Antibody-drug conjugate with a cleavable linker

Abstract

The present invention relates to a prodrug comprising a linker comprising a cyclic acetal having five or six members and an adjacent specific cleavage site, and to a precursor compound for synthesizing said prodrug. One aspect of the present invention relates to an amatoxin conjugate targeting an antibody comprising said linker, a method for synthesizing said amatoxin conjugate targeting said antibody, and the use of said amatoxin conjugate targeting said antibody. In a further aspect, the present invention relates to a pharmaceutical composition comprising said conjugate, and the use of said conjugate or composition for therapeutic purposes, particularly for cancer treatment and oncology.

Description

Technology Field

[0001] The present invention relates to a prodrug comprising a linker comprising a cyclic acetal having five or six members and an adjacent specific cleavage site, and to a precursor compound for synthesizing said prodrug. One aspect of the present invention relates to an amatoxin conjugate targeting an antibody comprising said linker, a method for synthesizing said amatoxin conjugate targeting said antibody, and the use of said amatoxin conjugate targeting said antibody. In a further aspect, the present invention relates to a pharmaceutical composition comprising said conjugate, and the use of said conjugate or composition for therapeutic purposes, particularly for cancer treatment and oncology. Background Technology

[0002] Prodrugs are pharmaceutically inactive agents that must be converted into an active form through biochemical or chemical reactions, such as hydrolysis or phosphorylation. In cancer treatment, the idea behind prodrugs is to reduce unintended side effects by designing compounds that react with specific targets. These prodrugs sometimes contain moietyes that bind to targets, which are linked to therapeutic or toxic components via linker components.

[0003] Prodrugs containing unstable acid components for the release of therapeutic or toxic components are frequently used in pharmaceutical research and application. In the field of antibody-drug conjugates (ADCs), unstable acid linkers enable the release of toxic payloads after targeting components into tumor cells by target-specific antibodies. This concept involves receptor-mediated endocytosis (cell uptake) of the ADC and subsequent intercellular trafficking into lysosomes. When the ADC binds to its surface tumor antigen, it is internalized into an endosome that subsequently matures and fuses with the lysosome; within the lysosome, the drug is released either by specific proteases, such as cathepsin B, or through the cleavage of the linker due to the degradation of the ADC. Lysosomes are characterized by an acidic pH of 4–5 (Chalouni and Doll, 2018) and are thought to possess sufficient acidity for the hydrolysis of the unstable acid binding. Linkers of unstable acids, such as acyl hydrazones, have been successfully used in ADCs and approved in the market (Chalouni and Doll, 2018).

[0004] Other unstable acid groups, such as acetals, have been considered as components of linkers for ADCs. While molecules containing cyclic acetals based on aliphatic aldehydes or ketones have been exemplified as being cleavable at pH 5, cyclic acetals of aliphatic carbonyl compounds have not been studied in this regard; these structures may possess high stability, and release by the acidic intercellular environment appears questionable.

[0005] WO2014 / 135282 discloses a method for preparing amatoxins comprising cyclic carbonates and cyclic acetals having improved properties for use in ADCs, but does not disclose compounds having a controllable release mechanism.

[0006] Gillies except(2004) describes cyclic acetals with five or six members included in low molecular weight prodrugs based on benzaldihydride derivatives. Unlike aliphatic acetals, acid stability can be controlled by additional substituents by introducing aromatic substituents to the acetal carbon atoms. Hammett's law makes it possible to predict the reactivity of benzyl acetals in relation to the substitution pattern of the aromatic ring. By following this principle, cyclic acetals can be synthesized with half-lives ranging from 17.6 hours at pH 5.0 to several weeks at pH 7.4.

[0007] WO2015 / 152182 (NOF Corporation) describes hydrophilic polymer derivatives having cyclic benzylidene acetal linkers capable of controlling the hydrolysis rate at pH in a weakly acidic in vivo environment through the position and characteristics of substituents in the benzyl structure; in particular, unstable acid linkers available for use in polymer drug conjugates based on cyclic acetals having five or six members from benzaldihydride derivatives are disclosed. This document does not disclose any specific examples of cytotoxic payloads and biological data. Furthermore, what WO2015 / 152182 teaches requires additional moieties for the covalent linkage of cyclic acetals to these drug compounds. It is presumed that these linker structures are not included in acid cleavage and remain attached to the released payload, thereby negatively affecting the pharmacokinetic activity of the same compound.

[0008] Since the cleavage of the cyclic acetal disclosed in the prior art requires an acidic environment, it is essential for the cell to take the components and transport them to a lysosome where only appropriate acidic conditions can be found. Therefore, the cyclic acetals disclosed in the prior art are limited to moieties that bind to targets resulting in efficient uptake after receptor binding.

[0009] However, other promising target structures remain on the cell surface upon binding to the target moiety and / or are not transported to late endosomes and lysosomes after uptake, and therefore are not suitable for this technology.

[0010] A combination of cleavable peptide structures and immolative groups between the antibody and drug binding to the ADC target has been described as applicable to payloads carrying primary or secondary amines. Jeffrey except (2005) and WO2016 / 142049 disclose a para-aminobenzyl ether linker applicable to phenolic moiety. The carboxylic acid of such payload can be handled by a similar para-aminobenzyl amide (WO 2017 / 149077).

[0011] Another type of related autosacrificial moiety frequently used for small molecule prodrugs is the Tranoy-Opalinski except This was disclosed in (2014), where a specific side chain contains β-glucuronide and undergoes spontaneous fragmentation starting from the terminal phenolic moiety after enzymatic cleavage by β-glucuronidase. This type of linker is also used in the field of ADCs, and for example , Jeffrey except It is described in (2006). The problem to be solved

[0012] The present invention provides an antibody-drug conjugate having a cleavable linker. means of solving the problem

[0013] Upon reviewing the prior art, one objective of the present invention is to provide a highly efficient drug release mechanism from a prodrug that allows the drug to be released without retention under controlled conditions, thereby increasing therapeutic efficiency and avoiding the loss of drug activity due to chemical residues remaining on the released therapeutic agent.

[0014] One object of the present invention is to provide a moiety amatoxin conjugate that binds to a target, comprising an efficient and pH-independent release mechanism to allow highly activated amatoxins to be released without retention.

[0015] Another objective of the present invention is to provide a modified amatoxin for the synthesis of the conjugate.

[0016] Another objective of the present invention is to provide a moiety amatoxin conjugate that binds to an improved target, particularly for therapeutic use in tumor treatment and oncology.

[0017] These objectives are achieved by methods and means according to the dependent claims of the present invention. The dependent claims relate to preferred embodiments. Effects of the invention

[0018] According to the present invention, an antibody-drug conjugate having a cleavable linker is provided. Brief explanation of the drawing

[0019] FIG. 1 schematically illustrates the drug release mechanism in the linker system of the present invention. Triangles represent structures that can be cleaved by enzymes, preferably peptide structures. A is an electron-donating group that may be selected from O, NH, or S, but is not limited thereto. Figure 2 shows the results of a cytotoxicity study on SKBR-3 cells in a BrdU assay after 96 hours of culture. Figure 3 shows the results of a cytotoxicity study on NCI-N87 cells in a BrdU assay after 96 hours of culture. Figure 4 shows the results of a cytotoxicity study on JIMT-1 cells in a BrdU assay after 96 hours of culture. Fig. 5 shows SKOV-3 In vivo This presents the results of a study on the efficacy of acetal linker conjugates in a xenograft tumor mouse model. Fig. 6 is In vivo conjugates T-D265C-30.2669 and T-D265C-30.2684 in NOD / SCID mice In vivo It shows the results of the tolerability study. Further details, features, characteristics, and advantages of the object of the present invention are disclosed in the dependent claims, and the following description of each drawing and example illustrates preferred embodiments of the present invention in an exemplary manner. However, these drawings should not be understood in any way as limiting the scope of the present invention. Specific details for implementing the invention

[0020] Before describing the invention in detail, it should be understood that the invention is not limited to the specific components of the apparatus described above or the steps of the process of the method described above, as such apparatus and methods may be modified. It should also be understood that the terms used herein are intended to describe specific embodiments only and are not intended to limit the invention. It should also be understood that the singular forms "one" and "above" used in this specification and the appended claims refer to singular and / or plural references unless the context clearly indicates otherwise. Furthermore, it should be understood that if a parameter range is given as a numerical limit, the range should be deemed to include such limiting value.

[0021] It should also be understood that the embodiments disclosed herein are not intended to be understood as separate embodiments unrelated to one another. Features discussed in one embodiment are intended to be disclosed in connection with other embodiments shown herein. In one instance, if a specific feature is disclosed in another embodiment rather than in one embodiment, a person skilled in the art will understand that this does not necessarily mean that said feature was intended to be disclosed in said other embodiment. A person skilled in the art will understand that while the core of this application is to disclose features for other embodiments as well, this work has not been done to clarify the description and keep the specification manageable in volume.

[0022] Furthermore, the contents of the prior art documents referenced in this specification are incorporated by reference into this specification. This refers particularly to prior art documents disclosing standard or conventional methods. In such cases, the incorporation by reference is intended primarily to provide sufficient disclosure to enable practice and to avoid verbose repetition.

[0023] The present invention provides a solution to the limitations described above regarding the controllable, non-residual release of a drug from a prodrug, particularly from an antibody-drug conjugate (ADC).

[0024] The present invention relates to a specific drug release mechanism for improving a prodrug system applied to targeted therapies, such as antibody-drug conjugates or peptide-drug conjugates. The prodrug according to the present invention comprises a benzyl cyclic acetal having five or six members comprising an electron donor group and a specific cleavage side, and / or including or binding to a side chain. Cleaving of the cleavage site triggers the release of the assisted acetal, resulting in the release of a drug that is not residual (Fig. 1).

[0025] Various cytotoxic drugs such as amatoxin, apidicolin, apoptoridin, calikimycin, digitoxin, digoxin, etoposide, glucopyricidin A, hypotemisin, isatropolone A, lactacystin, muscotoxin A, pancratistatin, phalloidin, fenfanstatin, phytospingosine, foscilaridin A, surotin A, rebecamycin, synefungin, G-strophanthin, swinsonine, turvostatin 1-4, and others contain 1,2- and 1,3-diol structures that can be used for cyclic acetal formulations. At least one of these 1,2- or 1,3-diol moiety is recovered when the drug is released from the compound according to the present invention.

[0026] The present invention relates to a compound comprising the introduction of a benzyl cyclic acetal, which has enhanced hydrolysis after cleavage of a specific side chain and consequently brings about a moiety at the 1-2 (orthogonal) or 1-4 (parallel) position of the acetal carbon, exhibiting a strong positive mesomeri effect, and a method for the same. A specific cleavage side ( for example Enzymatic cleavage of a side chain containing and / or representing (, peptide) an electron donor group having strong electron-donating properties ( for example It releases (2- or 4-amino groups), resulting in the release of a free payload through an ortho or paraquinone intermediate, leading to the spontaneous fragmentation of the benzylidene linker and diol.

[0027] Enzymatic cleavage is performed by a specific protease, for example, cathepsin, preferably cathepsin B, elastase, or matrix metalloprotease. In some embodiments, the protease is an enzyme specific to tumors within the extracellular matrix, such as matrix metalloprotease or β-glucuronidase.

[0028] Surprisingly, the inventors of the present invention have discovered that cleavable linker elements and self-sacrificial moiety can be combined and applied to cyclic acetals containing ADCs within prodrugs, particularly within amatoxins. The compounds and methods of the present invention enable the drug to be released from the prodrug, particularly from amatoxins containing ADCs, within cyclic acetals in a manner that is efficient, controllable, and pH-independent, without residues or structural elements from spacers, linkers, or moietyes binding to targets that could pose a risk of reducing drug activity and therapeutic efficiency in the released drug moiety.

[0029] According to one aspect of the present invention, the present invention relates to a compound of Formula I or II comprising a cytotoxic drug moiety.

[0030]

[0031] At this time

[0032] D is a cytotoxic drug moiety comprising at least one 1,2- or 1,3-diol moiety upon release from the above compound;

[0033] Z is CH2, CH2-CH2, or CHR3-CHR3, wherein R3 is independently H or an alkyl group and can optionally be substituted with a heteroatom;

[0034] R1 is H or a C1-C6 alkyl group;

[0035] A is an electronic donor group;

[0036] E is the amputation site;

[0037] R2 is an independent H or electronic receiving or donating group.

[0038] The cytotoxic drug according to the present invention is, for example, amatoxin, apidicolin, apoptoridin, calikimycin, digitoxin, digoxin, etoposide, glucopyricidin A, hypotemisin, isatropolone A, lactacystin, muscotoxin A, pancratistatin, phalloidin, fenfanstatin, phytospingosine, foscilaridin A, surotin A, rebecamycin, synefungin, G-strophanthin, swinesonine, and turvostatin 1-4 may be selected, but are not limited thereto.

[0039] Amatoxins are cyclic peptides composed of eight amino acids. They can be produced, for example, by isolating them from or synthesizing the Amanita phalloide mushroom. Amatoxins specifically inhibit RNA polymerase II, which is independent of the DNA in mammalian cells, thereby suppressing transcription and protein biosynthesis in affected cells. The inhibition of intracellular transcription leads to the cessation of growth and proliferation. Although not covalently bound, the complex between amanitin and RNA polymerase II is very tight (KD = 3 nM). The dissociation of amanitin from the enzyme is a very slow process, making recovery of affected cells difficult. If transcriptional inhibition persists for too long, the cells will undergo programmed cell death (apoptosis).

[0040] As used herein, the term “amatoxin” comprises all cyclic peptides composed of eight amino acids isolated from the genus Amanita and described in Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Biochem. 1978 Dec;5(3): 185-260), additionally all chemical derivatives thereof; additionally all semi-synthetic analogs thereof; additionally all synthetic analogs thereof constructed from components according to the master structure (cyclic, 8 amino acids) of the natural compound, additionally all synthetic or semi-synthetic analogs comprising non-hydroxylated amino acids rather than hydroxylated amino acids, additionally all synthetic or semi-synthetic analogs in which the thioether sulfoxide moiety is replaced by a carbon atom, such as in the carba-analogous derivative of Amanita, by a sulfide, sulfone, or by a non-sulfur atom, for example, as in the carba-analogous derivative of Amanita, and in each case, these derivatives or analogs are functionally activated by inhibiting mammalian RNA polymerase II.

[0041] As used herein, a “derivative” of a compound refers to a species having a chemical structure similar to that of the compound but having at least one chemical group not present in the compound, and / or a species lacking at least one chemical group present in the compound. The compound to which the derivative is compared is known as the “parent” compound. Generally, the “derivative” may be prepared from the parent compound through one or more chemical reaction steps.

[0042] As used herein, an "analogous" of a compound is structurally related to but not identical to the said compound and exhibits at least one of the activities of the compound. The compound against which the analog is compared is known as the "parent" compound. The aforementioned activities include, without limitation, binding activity to another compound; inhibitory activity, e.g., enzyme inhibitory activity; toxic effects; and activating activity, e.g., enzyme activating activity. It is not required that such an analog exhibit the same activity to the same degree as the parent compound. The compound exhibits at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%) of the activity of the parent compound. , An analog is considered within the context of this application if it exhibits related activity to an extent of at least 40% (more preferably, at least 50%). Accordingly, when used herein, "analogous to amatoxin" is structurally related to any one of α-amanitine, β-amanitine, γ-amanitine, ε-amanitine, amanin, amaninamide, amanulin, and amanulinic acid, and with respect to mammalian RNA polymerase II at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%) compared to at least one of α-amanitine, β-amanitine, γ-amanitine, ε-amanitine, amanin, amaninamide, amanulin, and amanulinic acid. , It refers to a compound exhibiting inhibitory activity of at least 40%, more preferably at least 50%. A "amatoxin analog" suitable for use in the present invention may exhibit greater inhibitory activity against mammalian RNA polymerase II than any one of α-amanitine, β-amanitine, γ-amanitine, ε-amanitine, amanin, amaninamide, amanulin, or amanulinic acid. Such inhibitory activity is the concentration at which 50% inhibition occurs (IC10).50 It is measured by determining the value. Inhibitory activity against mammalian RNA polymerase II can be indirectly determined by measuring inhibitory activity during cell proliferation.

[0043] "Semi-synthetic analogs" refer to analogs obtained by chemical synthesis using compounds from natural sources (e.g., plant material, bacterial culture, fungal culture, or cell culture) as starting materials. Generally, the "semi-synthetic analogs" of the present invention are synthesized starting from compounds isolated from mushrooms of the family Amanitaceae. On the other hand, "synthetic analogs" refer to analogs synthesized by so-called complete synthesis from small (generally petrochemical) components. Usually, such complete synthesis is carried out without the aid of biological processes.

[0044] Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferred amatoxins are those having functional groups (e.g., carboxyl groups, amino groups, hydroxyl groups, thiols, or thiol capture groups) capable of reacting with moietyes that bind to the linker molecule or target defined above. Amatoxins particularly suitable for the conjugates of the present invention are α-amanitine, β-amanitine, γ-amanitine, ε-amanitine, amanin, amaninamide, amanulin, and amanulinic acid, and their salts, chemical derivatives, semi-synthetic derivatives, and synthetic analogs. Amatoxins particularly suitable for use in the present invention are α-amanitine, β-amanitine, and amaninamide.

[0045] According to one aspect of the present invention, the amatoxin may be selected from the group consisting of α-amanitine, β-amanitine, amanin, amaninamide and analogs, derivatives and salts thereof.

[0046] As used herein, the term “electron donating group” refers to a substituent having a loan pair electron that can be donated to an aromatic π-electron system such as a thiol, along with nitrogen, oxygen, sulfur, or amine (NH2, NHR, NR2), phenol (OH) and its conjugated salt O-, alkoxy-group (OR), phenyl ester (OCOR), or an alkyl group having a positive inductive effect.

[0047] The term "electro withdrawing group" refers to a substituent that exhibits a negative mesomeri effect on an directional π-system, such as a halogen, nitro-, carbonyl-, cyano-, and sulfonyl group or a group having a negative inductive effect, such as a trifluoromethyl or trialkylammonium group.

[0048] According to one aspect of the present invention, the electron donor group A is selected from O, NH and S.

[0049] As used herein, the term "cutting site" refers to a moiety sensitive to a specific cut at a location defined under special conditions. The said conditions include: for example There are, for example, specific enzymes or reducing environments in specific body or cell compartments.

[0050] According to one aspect of the present invention, cleavage site E is a moiety cleaved by an enzyme comprising two or more amino acids. The enzyme-cleavable moiety preferably comprises valine-alanine (Val-Ala), valine-citrulline (Val-Cit), valine-lysine (Val-Lys), valine-arginine (Val-Arg) dipeptide, phenylalanine-lysine-glycine-proline-leucine-glycine (Phe Lys Gly Pro Leu Gly) or alanine-alanine-proline-valine (Ala Ala Pro Val) peptide, or β-glucuronide or β-galactoside.

[0051] According to one embodiment of the present invention, the cleavage site can be cleaved by at least one agent selected from the group consisting of cysteine ​​protease, metalloprotease, serine protease, threonine protease, and aspartate protease.

[0052] Cysteine ​​protease, also known as thiol protease, is a protease that shares a common catalytic mechanism involving nucleophilic cysteine ​​thiol within a catalytic triad or diad.

[0053] Metalloproteases are proteases whose catalytic mechanism involves metals. Most metalloproteases require zinc, but some use cobalt. Metal ions are coordinated to the protein by three ligands. The ligands coordinating the metal ions can vary as histidine, glutamate, aspartate, lysine, and arginine. A fourth coordination site is occupied by an unstable water molecule.

[0054] Serine proteases are enzymes that cleave peptide bonds within proteins; serine acts as the nucleophilic amino acid at the enzyme's active site. Serine proteases are divided into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.

[0055] Threonine proteases are a family of proteolytic enzymes containing threonine (Thr) residues within their active sites. While prototype members of this class of enzymes are catalytic subunits of the proteasome, acyltransferases have evolved by converging on the geometry and mechanism of the same active site.

[0056] Aspartate proteases are catalytic protease enzymes that use an activated water molecule bound to one or more aspartate residues for the catalytic action of their peptide substrates. Generally, they possess two highly conserved aspartate groups at their active site and are optimally activated at acidic pH. Almost all known aspartyl proteases are inhibited by pepstatin.

[0057] In a particular embodiment, the cleavable site is cleavable by at least one agent selected from the group consisting of cathepsin A or B, matrix metalloproteinase (MMP), elastase, β-glucuronidase, and β-galactosidase.

[0058] According to another aspect of the present invention, the cleavage site E is a disulfide bond and the specific cleavage is in a reducing environment, for example It is performed in a reducing environment within the cell.

[0059] In another aspect, the present invention relates to a conjugate comprising a compound according to the present invention and a TL moiety, wherein the TL moiety substitutes at least one residue R2 in Formula I or II, and

[0060] L is the linker and

[0061] T is a moiety that binds to the target.

[0062] The term "linker" used in the present invention refers to a group having two reactive groups that can be connected by covalent bonds to a moiety that binds to a target with a compound according to the present invention, which includes a cytotoxic drug moiety.

[0063] The linker L according to the present invention may comprise or be composed of an alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, arylkylene, or heteroarylkylene group formed from 1 to 4 heteroatoms selected from N, O, and S, and the linker is optionally substituted. The linker may also have only covalent bonds.

[0064] In one aspect according to the present invention, linker L comprises a moiety selected from at least one of the following moietyes: disulfide, ether, thioether, amine, ester, carboxamide, urethane, and urea moiety.

[0065] In the context of this application, "linker" refers to, for example, a moiety that binds to a target and a compound according to the present invention, for exampleIt refers to a molecule that increases the distance between two components to mitigate steric interference between amatoxin or an amatoxin derivative, otherwise the ability of amatoxin to react with RNA polymerase II would be reduced. The linker can be used for another purpose because it promotes the release of amatoxin within the target cell by a moiety that specifically binds to the target. It is desirable that the binding of the linker, and preferably the binding of the linker and the compound according to the present invention, preferably amatoxin on one side and the binding of the linker and the antibody on the other side, be stable under extracellular physiological conditions, e.g., blood, while being able to be cleaved within the cell, particularly within target cells, e.g., cancer cells or immune cells. To provide this selective stability, the linker may preferably include functionality that is sensitive to pH or sensitive to protease. Alternatively, a binding that connects the linker to a moiety that binds to the target may provide this selective stability. The linker preferably has at least one, preferably 1 to 30 atomic lengths (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 atoms), wherein one side of the linker reacted with amatoxin and the other side reacted with a moiety that binds to a target. In the context of the present invention, the linker is preferably a C1-30-alkyl, C1-30-heteroalkyl, C2-30-alkenyl, C2-30-heteroalkenyl, C2-30-alkynyl, C2-30-heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl group, which are optionally substituted. The linker may comprise one or more structural elements such as amides, esters, ethers, thioethers, disulfides, carbohydrate moietys, and similar ones.The linker may also include two or more combinations of these structural elements. Each of these structural elements may be present within the linker one or more times, for example, two, three, four, five, or six times. In some embodiments, the linker may include disulfide bonds. It should be understood that such a linker must be attached to a moiety that binds to the amatoxin and the target in a single step or in two or more subsequent steps. For this purpose, the linker must be attached to two groups, preferably at the near end and the far end, which may (i) form a covalent bond with an activated group on the group, preferably the amatoxin or the target-peptide, or (ii) form a covalent bond with a group on the amatoxin or be activated for this purpose. Accordingly, where the linker is present, it is preferable that chemical groups be located at the far end and the near end of the linker, which are the result of linking reactions such as, for example, esters, ethers, urethanes, peptide bonds, etc.

[0066] As used herein, the term “moiety binding to a target” refers to any molecule or portion of a molecule capable of specifically binding to a target molecule or a target epitope. Moietys binding to a target suitable for use in the present invention generally have a molecular weight of 40,000 Da (40 kDa) or greater.

[0067] As used herein, the terms “target molecule” and “target epitope” each refer to an antigen and an epitope of an antigen that are specifically bound by a moiety that binds to the target. The target molecule is preferably an antigen associated with a tumor, in particular an antigen or epitope present on the surface of one or more tumor cell types or tumor-associated cells at an increased concentration and / or different stereochemical composition compared to the surface of non-tumor cells. The antigen or epitope is preferably present on the surface of one or more tumor or tumor stromal cell types, but not on the surface of non-tumor cells. In other embodiments, the antigen or epitope is preferably expressed on cells associated with an autoimmune disease. In other embodiments, the antigen or epitope is preferably expressed on cells associated with an inflammatory disease.

[0068] According to one aspect of the present invention, a moiety T that binds to a target is selected from the group consisting of the following.

[0069] (i) fragments that bind to antibodies or their antigens;

[0070] (ii) antibody-like proteins, and

[0071] (iii) Nucleic acid aptamers.

[0072] The term "antibody or fragment binding to an antigen thereof" as used herein refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, in other words , refers to a molecule containing an antigen-binding site that specifically binds to an antigen. Also, target molecule, for example The immunoglobulin (Ig)-like protein selected by a technique including, for example, phage display, specifically binding to the target protein Her-2 / neu or EpCAM. The immunoglobulin molecule of the present invention is of any type ( for example, IgG, IgE, IgM, IgD, IgA, and IgY), any class ( for example It may have IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass of immunoglobulin molecules. The antibody and the fragment binding to its antigen suitable for use in the present invention may be polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized (particularly CDR-graft), immunocompromised, or chimeric antibody, monochain antibody ( for example scFv), Fab fragments, F(ab')2 fragments, fragments produced by Fab expression libraries, diabodies or tetrabodies (Poljak RJ, 1994), nanobodies, anti-idiotype (anti-Id) antibodies ( for example , including an anti-Id antibody as an antibody of the present invention), and any one of the epitope binding fragments, but not limited to these.

[0073] According to one aspect of the present invention, the fragment binding to the antibody or the antigen of the antibody is selected from a diabody, a tetrabody, a nanobody, a chimeric antibody, an immunocompromised antibody, a humanized antibody, or a human antibody.

[0074] According to one aspect of the present invention, the antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fd, Fv, single-chain Fv, disulfide-linked Fvs(dsFv), and fragments comprising at least one VL and / or VH domain.

[0075] As used herein, the term "antibody-like protein" refers to a protein engineered to specifically bind to a target molecule ( for exampleRefers to a protein (by mutagenesis of the Ig loop). Generally, these antibody-like proteins comprise at least one variable peptide loop attached to both ends of a protein backbone. This double structural restriction significantly enhances the binding affinity of the antibody-like protein to a level comparable to that of an antibody. The length of the variable peptide loop generally consists of 10 to 20 amino acids. The backbone protein can be any protein having excellent solubility characteristics. The backbone protein is preferably a small globular protein. Antibody-like proteins include affibodies, anticalins, and engineered ankyrin repeat proteins (Binz except Includes without limitation , 2005). These antibody-like proteins are derived from a large library of mutants, for example It can be induced by panning from a large-scale phage display library and can be isolated from analogs for common antibodies. Additionally, antibody-like binding proteins can be obtained by combination mutagenesis of residues exposed on the surface within globular proteins.

[0077] As used herein, the term "nucleic acid aptamer" refers to repeated rounds of binding to a target molecule. ex vivo It refers to nucleic acid molecules engineered through selection or SELEX (systematic evolution of ligands by exponential enrichment) (Wang except , 2019). The nucleic acid aptamer may be a DNA or RNA molecule. The aptamer is, for example It may include modifications such as modified nucleotides such as 2'-fluorine-substituted pyrimidines.

[0078] In one aspect, the present invention relates to a conjugate of Formula III or IV.

[0079]

[0080]

[0081] X is S, SO, or SO2;

[0082] R1 is H or a C1-C6 alkyl group;

[0083] R2 independently H or electronic receiving or donating group;

[0084] R4 is H, OH, OC 1- C8-alkyl, NO2, NH2, F, Cl, Br, or SH;

[0085] R5 is OH, NH2, or NHOH;

[0086] R6 and R7 are side chains of natural or non-natural amino acids;

[0087] L is the linker;

[0088] T is a conjugate that is a moiety binding to the target.

[0089] The conjugate of formula III or IV releases its payload (cytotoxic drug) after peptide cleavage by a peptidase such as cathepsin, elastase, or matrix metalloprotease at the anilide site, and said peptidase is not limited to said substances.

[0090] In another aspect, the present invention relates to a conjugate of formula V.

[0091]

[0092] X is S, SO, or SO2;

[0093] Y is CH2 or CO;

[0094] R1 is H or a C1-C6 alkyl group;

[0095] R2 independently serves as an H or electron receiving or donating group; at least one R2 group is substituted by the TL moiety, and

[0096] R4 is H, OH, OC 1- C8-alkyl, NO2, NH2, F, Cl, Br, or SH;

[0097] R5 is OH, NH2, or NHOH;

[0098] L is the linker, and

[0099] T is a conjugate that is a moiety binding to the target.

[0100] The conjugate of formula V releases its payload (cytotoxic drug) after glycoside cleavage by an enzyme such as β-glucuronidase or β-galactosidase, provided that the enzyme is not limited to the above substances.

[0101] In another aspect, the present invention relates to a conjugate of formula VI.

[0102]

[0103] X is S, SO, or SO2;

[0104] R1 is H or a C1-C6 alkyl group;

[0105] R2 is independently an H or electron receiving or donating group, provided that if R8 is not a TL-moiety, one R2 is substituted by said TL-moiety, and

[0106] R4 is H, OH, OC 1- C8-alkyl, NO2, NH2, F, Cl, Br, or SH;

[0107] R5 is OH, NH2, or NHOH;

[0108] R8 is a linear or branched alkyl group or, where no R2 is a TL-moiety, a TL-moiety;

[0109] L is the linker, and

[0110] T is a conjugate that is a moiety binding to the target.

[0111] The conjugate of Formula VI is, for example , release their payload (cytotoxic drug) after disulfide cleavage by a reducing environment such as a reducing intracellular environment.

[0112] In another aspect, the present invention relates to a conjugate of Formula VII, as

[0113]

[0114] X is S, SO, or SO2;

[0115] R1 is H or a C1-C6 alkyl group;

[0116] R2 is independently an H or electron receiving or donating group, provided that if R9 is not a TL-moiety, one R2 is substituted by said TL-moiety, and

[0117] R4 is H, OH, OC 1- C8-alkyl, NO2, NH2, F, Cl, Br, or SH;

[0118] R5 is OH, NH2, or NHOH;

[0119] R9 is H or a linear or branched alkyl group or, if none of R2 is a TL-moiety, a TL-moiety and

[0120] L is the linker, and

[0121] T is a conjugate that is a moiety binding to the target.

[0122] The conjugate of Formula VII releases its payload (cytotoxic drug) after enzymatic cleavage of the phosphate ester.

[0123] In another aspect, the present invention relates to a conjugate of Formula VIII.

[0124]

[0125] In an additional aspect, the present invention relates to a compound of formula IX or X.

[0126]

[0127] In another aspect, the present invention relates to a conjugate of the present invention for use as a pharmaceutical agent.

[0128] In another aspect, the present invention relates to a method for synthesizing a conjugate according to the present invention by reacting a 1,2- or 1,3-diol with dimethyl benzylidene acetal in an aprotic solvent under acidic conditions.

[0129] The present invention relates to a method for synthesizing a conjugate according to the present invention by reacting an amatoxin with a dimethyl benzylidene acetal in an aprotic solvent, preferably under acidic conditions. The amatoxin is selected from alpha-amanitine, beta-amanitine, amanin, amaninamide, and their respective thioethers.

[0130] In a preferred embodiment, the method according to the present invention relates to a method in which the aprotic solvent is DMF and / or the acid is trifluoroacetic acid.

[0131] The term "under acidic conditions" as used in the present invention refers to a pH of 5.0 or less. Particularly preferably, the method is performed at a pH of about 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, or 1.0.

[0132] Cyclic acetals can be formed by reacting aldehydes or ketones with diol compounds under acidic conditions. Generally, these reactions require raising the temperature and removing water formed by the product using means such as azeotropic distillation or water-binding agents like calcium sulfate or molecular filters. These methods may be impractical when using sensitive and expensive starting materials. In such cases, it is advantageous to pre-activate less sensitive and inexpensive components as acyl acetals. Subsequently, the formation of cyclic acetals can be achieved by the amount of catalytic acid, which is desirable in terms of the entropy effect in the formation of cyclic compounds.

[0133] For the cyclic acetal of the present invention, the diol is usually a more expensive and less stable compound. Therefore, the aromatic carbonyl group is preferably converted into a cyclic acetal having a low-boiling point alcohol, such as methanol, ethanol, propanol, butanol, or trifluoroethanol, in the presence of a water-binding agent. More preferably, the water-binding agent is an orthoester and the solvent is a low-boiling point alcohol. Preferred orthoesters are trimethyl orthoformic acid, triethyl orthoacetic acid, triethyl orthoformic acid, triethyl orthoacetic acid, or tripropyl orthoformic acid. Most preferred is the use of trimethyl orthoformic acid.

[0134] The acidic catalyst is preferably, for example , concentrated sulfuric acid in organic solvents such as HCl / EtOH, HCl / MeOH, HCl / 1,4-dioxane, and water-free acids such as hydrogen chloride, e.g., Dowex ® 50W X8, Amberlite ® These include 4-toluenesulfonic acid, trifluoroacetic acid, or polymer sulfonic acid in a strong cation exchanger in a protonated form such as IR-120. The most preferred method is to use hydrogen chloride in 1,4-dioxane and 4-toluenesulfonic acid.

[0135] The reaction between a cyclic acetal and a diol compound is preferably carried out in a high-boiling-point aprotic solvent such as DMSO, DMF, DMA, NMP, 1,4-dioxane xylene, or toluene. It is preferable to use DMF. In this reaction, an acidic catalyst can be selected from the above list. It is preferable to use an acid with a low boiling point, such as HCl / 1,4-dioxane or trifluoroacetic acid. It is most preferable to use trifluoroacetic acid.

[0136] In another aspect, the present invention relates to a conjugate of the present invention for use in the treatment of cancer in a patient, wherein, in particular, the cancer is selected from the group consisting of breast cancer, pancreatic cancer, gallbladder cancer, colon cancer, lung cancer, prostate cancer, uterine cancer, stomach cancer, kidney cancer, malignant melanoma, leukemia, and malignant lymphoma.

[0137] In another aspect, the present invention relates to a pharmaceutical composition comprising a conjugate according to the present invention, comprising one or more pharmaceutically acceptable diluents, carriers, excipients, fillers, binders, lubricants, disintegrants, absorbents; and / or optionally further comprising a preservative.

[0138] In certain embodiments, the pharmaceutical composition is used in the form of a drug administered by tissue. This includes, among other things, parenteral forms including infusion and administration. The infusion form is formulated in an ampoule or a form prepared for use as a so-called infusion, such as, for example, a form for use with a syringe or a single-use syringe and, in addition, a perforable flask capable of multiple draws. Infusion-possible administration may be in the form of subcutaneous (sc), intramuscular (im), intravenous (iv), or intracutaneous administration. In particular, it is possible to produce each suitable formulation for infusion, such as suspensions of crystals, solutions, nanoparticles, or colloidal dispersion systems such as hydrosols.

[0139] The specific combinations of elements and characteristics in the embodiments described in detail above are merely for illustrative purposes; it will be obvious to conceive of interchangeing or substituting such description with the present patent application and the patent / patent application incorporated by reference. Those skilled in the art will recognize that changes, modifications, and alternative implementations of the contents described herein may occur to those skilled in the art without departing from the spirit and scope of the claimed invention. Accordingly, the foregoing description is for illustrative purposes only and is not intended to limit the invention. The scope of the invention is defined by the following claims and their equivalents. Furthermore, the reference numerals used in the description and claims are not intended to limit the scope of the claimed invention.

[0140] yes

[0141] Although the present invention has been illustrated and described in the drawings and the description above, such illustrations and descriptions are illustrative or exemplary and are not intended to limit the invention, and the invention is not limited to the disclosed embodiments. A person of ordinary skill in the art will be able to understand and perform variations of the disclosed embodiments by studying the drawings, the disclosed content, and the appended claims in practicing the claimed invention. In the claims, the word "comprising" does not exclude other elements or steps, and "one," which corresponds to the English indefinite article, does not exclude multiple. The fact that specific measures are cited in mutually dependent claims does not mean that a combination of such measures cannot be used to an advantage. References in the claims should not be inferred to limit the scope of the invention.

[0142] Example 1: 4-Dimethoxymethyl-phenol (HDP 30.2628)

[0143]

[0144] 4-hydroxybenzaldihydride (1.221 g, 10 mmol) was dissolved in 30 ml of methanol, 250 μl (1 mmol) of 4 M hydrogen chloride solution in 10.9 ml (100 mmol) of trimethyl orthoformic acid and dioxane was added, and the solution was heated under reflux for 1 hour. The TLC control group (n-hexane / ethyl acetate 1:1) showed complete conversion of the aldehyde (Rf=0.36) to dimethylacetal (Rf=0.45). After rotary evaporation to 10 ml, the reaction mixture was quenched with 50 ml of saturated sodium bicarbonate solution, and the mixture was extracted with 50 ml of ethyl acetate. The organic layer was washed with 50 ml of brine, dried over MgSO4, and evaporated as oil with 1.739 mg of 4-dimethoxymethyl-phenol. The product was homogeneous by TLC and HPLC and was used in the next step without further purification.

[0146] Example 2: 3-Dimethoxymethyl-phenol (HDP 30.2629)

[0147]

[0148] By applying the procedure of Example 1 to 3-hydroxybenzaldihydride, 3-dimethoxymethyl-phenol was obtained in quantitative yield.

[0149] Example 3: 4-Dimethoxymethyl-2-fluorophenol (HDP 30.2630)

[0150]

[0151] By applying the procedure from Example 1 to 1 g (7.14 mmol) of 3-fluoro-4-hydroxybenzaldihydride of Example 4, 4-dimethoxymethyl-2-fluoro-phenol was obtained in quantitative yield.

[0152] Example 4: 4-Bromo-3-dimethoxymethyl-phenol (HDP 30.2631)

[0153]

[0154] By applying the procedure from Example 1 to 2-bromo-3-hydroxybenzaldihydride, 4-bromo-3-dimethoxymethyl-phenol was obtained as a quantitative yield.

[0155] Example 5: 1-[11-bromo-3,6,9-trioxanedicycloxy]-4-dimethoxymethyl-benzene (HDP 30.2637)

[0156]

[0157] The raw material 4-dimethoxymethyl-phenol (1,739 g, max. 10 mmol) and 6.400 g (20 mmol) 1,11-Dibromo-3,6,6-trioxaundecane from Example 1 were dissolved in 40 ml of dry DMF, 3.910 g (12 mmol) of cesium carbonate was added, and the mixture was heated at 50°C for 1 hour. Afterward, the DMF was evaporated, the residue was stirred in 50 ml of dichloromethane, and then filtered. The filtrate was evaporated and purified on a silica gel having a conversion rate from n-hexane to ethyl acetate to obtain 1.862 g (46%) of the product 1-[11-bromo-3,6,9-trioxanedicycloxy]-4-dimethoxymethyl-benzene as oil.

[0158] 1 H NMR (500 MHz, CDCl3) d 7.39 - 7.32 (m, 2H), 6.94 - 6.87 (m, 2H), 5.35 (s, 1H), 4.17 - 4.11 (m, 2H), 3.91 - 3.84 (m, 2H), 3.80 (t, J = 6.3 Hz, 2H), 3.76 - 3.71 (m, 2H), 3.71 - 3.66 (m, 6H), 3.46 (t, J = 6.3 Hz, 2H), 3.31 (s, 6H).

[0159] 13 C NMR (126 MHz, CDCl3) d 158.85, 130.53, 127.88, 114.21, 102.99, 71.19, 70.84, 70.70, 70.64, 70.53, 69.73, 67.42, 52.59, 30.33.

[0160] MS (ESI + ) Result: 429.17; [M+Na] + Calculation for: 429.09 (C 17 H 27 BrNaO6)

[0161] Example 6: 1-[11-bromo-3,6,9-trioxanedicycloxy]-3-dimethoxymethyl-benzene (HDP 30.2641)

[0162]

[0163] By applying the procedure from Example 5 to the raw material product of Example 2, 2.079 g (51%) of the title compound (HDP 30.2641) was obtained as oil.

[0164] 1 H NMR (500 MHz, CDCl3) d 7.30 - 7.23 (m, 1H), 7.06 - 7.00 (m, 2H), 6.88 (ddd, J = 8.3, 2.5, 1.1 Hz, 1H), 5.35 (s, 1H), 4.17 - 4.12 (m, 2H), 3.88 - 3.84 (m, 2H), 3.81 (t, J = 6.3 Hz, 2H), 3.77 - 3.71 (m, 2H), 3.71 - 3.66 (m, 6H), 3.46 (t, J = 6.3 Hz, 2H), 3.32 (s, 6H).

[0165] 13 C NMR (126 MHz, CDCl3) d 158.92, 139.76, 129.32, 119.38, 115.01, 112.74, 103.06, 71.31, 70.95, 70.82, 70.76, 70.65, 69.87, 67.54, 52.84, 30.44.

[0166] MS (ESI + ) Result: 375.17 / 377.17; [MH-OMe] + Calculation for: 375.08 / 377.08 (C 16 H 24 BrO5)

[0167] Example 7: 1-(11-bromo-3,6,6-trioxaundecyl)-4-dimethoxymethyl-2-fluorobenzene (HDP 30.2642)

[0168]

[0169] By applying the procedure from Example 5 to the raw material product of Example 3, 2.032 g (67%) of the title compound (HDP 30.2642) was obtained as oil.

[0170] MS (ESI + ) Result: 393.17 / 395.17; [MH-OMe] + Calculation for: 393.07 / 395.07 (C 16 H 23 BrFO5)

[0171] 1 H NMR (500 MHz, CDCl3) d 7.18 (dd, J = 12.0, 2.1 Hz, 1H), 7.13 (ddt, J = 8.4, 1.9, 0.8 Hz, 1H), 6.97 (t, J = 8.4 Hz, 1H), 5.33 (s, 1H), 4.23 - 4.17 (m, 2H), 3.90 - 3.86 (m, 2H), 3.81 (t, J = 6.3 Hz, 2H), 3.76 - 3.72 (m, 2H), 3.68 (d, J = 9.6 Hz, 6H), 3.46 (t, J = 6.3 Hz, 2H), 3.30 (s, 6H).

[0172] 13 C NMR (126 MHz, , CDCl3) d 152.60 (d, J = 246.1 Hz), 146.92 (d, J = 10.9 Hz), 131.90 (d, J = 5.7 Hz), 122.61 (d, J = 3.6 Hz), 114.93 (d, J = 4.0 Hz), 114.79, 102.20 (d, J = 1.6 Hz), 71.31, 71.06, 70.80, 70.74, 70.65, 69.71, 69.21, 52.67, 30.44.

[0173] Example 8: 1-Bromo-4-[11-Bromo-3,6,9-trioxanedicycloxy]-2-dimethoxymethyl-benzene (HDP 30.2643)

[0174]

[0175] By applying the procedure from Example 5 to the raw material product of Example 4, 2.757 g (57%) of the title compound (HDP 30.2643) was obtained as oil.

[0176] 1H NMR (500 MHz, CDCl3) d 7.42 (d, J = 8.7 Hz, 1H), 7.18 (d, J = 3.2 Hz, 1H), 6.79 (dd, J = 8.7, 3.1 Hz, 1H), 5.50 (s, 1H), 4.16 - 4.09 (m, 2H), 3.87 - 3.83 (m, 2H), 3.81 (t, J = 6.3 Hz, 2H), 3.74 - 3.71 (m, 2H), 3.70 - 3.66 (m, 6H), 3.47 (t, J = 6.3 Hz, 2H), 3.38 (s, 6H).

[0177] 13 C NMR (126 MHz, CDCL3) d 158.22, 137.89, 133.57, 117.11, 114.31, 113.52, 102.99, 71.34, 70.99, 70.83, 70.77, 70.67, 69.77, 67.89, 54.08, 30.44.

[0178] MS (ESI + ) Result: 502.00 / 504.00 / 506.00;

[0179] [M+NH4] + Calculation for: 502.04 / 504.04 / 506.04 (C 17 H 30 Br2NO6)

[0180] Result: 453.08 / 455.08 / 457.08;

[0181] [MH-OMe] + Calculation for: 452.99 / 454.99 / 456.99 (C 16 H 23 Br2O5)

[0182] Example 9: 1-[11-Fmoc-amino-3,6,9-trioxanedicycloxy]-4-dimethoxymethyl-benzene (HDP 30.2640)

[0183]

[0184] The product from Example 5, HDP 30.2637 (1.725 g, 4.24 mmol), was dissolved in 30 ml of dry DMF, 975 mg of sodium azide (12.7 mmol, 3 eq.) was added, and the suspension was stirred overnight at ambient temperature. Subsequently, the solvent was evaporated, the residue was taken in 50 ml of tert-butylmethyl ether (MTBE), and washed with sodium bicarbonate and brine (50 ml each). The organic phase was dried over MgSO4 and evaporated into a clear oil (1.435 g, yield 92%) containing the intermediate product 1-[11-azido-3,6,9-trioxanedicycloxy]-4-dimethoxy-methylbenzene.

[0185] MS (ESI + ) Result: 392.25; [M+Na] + Calculation for: 392.18 (C 17 H 27 N3NaO6)

[0186] Result: 338.25; [MH-OMe] + Calculation for: 338.17 (C 16 H 24 N3O5)

[0187] Subsequently, the azide intermediate was dissolved in THF, and 2.038 mg (2 eq.) of triphenylphosphine was added, followed by the addition of 2 ml of water. The mixture was stirred overnight at room temperature until gas evaporation ceased. After 21 hours, the volatile substances were evaporated, and a mixture of the remaining phosphine and the intermediate 1-[11-amino-3,6,9-trioxanedicycloxy]-4-dimethoxy-methylbenzene was dissolved in 20 ml of DMF.

[0188] 1.57 g (1.2 eq.) of Fmoc-N-hydroxysuccinimide ester and N-ethyldiisopropylamine (1320 μl; 2 eq.) were added. After stirring at room temperature for 17 hours, an additional 785 mg of Fmoc-OSu was added. After the complete conversion of the intermediate amine, the solvent was evaporated, the residue was dissolved in 100 ml of dichloromethane, washed with water (2 x 100 ml) and brine (100 ml), dried (MgSO4), and reduced to the dry product. The raw material product was purified on a silica gel with a change rate of 0 to 100% MTBE in n-hexane to obtain 617 mg (28%) of the title compound as a viscous oil.

[0189] 1 H NMR (500 MHz, CDCl3) d 7.75 (dt, J = 7.6, 0.9 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.39 (tt, J = 7.4, 0.9 Hz, 2H), 7.35 - 7.28 (m, 4H), 6.90 - 6.84 (m, 2H), 5.42 (t, J = 5.7 Hz, 1H), 5.33 (s, 1H), 4.40 (d, J = 6.9 Hz, 2H), 4.21 (t, J = 6.9 Hz, 1H), 4.08 (t, J = 4.9 Hz, 2H), 3.80 (t, J = 4.9 Hz, 2H), 3.72 - 3.58 (m, 8H), 3.55 (t, J = 5.1 Hz, 2H), 3.37 (q, J = 5.4 Hz, 2H), 3.29 (s, 6H).

[0190] 13 C NMR (126 MHz, CDCl3) d 158.77, 156.47, 143.97, 141.26, 127.84, 127.69, 127.60, 126.98, 125.02, 119.90, 114.14, 102.95, 70.79, 70.55, 70.30, 69.99, 69.85, 69.67, 67.32, 66.44, 52.55, 47.24, 40.90.

[0191] MS (ESI + ) Result: 588.33; [M+Na] + Calculation for: 588.26 (C 32 H 39 NNaO8)

[0192] Result: 534.33; [MH-OMe] + Calculation for: 534.25 (C 31 H 36 NO7)

[0193] Example 10: 1-[11-Fmoc-amino-3,6,9-trioxanedicycloxy]-3-dimethoxymethyl-benzene (HDP 30.2650)

[0194]

[0195] By applying the procedure from Example 9 to the 1.082 g raw material product of Example 6, 106 mg (7%) of the title compound (HDP 30.2650) was obtained as oil.

[0196] MS (ESI + ) Result: 588.33; [M+Na] + Calculation for: 588.26 (C 32 H 39 NNaO8)

[0197] Example 11: 1-(11-Fmoc-amino-3,6,6-trioxaundecyl)-4-dimethoxymethyl-2-fluorobenzene (HDP 30.2651)

[0198]

[0199] By applying the procedure from Example 9 to the 1.900 g raw material product of Example 7, 112 mg (4%) of the title compound (HDP 30.2651) was obtained as oil.

[0200] MS (ESI + ) Result: 606.33; [M+Na] + Calculation for: 606.25 (C 32 H 38 FNNaO8)

[0201] Result: 552.33; [MH-OMe] + Calculation for: 552.24 (C 31 H 35 FNO7)

[0202] Example 12: 1-Bromo-4-[11-Fmoc-amino-3,6,9-trioxanedicycloxy]-2-dimethoxymethyl-benzene (HDP 30.2652)

[0203]

[0204] By applying the procedure from Example 9 to the 2.625 g raw material product of Example 8, 193 mg (6%) of the title compound (HDP 30.2652) was obtained as oil.

[0205] MS (ESI + ) Result: 666.17 / 668.17

[0206] [M+Na] + Calculation for: 666.17 / 668.17 (C32H38BrNNaO8)

[0207] Example 13: Fmoc-Val-Ala-PAP-CHO (HDP 30.2623)

[0208]

[0209] In a three-necked flask equipped with a thermometer, a dropping funnel, and a rubber diaphragm, 20 ml of dry dichloromethane (446 μl, 5.2 mmol) was treated with oxalyl chloride, and the solution was cooled to -80°C under an argon atmosphere.

[0210] While maintaining the temperature below -70℃, DMSO (739 μl, 10.4 mmol) was added dropwise through a rubber diaphragm using a syringe. After stirring for 15 minutes, Fmoc-Val-Ala-PAB-OH dissolved in 20 ml of dichloromethane ( HDP 30.1419 , 2.062 g, 4.0 mmol) was added dropwise through a dropping funnel over a period of 30 minutes, and the mixture was stirred again for 30 minutes.

[0211] Next, triethylamine (2.634 ml, 19.0 mmol) was added via syringe, and after 5 minutes, the cooling bath was removed and the reaction was allowed to proceed to room temperature. Subsequently, 25 ml of water and 25 ml of dichloromethane were added, and the phases were separated. The liquid phase was extracted with 20 ml of dichloromethane, and the combined organic phase was washed with 20 ml of 0.2 M citric acid, 3 x 20 ml of water, and 20 ml of brine. After drying (MgSO4), the solvent was evaporated, and the raw material product (965 mg) was purified on silica gel with ethyl acetate at a change rate of 0-20% in dichloromethane to obtain 631 mg (31%) of the product as an amorphous solid.

[0212] 1 H NMR (500 MHz, D6-DMSO) d 10.36 (s, 1H), 9.89 (s, 1H), 8.22 (d, J = 6.8 Hz, 1H), 7.91 - 7.79 (m, 6H), 7.74 (t, J = 8.6 Hz, 2H), 7.45 - 7.29 (m, 5H), 4.47 (p, J = 7.0 Hz, 1H), 4.37 - 4.28 (m, 1H), 4.28 - 4.19 (m, 2H), 3.95 (dd, J = 8.9, 7.0 Hz, 1H), 2.02 (h, J = 6.7 Hz, 1H), 1.35 (d, J = 7.1 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H), 0.88 (d, J = 6.7 Hz, 3H).

[0213] 13C NMR (126 MHz, D6-DMSO) d 191.35, 171.75, 171.03, 156.05, 144.40, 143.79, 143.69, 140.61, 131.30, 130.68, 127.52, 127.50, 126.93, 125.22, 119.96, 119.95, 118.76, 65.63, 59.86, 49.20, 46.64, 30.32, 19.06, 18.12, 17.70.

[0214] MS (ESI + ) Result: 536.25; [M+Na] + Calculation for: 536.22 (C 30 H 31 N3NaO5)

[0215] Example 14: Fmoc-Val-Ala-PAP-CH(OMe) 2 (HDP 30.2677)

[0216]

[0217] 13-Step Product HDP 30.2623 (514 mg, 1.00 mmol) was dissolved in 20 ml of methanol. Trimethyl orthoformic acid (5.47 ml, 50 mmol), 4-toluenesulfonic acid monohydrate (21 mg, 0.1 mmol), and 20 ml of dichloromethane were added, and the mixture was refluxed under an argon atmosphere. After 5 hours, trimethyl orthoformic acid (5.47 ml) and 4-toluenesulfonic acid (210 mg) were further added, and heating was continued for an additional 2 hours. The reaction mixture was cooled and dropped into 50 ml of saturated sodium bicarbonate with vigorous stirring. After stopping the gas phase, 100 ml of dichloromethane was added, and the turbid mixture was centrifuged at 4000 xg in a 40 ml portion. The clear supernatant was removed, the organic layer was washed with 15 ml of water, and then centrifuged. The combined organic matter was evaporated, and the residue was evaporated together with 2 x 20 ml methanol to remove traces of water. The residue was taken with 100 ml dichloromethane, and insoluble material was removed by filtration. The filtrate was evaporated, and the residue was chewed with 50 ml MTBE at 40°C for 1 hour, after which it was cooled to room temperature and filtered by suction. The precipitate was washed with 20 ml MTBE and dried under vacuum to obtain 445 mg (79%) of the title product as an amorphous solid.

[0218] Example 15: Fmoc-Val-Ala-OAB-OH (HDP 30.2761)

[0219]

[0220] Dipeptide Fmoc-Val-Ala-OH (4.105 g, 10.0 mol) and 4-aminobenzyl alcohol (1.293 g, 1.05 eq.) were dissolved in 60 ml of isolated tetrahydrofuran (THF). 2-ethoxy-N-(ethoxycarbonyl)-1,2-dihydroquinoline (EEDQ, 2.597 g, 1.05 eq.) was added, and the mixture was stirred at room temperature while protected from light.

[0221] After 3 days, the reaction mixture forming the gelatinous substance was diluted with 140 ml MTBE and stirred until fine particles were formed; the mixture was filtered by suction, washed with 50 ml MTBE, and dried to obtain 4.480 g (87%) of the title product. HDP 30.2761 It was obtained as a colorless solid.

[0222] 1 H NMR (500 MHz, D6-DMSO) d 9.40 (s, 1H), 8.20 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.74 (t, J = 7.6 Hz, 2H), 7.53 (d, J = 8.0 Hz, 1H), 7.46 - 7.36 (m, 4H), 7.32 (tt, J = 7.5, 1.3 Hz, 2H), 7.23 (td, J = 7.6, 1.7 Hz, 1H), 7.15 (td, J = 7.4, 1.3 Hz, 1H), 5.24 (t, J = 5.6 Hz, 1H), 4.56 - 4.42 (m, 3H), 4.36 - 4.28 (m, 1H), 4.28 - 4.20 (m, 2H), 3.96 (dd, J = 9.1, 6.8 Hz, 1H), 2.04 (h, J = 6.8 Hz, 1H), 1.35 (d, J = 7.1 Hz, 3H), 0.90 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.8 Hz, 3H).

[0223] 13C NMR (126 MHz, D6-DMSO) d 171.09, 170.90, 156.09, 143.83, 143.72, 140.65, 140.63, 135.08, 134.74, 127.55, 127.54, 127.23, 126.97, 126.87, 125.27, 124.64, 123.66, 120.00, 119.98, 65.65, 60.03, 59.86, 48.90, 46.65, 40.01, 39.84, 39.67, 39.50, 39.34, 39.17, 39.00, 31.23, 30.33, 19.15, 18.04, 17.89.

[0224] MS (ESI + ) Result: 538.25 [M+Na] + Calculation for: 538.23 (C 30 H 33 N3NaO5)

[0225] Example 16: Fmoc-Val-Ala-OAP-CH(OMe)2 (HDP 30.2769)

[0226]

[0227] Benzyl alcohol from Step 15 HDP 30.276118 ml DMSO and 36 ml isolated dichloromethane were added to (4.48 g, 8.69 mmol), followed by the addition of 3.66 g, 1.0 eq.) Dess-Martin periodinan. After stirring for 2 hours, the resulting brownish solution was diluted with 90 ml chloroform and washed with 90 ml water. The liquid phase was extracted again with 2 x 40 ml chloroform, and the bound organic matter was washed by shaking with 90 ml saturated sodium bicarbonate until the pH reached 7-8. Subsequently, 30 ml of 20% sodium thiosulfate solution was added, and shaking was continued for 2 minutes. After separating the organic layer, it was washed with an additional 90 ml of water and dried (MgSO4) to remove volatile substances. The remaining 4.875g was stirred with 150 ml MTBE until fine particles were formed, filtered by suction to remove them, washed with 50 ml MTBE, and dried to 4.307 g (97%) to obtain the intermediate product aldehyde as a colorless solid.

[0228] MS (ESI + ) Result: 536.25 [M+Na] + Calculation for: 536.22 (C 30 H 31 N3NaO5)

[0229] 4.187 g (8.152 mmol) of the aldehyde Fmoc-Val-Ala-OAP-CHO was suspended in 80 ml of methanol, trimethyl orthoformic acid (45 ml, 50 eq.) and 4-toluenesulfonic acid monohydrate (1.551 g, 1 eq.) were added, and the mixture was refluxed under an argon atmosphere for 1 hour. After cooling to room temperature, the reaction mixture was added to 100 ml of saturated sodium bicarbonate and vigorously stirred.

[0230] 100 ml of chloroform was added to dissolve the gelatinous material and separate the phases. The liquid phase was re-extracted with 2 x 50 ml of chloroform, and the combined organic layer was washed with 50 ml of semi-saturated brine, dried (MgSO4), and evaporated. The raw material product was purified on silica gel with ethyl acetate at a change rate of 0 to 20% in dichloromethane to yield 3.279 g (72%) of the title product. HDP 30.2769 It was obtained as an amorphous solid.

[0231] 1 H NMR (500 MHz, D6-DMSO) d 9.27 (s, 1H), 8.30 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.75 (t, J = 7.2 Hz, 2H), 7.70 (d, J = 8.1 Hz, 1H), 7.48 - 7.38 (m, 4H), 7.32 (ddd, J = 8.7, 6.7, 1.4 Hz, 3H), 7.18 (td, J = 7.6, 1.3 Hz, 1H), 5.45 (s, 1H), 4.46 (p, J = 7.0 Hz, 1H), 4.37 - 4.29 (m, 1H), 4.28 - 4.20 (m, 2H), 3.99 (dd, J = 9.2, 6.8 Hz, 1H), 3.26 (s, 3H), 3.26 (s, 3H), 2.05 (h, J = 6.7 Hz, 1H), 1.35 (d, J = 7.1 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H).

[0232] 13C NMR (126 MHz, D6-DMSO) d 176.52, 176.05, 161.32, 149.06, 148.95, 145.89, 145.87, 140.68, 134.89, 133.97, 132.82, 132.80, 132.38, 132.23, 130.56, 130.55, 129.58, 129.11, 125.27, 106.40, 70.84, 65.01, 58.68, 58.37, 54.27, 51.85, 35.65, 24.41, 23.26, 22.69.

[0233] MS (ESI + ) Result: 582.33 [M+Na] + Calculation for: 582.26 (C 32 H 37 N3NaO6)

[0234] Example 17: BMP-Val-Ala-OAP-CH(OMe) 2 (HDP 30.2776)

[0235]

[0236] The product from Example 16, HDP 30.2769 (3.236 g, 5.782 mmol) was suspended in 30 ml of DMF. Diethylamine (8 ml) was added and stirred at room temperature for 30 minutes to form a clear solution. Volatile substances were evaporated, and the residue was evaporated together with 30 ml of fresh DMF and subsequently dried under high vacuum. The raw material product contained the intermediate amine H-Val-Ala-OAP-CH(OMe)2 and was used without further purification.

[0237] MS (ESI + ) Result: 360.25 [M+Na] + Calculation for: 360.19 (C 17 H 27 N3NaO4)

[0238] The intermediate product free amine was dissolved in 30 ml of DMF, 1.539 g (1 eq.) of 3-(maleimido)propionic acid N-hydroxysuccinimide ester (BMPS) and 1.967 ml (2 eq.) of N-ethyldiisopropylamine (DIPEA) were added, and the solution was stirred for 1 hour. The solvent was evaporated, and the residue was stirred with 100 ml of MTBE. Fine particles were formed, which were removed by filtration, washed with 20 ml of MTBE, and dried under vacuum. The raw material product was purified on silica gel with methanol at a change rate of 0 to 10% in dichloromethane to obtain 2.164 g (77%) of pure title product as a colorless, amorphous solid.

[0239] 1 H NMR (500 MHz, D6-DMSO) d 9.20 (s, 1H), 8.27 (d, J = 6.8 Hz, 1H), 8.00 (d, J = 9.0 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.43 (dd, J = 7.7, 1.6 Hz, 1H), 7.33 (td, J = 7.8, 1.7 Hz, 1H), 7.16 (td, J = 7.5, 1.3 Hz, 1H), 6.99 (s, 2H), 5.44 (s, 1H), 4.41 (p, J = 7.1 Hz, 1H), 4.22 (dd, J = 8.8, 6.6 Hz, 1H), 3.61 (dq, J = 17.1, 6.6 Hz, 2H), 3.27 (s, 3H), 3.26 (s, 3H), 2.48 - 2.40 (m, 2H), 2.01 (dh, J = 20.3, 6.8 Hz, 1H), 1.33 (d, J = 7.2 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H).

[0240] 13C NMR (126 MHz, D6-DMSO) d 171.03, 170.78, 170.63, 169.50, 135.51, 134.47, 129.50, 128.70, 127.15, 124.23, 123.70, 101.33, 57.27, 53.46, 53.18, 49.08, 33.99, 33.64, 30.32, 19.10, 17.91, 17.28.1

[0241] MS (ESI + ) Result: 511.25 [M+Na] + Calculation for: 511.22 (C 24 H 32 N4NaO7)

[0242] Example 18: Fmoc-Val-Ala-MAB-OH (HDP 30.2767)

[0243]

[0244] By performing the procedure of Example 15 with 3-aminobenzyl alcohol, 30.2767 g of the title product HDP, 4.698 g (91%), was obtained as an amorphous solid.

[0245] 1H NMR (500 MHz, D6-DMSO) d 9.95 (s, 1H), 8.17 (d, J = 7.0 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H), 7.75 (t, J = 7.8 Hz, 2H), 7.59 (d, J = 1.9 Hz, 1H), 7.50 - 7.37 (m, 4H), 7.32 (tt, J = 7.5, 1.6 Hz, 2H), 7.24 (t, J = 7.8 Hz, 1H), 6.99 (d, J = 7.8 Hz, 1H), 5.19 (t, J = 5.7 Hz, 1H), 4.45 (dd, J = 14.7, 6.5 Hz, (3H), 4.36 - 4.28 (m, 1H), 4.28 - 4.19 (m, 2H), 3.93 (dd, J = 9.0, 7.1 Hz, 1H), 2.01 (h, J = 6.8 Hz, 1H), 1.32 (d, J = 7.0 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H).

[0246] 13 C NMR (126 MHz, D6-DMSO) d 170.93, 156.08, 143.80, 143.72, 143.16, 140.63, 140.62, 138.78, 128.29, 127.57, 127.54, 126.98, 125.29, 121.21, 120.02, 120.00, 117.41, 117.11, 65.63, 62.77, 59.92, 48.95, 46.61, 30.32, 19.13, 18.19, 18.07, 3.26.

[0247] MS (ESI + ) 결과: 538.33 [M+Na] + 에 대한 계산: 538.23 (C 30 H 33 N3NaO5)

[0248] Example 19: Fmoc-Val-Ala-MAP-CH(OMe)2 (HDP 30.2778)

[0249]

[0250] Omitting the final silica gel chromatography, the compound of Example 18 ( HDP 30.2767 ) By performing the procedure of Example 16 as ), 6.28 g raw material title product sufficiently purified for use in the next step HDP 30.2778 It was obtained as an amorphous solid.

[0251] Example 20: BMP-Val-Ala-MAP-CH(OMe) 2 (HDP 30.2776)

[0252]

[0253] Compound of Example 19 ( HDP 30.2778 By performing the procedure of Example 17 with ), HDP 30.2767 Title product on the surface HDP 30.2781 It was obtained as an amorphous solid based on a 95% yield.

[0254] 1 H NMR (500 MHz, D6-DMSO) d 9.89 (s, 1H), 8.16 (d, J = 6.9 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.66 (t, J = 1.9 Hz, 1H), 7.64 - 7.54 (m, 1H), 7.30 (t, J = 7.9 Hz, 1H), 7.06 (dt, J = 7.6, 1.4 Hz, 1H), 7.00 (s, 2H), 5.35 (s, 1H), 4.38 (p, J = 7.1 Hz, 1H), 4.14 (dd, J = 8.4, 6.8 Hz, 1H), 3.68 - 3.57 (m, 2H), 3.24 (s, 6H), 2.50 - 2.40 (m, 2H), 2.00 - 1.90 (m, J = 6.8 Hz, 1H), 1.32 (d, J = 7.1 Hz, 3H), 0.87 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H).

[0255] 13C NMR (126 MHz, D6-DMSO) d 171.09, 170.77, 170.71, 169.76, 138.90, 138.79, 134.52, 128.46, 121.42, 118.99, 117.25, 102.42, 57.71, 52.44, 52.42, 49.08, 34.02, 33.70, 30.26, 19.11, 18.17, 17.83.

[0256] MS (ESI + ) Result: 511.25 [M+Na] + Calculation for: 511.22 (C 24 H 32 N4NaO7)

[0257] General procedure for cyclic acetals A

[0258] A dimethyl acetal compound (10 eq.) was added to a solution of the diol compound in dried DMF (100 μl / mg). The mixture was acidified with trifluoroacetic acid (10 μl / mg) and stirred at ambient temperature under an argon atmosphere until a clear solution was formed. Subsequently, the solvent was evaporated under high vacuum, and the residue was redissolved in fresh DMF. The sample was quenched in 1% triethylamine in methanol and analyzed by HPLC to show whether the starting material had been completely converted.

[0259] The DMF solution was dropped into a 0.1% triethylamine solution cooled with ice in 10 times the volume of tert-butylmethyl ether (MTBE), and the resulting precipitate was separated by centrifugation. The supernatant was removed, and the pellet was resuspended in an equal volume of 0.1% triethylamine MTBE and centrifuged again. The raw material product, dried under vacuum, was further purified by prep.-HPLC.

[0260] General procedure for cyclic acetals B

[0261] The diol compound (1 eq.) and the dimethyl acetal compound (8 eq.) were placed in a centrifuge tube and dissolved in DMF / TFA 4:1 (v / v, 20 μl / μmol diol compound). The tube was shaken at room temperature until the HPLC control group showed complete conversion of the diol compound (1–18 hours). Subsequently, 20 times the volume of ice-cooled MTBE was added, and the precipitate was separated by centrifugation. The first pellet was resuspended in an equal volume of MTBE containing 2 vol-% triethylamine and centrifuged again. The dried pellet was used for the next reaction without further purification.

[0262] Example 21: α-Amanitine-(Fmoc-Val-Ala-4-amino-benzylideneacetal) HDP 30.2681

[0263]

[0264] Alpha-amanitin (30.98 mg) and HDP 30.2677 were reacted according to a standard procedure. The raw material product was purified for 15 minutes with acetone ytrile with a change rate of 5 to 100% in water on Phenomenex Luna-C18 (2), 10 μm, 250 x 21.2 mm. The product containing a portion of 10.40-11.08 min was freeze-dried to obtain 37.07 mg (78%) of freeze-dried product.

[0265] MS (ESI + ) Result: 1414.67; [MH] + Calculation for: 1414.58 (C 69 H 84 N 13 O 18 S)

[0266] Example 22: α-Amanitine-[4-(11-Fmoc-amino-3,6,9-trioxanedicyfloxy)-benzylideneacetal] HDP 30.2665

[0267]

[0268] α-amanitine (20.51 mg) according to general procedure A and from Example 9 HDP 30.2640 20.96 mg (65%) of the title product was obtained as freeze-dried powder by reacting.

[0269] Example 23: α-Amanitine-[3-(11-Fmoc-amino-3,6,9-trioxanedicyproxy)-benzylideneacetal] HDP 30.2666

[0270]

[0271] α-amanitine (20.51 mg) according to general procedure B and from Example 10 HDP 30.2650 The original compound was reacted and used without further purification.

[0272] Example 24: α-Amanitine-[4-(11-Fmoc-amino-3,6,9-trioxanedicycloxy)-3-fluoro-benzylideneacetal] HDP 30.2667

[0273]

[0274] α-amanitine (20.51 mg) according to general procedure B and from Example 11 HDP 30.2651 The original compound was reacted and used without further purification.

[0275] Example 25: α-Amanitine-[2-Bromo-3-(11-Fmoc-amino-3,6,9-trioxanedicyproxy)-benzylideneacetal] HDP 30.2668

[0276]

[0277] α-amanitine (20.51 mg) according to general procedure B and from Example 11 HDP 30.2652 The reaction was performed, and the original compound was used without further purification.

[0278] Example 26: α-Amanitin-(BMP-Val-Ala-4-amino-benzylideneacetal) HDP 30.2684

[0279]

[0280] Cyclic acetal HDP 30.2681 (37.07 mg, 26.21 μmol) was dissolved in 2 ml of dry DMF. Diethylamine (92 μl, 891 μmol) was added, and the mixture was stirred at ambient temperature for 15 minutes. The volatile substances vacuum The residue was evaporated and redissolved in dried DMF. HPLC and MS analysis showed complete deprotection to free amines.

[0281] (MS-ESI + ) Result: 1192.67; [MH] + Calculation for: 1192.51 (C 54 H74 N 13 O 16 S)

[0282] 3-(maleimido)propionic acid N-hydroxysuccinimide ester (BMPS, 14 mg, 52.42 μmol = 2 eq.) was dissolved in 500 μl dry DMF, and then 8.92 μl (52.42 μmol = 2 eq.) N,N-diisopropylethylamine was added. After stirring at room temperature for 2 hours, the reaction mixture was dropped into 10 ml ice-cooled MTBE, and the precipitate was separated by centrifugation. The raw material pellet was purified by prep. HPLC on Luna-C18 (2) with a change rate of 5-50% acetonytrile in water for 15 minutes. The product fraction of 11.9-12.8 minutes was freeze-dried to obtain 20.85 mg (59%) of colorless freeze-dried product.

[0283] MS (ESI + ) Result: 1343.58; [MH] + Calculation for: 1343.54 (C 61 H 79 N 14 O 19 S)

[0284] Example 27: α-Amanitine-[4-(11-(3-maleimidopropyl-amido)-3,6,9-trioxanedichydroxy)-benzylideneacetal] HDP 30.2669

[0285]

[0286] By applying the procedure from Example 26 to 20.96 mg HDP 30.2665 from Example 22, the title product HDP 30.2669 (13.00 mg, 65%) was obtained as freeze-dried powder.

[0287] MS (ESI + ) Result: 1371.58; [MH] + Calculation for 1371.52 (C 61 H 80 N 12 NaO 21 S))

[0288] Example 28: α-Amanitine-[3-(11-(3-maleimidopropyl-amido)-3,6,9-trioxanedicyproxy)-benzylideneacetal] HDP 30.2670

[0289]

[0290] The procedure from Example 26 and the raw material from Example 23 HDP 30.2666 By applying to, the title product HDP 30.2670 (4.39 mg, α-amanitine-based 15%) was obtained as freeze-dried powder.

[0291] MS (ESI + ) Result: 1371.50; [MH] + Calculation for: 1371.52 (C 61 H 80 N 12 NaO 21 S))

[0292] Example 29: α-Amanitine-[4-(11-(3-maleimidopropyl-amido)-3,6,9-trioxanedicycloxy)-3-fluoro-benzylideneacetal] HDP 30.2671

[0293]

[0294] The procedure from Example 26 and the raw material from Example 24 HDP 30.2667 By applying to, the title product HDP 30.2671 (5.82 mg, α-amanitine-based 19%) was obtained as freeze-dried powder.

[0295] MS (ESI + ) Result: 1389.50; [MH] + Calculation for: 1389.51 (C 61 H 79 FN 12 NaO 21 S)

[0296] Example 30: α-Amanitine-[2-Bromo-3-(11-(3-maleimidopropyl-amido)-3,6,9-trioxanedichydroxy)-benzylideneacetal] HDP 30.2672

[0297]

[0298] The procedure from Example 26 and the raw material from Example 24 HDP 30.2668 By applying to, the title product HDP 30.2672 (2.43 mg, 8% based on α-amanitine) was obtained as freeze-dried powder.

[0299] MS (ESI + ) Result: 1449.42 / 1451.42;

[0300] [MH] + Calculation for: 1449.43 / 1451.43 (C 61 H 79 BrN 12 NaO 21 S)

[0301] Example 31: α-Amanitin-(BMP-Val-Ala-2-amino-benzylideneacetal) HDP 30.2792

[0302]

[0303] The title compound could be produced by reacting α-amanitine with compound HDP 30.2776 from Example 17.

[0304] Example 32: α-Amanitin-(BMP-Val-Ala-3-amino-benzylideneacetal) HDP 30.2793

[0305]

[0306] α-Amanitine compound HDP from Example 20 30.2781 The title compound could be produced by reacting with it.

[0307] Example 33: Amatoxin antibody conjugate synthesis

[0308] Derivatives of trastuzumab with the D265C mutation ("thiomav," a mutation from aspartic acid to cysteine ​​at amino acid position 265 of the Ig heavy chain), anti-Her2neu-specific antibodies were conjugated with amatoxin-linker derivatives HDP 30.2669, HDP 30.2670, HDP 30.2671, HDP 30.2672, and HDP 30.2684.

[0309] For each conjugation reaction, 10 mg of thiomab in PBS buffer adjusted to 1 mM EDTA was used. Cysteine ​​was opened by antibody incubation with 40 eq. TCEP on a shaker at 37°C for 3 hours, and two consecutive dialysis cycles were performed at 4°C in 1x PBS, 1 mM EDTA, pH 7.4 in a Slide-A-Lyzer dialysis cassette. Oxidation was performed for 3 hours by antibody incubation with 20 eq. dehydroascorbic acid (dhAA) on a shaker at RT.

[0310] Conjugation with amatoxin-linker derivatives was performed using 4 eq. HDP 30.2669, HDP 30.2670, HDP 30.2671, HDP 30.2672, and HDP 30.2684, respectively, to enable solubility in DMSO. The mixture was incubated at room temperature for 1 hour, 25 eq. N-acetyl-L-cysteine ​​was added, quenched, and incubated at RT for 15 minutes or overnight at 4°C. Each reaction mixture was purified by Sephadex G-25 gel filtration (PD-10 column, GE Healthcare Life Sciences) equilibrated to PBS, pH 7.4. Protein-containing portions were identified using Bradford reagent on Parafilm and pooled. The conjugate solutions were 4 in a Slide-A-Lyzer dialysis cassette (Thermo Scientific, 20,000 MWCO) against PBS. o Dialysis was performed overnight at C, pH 7.4. Protein concentration was determined using a RotiQuant assay (Carl Roth, Germany). The conjugate solution was 5 mg / ml (~3.44x10 -5 The protein concentration was adjusted to M), sterilized and filtered, and stored at 4°C. The conjugates were designated as "T-D265C-30.2669", "T-D265C-30.2670", "T-D265C 30.2671", "T-D265C-30.2672", and "T-D265C-30.2684".

[0311] In Coomassie staining under reducing conditions, the light and heavy chains of the amatoxin antibody conjugates and the naked antibody were expected to have apparent masses of approximately 25 and 50 kDa, respectively. For the amatoxin antibody conjugates, an upshift in the protein signal of the heavy chain was observed compared to the naked antibody, indicating toxin conjugation to the heavy chain. In the anti-amanitine Western Blot under reducing conditions, the thiomav conjugate showed a signal at approximately 55 kDa for the heavy chain and no signal for the light chain. This indicates that toxin conjugation occurs only in the heavy chain.

[0312] Example 34: ex vivo Cytotoxicity study on HER2-positive tumor cell lines

[0313] Cytotoxic activity of trastuzumab (Anti-Her2neu-specific antibody)-amatoxin conjugate using chemiluminescent BrdU-ELISA conjugate (Roche) on HER2-sheep tumor cell lines SKBR-3 (breast cancer), NCI-N87 (gastric cancer), and JIMT-1 (breast cancer) ex vivo The tested conjugates include one conjugate having a directional cyclic acetal of amatoxin (T-D265C-30.2684) according to the present invention with a dipeptide enzyme cleavage site linked to aa3, one conjugate having a dipeptide enzyme with a para-amino-benzyl ether linker with a cleavage site linked to aa4 of amatoxin (T-D265C-30.1699), and four conjugates having directional cyclic acetals with a poly-ethylene glycol (PEG) linker linked to aa3 of amatoxins (T-D265C-30.2669 with a p-PEG3 linker, -30.2670 with an m-PEG3 linker, -30.2671 with a p-PEG3 linker and aryl fluoride moiety, and -30.2672 with an o-PEG3 linker and aryl-bromide moiety).

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320] The results of the cytotoxicity study on SKBR-3 cells are shown in Fig. 2, the results of the cytotoxicity study on NCI-N87 cells are shown in Fig. 3, and the results for JIMT-1 cells are shown in Fig. 4.

[0321] EC of various trastuzumab-amatoxin conjugates on different Her2-positive cell lines 50 The values ​​are shown in Table 1:

[0322]

[0323] [( ), >50% cell viability; - , no cytotoxicity; VA, valine-aniline dipeptide; PEG, polyethylene glycol]

[0324] The cytotoxicity of the conjugate T-D265C-30.2684 according to the present invention was compared with the cytotoxicity of the conjugate T-D265C-30.1699. When compared to the conjugate T-D265C-30.2669 containing a directional cyclic acetal with a PEG linker, the cytotoxicity of the conjugate T-D265C-30.2684 according to the present invention was somewhat higher on SKBR-3 cells, but significantly higher on NCI-N87 cells and JIMT-1 cells.

[0325] ADCs T-D265C-30.2684 and T-D265C-30.1699, both possessing cleavable Val-Ala linkers, exhibited fully expressed cytotoxic potential in the tested cell lines. The unstable acidic ADCs T-D265C-30.2670, T-D265C-30.2671, and T-D265C-30.2672 showed only growth inhibition on SKBR-3 cells with residual cell viability of approximately 65%, 50%, and 60%, respectively, and showed no cytotoxicity on JIMT-1 cells.

[0327] Amatoxin derivative HDP 30.2684 is HDP 30.1699 ex vivo It was as effective as, but synthesized much faster. Three HPLC purifications were required during the synthesis of HDP 30.1699, whereas only one purification was required for the synthesis of HDP 30.2684.

[0329] Example 35: In vivo Efficacy of acetal linker conjugates in the SKOV-3 xenograft tumor mouse model

[0330] This study consisted of 7 experimental groups of 10 female NOD Scid mice, each with a subcutaneous Her2-positive SKOV-3 (human oocyte) tumor. The efficacy of two directional cyclic acetal conjugates with high-dose amatoxin aa3 linkages (T-D265C-30.2684, T-D265C-30.2669) was compared with T-D265C-30.1699, which has a para-amino-benzyl linker with a dipeptide cleavage site linked to amatoxin aa4, and an untreated control group. Mice were treated with a single IV dose.

[0331] The experimental setup is as shown in Table 2:

[0332]

[0333] The results of the study are shown in Figure 5.

[0334] In the group treated with the cyclic acetal amatoxin-drug conjugate, all animals were tumor-free. In the animal group treated with 1 mg / kg T-D265C-30.1699, no tumor-free animals were obtained. In the animal group treated with 2 mg / kg T-D265C-30.1699, 4 out of 10 animals were tumor-free.

[0335] Example 36: Tolerance of acetal linker conjugates in an in vivo mouse model

[0336] The tolerability of amatoxin ADCs T-D265C-30.2669 and T-D265C-30.2684, respectively, was evaluated in NOD / SCID mice after a single intravenous administration. A total of 21 animals were used. T-D265C-30.2669 and T-D265C-30.2684 were tolerable at effective doses up to 6 mg / kg. The results are shown in Figure 6.

[0337] References

[0338] Binz HK et al. (2005). Engineering treatment of novel binding proteins from non-immunoglobulin domains. Nat. Biotechnol. Vol. 10: 1257-1268.

[0339] Chalouni C. and Doll S. (2018). Fate of antibody-drug conjugates in cancer cells. Journal of Experimental & Clinical Cancer Research. Vol. 37: 20.

[0340] Gillies ER except (2004). Acetals as pH-sensitive linkages for drug delivery. Bioconjugate Chem. Vol. 15: 1254-1263.

[0341] Jeffrey except (2005). Design, synthesis, and in vitro evaluation of dipeptide-based antibody minor groove conjugates. J. Med. Chem. Vol. 48: 1344-1358.

[0342] *Jeffrey except (2006). Development and characterization of β-glucuronide linkers for monoclonal antibody-drug conjugates. Bioconjugate Chem. Vol. 17: 831-840.

[0343] Poljak RJ (1994). Production and structure of a diaphragm body. Structure Vol. 2: 1121-1123.

[0344] Tranoy-Opalinski except (2014). β-glucuronidase-responsive prodrugs for selective cancer chemotherapy: update. European Journal of Medicinal Chemistry Vol. 74: 302-313.

[0345] Wang except (2019). 30 Years of Nucleic Acid Aptamer Technology: Lessons Learned, Advancements, and Opportunities in Aptamer Development. Biotechnology Advances Vol. 37: 28-50.

[0346] Wieland T. and Faulstich H. (1978). Amatoxin, phallotoxin, phalloidin, and antamanide: Biologically active components of the toxic Amanita mushroom. CRC Crit Rev Biochem. Vol. 5(3): 185-260.

Claims

Claim 1 As a compound of Formula I or II containing a cytotoxic drug moiety D is a cytotoxic drug moiety comprising at least one 1,2- or 1,3-diol moiety upon release from the compound; Z is CH2, CH2-CH2 or CHR3-CHR3, wherein R3 is independently H or an alkyl group and may optionally be substituted with a heteroatom; R1 is H or a C1-C6 alkyl group; A is an electron-donating group; E is a cleavage site; R2 is independently H or an electron-receiving or donating group, wherein D is an amatoxin selected from the group consisting of α-amanitine, β-amanitine, amanin, and amaninamide, wherein the electron-donating group A is selected from O, NH, and S, and the cleavage site E is an enzyme-cleavable moiety comprising two or more amino acids, β-glucuronide, or β-galactoside, a compound. Claim 2 delete Claim 3 In claim 1, the enzyme-cleavable moiety comprising two or more amino acids is a compound that is a valine-alanine (Val-Ala), valine-citrulline (Val-Cit), valine-lysine (Val-Lys), valine-arginine (Val-Arg) dipeptide, phenylalanine-lysine-glycine-proline-leucine-glycine (Phe Lys Gly Pro Leu Gly) or alanine-alanine-proline-valine (Ala Ala Pro Val) peptide. Claim 4 A conjugate comprising a compound according to claim 1 and a TL moiety, wherein the TL moiety substitutes at least one residue R2 in formula I or II, L is a linker, and T is a moiety that binds to a target, wherein the linker L connects the moiety T that binds to the target to the compound, and the moiety T that binds to the target is a fragment of the antibody that binds to an antibody or antigen, or a nucleic acid aptamer. Claim 5 In claim 4, the linker L is a conjugate comprising alkylene, heteroalkylene, alkenylene, heteroalkenylene, alkynylene, heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aryllene, or heteroarylkylene group comprising 1 to 4 heteroatoms selected from N, O, and S. Claim 6 In claim 4, the linker L comprises a conjugate comprising a moiety selected from at least one moiety among disulfide, ether, thioether, amine, ester, carboxamide, urethane, and urea moiety. Claim 7 delete Claim 8 In claim 4, the fragment of the antibody that binds to the antibody or antigen is a conjugate selected from a diabody, tetrabody, nanobody, chimeric antibody, immunocompromised antibody, humanized antibody, or human antibody. Claim 9 In claim 8, the fragment of the antibody binding to the antigen is a conjugate selected from the group consisting of Fab, F(ab')2, Fd, Fv, single-chain Fv, and disulfide-linked Fvs(dsFv). Claim 10 As a conjugate having Formula III or IV in Clause 4 X is S, SO, or SO2; R1 is H or a C1-C6 alkyl group; R2 is independently H or a TL moiety; R4 is H, OH, OC 1- C8-alkyl, NO2, NH2, F, Cl, Br, or SH; R5 is OH, NH2, or NHOH; R6, R7 are side chains of amino acids; L is a linker; T is a conjugate that is a moiety binding to a target. Claim 11 As a composite having formula V in Clause 4 X is S, SO, or SO2; Y is CH2 or CO; R1 is H or a C1-C6 alkyl group; R2 is independently H or a TL moiety, R4 is H, OH, OC 1- C8-alkyl, NO2, NH2, F, Cl, Br, or SH; R5 is OH, NH2, or NHOH; L is a linker and T is a moiety that binds to a target, conjugate. Claim 12 As a conjugate having Formula VI in Clause 4 X is S, SO, or SO2; R1 is H or a C1-C6 alkyl group; R2 is independently H or a TL-moiety, R4 is H, OH, OC 1- A conjugate comprising a C8-alkyl, NO2, NH2, F, Cl, Br, or SH; R5 is OH, NH2, or NHOH; R8 is a linear or branched alkyl group, or a TL-moiety; L is a linker, and T is a moiety that binds to a target, provided that at least one of R2 and R8 is a TL moiety. Claim 13 As a compound having Formula VII in Clause 4 X is S, SO, or SO2; R1 is H or a C1-C6 alkyl group; R2 is independently H or a TL-moiety, R4 is H, OH, OC 1- A conjugate in which C8-alkyl, NO2, NH2, F, Cl, Br, or SH; R5 is OH, NH2, or NHOH; R9 is H or a linear or branched alkyl group, or a TL-moiety, L is a linker, and T is a moiety that binds to a target, provided that at least one of R2 and R9 is a TL moiety. Claim 14 A conjugate represented by Formula VIII or IX in Clause 4. Claim 15 A method for synthesizing a conjugate according to any one of claims 4 to 6 and claims 8 to 14 by reacting a 1,2- or 1,3-diol with dimethyl benzylidene acetal in an aprotic solvent under acidic conditions. Claim 16 A method for synthesizing a conjugate according to any one of claims 4 to 6 and claims 8 to 14 by reacting amatoxin with dimethyl benzylidene acetal in an aprotic solvent under acidic conditions. Claim 17 In claim 16, the above-mentioned amatoxin is selected from alpha-amanitine, beta-amanitine, amanin, amaninamide, and each of these thioethers. Claim 18 A method according to claim 16, wherein the aprotic solvent is DMF and the acidic conditions are provided by trifluoroacetic acid. Claim 19 In paragraph 4, a conjugate for use as a pharmaceutical agent. Claim 20 A conjugate for use in the treatment of a patient's cancer according to any one of claims 4 to 6, 8 to 14, and 19, wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, biliary cancer, colon cancer, lung cancer, prostate cancer, uterine cancer, stomach cancer, kidney cancer, malignant melanoma, leukemia, and malignant lymphoma. Claim 21 A pharmaceutical composition for treating cancer comprising a conjugate according to any one of claims 4 to 6 and claims 8 to 14, wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, biliary cancer, colon cancer, lung cancer, prostate cancer, uterine cancer, gastric cancer, kidney cancer, malignant melanoma, leukemia, and malignant lymphoma. Claim 22 delete Claim 23 delete

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