Synthesis of (S)-6-Hydroxytryptophan and Its Derivatives

Abstract

The present invention relates to novel methods and compounds for synthesizing amanitin derivatives. The present invention particularly relates to methods for synthesizing (S)-6-hydroxy-tryptophan derivatives, which can be used as synthetic building blocks for synthesizing amanitin derivatives or amatoxin drug conjugates. The present invention also relates to intermediate compounds of the synthetic pathways for the synthesis of amanitin derivatives and amatoxin drug conjugates, and the use of specific catalysts suitable for mediating the synthetic pathways.

Description

Field of the Invention

[0001] The present invention relates to novel methods and compounds for the synthesis of amanitin derivatives. The present invention particularly relates to methods for the synthesis of (S)-6-hydroxy-tryptophan derivatives, which can be used as synthetic building blocks for the synthesis of amanitin derivatives or amatoxin drug conjugates. The present invention also relates to intermediate compounds of the synthetic pathways, which are useful for the synthesis of amanitin derivatives and amatoxin drug conjugates, and to the use of specific catalysts suitable for mediating the synthetic pathways.

[0002] background

[0003] Amanita toxins are cyclic peptides composed of 8 amino acids found in the mushroom Amanita phalloides (see Figure 1 ). Amatoxins specifically inhibit the DNA-dependent RNA polymerase II of mammalian cells and thereby also inhibit transcription and protein biosynthesis of affected cells. Inhibition of intracellular transcription leads to cessation of growth and proliferation. Although not covalently bound, the complex between amanitin and RNA polymerase II is very tight (KD = 3 nM). Dissociation of amanitin from the enzyme is a very slow process, making it unlikely that affected cells will recover. When transcriptional inhibition persists long enough, cells undergo programmed cell death (apoptosis).

[0004] Amatoxins can be isolated from collected fruiting bodies of Amanita phalloides, or from pure cultures (see Zhang P et al., FEMS Microbiol Lett. 2005 Nov 15; 252(2): 223-8. Epub 2005 Sep 15). However, the amount of amatoxin that can be obtained is quite low (ranging from about 0.3-3 mg / g dry matter from natural fruiting bodies, and about 10% from pure culture dry matter), and the flexibility for further modification of naturally occurring amatoxin variants is limited. Alternatively, amatoxins can be obtained by fermentation using basidiomycetes (see Muraoka S and Shinozawa T., J Biosci Bioeng. 2000; 89(1): 73-6) or A. fissa (Guo XW et al., 2006 Jun; 46(3): 373-8). Again, the yield is low, and the flexibility for further modification of naturally occurring amatoxin variants is also limited. Finally, amatoxins have been prepared by partial or total synthesis (e.g. Zanotti G, C and Wieland T., lnt J Pept Protein Res. 1987 Oct; 30(4): 450-9; Zanotti G, Wieland T, Benedetti E, Di Blasio 8, Pavone V and Pedone C., lnt J Pept Protein Res. 1989 Sep; 34(3): 222-8). Alternatively, the application of the total synthetic route to amatoxins can provide a larger supply of amatoxins required for therapeutic use and can provide the construction of a variety of new amatoxin variants by using appropriate starting materials as synthetic building blocks.

[0005] Naturally occurring amanitin (e.g., α-amanitin, β-amanitin, or γ-amanitin) contains a phenolic hydroxyl (—OH) functional group at the 6′ position of the tryptophan (which represents amino acid 4 in the cyclic amanitin octapeptide), which allows for the coupling of a linker to amatoxin (see Figure 1 ). Target binding macromolecules (e.g. antibodies or aptamers) can then be coupled via the linker to produce a conjugate, such as an antibody-drug conjugate. Already in 1981, the use of a linker with tryptophan (Trp, amino acid 4; see Figure 1 ) was used to develop the use of amatoxin as a cytotoxic moiety for tumor therapy by coupling an anti-Thy 1.2 antibody to α-amanitin via diazotization (Davis & Preston, 1981, Science 213: 1385-1388).

[0006] With the fully synthetic amanitin compounds currently under development, the introduction of functionalized tryptophan for coupling to the linker element has only recently been achieved. Therefore, until now, the linker has been coupled to the fully synthetic amanitin mainly at the indole-nitrogen (N1) position of amino acid 1 (aspartic acid) or amino acid 4 (tryptophan). Since it has been shown that the biological activity spectrum of amanitin coupled via different anchoring positions of the amanitin framework is very different, the ability to provide a synthetic tryptophan hydroxylated at its 6′ position (i.e. corresponding to the natural α-amanitin, β-amanitin or γ-amanitin) as a building block that can be introduced during the synthesis of amanitin has attracted great attention.

[0007] The synthetic introduction of tryptophan into the amanitin framework can be carried out by the Savige-Fontana reaction (Savige & Fontana, 1980, Int J Pept Protein Res. 15 (3): 285-97). According to this reaction, tryptophan is converted into a mixture of Boc-protected cis-2-carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole and trans-2-carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole, and then introduced into the amino acid sequence of the linear amanitin precursor.

[0008] For the total synthetic preparation of amanitin, the synthesis of (S)-6-hydroxytryptophan and the synthesis of the respective building blocks are crucial. In particular, one of these important building blocks is (S)-6-acetoxy-N-tert-butyloxycarbonyl-tryptophan (HDP 30.2550).

[0009]

[0010] To date, no satisfactory and efficient synthetic routes for (S)-6-hydroxytryptophan and its synthetic building blocks have been described in the prior art. In particular, there are no synthetic routes that can produce the necessary purity of the enantiomers (L or S) of this amino acid derivative. Different basic options for the synthesis can mainly be considered. A first option can be crystallization of the racemic form with a chiral auxiliary base or acid. However, only a maximum yield of 50% can be achieved with this method. A second option is enzymatic production; however, this method is time-consuming, the results are uncertain, and the reproducibility is poor.

[0011] Therefore, it is an object of the present invention to provide an efficient, simple and reproducible process for the synthesis of (S)-6-hydroxytryptophan, its derivatives and their building blocks. Preferably, it is an object of the present invention to provide an efficient process for the synthesis of (S)-6-acetoxy-N-tert-butyloxycarbonyl-tryptophan (HDP 30.2550) with sufficiently high enantiomeric purity.

[0012] As another object of the present invention, the use of the (S)-6-hydroxytryptophan and its derivatives (preferably (S)-6-acetoxy-N-tert-butyloxycarbonyl-tryptophan) as synthetic building blocks for the total synthesis of amatoxin should be provided.

[0013] These and further objects are achieved by the method and the device according to the independent claims of the invention. The dependent claims relate to specific embodiments. SUMMARY OF THE INVENTION

[0015] The present invention provides a method for synthesizing (S)-6-hydroxytryptophan, (S)-6-acetoxy-N-tert-butyloxycarbonyl-tryptophan and derivatives thereof and a synthetic building block for the synthesis thereof. The present invention also provides a compound and a synthetic building block for synthesizing amanitin or amanitin derivatives or amanitin-drug conjugates.

[0016] The general advantages of the present invention and its features are discussed in detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The structural formulas of different amatoxins are shown. The bold numbers (1 to 8) indicate the standard numbering of the eight amino acids that form amatoxins. The standard designations of the atoms in amino acids 1, 3, and 4 are also shown (Greek letters α to γ, Greek letters α to δ, and numbers from 1' to 7', respectively).

[0018] Figure 2 The synthesis of compound HDP 30.2822 is described.

[0019] Figure 3 The synthesis of compound HDP 30.2758 is described.

[0020] Figure 4 The synthesis of compounds HDP 30.2550 and HDP 30.2555 is described.

[0021] Figure 5 The structural composition of the catalyst used for the conversion of HDP 30.2824 to HDP 30.2826 is shown.

[0022] Figure 6 The 1H NMR spectrum result from compound HDP 30.2826 (400 MHz, CDCl 3 , δ=ppm) is shown.

[0023] Figure 7 The 13C NMR spectrum result from compound HDP 30.2826 (100 MHz, CDCl3, δ=ppm) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0025] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific components of the device or the process steps of the method, because the device and method can be varied. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments and are not intended to be limiting. It must be noted that, unless the context clearly indicates otherwise, as used in the specification and the appended claims, the singular forms "a", "an" and "the" include singular and / or plural referents. In addition, it should be understood that where a parameter range defined by a numerical value is given, the range is considered to include these thresholds.

[0026] It should also be understood that the embodiments disclosed herein are not meant to be understood as individual embodiments that are unrelated to each other. Features discussed with respect to one embodiment are also meant to be disclosed in conjunction with other embodiments shown herein. If in one case, a particular feature is not disclosed with respect to one embodiment, but is disclosed with respect to another embodiment, a person skilled in the art will understand that this does not necessarily mean that the feature is not intended to be disclosed with respect to the other embodiment. A person skilled in the art will understand that the subject matter of the present invention is to disclose the feature also with respect to the other embodiment, but only for the purpose of clarity and to keep the specification at a manageable size, this has not yet been done.

[0027] Furthermore, the contents of the prior art documents cited in this article are incorporated by reference. This refers in particular to prior art documents that disclose standard or conventional methods. In this case, the main purpose of incorporating by reference is to provide sufficient disclosure and to avoid lengthy repetitions.

[0028] Large-scale, industrially viable production of (S)-6-hydroxytryptophan contained in naturally occurring α-, β- and γ-amanitin and / or its chemically protected forms requires an efficient, simple and reproducible synthetic process. The inventors were able to provide such a simple and efficient preparation process for the first time by using enantioselective hydrogenation of olefinic amino acid precursors. Using specific chiral catalysts, they surprisingly found that this type of reaction can give very high enantiomeric purity.

[0029] A simple synthetic route is disclosed to obtain (S)-6-hydroxytryptophan that is identical to the respective natural structure. The ability to use the compound and its protected forms for industrial large-scale preparation of amanitin would significantly reduce the cost and increase the efficiency of, for example, manufacturing synthetic α, β or γ-amanitin and amatoxin-based antibody-drug conjugates for therapeutic applications.

[0030] According to a first aspect, the invention relates to a process for the synthesis of (S)-6-acetoxy-N-tert-butoxycarbonyl-tryptophan (HDP30.2550) or (S)-6-hydroxytryptophan, wherein the process comprises at least one step of enantioselective hydrogenation of an olefinic precursor compound using at least one chiral catalyst.

[0031] In one embodiment of the claimed process, the olefinic precursor for the asymmetric hydrogenation is an olefinic unsaturated amino acid precursor. Preferably, it is the compound HDP 30.2824.

[0032]

[0033] In one embodiment of the claimed method, the olefinic precursor is synthesized by using the following compound:

[0034]

[0035] The olefinic precursor can further be synthesized by using any of compounds A, B or C.

[0036]

[0037] In another embodiment of the claimed method, the method comprises at least the following steps:

[0038]

[0039] In yet another embodiment of the claimed method, the method comprises at least the following steps:

[0040]

[0041] In the context of the present invention, the term "amanitin" includes all cyclic peptides consisting of 8 amino acids isolated from the genus Amanita and described in Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Biochem. 5 (1978) 185-260). Further include all chemical derivatives thereof; further all semi-synthetic analogs thereof; further all synthetic analogs thereof constructed from synthetic building blocks according to the main structure of the natural compound (cyclic, 8 amino acids), further all synthetic or semi-synthetic analogs containing non-hydroxylated amino acids instead of hydroxylated amino acids, further all synthetic or semi-synthetic analogs, wherein the thioethersulfoxide part is replaced by sulfide, sulfone, thioether or an atom other than sulfur (e.g., as in the carbon atom in the carbanalog of amanita toxin). Functionally, amanita toxin is defined as a peptide or ester peptide that inhibits mammalian RNA polymerase II. Preferred amatoxins are those having a functional group (eg a carboxyl, amino, hydroxyl, sulfhydryl or sulfhydryl capture group) that is reactive with a linker molecule or target binding group as defined below.

[0042] In the context of the present invention, the term "amanitin" refers in particular to a bicyclic structure 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 tryptophan or hydroxytryptophan residue at position 4, a glycine residue at positions 5 and 7, an isoleucine residue at position 6 and a cysteine ​​residue at position 8 (in particular a cysteine ​​derivative oxidized to a sulfoxide or sulfone derivative) (for numbering and representative examples of amanitin see Figure 1 ), and also includes all chemical derivatives thereof; further all semisynthetic analogs thereof; further all synthetic analogs thereof built from synthetic building blocks according to the primary structure of the natural compound (cyclic, 8 amino acids), further all synthetic or semisynthetic analogs containing non-hydroxylated amino acids instead of hydroxylated amino acids, further all synthetic or semisynthetic analogs, in each case, wherein any such derivative or analog has functional activity by inhibiting mammalian RNA polymerase II.

[0043] As used herein, the term "target binding group" refers to any molecule or portion of a molecule that can specifically bind to a target molecule or target epitope. Preferred target binding groups 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 groups" suitable for use in the present invention typically have a molecular weight of 40,000 Da (40 kDa) or higher.

[0044] In the context of the present application, "linker" refers to a molecule that increases the distance between two components, for example, to reduce the spatial interference between the target binding group and amatoxin, otherwise this interference may reduce the ability of amatoxin to interact with RNA polymerase II. The linker can be used for another purpose because it can promote the specific release of amatoxin in cells targeted by the target binding group. Preferably, the linker and the preferred linker and the bond between the amatoxin on one side and the bond between the linker and the antibody on the other side are stable under physiological conditions outside the cell (such as blood), and it can be inside the cell, particularly inside the target cell (such as cancer cell or immune cell) cleaved. In order to provide this selective stability, the linker can include a preferably pH-sensitive or protease-sensitive functional group. Alternatively, the bond connecting the linker to the target binding group can provide selective stability. Preferably, the linker has a length of at least 1, preferably 1-30 atoms in length (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 has been reacted with the amatoxin and the other side has been reacted with the target binding group. In the context of the present invention, the linker is preferably C 1-30 -alkyl, C 1-30 -heteroalkyl, C 2-30 -Alkenyl, C 2-30 -heteroalkenyl, C 2-30 -Alkynyl, C 2-30-heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl, which are optionally substituted. The linking group may contain one or more structural elements, such as amides, esters, ethers, thioethers, disulfides, hydrocarbon moieties, etc. The linking group may also contain a combination of two or more of these structural elements. Each of these structural elements may exist more than once (e.g., twice, three times, four times, five times, or six times) in the linking group. In some embodiments, the linking group may include a disulfide bond. It should be understood that the linking group must be connected to amatoxin and the target binding group in a separate step or in two or more subsequent steps. For this reason, the linking group to be connected will carry two groups, preferably at the proximal and distal ends, which can (i) form a covalent bond with a group (preferably an activated group) on amatoxin or a target binding peptide, or (ii) it is activated or can be activated to form a covalent bond with a group on amatoxin. Accordingly, if a linker is present, it is preferred that chemical groups are located at the distal and proximal ends of the linker which are the result of such a coupling reaction, such as esters, ethers, carbamates, peptide bonds, etc. The presence of a "linker" is optional, i.e., in some embodiments of the target binding group toxin conjugate, the toxin can be directly linked to the residue of the target binding group.

[0045] According to a second aspect, the present invention relates to (S)-6-acetoxy-N-tert-butyloxycarbonyl-tryptophan (HDP 30.2550), (S)-6-hydroxy-tryptophan or any one of the precursor compounds according to the present invention, which are used for the synthesis of amanitin or amanitin derivatives or amatoxin-drug conjugates.

[0046] In one embodiment, the present invention relates to a dehydrogenated amino acid compound selected from the group consisting of compounds I, II, III, IV and V,

[0047]

[0048]

[0049] in:

[0050] R1 is selected from: H, optionally substituted alkyl, alkenyl, aralkyl

[0051] R2 is selected from: Boc, Cbz, N protecting group

[0052] R3 is selected from: Boc, Cbz, N protecting group

[0053] R4 is an amino acid residue

[0054] It is used for the synthesis of amanitin or amanitin derivatives or amatoxin-drug conjugates.

[0055] Compounds III, IV and V are preferred.

[0056] Furthermore, the inventors surprisingly found that from a large group of catalytic compounds tested for asymmetric hydrogenation (see Table 1, Figure 5 ), the catalyst cyclooctadiene-1,5-[(R,R)-DIPAMP]rhodium tetrafluoroborate (HDP 30.2758) gave the highest enantiomeric purity of >98%.

[0057]

[0058] It was found that only the catalyst HDP 30.2758 gave a very high purity of the (S)-enantiomer of more than 98%. With the exception of (R,R)-Et-DUPHOS(BF4-), all other catalysts gave rather low (S)-enantiomer purities of 50-70%. Furthermore, the overall absolute compound conversions and yields were much lower than the results with HDP 30.2758.

[0059] The tested catalysts for the asymmetric hydrogenation and the various (S)-enantiomeric purity levels are compiled in Table 1.

[0060] Table 1: Comparative evaluation of various catalysts for enantioselective hydrogenation

[0061] catalyst# Logo Purity Level 1 HDP 30.2758 >98% chiral purity 2 <![CDATA[(R,R)-Et-DUPHOS(CF3SO 3- )]]> No conversion 3 <![CDATA[(R,R)-Et-DUPHOS(BF 4- )]]> 90-95% chiral purity 4 <![CDATA[(R,R)-DuPhos-ferrocene (BF 4- )]]> 65% chiral purity 5 <![CDATA[(R,R)-DuPhos-ferrocene-Et2(BF 4- )]]> 55% chiral purity 6 <![CDATA[(R,R)-DuPhos-alkyl(CF3SO 3- )]]> 73% chiral purity 7 <![CDATA[(R,R)-Phenyl-DuPhos-alkyl (BF 4- )]]> <60% chiral purity

[0062] Thus, according to a third aspect, the present invention relates to a compound selected from the group consisting of HDP 30.2758, (R,R)-Et-DUPHOS (BF 4- )、(R,R)-DuPhos-ferrocene(BF 4- )、(R,R)-DuPhos-ferrocene-Et2(BF 4- ), (R, R)-DuPhos-alkyl (CF3SO 3- ) and (R,R)-phenyl-DuPhos-alkyl (BF 4- ) as a hydrogenation catalyst in the following reaction:

[0063]

[0064] According to this aspect, the present invention preferably relates to the chiral catalyst cyclooctadiene-1,5-[(R,R)-DIPAMP]rhodium tetrafluoroborate (HDP 30.2758) or (R,R)-Et-DUPHOS (BF 4- ), the best catalyst cyclooctadiene-1,5-[(R,R)-DIPAMP] rhodium tetrafluoroborate (HDP30.2758), is used as a hydrogenation catalyst in the following reactions:

[0065]

[0066] The introduction of tryptophan derivatives as building blocks into amanitin precursors has been described in PCT / EP2018 / 071268, the contents of which are incorporated herein by reference. For example, HDP 30.2115 can be synthesized by introducing an unsubstituted Hpi building block into an amanitin peptide precursor molecule to obtain synthetic amanitin, and the 6-hydroxy-substituted building block HDP30.2555 can be used for the synthesis of 6-hydroxy-substituted amanitin according to the present invention.

[0067] Example

[0068] Although the invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description are to be considered illustrative or exemplary rather than restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments may be understood and effected by those skilled in the art in practicing the claimed invention by studying the drawings, the disclosure and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0069] Example 1: Synthesis of Phosphonium Precursor (Building Block) HDP 30.2822

[0070] The synthesis of the phosphonium precursor (building block) HDP 30.2822 was carried out as described in (CHEMISTRY A European Journal, 2018, Vol. 24, No. 7, pp 1544-1553):

[0071]

[0072] Example 1.1: Preparation of (R, S)-Boc-α-phosphonoglycine trimethyl ester HDP 30.2819

[0073]

[0074] 5.0 g (15.1 mmol) of (R, S)-N-Cbz-phosphonoglycine trimethyl ester (CAS: 88568-95-0) was hydrogenated with 1.4 g of 10% Pd.C in 100 ml of methanol at 1 atm until the reaction was complete as indicated by TLC (chloroform / methanol 15:1). The reaction was completed within 3 hours. The catalyst was filtered off through a plug of Celite (celite) and the methanolic solution of the free amine was concentrated in vacuo to a colorless oil (2.9 g). The crude oil was used in the next step without purification.

[0075] 2.9 g of the crude hydrogenation product was dissolved in 20 ml of dichloromethane and treated with 3.23 ml (15.1 mmol) of di-tert-butyl dicarbonate (Boc2O). After stirring at ambient temperature for 17 hours under argon, the reaction mixture was concentrated to dryness. The remaining colorless oil crystallized to give a white solid (4.3 g). The crude HDP 30.2819 was used in the next step without purification.

[0076] Example 1.2: Preparation of (R, S)-N-Boc-a-phosphonoglycine trimethyl ester HDP 30.2821

[0077]

[0078] 4.3 g (14.3 mmol, considered) of crude HDP 30.2819 were dissolved in 10 ml of 1,4-dioxane and rapidly treated with 14.5 ml of 1N KOH under argon at room temperature. After 85 minutes, the reaction mixture was diluted with 36 ml of water and extracted with 35 ml of ethyl acetate. The ethyl acetate extract was discarded and the aqueous solution was acidified to pH 3 by dropwise addition of 1N HCl. The reaction mixture was extracted with 60 ml of ethyl acetate (2x) and dried over MgSO4, and the resulting white solid (2.1 g of HDP 30.2821) was dried in vacuo and used directly in the next reaction step without purification.

[0079] MP: 148-150℃(Lit.JACS 111, 6244, 1989mp: 154-155℃)

[0080] MS (ESI - ) Measured value: 282.00[MH] - ; Calculated value: 283.08 (C9H 18 NO7P)

[0081] MS (ESI - ) Measured value: 238.17 [M-CO2] -

[0082] Example 1.3: Preparation of (R, S)-N-Boc-α-dimethylphosphonyl)-glycine benzyl ester HDP 30.2822

[0083]

[0084] 2.0 g (7.1 mmol) of HDP 30.2821 in 90 ml of dry dichloromethane were treated with 4.6 ml (44.1 mmol) of benzyl alcohol, 230 mg of DMAP and 2.2 g (10.6 mmol) of DCC dissolved in 7 ml of dichloromethane. The reaction mixture was stirred at ambient temperature for 24 hours under argon. The urea was then filtered off and the organic phase was washed with 5% citric acid and dried over MgSO4. After evaporation of the dichloromethane, the remaining semisolid was dissolved in ethyl acetate and filtered again to remove additional urea. The crude product was purified by flash chromatography (330 g silica gel column, detection wavelength 254 nm) with a gradient from n-hexane to n-hexane / ethyl acetate (1:2) to give, after evaporation, 1.94 g (73%) of HDP 30.2822 in the form of a white solid.

[0085] MS (ESI + ) Measured value: 373.92 [MH] + ; Calculated value: 373.13 (C 16 H 242 O7P)

[0086] MS (ESI + ) Measured value: 396.17[M+Na] +

[0087] Example 2: Synthesis of (S)-6-acetoxy-N-tert-butyloxycarbonyl-tryptophan HDP 30.2550 as a precursor of HDP 30.2555 (hydroxy-Hpi)

[0088] The synthetic routes are summarized in the following synthetic schemes.

[0089]

[0090] Example 2.1: Preparation of N-Cbz-6-benzyloxy-indole-3-carboxaldehyde HDP 30.2803

[0091] The starting material for the synthesis, 6-benzyloxyindole-3-carbaldehyde, is commercially available or can be prepared in high yields via the Vilsmeier reaction starting from 6-benzyloxyindole.

[0092]

[0093] No chromatography purification is required.

[0094]

[0095] Triethylamine (1.66 mL, 11.94 mmol, 1.50 eq) was added to a solution of 6-benzyloxy-3-formylindole (2.00 g, 7.96 mmol, 1 eq) and (DMAP)4-dimethylaminopyridine (97.23 mg, 796 μmol) in dichloromethane (20 mL) at 23 °C via syringe. Benzyl chloroformate (1.45 mL, 10.35 mmol, 1.30 eq) was added dropwise to the solution via syringe. After 1 h, another portion of benzyl chloroformate (223 μL, 1.59 mmol, 0.20 eq) was added via syringe. After 95 min, the reaction mixture was diluted with dichloromethane (85 mL) and washed with saturated aqueous sodium bicarbonate solution (85 mL). The aqueous layer was further extracted with dichloromethane (2×20 mL). The combined organic layers were washed with aqueous hydrogen chloride solution (1N, 85 mL), and the resulting aqueous layer was extracted with dichloromethane (2×20 mL). The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography (330 g silica gel column, detection wavelength 254 nm) with a gradient of n-hexane / ethyl acetate 4:1 to n-hexane / ethyl acetate (1:1) to give 2.33 g (76%) of HDP 30.2803 as a white solid after evaporation.

[0096] 1 H-NMR (400MHz, CDCl3, δ=ppm) δ=5.05 (s, 2H, OCH2); 5.47 (s, 2H, COOCH2); 7.06-8.14 (m, Ar-H, 14H); 10.01 (s, 1H, CHO)

[0097] Example 2.2: Preparation of [6-benzyloxy-1H-(benzyloxycarbonyl)-3-indole]-2-(tert-butyloxycarbonylamino)-acrylic acid benzyl ester HDP 30.2824

[0098]

[0099] 1.90 g (5.09 mmol) of (R, S)-N-Boc-α-dimethylphosphonyl)-glycine benzyl ester HDP30.2822 was suspended in 8 ml of dichloromethane under argon. 0.705 ml (4.73 mmol) of DBU was added. After stirring for 10 minutes, 1.66 g (4.31 mmol) of N-Cbz-6-benzyloxy-indole-3-carboxaldehyde HDP 30.2803 in 4.7 ml of dichloromethane was slowly added. The reaction mixture was stirred for 5 hours and the solvent was evaporated under reduced pressure. The residue was dissolved in 120 ml of ethyl acetate and the organic solution was washed twice with 50 ml of 1N HCl and 50 ml of brine, dried over MgSO4 and concentrated under reduced pressure to give 2.70 g of crude material. The crude product was purified by flash chromatography (330 g silica gel column, detection wavelength 254 nm) using a gradient from n-hexane to n-hexane / ethyl acetate (1:1) to give after evaporation 2.00 g (73%) of HDP 30.2824 as a white solid.

[0100] MS (ESI + ) Measured value: 632.92[MH] + ; Calculated value: 632.25(C 38 H 36 N2O7)

[0101] MS (ESI + ) Measured value: 655.25[M+Na] +

[0102] Example 2.3: Preparation of (S)-6-Benzyloxy-N-tert-butyloxycarbonyl-1-Cbz-L-tryptophan benzyl ester HDP 30.2826

[0103]

[0104] Example 2.3.1: Synthesis of cyclooctadiene-1,5-[(R,R)-DIPAMP] rhodium tetrafluoroborate catalyst HDP 30.2758

[0105]

[0106] 97.0 mg (0.20 mmol) of bis(cyclooctadiene-1,5)-dichlorodirhodium [Rh(COD)Cl]2 (CAS: 12092-47-6. Alfa Aesar) are added to a suspension of 180.0 mg (0.39 mmol) (R,R)-DIPAMP (CAS: 55739-58-7, Alfa Aesar) in 2.0 ml of methanol / water (1.5 ml / 0.5 ml). The orange slurry is stirred under argon for 1 hour to give an orange solution. The complex is precipitated by slowly adding (over 30 minutes) a solution of 65.0 mg (0.6 mmol) of sodium tetrafluoroborate in 0.5 ml of water. After stirring at room temperature for 2.5 hours, the orange crystals are filtered off, washed twice with small portions of water and dried under high vacuum. 240 mg (81%) of the catalyst cyclooctadiene-1,5-[(R,R)-DIPAMP]rhodium tetrafluoroborate HDP 30.2758 were obtained as a bright yellow powder. The catalyst was used without further purification.

[0107] Example 2.3.2: Synthesis of (S)-6-Benzyloxy-N-tert-butyloxycarbonyl-1-Cbz-L-tryptophan benzyl ester HDP 30.2826

[0108] A 250 ml stainless steel autoclave was charged with 35.0 mg (0.08 mmol) of cyclooctadiene-1,5-[(R,R)-DIPAMP] rhodium tetrafluoroborate HDP 30.2758, 1000 mg (1.8 mmol) of [6-benzyloxy-1H-(benzyloxycarbonyl)-3-indole]-2-(tert-butoxycarbonylamino)-acrylic acid benzyl ester HDP 30.2824 in 40 ml of dry methanol / 15 ml of dichloromethane. After four cycles of vacuum / Ar and H2, the reaction was pressurized to an initial pressure of 12 bar. The reaction was allowed to proceed at ambient temperature for 4 days. After evaporation of the solvent, the crude product was purified by flash chromatography (220 g silica gel column, detection wavelength 254 nm) using a gradient of n-hexane / ethyl acetate (3:1) to give, after evaporation, 0.79 g (79%) of HDP 30.2826 in the form of a white powder.

[0109] MS (ESI + )Calculated value: 634.26(C 38 H 38 N2O7)

[0110] MS (ESI + ) Measured value: 657.33[M+Na] + ;

[0111] Example 2.4: Preparation of N-tert-butyloxycarbonyl-(S)-tryptophan HDP 30.2832

[0112]

[0113] 700 mg (1.10 mmol) of (S)-6-benzyloxy-N-tert-butyloxycarbonyl-1-Cbz-tryptophan benzyl ester HDP30.2826 was hydrogenated with 100 mg of Pd-C 10% in a mixture of 7 ml of ethyl acetate and 4 ml of methanol. After hydrogenation for 3 hours at room temperature and 1 atm (TLC control, chloroform / methanol 19:1 + 1% AcOH), the mixture was stirred for 3 hours. The catalyst was removed by filtration through a plug of Celite®. The solvent was removed and the remaining 378 mg of the crude HDP 30.2832 residue was used in the next step without purification.

[0114] Example 2.5: Preparation of (S)-6-acetoxy-N-tert-butyloxycarbonyl-tryptophan HDP 30.2550

[0115]

[0116] 378 mg of crude HDP 30.2832 (1.10 mmol, assumed) were dissolved in 2.21 ml of 1 NaOH. 208.5 μL (2.20 mmol) of acetic anhydride were added in one portion under argon and ambient temperature. The mixture was stirred for 3.5 hours and acidified with 5% citric acid. The reaction mixture was extracted 3 times (3x) with ethyl acetate and the combined organic phases were washed with 5% sodium chloride and dried over anhydrous MgSO4. Filtration and evaporation to dryness gave 380 mg of crude product. The crude product was purified by flash chromatography (120 g silica column, detection wavelength 254 nm) with a gradient of dichloromethane + 2% AcOH / dichloromethane / methanol (15:1) + 2% AcOH to give after evaporation 270 mg (68%) of HDP 30.2550 as a white solid.

[0117] MS (ESI - ) Measured value: 361.17 [MH] - ; Calculated value: 362.15 (C 18 H 22 N2O6)

[0118] MS (ESI - ) Measured value: 723.08[2M-H] -

[0119] Example 3: Preparation of (S)-6-Hydroxy-Tryptophan by Asymmetric Hydrogenation of Dehydroamino Acids

[0120] The synthetic routes are summarized in the following synthetic schemes.

[0121]

[0122] Example 3.1: Preparation of 6-benzyloxy-1H-indole-3-carbaldehyde

[0123]

[0124] To a stirred solution of phosphorus oxychloride (10.0 mL, 107.0 mmol) in DMF (35 mL) at room temperature was added a solution of 6-benzyloxyindole (22.3 g, 100.0 mmol) in DMF (25 mL). After 45 minutes, the reaction mixture was poured into ice water (200 mL). To this mixture was added solid NaOH (19.0 g, 475.0 mmol) and water (100 mL). After 30 minutes, additional water (200 ml) was added and the entire mixture was refluxed for 3 minutes. The precipitate was collected, washed with 5 50 ml portions of cold water and dried to give 24.8 g (98.8%) of 6-benzyloxy-1H-indole-3-carboxaldehyde as a white powder. This compound was identical to the reference substance and was pure enough for the next reaction.

[0125] Example 3.2: Preparation of 6-benzyloxy-1H-1-tert-butyloxycarbonyl-indole-carboxaldehyde HDP 30.2738

[0126]

[0127] 10.0 g (39.8 mmol) of 6-benzyloxy-1H-indole-3-carbaldehyde was suspended in 100 ml of dichloromethane and treated with 0.56 g (4.5 mmol) of 4-dimethylaminopyridine DMAP and 10.5 g (47.3 mmol) of di-tert-butyl dicarbonate Boc2O dissolved in 10 ml of dichloromethane. After stirring for 2 hours, 100 ml of 1N KHSO4 was added and the dichloromethane was evaporated. The aqueous layer was extracted with several portions of ether (2×200 ml) and the combined organic extracts were washed with 250 ml of 1N KHSO4, 250 ml of 1N NaHCO3 and 250 ml of brine. The organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure to give 12.0 g (86%) of a reddish brown powder. The compound was pure enough for the next reaction step.

[0128] Example 3.3: Preparation of 3-[6-benzyloxy-1H-(1-tert-butyloxycarbonyl)-3-indole]-2-(benzyloxycarbonylamino)-methyl acrylate HDP 30.2739

[0129]

[0130] 5.12 g (15.44 mmol) (R, S)-benzyloxycarbonyl-δ-phosphono-glycine trimethyl ester (CAS: 88568-95-0, Alfa Aesar) were dissolved in 18 ml of dichloromethane under argon. 2.14 ml (14.31 mmol) of DBU were added. After stirring for 10 minutes, 4.60 g (13.07 mmol) of 6-benzyloxy-1H-1-tert-butoxycarbonyl-indole-3-carbaldehyde HDP 30.2738 in 14 ml of dichloromethane were slowly added. The reaction mixture was stirred for 6 hours and the solvent was evaporated under reduced pressure. The residue was dissolved in 300 ml of ethyl acetate and the organic solution was then washed twice with 120 ml of 1N HCl and 120 ml of brine, dried over MgSO4 and concentrated under reduced pressure to give 7.43 g of crude product. The crude product was purified by flash chromatography (330 g silica gel column, detection wavelength 254 nm) using a gradient from n-hexane to n-hexane / ethyl acetate (2:1) to give, after evaporation, 5.23 g (72%) of HDP30.2739 in the form of a white solid.

[0131] MS (ESI + ) Measured value: 557.17 [MH] + ; Calculated value: 557.22 (C 32 H 32 N2O7)

[0132] MS (ESI + ) Measured value: 579.25[M+Na] +

[0133] Example 3.4: Preparation of 6-benzyloxy-N-benzyloxycarbonyl-1-tert-butyloxycarbonyl-L-tryptophan methyl ester HDP 30.2760

[0134]

[0135] Example 3.4.1: Synthesis of cyclooctadiene-1,5-[(R,R)-DIPAMP]rhodium tetrafluoroborate HDP 30.2758

[0136] The catalyst cyclooctadiene-1,5-[(R,R)-DIPAMP]rhodium tetrafluoroborate HDP30.2758 was synthesized as described in Example 2.3.1.

[0137] Example 3.4.2: Synthesis of (S)-6-Benzyloxy-N-Benzyloxycarbonyl-1-tert-butyloxycarbonyl-tryptophan methyl ester HDP30.2760

[0138] In a 250 ml stainless steel autoclave were added 60.0 mg (0.08 mmol) of cyclooctadiene-1,5-[(R,R)-DIPAMP] rhodium tetrafluoroborate HDP 30.2758 and 1000 mg (1.8 mmol) of [6-benzyloxy-1H-(1-tert-butyloxycarbonyl)-3-indole]-2-(benzyloxycarbonyl-amino) acrylate methyl ester HDP 30.2739 in 40 ml of dry methanol. After four cycles of vacuum / Ar and H2, the reaction was pressurized to an initial pressure of 30 bar. The reaction was allowed to proceed at ambient temperature for 4 days. After evaporation of the solvent, the crude product was purified by flash chromatography (120 g silica gel column, detection wavelength 254 nm) with a gradient from n-hexane to n-hexane / ethyl acetate (2:1) to give, after evaporation, 0.85 g (86%) of HDP 30.2760 in the form of a white solid.

[0139] MS (ESI + )Calculated value: 558.23(C 33 H 34 N2O7)

[0140] MS (ESI + ) Measured value: 581.17[M+Na] + ; 1138.83[2M+Na] +

[0141] Example 3.5: Preparation of 6-benzyloxy-N-benzyloxycarbonyl-L-tryptophan methyl ester HDP 30.2790

[0142]

[0143] 100.0 mg (0.18 mmol) of (S)-6-benzyloxy N-benzyloxycarbonyl-1-tert-butoxycarbonyl-tryptophan methyl ester HDP30.2760 was dissolved in 5.0 ml of formic acid and stirred at 40° C. for 1 hour. The reaction mixture was evaporated to dryness and the residue was dissolved in ethyl acetate. The ethyl acetate solution was washed with water, saturated NaHCO3 and brine and dried over MgSO4. After evaporation of the solvent, the crude product was purified by flash chromatography (24 g silica gel column, detection wavelength 254 nm) using a gradient from n-hexane to n-hexane / ethyl acetate (1:1) to give 29 mg (35%) of HDP 30.2790 in the form of a white solid after evaporation.

[0144] MS (ESI + )Calculated value: 458.52(C 27 H 26 N2O5)

[0145] MS (ESI +) Measured value: 459.25[M+H] +

[0146] Example 3.6: Preparation of (s)-6-benzyloxy-N-benzyloxycarbonyl-tryptophan HDP 30.2782

[0147]

[0148] 2N LiOH aqueous solution (84.7 μl) was added to a solution of 25.9 mg (0.056 mmol) HDP30.2790 in 1000 μl tetrahydrofuran / water (10:1) at ambient temperature. The reaction mixture was stirred for 2.5 hours and partitioned between ethyl acetate and 5% citric acid. The aqueous layer was extracted with ethyl acetate and the organic layers were combined, dried (MgSO4) and concentrated. The resulting carboxylic acid HDP 30.2782 was purified on silica gel using dichloromethane / methanol (+1% acetic acid) as mobile phase. 13.7 mg (55%) of a white solid was obtained.

[0149] MS (ESI + )Calculated value: 444.17.23(C 26 H 24 N2O5)

[0150] MS (ESI + ) Measured value: 445.25[M+H] + ; 467.17[M+Na] +

[0151] Example 3.7: Preparation of (S)-6-hydroxy-N-(tert-butyloxycarbonyl)-tryptophan HDP 30.2832

[0152]

[0153] 10 mg (10 wt%) palladium on carbon was added to a solution of 50 mg (0.11 mmol) HDP 30.2782 in 800 ul methanol. The reaction mixture was purged with hydrogen three times and stirred at room temperature for 2.5 h. The suspension was washed with methanol. The mixture was filtered through a plug of 4% paraffin and concentrated to dryness. The solid residue (22.2 mg) (S)-6-hydroxytryptophan was dissolved in 1000 μl 1,4-dioxane / water (1:1) and treated with 101 μl (0.101 mmol) 1N NaOH and 21.57 μl (0.10 mmol) di-tert-butyl dicarbonate. The reaction mixture was stirred for 16 hours and adjusted to pH 2 with 1N hydrochloric acid. The aqueous solution was extracted three times with ethyl acetate and the combined organic phases were washed with brine, dried and evaporated to dryness. The crude HDP 30.2832 was purified on silica gel with dichloromethane / methanol (+1% acetic acid) as mobile phase. 9.9 mg (31%) of a white solid were obtained. This material was identical to the reference sample.

[0154] MS(ES - )Calculated value: 320.14(C 16 H 20 N2O5)

[0155] MS (ESI - ) Measured value: 319.08[MH] -

[0156] Example 4: Preparation of cis, trans-1-(tert-butyloxycarbonyl)-2-carboxy-3a-hydroxy-6-acetoxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole cis-HDP 30.2555 and trans-HDP 30.2555 (cis, trans-6-acetoxy-Hpi)

[0157] Example 4.1: Preparation of (S)-N-(tert-butyloxycarbonyl)-6-acetoxy-tryptophan HDP 30.2550

[0158]

[0159] 590.0 mg (2.68 mmol) of (S)-6-hydroxytryptophan from the hydrogenation step in Example 3.7 are suspended in 30 ml of a mixture of 1,4-dioxane / water 1:1 (v,v). Under argon, 2.68 ml (2.68 mmol) of 1N NaOH are added in one portion at ambient temperature. The resulting yellow solution is then treated with 574.6 ml (2.68 mmol) of Boc anhydride (Boc2O) and stirred at room temperature for 24 hours. The solution is acidified to pH 2.4 with 1N hydrochloric acid and extracted three times with 25 ml of ethyl acetate. The combined ethyl acetate extracts are washed with saturated NaCl solution and dried over MgSO4. Filtration and evaporation to dryness give 785.0 mg of crude material. The crude N-Boc-6-hydroxy-L-tryptophan was dissolved in 4.91 ml (4.91 mmol) of 1N NaOH and treated with 463.2 ml (500.3 mg, 4.90 mmol) of acetic anhydride. The reaction mixture was stirred under argon for 3 hours 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 MgSO4. Filtered and evaporated to give 635 mg of crude solid. The crude product was purified by flash chromatography (330 g silica gel column, detection wavelength 254 nm) with a gradient of CH2Cl2+1% acetic acid-CH2Cl2 / MeOH (15:1)+1% acetic acid to give 564.4 mg (56% yield) of white powder after coevaporation with toluene.

[0160] MS (ESI - ) Measured value: 361.08[MH] - ; Calculated value: 362.15 (C 18 H 22 N2O6)

[0161] Example 4.2: Preparation of cis, trans-6-acetoxy-Hpi

[0162]

[0163] Photo-oxidation was performed with a 400W high pressure sodium vapor lamp (Sirius X400 lamp 230V, 400W; 55,000 lumens at a distance of 1.3m). Rose Bengal was used as a dye sensitizer. The reaction was carried out in a 500ml cylindrical reaction vessel (with a heat exchange jacket made of borosilicate glass, a flat bottom and a flat laboratory flange (DN) with two GL 18 threaded connectors). The distance from the lamp to the reaction vessel was 15cm, and the reaction temperature range was 3-4°C.

[0164] The final product was purified on a Teledyne ISCO flash chromatography system with a 330 g Silica Redi Sept flash column (Teledyne ISCO cat. 69-2203-330). Solvents CH2Cl2, CH3OH, CH3COOH were standard HPLC or BP grade. Dry oxygen (99.5% purity) was bubbled into the reaction mixture at a rate of 2-4 1 / min.

[0165] 943.0 mg (2.60 mmol) of N-(tert-butyloxycarbonyl)-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. by using a Huber cryostat (with ethylene glycol / water as cooling medium). The reaction solution was irradiated with a 400 W high pressure sodium vapor lamp. During irradiation, a slow stream of oxygen was bubbled through the reaction solution. After 5 hours of irradiation, oxidation and cooling were stopped and the reaction medium was treated with 10 ml of dimethyl sulfide. The mixture was stirred for 2 hours and evaporated to dryness by using a rotary evaporator with a water bath temperature of 35° C. The dark red residue was further dried in a high vacuum to 1.20 g of a crystalline solid. The crude material was purified on a 330 g silica gel column (detection wavelength 254 nm) with a gradient from CH2Cl2+5% acetic acid to CH2Cl2 / 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 freeze-drying from tert-butanol, both isomers were obtained in the form of off-white powders.

[0166] cis-1-(tert-Butyloxycarbonyl)-2-carboxy-3a-hydroxy-6-acetoxy-1,2,3,3a,8,8a-hexahydropyrrole Indole (cis-HDP30.2555)

[0167] 380mg cis-HDP 30.2555 Yield: 39%

[0168] 1 H-NMR (400MHz, CD3OD, δ=ppm)

[0169] δ=1.22, 1.44, 1.54[s, 9H, C(CH3)3]; 2.23(s, 3H, OCOCH3); 2.46-2.63(m, 2H, CH2); 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)

[0170] 13 C-NMR (100MHz, CD3OD, δ=ppm)

[0171] 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

[0172] MS (ESI + ) Measured value: 378.92[MH] + ; Calculated value: 378.14 (C 18 H 22 N2O7)

[0173] MS (ESI + ) Measured value: 401.17[M+Na] + ; Calculated value: 401.14(C 18 H 22 N2NaO7)

[0174] UV / VIS(CH3OH):λ max =296nm, 239nm, 215nm

[0175] λ min =266nm, 227nm

[0176] trans-1-(tert-Butyloxycarbonyl)-2-carboxy-3a-hydroxy-6-acetoxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole (trans-HDP30.2555)

[0177] 290mg trans-HDP 30.2555 Yield: 30%

[0178] 1 H-NMR (400MHz, CD3OD, δ=ppm)

[0179] δ=1.22, 1.45, 1.54[s, 9H, C(CH3)3]; 2.22(s, 3H, OCOCH3); 2.55-2.73(m, 2H, CH2); 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)

[0180] 13 C-NMR (100MHz, CD3OD, δ=ppm)

[0181] 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

[0182] MS (ESI + ) Measured value: 379.00 [MH] + ; Calculated value: 378.14 (C 18 H 22 N2O7)

[0183] MS (ESI + ) Measured value: 401.17[M+Na] + ; Calculated value: 401.14(C 18 H 22 N2NaO7)

[0184] MS (ESI + ) Measured value: 779.00 [2M + Na] + ; Calculated value: 779.28 (C 36 H 44 N4Na2O 14 )

[0185] UV / VIS(CH3OH):λ max =299nm, 241nm, 215nm

[0186] λ min =268nm, 228nm

[0187] Example 4.3: Introduction of cis, trans-6-acetoxy-Hpi into the amanitin precursor

[0188] Mainly for the purpose of enabling implementation, the synthesis of amanitin was carried out using cis, trans-6-acetoxy-Hpi as described in PCT / EP2018 / 071268 (the contents of which are incorporated herein by reference).

[0189] References:

[0190] Muraoka S and Shinozawa T., J Biosci Bioeng.2000;89(1):73-6

[0191] Wieland T., Faulstich H. 1978. CRC Crit Rev Biochem. Vol. 5: 185-260.

[0192] Zanotti G, C and Wieland T., Int J Pept Protein Res. 1987 Oct; 30(4): 450 - 9;

[0193] Zanotti G, Wieland T, Benedetti E, Di Blasio 8, Pavone V and Pedone C., lnt JPept Protein Res. 1989 Sep; 34(3): 222 - 8

[0194] Zhang P et al., FEMS Microbiol Lett. 2005 Nov 15; 252(2): 223 - 8. Epub 2005 Sep 15

Claims

1. A process for the synthesis of (S)-6-acetoxy-N-tert-butyloxycarbonyl-tryptophan or (S)-6-hydroxytryptophan, wherein the process comprises at least one step of enantioselective hydrogenation of an olefinic amino acid precursor compound using at least one chiral catalyst, wherein the chiral catalyst is selected from the group consisting of compounds HDP 30.2758, (R,R)-Et-DUPHOS (BF 4- )、(R,R)-DuPhos-ferrocene(BF 4- )、(R,R)-DuPhos-ferrocene-Et2(BF 4- ), (R, R)-DuPhos-alkyl (CF3SO 3- ) and (R,R)-phenyl-DuPhos-alkyl (BF 4- ) of the compound: And wherein the olefinic amino acid precursor compound is a dehydrogenated amino acid compound selected from compounds I, II and III: in: R1 is selected from: H, optionally substituted alkyl, alkenyl, aralkyl, R2 is selected from: Boc, Cbz, N protecting group, R3 is selected from: Boc, Cbz, N protecting group, R4 is an amino acid residue.

2. The method according to claim 1, wherein the olefinic amino acid precursor compound is compound HDP30.2824 3. The method according to claim 1, wherein the chiral catalyst is compound HDP 30.2758 or (R,R)-Et-DUPHOS (BF 4- ).

4. The method according to claim 2, wherein the olefinic amino acid precursor compound is synthesized by using compound HDP30.2822 5. The method according to claim 4, wherein the olefinic amino acid precursor compound is further synthesized by using compound B 6. The method according to any one of claims 1 to 5, wherein the method comprises using at least one starting compound or intermediate compound selected from the group consisting of:

7. A method for synthesizing (S)-6-acetoxy-N-tert-butyloxycarbonyl-tryptophan according to any one of claims 1 to 5, wherein the method comprises at least the following steps:

8. A method for synthesizing (S)-6-hydroxytryptophan according to any one of claims 1 to 5, wherein the method comprises at least the following steps:

9. A precursor compound or a derivative compound selected from:

10. Use of any of the following precursor compounds for the synthesis of amanitin or amanitin derivatives or amatoxin-drug conjugates: The amatoxin-drug conjugate optionally comprises a linker.

11. A dehydrogenated amino acid compound selected from compounds I, II and III in: R1 is selected from: H, optionally substituted alkyl, alkenyl, aralkyl, R2 is selected from: Boc, Cbz, N protecting group, R3 is selected from: Boc, Cbz, N protecting group, R4 is an amino acid residue, It is used for the synthesis of amanitin or amanitin derivatives or amatoxin-drug conjugates.

12. Selected from compounds HDP 30.2758, (R,R)-Et-DUPHOS (BF 4- )、(R,R)-DuPhos-ferrocene(BF 4- )、(R,R)-DuPhos-ferrocene-Et2(BF 4- ), (R, R)-DuPhos-alkyl (CF3SO 3- ) and (R,R)-phenyl-DuPhos-alkyl (BF 4- ) as a catalyst for hydrogenation in the following reaction: Among them, compounds HDP 30.2758, (R,R)-Et-DUPHOS(BF 4- )、(R,R)-DuPhos-ferrocene(BF4-)、(R,R)-DuPhos-ferrocene-Et2(BF 4- ), (R,R)-DuPhos-alkyl (CF3SO 3- ) and (R,R)-phenyl-DuPhos-alkyl (BF 4- ) has the following structure:

13. Use of cyclooctadiene-1,5-[(R,R)-DIPAMP]rhodium tetrafluoroborate HDP 30.2758 having the structure shown below as a catalyst for hydrogenation in the following reaction:

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