Synthesis of (S)-6-hydroxytryptophan and its derivatives

Abstract

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

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to novel methods and compounds for synthesizing amanitin derivatives. The present invention relates in particular to a method for synthesizing (S)-6-hydroxy-tryptophan derivatives, which can be used as building blocks for synthesizing amanitin derivatives or amatoxin drug conjugates. The present invention further relates to intermediate compounds of said synthetic pathway for use in the synthesis of amanitin derivatives and amatoxin drug conjugates, and to the use of certain catalysts suitable for mediating said synthetic pathway.

[0002] background Amatoxin is a cyclic peptide consisting of eight amino acids found in the mushroom Amanita phalloides (see Figure 1). Amatoxins specifically inhibit DNA-dependent RNA polymerase II in mammalian cells, thereby also inhibiting transcription and protein biosynthesis in affected cells. Inhibiting transcription in cells causes growth and proliferation to cease. 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, and therefore recovery of affected cells is unlikely. If transcription is inhibited long enough, the cell will undergo programmed cell death (apoptosis).

[0003] Amatoxins can be isolated from collected fruit bodies of Amanita phalloides mushroom or from pure cultures (Zhang P, et al., FEMS Microbiol Lett. 2005 Nov 15;252(2):223-8), Epub 2005 Sep 15). However, the amounts of amatoxins obtained are rather low (ranging from about 0.3 to 3 mg / g dry matter from natural fruit bodies and about 10% of that from pure cultures), and the flexibility to further modify naturally occurring amatoxin variants is limited. Alternatively, amatoxins can be obtained from fermentation with basidiomycetes (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). However, again, the yields are low and the flexibility to further modify naturally occurring amatoxin variants is similarly limited. Finally, amatoxins can be obtained by partial or total synthesis (e.g., Zanotti G, Mahringer 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 use of a completely synthetic route to amatoxins may provide a supply of larger quantities of amatoxins required for therapeutic use and may provide for the construction of a variety of novel amatoxin variants by using appropriate starting materials as building blocks.

[0004] Naturally occurring amanitins, such as α-amanitin, β-amanitin or γ-amanitin, contain a phenolic hydroxy (-OH) functional group at the 6'-position of tryptophan, which represents amino acid 4 in the cyclic amanitin octapeptide, allowing the coupling of a linker to the amatoxin (see Figure 1). A target-binding macromolecule, such as an antibody or an aptamer, can then be attached via the linker to generate a conjugate, e.g., an antibody-drug conjugate. The use of amatoxins as cytotoxic moieties for tumor therapy was already explored in 1981 by coupling anti-Thy 1.2 antibody to α-amanitin using a linker attached to the indole ring of tryptophan (Trp, amino acid 4; see Figure 1) via diazotization (Davis & Preston, 1981, Science 213: 1385-1388).

[0005] With the fully synthesized amanitin compounds currently being explored, the incorporation of functionalized tryptophan for the coupling of linker elements has only recently been achieved. Thus, prior to that, linkers were primarily coupled to fully synthesized amanitin 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 profiles of amanitins attached via different anchoring positions in the amanitin backbone are very different, it would be of great interest 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 incorporated in the course of amanitin synthesis.

[0006] Synthetic introduction of tryptophan into the amanitin backbone can be achieved 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]indoles and trans-2-carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indoles, which are then incorporated into the amino acid sequence of the linear amanitin precursor.

[0007] For the complete synthetic production of amanitin, the synthesis of (S)-6-hydroxytryptophan and each of its building blocks is essential. In particular, one of these building blocks of essential importance is (S)-6-acetyloxy-N-tert-butoxycarbonyl-tryptophan (HDP 30.2550).

[0008] [ka] So far, no satisfactory and efficient synthetic route for (S)-6-hydroxytryptophan and its building blocks has been described in the prior art. In particular, no synthetic route was available that would yield the required purity of the enantiomer (L or S) of this amino acid derivative. Various basic options for synthesis can be primarily considered. The first option could be crystallization of the racemic form with a chiral auxiliary base or acid. However, this method only achieved a maximum yield of 50%. The second option is enzymatic production, but this method is time-consuming, the results are uncertain, and the reproducibility is poor.

[0009] Therefore, one object of the present invention was to provide an efficient, simple and reproducible method for the synthesis of (S)-6-hydroxytryptophan, its derivatives and its building blocks. It was one object of the present invention to provide an efficient method for synthesizing (S)-6-acetyloxy-N-tert-butoxycarbonyl-tryptophan (HDP 30.2550), preferably with sufficiently high enantiomeric purity.

[0010] As a further object of the present invention, there should be provided the use of said (S)-6-hydroxytryptophan and its derivatives, preferably (S)-6-acetyloxy-N-tert-butoxycarbonyl-tryptophan, as building blocks for the total synthetic production of amatoxins.

[0011] These and further objects are achieved by the methods and means described in the independent claims. The dependent claims relate to specific embodiments.

[0012] Summary of the Invention

[0013] The present invention provides a method for the synthesis of (S)-6-hydroxytryptophan, (S)-6-acetyloxy-N-tert-butoxycarbonyl-tryptophan, as well as derivatives thereof and building blocks for the synthesis thereof. The present invention further provides compounds and building blocks for use in the synthesis of amanitin or amanitin derivatives or amatoxin-drug conjugates.

[0014] The general advantages of the present invention and its features are explained in detail below. [Brief description of the drawings]

[0015] Description of the drawings [Figure 1] Figure 1 shows the structural formulas of different amatoxins. The bold numbers (1–8) indicate the standard numbering of the eight amino acids that form the amatoxins. The standard designations of the atoms in amino acids 1, 3, and 4 are also shown (Greek letters α–γ, Greek letters α–δ, and numbers 1′–7′, respectively).

[0016] [Diagram 2] Figure 2 shows the synthesis of compound HDP 30.2822.

[0017] [Diagram 3] Figure 3 shows the synthesis of compound HDP 30.2758.

[0018] [Figure 4] Figure 4 shows the synthesis of compounds HDP 30.2550 and HDP 30.2555.

[0019] [Diagram 5] Figure 5 shows the structural composition of the catalyst used for the conversion of HDP 30.2824 to HDP 30.2826.

[0020] [Figure 6] Figure 6 shows the results of 1H NMR spectroscopy from compound HDP 30.2826 (400 MHz, CDCl3, δ = ppm).

[0021] [Figure 7] Figure 7 shows the results of 13C NMR spectroscopy from compound HDP 30.2826 (100 MHz, CDCl3, δ = ppm).

[0022] Detailed Description of the Invention Before describing the present invention in detail, it is to be understood that the invention is not limited to the particular component parts of the described means, or process steps of the methods, as such means and methods may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include singular and / or plural references unless the context clearly dictates otherwise. It should further be understood that when a parameter range is given, bounded by numerical values, the range is deemed to be inclusive of these limiting values.

[0023] Further, it should be understood that the embodiments disclosed herein are not meant to be construed as separate embodiments unrelated to each other. Features discussed in one embodiment are intended to be disclosed in conjunction with the other embodiments disclosed herein. If, in one instance, a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, a person skilled in the art will understand that this does not necessarily mean that the feature is not disclosed in the other embodiment. Those skilled in the art will appreciate that it would be a purpose of this application to disclose such features for other embodiments as well, but that this has not been done for the sake of clarity and to keep the specification to a manageable volume.

[0024] Additionally, the contents of the prior art documents described herein are incorporated by reference. This refers specifically to prior art documents that disclose standard or routine methods. In that case, incorporation by reference has the primary objective of providing a sufficient possible disclosure and avoiding lengthy repetition.

[0025] Large-scale, industrially viable production of ( S )-6-hydroxytryptophan contained in naturally occurring α-, β-, and γ-amanitins and / or their chemically protected forms requires efficient, simple, and reproducible synthetic methods. For the first time, we have been able to provide such a simple and efficient preparation method by using enantioselective hydrogenation of olefinic amino acid precursors. Surprisingly, it has been found that when certain chiral catalysts are used, very high enantiomeric purity can be obtained in these types of reactions.

[0026] The simple synthetic route disclosed yields (S)-6-hydroxytryptophan that is completely identical to the respective natural structure. The ability to use said compound and its protected forms for industrial large-scale production of amanitin would considerably reduce the cost and increase the efficiency of, for example, the production of synthetic α-, β- or γ-amanitin, as well as amatoxin-based antibody-drug conjugates for therapeutic applications.

[0027] According to a first aspect, the present invention relates to a method for the synthesis of (S)-6-acetyloxy-N-tert-butoxycarbonyltryptophan (HDP 30.2550) or (S)-6-hydroxytryptophan, said method comprising at least one step of enantioselective hydrogenation of an olefinic precursor compound by the use of at least one chiral catalyst.

[0028] In one embodiment of the claimed method, the olefinic precursor used in the asymmetric hydrogenation is an olefinically unsaturated amino acid precursor. Preferably, it is the compound HDP 30.2824.

[0029] [ka]

[0030] In one embodiment of the method of the claims, the olefinic precursor is synthesized by use of the following compound: [ka]

[0031] The olefinic precursor can be further elaborated by the use of either compound A, B, or C.

[0032] [ka]

[0033] In a further embodiment of the claimed method, the method comprises at least the following steps:

[0034] [ka] In yet another embodiment of the claimed method, the method comprises at least the following steps: [ka] In the context of the present invention, the term "amatoxin" refers to the compounds isolated from the genus Amanita and described by Wieland, T. and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Biochem. 5 (1978) 185-260): Any cyclic peptide consisting of eight amino acids; plus all its chemical derivatives; Plus all its semi-synthetic analogues; Furthermore, all its synthetic analogues constructed from building blocks according to the master structure of the natural compound (cyclic, 8 amino acids); In addition, all synthetic or semisynthetic analogs containing non-hydroxylated amino acids in place of hydroxylated amino acids, Additionally, it includes all synthetic or semisynthetic analogues in which the thioether sulfoxide moiety is replaced by a sulfide, sulfone, thioether, or an atom different from sulfur (e.g., the carbon atom in the carbon analogue of amanitin). Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferred amatoxins are those that have a functional group (e.g., a carboxyl group, an amino group, a hydroxyl group, a thiol or a thiol capture group) that can be reacted with a linker molecule or a target binding moiety, as defined below.

[0035] In the context of the present invention, the term "amanitin" refers in particular to an aspartic acid or asparagine residue at position 1, Proline residues at position 2, especially hydroxyproline residues, Isoleucine, hydroxyisoleucine or dihydroxyisoleucine at position 3, A tryptophan or hydroxytryptophan residue at position 4, Glycine residues at positions 5 and 7, an isoleucine residue at position 6, and Refers to a bicyclic structure based on a cysteine ​​residue at position 8, specifically derivatives of cysteine ​​that are oxidized to sulfoxide or sulfone derivatives (see Figure 1 for numbering and representative examples), Additionally, all its chemical derivatives; In addition, all of its semisynthetic analogues; Additionally, all of its synthetic analogs are constructed from building blocks following the master structure of the natural compound (cyclic, 8 amino acids); Additionally, any synthetic or semi-synthetic analogues which contain non-hydroxylated amino acids instead of hydroxylated amino acids; In each case, any such derivative or analog is functionally active by inhibiting mammalian RNA polymerase II, including any further synthetic or semisynthetic analogs.

[0036] The term "target binding moiety," as used herein, refers to any molecule or portion of a molecule that can specifically bind to a target molecule or target epitope. Preferred target binding moieties in the context of the present application are: (i) an antibody or antigen-binding fragment thereof; (ii) an antibody-like protein; and (iii) It is a nucleic acid aptamer. "Target-binding moieties" suitable for use in the present invention typically have a molecular weight of 40,000 Da (40 kDa) or greater.

[0037] A "linker" in the context of this application refers to a molecule that increases the distance between a target binding moiety and an amatoxin, for example, to alleviate steric interference between the two components that might otherwise reduce the ability of the amatoxin to interact with RNA polymerase II. The linker may serve another purpose, facilitating release of the amatoxin specifically in cells targeted by the target binding moiety. The linker and, preferably, the bond between the linker and 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, e.g. in blood, but can be cleaved inside the cell, in particular inside the target cell, e.g. a cancer cell or an immune cell. To provide this selective stability, the linker may preferably contain a functional group that is pH-sensitive or protease-sensitive. Alternatively, the bond connecting the linker to the target binding moiety may provide the selective stability. Preferably, the linker has a length of at least 1, preferably 1 to 30 atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 atoms) in which one side of the linker has been reacted with the amatoxin and the other side has been reacted with the target binding moiety. In the context of the present invention, the linker is preferably an optionally substituted 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, It is an aralkyl or heteroaralkyl group. The linker may contain one or more structural elements such as an amide, ester, ether, thioether, disulfide, carbohydrate moiety, etc. The linker may also contain a combination of two or more of these structural elements. Each of these structural elements may be present more than once in the linker, e.g., 2, 3, 4, 5, or 6 times. In some embodiments, the linker may include a disulfide bond. It is understood that the linker must be attached to the amatoxin and the target binding moiety either in a single step or in two or more sequential steps. Thus, the linker to be present has two groups, preferably at the proximal and distal ends, that are either (i) capable of forming a covalent bond with a group, preferably an activated group, on the amatoxin or target binding peptide, or (ii) have been activated or can be activated to form a covalent bond with a group on the amatoxin. Thus, when a linker is present, chemical groups are preferably at the distal and proximal ends of the linker, which are the result of such coupling reactions, e.g., ester, ether, urethane, peptide bonds, etc. The presence of a "linker" is optional, i.e., in some embodiments of the target-binding moiety-toxin conjugate, the toxin may be directly linked to a residue of the target-binding moiety.

[0038] According to a second aspect, the present invention relates to either (S)-6-acetyloxy-N-tert-butoxy-carbonyltryptophan (HDP 30.2550), (S)-6-hydroxy-tryptophan or a precursor compound according to the invention for use in the synthesis of amanitin or an amanitin derivative or an amatoxin-drug conjugate.

[0039] In one embodiment, the present invention relates to a dehydroamino acid compound selected from the group consisting of compounds I, II, III, IV and V. [ka]

[0040] It is. Here, R1 is selected from H, alkyl, alkenyl, optionally substituted arylalkyl: R2 is selected from Boc, Cbz, N-protecting groups: R3 is selected from Boc, Cbz, N-protecting groups: R4 is an amino acid residue for use in the synthesis of amanitin or an amanitin derivative or an amatoxin-drug conjugate.

[0041] Compounds III, IV and V are preferred.

[0042] Furthermore, we surprisingly found that from a larger panel 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, yielded the highest enantiomeric purity, which was >98%. [ka]

[0043] It was found that only catalyst HDP 30.2758 produced a very high purity of the (S)-enantiomer, exceeding 98%. (R,R)-Et-DUPHOS(BF 4- ), all other catalysts yielded rather low (S)-enantiomeric purity of 50–70%. Furthermore, the overall absolute compound turnover and yield rates were much lower than those using HDP 30.2758.

[0044] The catalysts tested for asymmetric hydrogenation and their respective (S)-enantiomeric purity levels are summarized in Table 1 .

[0045] [Table 1] Thus, according to a third aspect, the present invention provides the compound 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- ) The use of a compound selected from the group consisting of as a catalyst for hydrogenation in the following reaction:

[0046] [ka]

[0047] According to this embodiment, the chiral catalyst cyclooctadiene-1,5-[(R,R)-DIPAMP] rhodium tetrafluoroborate (HDP 30.2758) or (R,R)-Et-DUPHOS (BF 4- ), most preferably relating to the use of the chiral catalyst cyclooctadiene-1,5-[(R,R)-DIPAMP] rhodium tetrafluoroborate (HDP 30.2758) as a catalyst for hydrogenation in the following reaction:

[0048] [ka]

[0049] The incorporation of tryptophan derivatives as building blocks into amanitin precursors has been previously described in PCT / EP2018 / 071268, the contents of which are incorporated herein by reference. Such as HDP 30.2115 can be synthesized by incorporating unsubstituted Hpi building blocks into amanitin peptidic precursor molecules to yield synthetic antitins, and the 6-hydroxy-substituted building block HDP 30.2555 can be used for the synthesis of 6-hydroxy-substituted amanitins according to the present invention.

[0050] Working Example

[0051] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered as illustrative or exemplary and not restrictive, and the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprise" does not exclude other elements or steps and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims shall not be construed as limiting the scope. Example 1: Synthesis of the phosphonium precursor (building block) HDP 30.2822 .

[0052] The synthesis of the phosphonium precursor (building block) HDP 30.2822 was carried out as described (CHEMISTRY A European Journal, 2018, Vol. 24, Issue 7, pp 1544-1553):

[0053] [ka]

[0054] Example 1.1: Preparation of (R,S)-Boc-α-phosphonoglycine trimethyl ester HDP 30.2819

[0055] [ka] 5.0 g (15.1 mmol) of (R,S)-N-Cbz-phosphonoglycine trimethyl ester (CAS: 88568-95-0) was hydrogenated in 100 mL of methanol at 1 atm over 1.4 g of 10% Pd-C until the reaction was complete by TLC (chloroform / methanol 15:1). The reaction was complete in 3 hours. The catalyst was filtered through a plug of Celite® (diatomaceous earth) and the methanolic solution of the free amine was concentrated in vacuo to give a colorless oil (2.9 g). The crude oil was used in the next step without purification.

[0056] 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 under argon for 17 h, the reaction mixture was concentrated to dryness. The remaining colorless oil crystallizes into a white solid (4.3 g). The crude HDP 30.2819 was used in the next step without purification.

[0057] Example 1.2: Preparation of (R,S)-N-Boc-α-phosphonoglycine trimethyl ester HDP 30.2821.

[0058] [ka]

[0059] 4.3 g (estimated 14.3 mmol) of crude HDP 30.2819 was dissolved in 10 mL of 1,4-dioxane and rapidly treated with 14.5 mL of 1 N KOH at room temperature under argon. After 85 min, 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. The resulting white solid, 2.1 g of HDP 30.2821, was dried in vacuum and used directly in the next reaction step without purification. Mp:148-150℃ (Reference. JACS 111,6244,1989 mp:154-155℃) MS(ESI - ) Measurement value: 282.00 [MH] - ; Calculated value: 283.08(C9H 18 NO7P) MS(ESI - )Measured value: 238.17 [M-CO2] -

[0060] Example 1.3: Preparation of (R,S)-N-Boc-α-dimethylphosphono)-glycine benzyl ester HDP 30.2822

[0061] [ka]

[0062] 2.0 g (7.1 mmol) of HDP 30.2821 in 90 mL of dry dichloromethane was 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 under argon at ambient temperature for 24 h. Then, the urea was filtered and the organic phase was washed with 5% citric acid and dried over MgSO4. After evaporation of the dichloromethane, the remaining semi-solid was taken up in ethyl acetate and filtered again to remove further urea. The crude product was purified by flash chromatography on a 330 g silica gel column (detection wavelength 254 nm) with a gradient from n-hexane to n-hexane / ethyl acetate (1:2), and after evaporation, 1.94 g (73%) of HDP 30.2822 was obtained as a white solid. MS(ESI + ) Measurement value: 373.92 [MH] + ; Calculated value: 373.13(C 16 H 242 O7P) MS(ESI + ) Measured value: 396.17 [M+Na] +

[0063] Example 2: Synthesis of (S)-6-acetyloxy-N-tert-butoxycarbonyl-tryptophan HDP 30.2550 as a precursor of HDP 30.2555 (hydroxy-Hpi)

[0064] The synthetic route is summarized in the following synthetic scheme. [ka]

[0065] Example 2.1: Preparation of N-Cbz-6-benzyloxy-indole-3-aldehyde HDP 30.2803

[0066] The starting material for the synthesis, 6-benzyloxyindole-3-aldehyde, is either commercially available or can be prepared in high yield starting from 6-benzyloxyindole by the Vilsmeier reaction.

[0067] [ka]

[0068] No chromatography is required for purification.

[0069] [ka]

[0070] Triethylamine (1.66 mL, 11.94 mmol, 1.50 equiv.) was added via syringe to a solution of 6-benzyloxy-3-formylindole (2.00 g, 7.96 mmol, 1 equiv.) and 4-dimethylaminopyridine (DMAP) (97.23 mg, 796 μmol) in dichloromethane (20 mL) at 23 °C. Benzyl chloroformate (1.45 mL, 10.35 mmol, 1.30 equiv.) was added dropwise to the solution via syringe. After 1 h, another portion of benzyl chloroformate (223 μL, 1.59 mmol, 0.20 equiv.) was added via syringe. After 95 min, the reaction mixture was diluted with dichloromethane (85 mL) and washed with saturated aqueous sodium bicarbonate (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 on a 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), and after evaporation, 2.33 g (76%) of HDP 30.2803 was obtained as a white solid. 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)

[0071] Example 2.2: Preparation of [6-benzyloxy-1H-(benzyloxycarbonyl)-3-indole]-2-(tert-butyloxycarbonylamino)-acrylic acid benzyl ester HDP 30.2824

[0072] [ka]

[0073] 1.90 g (5.09 mmol) of (R,S)-N-Boc-α-dimethylphosphono)-glycine benzyl ester HDP 30.2822 was suspended in 8 mL of dichloromethane under argon. 0.705 mL (4.73 mmol) of DBU was added. After stirring for 10 min, 1.66 g (4.31 mmol) of N-Cbz-6-benzyloxy-indole-3-aldehyde HDP 30.2803 in 4.7 mL of dichloromethane was slowly added. The reaction mixture was stirred for 5 h, 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 hydrochloric acid and 50 mL of brine, dried over MgSO4, and concentrated in vacuo to give 2.70 g of crude material. The crude product was purified by flash chromatography on a 330 g silica gel column (detection wavelength 254 nm) with a gradient from n-hexane to n-hexane / ethyl acetate (1:1), and after evaporation, 2.00 g (73%) of HDP 30.2824 was obtained as a white solid. MS(ESI + ) Measurement value: 632.92 [MH] + ; Calculated value: 632.25 (C 38 H 36 N2O7) MS(ESI + ) Measured value: 655.25 [M+Na] +

[0074] [ka]

[0075] [ka]

[0076] 97.0 mg (0.20 mmol) of bis(cyclooctadiene-1,5)-dichlorodirhodium [Rh(COD)Cl]2 (CAS: 12092-47-6, Alfa Aesar) was added to a suspension of 180.0 mg (0.39 mmol) of (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 was stirred under argon for 1 h to give an orange solution. The complex was precipitated by the slow addition (over 30 min) 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 were filtered off, washed twice with a small amount of water, and dried under high vacuum. 240 mg (81%) of the catalyst Cyclooctadiene-1,5-[(R,R)-DIPAMP]rhodium tetrafluoroborate HDP 30.2758 was obtained as a light yellow powder. The catalyst was used without further purification.

[0077] Example 2.3.2: Synthesis of (S)-6-benzyloxy-N-tert-butoxycarbonyl-1-Cbz-L-tryptophan benzyl ester HDP 30.2826 In a 250 mL stainless steel autoclave, 35.0 mg (0.08 mmol) of cyclooctadiene-1,5-[(R,R)-DIPAMP] rhodium tetrafluoroborate HDP 30.2758, 1000 mg (1.8 mmol), [6-benzyloxy-1H-(benzyloxycarbonyl)-3-indole]-2-(tert-butyloxycarbonylamino)-acrylic acid benzyl ester HDP 30.2824 were placed in 40 mL of dry methanol / 15 mL of dichloromethane. After four vacuum / Ar and H2 cycles, the reaction was pressurized to an initial pressure of 12 bar. The reaction was allowed to proceed at ambient temperature for four days. After evaporating the solvent, the crude product was purified by flash chromatography on a 220 g silica gel column (detection wavelength 254 nm) with n-hexane / ethyl acetate (3:1), and after evaporation, 0.79 g (79%) of HDP 30.2826 was obtained as a white powder. MS(ESI + ) Calculated value: 634.26(C 38 H 38 N2O7) MS(ESI + ) Measured value: 657.33 [M+Na] + ;

[0078] Example 2.4: Preparation of N-tert-butoxycarbonyl-(S)-tryptophan HDP 30.2832

[0079] [ka] 700 mg (1.10 mmol) of (S)-6-benzyloxy-N-tert-butoxycarbonyl-1-Cbz-tryptophan benzyl ester HDP 30.2826 was hydrogenated over 100 mg of Pd-C 10% in a mixture of 7 mL of ethyl acetate and 4 mL of methanol. After 3 h of hydrogenation (TLC control with chloroform / methanol 19:1 + 1% AcOH) at room temperature and 1 atm, the catalyst was removed by filtration over a plug of Celite®. The solvent was removed and the remaining crude residue, 378 mg of HDP 30.2832, was used in the next step without purification.

[0080] Example 2.5: Preparation of (S)-6-acetyloxy-N-tert-butoxycarbonyl-tryptophan HDP 30.2550

[0081] [ka] 378 mg of crude HDP 30.2832 (estimated 1.10 mmol) was dissolved in 2.21 mL of 1 NaOH. Under argon and ambient temperature, 208.5 μL (2.20 mmol) of acetic anhydride was added in one portion. The mixture was stirred for 3.5 h and acidified with 5% citric acid. It was extracted three times with ethyl acetate, and the combined organic phase was washed with 5% sodium chloride and dried over MgSO4. Filtration and evaporation to dryness gave 380 mg of crude material. The crude product was purified by flash chromatography on a 120 g silica gel column (detection wavelength 254 nm) with a gradient of dichloromethane + 2% AcOH / dichloromethane / methanol (15:1) + 2% AcOH to give 270 mg (68%) of HDP 30.2550 as a white solid after evaporation. MS(ESI - ) Measurement value: 361.17 [MH] - Calculated value: 362.15(C 18 H 22 N2O6) MS(ESI -) Measurement value: 723.08 [2M-H] -

[0082] Example 3: Preparation of (S)-6-hydroxy-tryptophan by asymmetric hydrogenation of dehydrogenated amino acids The synthetic route is summarized in the following synthetic scheme.

[0083] [ka] Example 3.1: Preparation of 6-benzyloxy-1H-indole-3-carbaldehyde

[0084] [ka] To a stirred solution of phosphorus oxychloride (10.0 mL, 107.0 mmol) in DMF (35 mL) was added a solution of 6-benzyloxyindole (22.3 g, 100.0 mmol) in DMF (25 mL) at room temperature. After 45 min, the reaction mixture was poured into ice water (200 mL). To this mixture, solid NaOH (19.0 g, 475.0 mmol) and water (100 mL) were added. After 30 min, more water (200 mL) was added, and the whole mixture was refluxed for 3 min. The precipitate was collected, washed five times with 50 mL of cold water, and dried to give 24.8 g (98.8%) of 6-benzyloxy-1H-indole-3-carbaldehyde as a white powder. The compound was identical to the reference material and was sufficiently pure for the next reaction.

[0085] Example 3.2: Preparation of 6-benzyloxy-1H-1-tert-butoxycarbonyl-indole-carbaldehyde HDP 30.2738

[0086] [ka] 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 diethyl 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 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.

[0087] Example 3.3: Preparation of 3-[6-benzyloxy-1H-(1-tert-butoxycarbonyl)-3-indole]-2-(benzyloxycarbonylamino)-acrylic acid methyl ester HDP 30.2739

[0088] [ka]

[0089] 5.12 g (15.44 mmol) of (R,S)-benzyloxycarbonyl-δ-phosphono-glycine trimethyl ester (CAS: 88568-95-0, Alfa Aesar) was dissolved in 18 mL of dichloromethane under argon. 2.14 mL (14.31 mmol) of DBU was added. After stirring for 10 min, 4.60 g (13.07 mmol) of 6-benzyloxy-1H-1-tert-butyloxycarbonyl-indole-3-carbaldehyde HDP 30.2738 in 14 mL of dichloromethane was slowly added. The reaction mixture was stirred for 6 h 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 material. The crude product was purified by flash chromatography on a 330 g silica gel column (detection wavelength 254 nm) with a gradient from n-hexane to n-hexane / ethyl acetate (2:1), giving 5.23 g (72%) of HDP 30.2739 as a white solid after evaporation. MS(ESI + ) Measurement value: 557.17 [MH] + Calculated value: 557.22(C 32 H 32 N2O7) MS(ESI + ) Measured value: 579.25 [M+Na] +

[0090] Example 3.4: Preparation of 6-benzyloxy-N-carbobenzyloxy-1-tert-butoxycarbonyl-L-tryptophan methyl ester HDP 30.2760

[0091] [ka]

[0092] Example 3.4.1: Synthesis of Cyclooctadiene-1,5-[(R,R)-DIPAMP]Rhodium Tetrafluoroborate HDP 30.2758 The catalyst Cyclooctadiene-1,5-[(R,R)-DIPAMP] rhodium tetrafluoroborate HDP 30.2758 was synthesized as described in Example 2.3.1.

[0093] Example 3.4.2: Synthesis of (S)-6-benzyloxy-N-carbobenzyloxy-1-tert-butoxycarbonyl-tryptophan methyl ester HDP 30.2760 In a 250 mL stainless steel autoclave, 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-butoxycarbonyl)-3-indole]-2-(benzyloxycarbonyl-amino)acrylic acid methyl ester HDP 30.2739 were placed in 40 mL of dry methanol. After four vacuum / Ar and H2 cycles, the mixture 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 on a 120 g silica gel column (detection wavelength 254 nm) with a gradient from n-hexane to n-hexane / ethyl acetate (2:1), giving 0.85 g (86%) of HDP 30.2760 as a white solid after evaporation. MS(ESI + ) Measurement value: 558.23(C 33 H 34 N2O7) MS(ESI + ) Measured value: 581.17 [M+Na] + ;1138.83[2M+Na] +

[0094] Example 3.5: Preparation of 6-benzyloxy-N-carbobenzyloxy-L-tryptophan methyl ester HDP 30.2790

[0095] [ka]

[0096] 100.0 mg (0.18 mmol) of (S)-6-benzyloxy-N-carbobenzyloxy-1-tert-butoxycarbonyl-tryptophan methyl ester HDP 30.2760 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 on a 24 g silica gel column (detection wavelength 254 nm) with a gradient from n-hexane to n-hexane / ethyl acetate (1:1), giving 29 mg (35%) of HDP 30.2790 as a white solid after evaporation. MS(ESI + ) Calculated value: 458.52(C 27 H 26 N2O5) MS(ESI + ) Measured value: 459.25 [M+H] +

[0097] [ka]

[0098] A 2N aqueous solution of LiOH (84.7 μL) was added to a solution of 25.9 mg (0.056 mmol) of HDP 30.2790 in 1000 μL of tetrahydrofuran / water (10:1) at ambient temperature. The reaction mixture was stirred for 2.5 h 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 the mobile phase. 13.7mg (55%) white solid. MS(ESI + ) Calculated value: 444.17.23(C 26 H 24 N2O5) MS(ESI + ) Measured value: 445.25 [M+H] + ;467.17[M+Na] +

[0099] Example 3.7: Preparation of (S)-6-hydroxy-N-(tert-butoxycarbonyl)-tryptophan, HDP 30.2832

[0100] [ka] 10 mg (10 wt%) of palladium on carbon was added to a solution of 50 mg (0.11 mmol) of HDP 30.2782 in 800 μL of methanol. The reaction mixture was purged with hydrogen three times and stirred at room temperature for 2.5 h. The suspension was filtered through a plug of Celite®, washed with methanol, and concentrated to dryness. The solid residue (22.2 mg) of (S)-6-hydroxytryptophan was dissolved in 1000 μL of 1,4-dioxane / water (1:1) and treated with 101 μL (0.101 mmol) of 1N NaOH and 21.57 μL (0.10 mmol) of di-tert-butyl dicarbonate. The reaction mixture was stirred for 16 h and then 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 using dichloromethane / methanol (+1% acetic acid) as the mobile phase. 9.9 mg (31%) of a white solid. The material was identical to the reference sample. MS(ES - ) Calculated value: 320.14(C 16 H 20 N2O5) MS(ESI - ) Measurement value: 319.08 [MH] -

[0101] Example 4: Preparation of cis,trans-1-(tert-butoxycarbonyl)-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)

[0102] Example 4.1: Preparation of (S)-N-(tert-butoxycarbonyl)-6-acetoxy-tryptophan HDP 30.2550

[0103] [ka]

[0104] 590.0 mg (2.68 mmol) of (S)-6-hydroxytryptophan from the hydrogenation step of Example 3.7 was suspended in 30 mL of a 1:1 (v,v) mixture of 1,4-dioxane / water. Under argon, 2.68 mL (2.68 mmol) of 1N NaOH was added in one portion at ambient temperature. The resulting yellow solution was then treated with 574.6 mL (2.68 mmol) of di-tert-butyl dicarbonate (Boc2O) and stirred at room temperature for 24 h. The solution was acidified to pH 2.4 with 1N hydrochloric acid and extracted three times with 25 mL of ethyl acetate. The combined ethyl acetate extracts were washed with saturated brine and dried over MgSO4. Filtration and evaporation to dryness gave 785.0 mg of crude material. 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 h 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. Filtration and evaporation gave 635 mg of crude solid. The crude product was purified by flash chromatography on a 330 g silica gel column (detection wavelength 254 nm) with a gradient from CH2Cl2 + 1% acetic acid to CH2Cl2 / MeOH (15:1) + 1% acetic acid, and after coevaporation with toluene, 564,4 mg (56% yield) of white powder was obtained. MS(ESI - ) Measurement value: 361.08 [MH] - Calculated value: 362.15(C 18 H 22 N2O6) Example 4.2: Preparation of cis,trans-6-acetoxy-Hpi

[0105] [ka] Photooxygenation was performed using a 400-W high-pressure sodium vapor lamp (Sirius X400 lamp 230 V, 400 W; 55,000 lumens at a distance of 1.3 m). Rose Bengal is used as the dye sensitizer. The reaction was carried out in a 500 mL cylindrical reaction vessel with a heat exchange jacket made of borosilicate glass, a flat bottom, and a flat laboratory flange (DN) with two connectors with GL18 threads. The distance from the lamp to the reaction vessel was 15 cm, and the reaction temperature ranged from 3 to 4 °C.

[0106] The final product was purified on a Teledyne ISCO Flash chromatography system using a 330 g Silica Redi Sept Flash column (Teledyne ISCO cat. 69-2203-330). The solvents, CH2Cl2, CH3OH, and CH3COOH, were all standard HPLC and BP grade. Dry oxygen (99.5% purity) was bubbled through the reaction mixture at a rate of 2–4 liters / min.

[0107] 943,0mg (2,60mmol) of N-(tert-butoxycarbonyl)-L-6-acetoxy-tryptophan HDP 30.2550 and 100mg of Rose Bengal were dissolved in 500mL of methanol and cooled to 3°C by using a Huber cryostat with polyethylene glycol / water as the cooling medium. The reaction solution was irradiated with a 400W high-pressure sodium vapor lamp. During irradiation, a slow stream of oxygen was bubbled through the reaction solution. After 5 h of irradiation, the oxidation and cooling were stopped, and the reaction medium was treated with 10 mL of dimethyl sulfide. The mixture was stirred for 2 h and evaporated to dryness using a rotary evaporator with a water bath temperature of 35 °C. The dark red residue was further dried under high vacuum to give 1.20 g of a crystalline solid. The crude product was purified on a 330 g silica gel column (detection wavelength 254 nm) with a gradient from CH2Cl2+5% acetic acid to CH2Cl2 / MeOH (30:1)+5% acetic acid. cis-HDP 30.2555 380mg and trans-HDP 30.2555 290mg were eluted and coevaporated with toluene. After lyophilization in tert-butanol, both isomers were obtained as off-white powders.

[0108] cis-1-(tert-butoxycarbonyl)-2-carboxy-3a-hydroxy-6-acetoxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole (cis-HDP 30.2555) cis-HDP 30.2555 380mg Yield: 39%. 1 H-NMR (400MHz, CD3OD, δ=ppm) 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) 13 C-NMR (100MHz, CD3OD, δ=ppm) δ=20.93,28.45,31.12,42.80,61.12,69.44, 82,21,85.82,87.93,104.97,112.98,124.84,129.42,151.51,154.04,155.97,171.34,175.79 MS(ESI + ) Measurement value: 378.92 [MH] + Calculated value: 378.14(C 18 H 22 N2O7) MS(ESI + ) Measured value: 401.17 [M+Na] + Calculated value: 401.14(C 18 H 22 N2NaO7) UV / VIS(CH3OH):λ max = 296nm, 239nm, 215nm λ min=266nm,227nm

[0109] trans-1-(tert-butoxycarbonyl)-2-carboxy-3a-hydroxy-6-acetoxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole (trans-HDP 30.2555) trans-HDP 30.2555 290mg Yield: 30%. 1 H-NMR (400MHz, CD3OD, δ=ppm) 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) 13 C-NMR (100MHz, CD3OD, δ=ppm) δ=20.95,28.50,31.12,42.47,60.97,69.44, 82.06,84.84,87.54,104.74,112.67,125.03,128.70,152.31,154.22,156.00,171.23,174.67 MS (ESI + )Measurement value:379.00[MH] + ; Calculated value: 378.14(C 18 H 22 N2O7) MS (ESI + )Measurement value:401.17[M+Na] + ; Calculated value: 401.14(C 18 H 22 N2NaO7) MS (ESI + )Measurement value:779.00[2M+Na] + ;Calculated value:779.28(C 36 H 44 N4Na2O 14 ) UV / VIS(CH3OH):λ max =299nm,241nm,215nm λ min =268nm,228nm

[0110] Example 4.3: Introduction of cis,trans-6-acetoxy-Hpi into amanitin precursor The synthesis of amanitin using cis,trans-6-acetoxy-Hpi was carried out as described in PCT / EP2018 / 071268, the contents of which are incorporated herein by reference primarily for enabling purposes.

[0111] References Muraoka S, and Shinozawa T., J Biosci Bioeng. 2000;89(1):73-6 Wieland T., Faulstich H. 1978. CRC Crit Rev Biochem. Vol. 5: 185-260. Zanotti G, Mahringer 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 Zhang P, et al., FEMS Microbiol Lett. 2005 Nov 15;252(2):223-8. Epub 2005 Sep 15

Claims

1. A method for the synthesis of (S)-6-acetyloxy-N-tert-butoxycarbonyltryptophan (HDP 30.2550) or (S)-6-hydroxytryptophan, said method comprising at least one step of enantioselective hydrogenation of an olefinic amino acid precursor by use of at least one chiral catalyst, The chiral catalyst is the following compound HDP 30.2758, (R,R)-Et-DUPHOS-Ferrocene (BF 4- ), (R,R)-DuPhos-Ferrocene (BF 4- ), (R,R)-DuPhos-Ferrocene-Et 2 (B.F. 4- ), (R,R)-DuPhos-Alkyl (CF 3 SO 3- ), and (R,R)-Phenyl-DuPhos-Alkyl (BF 4- ) is a compound selected from the group consisting of 【Chemistry 1】 said olefinic amino acid precursor is a dehydroamino acid compound selected from the group consisting of compounds I and III; 【Chemistry 2】 Where: R 1 is selected from H, alkyl, alkenyl, optionally substituted arylalkyl: R 2 is selected from Boc, Cbz, N-protecting groups: R 3 is selected from Boc, Cbz, N-protecting groups.

2. 2. The method of claim 1, wherein the olefinic amino acid precursor is the compound HDP 30.2824. 【Chemistry 3】

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

4. The method according to any one of claims 1 to 3, wherein the olefinic amino acid precursor is synthesized by use of the compound HDP 30.2822.

5. 5. The method of claim 4, wherein the olefinic amino acid precursor is further synthesized by use of compound B. 【Chemistry 4】

6. 6. The method according to any one of claims 1 to 5, wherein the method comprises the use of at least one starting or intermediate compound selected from the group consisting of: 【Chemistry 5】

7. A method for the synthesis of (S)-6-acetyloxy-N-tert-butoxycarbonyltryptophan (HDP 30.2550) according to any one of claims 1 to 6, said method comprising at least the following steps: 【Chemistry 6】

8. A method for synthesizing (S)-6-hydroxytryptophan according to any one of claims 1 to 6, comprising at least the following steps: 【Chemistry 7】

9. A method for the synthesis of amanitin or an amanitin derivative or an amatoxin-drug conjugate comprising the synthesis of (S)-6-acetyloxy-N-tert-butoxycarbonyl-tryptophan (HDP 30.2550) or (S)-6-hydroxy-tryptophan by the method according to any one of claims 1 to 8.

10. A catalyst for hydrogenation in the following reaction according to the method of any one of claims 1 to 8, comprising the compound HDP 30.2758, (R,R)-Et-DUPHOS(BF 4- ), (R,R)-DuPhos-Ferrocene (BF 4- ), (R,R)-DuPhos-Ferrocene-Et 2 (B.F. 4- ), (R,R)-DuPhos-Alkyl (CF 3 SO 3- ), and (R,R)-Phenyl-DuPhos-Alkyl (BF 4- Catalyst comprising a compound selected from the group consisting of: 【Chemistry 8】

11. A catalyst for the hydrogenation in the following reaction in the process according to any one of claims 1 to 8, comprising cyclooctadiene-1,5-[(R,R)-DIPAMP] rhodium tetrafluoroborate (HDP 30.2758). 【Chemistry 9】

Images