) [ka]

[0131] The product of Example 16, HDP 30.2769 (3.236 g, 5.782 mmol), was suspended in 30 ml of DMF. Diethylamine (8 ml) was added and stirred at room temperature for 30 min to form a clear solution. The volatiles were removed and the residue was co-evaporated with 30 ml of fresh DMF and then dried under high vacuum. The crude product was the intermediate amine H-Val-Ala-OAP-CH(OMe). 2 and was used without further purification. MS (ESI + ) Measured value: 360.25 Calculated value [M+Na] + :360.19 (C 17 H 27 N 3 NaO 4 )

[0132] The intermediate free amine was dissolved in 30 ml of DMF, 1.539 g (1 eq.) of 3-(maleimido)propionic acid N-hydroxysuccinimide ester (BMPS) and 1.967 ml (2 eq.) of N-ethyldiisopropylamine (DIPEA) were added and the solution was stirred for 1 h. The solvent was evaporated and the residue was stirred with 100 ml of MTBE. The fine precipitate formed was filtered off, washed with MTBE (20 ml) and dried in vacuum. The crude product was purified on silica gel with a gradient of 0-10% methanol in dichloromethane to give 2.164 g (77%) of pure title product as a colorless amorphous solid.

[0133] 1 H NMR (500 MHz, D 6 -DMSO) d 9.20 (s, 1H), 8.27 (d, J = 6.8 Hz, 1H), 8.00 (d, J = 9.0 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.43 (dd, J = 7.7, 1.6 Hz, 1H), 7.33 (td, J = 7.8, 1.7 Hz, 1H), 7.16 (td, J = 7.5, 1.3 Hz, 1H), 6.99 (s, 2H), 5.44 (s, 1H), 4.41 (p, J = 7.1 Hz, 1H), 4.22 (dd, J = 8.8, 6.6 Hz, 1H), 3.61 (dq, J = 17.1, 6.6 Hz, 2H), 3.27 (s, 3H), 3.26 (s, 3H), 2.48-2.40 (m, 2H), 2.01 (dh, J = 20.3, 6.8 Hz, 1H), 1.33 (d, J = 7.2 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H). 13 C NMR (126 MHz, D 6-DMSO) d 171.03, 170.78, 170.63, 169.50, 135.51, 134.47, 129.50, 128.70, 127.15, 124.23, 123.70, 101.33, 57.27, 53.46, 53.18, 49.08, 33.99, 33.64, 30.32, 19.10, 17.91, 17.28.1 MS (ESI + ) Measured value: 511.25 Calculated value [M+Na] + :511.22 (C 24 H 32 N 4 NaO 7 )

[0134] Example 18: Fmoc-Val-Ala-MAB-OH (HDP 30.2767) [ka]

[0135] Following the procedure of Example 15 with 3-aminobenzyl alcohol gave 4.698 g (91%) of the title product, HDP 30.2767, as an amorphous solid. 1 H NMR (500 MHz, D 6-DMSO) d 9.95 (s, 1H), 8.17 (d, J = 7.0 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H), 7.75 (t, J = 7.8 Hz, 2H), 7.59 (d, J = 1.9 Hz, 1H), 7.50-7.37 (m, 4H), 7.32 (tt, J = 7.5, 1.6 Hz, 2H), 7.24 (t, J = 7.8 Hz, 1H), 6.99 (d, J = 7.8 Hz, 1H), 5.19 (t, J = 5.7 Hz, 1H), 4.45 (dd, J = 14.7, 6.5 Hz, 3H), 4.36-4.28 (m, 1H), 4.28-4.19 (m, 2H), 3.93 (dd, J = 9.0, 7.1 Hz, 1H), 2.01 (h, J = 6.8 Hz, 1H), 1.32 (d, J = 7.0 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H). 13 C NMR (126 MHz, D 6 -DMSO) d 170.93, 156.08, 143.80, 143.72, 143.16, 140.63, 140.62, 138.78, 128.29, 127.57, 127.54, 126.98, 125.29, 121.21, 120.02, 120.00, 117.41, 117.11, 65.63, 62.77, 59.92, 48.95, 46.61, 30.32, 19.13, 18.19, 18.07, 3.26. MS (ESI + ) Measured value: 538.33 Calculated value [M+Na] + :538.23(C 30 H 33 N 3 NaO 5 )

[0136] Example 19: Fmoc-Val-Ala-MAP-CH(OMe)2 (HDP 30.2778) [ka] The compound of Example 18 (HDP 30.2767) was subjected to the procedure of Example 16, excluding the final silica gel chromatography, to give 6.28 g of crude title product, HDP 30.2778, sufficiently pure for use in the next step as an amorphous solid.

[0137] Example 20: BMP-Val-Ala-MAP-CH(OMe) 2 (HDP 30.2776) [ka] The compound of Example 19 (HDP 30.2778) was subjected to the procedure of Example 17 to afford the title product HDP 30.2781 in 95% yield based on HDP 30.2767 as an amorphous solid.

[0138] 1 H NMR (500 MHz, D 6 -DMSO) d 9.89 (s, 1H), 8.16 (d, J = 6.9 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.66 (t, J = 1.9 Hz, 1H), 7.64-7.54 (m, 1H), 7.30 (t, J = 7.9 Hz, 1H), 7.06 (dt, J = 7.6, 1.4 Hz, 1H), 7.00 (s, 2H), 5.35 (s, 1H), 4.38 (p, J = 7.1 Hz, 1H), 4.14 (dd, J = 8.4, 6.8 Hz, 1H), 3.68-3.57 (m, 2H), 3.24 (s, 6H), 2.50-2.40 (m, 2H), 2.00-1.90 (m, J = 6.8 Hz, 1H), 1.32 (d, J = 7.1 Hz, 3H), 0.87 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H). 13 C NMR (126 MHz, D6 -DMSO) d 171.09, 170.77, 170.71, 169.76, 138.90, 138.79, 134.52, 128.46, 121.42, 118.99, 117.25, 102.42, 57.71, 52.44, 52.42, 49.08, 34.02, 33.70, 30.26, 19.11, 18.17, 17.83. MS (ESI + ) Measured value: 511.25 Calculated value [M+Na] + :511.22 (C 24 H 32 N 4 NaO 7 )

[0139] General Procedure A for Cyclic Acetals To a solution of the diol compound in dry DMF (100 μl / mg) was added the dimethyl acetal compound (10 equiv.). The mixture was acidified with trifluoroacetic acid (10 μl / mg) and stirred at ambient temperature under argon until a clear solution was formed. The solvent was then removed under high vacuum and the residue was redissolved in fresh DMF. A sample was quenched with 1% triethylamine in methanol and analyzed by HPLC, which showed complete conversion of the starting material.

[0140] The DMF solution was added dropwise to a 10-fold volume of an ice-cold solution of 0.1% triethylamine in tert-butyl methyl ether (MTBE) and the resulting precipitate was isolated by centrifugation. The supernatant was drained and the pellet was resuspended in an equal volume of 0.1% triethylamine MTBE and centrifuged again. The vacuum-dried crude product was further purified by preparative HPLC.

[0141] General Procedure B for Cyclic Acetals The diol compound (1 equiv.) and the dimethyl acetal compound (8 equiv.) were placed in a centrifuge tube and dissolved in DMF / TFA 4:1 (v / v, 20 μl / μmol diol compound). The tube was shaken at room temperature until HPLC control showed complete conversion of the diol compound (1–18 h). Then, 20 volumes of ice-cold MTBE were added and the precipitate was isolated by centrifugation. The first pellet was resuspended in the same volume of MTBE containing 2 vol-% triethylamine and centrifuged again. The dried pellet was used in the next reaction without further purification.

[0142] Example 21: α-Amanitin-(Fmoc-Val-Ala-4-amino-benzylidene acetal) HDP 30.2681 [ka]

[0143] Alpha-amanitin (30.98 mg) and HDP 30.2677 were reacted according to general procedure A. The crude product was purified on a Phenomenex Luna-C18(2), 10 μm, 250x21.2 mm, with a gradient of 5-100% acetonitrile in water for 15 min. The product, containing the fraction from 10.40-11.08 min, was lyophilized to give 37.07 mg (78%) of lyophilizate. MS (ESI + ) Measured value: 1414.67; Calculated value [MH] + :1414.58 (C 69 H 84 N 13 O 18 S)

[0144] Example 22: α-Amanitin-[4-(11-Fmoc-amino-3,6,9-trioxaundecyloxy)-benzylidene acetal] HDP 30.2665 [ka]

[0145] According to general procedure A, α-amanitin (20.51 mg) and HDP 30.2640 from Example 9 were reacted to give 20.96 mg (65%) of the title product as a lyophilized powder.

[0146] Example 23: α-Amanitin-[3-(11-Fmoc-amino-3,6,9-trioxaundecyloxy)-benzylidene acetal] HDP 30.2666 [ka]

[0147] According to general procedure B, α-amanitin (20.51 mg) and HDP 30.2650 from Example 10 were reacted and the crude title product was used without further purification.

[0148] Example 24: α-Amanitin-[4-(11-Fmoc-amino-3,6,9-trioxaundecyloxy)-3-fluoro-benzylidene acetal] HDP 30.2667 [ka]

[0149] According to general procedure B, α-amanitin (20.51 mg) and HDP 30.2651 from Example 11 were reacted and the crude title product was used without further purification.

[0150] Example 25: α-Amanitin-[2-bromo-3-(11-Fmoc-amino-3,6,9-trioxaundecyloxy)-benzylidene acetal] HDP 30.2668 [ka]

[0151] According to general procedure B, α-amanitin (20.51 mg) and HDP 30.2652 from Example 11 were reacted and the crude title product was used without further purification.

[0152] Example 26: α-Amanitin-(BMP-Val-Ala-4-amino-benzylidene acetal) HDP 30.2684 [ka]

[0153] The cyclic acetal HDP30.2681 (37.07 mg, 26.21 μmol) was dissolved in 2 ml of dry DMF. Diethylamine (92 μl, 891 μmol) was added and the mixture was stirred at ambient temperature for 15 min. The volatiles were removed in vacuo and the residue was redissolved in dry DMF. HPLC and MS analysis showed complete deprotection to the free amine. (MS-ESI + ) Measured value: 1192.67; Calculated value [MH] + :1192.51 (C 54 H 74 N 13 O 16 S) 3-(Maleimido)propionic acid N-hydroxysuccinimide ester (BMPS, 14 mg, 52.42 μmol = 2 equiv.) dissolved in 500 μl dry DMF was added, followed by 8.92 μl (52.42 μmol = 2 equiv.) N,N-diisopropylethylamine. After stirring for 2 h at room temperature, the reaction mixture was added dropwise to 10 ml ice-cold MTBE and the precipitate was isolated by centrifugation. The crude pellet was purified by preparative HPLC on a Luna-C18(2) using a gradient of 5-50% acetonitrile in water in 15 min. The product fraction from 11.9-12.8 min was lyophilized to give 20.85 mg (59%) of colorless lyophilizate. MS (ESI + ) Measured value: 1343.58; Calculated value [MH] + :1343.54 (C 61 H 79 N 14 O 19 S)

[0154] Example 27: α-Amanitin-[4-(11-(3-maleimidopropyl-amido)-3,6,9-trioxaundecyloxy)-benzylidene acetal] HDP 30.2669 [ka]

[0155] The procedure of Example 26 was applied to 20.96 mg of HDP 30.2665 from Example 22 to give the title product HDP 30.2669 (13.00 mg, 65%) as a lyophilized powder. MS (ESI + ) Measured value: 1371.58; Calculated value [MH] + :1371.52 (C 61 H 80 N 12 NaO 21 S))

[0156] Example 28: α-Amanitin-[3-(11-(3-maleimidopropyl-amido)-3,6,9-trioxaundecyloxy)-benzylidene acetal] HDP 30.2670 [ka]

[0157] The procedure of Example 26 was applied to the crude product of HDP 30.2666 from Example 23 to give the title product HDP 30.2670 (4.39 mg, 15% based on α-amanitin) as a lyophilized powder. MS (ESI + ) Measured value: 1371.50; Calculated value [MH] + :1371.52 (C 61 H 80 N 12 NaO 21 S))

[0158] Example 29: α-Amanitin-[4-(11-(3-maleimidopropyl-amido)-3,6,9-trioxaundecyloxy)-3-fluoro-benzylidene acetal] HDP 30.2671 [ka]

[0159] The procedure of Example 26 was applied to the crude product of HDP 30.2667 from Example 24 to give the title product HDP 30.2671 (5.82 mg, 19% based on α-amanitin) as a lyophilized powder. MS (ESI + ) Measured value: 1389.50; Calculated value [MH] + :1389.51 (C 61 H 79 FN 12 NaO 21 S)

[0160] Example 30: α-Amanitin-[2-bromo-3-(11-(3-maleimidopropyl-amido)-3,6,9-trioxaundecyloxy)-benzylidene acetal] HDP 30.2672 [ka]

[0161] The procedure of Example 26 was applied to the crude product of HDP 30.2668 from Example 24 to give the title product HDP 30.2672 (2.43 mg, 8% based on α-amanitin) as a lyophilized powder. MS (ESI + ) Actual value: 1449.42 / 1451.42; Calculated value [MH] + :1449.43 / 1451.43 (C 61 H 79 BrN 12 NaO 21 S)

[0162] Example 31: α-Amanitin-(BMP-Val-Ala-2-amino-benzylidene acetal) HDP 30.2792 [ka] α-Amanitin could be reacted with HDP 30.2776, the compound of Example 17, to give the title compound.

[0163] Example 32: α-Amanitin-(BMP-Val-Ala-3-amino-benzylidene acetal) HDP 30.2793 [ka] α-Amanitin could be reacted with HDP 30.2781, the compound of Example 20, to give the title compound.

[0164] Example 33: Synthesis of amatoxin-antibody conjugates Derivatives of trastuzumab, an anti-Her2neu specific antibody with a D265C mutation ("thiomab", an aspartic acid to cysteine ​​mutation at amino acid position 265 of the Ig heavy chain), were conjugated to the amatoxin linker derivatives HDP 30.2669, HDP 30.2670, HDP 30.2671, HDP 30.2672 and HDP 30.2684.

[0165] For each conjugation reaction, 10 mg of thiomab in PBS buffer adjusted to 1 mM EDTA was used. Cysteines were removed by reaction of the antibody with 40 equivalents of TCEP for 3 hours at 37°C on a shaker and two successive dialysis steps at 4°C in 1xPBS, 1 mM EDTA, pH 7.4 in a Slide-A-Lyzer dialysis cassette. Oxidation was performed by incubation of the antibody with 20 equivalents of dehydroascorbic acid (dhAA) for 3 hours at room temperature on a shaker.

[0166] Conjugation with amatoxin-linker derivatives was performed with 4 equivalents of HDP 30.2669, HDP 30.2670, HDP 30.2671, HDP 30.2672 and HDP 30.2684, respectively, dissolved in DMSO, by incubation at room temperature for 1 h, quenching by addition of 25 equivalents of N-acetyl-L-cysteine, and incubation at room temperature for 15 min or at 4°C overnight. Each reaction mixture was purified by Sephadex G-25 gel filtration (PD-10 column, GE Healthcare Life Sciences) equilibrated with PBS, pH 7.4. Protein-containing fractions were identified with Bradford reagent on parafilm and pooled. The conjugate solution was dialyzed overnight at 4°C in a Slide A-Lyser dialysis cassette (Thermo Scientific, 20.000 MWCO) against PBS, pH 7.4. Protein concentrations were measured using RotiQuant-Assay (Carl Roth, Germany). The complex solutions were diluted to 5 mg / ml (~3.44 × 10 -5 The protein concentration was adjusted to 100 mM M, sterile filtered, and stored at 4° C. The conjugates were designated “T-D265C-30.2669”, “T-D265C-30.2670”, “T-D265C 30.2671”, “T-D265C-30.2672”, and “T-D265C-30.2684”.

[0167] In Coomassie staining under reducing conditions, the light and heavy chains of the amatoxin-antibody conjugate and the naked antibody had the expected apparent masses of approximately 25 and 50 kDa. In the case of the amatoxin-antibody conjugate, an upshift of the heavy chain protein signal was observed compared to the naked antibody, indicating binding of the toxin to the heavy chain. In anti-amanitin Western blots under reducing conditions, the thiomab conjugate showed a heavy chain signal of approximately 55 kDa and no light chain signal, indicating binding of the toxin exclusively to the heavy chain.

[0168] Example 34: In vitro cytotoxicity test against HER2-positive tumor cell lines The cytotoxic activity of trastuzumab (anti-HER2neu specific antibody)-amatoxin conjugates was evaluated in vitro on the HER2 positive tumor cell lines SKBR-3 (breast cancer), NCI-N87 (gastric cancer) and JIMT-1 (breast cancer) using a chemiluminescent BrdU-ELISA incorporation assay (Roche). The conjugates tested were one conjugate with an aromatic cyclic acetal having a dipeptidine cleavage site according to the invention linked to aa3 of amatoxin (T-D265C-30.2684), one conjugate with a para-aminobenzyl ether linker having a dipeptidine cleavage site linked to aa4 of amatoxin (T-D265C-30.1699), and four conjugates with aromatic cyclic acetals having a polyethylene glycol (PEG) linker linked to aa3 of amatoxin (p-PEG 3 T-D265C-30.2669 with linker, m-PEG 3 -30.2670, p-PEG with linker 3 -30.2671, o-PEG with linker and fluoroaryl moiety 3 The compound contained a linker and an aryl bromide moiety (-30.2672).

[0169] [ka]

[0170] [ka]

[0171] The results of the cytotoxicity test on SKBR-3 cells are shown in FIG. 2, the results of the cytotoxicity test on NCI-N87 cells are shown in FIG. 3, and the results on JIMT-1 cells are shown in FIG. EC of different trastuzumab-amatoxin conjugates on different Her2-positive cell lines 50 The values ​​are shown in Table 1:

[0172] [Table 1]

[0173] The cytotoxicity of the inventive conjugate T-D265C-30.2684 was comparable to that of the conjugate T-D265C-30.1699. The cytotoxicity of the inventive conjugate T-D265C-30.2684 was slightly higher in SKBR-3 cells but significantly higher in NCI-N87 cells and JIMT-1 cells when compared to the conjugate T-D265C-30.2669 containing an aromatic cyclic acetal with a PEG linker.

[0174] The ADCs T-D265C-30.2684 and T-D265C-30.1699, both of which possess a cleavable Val-Ala linker, showed full cytotoxicity against the cell lines tested. The acid-labile ADCs T-D265C-30.2670, T-D265C-30.2671 and T-D265C-30.2672 showed only growth inhibition against SKBR-3 cells with residual cell viability of approximately 65%, 50% and 60%, respectively, and no cytotoxicity against JIMT-1 cells.

[0175] The amatoxin derivative HDP 30.2684 is as effective in vitro as HDP 30.1699, but is synthesized much faster: three HPLC purifications are required during the synthesis of HDP 30.1699, whereas the synthesis of HDP 30.2684 requires only one.

[0176] Example 35: Efficacy of acetal linker conjugates in an in vivo SKOV-3 xenograft tumor mouse model The study consisted of seven experimental groups of 10 female NOD Scid mice each bearing a subcutaneous Her2-positive SKOV-3 (human ovarian) tumor. The efficacy of increasing doses of two aromatic cyclic acetal conjugates (T-D265C-30.2684, T-D265C-30.2669) with an amatoxin a3 linkage was compared with T-D265C-30.1699, which has a para-amino-benzyl linker with a dipeptidine cleavage site attached to aa4 of the amatoxin, and untreated controls. Mice were administered a single intravenous dose.

[0177] The experimental setup is shown in Table 2. [Table 2] The results of this study are shown in FIG.

[0178] All animals treated with cyclic acetal amatoxin drug conjugate were tumor free. No animals were tumor free in the group treated with 1 mg / kg T-D265C-30.1699. Four of 10 animals were tumor free in the group treated with 2 mg / kg T-D265C-30.1699.

[0179] Example 36: Tolerability of acetal linker conjugates in an in vivo mouse model The tolerability of amatoxin ADCs T-D265C-30.2669 and T-D265C-30.2684, respectively, was evaluated in NOD / SCID mice after a single intravenous application. A total of 21 animals were used. T-D265C-30.2669 and T-D265C-30.2684 were well tolerated at effective doses up to 6 mg / kg. The results are shown in Figure 6.

[0180] References Binz HK et al. (2005). Engineering novel binding proteins from non-immunoglobulin domains. Nat. Biotechnol. Vol. 10: 1257-1268. Chalouni C. and Doll S. (2018). Fate of Antibody-Drug Conjugates in Cancer Cells. Journal of Experimental & Clinical Cancer Research. Vol. 37: 20. Gillies E.R. et al. (2004). Acetals as pH-Sensitive Linkages for Drug Delivery. Bioconjugate Chem. Vol. 15: 1254-1263. Jeffrey et al. (2005). Design, Synthesis, and in Vitro Evaluation of Dipeptide-Based Antibody Minor Groove Binder Conjugates. J. Med. Chem. Vol. 48: 1344-1358. Jeffrey et al. (2006). Development and Properties of β-Glucuronide Linkers for Monoclonal Antibody-Drug Conjugates. Bioconjugate Chem. Vol. 17: 831-840. Poljak R.J. (1994). Production and structure of diabodies. Structure Vol. 2: 1121-1123. Tranoy-Opalinski et al. (2014). β-Glucuronidase-responsive prodrugs for selective cancer chemotherapy: An update. European Journal of Medicinal Chemistry Vol. 74: 302-313. Wang et al. (2019). Three decades of nucleic acid aptamer technologies: Lessons learned, progress and opportunities on aptamer development. Biotechnology Advances Vol. 37: 28-50. Wieland T. and Faulstich H. (1978). Amatoxins, Phallotoxins, Phallolysin, and Antamanide: The biologically active components of poisonous Amanita mushrooms. CRC Crit Rev Biochem. Vol. 5(3): 185-260.

Claims

1. A conjugate comprising an amatoxin and a T-L moiety having formula III or IV; 【Chemistry 1】 During the ceremony, X is S, SO, or SO ２ and R1 is H or C １ -C ６ is an alkyl group; R2 is independently H or an electron withdrawing or donating group; R4 is H, OH, O-C １ -C ８ -Alkyl, NO ２ , N.H. ２ , F, Cl, Br, or SH; R5 is OH, NH ２ or NHOH; R6, R7 are the side chains of a natural or unnatural amino acid; L is a linker, and T is a target binding moiety.

2. A conjugate comprising an amatoxin and a T-L moiety having formula V; 【Chemistry 2】 During the ceremony, X is S, SO, or SO ２ and Y is CH ２ or CO; R1 is H or C １ -C ６ is an alkyl group; R2 is independently H or an electron withdrawing or donating group; at least one R2 group is substituted with said T-L moiety. R4 is H, OH, O-C １ -C ８ -Alkyl, NO ２ , N.H. ２ , F, Cl, Br, or SH; R5 is OH, NH ２ or NHOH; L is a linker, and T is a target binding moiety.

3. A conjugate comprising an amatoxin and a T-L moiety having formula VI; 【Chemistry 3】 During the ceremony, X is S, SO, or SO ２ and R1 is H or C １ -C ６ is an alkyl group; R2 is independently H or an electron withdrawing or donating group, with the proviso that if R8 is not a T-L moiety, then one R2 is replaced with said T-L moiety; R4 is H, OH, O-C １ -C ８ -Alkyl, NO ２ , N.H. ２ , F, Cl, Br, or SH; R5 is OH, NH ２ or NHOH; R8 is a straight or branched chain alkyl group, or a T-L moiety if neither R2 is a T-L moiety; L is a linker, and T is a target binding moiety.

4. A conjugate comprising an amatoxin and a T-L moiety having formula VII; 【Chemistry 4】 During the ceremony, X is S, SO, or SO ２ and R1 is H or C １ -C ６ is an alkyl group; R2 is independently H or an electron withdrawing or donating group, with the proviso that if R9 is not a T-L moiety, then one R2 is replaced with said T-L moiety; R4 is H, OH, O-C １ -C ８ -Alkyl, NO ２ , N.H. ２ , F, Cl, Br, or SH; R5 is OH, NH ２ or NHOH; R9 is H or a straight or branched chain alkyl group, or a T-L moiety if neither R2 is a T-L moiety; L is a linker, and T is a target binding moiety.

5. A conjugate comprising an amatoxin and a target binding moiety, represented by formula VIII or IX. 【Chemistry 5】

6. The amatoxin is selected from the group consisting of α-amanitin, β-amanitin, amanin, amaninamide, their respective thioethers, and salts; The complex according to any one of claims 1 to 4.

7. The conjugate of any one of claims 1 to 4 and 6, wherein the linker L is an alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, or heteroaralkylene group containing from 1 to 4 heteroatoms selected from N, O, and S, and wherein the linker is optionally substituted.

8. 8. The conjugate of claim 7, wherein the linker L comprises a moiety selected from at least one of the following moieties: disulfide, ether, thioether, amine, ester, carboxamide, urethane, and urea moieties.

9. The conjugate according to any one of claims 1 to 8, wherein the target binding moiety is selected from the group consisting of: (i) an antibody or antigen-binding fragment thereof, and (ii) Nucleic acid aptamers.

10. 10. The conjugate of claim 9, wherein the antibody or antigen-binding fragment thereof is selected from a diabody, a tetrabody, a nanobody, a chimeric antibody, a deimmunized antibody, a humanized antibody or a human antibody.

11. 11. The conjugate of claim 10, wherein the antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fd, Fv, single-chain Fv, and disulfide-linked Fv (dsFv).

12. A method for the synthesis of the conjugate according to any one of claims 1 to 11 by reacting the 1,2-diol of amatoxin with dimethyl benzylidene acetal in an aprotic solvent under acidic conditions.

13. 13. The method of claim 12, wherein the amatoxin is selected from alpha-amanitin, beta-amanitin, amanine, amaninamide and their respective thioethers.

14. 14. The method of claim 12 or 13, wherein the aprotic solvent is DMF and / or the acidic conditions are with trifluoroacetic acid.

15. A pharmaceutical comprising the conjugate according to any one of claims 1 to 11.

16. 16. A medicament as claimed in claim 15 for use in the treatment of cancer in a patient.

17. The pharmaceutical composition of claim 16, wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, bile duct cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, gastric cancer, renal cancer, malignant melanoma, leukemia, and malignant lymphoma.

18. 12. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 11, further comprising one or more pharma- ceutically acceptable diluents, carriers, excipients, fillers, binders, lubricants, disintegrants, adsorbents, and / or preservatives.

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