Synthesis of Amanitin and Its Derivatives

Abstract

The present invention relates to the chemical synthesis of amanitin and its derivatives and also to intermediates of amanitin synthesis.

Description

[0001] The present invention relates to the chemical synthesis of amanitin and its derivatives and also to intermediates in the synthesis of amanitin. Background of the Invention

[0003] The object of the present invention is to provide means and methods for chemically synthesizing amanitin or its derivatives. This object is achieved by the subject matter of the independent claims of this specification. SUMMARY OF THE INVENTION

[0005] The first aspect of the present invention relates to a method for preparing a compound of formula (I)

[0006]

[0007] Other aspects relate to intermediates in the synthesis of amanitin.

[0008] BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Synthesis of the dipeptide building block H-Asp(OAll)-Hyp-OFm°HCl(3).

[0010] Figure 2 Synthesis of deoxyamanitin. Detailed Description of the Invention

[0012] Terms and Definitions

[0013] The amino acid sequences are given from amino terminus to carboxyl terminus. Capital letters at sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3rd ed. p. 21). Lowercase letters at amino acid sequence positions refer to the corresponding D- or (2R)-amino acids.

[0014] The term AA in the context of this specification relates to an amino acid.

[0015] The term "protecting group" in the context of this specification relates to a moiety covalently attached to a functional group (particularly the carboxylic acid moiety, amino moiety or hydroxyl moiety of the molecules discussed herein), which can be selectively attached to the functional group and selectively removed without affecting the integrity or chiral orientation of the carbon backbone of the molecule to which the protecting group is attached, nor without cleaving specific other protecting groups attached to other protecting groups attached to the molecule.

[0016] The term "deprotecting agent" in the context of this specification refers to an agent that is capable of cleaving a specific protecting group. A person skilled in the art will be able to select a deprotecting agent based on the protecting group. The deprotecting agent is determined by the conditions under which the protecting group is cleavable. For example, if the protecting group is cleavable under acidic conditions, the deprotecting agent is an acid.

[0017] In the context of the present specification, the term "pre-activated carboxyl" relates to a carboxyl moiety that is reacted to form an active ester susceptible to nucleophilic attack by an amine group to form a peptide bond.

[0018] In the context of the present specification, the term "preactivated amino group" relates to an amino group which reacts with N-trimethylsilylamine with increased nucleophilicity to attack the carboxylic acid moiety in order to form a peptide bond.

[0019] A comprehensive review of modern protecting group chemistry, particularly as it relates to the compounds disclosed herein, is available in Peter G. M. Wuts, Greene's Protective Groups in Organic Synthesis, 5th edition, Wiley 2014.

[0020] US 6693178 B2 - "Protecting groups useful in the synthesis of polysaccharides, natural products, and combinatorial libraries" and US 20160024143A1 - "Deprotection method" are incorporated herein by reference.

[0021] Standard conventions in organic chemistry, in which unspecified positions in a chemical formula are assumed to be saturated carbons, are followed herein.

[0022] The first aspect of the present invention relates to a method for preparing a compound of formula (I)

[0023]

[0024] in

[0025] a) In reaction step (a), a compound of formula (IIa)

[0026]

[0027] reacts with peptide bond-forming reagents,

[0028] in particular with a coupling reagent selected from carbodiimides, imidazolinium reagents, phosphonium salts, organophosphorus reagents, uronium salts, pyridinium reagents and phosphonic acids,

[0029] More particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU or HCTU,

[0030] and, where applicable, making said compound with removal of RPGP and / or RP GOH and / or R AL and / or R AMIN The deprotection reaction

[0031] or among them

[0032] b) In the reaction step (b), the compound of formula (IIb)

[0033]

[0034] reacts with peptide bond-forming reagents,

[0035] in particular with a coupling reagent selected from carbodiimides, imidazolinium reagents, phosphonium salts, organophosphorus reagents, uronium salts, pyridinium reagents and phosphonic acids,

[0036] More particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU or HCTU,

[0037] and, where applicable, making said compound with removal of R PGP and / or R PGOH and / or R AL and / or R AMIN The deprotection reaction

[0038] or among them

[0039] c) In the reaction step (c), the compound of formula (IIc)

[0040]

[0041] reacts with peptide bond-forming reagents,

[0042] in particular with a coupling reagent selected from carbodiimides, imidazolinium reagents, phosphonium salts, organophosphorus reagents, uronium salts, pyridinium reagents and phosphonic acids,

[0043] More particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU or HCTU,

[0044] and, where applicable, making said compound with removal of R PGP and / or R PGOH and / or R AL and / or R AMIN The deprotection reaction

[0045] or among them

[0046] d) In the reaction step (d), the compound of formula (IId)

[0047]

[0048] reacts with peptide bond-forming reagents,

[0049] in particular with a coupling reagent selected from carbodiimides, imidazolinium reagents, phosphonium salts, organophosphorus reagents, uronium salts, pyridinium reagents and phosphonic acids,

[0050] More particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU or HCTU,

[0051] and, where applicable, making said compound with removal of R PGP and / or R PGOH and / or R AL and / or R AMIN The deprotection reaction

[0052] in

[0053] ·X 1 、X 2 、X 3 and X 4 , and Y 1 、Y 2 、Y 3 and Y 4 is H, or

[0054] Y 1 、Y 2 、Y 3 and Y 4 One, two, three or all of are OH or NH2 and all other Y are H, and the corresponding X is NHR AMIN OR PGP and all other X are H, where R PGP is a protecting group for the phenolic OH group, particularly a non-acid or base labile phenolic OH protecting group, more particularly cleavable under reducing conditions, most particularly Cbz,

[0055] or R AMIN is a protecting group for a phenolamino group, particularly a non-acid or base labile phenolamino protecting group, more particularly one that is cleavable under reducing conditions, most particularly Cbz, or

[0056] Y 1 、Y 2 、Y 3 and Y 4 One, two, three or all of and the corresponding X are selected from F, Cl, Br, I, CN, NO2, acyl, N3 or alkin, and all other X and Y are H,

[0057] In particular X and Y are H, or Y 1 、Y 2 、Y 3 and Y 4 One is OH, and the corresponding X is OR PGP , and all other X and Y are H;

[0058] Z and W are H, or

[0059] Z is OH and W is OR PGOH , where R PGOH is a protecting group for a hydroxyl group, in particular a protecting group for a hydroxyl group that can be cleaved by fluoride ion,

[0060] ·R AL is a protected carboxyl or 1-5AA peptide, particularly R AL is an O-allyl or O-methyl ester or a 1-5AA peptide, more particularly R AL is O-allyl or O-methyl, most particularly R AL is O-allyl;

[0061] V is OH or a 1-5AA peptide, in particular V is OH;

[0062] Q is S or SO.

[0063] In certain embodiments, the method is performed by step a) or step b).

[0064] In certain embodiments, the 1-5 AA peptide consists of proteinogenic amino acids.

[0065] In certain embodiments, the compound of formula (III)

[0066]

[0067] Reaction with a compound of formula (IV)

[0068]

[0069] in

[0070] ·X 1 、X 2 、X 3 、X 4 , W, Q and R AL has the same meaning as defined above;

[0071] ·R COOY is a carboxyl-protecting group, in particular fluorenylmethyl or benzyl, more in particular fluorenylmethyl;

[0072] wherein in reaction step (e), (III) and (IV) are reacted with a peptide bond-forming reagent, in particular with HATU, COMU, HBTU, TBTU, TOMBU, COMBU or HCTU,

[0073] To obtain the compound characterized by (IIa).

[0074] In certain embodiments, the compound of formula (V)

[0075]

[0076] Reaction with a compound of formula (VI)

[0077]

[0078] in

[0079] ·X 1 、X 2 、X 3 、X 4 , Q and W have the same meanings as defined above;

[0080] ·R NHB is an amino protecting group, in particular an amino protecting group cleavable under alkaline conditions, more particularly Fmoc,

[0081] wherein in reaction step (f), the amino group of (V) is preactivated, in particular with MSA, and the preactivated (V) and (VI) are reacted with a peptide bond-forming reagent, in particular with HATU, COMU, HBTU, TBTU, TOMBU, COMBU or HCTU, more in particular with COMU,

[0082] to obtain compound (III).

[0083] In certain embodiments, the compound of formula (V)

[0084]

[0085] Reaction with a compound of formula (VII)

[0086]

[0087] in

[0088] ·X 1 、X 2 、X 3 、X 4 , W, Q, R NHB and R AL has the same meaning as defined above;

[0089] wherein in the reaction step (g)

[0090] The amino group of (V) is preactivated, in particular with MSA, and the preactivated (V) and (VII) are reacted with a peptide bond forming agent, in particular with HATU, or

[0091] The amino group of (V) is preactivated, in particular with MSA, and the carboxyl group of compound (VII) is preactivated, in particular with O-PFP-ester, O-PCP-ester or OSu-ester, and the preactivated (V) and the preactivated (VII) are reacted,

[0092] to obtain compound (IIb).

[0093] For coupling compounds (V) and (VII), the acid-COOH group of compound (V) does not need to be protected. No significant side reactions were observed in the absence of a protecting group.

[0094] In certain embodiments, the compound of formula (V)

[0095]

[0096] Reaction with a compound of formula (VII)

[0097]

[0098] in

[0099] ·X 1 、X 2 、X 3 、X 4 , W, Q, R NHB and R AL has the same meaning as defined above;

[0100] wherein (V) and (VII) are reacted with a peptide bond-forming reagent, in particular with HATU, COMU, HBTU, TBTU, TOMBU, COMBU or HCTU,

[0101] to obtain compound (IIc).

[0102] In certain embodiments, the compound of formula (XVI)

[0103]

[0104] Reaction with a compound of formula (XVII)

[0105]

[0106] in

[0107] ·X 1 、X 2 、X 3 、X 4 , W, Q and R AL has the same meaning as defined above;

[0108] ·R NHB2 is an amino protecting group, in particular an amino protecting group cleavable under acidic conditions, more particularly Boc;

[0109] in

[0110] The amino group of (XVI) is preactivated, in particular with MSA, and the preactivated (XVI) and (XVII) are reacted with a peptide bond-forming agent, in particular with HATU, or

[0111] The amino group of (XVI) is preactivated, in particular with MSA, and the carboxyl group of compound (XVII) is preactivated, in particular with O-PFP-ester, O-PCP-ester or OSu-ester, and the preactivated (XVI) and the preactivated (XVII) are reacted,

[0112] to obtain compound (IId).

[0113] In certain embodiments, the compound of formula (V)

[0114]

[0115] Reaction with a compound of formula (XVIII)

[0116]

[0117] in

[0118] ·X 1 、X 2 、X 3 、X 4 , W, Q, R NHB and R AL has the same meaning as defined above;

[0119] in

[0120] The amino group of (V) is preactivated, in particular with MSA, and the preactivated (V) and (XVIII) are reacted with a peptide bond forming agent, in particular with HATU, or

[0121] The amino group of (V) is preactivated, in particular with MSA, and the carboxyl group of compound (XVIII) is preactivated, in particular with O-PFP-ester, O-PCP-ester or OSu-ester, and the preactivated (V) and preactivated (XVIII) are reacted,

[0122] to obtain compound (XVI).

[0123] In certain embodiments, for compounds of formula (Idesox), the sulfur atom is oxidized,

[0124]

[0125] in

[0126] ·Y 1 、Y 2 、Y 3 、Y 4 , Z, Q and V have the same meanings as defined above.

[0127] In certain embodiments, the oxidation of the sulfur atom is carried out using manganese ions.

[0128] In certain embodiments, the compound is reacted with a compound of formula (XV)

[0129]

[0130] And react with Mn(OTf)2 and H2O2.

[0131] In certain embodiments, the oxidation of the sulfur atom is carried out using PPO, dibenzoyl peroxide, t-butyl peroxybenzoate, or lauroyl peroxide. The preparation of PPO is described in (S. Gan, J. Yin, Y. Yao, Y. Liu, D. Chang, D. Zhu, L. Shi, Org. Biomol. Chem. 2017, 15, 2647-2654.).

[0132] In certain embodiments, oxidation of the sulfur atom is performed with mCPBA (meta-chloroperbenzoic acid) in isopropanol / ethanol (8:3).

[0133] In certain embodiments, oxidation of the sulfur atom is performed with an oxaziridine salt as described in (Rio et al., Org. Lett. 2007, 9, 12, 2265-2268).

[0134] In certain embodiments, oxidation of the sulfur atom is performed with a diastereoselective reagent or simply with oxygen or hydrogen peroxide.

[0135] In certain embodiments, oxidation of the sulfur atom is performed using iodine and oxygen.

[0136] In reaction step (h), oxidation of the sulfur atom is carried out to obtain compound (Iox)

[0137]

[0138] In certain embodiments, for compounds of formula (Vdesox), the sulfur atom is oxidized,

[0139]

[0140] in

[0141] ·X 1 、X 2 、X 3 and X 4 has the same meaning as defined above.

[0142] In certain embodiments, the oxidation of the sulfur atom is carried out using manganese ions.

[0143] In certain embodiments, the compound is reacted with a compound of formula (XV)

[0144]

[0145] And react with Mn(OTf)2 and H2O2.

[0146] In certain embodiments, the oxidation of the sulfur atom is carried out using PPO, dibenzoyl peroxide, t-butyl peroxybenzoate, or lauroyl peroxide. The preparation of PPO is described in (S. Gan, J. Yin, Y. Yao, Y. Liu, D. Chang, D. Zhu, L. Shi, Org. Biomol. Chem. 2017, 15, 2647-2654.).

[0147] In certain embodiments, oxidation of the sulfur atom is performed using mCPBA (meta-chloroperbenzoic acid) in isopropanol / ethanol (8:3).

[0148] In certain embodiments, oxidation of the sulfur atom is performed with an oxaziridine salt as described in (Rio et al., Org. Lett. 2007, 9, 12, 2265-2268).

[0149] In certain embodiments, oxidation of the sulfur atom is performed with a diastereoselective reagent or simply with oxygen or hydrogen peroxide.

[0150] In certain embodiments, oxidation of the sulfur atom is performed using iodine and oxygen.

[0151] In reaction step (i), oxidation of the sulfur atom is carried out to obtain compound (Vox)

[0152]

[0153] In certain embodiments, in reaction step (j), the compound of formula (VIII)

[0154]

[0155] in

[0156] -R NHF is an amino protecting group, in particular an amino protecting group cleavable by fluoride ion or a strong acid, more particularly Teoc, or an amino protecting group cleavable by alkaline conditions, more particularly Fmoc,

[0157] -R COOA is a carboxyl protecting group, in particular a carboxyl protecting group cleavable under strongly acidic conditions, more particularly a tert-butyl group,

[0158] -X 1 、X 2 、X 3 and X 4 have the same meanings as set out above,

[0159] reaction with a peptide bond-forming reagent, in particular with a coupling reagent selected from carbodiimides, imidazolinium reagents, phosphonium salts, organophosphorus reagents, uronium salts, pyridinium reagents and phosphonic acids, more particularly with T3P, HATU, COMU, HBTU, TBTU, TOMBU, COMBU or HCTU,

[0160] And in the reaction step (k), the compound is reacted with the removal of R NHF and R COOA Deprotection reaction, in particular with TFA,

[0161] To obtain a compound characterized by (Vdesox) or (V).

[0162] In certain embodiments, the compound of formula (IX)

[0163]

[0164] Reaction with a compound of formula (X)

[0165]

[0166] in

[0167] -R NHA is an amino protecting group, in particular an amino protecting group cleavable under acidic conditions, more particularly Boc,

[0168] -R COOA 、R NHF and X 1 、X 2 、X 3 and X 4 have the same meanings as set out above,

[0169] wherein in reaction step (1), compound (VII)

[0170] preactivated with a peptide bond-forming reagent, in particular HATU, COMU, HBTU, TBTU, TOMBU, COMBU or HCTU, and subsequently reacted with the silylated compound (VIII), or

[0171] preactivated to the OSu-ester form and subsequently reacted with the compound (VII),

[0172] And in the reaction step (m), the compound is reacted with the removal of R NHA in the presence of a deprotecting agent, particularly under acidic conditions, more particularly at a pH of from -3 to 0, even more particularly with HCl or p-toluenesulfonic acid, most particularly with 2M HCl in dioxane,

[0173] To obtain the compound characterized by (VIII).

[0174] In certain embodiments, the compound of formula (XI)

[0175]

[0176] Reaction with a compound of formula (XII)

[0177]

[0178] in

[0179] -R COOZ is a carboxyl protecting group, in particular a carboxyl protecting group cleavable by Zn, more particularly Tce, or R COOZ It's H.

[0180] -R COOA 、R NHF 、R NHA and X 1 、X 2 、X 3 and X 4 have the same meanings as set out above,

[0181] reacts in reaction step (n), and if R COOZ is a carboxyl protecting group, then in the reaction step (o), the compound is reacted with aCOOZ reacting with a deprotecting agent, in particular with Zn,

[0182] To obtain the compound characterized by (IX).

[0183] A protecting group strategy that relies on acid stability was applied. Lowering the pH value was used for deprotection. First, the Tce group of tryptophan (R of compound VIII) was removed using Zn under reducing conditions with a mild acidic pH. COOZ ). Then, the Boc group of cysteine ​​(R NHA Finally, the Teoc (R) of compound (V) was removed simultaneously with 95% TFA. NHF ) and tert-butyl (R COOA ).

[0184] In certain embodiments, the compound of formula (VI)

[0185]

[0186] and a compound of formula (XIII)

[0187]

[0188] and react with a compound of formula (XIV)

[0189]

[0190] in

[0191] -R AL has the same meaning as above,

[0192] -R Pep is an active ester, in particular O-pentafluorophenol or OSu-ester,

[0193] -R NHB is an amino protecting group, in particular an amino protecting group cleavable under alkaline conditions, more particularly Fmoc,

[0194] In the reaction step (p), the reaction is carried out using solid phase peptide synthesis, wherein the carboxyl group of compound (XIV) may be protected.

[0195] To obtain the compound characterized by (VII).

[0196] Another aspect of the present invention relates to compounds of formula (I)

[0197]

[0198] in

[0199] ·Y1 、Y 2 and Y 4 One, two or three of are OH, and all other Y's are H, or

[0200] Y 1 、Y 2 、Y 3 and Y 4 One, two, three or all of Y are selected from F, Cl, Br and I, and all other Y are H, in particular Y 1 、Y 2 and Y 4 One of Y is OH, and all other Y are H;

[0201] Z is H, or

[0202] Z is OH,

[0203] V is OH or a 1-5AA peptide, in particular V is OH,

[0204] Q is S or SO.

[0205] Another aspect of the present invention relates to compounds of formula (IIa)

[0206]

[0207] in

[0208] ·X 1 、X 2 、X 3 and X 4 is H, or

[0209] X 1 、X 2 、X 3 and X 4 One, two, three or all of are OH, and all other X are H, or

[0210] X 1 、X 2 、X 3 and X 4 One, two, three or all of are selected from F, Cl, Br and I, and all other X are H,

[0211] Especially X 1 、X 2 、X 3 and X 4 Is H, or X 1 、X 2 、X 3 and X 4 One is OH and all other Xs are H;

[0212] W is H, or

[0213] W is OH,

[0214] V is OH or a 1-5AA peptide, in particular V is OH,

[0215] Q is S or SO.

[0216] Another aspect of the present invention relates to compounds of formula (IIb)

[0217]

[0218] in

[0219] ·X 1 、X 2 、X 3 and X 4 is H, or

[0220] X 1 、X 2 、X 3 and X 4 One, two, three or all of are OH, and all other X are H, or

[0221] X 1 、X 2 、X 3 and X 4 One, two, three or all of are selected from F, Cl, Br and I, and all other X are H,

[0222] Especially X 1 、X 2 、X 3 and X 4 Is H, or X 1 、X 2 、X 3 and X 4 One is OH and all other Xs are H;

[0223] W is H, or

[0224] W is OH,

[0225] V is OH or a 1-5AA peptide, in particular V is OH,

[0226] Q is S or SO.

[0227] Another aspect of the present invention relates to compounds of formula (III)

[0228]

[0229] in

[0230] ·X 1 、X 2 and X 4 One, two or all of are OH, and all other X are H, or

[0231] X 1 、X 2 and X 4 One, two or all of are selected from F, Cl, Br and I, and all other X are H,

[0232] Especially X 1 、X 2 and X 4 One of X is OH, and all other X are H;

[0233] W is H, or

[0234] W is OH,

[0235] Q is S or SO.

[0236] Another aspect of the present invention relates to compounds of formula (V)

[0237]

[0238] in

[0239] ·X 1 、X 2 and X 4 One, two or all of are OH, and all other X are H, or

[0240] X 1 、X 2 and X 4 One, two or all of are selected from F, Cl, Br and I, and all other X are H,

[0241] Especially X 1 、X 2 and X 4 One of X is OH, and all other X are H,

[0242] Q is S or SO.

[0243] The present invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be derived. These examples are intended to illustrate the present invention but not to limit its scope. BRIEF DESCRIPTION OF THE DRAWINGS

[0244] Figure 1 Synthesis of the dipeptide building block H-Asp(OAll)-Hyp-OFm°HCl(3).

[0245] Figure 2 Synthesis of deoxyamanitin. Example

[0246] Synthesis of (N-Boc)2-cystine-(OtBu)2(35)

[0247]

[0248] A solution of L-cystine-(OtBu)2 (34, 10 g, 24 mmol, 1.0 equiv) in a 1:1 mixture of H2O / dioxane (240 mL) was treated with NaHCO3 (8.06 g, 96.0 mmol, 4.00 equiv) and Boc2O (10.1 mL, 47.0 mmol, 2.00 equiv), and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure, and the aqueous layer was extracted with EtOAc (3x120 mL). The organic layer was washed with brine (100 mL), dried over Na2SO4, and the solvent was removed under reduced pressure to give 35 (13.2 g, 24.0 mmol, quantitative) as a pale yellow solid.

[0249] HRMS(ESI):C 24 H 44 N2O8S2(M+H) + The calculated m / z value is 553.2612, the found value is 553.2615.

[0250] S-(6-(Benzyloxy)-3-((S)-3-oxo-3-(2,2,2-trichloroethoxy)-2-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)propyl)-1H-indol-2-yl)-N-(tert-butoxycarbonyl)-L-cysteine ​​tert-butyl ester (40)

[0251]

[0252] To a solution of (N-Boc)2-1-cystine-(OtBu)2 (35, 900 mg, 1.63 mmol, 1.00 equivalent) in CHCl3 (16.3 mL) was added SO2Cl2 (263 μL, 3.26 mmol, 2.00 equivalent). After the reaction mixture was stirred at room temperature for 1 hour, the solvent was removed under reduced pressure. The residue was redissolved in CHCl3 (16.3 mL) and cooled to 0°C and added dropwise to an ice-cold solution of 39 (800 mg, 1.67 mmol, 1.00 equivalent) and NaHCO3 (420 mg, 5.00 mmol, 3.00 equivalent) in CHCl3 (16.7 mL) over a period of 10 minutes. The reaction mixture was then stirred at 0°C for 15 minutes and at room temperature for 1 hour. The organic layer was washed with H2O (10 mL) and saturated NaHCO3 solution (10 mL). After drying the organic layer over Na 2 SO 4 and removing the solvent under reduced pressure, the crude product of 40 was used in the next step without further purification.

[0253] HRMS(ESI):C 38 H 52 Cl3N3O9SSi(M+H) + The calculated m / z value is 860.2332, the found value is 860.2323.

[0254] N-(tert-Butoxycarbonyl)-S-(3-((S)-3-oxo-3-(2,2,2-trichloroethoxy)-2-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)propyl)-1H-indol-2-yl)-L-cysteine ​​tert-butyl ester (41)

[0255]

[0256] To (N-Boc) 2-L-cystine-(OtBu) 2 (39, 5.06 g, 9.15 mmol, 1.00 equivalent) in CHCl 3 (92 mL) in solution add SO 2 Cl 2 (1.48 mL, 18.3 mmol, 2.00 equivalent). After the reaction mixture was stirred at room temperature for 1 hour, the solvent was removed under reduced pressure. The residue was redissolved in CHCl 3 (92 mL), cooled to 0 ℃ and added dropwise to an ice-cold solution of 38 (4.4 g, 9.17 mmol, 1.00 equivalent) and NaHCO 3 (2.31 g, 27.5 mmol, 3.00 equivalent) in CHCl 3 (92 mL) over a period of 10 minutes. The reaction mixture was then stirred at 0 ℃ for 15 minutes and at room temperature for 1 hour. With organic layer H 2 O (2x100 mL) and saturated NaHCO 3 solution (2x80 mL) washing. After drying the organic layer over Na 2 SO 4 and removing the solvent under reduced pressure, the crude product of 41 was used for the next step without further purification.

[0257] HRMS(ESI):C 31 H 46 Cl3N3O8SSi(M+H) + The calculated m / z value is 754.1913, the found value is 754.1917.

[0258] (S)-3-(6-(Benzyloxy)-2-(((R)-3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)thio)-1H-indol-3-yl)-2-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)propanoic acid (49):

[0259]

[0260] A solution of aminocarboxyethylthiotryptophan (tryptathionine) derivative 39 (1.63 mmol, 1.00 equiv) in DMF (8.4 mL) was treated with CHCOOH (0.8 mL) and zinc (3.51 g, 53.6 mmol, 33.0 equiv) at 45 ° C for 2 hours. The reaction mixture was filtered through celite and the solvent was removed under reduced pressure. The crude product was dissolved in EtOAc (50 mL) and washed with 10% KHSO solution (2x25 mL) and brine (2x25 mL). After drying over NaSO and removing the solvent under reduced pressure, the crude product was purified by C18 reverse phase chromatography (AcN / H O 50% to 100% gradient) to obtain compound 49 (840 mg, 83% over 2 steps) as a yellow solid.

[0261] HRMS(ESI):C 36 H51 N3O9SSi(M+H) + The calculated m / z value is 730.3183, the found value is 730.3188.

[0262] (S)-3-(2-(((R)-3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)thio)-1H-indol-3-yl)-2-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)propanoic acid (50)

[0263]

[0264] A solution of aminocarboxyethylthiotryptophan derivative 38 (9.15 mmol, 1.00 equiv) in DMF (40 mL) was treated with CHCOOH (4 mL) and zinc (20.0 g, 302 mmol, 33.0 equiv) at 45 ° C for 2 hours. The reaction mixture was filtered through celite and the solvent was removed under reduced pressure. The crude product was dissolved in EtOAc (200 mL) and washed with 10% KHSO solution (2x50 mL) and brine (2x50 mL). After drying over NaSO and removing the solvent under reduced pressure, the crude product was purified by C18 reverse phase chromatography (AcN / H O 50% to 100% gradient) to obtain compound 50 (5.0 g, 88% over 2 steps) as a yellow oil.

[0265] HRMS(ESI):C 31 H 46 Cl3N3O8SSi(M+H) + The calculated m / z value is 624.2769, the found value is 624.2775.

[0266] ((Benzyloxy)carbonyl)glycyl-L-isoleucine (44)

[0267]

[0268] To a solution of Cbz-glycine (42, 10.0 g, 32.7 mmol, 1.00 equiv) in acetone (100 mL) was added a suspension of L-isoleucine (4.71 g, 35.9 mmol, 1.10 equiv) and NaHCO (8.23 g, 87.9 mmol, 3.00 equiv) in water (100 mL). The reaction mixture was stirred at room temperature for 3 hours and concentrated under reduced pressure. The aqueous layer was carefully acidified to pH = 4 by the addition of 1 M HCl and extracted with EtOAc (3 x 150 mL). The organic phase was then washed with brine (2 x 100 mL), dried over Na SO and evaporated under reduced pressure to give the product 44 (10.1 g, 96%) as a colorless oil.

[0269] HRMS(ESI):C 16 H 22 N2O5(M+H) + The calculated m / z value is 323.1601, the found value is 323.1606.

[0270] Glycyl-L-isoleucylglycine (45):

[0271]

[0272] Dipeptide 44 (10.1 g, 31.3 mmol, 1.00 equiv) and glycine benzyl ester (8.21 g, 40.7 mmol, 1.30 equiv) were dissolved in anhydrous DMF (125 mL). COMU (17.4 g, 40.7 mmol, 1.30 equiv) and DIPEA (12.6 mL, 72.1 mmol, 3.00 equiv) were then added at 0°C. The reaction mixture was allowed to warm to room temperature overnight and then diluted with EtOAc (300 mL). After washing with 10% KHSO4 solution (2 x 100 mL), the fully protected tripeptide precipitated in the organic phase. The organic phase was cooled to 4°C for 4 hours to precipitate the peptide, and the precipitate was then filtered and washed with cold EtOAc. The precipitate was redissolved in a 1:1 mixture of water and THF (260 mL). After degassing with N2 for 30 minutes, Pd / C (1 g) was added to the solution. Then, the reaction mixture was degassed with hydrogen for 1 hour. After vigorous stirring overnight at room temperature and 1.0 atm of hydrogen, the catalyst was filtered through a celite pad. Then, the mixture was concentrated under reduced pressure to obtain the product 45 (5.71 g, 74%) as a white solid.

[0273] HRMS(ESI):C 10 H 19 N3O4(M+H) + The calculated m / z value is 246.1448, the found value is 246.1440.

[0274] Synthesis of Pentapeptide 51:

[0275]

[0276] A solution of thioether building block 49 (111 mg, 0.14 mmol, 1.00 equiv) in AcN (0.7 mL) was treated with collidine (37 μL, 0.27 mmol, 2.0 equiv) and N,N'-disuccinimidyl carbonate (39 mg, 0.15 mmol, 1.1 equiv) and stirred at room temperature for 1 hour. A solution of tripeptide 45 (44 mg, 0.18 mmol, 1.3 equiv) in a 1:4 mixture of AcN / H2O (1 mL) was added, and the reaction mixture was stirred at room temperature for 2 hours. Afterwards, the mixture was diluted with EtOAc (20 mL), 10% KHSO4 solution (20 mL) was added, and the aqueous layer was extracted with EtOAc (2 x 20 mL). The organic layer was washed with brine (2 x 20 mL), dried over Na2SO4, and evaporated under reduced pressure to provide pentapeptide 51 (115 mg, 90%) as a yellow solid.

[0277] HRMS(ESI):C 46 H 68 N6O 12 SSi(M+H) + The calculated m / z value is 957.4458, the found value is 957.4457.

[0278] Synthesis of Pentapeptide 52:

[0279]

[0280] A solution of aminocarboxyethylthiotryptophan building block 50 (2.0 g, 2.5 mmol, 1.0 equiv) in AcN (10 mL) was treated with collidine (659 μL, 4.95 mmol, 2.00 equiv) and N,N'-disuccinimidyl carbonate (697 mg, 2.72 mmol, 1.10 equiv) and stirred at room temperature for 1 hour. A solution of tripeptide 45 (790 mg, 3.22 mmol, 1.30 equiv) in a 1:4 mixture of AcN / H2O (18 mL) was added, and the reaction mixture was stirred at room temperature for 2 hours. Afterwards, the mixture was diluted with EtOAc (100 mL), 10% KHSO4 solution (20 mL) was added, and the aqueous layer was extracted with EtOAc (2 x 50 mL). The organic layer was washed with brine (2×50 mL), dried over Na 2 SO 4 and evaporated under reduced pressure to give pentapeptide 52 (2.15 g, 93%) as a yellow solid.

[0281] HRMS(ESI):C 36 H 51 Cl3N6O 11 The m / z calculated for S was 851.4039 and found to be 851.4058.

[0282] Fully protected cyclic pentapeptide 53:

[0283]

[0284] Pentapeptide 51 (151 mg, 0.180 mmol, 1.00 equiv) was dissolved in 1 mL of p-toluenesulfonic acid in THF (1.8 M) and stirred at room temperature for 4 h. The reaction mixture was then neutralized by the addition of DIPEA (320 μL, 1.84 mmol, 10 equiv) and diluted with DCM (180 mL). DIPEA (60.2 μL, 354 μmol, 2.00 equiv) and T3P (50% in EtOAc, 210 μL, 354 μmol, 0.34 equiv) were then added. After the solution was stirred at room temperature for 16 h, 2 / 3 of the solvent was concentrated under reduced pressure. The organic phase was washed with 10% KHSO4 solution (20 mL), saturated NaHCO3 solution (20 mL), water (20 mL) and brine (20 mL). The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified by C18 reverse phase chromatography (AcN / H2O 50% to 100% gradient) to give cyclic pentapeptide 53 as a yellow solid (82 mg, 70%).

[0285] HRMS(ESI):C 41 H 58 N6O9SSi(M+H) + The calculated m / z value is 839.3828, the found value is 839.3839.

[0286] Fully protected cyclic pentapeptide (54):

[0287]

[0288] Pentapeptide 52 (700 mg, 0.822 mmol, 1.00 equiv) was dissolved in 10 mL of dioxane containing 2M HCl and stirred at room temperature for 40 minutes. The reaction mixture was then diluted with 40 mL of dioxane and the solvent was evaporated under reduced pressure. The precipitate was dissolved in 8 mL of DMF and diluted with 82 mL of DCM. DIPEA (279 μL, 1.64 mmol, 2.00 equiv) and T3P (50% in EtOAc, 977 μL, 1.64 mmol, 2.00 equiv) were then added. After the solution was stirred at room temperature for 5 hours, 1 / 3 of the solvent was concentrated under reduced pressure. The organic phase was washed with 10% KHSO4 solution (20 mL), saturated NaHCO3 solution (20 mL), water (20 mL) and brine (20 mL). The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified by C18 reverse phase chromatography (AcN / H2O 50% to 100% gradient) to give cyclic pentapeptide 54 (420 mg, 72%) as a yellow solid.

[0289] HRMS(ESI):C 34 H 52 N6O8SSi(M+H) + The calculated m / z value is 733.3409, the found value is 733.3409.

[0290] Completely deprotected monocyclic pentapeptide 55:

[0291]

[0292] Monocyclic pentapeptide 53 (125 mg, 0.17 mmol, 1.00 equiv) was stirred in TFA / DCM / TIPS (8:1.5:0.5) at room temperature for 2 h. The solvent was removed under reduced pressure, and the crude product was purified by C18 reverse phase chromatography (AcN / H2O 20% to 100%) to give the fully deprotected monocyclic pentapeptide 55 (100 mg, quantitative) in the form of a white powder.

[0293] HRMS(ESI):C 31 H 38 N6O7S(M+H) + The calculated m / z value is 639.2595, the found value is 639.2590.

[0294] Completely deprotected monocyclic pentapeptide 56:

[0295]

[0296] Monocyclic pentapeptide 54 (250 mg, 0.34 mmol, 1.00 equiv) was stirred in TFA / DCM / TIPS (8:1.5:0.5) at room temperature for 2 h. The solvent was removed under reduced pressure, and the crude product was purified by C18 reverse phase chromatography (AcN / H2O 10% to 30%) to give the fully deprotected monocyclic pentapeptide 56 (200 mg, quantitative) in the form of a white powder.

[0297] HRMS(ESI):C 24 H 32 N6O6S(M+H) + The calculated m / z value is 533.2177, the found value is 533.2188.

[0298] Monocyclic hexapeptide 67:

[0299]

[0300] A solution of the fully deprotected monocyclic pentapeptide 55 (42 mg, 0.66 mmol, 1.00 equiv) and MSA (11.6 μL, 0.723 mmol, 1.10 equiv) in DMA (2 mL) was stirred at 50°C for 2 hours. Simultaneously, a solution of Fmoc-DHIle(TBS)2-OH (13, 52 mg, 0.85 mmol, 1.30 equiv), COMU (36 mg, 0.85 mmol, 1.30 equiv) and DIPEA (15 μL, 0.85 mmol, 1.30 equiv) in DMA (0.4 mL) was stirred at 0°C for 30 minutes. The silylated monocyclic peptide was then added to the activated dihydroxyisoleucine derivative and stirred at 0°C for 1 hour, then at room temperature overnight. The mixture was then diluted with EtOAc (50 mL) and washed with 10% KHSO4 solution (3 x 5 mL). The organic phase was washed with brine (2 x 20 mL), dried over NaSO 4 and evaporated under reduced pressure. The crude product of 67 was used in the next step without any further purification.

[0301] HRMS(ESI):C 64 H 87 N7O 12 SSi2(M+H) + The calculated m / z value is 1234.5745, and the found value is 1234.5745.

[0302] Monocyclic hexapeptide 68:

[0303]

[0304] A solution of the fully deprotected monocyclic pentapeptide 56 (100 mg, 0.188 mmol, 1.00 equiv) and MSA (33.2 μL, 0.207 mmol, 1.10 equiv) in DMA (4 mL) was stirred at 50° C. for 2 h. Simultaneously, a solution of Fmoc-DHIle(TBS)2-OH (13, 149 mg, 0.244 mmol, 1.30 equiv), COMU (104 mg, 0.244 mmol, 1.30 equiv) and DIPEA (42.5 μL, 0.244 mmol, 1.30 equiv) in DMA (1.25 mL) was stirred at 0° C. for 30 min. The silylated monocyclic peptide was then added to the activated dihydroxyisoleucine derivative and stirred at 0° C. for 1 h and then at room temperature overnight. Then, the mixture was diluted with EtOAc (100 mL) and washed with 10% KHSO solution (3x10 mL). The organic phase was washed with salt water (2x25 mL), dried over NaSO and evaporated under reduced pressure. The crude product of 68 was used for the next step without any further purification.

[0305] HRMS(ESI):C 57 H 81 N7O 11 SSi2(M+H) + The calculated m / z value is 1128.5326, and the found value is 1128.5316.

[0306] Synthesis of (9H-fluoren-9-yl)methyl (2S, 4R)-4-hydroxypyrrolidine-2-carboxylate hydrochloride (2):

[0307]

[0308] A solution of N-Boc-protected (2S,4R)-4-hydroxyproline 1 (5.0 g, 22 mmol, 1.0 equiv) in DMF (20 mL) was added dropwise to a solution of 9-fluorenylmethanol (8.5 mg, 43 mmol, 2.0 equiv), EDC*HCl (8.3 g, 43 mmol, 2.0 equiv), and DMAP (396 mg, 3.24 mmol, 0.150 equiv) in DCM (220 mL). The reaction mixture was stirred at room temperature for 2 hours. 10% KHSO₄ solution (50 mL) was then added. The organic phase was washed with brine (50 mL) and dried over NaSO₄. The solvent was then removed under reduced pressure, and the crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 1:1) and treated with 4M HCl in dioxane for 30 minutes. The solvent was evaporated under reduced pressure to give the product 2 (3.7 g, 56%) as a white solid.

[0309] HRMS(ESI):C19 H 19 NO3(M+H-HCl) + The calculated m / z value is 310.1438, the found value is 310.1426.

[0310] Synthesis of (9H-fluoren-9-yl)methyl (2S,4R)-1-((S)-4-(allyloxy)-2-amino-4-oxobutanoyl)-4-hydroxypyrrolidine-2-carboxylate (3):

[0311]

[0312] Boc-L-aspartic acid 4-allyl ester (287 mg, 1.05 mmol, 1.30 equiv), compound 2 (250 mg, 0.808 mmol, 1.0 equiv) and HATU (614 mg, 1.62 mmol, 2.0 equiv) were dissolved in DMF (2 mL) at 0°C. DIPEA (563 μL, 3.23 mmol, 4 equiv) was then added and the reaction mixture was stirred at room temperature for 2 hours. Subsequently, the reaction mixture was diluted with EtOAc (50 mL). The organic phase was washed with 10% KHSO solution (2 x 10 mL), saturated NaHCO (10 mL) and brine (10 mL). After drying over NaSO and removing the solvent under reduced pressure, the crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 2: 1) and thereafter treated with dioxane containing 4M HCl for 30 minutes. The solvent was evaporated under reduced pressure to give the product 3 (344 mg, 85%) as a yellow oil.

[0313] HRMS(ESI):C 26 H 28 N2O6(M+H-HCl) + The calculated m / z value is 465.2020, the found value is 465.2017.

[0314] Synthesis of monocyclic octapeptide 5:

[0315]

[0316] The fully deprotected monocyclic pentapeptide 4 (50 mg, 0.09 mmol, 1.00 equiv) was dissolved in DMA (1 mL) and MSA (17 μL, 0.1 mmol, 1.1 equiv) was added. The solution was stirred at 50°C for 2 hours. A solution of Fmoc-DhI(OTBS)2-OH (75 mg, 0.12 mmol, 1.3 equiv), COMU (52 mg, 0.12 mmol, 1.3 equiv) and DIPEA (43 μL, 0.24 mmol, 2.6 equiv) in DMA (0.5 mL) was stirred at 0°C for 30 minutes. The silylated monocyclic pentapeptide was added to the preactivated amino acid and stirred for 1 hour. At 0 ℃, H-Asp (OAll) -Hyp-OFm * HCl (70mg, 0.14mmol, 1.5 equivalents) and HATU (53mg, 0.14mmol, 1.5 equivalents) are added into reaction mixture. Add DIPEA (49 μ L, 0.28mmol, 3.0 equivalents), and reaction mixture is stirred 2 hours, then diluted with EtOAc (50mL) and with 10% citric acid (2x10mL) and saturated NaHCO (2x10mL) washing. Organic phase is washed with salt water (2x20mL), through NaSO dry and evaporated under reduced pressure. Crude product 5 is used for next step without the need for any further purification.

[0317] HRMS(ESI):C 83 H 107 N9O 16 SSi2(M+H) + The calculated m / z value is 1574.7167, the found value is 1574.7134.

[0318] Synthesis of monocyclic C-terminal and N-terminal deprotected octapeptide 6:

[0319]

[0320] The monocyclic octapeptide 5 obtained from the above reaction without further chromatography purification step was dissolved in DMF / ACN (2mL). Then Et2NH (191 μ L, 1.85mmol, 20.0 equivalents) was added, and stirred at room temperature for 30 minutes. The solvent was removed under reduced pressure, and the precipitate was redissolved in THF (1mL). Then, a solution of TBAF in THF (1m, 0.85mL, 10eq) was added, and the reaction mixture was stirred at room temperature for 4 hours. The solvent was evaporated under vacuum, and the crude product was passed through C18 reverse phase chromatography (AcN / H2O5% to 70%) purifying, to obtain the product 6 (51mg, 64% after four steps) as a white solid.

[0321] HRMS(ESI):C42 H 59 N9O 14 S(M+H) + The calculated m / z value is 946.3975, the found value is 946.3972.

[0322] Synthesis of allyl ester protected deoxyamanitin precursor (7):

[0323]

[0324] The monocyclic octapeptide 6 (10.0 mg, 10.6 μmol, 1.00 eq.) was dissolved in DMF (5 mL). DIPEA (3.68 μL, 21.4 μmol, 2.00 eq.) and HATU (8.04 mg, 21.1 μmol, 2.00 eq.) were then added at 0°C. The reaction mixture was stirred for 5 hours, during which time it was allowed to warm to room temperature. The crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10 μm, 50 x 150 mm column, gradient A) to yield the allyl-protected deoxyamanitin precursor 7 (6 mg, 68%) as a white powder.

[0325] HRMS(ESI):C 42 H 57 N9O 13 S(M+H) + The calculated m / z value is 928.3869, the found value is 928.3885.

[0326] Synthesis of (S)-deoxyamanitin (8):

[0327]

[0328] Deoxyamanitin precursor 7 (3.0 mg, 3.2 μmol, 1.0 eq.) was dissolved in THF (0.3 mL). Then, morpholine (5.58 μL, 64.6 μmol, 20.0 eq.) and Pd(PPh 3 ) 4 (747 μg, 0.64 μmol, 0.2 eq.) were added. The reaction mixture was stirred vigorously for 3 hours. The crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10 μm, 50×150 mm column, gradient B) to give (S)-deoxyamanitin (8) (1.5 mg, 50%) as a white powder.

[0329] HRMS(ESI):C 39 H 53 N9O 13 S(M+H) +The calculated m / z value is 888.3556, the found value is 888.3529.

[0330] Preparative HPLC purification gradient:

[0331] Gradient A:

[0332] 0-30min 10%-30%B, 30-35min 30-100%B; 40-45min 100%B; 45-50min, 100-10%B

[0333] 0.1% formic acid in water (solvent A) and 0.1% formic acid in ACN (solvent B).

[0334] Gradient B:

[0335] 0-30min 15%-40%B, 30-35min 40-100%B; 35-40min 100%B, 40-15min 15%B

[0336] 0.1% formic acid in water (solvent A) and 0.1% formic acid in ACN (solvent B).

Claims

1. Method for preparing the compound of formula (I) The method comprises the following steps: The compound of formula (IIa) reacts with peptide bond-forming reagents, And when R PGP and / or R PGOH and / or R AL and / or R AMIN When present, the compound obtained from the above reaction is reacted with R PGP and / or R PGOH and / or R AL and / or R AMIN The deprotection reaction in ·X 1 、X 2 、X 3 and X 4 , and Y 1 、Y 2 、Y 3 and Y 4 is H, or Y 1 、Y 2 、Y 3 and Y 4 One, two, three or all of are OH or NH2 and all other Y are H, and the corresponding X is NHR AMIN OR PGP and all other X are H, where R PGP is a protecting group for the phenolic OH group, or R AMIN is a protecting group for a phenol amino group, or Y 1 、Y 2 、Y 3 and Y 4 One, two, three or all of and the corresponding X are selected from F, Cl, Br, I, CN, NO2, acyl, N3 or alkyne, and all other X and Y are H, Z and W are H, or Z is OH and W is OR PGOH , where R PGOH is a protecting group for hydroxyl, ·R AL is a protected carboxyl group or a 1-5AA peptide; V is OH or 1-5AA peptide; Q is S.

2. The method according to claim 1, wherein the peptide bond forming reagent is a coupling reagent selected from carbodiimide, imidazolinium reagent, phosphonium salt, organophosphorus reagent, uronium salt, pyridinium reagent and phosphonic acid.

3. The method according to claim 1 or 2, wherein the peptide bond forming reagent is HATU, COMU, HBTU, TBTU, TOMBU, COMBU or HCTU.

4. The method according to any one of claims 1 to 2, wherein R PGP It is a non-acid or base labile phenol OH protecting group.

5. The method according to claim 4, wherein R PGP It is a phenolic OH protecting group that can be cleaved under reducing conditions.

6. The method according to any one of claims 1 to 2, wherein R AMIN It is a non-acid or base labile phenol amino protecting group.

7. The method according to claim 6, wherein R AMIN It is a phenol amino protecting group that can be cleaved under reducing conditions.

8. The method according to any one of claims 1 to 2, wherein R PGOH It is a hydroxyl protecting group that can be cleaved by fluoride ion.

9. The method according to any one of claims 1 to 2, wherein R AL It is an O-allyl or O-methyl ester or a 1-5AA peptide.

10. The method according to claim 9, wherein R AL It is O-allyl or O-methyl ester.

11. The method according to claim 10, wherein R AL It is O-allyl.

12. The method of any one of claims 1-2, wherein V is OH.

13. The method according to any one of claims 1 to 2, wherein X and Y are H, or Y 1 、Y 2 、Y 3 and Y 4 One is OH, and the corresponding X is OR PGP , and all other X and Y are H.

14. The method according to any one of claims 1 to 2, further comprising the following steps: The compound of formula (III) Reaction with a compound of formula (IV) in ·X 1 、X 2 、X 3 、X 4 , W, Q and R AL has the same meaning as defined in claim 1; ·R COOY is a carboxyl protecting group; Wherein (III) and (IV) are reacted with a peptide bond forming reagent to obtain a compound characterized as (IIa).

15. The method according to any one of claims 14, wherein the carboxyl protecting group is fluorenylmethyl or benzyl. The method according to claim 15 , wherein the carboxyl protecting group is a fluorenylmethyl group.

17. The method of claim 14, wherein (III) and (IV) are reacted with HATU, COMU, HBTU, TBTU, TOMBU, COMBU or HCTU.

18. The method according to any one of claims 1 to 2, further comprising the following steps: The compound of formula (V) Reaction with a compound of formula (VI) in ·X 1 、X 2 、X 3 、X 4 , Q and W have the same meanings as defined in claim 1; ·R NHB is an amino protecting group, wherein the amino group of (V) is preactivated, and the preactivated (V) and (VI) are reacted with a peptide bond forming reagent, to obtain compound (III). The method according to claim 18 , wherein the amino protecting group is an amino protecting group cleavable under basic conditions.

20. The method of claim 19, wherein the amino protecting group is Fmoc.

21. The method according to claim 18, wherein the amino group of (V) is preactivated with MSA.

22. The method of claim 18, wherein preactivated (V) and (VI) are reacted with HATU, COMU, HBTU, TBTU, TOMBU, COMBU or HCTU.

23. The method according to claim 22, wherein preactivated (V) and (VI) are reacted with COMU.

24. The method according to claim 1, wherein the compound of formula (I) is a compound of formula (Idesox), in Y 1 、Y 2 、Y 3 、Y 4 , Z and V have the same meanings as defined in claim 1.

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