RU2792210C2 - New method for synthesis of amanitines - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention describes the new methods for the synthesis of amanitin derivatives having a hydroxy group attached to the central tryptophan fragment, as well as to a hydroxy-substituted derivative of 2-carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole according to formula I

in which R1 is chosen from C_1-2-alkyl; P₁ represents hydrogen or a protective group; P₂ is hydrogen or a protecting group and R 2 is chosen from OH, OR 1 and a polypeptide chain of 1-7 amino acid residues, where the protecting group, if present, is independently chosen from Boc, PhCH₂ OCO-, CH₂ =CHCH₂ O-CO- and trityl.

EFFECT: new derivatives of amanitin are obtained, having a hydroxy group attached at position 4', 5'', 6' or 7' a central tryptophan moiety, novel conjugates of such amanitin derivatives, and pharmaceutical compositions comprising such conjugates for the treatment of cancer.

19 cl, 1 ex, 15 dwg

Description

The field of technology to which the present invention relates

The present invention relates to novel methods for the synthesis of amanitin derivatives having a hydroxyl group attached to the central tryptophan moiety. The present invention further relates to novel amanitin derivatives having a hydroxy group attached at the 4', 5' or 7' position of the central tryptophan moiety, novel conjugates of such amanitin derivatives, and pharmaceutical compositions comprising such conjugates.

Background of the Invention

Amatoxins are 8 amino acid cyclic peptides found in the fungus Amanita phalloides (see FIG. 1). Amatoxins specifically inhibit the DNA-dependent RNA polymerase II of mammalian cells and thereby also the transcription and protein biosynthesis of damaged cells. Inhibition of transcription in a cell causes cessation of growth and proliferation. Although not covalently bound, the complex between amanitin and RNA polymerase II is very strong (K _D =3 nM). The dissociation of amanitin from the enzyme is a very slow process, making repair of the damaged cell unlikely. If transcription inhibition lasts too long, the cell undergoes programmed cell death (apoptosis).

The use of amatoxins as cytotoxic moieties for antitumor therapy was already explored in 1981 by coupling an anti-Thy 1,2 antibody to α-amanitin using a linker attached to the indole ring of Trp (amino acid 4; see Fig. 1) via diazotization (Davis & Preston, Science 213 (1981) 1385-1388). Davis and Preston identified the site of attachment as position 7'. Morris and Venton also demonstrated that substitution at the 7' position produces a derivative that retains cytotoxic activity (Morris & Venton, Int. J. Peptide Protein Res. 21 (1983) 419-430).

European Patent Application No. EP1859811 A1 (published Nov. 28, 2007) describes conjugates in which the amatoxin γ atom of amino acid 1 of β-amanitine was directly linked, i. without linker structure, with albumin or with monoclonal antibody HEA125, OKT3 or PA-1. Moreover, these conjugates have been shown to have an inhibitory effect on the proliferation of breast cancer (MCF-7), Burkitt's lymphoma (Raji) and T-lymphoma (Jurkat) cells. The use of linkers, including linkers including elements such as amide, ester, ether, thioether, disulfide, urea, thiourea, hydrocarbon moieties and the like, has been contemplated, but such constructs have not actually been shown and no more details have been provided. such as attachment sites on amatoxins.

Patent applications WO 2010/115629 and WO 2010/115630 (both published October 14, 2010) describe conjugates in which antibodies, such as anti-EpCAM antibodies, such as the humanized huHEA125 antibody, are linked to amatoxins via (i) γ C - atom of amino acid 1 of amatoxin, (ii) 6' C-atom of amino acid 4 of amatoxin, or (iii) via the δ C-atom of amino acid 3 of amatoxin, in each case either directly or via a linker between the antibody and amatoxins. Contemplated linkers include elements such as amide, ester, ether, thioether, disulfide, urea, thiourea, hydrocarbon moieties, and the like. Moreover, such conjugates have been shown to have inhibitory effects on the proliferation of breast cancer (MCF-7 cell line), pancreatic carcinoma (Capan-1 cell line), colon cancer (Co10205 cell line) and cholangiocarcinoma (OZ cell line).

Patent application WO 2012/119787 describes that target-binding moieties can be attached to amatoxins via linkers at additional attachment sites on amino acid 4 of tryptophan, namely at positions 1'-N, without affecting the interaction of such amatoxins with their target. - DNA-dependent RNA polymerase II of mammalian cells.

Patent application WO 2014/009025 describes the total synthesis of amanitin derivatives using a novel synthon for γ,δ-dihydroxyisoleucine as one of the starting materials. In addition, patent application PCT/EP2016/078984 [published as WO 2017/089607] describes the total synthesis of γ- and ε-amanitine derivatives using readily available (2S,3R,4S)-L-4-hydroxyisoleucine as one from source materials. However, these approaches and all other wholly or partially synthetic approaches used to date have been to incorporate a central tryptophan moiety into the amatoxin core using a method according to the Savige-Fontana reaction. In this method, cis-2-carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole ("Hpi") is inserted into the linear amanitin precursor structure. The acid-catalyzed Hpi-cysteine coupling reaction produces an amanitine ring system with a central tryptophan moiety.

Hpi, however, does not have any functional moiety attached to the phenyl ring of the indole moiety in Hpi. Thus, the amanitin products obtained from the Savige-Fontana method contain the central tryptophan moiety without any additional substituents. Naturally occurring α-, β-, γ and ε-amanitins, however, contain a central 6'-hydroxy-tryptophan moiety, and this 6'-hydroxy group has been successfully used as a functional group to functionalize amanitins, for example, by attaching targeting moieties, either directly or via linkers (see, for example, WO 2010/115629, WO 2010/115630 and WO 2012/041504). Thus, in the case of synthetic amanitins, the functionalization must have been performed (i) through the γ C atom of amino acid 1 of amatoxin, or (ii) through the δ C atom of amino acid 3 of amatoxin, as described above, or through the nitrogen atom of the tryptophan moiety, as described in WO 2012/119787.

Hpi can be obtained by reacting tryptophan with peracetic acid or photochemically with singlet oxygen. However, no derivatives with substituents attached to the phenyl ring of the indole moiety Hpi have been described so far.

While the use of fully synthetic pathways for amatoxins may provide an option to supply larger amounts of amatoxins needed for therapeutic applications and may allow the creation of many new amatoxin variants using the appropriate starting materials as building blocks, previous approaches have been limited by the fact that the native structure of α-, β-, γ and/or ε-amanitin could not be obtained because the 6'-hydroxy moiety attached to the tryptophan moiety as a core could not be included in such amanitins. Thus, the options for the functionalization of synthetic amanitins have been rather limited so far. In addition, it would be highly desirable to expand the range of options available for functionalization, since factors such as steric hindrance and reactivity can greatly influence the reactivity, biological activity and/or stability of synthetic amanitins and their conjugates. However, the stability and efficacy of conjugates comprising highly toxic amanitins are of paramount importance for intended use as therapeutic molecules for administration to humans.

Purpose of the present invention

Thus, there is still a great need for a cost-effective and reliable method for the synthesis of amatoxins with a hydroxy group attached to the phenyl ring of the central tryptophan moiety. In particular, there is a strong need to identify starting materials that could be used in the established Savige-Fontana reaction and that are provided in such a way that they can cause the incorporation of such hydroxyl groups.

Brief summary of the present invention

The present invention is based on the surprising observation that Hpi variants can be synthesized that allow the introduction of hydroxyl groups during the synthesis of amanitine derivatives.

Thus, according to one aspect, the present invention relates to a hydroxy-substituted derivative of 2-carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole in accordance with formula I

in which R1 is selected from alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl and substituted heteroaryl;

P ₁ represents hydrogen or a protecting group, in particular a protecting group selected from Boc, PhCH ₂ OCO-, CH ₂ =CHCH ₂ O-CO- and trityl;

P ₂ represents hydrogen or a protecting group, in particular a protecting group, in particular a protecting group selected from Boc, PhCH ₂ OCO-, CH ₂ =CHCH ₂ O-CO- and trityl; And

R2 is selected from OH, OR1 and a polypeptide chain consisting of 1-7 amino acid residues.

According to a second aspect, the present invention relates to a method for the synthesis of a linear precursor comprising eight amino acid residues of an amanitin derivative comprising a hydroxylated tryptophan fragment, comprising the step of using a hydroxy-substituted derivative of 2-carboxy-3a-hydroxy-1,2,3,3a ,8,8a-hexahydropyrrolo[2,3-b]indole according to the present invention in the peptide synthesis of said precursor.

According to a third aspect, the present invention relates to a process for the synthesis of an amanitin derivative comprising a hydroxylated tryptophan moiety, comprising the steps of:

(i) initiating or causing the formation of a bond between a cysteine residue and a tryptophan fragment of a linear precursor in accordance with the present invention; And

(ii) initiating or causing the formation of said amanitin derivative by reacting an N-terminal linear precursor according to the present invention with the C-terminus of said precursor.

According to a fourth aspect, the present invention relates to an amanitine derivative comprising a hydroxylated tryptophan moiety which is selected from (i) S-deoxy-4'-hydroxy-amanin, 4'-hydroxy-amanin, S-deoxy-5'-hydroxy- amanine, 5'-hydroxy-amanin, S-deoxy-7'-hydroxy-amanin, 7'-hydroxy-amanin, (ii) S-deoxy-4'-hydroxy-amaninamide, 4'-hydroxy-amaninamide, S- deoxy-5'-hydroxy-amaninamide, 5'-hydroxy-amaninamide, S-deoxy-7'-hydroxy-amaninamide and 7'-hydroxy-amaninamide, (iii) an amanitin derivative according to (i), in which the moiety of the free carboxylic acid of amino acid 1 is converted to a -C(=O)OR1 carboxylic acid ester or to a -C(=O)NH-OR1 moiety, wherein R1 is selected from alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl, and substituted heteroaryl.

According to a fifth aspect, the present invention relates to a conjugate comprising (a) an amanitin derivative comprising a hydroxylated tryptophan moiety according to the present invention; (b) a target-binding moiety; and (c) optionally a linker linking said amanitin derivative and said target-binding moiety.

According to a sixth aspect, the present invention relates to a pharmaceutical composition comprising amanitin according to the present invention or a conjugate according to the present invention.

According to a seventh aspect, the present invention relates to an amanitin derivative according to the present invention, a conjugate according to the present invention, or a pharmaceutical composition according to the present invention for use in the treatment of a cancer in a patient, in particular, the cancer is selected from the group consisting of of breast cancer, pancreatic cancer, cholangiocarcinoma, colon and rectal cancer, lung cancer, prostate cancer, ovarian cancer, gastric cancer, kidney cancer, malignant melanoma, leukemia and malignant lymphoma.

According to an eighth aspect, the present invention relates to a construct comprising (a) an amanitine derivative according to the present invention and (b) a linker moiety bearing a Y reactive group for linking said amanitine derivative to a target-binding moiety.

Brief description of graphic materials

In FIG. 1 shows the structural formulas of various amatoxins. The numbers in bold (from 1 to 8) indicate the standard numbering of the eight amino acids that form amatoxin. Also shown are the standard atomic designations in amino acids 1, 3, and 4 (Greek letters α to γ, Greek letters α to δ, and numbers 1' to 7', respectively).

In FIG. 2 shows the structure of the compound S-deoxy-α-amanitin (HDP 30.0735) and α-amanitin.

In FIG. 3 shows that Hpi (HDP 30.0079) is formed in a one-step synthesis starting from Boc-L-tryptophan.

In FIG. 4 shows that the compound S-deoxy-α-amanitin (HDP 30.0735) is formed by solid phase peptide synthesis involving the Hpi derivative HDP 30.2555.

In FIG. 5 shows the cytotoxicity of S-deoxy-α-amanitin (HDP 30.0735) and S-deoxy-5'-hydroxy-amaninamide HDP 30.2548 to HEK293 cells and to HEK293 OATP1B3 cells.

In FIG. 6 shows an S-deoxy-α-amanitine (HDP 30.0735) construct with a cleavable linker attached to the 6'-hydroxy group and an exemplary Y reactive maleimide group to link the construct to a target-binding moiety.

In FIG. 7 shows an alternative construct based on S-deoxy-α-amanitin (HDP 30.0735) with a cleavable linker attached to the carboxyl group of amino acid residue 1 and the terminal maleimide group as an example of a Y reactive group to link the construct to a target-binding moiety.

In FIG. 8 shows that the hydroxylated Hpi derivative HDP 30.2536 is formed in a multi-step synthesis starting from 5'-hydroxy-L-tryptophan.

In FIG. 9 shows that the amanitin precursor compound HDP 30.2544 is formed by solid phase peptide synthesis involving the Hpi derivative HDP 30.2536.

In FIG. 10 shows that the amanitin compound HDP 30.2546 is formed by ring closure from the amanitin precursor HDP 30.2544.

In FIG. 11 shows that the amanitin derivative HDP 30.2548 (S-deoxy-5'-hydroxy-amaninamide) is formed by deprotection of the amanitin derivative HDP 30.2546.

In FIG. 12 shows a construct based on the amanitin derivative HDP 30.2548 (S-deoxy-5'-hydroxy-amaninamide) with a cleavable linker attached to the 5'-hydroxy group and a terminal maleimide group as an example of a reactive Y group to link said construct to a target-binding fragment.

In FIG. 13 shows the cytotoxicity of HDP 30.2347, HDP 30.2371 and HDP 30.2602 ADCs targeting A) HER-2/neu in SKBR-3 (HER-2/neu+++) cells and in JIMT-1 (HER-2/neu+) cells, B) PSMA in LnCap (PSMA+++) cells and 22rv1 (PSMA++) cells, and C) CD19 in Raji (CD19+++) cells and Nalm-6 (CD19++) cells, compared to ADC HDP 30.1699 (comprising the same cleavable linker, same as HDP 30.2347, HDP 30.2371 and HDP 30.2602, but alpha-amanitin instead of the amanitin derivatives described above).

Detailed disclosure of the present invention

Before the present invention is described in detail below, it should be noted that the present invention is not limited to the specific methodology, protocols, and reagents described herein, as they may vary. It should also be noted that the terminology used herein is for the sole purpose of describing specific embodiments and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise indicated, all technical and scientific terms used in this document have the same meanings as commonly understood by a person skilled in the art.

In particular, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kölbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

Throughout this specification and the following claims, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" are intended to include the specified integer, compositions or steps, or groups of integers, compositions or steps, while any additional integer, composition or step, or group of integers, compositions or steps may also optionally be present, including embodiments in which there are no additional an integer, composition or stage, or a group of integers, compositions or stages. In such latter embodiments, the term "comprising" is used in conjunction with "consisting of".

Some documents are cited throughout the text of this description. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, sequence representations as GenBank Access Numbers, etc.), whether higher or lower, thereby incorporated by reference in its entirety to the extent possible under the relevant patent law. Nothing herein should be construed as an admission that the invention is not entitled to precede such disclosure by virtue of the existence of prior invention.

Next, the present invention will be described. In the following parts, various aspects of the present invention are defined in more detail. Each aspect thus defined may be combined with any other aspect or aspects, unless expressly stated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The present invention is based on the surprising observation that Hpi variants can be synthesized that allow the introduction of hydroxyl groups during the synthesis of amanitine derivatives.

Thus, according to one aspect, the present invention relates to a hydroxy-substituted derivative of 2-carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole in accordance with formula I

in which R1 is selected from alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl and substituted heteroaryl;

P ₁ represents hydrogen or a protective group;

R ₂ represents hydrogen or a protective group; And

R2 is selected from OH, OR1 and a polypeptide chain consisting of 1-7 amino acid residues.

In the context of the present invention, the term "protecting group" refers to a group that attaches to the nitrogen atom at positions 1 or 8 of the central hexahydropyrrolo[2,3-b]indole moiety to block the nitrogen atom from reacting with other reactants used for synthesis and/ or for further functionalization of compounds according to formula I. The person of ordinary skill in the art is well aware of the various protecting groups that are available in the art, which can be attached to the appropriate nitrogen atom when the nitrogen atom is to be protected, and which can then be cleaved off when N -Protection is no longer required. In specific embodiments, an N-acylating reagent is used for N-protection. Thus, in such embodiments, P1 and/or P2 are acyl groups. In other specific embodiments, an N-alkylating reagent is used for N-protection. Thus, in such embodiments, P1 and/or P2 is an alkyl group.

In specific embodiments, the protecting group P1 or P2, if present, is independently selected from Boc, PhCH ₂ OCO-, CH ₂ =CHCH ₂ O-CO-, and trityl.

In a particular embodiment, the hydroxy group at position 3a and the hydrogen atom at position 8a are in cis configuration with respect to the functional group attached at position 2. In another specific embodiment, the hydroxy group at position 3a and the hydrogen atom at position 8a are in trans configuration with respect to the functional group a group attached at the 2-position. In a specific embodiment, the amino-substituted derivative according to the present invention is a mixture of cis- and trans-configuration compounds.

In a specific embodiment, the R1-C(=O)-O-substituent is attached at position 4 in formula I.

In a specific embodiment, the substituent R1-C(=O)-O- is attached at position 5 in formula I.

In a specific embodiment, the R1-C(=O)-O-substituent is attached at position 6 in Formula I.

In a specific embodiment, the R1-C(=O)-O-substituent is attached at position 7 in Formula I.

According to a second aspect, the present invention relates to a method for the synthesis of a linear precursor comprising eight amino acid residues of an amanitin derivative comprising a hydroxylated tryptophan fragment, comprising the step of using a hydroxy-substituted derivative of 2-carboxy-3a-hydroxy-1,2,3,3a ,8,8a-hexahydropyrrolo[2,3-b]indole according to the present invention in the peptide synthesis of said precursor.

According to a third aspect, the present invention relates to a process for the synthesis of an amanitin derivative comprising a hydroxylated tryptophan moiety, comprising the steps of:

(i) initiating or causing the formation of a bond between a cysteine residue and a tryptophan fragment of a linear precursor in accordance with the present invention; And

(ii) initiating or causing the formation of said amanitin derivative by reacting an N-terminal linear precursor according to the present invention with the C-terminus of said precursor.

According to additional aspects, the present invention relates to individual amanitin precursors synthesized as shown in the examples, in particular to compounds HDP 30.2569, HDP 30.2572 and solid phase intermediates synthesized in accordance with [00145].

According to a particular embodiment, the method of the present invention further comprises oxidizing the sulfur atom of the cysteine moiety to form a sulfoxide or sulfone, in particular a sulfoxide.

According to a fourth aspect, the present invention relates to an amanitine derivative comprising a hydroxylated tryptophan moiety which is selected from (i) S-deoxy-4'-hydroxy-amanin, 4'-hydroxy-amanin, S-deoxy-5'-hydroxy- amanine, 5'-hydroxy-amanin, S-deoxy-7'-hydroxy-amanin, 7'-hydroxy-amanin, (ii) S-deoxy-4'-hydroxy-amaninamide, 4'-hydroxy-amaninamide, S- deoxy-5'-hydroxy-amaninamide, 5'-hydroxy-amaninamide, S-deoxy-7'-hydroxy-amaninamide and 7'-hydroxy-amaninamide, (iii) amanitine derivative according to (i), wherein the moiety is free carboxylic acid of amino acid 1 is converted to a -C(=O)OR1 carboxylic acid ester or to a -C(=O)NH-OR1 moiety, wherein R1 is selected from alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl, and substituted heteroaryl.

In a specific embodiment, the amanitine derivative of the present invention is selected from S-deoxy-5'-hydroxy-amaninamide, 5'-hydroxy-amaninamide, S-deoxy-5'-hydroxy-amaninamide, and 5'-hydroxy-amaninamide.

According to a fifth aspect, the present invention relates to a conjugate comprising (a) an amanitin derivative comprising a hydroxylated tryptophan moiety according to the present invention; (b) a target-binding moiety; and (c) optionally a linker linking said amanitin derivative and said target-binding moiety.

According to a sixth aspect, the present invention relates to a pharmaceutical composition comprising amanitin according to the present invention or a conjugate according to the present invention.

According to a seventh aspect, the present invention relates to an amanitin derivative according to the present invention, a conjugate according to the present invention, or a pharmaceutical composition according to the present invention for use in the treatment of a cancer in a patient, in particular, the cancer is selected from the group consisting of of breast cancer, pancreatic cancer, cholangiocarcinoma, colon and rectal cancer, lung cancer, prostate cancer, ovarian cancer, gastric cancer, kidney cancer, malignant melanoma, leukemia and malignant lymphoma.

According to an eighth aspect, the present invention relates to a construct comprising (a) an amanitine derivative according to the present invention and (b) a linker moiety bearing a Y reactive group for linking said amanitine derivative to a target-binding moiety.

In the context of the present invention, the term "amanitin" refers to a particular group of amatoxins. In the context of the present invention, the term "amatoxin" includes all cyclic peptides consisting of 8 amino acids isolated from the genus Amanita and described by Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Biochem. 5 ( 1978) 185-260). In the context of the present invention, the term "amanithins" refers to a bicyclic structure which is based on an aspartic acid or asparagine residue at position 1, a proline residue, in particular a hydroxyproline residue at position 2, an isoleucine, hydroxyisoleucine or dihydroxyisoleucine at position 3, a hydroxytryptophan residue at position 4, glycine residues at positions 5 and 7, an isoleucine residue at position 6, and a cysteine residue at position 8, in particular, to a cysteine derivative that is oxidized to a sulfoxide or sulfone derivative (see figure 1 for numbering and illustrative examples of amanitins), and, in addition, includes all its chemical derivatives; in addition, all its semi-synthetic analogues; in addition, all its synthetic analogues built from building blocks in accordance with the basic structure of natural compounds (cyclic, 8 amino acids), in addition, all synthetic or semi-synthetic analogues that include non-hydroxylated amino acids instead of hydroxylated amino acids (provided that according to at least one hydroxyl group is present on the phenyl ring of the tryptophan moiety), in addition, all synthetic or semi-synthetic analogs, in which case any such derivative or analog is functionally active in inhibition of mammalian RNA polymerase II.

Thus, in the context of the present invention, the term "eight amino acid residues of an amanitin derivative" refers to certain amino acids that form the bicyclic structure of an amanitin polypeptide.

Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferred amatoxins are those with a functional group (for example, a derivative with a carboxyl group or a carboxylic acid, such as carboxamide or hydroxamic acid, an amino group, a hydroxy group, a thiol or thiol capturing group) that can be reacted with linker molecules or target-binding moieties, defined above. Amatoxins which are particularly suitable for the conjugates according to the present invention are the di-deoxy variants of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanullin or amanullic acid, or the mono-deoxy variants of amanin, amaninamide, γ- amanin or γ-amaninamide as shown in FIG. 1, as well as their salts, chemical derivatives, semi-synthetic analogues and synthetic analogues.

In a specific embodiment, the hydroxy-substituted derivative, the amanitin derivative comprising a hydroxylated tryptophan moiety, and/or the conjugate of the present invention is greater than 90% pure, in particular greater than 95%, more particularly greater than 98% or even more than 99%.

In the context of the present invention, the term "purity" refers to the total amount of, for example, conjugates present. A purity of more than 90%, for example, means that in 1 mg of a composition comprising a conjugate according to the present invention, there is more than 90%, i. e. more than 900 μg of such a conjugate. The rest, i.e. impurities may include unreacted starting material and other reactants, solvents, cleavage products and/or by-products.

According to a specific embodiment, a composition comprising a hydroxy-substituted derivative, an amanitin derivative comprising a hydroxylated tryptophan moiety, and/or a conjugate according to the present invention comprises more than 100 mg, in particular more than 500 mg, and more specifically, more than 1 g of such a hydroxy-substituted derivative, an amanitin derivative comprising a hydroxylated tryptophan moiety, and/or a conjugate. Thus, a trace amount of, for example, a conjugate according to the present invention, which may possibly be present in complex preparations of prior art conjugates, is explicitly excluded.

As used herein, the term "target-binding fragment" refers to any molecule or portion of a molecule that can specifically bind to a target molecule or target epitope. Preferred target-binding fragments in the context of the present application are (i) antibodies or antigen-binding fragments thereof; (ii) antibody-like proteins; and (iii) nucleic acid aptamers. "Target-binding moieties" suitable for use in accordance with the present invention typically have a molecular weight of 40,000 Da (40 kDa) or greater.

As used herein, a first compound (e.g., an antibody) is considered to "specifically bind" to a second compound (e.g., an antigen such as a target protein) if it has a dissociation constant K _D with said second compound of 100 μM or less, in particular 50 µM or less, in particular 30 µM or less, in particular 20 µM or less, in particular 10 µM or less, in particular 5 µM or less, more specifically 1 µM or less, more specifically 900 nM or less, more specifically 800 nM or less, more specifically 700 nM or less, more specifically 600 nM or less, more specifically 500 nM or less, more specifically 400 nM or less, more specifically 300 nM or less, more specifically 200 nM or less, still more specifically 100 nM or less, even more specifically 90 nM or less, even more specifically 80 nM or less, even more specifically 70 nM or less, even more specifically 60 nM or less, even more specifically 50 nM or less, even more specifically 40 nM or less, even more specifically 30 nM or less, even more specifically 20 nM or less, and even more specifically 10 nM or less.

As used herein, the terms "target molecule" and "target epitope", respectively, refer to an antigen and an antigen epitope, respectively, that are specifically bound by a target-binding moiety. In particular, the target molecule is a tumor-associated antigen, in particular an antigen or epitope, which is present on the surface of one or more types of tumor cells in an increased concentration and/or in a different steric configuration compared to the surface of non-tumor cells. In particular, the specified antigen or epitope is present on the surface of one or more types of tumor cells, but not on the surface of non-tumor cells. In specific embodiments, the target-binding fragment specifically binds to an epitope of an antigen selected from PSMA, CD 19, CD269, Lewis ^a sialyl, HER-2/neu, and epithelial cell adhesion molecule (EpCAM). In other embodiments, said antigen or epitope is predominantly expressed on cells involved in autoimmune diseases. In such specific embodiments, the target-binding moiety specifically binds to an epitope of the IL-6 receptor (IL-6R).

As used herein, the term "antibody or antigen-binding fragment thereof" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. to molecules that contain an antigen-binding site that immunospecifically binds an antigen. Thus, the term "antigen-binding fragments thereof" refers to an antibody fragment that includes at least a functional antigen-binding domain. Also included are immunoglobulin-like proteins that are selected by techniques including, for example, phage display, for specific binding to a target molecule, for example, a target protein selected from PSMA, CD19, CD269, Lewis ^a sialyl, HER-2/neu and ErSAM. The immunoglobulin molecules of the present invention may 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 the immunoglobulin molecule. "Antibodies and antigen-binding fragments thereof" suitable for use in accordance with the present invention include, without limitation, 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 fragments, fragments generated with a Fab expression library, diabody or tetrabody (Holliger P. et al., Proc Natl Acad Sci USA. 90 (1993) 6444-8), nonotela, anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against antibodies according to the present invention), and epitope-binding fragments of any of the above.

In some embodiments, antigen binding fragments are human antigen binding fragments of antibodies according to the present invention and include, but are not limited to, Fab, Fab' and F(ab') ₂ , Fd, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (dsFv) and fragments that include either the VL or VH domain. Antigen binding fragments of antibodies, including single chain antibodies, may include variable domain(s) alone or in combination with all or part of the following: hinge, CL, CH1, CH2 and CH3 domains. Also included in the present invention are antigen binding fragments also comprising any combination of variable domain(s) with the hinge, CL, CH1, CH2 and CH3 domains.

Antibodies useful in accordance with the present invention may be derived from any animal, including birds and mammals. In particular, the antibody source is human, rodent (eg, mouse, rat, guinea pig, or rabbit), chicken, pig, sheep, goat, camel, cow, horse, donkey, cat, or dog. Particularly preferred is the origin of the antibodies from a human or a mouse. As used herein, the term "human antibodies" includes antibodies having the amino acid sequence of human immunoglobulin and includes antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and which do not express endogenous immunoglobulins, as described, for example, in US patent No. 5939598 Kucherlapati and Jakobovits.

The term "antibody-like protein" refers to a protein that has been genetically engineered (eg, by loop mutagenesis) to specifically bind to a target molecule. Typically, such an antibody-like protein includes at least one variable peptide loop attached to both ends of the protein backbone. This dual structural constraint greatly enhances the binding affinity of the antibody-like protein to levels comparable to those of the antibody. The length of the variable peptide loop, as a rule, is 10-20 amino acids. The scaffold protein can be any protein having good solubility properties. In particular, the scaffold protein is a small globular protein. Antibody-like proteins include, but are not limited to, affitels, anticalins, and engineered ankyrin repeat proteins (for a review, see Binz et al., Nat Biotechnol. 2005, 1257-68). Antibody-like proteins can be obtained from large mutant libraries, for example, can be obtained from large phage display libraries, and can be isolated in analogy to conventional antibodies. Also, antibody-like binding proteins can be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins.

The term "nucleic acid aptamer" refers to a nucleic acid molecule that has been genetically engineered through repeated cycles of in vitro selection or SELEX (systematic ligand evolution by exponential enrichment) to bind to a target molecule (for a review, see Brody and Gold, J Biotechnol 74 (2000) 5-13). The nucleic acid aptamer can be a DNA or RNA molecule. Aptamers may contain modifications, for example, modified nucleotides such as 2'-fluoro-substituted pyrimidines.

The term "linker" in the context of the present invention refers to a structure that connects two components, each of which is attached to one end of the linker. In case the linker is a bond, direct binding of the amatoxin to the antibody may decrease the ability of the amatoxin to interact with RNA polymerase II. In specific embodiments, the linker increases the distance between the two components and reduces the steric interaction between these components, such as in this case between the antibody and amatoxin. In specific embodiments, the linker has a continuous chain of 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 or 30 atoms) in its backbone, i.e. linker length is defined as the shortest connection, as measured by the number of atoms or bonds between the amatoxin moiety and the antibody, where one side of the linker backbone has reacted with the amatoxin and the other side is available for reaction or has reacted with the antibody. In the context of the present invention, the linker is in particular C _1-20 alkylene, C _1-20 heteroalkylene, C _2-20 alkenylene, C _2-20 heteroalkenylene, C _2-20 alkynylene, C _2-20 -heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene or heteroaralkylene group, optionally substituted. The linker may include one or more building blocks such as carboxamide, ester, ether, thioether, disulfide, urea, thiourea, hydrocarbon moieties, and the like. The linker may also include combinations of two or more of these building blocks. Each of these structural elements can be present in the linker more than once, for example, two times, three times, four times, five times or six times. In some embodiments, the linker may include a disulfide bond. It will be appreciated that the linker must be attached either in one step or in two or more successive steps to the amatoxin and the antibody. To this end, this linker will carry two groups, in particular at the proximal and distal ends, which (i) can form a covalent bond with a group present in one of the components to be linked, in particular to an activated group on the amatoxin or a target-binding peptide; or (ii) which are or can be activated to form a covalent bond with a group on the amatoxin. Therefore, it is preferred that the chemical groups are at the distal and proximal end of the linker as a result of such a coupling reaction, eg ester, ether, urethane, peptide bond, etc.

In particular embodiments, the linker L is a linear chain of 1 to 20 atoms independently selected from C, O, N, and S, in particular 2 to 18 atoms, more in particular 5 to 16 atoms, and even more in particular 6 to 15 atoms. In specific embodiments, at least 60% of the atoms in the linear chain are C atoms. In specific embodiments, the atoms in the linear chain are linked by single bonds.

In specific embodiments, linker L is an alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, or heteroaralkylene group comprising from 1 to 4 heteroatoms selected from N, O, and S, wherein said linker is optionally substituted.

The term "alkylene" refers to divalent straight chain saturated hydrocarbon groups having from 1 to 20 carbon atoms, including groups having from 1 to 10 carbon atoms. In some embodiments, the alkylene groups may be lower alkylene groups. The term "lower alkylene" refers to alkylene groups having from 1 to 6 carbon atoms, and in some embodiments from 1 to 5 or from 1 to 4 carbon atoms. Examples of alkylene groups include, without limitation, methylene (-CH ₂ -), ethylene (-CH ₂ -CH ₂ -), n-propylene, n-butylene, n-pentylene, and n-hexylene.

The term "alkenylene" refers to divalent straight chain groups having 2 to 20 carbon atoms in which at least one of the carbon-carbon bonds is a double bond, while the other bonds may be single bonds or additional double bonds. The term "alkynylene" as used herein refers to groups having 2 to 20 carbon atoms in which at least one of the carbon-carbon bonds is a triple bond, while the other bonds may be single, double or additional triple bonds. Examples of alkenylene groups include ethenylene (-CH=CH-), 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 3-butenylene, and the like. Examples of alkynylene groups include ethynylene, 1-propynylene, 2-propynylene, etc.

As used herein, the term "cycloalkylene" refers to a divalent ring that is part of any stable monocyclic or polycyclic system in which such a ring has 3 to 12 carbon atoms but not a heteroatom, and in which such ring is fully saturated, and the term " "cycloalkenylene" refers to a divalent ring that is part of any stable monocyclic or polycyclic system in which such ring has 3 to 12 carbon atoms but not a heteroatom, and in which such ring is at least partially unsaturated (but excluding any arylene ring) . Examples of cycloalkylenes include, without limitation, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene. Examples of cycloalkenylenes include, without limitation, cyclopentenylene and cyclohexenylene.

As used herein, the terms "heterocycloalkylene" and "heterocycloalkenylene" refer to a divalent ring that is part of any stable monocyclic or polycyclic system in which such a ring has from 3 to about 12 atoms, and in which such ring consists of carbon atoms and at least at least one heteroatom, in particular at least one heteroatom independently selected from the group consisting of N, O and S, wherein heterocycloalkylene refers to such a ring which is fully saturated and heterocycloalkenylene refers to such a ring which is at least least partially unsaturated (but excluding any arylene or heteroarylene ring).

The term "arylene" means a divalent ring or ring system that is part of any stable monocyclic or polycyclic system in which such a ring or ring system has from 3 to 20 carbon atoms, but no heteroatoms, while the ring or ring system consists of an aromatic moiety , as determined by the "4n+2" rule of π-electrons, including phenylene.

As used herein, the term "heteroarylene" refers to a divalent ring or ring system that is part of any stable monocyclic or polycyclic system in which such ring or ring system has from 3 to 20 atoms, and the ring or ring system consists of an aromatic moiety , as defined by the "4n+2" π-electron rule, and contains carbon atoms and one or more nitrogen, sulfur and/or oxygen heteroatoms.

In the context of the present invention, the term "substituted" means an indication that one or more hydrogens present in the backbone of the linker are substituted with a choice from the specified group(s), provided that the specified normal valency of the atom or that of the corresponding atom of the group that is substituted , is not exceeded, and that the substitution results in a stable connection. The term "optionally substituted" means that the linker is either unsubstituted or substituted, as defined herein, with one or more substituents as defined herein. When the substituent is a keto (or oxo, ie =O) group, a thio or imino group or the like, the two hydrogens on the linker backbone are replaced. Illustrative substituents include, for example, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, acyl, aroyl, heteroaroyl, carboxyl, alkoxy, aryloxy, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, halogen, complex ( thio)ether, cyano, phosphoryl, amino, imino, (thio)amido, sulfhydryl, alkylthio, acylthio, sulfonyl, sulfate, sulfonate, sulfamoyl, sulfonamido, nitro, azido, haloalkyl, including perfluoroalkyl (such as trifluoromethyl), haloalkoxy , alkylsulfanyl, alkylsulfinyl, alkyl sulfonyl, alkylsulfonylamino, arylsulfonoamino, phosphoryl, phosphate, phosphonate, phosphinate, alkylcarboxy, alkylcarboxyamide, oxo, hydroxy, mercapto, amino (optionally mono- or disubstituted, e.g., alkyl, aryl, or heteroaryl), imino , carboxamide, carbamoyl (optionally mono- or disubstituted, for example, alkyl, aryl, or heteroaryl), amidino, aminosulfonyl, acylamino, aroylamino, (thio)ureido, (arylthio)ureido, alkyl(thio)y reido, cycloalkyl(thio)ureido, aryloxy, aralkoxy, or -O(CH ₂ ) _n -OH, -O(CH ₂ ) _n -NH ₂ , -O(CH ₂ ) _n COOH, -(CH ₂ ) _n COOH, -C(O)O(CH ₂ ) _n R, -(CH ₂ ) _n N(H)C(O)OR or -N(R)S(O) ₂ R, wherein n is 1-4, and R is independently selected from hydrogen, -alkyl, -alkenyl, -alkynyl, -cycloalkyl, -cycloalkenyl, -(C-linked-heterocycloalkyl), -(C-linked-heterocycloalkenyl), -aryl, and -heteroaryl, with the possibility of multiple degrees of substitution . One skilled in the art will appreciate that substituents such as heterocycloalkyl, aryl, heteroaryl, alkyl, etc., or functional groups such as -OH, -NHR, etc., may themselves be substituted if necessary. It will also be appreciated by those skilled in the art that substituted moieties themselves may also be substituted if desired.

In specific embodiments, linker L includes a moiety selected from one of the following moieties: disulfide (-S-S-), ether (-O-), thioether (-S-), amine (-NH-), ester (-O -C(=O)- or -C(=O)-O-), carboxamide (-NH-C(=O)- or -C(=O)-NH-), urethane (-NH-C(= O)-O- or -O-C(=O)-NH-) and the urea moiety (-NH-C(=O)-NH-).

According to specific embodiments of the present invention, the linker L includes a number m of groups selected from the list: alkylene, alkenylene, alkynylene, cycloalkylene, heteroalkylene, heteroalkenylene, heteroalkynylene, heterocycloalkylene, arylene, heteroarylene, aralkylene and heteroaralkylene group, each group optionally be independently substituted, the linker further comprises a number n of moieties independently selected from one of the following moieties: disulfide (-S-S-), ether (-O-), thioether (-S-), amine (-NH- ), ester (-O-C(=O)- or -C(=O)-O-), carboxamide (-NH-C(=O)- or -C(=O)-NH-), urethane ( -NH-C(=O)-O- or -O-C(=O)-NH-) and urea fragment (-NH-C(=O)-NH-), while m=n+1. In specific embodiments, m is 2 and n is 1, or m is 3 and n is 2. In specific embodiments, the linker includes 2 or 3 unsubstituted alkylene groups and 1 or 2, respectively, disulfide, ether, thioether, amine, ester, carboxamide, urethane or urea moieties linking unsubstituted alkylene groups.

In a specific embodiment, linker L does not contain a heteroarylene group.

In specific embodiments, the C atoms in the straight chain are independently part of optionally substituted methylene groups (-CH ₂ -). In such specific embodiments, the optional substituents are independently selected from halo and C _1-6 alkyl, in particular methyl.

In specific embodiments, linker L is a stable linker.

In the context of the present invention, the term "stable linker" refers to a linker that is stable (i) in the presence of enzymes and (ii) in an intracellular reducing environment.

In specific embodiments, the stable linker does not contain (i) an enzyme-cleavable substructure and/or (ii) a disulfide group. In such specific embodiments, the linker is up to 12 atoms in length, in particular 2 to 10, more in particular 4 to 9, and most in particular 6 to 8 atoms.

In other specific embodiments, the linker is a cleavable linker.

In the context of the present invention, the term "cleavable linker" refers to (i) a linker that is cleavable by an enzyme, or (ii) a reducible linker. In specific embodiments, the term refers exclusively to a linker that is cleavable by an enzyme (and not to a reducible linker).

In the context of the present invention, the term "linker that is cleavable ... by an enzyme" refers to a linker that can be cleaved by an enzyme, in particular a lysosomal peptidase such as cathepsin B, which results in the intracellular release of a toxin cargo conjugated to the targeting antibody, after internalization (see Dubowchik et al., Bioconjug Chem. 13 (2002) 855-69). In specific embodiments, the cleavable linker comprises a dipeptide selected from Phe-Lys, Val-Lys, Phe-Ala, Val-Ala, Phe-Cit, and Val-Cit, in particular, wherein the cleavable linker further comprises p- aminobenzyl (PAB) spacer between dipeptides and amatoxin.

According to such specific embodiments, the cleavable linker includes the structure L ¹ -L*-L ² in which L* is a p-aminobenzyl dipeptide fragment, L ¹ is the part of the linker that connects L* to amatoxin, in particular, in which L ¹ is connected to L* through -NH- or -O- group, in particular, the group -C (=O) -NH-, -C (=O) -NH-O- or -C (=O) - O-, and in which L ² is the part of the linker that connects L* to the target-binding fragment, in particular, in which L ² is connected to L* through the fragment -(CH ₂ ) _m -, while m is an integer selected from 1-8, in particular 1-5, or through the fragment -(CH ₂ -CH ₂ O) _n -, while n is an integer selected from 1-3, in particular 1-2. For example, in the case of a cleavable linker comprising a Val-Ala dipeptide, the structure of L ¹ -L*-L ² is as follows

In other such specific embodiments, L* includes a Val-Lys dipeptide and has the following structure

In specific embodiments, the L ¹ linker is a linear chain of 1 to 4 atoms independently selected from C, O, N, and S, in particular 1 to 3 atoms, more in particular 1 to 2 atoms, and more specifically only 1 atom. In specific embodiments, at least 50% of the atoms in the linear chain are C atoms. In specific embodiments, the atoms in the linear chain are linked by single bonds. In a particular embodiment, L is a -NH- or -O- group which is part of the amatoxin. In specific embodiments, L1 is a -O- group derived from a hydroxy group attached at the 4', 5', 6', or 7' position of the central tryptophan moiety. In specific embodiments, L ¹ is a -O- group derived from a hydroxyl group that is part of the carboxylic acid group of amino acid residue 1 of the amanine derivative according to the present invention. In specific embodiments, L ¹ is an -NH- group derived from an amino group that is part of the carboxamide group of amino acid residue 1 of the amaninamide derivative according to the present invention. In specific embodiments, L ¹ is a -O- group derived from a hydroxyl group that is part of amino acid residue 3 of an amanine or amaninamide derivative according to the present invention.

In the context of the present invention, the term "reducing linker" refers to a linker that can be cleaved in an intracellular reducing environment, in particular, a linker that contains disulfide groups, which results in the intracellular release of a toxin cargo conjugated to a target-binding moiety, after internalization with intracellular recovery medium (see Shen et al., J. Biol. Chem. 260 (1985) 10905-10908). In specific embodiments, the reducible linker includes a moiety

in which R1 - R4 are independently selected from H and methyl.

In such specific embodiments, such a cleavable linker is up to 20 atoms in length, in particular 6 to 18, more in particular 8 to 16, and most in particular 10 to 15 atoms. In such specific embodiments, the portion of the linker linking the amatoxin of the present invention and the cleavable disulfide group is a linear chain of 3 or 4 atoms, in particular 3 C atoms. In specific embodiments, 3 or 4 C atoms in a linear chain linked by single bonds. In specific embodiments, the linker is an n-propylene group.

In specific embodiments, said linker is present and attached on one side to the hydroxyl group attached to the phenyl ring of the central tryptophan moiety, i.e. to the 4', 5' or 7' hydroxy substituent.

In other specific embodiments, said linker is present and attached on one side at the position of the amanitin derivative of the present invention, said position being selected.

(i) in the case of S-deoxy-4'-hydroxy-amaninamide, 4'-hydroxy-amaninamide, S-deoxy-5'-hydroxy-amaninamide, 5'-hydroxy-amaninamide, S-deoxy-7'-hydroxy- amaninamide and 7'-hydroxy-amaninamide from the nitrogen atom of the carboxamide group at the γ C atom of amino acid 1 of amatoxin (amide bond);

(ii) in the case of S-deoxy-4'-hydroxy-amanin, 4'-hydroxy-amanin, S-deoxy-5'-hydroxy-amanin, 5'-hydroxy-amanin, S-deoxy-7'-hydroxy- amanine, T-hydroxy-amanine from the oxygen atom of the acidic group at the γ C atom of amino acid 1 of amatoxin (ester bond);

(iii) in the case of an amanitin derivative according to the present invention in which the free carboxylic acid moiety of amino acid 1 has been converted to a -C(=O)NH-OR1 moiety, from the oxygen atom of the hydroxamic acid group at the γ C atom of amino acid 1 of amatoxin;

(iv) from the oxygen atom of the hydroxy group at the δ C atom of the amino acid 3 of amatoxin, in particular via an ester bond, an ether bond or a urethane bond; or

(v) from the ring nitrogen of amino acid 4.

Linking a linker to a target-binding moiety can be achieved by a number of methods well known to one of ordinary skill in the art, particularly in the art of antibody-drug conjugates (ADCs).

In specific embodiments, said linker is coupled to the target-binding moiety via a urea moiety (...-linker-NH-C(=O)-NH-target-binding moiety). In such specific embodiments, the urea moiety is the result of the reaction of a primary amine originally present in the target-binding moiety, such as the amino group of a lysine side chain, with a carbamic acid derivative ... -linker-NH-C(O)-Z, wherein Z is a leaving group which may be replaced by a primary amine.

In other specific embodiments, said linker is present and coupled to the target-binding moiety via a thioether moiety (...-linker-S-target-binding moiety). Thus, according to such embodiments, the present invention relates to a conjugate of the general formula:

amanitin-L-X*-S-Tbm,

in which amanitin is an amanitin derivative according to the present invention, L is a linker, X* is a moiety resulting from the combination of a thiol group with a thiol-reactive group, S is a sulfur atom of said thiol group, in particular a thiol group a cysteine amino acid residue, and Tbm is a target-binding fragment, in particular an antibody or a functional antibody fragment, comprising said cysteine amino acid residue. In specific embodiments, said cysteine amino acid residue (i) is located in an antibody domain selected from CL, CH1, CH2, and CH3; (ii) is located at a position in which the germline sequence showing the closest homology to the sequence of the specified antibody domain contains an amino acid residue other than cysteine; and (iii) located in a position that is exposed to the solvent.

In the context of the present invention, the term "thiol-reactive group" refers to a group that selectively reacts with a thiol group, for example, free cysteine in an antibody, in particular at a pH value in the range from 6.0 to 8.0, more specifically at a value of pH in the range of 6.5 to 7.5. In particular, the term "selectively" means that less than 10% of the coupling reactions of a molecule comprising a thiol-reactive group with an antibody comprising at least one free cysteine residue are coupling reactions with non-cysteine residues, in the antibody, such as lysine residues, in particular less than 5%, more specifically less than 2%. In specific embodiments, the thiol-reactive group is selected from bromoacetamide, iodoacetamide, maleimide, maleimide having a leaving group at position 3, in particular a leaving group selected from -Br and a substituted thiol (see, for example, US patent No. 9295729), 1,2-dihydropyridazine-3,6-dione having a leaving group at position 4, in particular a leaving group selected from -Br and a substituted thiol (see, for example, US patent No. 9295729), methylsulfonylbenzothiazole, methylsulfonylphenyltetrazole, methylsulfonylphenyloxadiazole ( see Toda et al., Angew. Chem. Int. Ed. Engl., 52 (2013) 12592-6), 3-aryl propionitrile (see Kolodych et al, Bioconjugate Chem. 2015, 26, 197-200) and 5 -nitro-pyridin-2-yl-disulfide (...-L-S-S-(5-nitro-pyridin-2-yl).

In particular embodiments, said position or functional group, which is connected to the linker on one side and which can be connected directly or indirectly to a position or functional group present in the target-binding moiety, is a moiety that can react with two thiol groups present in one target-binding fragment or in two target-binding fragments. In particular embodiments, the thiol-reactive groups are maleimide having two leaving groups at positions 3 and 4, in particular selected from 3,4-dibromomaleimide, 3,4-bis(arylthio)-maleimide, in particular 3,4 -diphenylthio-maleimide and 3,4-bis(heteroarylthio)-maleimide, in particular 3,4-bis(2-pyridinylsulfanyl)-maleimide. In other specific embodiments, the thiol-reactive groups are 1,2-dihydropyridazine-3,6-dione having two leaving groups at positions 4 and 5, specifically selected from 4,5-bromo-1,2-dihydropyridazine- 3,6-dione, 4,5-bis(arylthio)-1,2-dihydropyridazin-3,6-dione, in particular 4,5-diphenylthio-1,2-dihydropyride azine-3,6-dione and 4 ,5-bis(heteroarylthio)-1,2-dihydropyride of azine-3,6-dione, in particular 4,5-bis(2-pyridinylsulfanyl)-1,2-dihydropyridazine-3,6-dione.

In specific embodiments, the moiety resulting from the coupling of the thiol group with the thiol-reactive group is selected from thiol-substituted acetamide; thiol-substituted succinimide; thiol-substituted succinamic acid; thiol-substituted heteroaryl, in particular thiol-substituted benzothiazole, thiol-substituted phenyltetrazole and thiol-substituted phenyloxadiazole and a disulfide in which one sulfur atom comes from a cysteine residue of an antibody. In specific embodiments, the fragment resulting from the combination of the thiol group with the thiol-reactive group is a thiol-substituted succinimide.

In specific embodiments, the linker L in the L-X*-S moiety present in the general formula of paragraph [00101] is selected from the following group of moieties:

(amanitin side) -(CH ₂ ) ₂ -SS-(CH ₂ ) ₂ -XS- (Tbm side);

(amanitin side) -(CH ₂ ) ₃ -SS-(CH ₂ ) ₂ -XS- (Tbm side);

(amanitin side) -(CH ₂ ) ₂ -SS-(CH ₂ ) ₃ -XS- (Tbm side);

(amanitin side) -(CH ₂ ) ₃ -SS-(CH ₂ ) ₃ -XS- (Tbm side);

(amanitin side) -(CH ₂ ) ₄ -SS-(CH ₂ ) ₄ -XS- (Tbm side);

(amanitin side) -(CH ₂ ) ₂ -CMe ₂ -SS-(CH ₂ ) ₂ -XS- (Tbm side);

(amanitin side) -(CH ₂ ) ₂ -SS-CMe ₂ -(CH ₂ ) ₂ -XS- (Tbm side);

(amanitin side) -(CH ₂ ) ₃ -SS- (Tbm side);

(amanitin side) -CH ₂ -C ₆ H ₄ -NH-Cit-Val-CO(CH ₂ ) ₅ -XS- (Tbm side)

(amanitin side) -CH ₂ -C ₆ H ₄ -NH-Ala-Val-CO(CH ₂ ) ₅ -XS- (Tbm side);

(amanitin side) -CH ₂ -C ₆ H ₄ -NH-Ala-Val-CO(CH ₂ ) ₂ -XS- (Tbm side);

(amanitin side) -CH ₂ -C ₆ H ₄ -NH-Ala-Phe-CO(CH ₂ ) ₂ -XS- (Tbm side);

(amanitin side) -CH ₂ -C ₆ H ₄ -NH-Lys-Phe-CO(CH ₂ ) ₂ -XS- (Tbm side);

(amanitin side) -CH ₂ -C ₆ H ₄ -NH-Cit-Phe-CO(CH ₂ ) ₂ -XS- (Tbm side);

(amanitin side) -CH ₂ -C ₆ H ₄ -NH-Val-Val-CO(CH ₂ ) ₂ -XS- (Tbm side);

(amanitin side) -CH ₂ -C ₆ H ₄ -NH-Ile-Val-CO(CH ₂ ) ₂ -XS- (Tbm side);

(amanitin side) -CH ₂ -C ₆ H ₄ -NH-His-Val-CO(CH ₂ ) ₂ -XS- (Tbm side);

(amanitin side) -CH ₂ -C ₆ H ₄ -NH-Met-Val-CO(CH ₂ ) ₂ -XS- (Tbm side);

(amanitin side) -CH ₂ -C ₆ H ₄ -NH-Asn-Lys-CO(CH ₂ ) ₂ -XS- (Tbm side);

in which -NH- and -CO- flanking the dipeptide sequences represent the amino and carbonyl fragments of the linker forming amide bonds with the carboxy and amino ends of the dipeptide, respectively.

In the context of the present invention, the term "moiety resulting from the combination of a thiol group with a thiol-reactive group" refers to a structure that results from (i) nucleophilic substitution of a Y leaving group present on a thiol-reactive group with a sulfur atom of a cysteine residue, e.g. , bromoacetamide group, iodoacetamide, 4,6-dichloro-1,3,5-triazin-2-ylamino group, alkylsulfone or heteroarylsulfone; (ii) adding the HS group of the cysteine residue to an activated double bond of a thiol-reactive group, e.g., maleimide, or (iii) disulfide exchange of the activated disulfide or methanethiosulfonate with the sulfur atom of the cysteine residue, e.g., pyridine-2-thiol, 5-nitropyridine -2-thiol or methanesulfinate as leaving group; or (iv) any other chemical reaction that produces a stable bond between the sulfur atom of a cysteine residue and a reactive moiety that is part of a thiol-reactive group.

The initial fragment resulting from the coupling of the thiol group may optionally be further derivatized, for example, the succinimidyl thioester derived from maleimide may be hydrolyzed to succinamic acid thioesters with the following general structures

In other specific embodiments, site-specific coupling can be achieved by reducing the disulfide bridge present in the target-binding moiety and by reacting the two cysteine residues with the X* bridging moiety present in the amanitin-L-X* construct (see Badescu et al. Bridging disulfides for stable and defined antibody drug conjugates Bioconjugate Chemistry 25 (2014) 1124-1136.

In such an embodiment, site-specific coupling can be achieved by reducing the disulfide bridge present in the target-binding moiety and by reacting the two cysteine residues with the X* bridging moiety present in the amanitin-L-X* construct, specifically in which X * represents

(See Bryden et al., Bioconjug Chem, 25 (2014) 611-617; Schumacher et al., Org Biomol Chem, 2014, 7261-7269).

According to another specific embodiment, the coupling is achieved by regiospecific coupling of the amino group present in the linker with the glutamine residue present in the target-binding moiety via transaminase, in particular by coupling with the glutamine Q295 antibody.

In a specific embodiment, the coupling is achieved by site-specific conjugation to target-binding moieties that include N-glycan side chains. In particular, the N-glycan side chain can be degraded enzymatically, followed by trans-glycosylation with azido-galactose. Using click chemistry, such a modified target-binding moiety can be coupled to suitably modified amanitin-LX* constructs wherein X* is, for example, dibenzocyclooxine (DIBO), or a similar moiety including a C triple bond. -WITH. For example, the amanitin-L-NH ₂ construct can be coupled to DIBO-SE,

by nucleophilic substitution of the hydroxysuccinimide moiety. The resulting DIBO-modified linker construct can then be coupled to the azido derivative mentioned above. In an alternative embodiment, the target-binding moiety can be modified by incorporating a non-naturally occurring amino acid that allows click chemistry, in particular by incorporating para-azidomethyl-L-phenylalanine (pAMF).

In particular embodiments, the L to -L-X* linker is a linear chain of at least 5, in particular at least 10, more in particular 10 to 20 atoms independently selected from C, O, N and S, in particular 10 to 18 atoms, more specifically 10 to 16 atoms, and even more specifically 10 to 15 atoms. In specific embodiments, at least 60% of the atoms in the linear chain are C atoms. In specific embodiments, the atoms in the linear chain are linked by single bonds.

In alternative embodiments, the position or functional group that can be directly or indirectly attached to the position or functional group present on the target-binding moiety is not an ethynyl group, or, more broadly, is not an alkynyl group, or is not are a group that can be reacted with a 1,3-dipole in a 1,3-dipolar cycloaddition (click chemistry).

In other specific embodiments, the site-specific coupling of the amanitin-L-X* construct to the target-binding moiety can be achieved by incorporating a non-naturally occurring amino acid containing a keto group, in particular p-acetylphenylalanine (pAcPhe), into the target-binding moiety. , and by reacting such a modified target-binding moiety with an amanitin-L-X* construct wherein X* is a hydroxylamine moiety.

In a further embodiment, the formyl group can be introduced by a formylglycine generating enzyme (FGE) that is highly selective for the cysteine group in recognizing the CxPxR sequence to form an aldehyde tag. Such an aldehyde label can be reacted with the appropriate X* group present in the amanitin-L-X* construct, in particular where X* is

(See Agarwal et al., Bioconjugate Chem 24 (2013) 846-851).

According to a second aspect, the present invention relates to a pharmaceutical composition comprising a conjugate according to the present invention.

According to a third aspect, the present invention relates to a conjugate according to the present invention for use in the treatment of a cancer in a patient, in particular, wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, cholangiocarcinoma, colon and rectal cancer. , lung cancer, prostate cancer, ovarian cancer, gastric cancer, kidney cancer, malignant melanoma, leukemia, and malignant lymphoma.

As used herein, the terms "treating", "treating" or "treating" a disease or disorder means one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing the development of characteristic symptoms of the disorder(s) being treated; (c) inhibiting worsening of characteristic symptoms of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients who previously had the disorder(s); and (e) limiting or preventing the recurrence of symptoms in patients who previously had symptoms of the disorder(s).

As used herein, the term "treatment" may include administration of a conjugate or pharmaceutical composition according to the present invention to a patient, where "administration" includes in vivo administration as well as administration directly to ex vivo tissue, such as venous grafts.

In specific embodiments, a therapeutically effective amount of a conjugate according to the present invention is used.

The term "therapeutically effective amount" is an amount of a therapeutic agent sufficient to achieve the desired goal. The effective amount of a given therapeutic agent will vary depending on factors such as the nature of the agent, the route of administration, the size and species of the animal receiving the therapeutic agent, and the purpose of administration. The effective amount in each individual case can be empirically determined by a person skilled in the art in accordance with established methods in the prior art.

According to another aspect, the present invention relates to a pharmaceutical composition comprising an amanitin derivative according to the present invention or a conjugate according to the present invention of an amanitin derivative with a target-binding moiety, and further comprising one or more pharmaceutically acceptable diluents, carriers, auxiliaries , fillers, binders, lubricants, glidants, disintegrants, adsorbents and/or preservatives.

The term "pharmaceutically acceptable" means approved by federal or state government regulators or listed in the USP or other generally accepted pharmacopoeia for use in animals, and more specifically in humans.

In specific embodiments, the pharmaceutical composition is used in the form of a systemically administered drug. It includes parenterals, which include but are not limited to injectables and infusions. The injectable preparations are either in the form of ampoules or in the form of so-called ready-to-use injectable preparations, for example ready-to-use syringes or disposable syringes, and furthermore in multi-use pierceable vials. Administration of injectable preparations may be in the form of subcutaneous (s.c), intramuscular (i.m.), intravenous (i.v.) or intradermal (i.e.) administration. In particular, suitably suitable injectable formulations can be prepared in the form of a suspension of crystals, solutions, nanoparticle systems or colloidal dispersed systems, such as, for example, hydrosols.

Injectable formulations may also be prepared as concentrates which may be dissolved or dispersed in aqueous isotonic diluents. The infusion can also be given in the form of isotonic solutions, fatty emulsions, liposome formulations, and microemulsions. Like injectables, infusion formulations can also be prepared in the form of diluted concentrates. The injectable formulations may also be administered in the form of continuous infusions or in inpatient or outpatient therapy, for example with mini-pumps.

Parenteral drug formulations, e.g. albumin, plasma, dilator, surfactants, organic diluents, pH influencing agents, complexing agents or polymeric agents, can be added, particularly as agents to influence the adsorption of the target-binding conjugate. fragment and toxin of the present invention with proteins or polymers, or they may be added to reduce the adsorption of the targeting fragment and toxin conjugates of the present invention to materials such as injectables or packaging materials such as plastic or glass.

The amanitin derivatives of the present invention, comprising a target-binding moiety, can be coupled to microcarriers or nanoparticles in parenteral formulations, such as finely divided particles based on poly(meth)acrylates, polylactates, polyglycolates, polyamino acids, or polyetherurethanes. Parenteral formulations can also be modified as depot formulations, for example based on a "multiple particle delivery system" if the target-binding moiety-toxin conjugates of the present invention are administered in finely divided, dispersed and suspended form, respectively, or as a suspension of crystals in a drug formulation or based on a "single particle delivery system" if the target-binding fragment-toxin conjugate of the present invention is formulated into, for example, a tablet or rod, which is subsequently implanted. Such implants or depot drugs in single-particulate and multi-particulate formulations often consist of so-called biodegradable polymers such as, for example, polyesters of lactic acid and glycolic acid, polyether urethanes, polyamino acids, poly(meth)acrylates or polysaccharides.

The adjuvants and carriers added during the preparation of the pharmaceutical compositions according to the invention formulated as parenteral preparations are preferably aqua sterilisata (sterile water), substances that affect the pH value, such as organic or inorganic acids or bases, and also their salts, pH buffering agents, isotonizing agents such as sodium chloride, sodium hydrogen carbonate, glucose and fructose, tensides and surfactants, respectively, and emulsifiers, such as partial fatty acid esters and polyoxyethylene sorbitols (eg Tween®) or, for example, fatty acid esters of polyoxyethylenes (eg Cremophor®), fatty oils such as, for example, peanut oil, soybean oil or castor oil, synthetic fatty acid esters, such as, e.g. ethyl oleate, isopropyl myristate and neutral oil (e.g. Miglyol) as well as polymer auxiliaries substances such as gelatin, dextran, polyvinylpyrrolidone, additives which increase the solubility of organic solvents such as propylene glycol, ethanol, N,N-dimethylacetamide, propylene glycol or complexing agents such as citrate and urea, preservatives, such as hydroxypropyl ester and benzoic acid methyl ester, benzyl alcohol, antioxidants such as sodium sulfite and stabilizers such as EDTA.

When formulating pharmaceutical compositions according to the present invention as suspensions according to a preferred embodiment, thickeners are added to prevent settling of the target-binding moiety-toxin conjugates according to the present invention, or tensides and polyelectrolytes to ensure resuspendability of the precipitates and/or complexing agents, such as for example , EDTA. It is also possible to obtain complexes of the active ingredient with various polymers. Examples of such polymers are polyethylene glycol, polystyrene, carboxymethylcellulose, Pluronics® or fatty acid esters, and polyethylene glycol sorbitol. Target-binding moiety-toxin conjugates of the present invention may also be incorporated into liquid formulations in the form of inclusion compounds, for example with cyclodextrins. In particular embodiments, dispersants may be added as additional adjuvants. Dispersants such as mannitol, dextran, sucrose, human albumin, lactose, PVP, or a variety of gelatins can be used to prepare lyophilisates.

EXAMPLES

Hereinafter, the present invention is explained in more detail by non-limiting examples.

A. Total synthesis of S-deoxy-α-amanitine HDP 30.0735

1. Preparation of N-(tert-butoxycarbonyl)-L-6-acetoxy-tryptophan HDP 30.2550

590.0 mg (2.68 mmol) of 6-hydroxy-L-tryptophan (CAS: 13567-14-1) were suspended in 30 ml of 1,4-dioxane/water 1:1 (v/v). 2.68 ml (2.68 mmol) of 1 N hydrochloric acid were added under argon. NaOH once at ambient temperature. The resulting yellow solution was then treated with 574.6 μl (2.68 mmol) Boc anhydride (Boc ₂ O) and shaken for 24 hours at room temperature. The solution was acidified with 1 N. hydrochloric acid to pH 2.4 and was extracted 3 times with 25 ml of ethyl acetate. The combined ethyl acetate extracts were washed with saturated NaCl solution and dried over MgSO ₄ . Filtration and evaporation to dryness gave 785.0 mg of crude material. The crude N-Boc-6-hydroxy-L-tryptophan was dissolved in 4.91 ml (4.91 mmol) 1 H. NaOH and treated with 463.2 ml (500.3 mg, 4.90 mmol) acetic anhydride. The reaction mixture was agitated for 3 hours under argon and acidified with 5% citric acid. The aqueous phase was extracted three times with 25 ml of ethyl acetate, washed with saturated NaCl solution and dried over MgSO ₄ . Filtration and evaporation gave 635 mg of a crude solid.

The crude product was purified by flash chromatography on a 330 g silica gel column (detection wavelength 254 nm) with a gradient of CH ₂ Cl ₂ + 1% acetic acid - CH ₂ Cl ₂ /MeOH (15:1) + 1% acetic acid and after evaporation with toluene, 564.4 mg (56% yield) of a white powder were obtained.

MS (ESI ^- ) - revealed: 361.08 [M-H] ^- ; calculated: 362.15 (C ₁₈ H ₂₂ N ₂ O ₆ ).

2. Preparation of cis,trans-1-(tert-butoxycarbonyl)-2-carboxy-3а-hydroxy-6-acetoxy-1,2,3,3а,8,8а-hexahydropyrrolo[2,3-b]indole, cis -HDP 30.2555 and trans-HDP 30.2555 (cis,trans-6-acetoxy-Hpi)

Photooxidation

Photooxidation was carried out with a 400 W high pressure sodium lamp (Sinus X400 lamp, 230 V, 400 W; 55,000 lumens at 1.3 m) or alternatively with a tungsten-halogen lamp (500 W). A filter solution (CuCl ₂ -CaCO ₃ ) was used, which cut off light with λ < 490 nm, for a tungsten-halogen lamp.

Methylene blue or rose bengal was used as a sensitizing dye.

The reaction was carried out in a 500 ml cylindrical reaction vessel with a heat exchange jacket made of ^DURAN® borosilicate glass, with a flat bottom and a flat laboratory flange (DN) with two connectors with GL 18 thread. The distance from the lamp to the reaction vessel was 15 cm, and the reaction the temperature was in the range of 3-4°C.

The final product was purified on a Teledyne ISCO flash chromatography system with a 330 g Silica Redi Sept Flash column (Teledyne ISCO part no. 69-2203-330). The solvents CH ₂ Cl ₂ , CH ₃ OH, CH ₃ COOH were of standard purity for HPLC or BP.

Dry oxygen (99.5% pure) was bubbled through the reaction mixture at a rate of 2-4 liters per minute.

943.0 mg (2.60 mmol) of N-(tert-butoxycarbonyl)-L-6-acetoxy-tryptophan HDP 30.2550 and 100 mg of rose bengal were dissolved in 500 ml of methanol and cooled to 3°C using a Huber cryostat with glycol/ water as a cooling medium. The reaction solution was illuminated with a 400 W high pressure sodium lamp. During illumination, a slow stream of oxygen was bubbled through the reaction solution. After 5 hours of illumination, the oxidation and cooling were stopped and the reaction medium was treated with 10 ml of dimethyl sulfide. The mixture was shaken for 2 hours and evaporated to dryness using a rotary evaporator with a water bath temperature of 35°C. The dark red residue was further dried under high vacuum to 1.20 g of a crystalline solid. The crude product was purified on a 330 g silica gel column (detection wavelength 254 nm) with a gradient of CH ₂ Cl ₂ + 5% acetic acid - CH ₂ Cl ₂ /MeOH (30:1) + 5% acetic acid. 380 mg of cis-HDP 30.2555 and 290 mg of trans-HDP 30.2555 were eluted and co-evaporated with toluene. After lyophilization in tert-butanol, both isomers were obtained as off-white powders.

Cis-1-(tert-butoxycarbonyl)-2-carboxy-3а-hydroxy-6-acetoxy-1,2,3,3а,8,8а-hexahydropyrrolo[2,3-b]indole (cis-HDP 30.2555)

380 mg cis-HDP 30.2555, yield: 39%.

¹ H-NMR (400 MHz, CD3OD, δ = ppm)

δ = 1.22, 1.44, 1.54 [s, 9H, C(CH ₃ ) ₃ ]; 2.23 (s, 3H, OCOCH ₃ ); 2.46-2.63 (m, 2H, CH ₂ ); 4.14-4.29 (m, 1H, 2-H); 5.35 (s, 1H, 8a-H); 6.39-6.46 (m, 2H, 7-H, 5-H); 7.20-7.24 (m, 1H, 4-H).

¹³ C-NMR (100 MHz, CD ₃ OD, δ = ppm)

δ = 20.93, 28.45, 31.12, 42.80, 61.12, 69.44, 82.21, 85.82, 87.93, 104.97, 112.98, 124.84, 129.42, 151.51, 154.04, 155.97, 171.34, 175.79.

MS (ESI ⁺ ) - detected: 378.92 [MN] ⁺ ; calculated: 378.14 (C ₁₈ H ₂₂ N ₂ O ₇ ).

MS (ESI ⁺ ) - detected: 401.17 [M+Na] ⁺ ; calculated: 401.14 (C ₁₈ H ₂₂ N ₂ NaO ₇ ).

UV/VIS (CH ₃ OH): λ _max = 296 nM, 239 nM, 215 nM.

_λmin = 266 nM, 227 nM.

Trans-1-(tert-butoxycarbonyl)-2-carboxy-3а-hydroxy-6-acetoxy-1,2,3,3а,8,8а-hexahydropyrrolo[2,3-b]indole (trans-HDP 30.2555)

290 mg trans-HDP 30.2555, yield: 30%.

¹ H-NMR (400 MHz, CD ₃ OD, δ = ppm)

δ = 1.22, 1.45, 1.54 [s, 9H, C(CH ₃ ) ₃ ]; 2.22 (s, 3H, OCOCH ₃ ); 2.55-2.73 (m, 2H, CH ₂ ); 4.51-4.57 (m, 1H, 2-H); 5.21-5.24 (s, 1H, 8a-H); 6.36-6.41 (m, 2H, 7-H, 5-H); 7.17-7.18 (m, 1H, 4-H).

¹³ C-NMR (100 MHz, CD ₃ OD, δ = ppm)

δ = 20.95, 28.50, 31.12, 42.47, 60.97, 69.44, 82.06, 84.84, 87.54, 104.74, 112.67, 125.03, 128.70, 152.31, 154.22, 156.00, 171.23, 174.67.

MS (ESI ⁺ ) - detected: 379.00 [MH] ⁺ ; calculated: 378.14 (C ₁₈ H ₂₂ N ₂ O ₇ ).

MS (ESI ⁺ ) - detected: 401.17 [M+Na] ⁺ ; calculated: 401.14 (C ₁₈ H ₂₂ N ₂ NaO ₇ ).

MS (ESI ⁺ ) - detected: 779.00 [M+Na] ⁺ ; calculated: 779.28 (C ₃₆ H ₄₄ N ₄ Na ₂ O ₁₄ ).

UV/VIS (CH ₃ OH): λ _max = 299 nM, 241 nM, 215 nM.

_λmin = 268 nM, 228 nM.

3. Preparation of S-deoxy-α-amanitine HDP 30.0735

Stage 1 HDP 30.0013

FmocHypOH (10.0 g, 28.3 mmol) was suspended in 100 ml of 80% MeOH and CS ₂ CO ₃ (4.6 g, 14.1 mmol) was added. The suspension was shaken at 50°C for 30 minutes until complete dissolution. The reaction mixture was concentrated to dryness and redissolved in 100 ml DMF. Allyl bromide (1.6 ml, 3.6 g, 29.7 mmol) was added dropwise and the reaction mixture was agitated overnight at room temperature. DMF was distilled off and the residue was dissolved in tert-butyl methyl ether. The precipitates were filtered off and the clear solution was absorbed onto celite before column chromatography. The compound was purified on 220 g silica gel with an n-hexane/ethyl acetate gradient. Yield: 11.5 g, 100%.

Stage 2 HDP 30.0400

HDP 30.0013 (5.0 g, 14.1 mmol), pyridinium 4-toluenesulfonate (1.33 g, 5.3 mmol) was added to a suspension of 1,3-dihydro-2H-pyran-2-yl-methoxymethyl resin (5 .0 g, 1.0 mmol/g THP resin) in 40 ml dichlopentane. The reaction mixture was shaken at 80° C. overnight. After cooling, the resin was filtered and thoroughly washed with dichlopentane, dimethylformamide, acetonitrile, dichloromethane and tert-butyl methyl ether.

The loading was 0.62 mmol/g (as determined by UV spectroscopy of the fluorenemethyl group after deprotection).

Stage 3. HDP 30.2569 (solid phase synthesis)

Resin Pretreatment

HDP 30.0400 (0.31 g, 0.25 mmol) was treated with N,N-dimethylbarbituric acid (241 mg, 1.55 mmol) and Pd(PPh ₃ ) ₄ (35 mg, 0.03 mmol). The resin was shaken overnight at room temperature. Thereafter, the resin was thoroughly washed with dichloromethane, DMF, acetonitrile, dichloromethane and tert-butyl methyl ether and dried under reduced pressure.

Combination procedure

All reactants and reagents were dissolved in dichloromethane/DMF (1:1, v/v). HDP 30.0477 [see WO 2014/009025] (102 mg, 0.30 mmol) was dissolved in 6.0 ml of dichloromethane/N,N-dimethylformamide and treated with 4.0 ml of 0.2N. PyBOP/HOBt solution and 2 ml DIEA (40% in DMF). After adding 2.0 ml of N,N-dimethylformamide, the reaction mixture was heated to 50°C for 8 minutes using microwave radiation (20 W, CEM microwave reactor) and washed with N,N-dimethylformamide after coupling.

Removing the Fmoc protecting group

Deprotection was performed by adding 6.0 ml of 20% piperidine in N,N-dimethylformamide at 50° C. over 10 minutes. The resin was washed with N,N-dimethylformamide (no deprotection after final amino acid coupling).

All other amino acids were combined according to the above protocol, the weights are shown below.

After completion, the resin was finally transferred to a frit-bottomed syringe, washed with DCM, and dried under reduced pressure.

Resin release and B-ring formation

A solution of 5 ml TFA, 5 ml DCM plus 10% MeOH was aspirated into the resin and shaken for 15 minutes at ambient temperature. The solution was dispensed into a 50 ml reaction flask and the resin was washed with TFA/DCM 1:1 plus 10% MeOH once and poured into the same flask. The reaction flask was shaken for 16 hours. Triisopropylsilane (0.5 ml) was added and the reaction mixture was concentrated in vacuo. The residue was dissolved in 500 μl of MeOH and the peptide was precipitated in 50 ml of ice-cold TBME. After centrifugation, the supernatant was decanted, the pellet was washed once with 50 ml of TBME and dried under reduced pressure.

The precipitate was solubilized in 2 ml of methanol and purified by reverse phase preparative column chromatography. The methanol was distilled off under reduced pressure and the remaining aqueous phase was freeze-dried.

Yield: 136.5 mg, 62.5%.

MS (ESI+) - detected: 1047.4 [M+H] ⁺ ; calculated: 1047.4 (C ₄₅ H ₆₃ N ₁₀ O ₁₇ S).

HPLC: 91.9% area.

Step 4: Cyclization (A-ring formation, HDP 30.2572)

The above freeze-dried monocyclic intermediate (136 mg, 130 µmol) was dissolved in 16 ml DMF and treated with diphenylphosphoryl azide (DPPA, 131 µl, 1300 µmol, 10 equiv.) and diisopropylethylamine (DIEA, 162 µl, 1300 µmol, 10 equiv.) . The reaction mixture was agitated for 16 hours and quenched with 500 μl of water upon completion. The transformation was monitored by HPLC. The mixture was concentrated under reduced pressure, redissolved in 1 ml of methanol and purified by reverse phase preparative column chromatography.

Yield: 55.3 mg, 41%.

MS (ESI+) - detected: 1029.33 [M+Na] ⁺ ; calculated: 1030.10 (C ₄₅ H ₆₁ N ₁₀ O ₁₆ S).

HPLC: 99.2% area.

Step 5: Deprotection of the acetate (HDP 30.0735)

[001] HDP 30.2572 (55.3 mg, 53.7 µmol) was dissolved in 7N. methanol solution of NH ₃ (3.0 ml) and shaken. The conversion was checked using HPLC/MS. After completion (6-8 hours), the reaction mixture was concentrated in vacuo, resuspended in 100 μl of MeOH and purified using preparative HPLC.

Yield: 14.1 mg, 29%.

HPLC: 100%.

MS (ESI+) detected: 903.3 [M+H] ⁺ ; calculated: 902.9 (C ₃₉ H ₅₄ N ₁₀ O ₁₃ S).

revealed: 925.33 [M+Na] ⁺ .

4. Preparation of 6'-((3-maleidopropanamido)-Val-Ala-PAB)-S-deoxy-α-amanitine (HDP 30.2371)

Stage 1 HDP 30.2364

S-Deoxy-α-amanitin HDP 30.0735 (30 mg, 33.2 µmol) and HDP 30.1690 (WO 2016/142049, 78 mg, 133 µmol = 4.0 equiv.) were dissolved in 1500 µl of dry dimethylacetamide (DMA). A 0.2 M solution of cesium hydrogen carbonate in water (199 µl, 1.2 equiv.) was added in one portion and the mixture was shaken at room temperature. After 1.5 and 4 hours, additional portions of 99 μl (0.6 equiv.) of cesium carbonate solution were added.

After 18 hours the solvent was evaporated using high vacuum.

The residue was dissolved in 400 μl of methanol and added dropwise to ice-cold MTBE (10 ml). After settling at 0° C. for 10 minutes, the resulting precipitate was isolated by centrifugation (4 minutes, 4000×g). The supernatant was discarded, the pellet was resuspended in additional MTBE (10 ml) and centrifugation was repeated. The vacuum dried crude product was dissolved in 400 μl methanol and purified by preparative HPLC on a Phenomenex Luna-C ₁₈ (2) 10 μm (250 x 21.2 mm) column with a gradient of 5% - 100% methanol in water. The product fractions were combined and brought to 23 mg (70%) of the product as an amorphous solid.

MS (ESI+) [M+Na] ⁺ - detected: 1430.58; calculated: 1430.65 (C ₆₅ H ₉₇ N ₁₃ NaO ₁₈ SSi).

By evaporating the early elution peak, 5 mg (17%) of starting material was recovered.

Stage 2 HDP 30.2366

HDP 30.2364 (9.47 mg, 6.72 µmol) was dissolved in 750 µl of trifluoroacetic acid (TFA). After 2 minutes, the volatiles were removed in vacuo, the residue was dissolved in 750 μl of water and 3% ammonia was added dropwise until pH 10 and precipitation were reached.

The solution was freeze dried and purified by preparative HPLC sequentially (Phenomenex Luna-C ₁₈ (2), 10 µm, 250×21.2 mm, gradient 5-100% methanol (0.05% TFA) in water (0.05 % TFA)) to give 7.72 mg (89% based on TFA salt) of product.

MS (ESI ⁺ ) [MH] ⁺ - detected: 1178.42; calculated: 1178.53 (C ₅₄ H ₇₆ N ₁₃ O ₁₅ S).

Stage 3 HDP 30.2371

HDP 30.2366 (4.01 mg, 3.10 µmol) was dissolved in 500 µl dry DMF.

An ester of 3-(maleimido)propionic acid and N-hydroxysuccinimide (BMPS, 1.65 mg, 2 equiv.) was dissolved in 100 μl of DMF followed by the addition of 2.1 μl of DIPEA.

After shaking for 1 hour, the reaction mixture was dropped into 10 ml of ice-cold methyl tert-butyl ether. The tube was kept on ice for 10 minutes and centrifuged at 4000×g. The supernatant liquid was removed and the precipitate was washed with 10 ml of fresh methyl tert-butyl ether.

The vacuum dried precipitate was purified by RP-18 HPLC with a gradient of 5-100% methanol in water. The pure fractions were evaporated to dryness and lyophilized from 1 ml t-butanol/water to give 2.70 mg (65%) of the product as a colorless powder.

MS (ESI+) [MH] ⁺ - detected: 1329.3; calculated: 1329.6 (C ₆₁ H ₈₁ N ₁₄ O ₁₈ S).

[M+Na] ⁺ - revealed: 1351.5; calculated: 1351.5 (C ₆₁ H ₈₀ N ₁₄ NaO ₁₈ S).

B. Total synthesis of S-deoxy-5'-hydroxy-amaninamide HDP 30.2548

1. Obtaining N-(tert-butoxycarbonyl)-L-5-acetoxy-tryptophan HDP 30.2531

800.0 mg (3.63 mmol) of 5-hydroxy-L-tryptophan (CAS: 4350-09-8) were suspended in 40 ml of 1,4-dioxane/water 1:1 (v/v). Under argon, 3.64 ml (3.64 mmol) of 1 N hydrochloric acid were added. NaOH at a time at ambient temperature. The resulting yellow solution was then treated with 779.0 μl (794.0 mg, 3.63 mmol) Boc anhydride (Boc ₂ O) and shaken for 24 hours at room temperature. The solution was acidified with 1 N. hydrochloric acid to pH 2.4 and extracted 3 times with 35 ml of ethyl acetate. The combined ethyl acetate extracts were washed with saturated NaCl solution and dried over MgSO ₄ . Filtration and evaporation to dryness gave 1.26 g of crude material. The crude N-Boc-6-hydroxy-L-tryptophan was dissolved in 7.87 ml (7.87 mmol) 1N. NaOH and treated with 743.0 μl (802.4 mg, 7.86 mmol) of acetic anhydride. The reaction mixture was agitated for 3 hours under argon and acidified with 5% citric acid. The aqueous phase was extracted three times with 25 ml of ethyl acetate, washed with saturated NaCl and dried over MgSO ₄ . Filtered and evaporated.

The crude solid was purified by flash chromatography on a 330 g silica gel column (detection wavelength 254 nm) with a gradient of CH ₂ Cl ₂ + 1% acetic acid - CH ₂ Cl ₂ /MeOH (15:1) + 1% acetic acid and after coevaporation with toluene, 1020.0 mg (78% yield) of a white powder was obtained.

MS (ESI ^- ) - revealed: 361.08 [M-H] ^- ; calculated: 362.15 (C ₁₈ H ₂₂ N ₂ O ₆ ).

¹ H-NMR (400 MHz, CD ₃ OD, δ=ppm).

δ = 1.39 (s, 9H, C(CH ₃ ) ₃ ); 2.27 (s, 3H, OCOCH ₃ ); 3.08-3.31 (m, 2H, CH ₂ ); 4.39-4.41 (m, 1H, 2-H); 6.81-6.83; 7.13; 7.24-7.26 (m, 2H, 6-H, 7-H); 7.30-7.32 (1H, 4-H).

2. Preparation of cis,trans-1-(tert-butoxycarbonyl)-2-carboxy-3а-hydroxy-5-acetoxy-1,2,3,3а,8,8а-hexahydropyrrolo[2,3-b]indole, cis -HDP 30.2536 and trans-HDP 30.2536 (cis,trans-5-acetoxy-Hpi)

Photooxidation

Photooxidation was carried out with a 400 W high pressure sodium lamp (Sirius X400 lamp, 230 V, 400 W; 55,000 lumens at 1.3 m) or alternatively with a tungsten-halogen lamp (500 W). A filter solution (Cucl ₂ -CaCO ₃ ) was used, which cut off light with λ < 490 nm, for a tungsten-halogen lamp.

Methylene blue or rose bengal was used as a sensitizing dye. The reaction was carried out in a 500 ml cylindrical reaction vessel with a heat exchange jacket made of ^DURAN® borosilicate glass, with a flat bottom and a flat laboratory flange (DN) with two connectors with GL 18 thread. The distance from the lamp to the reaction vessel was 15 cm, and the reaction the temperature was in the range of 3-4°C.

The final product was purified on a Teledyne ISCO flash chromatography system with a 330 g Silica Redi Sept Flash column (Teledyne ISCO part no. 69-2203-330). The solvents CH ₂ Cl ₂ , CH ₃ OH, CH ₃ COOH were of standard purity for HPLC or BP. Dry oxygen (99.5% pure) was bubbled through the reaction mixture at a rate of 2-4 liters per minute.

1.20 g (3.17 mmol) of N-(tert-butoxycarbonyl)-L-5-acetoxy-tryptophan HDP 30.2531 and 100 mg of rose bengal were dissolved in 500 ml of methanol and cooled to 3°C using a Huber cryostat with glycol/ water as a cooling medium. The reaction solution was illuminated with a 400 W high pressure sodium lamp. During illumination, a slow stream of oxygen was bubbled through the reaction solution. After 4 hours of illumination, the oxidation and cooling were stopped and the reaction medium was treated with 20 ml of dimethyl sulfide. The mixture was shaken for 2 hours and evaporated to dryness using a rotary evaporator with a water bath temperature of 35°C. The dark red residue was further dried under high vacuum to a crystalline solid. The crude product was purified on a 330 g silica gel column (detection wavelength 254 nm) with a gradient of CH ₂ Cl ₂ + 5% acetic acid - CH ₂ Cl ₂ /MeOH (30:1) + 5% acetic acid.

178 mg cis-HDP 30.2536 and 132 mg trans-HDP 30.2536 were eluted and co-evaporated with toluene. After lyophilization in tert-butanol, both isomers were obtained as off-white powders.

Cis-1-(tert-butoxycarbonyl)-2-carboxy-3а-hydroxy-5-acetoxy-1,2,3,3а,8,8а-hexahydropyrrolo[2,3-b]indole (cis-HDP 30.2536)

178 mg cis-HDP 30.2536 yield: 15%.

¹ H-NMR (400 MHz, CD ₃ OD, δ=ppm).

δ = 1.44, 1.54 (s, 9H, C(CH ₃ ) ₃ ); 2.23 (s, 3H, OCOCH ₃ ); 2.45-2.62 (m, 2H, CH ₂ ); 4.18-4.33 (m, 1H, 2-H); 5.37 (s, 1H, 8a-H); 6.63-6.67; 6.82-6.84; (m, 2H, 7-H, 6-H); 6.96-6.98 (m, 1H, 4-H).

¹³ C-NMR (100 MHz, CD ₃ OD, δ=ppm).

δ = 20.86, 28.46, 31.12, 42.81, 61.21, 82.14, 85.60, 111.49, 117.83, 123.98, 132.77, 144.99, 148.03, 156.07, 172.01, 175.89.

MS (ESI ⁺ ) - detected: 379.00 [MN] ⁺ ; calculated: 378.14 (C ₁₈ H ₂₂ N ₂ O ₇ ).

MS (ESI ⁺ ) - detected: 401.17 [M+Na] ⁺ ; calculated: 401.14 (C ₁₈ H ₂₂ N ₂ NaO ₇ ).

MS (ESI ^- ) - detected: 377.17 [MH] ^- ; calculated: 378.14 (C ₁₈ H ₂₂ N ₂ O ₇ ).

UV/VIS (CH ₃ OH): λ _max = 299 nm, 242 nm, 207 nm.

_λmin = 270 nm, 222 nm.

Trans-1-(tert-butoxycarbonyl)-2-carboxy-3а-hydroxy-5-acetoxy-1,2,3,3а,8,8а-hexahydropyrrolo[2,3-b]indole (trans-HDP 30.2536)

132 mg trans-HDP 30.2536, yield: 11%.

¹ H-NMR (400 MHz, CD ₃ OD, δ=ppm).

δ = 1.47, 1.54 [s, 9H, C(CH ₃ ) ₃ ]; 2.21 (s, 3H, OCOCH ₃ ); 2.50-2.70 (m, 2H, CH ₂ ); 4.51-4.56 (m, 1H, 2-H); 5.22-5.26 (s, 1H, 8a-H); 6.58-6.63; 6.80-6.81 (m, 6-H, 7-H); 6.92 (s, 1H, 4-H).

¹³ C-NMR (100 MHz, CD ₃ OD, δ=ppm).

δ = 20.88, 28.50, 31.12, 42.60, 61.11, 82.05, 85.38, 111.26, 117.88, 124.07, 131.94 144.66, 148 .95, 156.11, 172.01, 174.81.

MS (ESI ⁺ ) - detected: 379.00 [MH] ⁺ ; calculated: 378.14 (C ₁₈ H ₂₂ N ₂ O ₇ ).

MS (ESI ⁺ ) - detected: 401.17 [M+Na] ⁺ ; calculated: 401.14 (C ₁₈ H ₂₂ N ₂ NaO ₇ ).

MS (ESI ^- ) - detected: 377.17 [MH] ^- ; calculated: 378.14 (C ₁₈ H ₂₂ N ₂ O ₇ ).

UV/VIS (CH ₃ OH): λ _max = 302 nm, 244 nm, 208 nm.

_λmin = 272 nm, 224 nm.

3. Preparation of S-deoxy-5'-hydroxy-amaninamide HDP 30.2548

Stage 1 HDP 30.0013

FmocHypOH (10.0 g, 28.3 mmol) was suspended in 100 ml of 80% MeOH and Cs ₂ CO ₃ (4.6 g, 14.1 mmol) was added. The suspension was shaken at 50°C for 30 minutes until complete dissolution. The reaction mixture was concentrated to dryness and redissolved in 100 ml DMF. Allyl bromide (1.6 ml, 3.6 g, 29.7 mmol) was added dropwise and the reaction mixture was agitated overnight at room temperature. DMF was distilled off and the residue was dissolved in tert-butyl methyl ether. The precipitates were filtered and the clear solution was absorbed onto celite before column chromatography. The compound was purified on 220N silica gel with an n-hexane/ethyl acetate gradient.

Yield: 11.5 g, 100%.

Stage 2 HDP 30.0400

HDP 30.0013 (5.0 g, 14.1 mmol), pyridinium 4-toluenesulfonate (1.33 g, 5.3 mmol) was added to a suspension of 1,3-dihydro-2H-pyran-2-yl-methoxymethyl resin (5 .0 g, 1.0 mmol/g THP resin) in 40 ml of dichlopentane. The reaction mixture was shaken at 80° C. overnight. After cooling, the resin was filtered and washed thoroughly with dichlopentane, dimethylformamide, acetonitrile, dichloromethane and tert-butyl methyl ether.

The loading was 0.62 mmol/g (as determined by UV spectroscopy of the fluorenemethyl group after deprotection).

Stage 3. HDP 30.2544 (solid phase synthesis)

Resin Pretreatment

HDP 30.0400 (0.31 g, 0.25 mmol) was treated with N,N-dimethylbarbituric acid (241 mg, 1.55 mmol) and Pd(PPh ₃ ) ₄ (35 mg, 0.03 mmol). The resin was shaken overnight at room temperature. Thereafter, the resin was thoroughly washed with dichloromethane, DMF, acetonitrile, dichloromethane and tert-butyl methyl ether and dried under reduced pressure.

Combination procedure

All reactants and reagents were dissolved in dichloromethane/DMF (1:1, v/v). HDP 30.0477 [see WO 2014/009025] (102 mg, 0.30 mmol) was dissolved in 6.0 ml of dichloromethane/N,N-dimethylformamide and treated with 4.0 ml of 0.2N. PyBOP/HOBt solution and 2 ml DIEA (40% in DMF). After adding 2.0 ml of N,N-dimethylformamide, the reaction mixture was heated to 50°C for 8 minutes using microwave radiation (20 W, CEM microwave reactor) and washed with N,N-dimethylformamide after coupling.

Removing the Fmoc protecting group

Deprotection was performed by adding 6.0 ml of 20% piperidine in N,N-dimethylformamide at 50° C. over 10 minutes. The resin was washed with N,N-dimethylformamide (no deprotection after final amino acid coupling).

All other amino acids were combined according to the above protocol, the weights are shown below.

After completion, the resin was finally transferred to a frit-bottomed syringe, washed with DCM, and dried under reduced pressure.

Resin release and B-ring formation

A solution of 5 ml TFA, 5 ml DCM plus 10% MeOH was aspirated into the resin and shaken for 15 minutes at ambient temperature. The solution was dispensed into a 50 ml reaction flask and the resin was washed with TFA/DCM 1:1 plus 10% MeOH once and poured into the same flask. The reaction flask was shaken for 16 hours. Triisopropylsilane (0.5 ml) was added and the reaction mixture was concentrated in vacuo. The residue was dissolved in 500 μl of MeOH and the peptide was precipitated in 50 ml of ice-cold TBME. After centrifugation, the supernatant was decanted, the pellet was washed once with 50 ml of TBME and dried under reduced pressure.

The precipitate was solubilized in 2 ml of methanol and purified by reverse phase preparative column chromatography. The methanol was distilled off under reduced pressure and the remaining aqueous phase was freeze-dried.

Yield: 75.1 mg, 35.9%.

MS (ESI ⁺ ) - detected: 1047.4 [M+H] ⁺ ; calculated: 1047.4 (C ₄₅ H ₆₃ N ₁₀ O ₁₇ S).

HPLC: 99.3% area.

Step 4: Cyclization (A-ring formation, HDP 30.2546)

The above freeze-dried monocyclic intermediate (49.4 mg, 47.2 µmol) was dissolved in 3 ml DMF and treated with diphenylphosphoryl azide (DPPA, 13 µl, 237 µmol, 5 equiv.) and diisopropylethylamine (DIEA, 40 µl, 237 µmol, 5 equiv.). The reaction mixture was agitated for 16 hours and quenched with 500 μl of water upon completion. The transformation was monitored by HPLC. The mixture was concentrated under reduced pressure, redissolved in 1 ml of methanol and purified by reverse phase preparative column chromatography.

Yield: 32.5 mg, 67.9%.

MS (ESI ⁺ ) - revealed: [M+H] ⁺ 1029.33, [MH] ⁺ 1051.3, [M+Na] ⁺ ; calculated: 1029.39; 1051.42 (C ₄₅ H ₆₁ N ₁₀ O ₁₆ S; C ₄₅ H ₆₀ N ₁₀ O ₁₆ SNa).

HPLC: 88.3% area.

Step 5: Deprotection of the acetate (HDP 30.2548)

HDP 30.2546 (23.4 mg, 22.7 µmol) was dissolved in 7N. methanol solution (2.3 ml) and shaken. The conversion was checked using HPLC/MS. After completion (6-8 hours), the reaction mixture was concentrated in vacuo, resuspended in 100 μl of MeOH and purified using preparative HPLC.

Yield: 12.5 mg, 53.3%.

HPLC: 99%.

MS (ESI ⁺ ) revealed: 903.4 [M+H] ⁺ ; calculated: 902.9 (C ₃₉ H ₅₄ N ₁₀ O ₁₃ S).

revealed: 925.4 [M+Na] ⁺ .

5. Preparation of 5'-((3-maleidopropanamido)-Val-Aka-n-amininobenzyloxy)-S-deoxy-amaninamide (HDP 30.2602)

Stage 1 HDP 30.2563

S-deoxy-5'-hydroxy-amaninamide HDP 30.2548 (9.66 mg, 10.7 µmol) and HDP 30.1690 (WO 2016/142049, 50.2 mg, 85.6 µmol = 8.0 equiv) were dissolved in 500 µl dry dimethylacetamide (DMA). A 1.0 M solution of cesium hydrogen carbonate in water (17.12 μl, 1.6 equiv.) was added in one portion and the mixture was shaken at room temperature.

After 1.5 hours, additional portions of 17.12 μl (1.6 equiv.) of cesium carbonate solution were added.

After 8 hours, the reaction mixture was neutralized with acetic acid, passed through a centrifuge filter (0.2 μM) and purified by HPLC on a Phenomenex Luna-C ₁₈ (2) 10 μm (250×21.2 mm) column with a gradient of (5-100 %) acetonitrile in water. The product fractions were combined and brought to 4.00 mg (26%) of the product as an amorphous solid.

MS (ESI ⁺ ) [M+Na] ⁺ - detected: 1430.58; calculated: 1430.65 (C ₆₅ H ₉₇ N ₁₃ NaO ₁₈ SSi).

Stage 2 HDP 30.2378

HDP 30.2563 (7.53 mg, 5.34 µmol) was dissolved in 500 µl of a mixture of trifluoroacetic acid/water/triisopropylsilane 95:5:5. After 5 minutes, the volatiles were removed in vacuo, the residue was dissolved in 1000 μl of water and 3% ammonia was added dropwise until pH 10 and precipitation were reached.

The solution was freeze-dried and purified by preparative HPLC sequentially (Phenomenex Luna-C ₁₈ (2) column, 10 µm, 250×21.2 mm, gradient 20-30% acetonitrile in water (0.05% TFA) over 16 minutes with yielding 3.95 mg (57% based on TFA salt) of product.

MS (ESF) [M+H] ⁺ - detected: 1178.50; calculated: 1178.53 (C ₅₄ H ₇₆ N ₁₃ O ₁₅ S).

Stage 3 HDP 30.2602

HDP 30.2578 (3.95 mg, 3.06 µmol) was dissolved in 400 µl dry DMF.

An ester of 3-(maleimido)propionic acid and N-hydroxysuccinimide (BMPS, 1.63 mg, 2 equiv.) was dissolved in 81.4 μl of DMF followed by the addition of 2.1 μl (2 equiv.) of DIPEA.

After agitation for 2 hours, the reaction mixture was passed through a centrifuge filter (0.2 μm) and purified using RP-18 HPLC with a gradient of 5-70% acetonitrile in water + 0.05% TFA. Pure fractions were evaporated to dryness and lyophilized from 2 ml of acetonitrile/water 1:1 to give 2.73 mg (67%) of the product as a colorless powder.

MS (ESI+) [M+H] ⁺ - detected: 1329.33; calculated: 1329.56 (C ₆₁ H ₈₁ N ₁₄ O ₁₈ S).

C. General synthesis of S-deoxy-4'-hydroxy-amaninamide

1. Schematic view of the synthesis of cis,trans-1-(tert-butoxycarbonyl)-2-carboxy-3а-hydroxy-4-acetoxy-1,2,3,3а,8,8а-hexahydropyrrolo[2,3-b]indole ; cis- and trans (cis,trans-4-acetoxy-Hpi)

2. Schematic view of the synthesis of S-deoxy-4'-hydroxy-amaninamide

D. Total synthesis of S-deoxy-7'-hydroxy-amaninamide

1. Synthesis of cis,trans-1-(tert-butoxycarbonyl)-2-carboxy-3a-hydroxy-4-acetoxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole; (cis,trans-4-acetoxy-Hpi)

The synthesis of cis,trans-7-acetoxy-Hpi was performed similarly to the synthesis of cis,trans-4-acetoxy-Hpi, starting from commercially available 7-hydroxy-L-tryptophan instead of 4-hydroxy-L-tryptophan.

2. Schematic view of the synthesis of S-deoxy-7'-hydroxy-amaninamide

The synthesis of S-deoxy-7'-hydroxy-amaninamide was performed similarly to the synthesis of S-deoxy-4'-hydroxy-amaninamide using cis,trans-7-acetoxy-Hpi instead of cis,trans-4-acetoxy-Hpi.

6. In vitro cytotoxicity of S-deoxy-α-amanitin HDP 30.0735 and S-deoxy-5'-hydroxy-amanitin HDP 30.2548

BrdU cell proliferation assay on HEK293 and HEK293 OATP1B3 cells

HEK293-OATP1B3 cell culture plates were coated with poly-D-lysine.

Coating with poly-D-lysine

5 mg poly-D-lysine in 50 ml sterile water

○ 50 µl per well of a 96-well plate;

○ 1 hour incubation at RT;

○ wash the wells twice with 200 µl of sterile water;

○ drying for at least 2 hours (RT).

Black 96-well clear bottom plates were prepared with 2.0×10 ³ HEK293 and HEK293 OATP1B3 cells/well and 90 μl of growth medium per well containing 10% FCS and supplements. Controls: “blank” was prepared with 100 µl of medium without cells, “background” and “100%” were prepared with cells in 100 µl of medium.

• Incubation for 24 hours at 37°C and 5% CO ₂ .

Dilution scheme for HDP 30.0735, HDP 30.2548 and alpha-amanitin Stock solutions were diluted 1:1000 (1:10 and 1:100 dilutions) in medium:

10 µl stock solution of amanitin derivative (1.0×10 ^-2 M) + 90 µl PBS=100 µl 1.0×10 ^-3 M;

2 µl dilution + 198 µl medium = 200 µl 1×10 ^-5 M.

Additional dilutions

A: 200 µl 1.0×10 ^-5 M;

B: 80 µl growth medium + 20 µl solution A (1:5 dilution);

C: 80 µl growth medium + 20 µl solution B (1:5 dilution);

D: 80 µl growth medium + 20 µl solution C (1:5 dilution);

E: 80 µl growth medium + 20 µl solution D (1:5 dilution);

F: 80 µl growth medium + 20 µl solution E (1:5 dilution);

G: 80 µl growth medium + 20 µl solution F (1:5 dilution);

H: 80 µl growth medium + 20 µl solution G (1:5 dilution).

10 μl of each solution was added to the well in triplicate.

Final volume: 100 µl/well.

Final dose: initial 1×10 ^-6 M; dilution series 1:5.

• Incubation for 96 hours at 37°C and 5% CO ₂ .

• After 96 hours: Roche Cell proliferation assay, fluorescent, according to the manufacturer's instructions.

• EC ₅₀ concentrations were determined using Graphpad Prism 4.0 data analysis software.

7. Synthesis of HDP 30.2347, HDP 2371 and conjugates 30.2602

Example. Synthesis T-D265C-30.2371

10 mg Thiomab T-D265C in PBS buffer was used to conjugate HDP 30.2371.

Bringing the antibody solution to 1 mM EDTA

2 ml antibody solution (10.0 mg) + 20 µl 100 mM EDTA, pH 8.0; amount of antibody: 10 mg = 6.9×10 ^-8 mol.

Removal of cysteine capping by antibody reaction with 40 equiv. TSER:

- 2 ml of antibody solution (6.9×10 ^-8 mol) + 55.2 μl of 50 mM TCEP solution (2.76×10 ^-6 mol);

- incubated for 3 hours at 37°C on a shaker;

- two consecutive dialysis at 4°C in 2.0 L 1 × PBS, 1 mM EDTA, pH 7.4, in Slide-A-Lyzer 20,000 MWCO dialysis cassettes, first dialysis approximately 4 hours, second dialysis for night.

Oxidation by antibody reaction with 20 equiv. dehydroascorbic acid (dhAA):

- approximately 2 ml of antibody solution (6.9×10 ^-8 mol) + 27.6 µl of fresh 50 mM dhAA solution (1.38×10 ^-6 mol);

- incubated for 3 hours at RT on a shaker.

Conjugation with amanitin using 6 equiv. HDP 30.2371 and blanking 25 equiv. N-acetyl-L-cysteine.

Solubilized 2 mg HDP 30.2371 in 200 µl DMSO = 10 µg/µl;

- approximately 2 ml of antibody solution (= 9.5 mg; 6.53×10 ^-8 mol) + 52.1 μl of HDP 30.2371 (= 520.8 μg; 3.92×10 ^-7 mol);

- incubated for 1 hour at RT;

- extinguished by adding 16.3 μl of 100 mm N-acetyl-L-cysteine (1.63×10 ^-6 mol);

- incubated for 15 minutes at RT (or overnight at 4°C);

- purified the reaction mixture using columns 1 × PD-10, balanced with 1 × PBS, pH 7.4; protein-containing fractions were identified with Bradford's reagent on parafilm and the protein-containing fractions were pooled together;

dialysis of the antibody solution at 4° C. overnight in 2.0 L of PBS, pH 7.4, and in Slide-A-Lyzer 20,000 MWCO dialysis cassettes;

- DAR determination by LC-ESI-MS analysis.

The protein concentration was brought to 5.0 mg/ml (3.4×10 ^-5 M) and brought to sterile conditions using filtration. Stored at 4°C.

Received ADC with a different antibody or with a different derivative of amanitin, respectively. The molecular amount of the antibody was calculated from the MW of the corresponding antibody. The amounts of linker toxin, TCEP, dhAA, N-acetyl-L-cysteine were adjusted accordingly to achieve appropriate equivalents.

8. In vitro cytotoxicity of HDP 30.2347, HDP 2371 and 30.2602 conjugates

BrdU cell proliferation assay on SKBR-3, JIMT-1, LnCap and 22rv1 cells

The analysis was performed as described above (6) with the following modifications.

Cell culture plates were not coated with poly-D-lysine.

• ADC dilution scheme.

• Stock solutions were diluted to 1.0×10 ^-6 M with growth medium.

Additional dilutions

A: 100 µl 1.0×10 ^-6 M;

B: 80 µl growth medium + 20 µl solution A (1:5 dilution);

C: 80 µl growth medium + 20 µl solution B (1:5 dilution);

D: 80 µl growth medium + 20 µl solution C (1:5 dilution);

E: 80 µl growth medium + 20 µl solution D (1:5 dilution);

F: 80 µl growth medium + 20 µl solution E (1:5 dilution);

G: 80 µl growth medium + 20 µl solution F (1:5 dilution);

H: 80 µl growth medium + 20 µl solution G (1:5 dilution).

• Add 10 µl of each solution per well in triplicate. Final volume: 100 µl/well.

Final dose: initial 1×10 ^-7 M; dilution series 1:5.

WST-I analysis on Raji cells and Nalm-6 cells

• 96-well F-bottom clear-bottom plates were prepared at 2.0 x 10 ³ cells/well and 90 µl of appropriate growth medium per well containing 10% FCS and supplements. Controls: "blank" was prepared with 100 μl of medium without cells;

"with cells only" was prepared with cells in 100 μl of medium.

• Incubation for 24 hours at 37°C and 5% CO ₂ .

• ADC Dilution Scheme:

• Stock solutions were diluted to 1.0×10 ^-6 M with growth medium.

Additional dilutions

A: 100 µl 1.0×10 ^-6 M;

B: 80 µl growth medium + 20 µl solution A (1:5 dilution);

C: 80 µl growth medium + 20 µl solution B (1:5 dilution);

D: 80 µl growth medium + 20 µl solution C (1:5 dilution);

E: 80 µl growth medium + 20 µl solution D (1:5 dilution);

F: 80 µl growth medium + 20 µl solution E (1:5 dilution);

G: 80 µl growth medium + 20 µl solution F (1:5 dilution);

H: 80 µl growth medium + 20 µl solution G (1:5 dilution).

• 10 µl of each solution was added to the well in triplicate. Final volume: 100 µl/well.

Final dose: initial 1×10 ^-7 M; dilution series 1:5.

• Incubation for 96 hours at 37°C and 5% CO ₂ .

• After 96 hours: Roche Cell proliferation assay, fluorescent, according to the manufacturer's instructions.

• EC ₅₀ concentrations were determined using Graphpad Prism 4.0 data analysis software.

Claims (36)

1. Hydroxy-substituted derivative of 2-carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole according to formula I

in which R1 is selected from C _1-2 -alkyl; P ₁ represents hydrogen or a protective group; P ₂ is hydrogen or a protecting group and R2 is selected from OH, OR1 and a polypeptide chain of 1-7 amino acid residues, with the protecting group, if present, independently selected from Boc, PhCH ₂ OCO-, CH ₂ =CHCH ₂ O-CO- and trityl. 2. Hydroxy-substituted derivative of 2-carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole according to claim 1, in which the substituent R1-C(=O)-O - is attached at position 5 in formula I. 3. Hydroxy-substituted derivative of 2-carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole according to claim 1, in which the substituent R1-C(=O)-O - is attached at position 6 in formula I. 4. A method for the synthesis of a linear precursor comprising eight amino acid residues of an amanitin derivative comprising a hydroxylated tryptophan fragment, comprising the step of using 4-, 5-, 6-, or 7-hydroxy-L-tryptophan to synthesize a hydroxy-substituted 2-carboxy- 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole according to any one of paragraphs. 1-3 in the peptide synthesis of said precursor. 5. A method for the synthesis of an amanitin derivative, including a hydroxylated tryptophan fragment, comprising the steps: (i) obtaining a linear precursor consisting of 8 amino acid residues of an amanitin derivative using 4-, 5-, 6- or 7-hydroxy-L-tryptophan to synthesize a hydroxy-substituted derivative of 2-carboxy-3a-hydroxy-1,2, 3,3a,8,8a-hexahydropyrrolo[2,3-b]indole according to any one of paragraphs. 1-3; (ii) initiating or ensuring the formation of a bond between the cysteine residue and the tryptophan fragment of the linear precursor of step (i); And (iii) initiating or causing the formation of said amanitin derivative by reacting the N-terminal linear precursor of step (ii) with the C-terminus of said precursor. 6. The method of claim 5, further comprising oxidizing the sulfur atom of the cysteine moiety to form a sulfoxide or sulfone, in particular a sulfoxide. 7. An amanitin derivative comprising a hydroxylated tryptophan moiety which is selected from (i)

(ii)

(iii) an amanitin derivative according to (i), wherein the free carboxylic acid moiety of amino acid 1 is converted to a -C(=O)OR1 carboxylic acid ester or to a -C(=O)NH-OR1 moiety in which R1 is selected from alkyl, aryl, heteroaryl. 8. An amanitine derivative according to claim 7 which is selected from S-deoxy-5'-hydroxy-amaninamide, 5'-hydroxy-amaninamide, S-deoxy-5'-hydroxy-amaninamide and 5'-hydroxy-amaninamide. 9. The use of an amanitin derivative according to claim 7 or 8 for the treatment of a malignant tumor in a patient, in particular, the malignant tumor is selected from the group consisting of breast cancer, pancreatic cancer, cholangiocarcinoma, colon and rectal cancer, lung cancer, prostate cancer, ovarian cancer, gastric cancer, kidney cancer, malignant melanoma, leukemia, and malignant lymphoma to achieve one or more of: (a) reducing the severity of the malignant tumor; (b) limiting or preventing the development of symptoms characteristic of a malignant tumour; (c) inhibiting the worsening of symptoms characteristic of a malignant tumor; (d) limiting or preventing the recurrence of a malignant tumor in patients in whom it has already been detected; and (e) limiting or preventing the recurrence of symptoms in patients. 10. A conjugate comprising (a) an amanitin derivative comprising a hydroxylated tryptophan moiety according to claim 7 or 8; (b) a target-binding fragment, where the target is a target on cancer cells; and (c) optionally a linker linking said amanitin derivative and said target-binding moiety, wherein said linker has a continuous chain of 1 to 30 atoms, and wherein said linker is an alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, cycloalkylene, a heterocycloalkylene, arylene, heteroarylene, aralkylene or heteroaralkylene group. 11. The conjugate according to claim 10, where the linker includes a structure selected from the group consisting of

12. The use of a conjugate according to claim 10 for the treatment of a cancer in a patient, in particular, the cancer is selected from the group consisting of breast cancer, pancreatic cancer, cholangiocarcinoma, colon and rectal cancer, lung cancer, prostate cancer , ovarian cancer, gastric cancer, kidney cancer, malignant melanoma, leukemia, and malignant lymphoma to achieve one or more of: (a) reducing the severity of the malignant tumor; (b) limiting or preventing the development of symptoms characteristic of a malignant tumour; (c) inhibiting the worsening of symptoms characteristic of a malignant tumor, (d) limiting or preventing the recurrence of a malignant tumor in patients in whom it has already been detected; and (e) limiting or preventing the recurrence of symptoms in patients. 13. Pharmaceutical composition for the treatment of a malignant tumor, including the amanitin derivative according to claim 7 or 8 in an effective amount and further comprising one or more pharmaceutically acceptable diluents, carriers, adjuvants, excipients, binders, lubricants, glidants, disintegrating agents, adsorbents and/or preservatives, wherein the malignant tumor is selected from the group consisting of breast cancer, pancreatic cancer, cholangiocarcinoma, colon and rectal cancer, lung cancer, prostate cancer, ovarian cancer, gastric cancer, kidney cancer , malignant melanoma, leukemia and malignant lymphoma. 14. The use of a pharmaceutical composition according to claim 13 for the treatment of a malignant tumor in a patient, in particular, the malignant tumor is selected from the group consisting of breast cancer, pancreatic cancer, cholangiocarcinoma, colon and rectal cancer, lung cancer, prostate cancer gland, ovarian cancer, gastric cancer, kidney cancer, malignant melanoma, leukemia and malignant lymphoma, to achieve one of the following goals: (a) reduce the severity of the malignant tumor; (b) limiting or preventing the development of symptoms characteristic of a malignant tumour; (c) inhibiting the worsening of symptoms characteristic of a malignant tumor; (d) limiting or preventing the recurrence of a malignant tumor in patients in whom it has already been detected; and (e) limiting or preventing the recurrence of symptoms in patients. 15. A pharmaceutical composition for the treatment of a malignant tumor, comprising the conjugate of claim 10 in an effective amount and further comprising one or more pharmaceutically acceptable diluents, carriers, adjuvants, excipients, binders, lubricants, glidants, disintegrants, adsorbents and/or preservatives, wherein the malignant tumor is selected from the group consisting of breast cancer, pancreatic cancer, cholangiocarcinoma, colon and rectal cancer, lung cancer, prostate cancer, ovarian cancer, gastric cancer, kidney cancer, malignant melanoma , leukemia and malignant lymphoma. 16. The use of a pharmaceutical composition according to claim 15 for use in the treatment of a malignant tumor in a patient, in particular, the malignant tumor is selected from the group consisting of breast cancer, pancreatic cancer, cholangiocarcinoma, colon and rectal cancer, lung cancer, prostate cancer, ovarian cancer, gastric cancer, kidney cancer, malignant melanoma, leukemia and malignant lymphoma. 17. A construct comprising (a) an amanitin derivative according to claim 7 or 8 and (b) a linker moiety bearing a Y reactive group for linking said amanitin derivative to a target-binding moiety, wherein one side of the backbone of the linker moiety has reacted with derivative of amanitin, and the other side is available for reaction with the specified target-binding fragment, and after binding of the specified derivative of amanitin with the specified target-binding fragment, this linker has a continuous chain of 1 to 30 atoms. 18. The construction according to claim 17, where the linker includes a structure selected from the group consisting of

19. The construction of claim 17, wherein said reactive group Y is a maleimide group.

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