Preparation method, intermediates and application of cyclic peptide toxin α-Amanitin and / or Amaninamide

Abstract

The present invention provides a preparation method, intermediates and application of cyclic peptide toxins α-Amanitin and / or Amaninamide, and belongs to the field of organic synthesis. The present invention provides a total synthesis method of cyclic peptide toxin compounds α-Amanitin and Amaninamide, the raw materials and reagents used are easy to purchase through commercial channels, the intermediates are stable, the reaction conditions are mild, the operation process is simple, the separation and purification process has good operability, high yield, and has important reference and practical value. It can realize the gram-scale preparation of α-Amanitin and Amaninamide, has good industrial prospects, and the preparation method of the present invention has significant application value in the field of cyclic peptide toxin synthesis.

Description

[0001] This application is a divisional application of the Chinese patent application filed with the China Patent Office on March 7, 2024, with application number CN202410257131.1 and invention name “Preparation method, intermediates and applications of cyclic peptide toxin α-Amanitin and / or Amaninamide”, all contents of which are incorporated by reference in this application. Technical Field

[0002] The present invention relates to the technical field of organic synthesis, and in particular to a preparation method, intermediates and application of cyclic peptide toxin alpha-Amanitin and / or Amaninamide. Background Art

[0003] Amanitin is a cyclic peptide substance isolated from highly poisonous mushrooms and belongs to the amanitin toxin class. Amanitin is a bicyclic octapeptide structure, including nine different peptides, namely α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, amanullinic acid and proamanullin. α-Amanitin is the most abundant bicyclic octapeptide in the amanitin series, and its antibody-drug conjugates (ADCs) can be used for cancer treatment. Therefore, the study of its chemical properties and applications is very popular.

[0004] α-Amanitin is a highly effective inhibitor of eukaryotic RNA polymerase II, which can block mRNA transcription and protein synthesis, while killing fast-growing and quiescent cells, causing them to rapidly degrade and eventually apoptosis. At present, α-Amanitin antibody-drug conjugates have cured pancreatic cancer in xenotransplanted mice and are moving towards human trials. Medical researchers need sufficient α-Amanitin to prepare their ADCs drugs for the treatment of cancer. α-Amanitin is mainly isolated and extracted from natural poisonous mushrooms, and is also synthesized by biological fermentation. Its natural product extraction and fermentation yield is low, and its market price is extremely expensive (about 12,000 / mg), which seriously restricts the research on its antibody-drug conjugates for cancer treatment. Chemical synthesis of α-Amanitin is an effective method that is expected to achieve its large-scale production. Although Heinrich Wieland et al. have begun to characterize the structure and properties of α-Amanitin and its derivatives since the 1940s, chemists have also been trying to chemically synthesize α-Amanitin.

[0005] So far, there is still a lack of efficient and large-scale methods for synthesizing α-Amanitin and its derivatives. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a preparation method, intermediates and applications of cyclic peptide toxins α-Amanitin and / or Amaninamide. The preparation method provided by the present invention is a total synthesis method, with readily available raw materials, stable intermediates, mild reaction conditions, simple operation process, good operability of separation and purification process, and high yield.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing cyclic peptide toxin compounds α-Amanitin and / or Amaninamide, wherein the α-Amanitin has a structure shown in Formula 1a, and the Amaninamide has a structure shown in Formula 1b:

[0009]

[0010] The following steps are involved:

[0011] Step 1: subjecting compound 2 to acetylation and esterification to obtain compound 3;

[0012] Step 2: subjecting the compound 3 to condensation reaction and deprotection reaction to obtain compound 4;

[0013] Step 3: subjecting the compound 4 to condensation reaction and deprotection reaction to obtain compound 5;

[0014] Step 4: subjecting the compound 5 to condensation reaction and deprotection reaction to obtain compound 6;

[0015] Step 5: subjecting the compound 6 to condensation reaction and deprotection reaction to obtain compound 7;

[0016] Step 6: subjecting the compound 7 to condensation reaction and deprotection reaction to obtain compound 8;

[0017] Step 7: subjecting the compound 8 to condensation reaction and deprotection reaction to obtain compound 9;

[0018] Step 8: subjecting the compound 9 to condensation reaction and deprotection reaction to obtain compound 10;

[0019] Step 9: The compound 10 is subjected to carbon-sulfur bond cyclization in an iodine-mediated manner to obtain compound 11;

[0020] Step 10: subjecting the compound 11 to a deprotection reaction to obtain a compound 12;

[0021] Step 11: subjecting the compound 12 to a deprotection reaction and a condensation reaction to obtain a compound 13;

[0022] Step 12: subjecting the compound 13 to an oxidation reaction to obtain a compound 14;

[0023] Step 13: subjecting the compound 14 to a deprotection reaction to obtain the cyclic peptide toxin compounds α-Amanitin and Amaninamide;

[0024] The structures of compounds 2 to 14 are as follows:

[0025]

[0026] In the compounds 9 to 14, R is -H or -OBn.

[0027] Preferably, the preparation method comprises the following steps:

[0028] Step 1: mixing the compound 2, pyridine, acetyl chloride and an organic solvent for acetylation reaction, and mixing the obtained acetylated product with N,N-dimethylpyridine and di-tert-butyl dicarbonate for esterification reaction to obtain the compound 3;

[0029] Step 2: mixing the compound 3, N-Fmoc-asparagine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent for condensation reaction and deprotection reaction to obtain the compound 4;

[0030] Step 3: mixing the compound 4, N-Fmoc-cysteine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain the compound 5;

[0031] Step 4: mixing the compound 5, N-Fmoc-glycine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain the compound 6;

[0032] Step 5: mixing the compound 6, N-Fmoc-isoleucine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain the compound 7;

[0033] Step 6: mixing the compound 7, N-Fmoc-glycine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain the compound 8;

[0034] Step 7: Mixing the compound 8, the compound 15, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent for condensation reaction and deprotection reaction to obtain the compound 9; the compound 15 is N-Fmoc-6-benzyloxytryptophan or N-Fmoc-tryptophan;

[0035] Step 8: Mixing the compound 9, (2S, 3R, 4R)-4,5-dihydroxyisoleucine derivative, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain compound 10;

[0036] Step 9: Under a protective atmosphere, the compound 10 and an organic solution of iodine are mixed to perform carbon-sulfur bond cyclization to obtain the compound 11;

[0037] Step 10: Mix the compound 11 and an organic solvent to carry out a deprotection reaction to obtain the compound 12;

[0038] Step 11: mixing the compound 12, diisopropylethylamine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and an organic solvent to carry out a deprotection reaction and a condensation reaction to obtain a compound 13;

[0039] Step 12: Mix the compound 13, m-chloroperbenzoic acid and an organic solvent to carry out an oxidation reaction to obtain the compound 14;

[0040] Step 13:

[0041] When R is -OBn, the compound 14, ethanethiol and boron trifluoride ether solution are reacted to obtain a crude product, and the crude product is mixed with an alcohol solution of ammonia to perform a deprotection reaction to obtain the α-Amanitin;

[0042] When R is -H, the compound 14 and an alcoholic solution of ammonia are mixed to carry out a deprotection reaction to obtain the Amaninamide.

[0043] Preferably, in step 1, the molar ratio of compound 2, acetyl chloride and pyridine is 1:1-3:1-5; the molar ratio of compound 2, N,N-dimethylpyridine and di-tert-butyl dicarbonate is 1:0.1-1:1-5.

[0044] Preferably, in step 2, the molar ratio of compound 3, N-Fmoc-asparagine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1:1.1;

[0045] In the step 3, the molar ratio of compound 4, N-Fmoc-cysteine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1:1.1;

[0046] In step 4, the molar ratio of compound 5, N-Fmoc-glycine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1:1.1;

[0047] In the step 5, the molar ratio of compound 6, N-Fmoc-isoleucine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1:1.1;

[0048] In step 6, the molar ratio of compound 7, N-Fmoc-glycine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1:1.1;

[0049] In step 7, the molar ratio of compound 8, compound 15, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1:1.1;

[0050] In step 8, the molar ratio of compound 9, (2S, 3R, 4R)-4,5-dihydroxyisoleucine derivative, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1:1.1.

[0051] Preferably, in step 9, the molar ratio of compound 10 to iodine in the organic solution of iodine is 1:4, and the concentration of the organic solution of iodine is 2 mg / mL.

[0052] Preferably, in step 10, the organic solvent is a diethylamine dichloromethane solution, the mass fraction of diethylamine in the diethylamine dichloromethane solution is 50%, and the concentration of compound 11 in the diethylamine dichloromethane solution is 0.1 g / mL.

[0053] Preferably, in step 11, the molar ratio of compound 12, diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:2.2:2.

[0054] Preferably, the concentration of the alcoholic ammonia solution in step 13 is 7 mol / L.

[0055] The present invention also provides an intermediate obtained by the preparation method described in the above technical solution, the structure of which is as follows:

[0056]

[0057] In the compounds 9 to 14, R is -H or -OBn.

[0058] The present invention also provides the use of the intermediate described in the above technical solution in the preparation of cyclopeptide toxin compounds.

[0059] The present invention provides a method for preparing cyclic peptide toxin compounds α-Amanitin and Amaninamide. Compared with the prior art, the present invention has the following beneficial effects:

[0060] The invention provides a total synthesis method of cyclic peptide toxin compounds α-Amanitin and Amaninamide. The raw materials and reagents used are easy to purchase through commercial channels, and the raw material cost is low. The intermediates obtained in the synthesis method are stable, the reaction conditions are mild, the operation process is simple, the separation and purification process has good operability and high yield, and has important reference and practical value. The gram-scale preparation of α-Amanitin and Amaninamide can be achieved. The preparation method of the invention has significant application value in the field of cyclic peptide toxin synthesis.

[0061] The present invention also provides a series of intermediates, which are helpful to promote the synthesis of cyclopeptide toxins and other polypeptide compounds and have significant application value in the field of polypeptide synthesis. DETAILED DESCRIPTION

[0062] The present invention provides a method for preparing cyclic peptide toxin compounds α-Amanitin and / or Amaninamide, wherein the α-Amanitin has a structure shown in Formula 1a, and the Amaninamide has a structure shown in Formula 1b:

[0063]

[0064] The following steps are involved:

[0065] Step 1: subjecting compound 2 to acetylation and esterification to obtain compound 3;

[0066] Step 2: subjecting the compound 3 to condensation reaction and deprotection reaction to obtain compound 4;

[0067] Step 3: subjecting the compound 4 to condensation reaction and deprotection reaction to obtain compound 5;

[0068] Step 4: subjecting the compound 5 to condensation reaction and deprotection reaction to obtain compound 6;

[0069] Step 5: subjecting the compound 6 to condensation reaction and deprotection reaction to obtain compound 7;

[0070] Step 6: subjecting the compound 7 to condensation reaction and deprotection reaction to obtain compound 8;

[0071] Step 7: subjecting the compound 8 to condensation reaction and deprotection reaction to obtain compound 9;

[0072] Step 8: subjecting the compound 9 to condensation reaction and deprotection reaction to obtain compound 10;

[0073] Step 9: The compound 10 is subjected to carbon-sulfur bond cyclization in an iodine-mediated manner to obtain compound 11;

[0074] Step 10: subjecting the compound 11 to a deprotection reaction to obtain a compound 12;

[0075] Step 11: subjecting the compound 12 to a deprotection reaction and a condensation reaction to obtain a compound 13;

[0076] Step 12: subjecting the compound 13 to an oxidation reaction to obtain a compound 14;

[0077] Step 13: subjecting the compound 14 to a deprotection reaction to obtain the cyclic peptide toxin compounds α-Amanitin and Amaninamide;

[0078] The structures of compounds 2 to 14 are as described above.

[0079] In the present invention, unless otherwise specified, the raw materials used are commercially available products in the art or are prepared by preparation methods well known to those skilled in the art.

[0080] In the present invention, the principle of the preparation method is shown in the following formula:

[0081]

[0082] In the present invention, Fmoc in the compound is fluorenylmethoxycarbonyl, Ac is acetyl, Trt is trityl, and Bn is benzyl.

[0083] In the present invention, the preparation method preferably comprises the following steps:

[0084] Step 1: mixing the compound 2, pyridine, acetyl chloride and an organic solvent for acetylation reaction, and mixing the obtained acetylated product, N,N-dimethylpyridine and di-tert-butyl dicarbonate for esterification reaction to obtain the compound 3;

[0085] Step 2: mixing the compound 3, N-Fmoc-asparagine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent for condensation reaction and deprotection reaction to obtain the compound 4;

[0086] Step 3: mixing the compound 4, N-Fmoc-cysteine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain the compound 5;

[0087] Step 4: mixing the compound 5, N-Fmoc-glycine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain the compound 6;

[0088] Step 5: mixing the compound 6, N-Fmoc-isoleucine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain the compound 7;

[0089] Step 6: mixing the compound 7, N-Fmoc-glycine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain the compound 8;

[0090] Step 7: Mixing the compound 8, the compound 15, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent for condensation reaction and deprotection reaction to obtain the compound 9; the compound 15 is N-Fmoc-6-benzyloxytryptophan or N-Fmoc-tryptophan;

[0091] Step 8: Mixing the compound 9, (2S, 3R, 4R)-4,5-dihydroxyisoleucine derivative, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain compound 10;

[0092] Step 9: Under a protective atmosphere, the compound 10 and an organic solution of iodine are mixed to perform carbon-sulfur bond cyclization to obtain the compound 11;

[0093] Step 10: Mix the compound 11 and an organic solvent to carry out a deprotection reaction to obtain the compound 12;

[0094] Step 11: mixing the compound 12, diisopropylethylamine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and an organic solvent to carry out a deprotection reaction and a condensation reaction to obtain a compound 13;

[0095] Step 12: Mix the compound 13, m-chloroperbenzoic acid and an organic solvent to carry out an oxidation reaction to obtain the compound 14;

[0096] Step 13:

[0097] When R is -OBn, the compound 14, ethanethiol and boron trifluoride ether solution are reacted to obtain a crude product, and the crude product is mixed with an alcohol solution of ammonia to perform a deprotection reaction to obtain the α-Amanitin;

[0098] When R is -H, the compound 14 and an alcoholic solution of ammonia are mixed to carry out a deprotection reaction to obtain the Amaninamide.

[0099] In the present invention, unless otherwise specified, the raw materials used are all commercially available products in the art.

[0100] The present invention mixes the compound 2, pyridine, acetyl chloride and an organic solvent for acetylation reaction, and mixes the obtained acetylation product, N,N-dimethylpyridine and di-tert-butyl dicarbonate for esterification reaction to obtain the compound 3.

[0101] In the present invention, the molar ratio of compound 2, acetyl chloride and pyridine in step 1 is preferably 1:1-3:1-5, more preferably 1:1.5:1.5.

[0102] In the present invention, the organic solvent is preferably dichloromethane, more preferably dry dichloromethane.

[0103] In the present invention, the temperature of the acetylation reaction is preferably 0° C., and the time is preferably 2 h.

[0104] In the present invention, the compound 2 is preferably dissolved in dry dichloromethane, and the pyridine and acetyl chloride are added under ice bath conditions.

[0105] After the acetylation reaction is completed, the present invention preferably uses a saturated ammonium chloride solution to quench the reaction, adjusts the pH of the resulting reaction solution to 2 with dilute hydrochloric acid, extracts three times with dichloromethane, combines the organic phases, dries over anhydrous sodium sulfate, and removes the solvent under reduced pressure to obtain a crude product, which is recorded as the acetylation reaction.

[0106] In the present invention, the molar ratio of the compound 2, N,N-dimethylpyridine and di-tert-butyl dicarbonate is preferably 1:0.1 to 1:1 to 5, and more preferably 1:0.3:2.

[0107] In the present invention, the temperature of the esterification reaction is preferably room temperature, and the time is preferably 0.5 h.

[0108] In the present invention, the crude product is preferably dissolved in tert-butyl alcohol, and N,N-dimethylpyridine and di-tert-butyl dicarbonate are added to carry out the esterification reaction.

[0109] After the esterification reaction is completed, the present invention preferably removes tert-butanol from the obtained product under reduced pressure to obtain a crude product, and dissolves the crude product in a dichloromethane / diethylamine solution (the mass fraction of dichloromethane in the dichloromethane / diethylamine solution is preferably 50%), stirs at room temperature for 1 hour, removes the solvent under reduced pressure, and separates by column chromatography to obtain the compound 3.

[0110] After obtaining the compound 3, the present invention mixes the compound 3, N-Fmoc-asparagine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain the compound 4.

[0111] In the present invention, the molar ratio of the compound 3, N-Fmoc-asparagine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is preferably 1:1.1:1.1:1.1;

[0112] In the present invention, the organic solvent is preferably dry dichloromethane.

[0113] In the present invention, the temperature of the condensation reaction and the deprotection reaction is preferably room temperature, and the time is preferably 12 hours.

[0114] The present invention preferably dissolves the compound 3 in dry dichloromethane, adds N-Fmoc-asparagine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride under ice bath conditions, reacts at room temperature for 12 hours, washes the organic phase with a saturated sodium carbonate solution, and dries over anhydrous sodium sulfate. The solvent is removed under reduced pressure to obtain a crude product, and the crude product is dissolved in a dichloromethane / diethylamine solution (the mass fraction of dichloromethane in the dichloromethane / diethylamine solution is preferably 50%), stirred at room temperature for 1 hour, and the solvent is removed under reduced pressure. The product is separated by column chromatography to obtain the compound 4.

[0115] After obtaining compound 4, the present invention mixes compound 4, N-Fmoc-cysteine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain compound 5.

[0116] In the present invention, the molar ratio of compound 4, N-Fmoc-cysteine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is preferably 1:1.1:1.1:1.1;

[0117] In the present invention, the organic solvent is preferably dry dichloromethane.

[0118] In the present invention, the temperature of the condensation reaction and the deprotection reaction is preferably room temperature, and the time is preferably 12 hours.

[0119] The present invention preferably dissolves the compound 4 in dry dichloromethane, adds N-Fmoc-cysteine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride under ice bath conditions, reacts at room temperature for 12 hours, washes the organic phase with a saturated sodium carbonate solution, and dries over anhydrous sodium sulfate. The solvent is removed under reduced pressure to obtain a crude product, and the crude product is dissolved in a dichloromethane / diethylamine solution (the mass fraction of dichloromethane in the dichloromethane / diethylamine solution is preferably 50%), stirred at room temperature for 1 hour, and the solvent is removed under reduced pressure. The product is separated by column chromatography to obtain the compound 5.

[0120] After obtaining compound 5, the present invention mixes the compound 5, N-Fmoc-glycine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain compound 6.

[0121] In the present invention, the molar ratio of the compound 5, N-Fmoc-glycine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is preferably 1:1.1:1.1:1.1.

[0122] In the present invention, the organic solvent is preferably dry dichloromethane.

[0123] In the present invention, the temperature of the condensation reaction and the deprotection reaction is preferably room temperature, and the time is preferably 12 hours.

[0124] The present invention preferably dissolves the compound 5 in dry dichloromethane, adds N-Fmoc-glycine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride under ice bath conditions, reacts at room temperature for 12 hours, washes the organic phase with a saturated sodium carbonate solution, and dries over anhydrous sodium sulfate. The solvent is removed under reduced pressure to obtain a crude product, and the crude product is dissolved in a dichloromethane / diethylamine solution (the mass fraction of dichloromethane in the dichloromethane / diethylamine solution is preferably 50%), stirred at room temperature for 1 hour, and the solvent is removed under reduced pressure. The product is separated by column chromatography to obtain the compound 6.

[0125] After obtaining compound 6, the present invention mixes the compound 6, N-Fmoc-isoleucine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain compound 7.

[0126] In the present invention, the molar ratio of the compound 6, N-Fmoc-isoleucine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is preferably 1:1.1:1.1:1.1.

[0127] In the present invention, the organic solvent is preferably dry dichloromethane.

[0128] In the present invention, the temperature of the condensation reaction and the deprotection reaction is preferably room temperature, and the time is preferably 12 hours.

[0129] The present invention preferably dissolves the compound 6 in dry dichloromethane, adds N-Fmoc-isoleucine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride under ice bath conditions, reacts at room temperature for 12 hours, washes the organic phase with a saturated sodium carbonate solution, and dries over anhydrous sodium sulfate. The solvent is removed under reduced pressure to obtain a crude product, and the crude product is dissolved in a dichloromethane / diethylamine solution (the mass fraction of dichloromethane in the dichloromethane / diethylamine solution is preferably 50%), stirred at room temperature for 1 hour, and the solvent is removed under reduced pressure. The product is separated by column chromatography to obtain compound 7.

[0130] After obtaining compound 7, the present invention mixes the compound 7, N-Fmoc-glycine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain compound 8.

[0131] In the present invention, the molar ratio of compound 7, N-Fmoc-glycine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is preferably 1:1.1:1.1:1.1.

[0132] In the present invention, the organic solvent is preferably dry dichloromethane.

[0133] In the present invention, the temperature of the condensation reaction and the deprotection reaction is preferably room temperature, and the time is preferably 12 hours.

[0134] The present invention preferably dissolves the compound 7 in dry dichloromethane, adds N-Fmoc-glycine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride under ice bath conditions, reacts at room temperature for 12 hours, washes the organic phase with a saturated sodium carbonate solution, and dries over anhydrous sodium sulfate. The solvent is removed under reduced pressure to obtain a crude product, and the crude product is dissolved in a dichloromethane / diethylamine solution (the mass fraction of dichloromethane in the dichloromethane / diethylamine solution is preferably 50%), stirred at room temperature for 1 hour, and the solvent is removed under reduced pressure. The product is separated by column chromatography to obtain compound 8.

[0135] After obtaining compound 8, the present invention mixes the compound 8, compound 15, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent for condensation reaction and deprotection reaction to obtain the compound 9; the compound 15 is N-Fmoc-6-benzyloxytryptophan or N-Fmoc-tryptophan.

[0136] In the present invention, the molar ratio of compound 8, compound 15, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is preferably 1:1.1:1.1:1.1.

[0137] In the present invention, the organic solvent is preferably dry dichloromethane.

[0138] In the present invention, the temperature of the condensation reaction and the deprotection reaction is preferably room temperature, and the time is preferably 12 hours.

[0139] In the present invention, the structure of the compound 15 is shown below:

[0140]

[0141] Wherein R is -OBn or -H. When R is -OBn, the compound 15 is N-Fmoc-6-benzyloxytryptophan. When R is -H, the compound 15 is N-Fmoc-tryptophan.

[0142] The present invention preferably dissolves the compound 8 in dry dichloromethane, adds N-Fmoc-6-benzyloxytryptophan, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride under ice bath conditions, reacts at room temperature for 12 hours, washes the organic phase with a saturated sodium carbonate solution, and dries over anhydrous sodium sulfate. The solvent is removed under reduced pressure to obtain a crude product, and the crude product is dissolved in a dichloromethane / diethylamine solution (the mass fraction of dichloromethane in the dichloromethane / diethylamine solution is preferably 50%), stirred at room temperature for 1 hour, and the solvent is removed under reduced pressure. The product is separated by column chromatography to obtain the compound 9a.

[0143] The present invention preferably dissolves the compound 8 in dry dichloromethane, adds N-Fmoc-tryptophan, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride under ice bath conditions, reacts at room temperature for 12 hours, washes the organic phase with a saturated sodium carbonate solution, and dries over anhydrous sodium sulfate. The solvent is removed under reduced pressure to obtain a crude product, and the crude product is dissolved in a dichloromethane / diethylamine solution (the mass fraction of dichloromethane in the dichloromethane / diethylamine solution is preferably 50%), stirred at room temperature for 1 hour, and the solvent is removed under reduced pressure. The product is separated by column chromatography to obtain the compound 9b.

[0144] After obtaining compound 9, the present invention mixes the compound 9, (2S, 3R, 4R)-4,5-dihydroxyisoleucine derivative, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and an organic solvent to carry out condensation reaction and deprotection reaction to obtain compound 10.

[0145] In the present invention, the molar ratio of the compound 9, (2S,3R,4R)-4,5-dihydroxyisoleucine derivative, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is preferably 1:1.1:1.1:1.1.

[0146] In the present invention, the organic solvent is preferably dry dichloromethane.

[0147] In the present invention, the temperature of the condensation reaction and the deprotection reaction is preferably room temperature, and the time is preferably 12 hours.

[0148] In the present invention, the structure of the (2S, 3R, 4R)-4,5-dihydroxyisoleucine derivative is shown in Formula 16 (Compound 16):

[0149]

[0150] The present invention preferably dissolves compound 9 in dry dichloromethane, adds (2S, 3R, 4R)-4,5-dihydroxyisoleucine derivative, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride under ice bath conditions, reacts at room temperature for 12 hours, washes the organic phase with saturated sodium carbonate solution, and dries over anhydrous sodium sulfate. The solvent is removed under reduced pressure to obtain a crude product, and the crude product is separated by column chromatography to obtain compound 10.

[0151] After obtaining compound 10, the present invention mixes compound 10 and an organic solution of iodine under a protective atmosphere to perform carbon-sulfur bond cyclization to obtain compound 11.

[0152] In the present invention, the protective atmosphere is preferably nitrogen.

[0153] In the present invention, the molar ratio of the compound 10 to the iodine element in the iodine organic solution is preferably 1:4, and the concentration of the iodine organic solution is preferably 2 mg / mL.

[0154] In the present invention, the solvent of the organic solution of iodine is preferably N,N-dimethylformamide.

[0155] In the present invention, the temperature for the carbon-sulfur ring closure is preferably room temperature, and the time is preferably 1 hour.

[0156] In the present invention, the compound 10 is preferably added to an iodine-containing N,N-dimethylformamide solution under a nitrogen environment, reacted at room temperature for 1 hour, and the N,N-dimethylformamide is removed and recovered under reduced pressure to obtain a crude product, and the crude product is separated by column chromatography to obtain compound 11.

[0157] After obtaining compound 11, the present invention mixes the compound 11 with an organic solvent to carry out a deprotection reaction to obtain compound 12.

[0158] In the present invention, the organic solvent is preferably a diethylamine dichloromethane solution, the mass fraction of diethylamine in the diethylamine dichloromethane solution is preferably 50%, and the concentration of the compound 11 in the diethylamine dichloromethane solution is preferably 0.1 g / mL.

[0159] In the present invention, the temperature of the deprotection reaction is preferably room temperature, and the time is preferably 1 hour.

[0160] In the present invention, the compound 11 is preferably dissolved in a dichloromethane / diethylamine solution, stirred at room temperature for 1 hour, the solvent is removed under reduced pressure, and the obtained crude product is separated by column chromatography to obtain the compound 12.

[0161] After obtaining compound 12, the present invention mixes the compound 12, diisopropylethylamine, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and an organic solvent to carry out deprotection reaction and condensation reaction to obtain compound 13.

[0162] In the present invention, the organic solvent is preferably N,N-dimethylformamide.

[0163] In the present invention, the molar ratio of the compound 12, diisopropylethylamine, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is preferably 1:2.2:2.

[0164] In the present invention, the temperature of the deprotection reaction and the condensation reaction is preferably room temperature, and the time is preferably 12 hours.

[0165] The present invention preferably dissolves the compound 12 in trifluoroacetic acid, the concentration of compound 12 in the obtained system is 0.1 g / mL, stirs at room temperature for 1 hour, removes trifluoroacetic acid under reduced pressure to obtain a crude product, dissolves the crude product in dry N,N-dimethylformamide, adds diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, reacts at room temperature for 12 hours, removes and recovers N,N-dimethylformamide under reduced pressure to obtain a crude product, and separates by column chromatography to obtain compound 13.

[0166] After obtaining compound 13, the present invention mixes compound 13, meta-chloroperbenzoic acid and an organic solvent to carry out an oxidation reaction to obtain compound 14.

[0167] In the present invention, the molar ratio of compound 13 to meta-chloroperbenzoic acid is preferably 1:1.

[0168] In the present invention, the organic solvent is preferably a mixed solution of isopropanol and ethanol, and the volume ratio of isopropanol to ethanol in the mixed solution of isopropanol and ethanol is preferably 2:1.

[0169] In the present invention, the temperature of the oxidation reaction is preferably room temperature, and the time is preferably 0.5 h.

[0170] The present invention preferably dissolves the compound 13 in a mixed solution of isopropanol and ethanol, wherein the concentration of the compound 13 in the obtained system is 0.2 g / mL, adds m-chloroperbenzoic acid, reacts at room temperature for 0.5 h, removes the solvent under reduced pressure to obtain a crude product, and separates the crude product by column chromatography to obtain the compound 14.

[0171] After obtaining compound 14, when R is -OBn (ie compound 14a), the present invention reacts the compound 14, ethanethiol and boron trifluoride ether solution to obtain a crude product, and mixes the crude product with an alcohol solution of ammonia to perform a deprotection reaction to obtain the α-Amanitin.

[0172] The present invention preferably dissolves the compound 14a in ethyl mercaptan, adds a boron trifluoride ether solution, reacts at room temperature for 1 hour, removes ethyl mercaptan under reduced pressure to obtain a crude product, dissolves the crude product in ammonia methanol solution, reacts at room temperature for 2 hours, removes the solvent under reduced pressure, and separates the obtained crude product by column chromatography to obtain compound 1a.

[0173] In the present invention, the molar ratio of the compound 14a to boron trifluoride in the boron trifluoride ether solution is preferably 1:20; the concentration of the compound 14a in ethanethiol is preferably 8 mg / mL; and the concentration of the crude product in the methanol solution of ammonia is preferably 0.01 g / mL.

[0174] When R is -H (ie, compound 14b), the present invention mixes the compound 14 with an alcohol solution of ammonia to carry out a deprotection reaction to obtain the Amaninamide.

[0175] In the present invention, the compound 14b is preferably dissolved in an alcohol solution of ammonia, reacted at room temperature for 2 hours, and the solvent is removed under reduced pressure to obtain a crude product. The crude product is separated by column chromatography to obtain compound 1b.

[0176] In the present invention, the concentration of the compound 14b in the alcoholic ammonia solution is preferably 0.01 g / mL.

[0177] In the present invention, the concentration of the alcoholic ammonia solution is preferably 7 mol / L; the alcoholic ammonia solution is preferably a methanolic ammonia solution.

[0178] The present invention also provides a series of intermediates, the structures of which are as follows:

[0179]

[0180] In the compounds 9 to 14, R is -H or -OBn.

[0181] The present invention also provides a method for preparing the intermediate described in the above technical scheme. The method for preparing the intermediate is consistent with the method for preparing compounds 4 to 14 in the above scheme, and will not be repeated here.

[0182] The present invention also provides the use of the intermediate described in the above technical solution in the preparation of cyclopeptide toxin compounds.

[0183] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0184] Example 1: Synthesis of compound 3, the principle is as follows:

[0185]

[0186] Take a 2500mL round-bottom flask and put in a magnetic stirrer. Add compound 2 (N-Fmoc-4-hydroxyproline) (35.31g, 100mmol) to the 2500mL round-bottom flask, add 200mL dry dichloromethane and stir to dissolve, and stir in an ice-water bath for 5min. Add pyridine (11.86g, 150mmol) with a syringe and stir for 5min. Slowly add acetyl chloride (11.70g, 150mmol) dropwise with a syringe, ice-water bath for 30min, and react at room temperature for 1h. After the reaction is completed, adjust the pH to 2 with 1N hydrochloric acid, extract three times with 500mL dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter, and remove dichloromethane under reduced pressure to obtain a crude product. The crude product is directly used in the next synthesis reaction without further purification. The crude product is dissolved in 400mL tert-butanol, DMAP (1.84g, 30mol%) is added, and stirred at room temperature. A solution of (Boc)2O (43.62 g, 200 mmol) dissolved in 100 mL of tert-butanol was added dropwise to the reaction solution, and the reaction was carried out at room temperature. The reaction was monitored by TLC until the raw material was consumed. After the reaction was completed, the solvent tert-butanol was removed under reduced pressure to obtain a crude product. The crude product was dissolved in 150 mL of dichloromethane, and 150 mL of diethylamine was added to remove the Fmoc protecting group. TLC monitoring was performed until the raw material was consumed and the reaction was stopped. The mixed solution of dichloromethane and diethylamine was removed under reduced pressure to obtain a crude product, which was chromatographed on a 200-300 mesh silica gel column (volume ratio of ethyl acetate / petroleum ether = 40%-50%) to obtain 15.46 g of a yellow oily liquid with a yield of 67.5%. 1 HNMR (600MHz, DMSO) δ5.07 (d, J=2.7Hz, 1H), 3.65 (t, J=7.6Hz, 1H), 3.15 (dd, J= 12.0,5.2Hz,1H),2.76(dd,J=12.0,1.5Hz,1H),2.00-1.98(m,5H),1.40(s,9H). 13 C NMR(151MHz,DMSO)δ173.1,170.1,80.2,75.0,59.1,52.2,36.4,27.6,20.9.HRMS(ESI+)calcd.forC 11 H 20 NO4 + (M+H) + :230.1387; found:230.1399.

[0187] Example 2: Synthesis of compound 4, the principle is as follows:

[0188]

[0189] Take a 2500mL round-bottom flask and put in a magnetic stirrer. Add compound N-Fmoc-asparagine (65.60g, 0.11mol) to the 2500mL round-bottom flask, add 500mL dry dichloromethane and stir to dissolve, and ice-water bath for 5min. Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (12.70g, 0.11mol) and 1-hydroxybenzotriazole (14.90g, 0.11mol) and ice-water bath for 30min. Then add compound 3 (35.70g, 0.1mol) dropwise and react in an ice-water bath for 30min. React at room temperature overnight, and monitor the reaction by TLC until the raw material is consumed. Wash the organic phase three times with saturated sodium bicarbonate, extract three times with 1000mL dichloromethane, combine the organic phases, wash with salt, dry with anhydrous sodium sulfate, filter, and remove the solvent under reduced pressure to obtain a crude product. The unpurified crude product is directly used in the next step of deprotection reaction. The crude product was dissolved in a 50 wt% diethylamine dichloromethane solution, stirred at room temperature, and the reaction was stopped after the raw material was completely consumed by TLC monitoring. The solvent and diethylamine were removed under reduced pressure, and chromatographed on a 200-300 mesh silica gel column (volume ratio of ethyl acetate / petroleum ether = 50%-60%) to obtain a white solid compound 4, 52.00 g, with a yield of 89%. 1 H NMR (600MHz, DMSO) δ9.27(s,1H),7.31-7.15(m,15H),5.25(s,1H),4.24(t,J=8.1Hz,1H),3.79(dd,J=16.9,11.3Hz,2H),3.68(dd,J=11.3,3.8Hz,1 H),2.41(dd,J=14.5,10.9Hz,1H),2.34-2.26(m,1H),2.20(d,J=14.7Hz, 1H),2.11-2.03(m,1H),2.01(s,3H),1.89(d,J=10.8Hz,2H),1.38(s,9H). 13 C NMR (151MHz, DMSO) δ173.1,170.3,169.9,169.6,145.0,128.5,127.4,126.3,80 .6,72.7,69.4,58.2,51.5,49.9,40.8,33.9,27.5,20.9.HRMS(ESI+)calcd.for C 34 H 40 N3O6 + (M+H) + :586.2912; found:586.2896.

[0190] Example 3: Synthesis of compound 5, the principle is as follows:

[0191]

[0192] Take a 2500mL round-bottom flask and put in a magnetic stirrer. Add compound N-Fmoc-cysteine ​​(64.40g, 0.11mol) to the 2500mL round-bottom flask, add 500mL dry dichloromethane and stir to dissolve, and react in an ice-water bath for 5min. Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (12.70g, 0.11mol) and 1-hydroxybenzotriazole (14.90g, 0.11mol) and react in an ice-water bath for 30min. Then add compound 4 (70.70mg, 0.11mol) dropwise and react in an ice-water bath for 30min. React at room temperature overnight, and monitor the reaction by TLC until the raw material is consumed. Wash the organic phase with saturated sodium bicarbonate, extract three times with 1000mL dichloromethane, combine the organic phases, wash with salt, dry with anhydrous sodium sulfate, filter, and remove the solvent under reduced pressure to obtain a crude product. The unpurified crude product is directly used in the next step of deprotection reaction. The crude product was dissolved in a 50 wt% diethylamine dichloromethane solution, stirred at room temperature, and the reaction was stopped after the raw material was completely consumed by TLC monitoring. The solvent and diethylamine were removed under reduced pressure, and chromatographed on a 200-300 mesh silica gel column (volume ratio of ethyl acetate / petroleum ether = 75%-80%) to obtain a white solid compound 5, 76.30 g, with a yield of 82%. 1 HNMR(600MHz,DMSO)δ8.73(s,1H),8.25(d,J=7.8Hz,1H),7.34-7.29(m,12H),7.23(dd,J=15.4,7.2Hz,1 0H),7.16(d,J=7.4Hz,8H),5.20(s,1H),4.65(dd,J=13.0,8.0Hz,1H),4.21(t,J=8.1Hz,1H),3.74(dt,J =11.6,7.9Hz,2H),3.13(dd,J=7.7,5.0Hz,1H),2.65(dd,J=14.8,8.9Hz,1H),2.50-2.48(m,2H),2.25(d dd,J=20.0,11.6,7.1Hz,2H),2.05(ddd,J=13.4,7.8,5.3Hz,1H),1.93(s,3H),1.70(s,2H),1.39(s,9H). 13C NMR (151MHz, DMSO) δ172.8,170.1,169.9,169.6,168.3,144.7,144.5,129.1,128.5,127.9,127.4,126.7, 126.3,80.7,72.4,69.5,65.9,58.1,53.8,51.7,47.9,38.0,37.1,33.9,27.5,20.8.HRMS(ESI+)calcd.for C 34 H 40 N3O6 + (M+H) + :931.4099; found:931.4084.

[0193] Example 4: Synthesis of compound 6, the principle is as follows:

[0194]

[0195] Take a 2500mL round-bottom flask and put in a magnetic stirrer. Add compound N-Fmoc-glycine (32.70g, 0.11mol) to the 2500mL round-bottom flask, add 500mL dry dichloromethane and stir to dissolve, and react in an ice-water bath for 5min. Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (12.70g, 0.11mol) and 1-hydroxybenzotriazole (14.90g, 0.11mol) and react in an ice-water bath for 30min. Then add compound 5 (108.70g, 0.11mol) dropwise and react in an ice-water bath for 30min. React at room temperature overnight, and monitor the reaction by TLC until the raw material is consumed. Wash the organic phase with saturated sodium bicarbonate, extract three times with 1000mL dichloromethane, combine the organic phases, wash with salt, dry with anhydrous sodium sulfate, filter, and remove the solvent under reduced pressure to obtain a crude product. The unpurified crude product is directly used in the next step of deprotection reaction. The crude product was dissolved in a 50 wt% diethylamine dichloromethane solution, stirred at room temperature, and the reaction was stopped after the raw material was completely consumed by TLC monitoring. The solvent and diethylamine were removed under reduced pressure, and chromatographed on a 200-300 mesh silica gel column (volume ratio methanol / dichloromethane = 0%-0.5%) to obtain a white solid compound 6, 68.10 g, with a yield of 69%. 1H NMR (600MHz, DMSO) δ8.66 (s, 1H), 8.55 (d, J = 7.7Hz, 1H), 8.13 (s, 1H), 7.39-7.29 (m, 12H), 7.28-7.21 (m, 10H),7.20-7.13(m,8H),5.21(s,1H),4.73(d,J=5.6Hz,1H),4.53(t,J=5.7Hz,1H),4.23(t,J=8.0Hz,1H) ,3.75(dt,J=11.5,8.1Hz,2H),3.13-3.05(m,2H),2.63(dd,J=15.2,8.5Hz,1H),2.56(dd,J=15.1,4.9Hz, 1H), 2.40 (d, J = 5.9Hz, 2H), 2.28-2.22 (m, 1H), 2.10-2.03 (m, 1H), 1.93 (s, 3H), 1.86 (s, 2H), 1.41 (s, 9H). 13 C NMR (151MHz, DMSO) δ172.4,170.1,170.0,169.4,169.3,168.2,144.7,144.272,129.1,128.5,128.0,127.4,126.7,1 26.3,80.6,72.4,69.5,65.7,58.2,51.7,50.2,47.9,44.5,39.5,37.8,34.5,33.8,27.6,20.7.HRMS(ESI+)calcd.for C 58 H 62 N5O8S + (M+H) + :988.4314; found:988.4307.

[0196] Example 5: Synthesis of compound 7, the principle is as follows:

[0197]

[0198] Take a 2500mL round-bottom flask and put in a magnetic stirrer. Add compound N-Fmoc-isoleucine (35.30g, 0.11mol) to the 2500mL round-bottom flask, add 500mL dry dichloromethane and stir to dissolve, and react in an ice-water bath for 5min. Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (12.70g, 0.11mol) and 1-hydroxybenzotriazole (14.90g, 0.11mol) and react in an ice-water bath for 30min. Then add compound 6 (98.70g, 0.10mol) dropwise and react in an ice-water bath for 30min. React at room temperature overnight, and monitor the reaction by TLC until the raw material is consumed. Wash the organic phase with saturated sodium bicarbonate, extract three times with 1000mL dichloromethane, combine the organic phases, wash with salt, dry with anhydrous sodium sulfate, filter, and remove the solvent under reduced pressure to obtain a crude product. The unpurified crude product is directly used in the next step of deprotection reaction. The crude product was dissolved in a 50 wt% diethylamine solution in dichloromethane, stirred at room temperature, and the reaction was stopped after the raw material was completely consumed by TLC monitoring. The solvent and diethylamine were removed under reduced pressure, and chromatographed on a 200-300 mesh silica gel column (volume ratio of methanol / dichloromethane = 0%-1%) to obtain a white solid compound 7, 80.30 g, with a yield of 73%. 1 HNMR (600MHz, DMSO) δ8.67(s,1H),8.45(d,J=7.1Hz,1H),8.16(s,1H),8.10(d,J=7.9Hz,1H),7.33-7.30(m,12H),7.25-7.23(m ,10H),7.18(d,J=6.7Hz,8H),5.19(s,1H),4.72(d,J=6.4Hz,1H),4.48(d,J=6.4Hz,1H),4.21(t,J=7.6Hz,1H),3.76-3.73(m,4 H),3.36(s,1H),3.06(d,J=4.2Hz,1H),2.68-2.54(m,2H),2.42(d,J=5.6Hz,1H),2.38-2.28(m,1H),2.23-2.21(m,1H),2.11-2 .01(m,1H),1.91(s,3H),1.69(d,J=30.6Hz,2H),1.41(s,9H),1.12-1.02(m,1H),0.85(d,J=6.4Hz,3H),0.79(t,J=7.0Hz,3H). 13C NMR (151MHz, DMSO) δ174.9,170.1,169.9,169.3,169.1,168.6,168.3,144.6,144.2,129.0,128.5,127.9,127.4,126.6,126.3,8 0.6,72.2,69.5,65.7,59.3,58.1,51.6,50.9,47.9,41.9,38.1,34.1,33.8,27.6,23.7,20.7,15.8,11.6.HRMS(ESI+)calcd.for C 64 H 73 N6O9S + (M+H) + :1101.5104; found:1101.5122.

[0199] Example 6: Synthesis of Compound 8, the principle is as follows:

[0200]

[0201] Take a 2500mL round-bottom flask and put in a magnetic stirrer. Add compound N-Fmoc-glycine (32.60g, 0.11mol) to the 2500mL round-bottom flask, add 500mL dry dichloromethane and stir to dissolve, and react in an ice-water bath for 5min. Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (12.70g, 0.11mol) and 1-hydroxybenzotriazole (14.90g, 0.11mol) and react in an ice-water bath for 30min. Then add compound 7 (110.00g, 0.10mol) dropwise and react in an ice-water bath for 30min. React at room temperature overnight, and monitor the reaction by TLC until the raw material is consumed. Wash the organic phase with saturated sodium bicarbonate, extract three times with 1000mL dichloromethane, combine the organic phases, wash with salt, dry with anhydrous sodium sulfate, filter, and remove the solvent under reduced pressure to obtain a crude product. The unpurified crude product is directly used in the next step of deprotection reaction. The crude product was dissolved in a 50 wt% diethylamine dichloromethane solution, stirred at room temperature, and the reaction was stopped after the raw material was completely consumed by TLC monitoring. The solvent and diethylamine were removed under reduced pressure, and chromatographed on a 200-300 mesh silica gel column (volume ratio methanol / dichloromethane = 0%-1.5%) to obtain a white solid compound 8, 70.60 g, with a yield of 61%. 1HNMR(600MHz,DMSO)δ8.68(s,1H),8.46(d,J=7.4Hz,1H),8.32(d,J=5.3Hz,1H),8.07 (d,J=8.0Hz,1H),7.97(s,1H),7.35-7.28(m,12H),7.25-7.23(m,10H),7.18(d,J=7. 7Hz,8H),5.19(s,1H),4.72(dd,J=13.7,7.0Hz,1H),4.46(dd,J=13.8,7.0Hz,1H),4. 26(s,1H),4.21(t,J=7.8Hz,1H),3.79-3.73(m,4H),3.45-3.30(m,1H),3.11(d,J=6. 6Hz,2H),2.65(dd,J=15.1,5.1Hz,1H),2.56(dd,J=15.2,7.4Hz,1H),2.50(s,1H),2. 40(dd,J=11.0,5.1Hz,1H),2.37-2.28(m,1H),2.22(t,J=9.3Hz,1H),2.10-2.03(m,1 H),1.98(d,J=8.6Hz,1H),1.90(d,J=6.2Hz,3H),1.76(t,J=14.7Hz,1H),1.50-1.43( m,1H),1.41(s,9H),1.10-1.00(m,1H),0.84(d,J=6.7Hz,3H),0.81(t,J=7.3Hz,3H). 13 C NMR (151MHz, DMSO) δ172.7,171.4,170.1,169.9,169.4,169.7,168.4,168.3,144.7,144.2,129.1,128.6,128.5,128.0,127.4,126.7,126.3, 80.6,72.3,69.5,65.7,58.1,56.5,51.7,51.0,47.8,44.5,41.8,37.9,37.1,34.2,33.8,27.6,24.3,20.7,15.4,11.2.HRMS(ESI+)calcd.for C 66 H 76 N7O 10 S + (M+H) + :1158.5369; found:1158.5354.

[0202] Example 7: Synthesis of compounds 9a and 9b, the principle is as follows:

[0203]

[0204] Take a 2500mL round-bottom flask and put in a magnetic stirrer. Add N-Fmoc-6-benzyloxytryptophan (68.90g, 0.10mol) to the 2500mL round-bottom flask, add 500mL dry dichloromethane and stir to dissolve, and react in an ice-water bath for 5min. Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (12.70g, 0.11mol) and 1-hydroxybenzotriazole (14.90g, 0.11mol) and react in an ice-water bath for 30min. Then add compound 8 (143.80g, 0.11mol) dropwise and react in an ice-water bath for 30min. React at room temperature overnight, and monitor the reaction by TLC until the raw material is consumed. Wash the organic phase with saturated sodium bicarbonate, extract three times with 1000mL dichloromethane, combine the organic phases, wash with salt, dry over anhydrous sodium sulfate, filter, and remove the solvent under reduced pressure to obtain a crude product. The crude product without purification was directly used for the next step of deprotection reaction. The crude product was dissolved in a 50wt% diethylamine dichloromethane solution, stirred at room temperature, and the reaction was stopped after the raw material was consumed by TLC monitoring. The solvent and diethylamine were removed under reduced pressure, and chromatographed on a 200-300 mesh silica gel column (volume ratio methanol / dichloromethane = 0%-2%) to obtain a white solid compound 9a, 100.00 g, with a yield of 69%. 1H NMR(600MHz,DMSO)δ10.65(s,1H),8.64(s,1H),8.46(d,J=7.4Hz,1H),8.24(d,J=5.5Hz,1H),8.18(s,1H),8.02(d,J=7.9Hz,1H),7.92(d,J=8.5Hz,1H),7.46(d,J=7.4Hz,2H),7.42(d,J=8.6Hz,1H),7.38(t,J=7.5Hz,2H),7.32-7.28(m,12H),7.24-7.22(m,10H),7.18-7.15(m,8H),7.10(d,J=7.8Hz,1H),7.04(s,1H),6.91(s,1H),6.72(d,J=8.6Hz,1H),5.17(s,1H),5.10-5.08(m,2H),4.73-4.67(m,1H),4.48-4.42(m,1H),4.24(t,J=7.7Hz,1H),4.19(t,J=7.9Hz,1H),3.82-3.78(m,1H),3.78-3.74(m,2H),3.70(s,2H),3.46(dd,J=8.6,4.1Hz,1H),3.07(dd,J=14.1,3.7Hz,1H),2.72-2.58(m,2H),2.56-2.52(m,1H),2.37(dd,J=11.1,5.1Hz,1H),2.33-2.27(m,1H),2.25-2.15(m,1H),2.05(dd,J=13.6,6.6Hz,1H),1.88(s,3H),1.73(s,2H),1.45(s,1H),1.40(s,9H),1.12-1.02(m,1H),0.83(d,J=6.7Hz,3H),0.78(t,J=7.2Hz,3H). 13C NMR (151MHz, DMSO) δ174.9,171.2,170.1,169.9,169.3,169.1,168.7,168.3,168.2,154.4 ,144.6,144.2,137.7,136.8,129.0,128.5,128.3,127.9,127.5,127.4,126.6,126.3,122. 5,118.9,110.6,109.1,96.0,80.6,72.2,69.5,65.7,58.1,56.9,55.2,51.6,50.9,47.8,41 .9,41.8,37.9,36.7,34.2,33.8,30.9,27.6,24.2,20.7,15.3,11.1.HRMS(ESI+)calcd.for C 84 H 92 N9O 12 S + (M+H) + :1450.6581; found:1450.6591.

[0205] Referring to the synthesis method of compound 9a, compound 8 (143.80 g, 0.11 mol) was reacted with N-Fmoc-tryptophan (42.60 g, 0.10 mol), and the Fmoc protecting group was removed to obtain white solid compound 9b, 104.80 g, 78%. 1H NMR(600MHz,DMSO)δ10.87(s,1H),8.67(s,1H),8.49(d,J=6.8Hz,1H),8.28(s,1H),8.22(s,1H),8.06(d,J=7.5Hz,1H),7.96(d,J=8.1Hz,1H),7.56(d,J=7.6Hz,1H),7.36-7.31(m,13H),7.24-7.23(m,8H),7.18(d,J=6.9Hz,10H),7.12(d,J=7.4Hz,1H),7.07(t,J=7.2Hz,1H),6.97(t,J=7.1Hz,1H),5.18(s,1H),4.73(d,J=6.4Hz,1H),4.48(d,J=6.4Hz,1H),4.26(t,J=7.3Hz,1H),4.21(t,J=7.7Hz,1H),3.82-3.73(m,5H),3.54-3.47(m,1H),3.38(s,2H),3.19-3.08(m,1H),2.74(dd,J=13.8,9.0Hz,1H),2.64(d,J=10.5Hz,1H),2.56(dd,J=14.5,7.0Hz,1H),2.39(d,J=5.8Hz,1H),2.37-2.27(m,1H),2.22(s,1H),2.11-2.02(m,1H),1.94-1.84(m,3H),1.76(s,2H),1.47(s,1H),1.41(s,9H),1.15-1.04(m,1H),0.85(d,J=6.2Hz,3H),0.80(t,J=7.0Hz,3H). 13 C NMR(151MHz,DMSO)δ174.9,171.3,170.1,169.9,169.4,169.2,168.8,168.3,168.2,144.7,144.2,136.3,129.1,128.6,128.5,128.0,127.4,126.7,126.3,123.8,120.9,118.4,118.3,111.3,110.7,80.7,72.3,69.5,65.7,58.1,56.9,55.3,51.7,50.9,47.8,41.9,41.8,37.9,36.8,34.3,33.8,30.9,27.6,24.2,20.7,15.4,11.2.HRMS(ESI+)calcd.for C 77 H 86 N9O 11 S +(M+H) + :1344.6162; found:1344.6189.

[0206] Example 8: Synthesis of compounds 10a and 10b, the principle is as follows:

[0207]

[0208] Take a 2500mL round-bottom flask and put in a magnetic stirrer. Add compound 16 (2S, 3R, 4R)-4,5-dihydroxyisoleucine derivative (46.90g, 0.10mol) to the 2500mL round-bottom flask, add 500mL dry dichloromethane and stir to dissolve, and react in an ice-water bath for 5min. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDC (12.70g, 0.11mol) and 1-hydroxybenzotriazole (14.90g, 0.11mol) and react in an ice-water bath for 30min. Then add compound 9a (183.10g, 0.11mol) dropwise and react in an ice-water bath for 30min. React at room temperature overnight, and monitor the reaction by TLC until the raw materials are consumed. The organic phase was washed with saturated sodium bicarbonate, extracted three times with 1000 mL of dichloromethane, the organic phases were combined, washed with salt, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to obtain a crude product. A white solid compound 10a was obtained by chromatography on a 200-300 mesh silica gel column (volume ratio methanol / dichloromethane = 0%-2%), 152.00 g, yield 80%. 1HNMR(600MHz,DMSO)δ10.66(s,1H),8.68(s,1H),8.51(s,1H),8.33(s,1H),8.28(s,1H),8.16-8.05(m,1H),8.07-8.03(m,2H),7.90-7.89(m,3H),7.75(dd,J=13.3,7.3Hz,2H),7.58(d,J=9.0Hz,1H),7.55-7.49(m,2H),7.47(d,J=7.2Hz,2H),7.45-7.37(m,5H),7.35-7.30(m,13H),7.26-7.24(d,J=6.7Hz,10H),7.20(d,J=7.0Hz,8H),7.14(d,J=7.5Hz,1H),7.10(s,1H),6.94(s,1H),6.76(d,J=8.2Hz,1H),5.20(s,1H),5.11-5.08(d,J=16.4Hz,3H),4.84-4.74(m,2H),4.71(d,J=5.5Hz,1H),4.55-4.46(m,2H),4.38(dd,J=24.5,10.2Hz,2H),4.31(s,1H),4.24(dt,J=14.8,7.4Hz,2H),4.19-4.11(m,1H),4.04(d,J=7.6Hz,1H),3.81(s,4H),3.75(s,2H),3.41(s,1H),3.17(d,J=11.0Hz,1H),3.05(s,1H),2.67(d,J=10.7Hz,1H),2.60(s,1H),2.42(s,1H),2.35(s,1H),2.23(s,1H),2.13-2.04(m,1H),1.98(dd,J=16.9,6.9Hz,6H),1.91(d,J=8.7Hz,3H),1.79(s,1H),1.47(d,J=26.7Hz,1H),1.42(s,9H),1.14(d,J=26.1Hz,1H),0.87(s,3H),0.80(t,J=20.4Hz,3H),0.76(d,J=5.5Hz,3H). 13C NMR (151MHz, DMSO) δ171.6,171.3,170.5,170.2,170.1,169.9,169.7,169.4,169.2,168.5,168.4,168.3,156.3,154.5,144.7,144.3,1 44.0,143.7,140.7,140.6,137.7,136.7,129.1,128.6,128.5,128.3,128.0,127.7,127.6,127.4,127.2,126.7,126.3,125.6,125.3,12 2.5,122.1,120.1,119.0,109.8,109.1,95.9,80.7,72.3,71.6,69.6,69.5,66.2,65.7,62.9,58.1,56.9,54.9,54.7,53.5,51.7,50.9, 47.9,46.7,42.1,41.8,39.5,37.9,36.8,35.3,34.3,33.9,27.6,27.5,24.2,20.8,20.7,20.6,15.4,11.2,10.3.HRMS(ESI+)calcd.forC 109 H 116 N 10 NaO 19 S + (M+Na) + :1923.8031; found:1923.8068.

[0209] Referring to the synthesis method of compound 10a, compound 9b (180.00 g, 0.11 mol) and compound 16 (2S, 3R, 4R)-4,5-dihydroxyisoleucine derivative (46.90 g, 0.10 mol) were reacted, and the Fmoc protecting group was removed to obtain white solid compound 10b, 147.20 g, with a yield of 82%. 1H NMR(600MHz,DMSO)δ10.86(s,1H),8.70(s,1H),8.53(s,1H),8.36(s,1H),8.30(s,1H),8.09(s,1H),7.90(d,J=6.5Hz,3H),7.77(dd,J=16.4,6.9Hz,2H),7.67(d,J=7.2Hz,1H),7.60(d,J=8.0Hz,1H),7.43(s,2H),7.34(s,14H),7.28-7.19(m,20H),7.08(d,J=6.8Hz,1H),7.01(s,1H),5.74(s,2H),5.22(s,1H),4.86-4.69(m,3H),4.53(s,2H),4.42(d,J=11.0Hz,2H),4.33(s,1H),4.26(d,J=7.4Hz,2H),4.17(s,1H),4.05(d,J=7.6Hz,1H),3.81(d,J=33.7Hz,6H),3.44(s,1H),3.24(d,J=10.3Hz,1H),3.12(s,1H),2.66(d,J=39.3Hz,2H),2.57-2.50(m,1H),2.42(d,J=46.2Hz,2H),2.24(s,1H),2.08(s,1H),2.00(d,J=11.4Hz,6H),1.91(s,3H),1.82(s,1H),1.51(s,1H),1.43(s,9H),1.14(s,1H),0.82(m,9H). 13C NMR (151MHz, DMSO) δ171.7,171.3,170.6,170.2,170.1,169.9,169.7,169.4,169.2,168.6,168.4,168.3,156.3,144.7,144.3, 144.1,143.7,140.8,140.7,136.2,129.1,128.6,128.0,127.7,127.6,127.4,127.2,126.7,126.4,125.6,125.3,123.7,120.8 ,120.1,118.4,118.2,111.3,109.9,80.7,72.3,71.6,69.6,66.2,65.8,63.0,58.2,56.9,54.9,54.7,53.5,51.7,51.0,47.9,4 6.7,42.2,41.9,39.5,38.0,36.8,35.4,34.3,33.9,28.0,27.6,24.3,20.8,20.7,20.6,15.4,11.2,10.4.HRMS(ESI+)calcd.for C 102 H 110 N 10 NaO 18 S + (M+Na) + :1817.7612; found:1817.7625.

[0210] Example 9: Synthesis of compounds 11a and 11b, the principle is as follows:

[0211]

[0212] Take a 2500mL round-bottom flask, put in a magnetic stirrer, and replace the gas three times using Schlenk technology. Add compound 10a (57.00g, 0.03mol) to a 2500mL round-bottom flask under N2 atmosphere, add 500mL dry N,N-dimethylformamide and stir to dissolve, and stir at room temperature. Prepare a 2mg / mL DMF solution of iodine element, and slowly add 4equiv of iodine solution to the reaction bottle with a syringe. React at room temperature for 2h, and add excess sodium thiosulfate solution to quench the reaction when the reaction stops. Remove DMF under reduced pressure, extract three times with 2000mL ethyl acetate, combine the organic phases, wash with salt, dry over anhydrous sodium sulfate, filter, and remove the solvent under reduced pressure to obtain a crude product. Use 200-300 mesh silica gel column chromatography (volume ratio methanol / dichloromethane = 0%-2%) to obtain white solid compound 11a, 30.60g, yield 62%. 1H NMR(600MHz,DMSO)δ11.06(d,J=49.8Hz,1H),8.63(s,1H),8.40(s,1H),8.28(d,J=45.1Hz,1H),8.18-7.98(m,3H),7.89(s,2H),7.74-7.74(m,2H),7.65-7.60(m,1H),7.52-7.32(m,10H),7.26-7.24(m,6H),7.16(t,J=13.4Hz,9H),6.85(s,1H),6.77(d,J=9.1Hz,1H),5.20(s,1H),5.13-5.09(m,2H),4.77(s,1H),4.69(s,1H),4.48-4.26(m,3H),4.25-4.21(m,2H),4.17-4.09(m,1H),4.03(s,2H),3.88(s,2H),3.71(d,J=20.8Hz,4H),3.32(s,2H),2.94(d,J=8.9Hz,2H),2.61(s,2H),2.53-2.46(m,1H),2.37(s,1H),2.28(s,1H),2.06(s,1H),2.01-1.83(m,10H),1.54(s,1H),1.39(s,9H),1.20-1.06(m,1H),0.96-0.60(m,9H). 13C NMR (151MHz, DMSO) δ172.1,172.1,171.4,170.5,170.4,170.2,170.1,169.9,169.9,169.6,168.7,168.6,168.3,156.3,155.3,152.3,144.7 ,144.6,144.0,143.7,143.6,140.7,140.6,139.4,137.7,137.5,132. 2,128.9,128.6,128.4,128.3,127.8,127.6,127.5,127.4,127.3,127 .1,126.3,125.5,125.3,123.8,121.8,121.4,120.1,116.8,111.6,10 9.7,95.3,80.7,72.5,71.5,70.0,69.5,69.4,66.2,62.9,59.7,58.8, 58.2,54.9,54.7,51.8,48.1,46.7,42.9,42.4,39.5,37.7,35.2,33.9,27.6,24.5,20.7,20.5,15.5,11.0,10.2,10.0.HRMS(ESI+)calcd.for C 90 H 100 N 10 NaO 19 S + (M+Na) + :1679.6779; found:1679.6795.

[0213] Referring to the synthesis method of compound 11a, a white solid compound 11b was obtained, 34.40 g, with a yield of 74%. 1H NMR(600MHz,DMSO)δ11.18(d,J=8.8Hz,1H),8.63(s,1H),8.42(s,1H),8.25(d,J=40.3Hz,1H),8.10(d,J=34.1Hz,1H),7.96(d,J=14.3Hz,1H),7.89(s,2H),7.73(d,J=6.4Hz,2H),7.47(d,J=9.2Hz,1H),7.41(d,J=3.0Hz,2H),7.37-7.28(m,3H),7.26-7.24(m,7H),7.18-7.15(m,9H),7.12-7.09(m,1H),7.03-6.94(m,1H),5.18(s,1H),4.98-4.83(m,1H),4.75(s,1H),4.67(d,J=5.9Hz,1H),4.41-4.29(m,3H),4.26-4.16(m,3H),4.16-4.04(m,1H),4.05-4.01(m,3H),3.89-3.86(d,J=15.7Hz,2H),3.70(d,J=18.8Hz,4H),3.18-3.16(m,1H),3.04-2.90(m,2H),2.62-2.50(m,2H),2.31(d,J=54.1Hz,2H),2.07(s,1H),1.99-1.97(m,5H),1.94-1.92(m,4H),1.89-1.84(m,1H),1.53(s,1H),1.37(s,9H),1.15-1.12(m,1H),0.90-0.81(m,9H). 13 C NMR(151MHz,DMSO)δ172.0,171.4,170.4,170.3,170.2,170.1,169.9,169.8,169.7,169.7,168.6,168.3,162.3,144.7,143.9,140.7,136.8,128.6,127.6,127.4,127.1,126.3,125.3,121.9,120.0,118.6,116.2,110.9,80.7,72.5,71.9,71.5,69.5,66.2,65.9,62.9,62.9,59.7,58.8,58.1,54.8,51.8,48.1,46.6,42.8,42.4,37.9,37.7,35.76,35.2,33.9,30.7,27.6,24.5,20.7,20.6,15.4,14.1,11.0,10.0.HRMS(ESI+)calcd.for C83 H 94 N 10 NaO 18 S + (M+Na) + :1573.6360; found:1573.6369.

[0214] Example 10: Synthesis of compounds 12a and 12b, the principle is shown in the following formula:

[0215]

[0216] Take a 2500mL round-bottom flask and put in a magnetic stirrer. Add compound 11a (49.70g, 0.03mol) to the 2500mL round-bottom flask, add an excess of 50wt% diethylamine in dichloromethane solution, react at room temperature for 2h, and remove the solvent under reduced pressure to obtain a crude product. Use 200-300 mesh silica gel column chromatography (volume ratio methanol / dichloromethane = 2%-4%) to obtain a white solid compound 12a, 34.80g, with a yield of 81%.

[0217] 1H NMR(600MHz,DMSO)δ11.10-10.94(m,1H),11.00(s,1H),8.60(s,1H),8.41-8.35(m,1H),8.05-8.02(m,1H),7.94(d,J=37.2Hz,1H),7.88-7.80(m,1H),7.78-7.68(m,1H),7.57(d,J=22.5Hz,1H),7.51(d,J=12.6Hz,1H),7.47(d,J=7.2Hz,1H),7.40(d,J=7.1Hz,2H),7.33(dd,J=13.5,6.7Hz,2H),7.26-724(m,6H),7.18-7.13(m,9H),6.84(s,1H),6.78-6.71(m,1H),5.17(s,1H),5.14-5.01(m,2H),4.71-4.59(m,2H),4.44-4.25(m,1H),4.21(t,J=7.9Hz,1H),4.06(dd,J=15.8,9.1Hz,2H),3.96-3.82(m,3H),3.69(d,J=16.5Hz,4H),3.28(s,2H),3.18(s,1H),2.98-2.81(m,2H),2.58(s,2H),2.28(s,1H),2.10-2.03(m,1H),2.02-1.86(m,10H),1.83-1.74(m,2H),1.53(s,1H),1.39(s,9H),1.14(d,J=6.8Hz,2H),0.76(m,9H). 13 C NMR(151MHz,DMSO)δ172.4,171.3,170.9,170.4,170.3,170.0,169.9,169.8,169.6,169.5,168.6,168.3,155.2,144.7,137.5,128.6,128.4,128.3,127.8,127.6,127.4,127.3,126.3,123.7,117.0,111.5,109.7,95.3,80.7,72.4,70.0,69.5,66.3,58.6,58.1,54.8,51.7,48.1,42.3,39.5,35.2,33.9,27.6,24.3,22.4,20.7,15.5,11.1,10.4,9.5.HRMS(ESI+)calcd.for C 75 H 91 N 10 O 17 S +(M+H) + :1435.6279; found:1435.6289.

[0218] Referring to the synthesis method of compound 12a, a white solid compound 12b was obtained, 22.32 g, with a yield of 84%. 1 H NMR (600MHz, DMSO) δ11.23(d,J=31.1Hz,1H),8.73(s,1H),8.61(d,J=30.2Hz,1H),8.41(s ,1H),8.33-8.24(m,1H),8.05-7.97(m,2H),7.89-7.79(m,1H),7.76-7.72(m,1H),7.68(d ,J=7.3Hz,1H),7.57(s,1H),7.30-7.24(m,6H),7.20-7.12(m,9H),7.04-6.98(m,1H),5.1 9(s,1H),4.89-4.79(m,1H),4.73(s,1H),4.69-4.51(m,2H),4.28(d,J=11.4Hz,1H),4.21( d,J=3.5Hz,1H),4.15(d,J=7.1Hz,1H),4.09-3.99(m,1H),3.97-3.81(m,3H),3.78-3.59( m,4H),3.53-3.40(m,1H),3.27(d,J=11.6Hz,1H),3.23-3.11(m,1H),3.00-2.84(m,1H),2 .74(s,1H),2.59(s,1H),2.36-2.24(m,1H),2.15-1.99(m,4H),1.98-1.90(m,8H),1.84(d ,J=14.4Hz,1H),1.57(d,J=7.2Hz,2H),1.39(s,9H),1.20-1.08(m,1H),0.96-0.64(m,9H). 13C NMR (151MHz, DMSO) δ173.6,171.8,171.6,171.4,170.8,170.6,170.6,170.5,170.31,170.2,170.1, 170.0,169.1,168.9,168.8,168.7,145.2,145.2,137.6,129.1,127.9,126.8,125.7,122.6,119.5,1 18.9,117.4,111.4,81.2,73.0,72.5,69.9,68.6,66.9,63.5,60.2,58.6,55.4,54.4,52.3,48.6,44. 2,42.6,38.1,35.9,35.6,34.4,28.1,24.4,22.9,21.2,16.1,11.7,10.0,8.7.HRMS(ESI+)calcd.for C 68 H 85 N 10 O 16 S + (M+H) + :1329.5860; found:1329.5867.

[0219] Example 11: Synthesis of compounds 13a and 13b, the principle is shown in the following formula:

[0220]

[0221] Take a 2500mL round-bottom flask and put in a magnetic stirrer. Add compound 12a (28.70g, 0.02mol) to the 2500mL round-bottom flask, add 200mL trifluoroacetic acid, stir at room temperature for 0.5h, and remove trifluoroacetic acid under reduced pressure to obtain a crude product. The crude product was not further purified and was directly used in the next step of synthesis. The crude product was dissolved in dry N,N-dimethylformamide, stirred at zero degree for 5min, and then diisopropylethylamine (6.90g, 2equiv) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (15.20g, 2equiv) were added. Warm up to room temperature and react for 12h. After the reaction is completed, remove N,N-dimethylformamide under reduced pressure to obtain a crude product. The crude product was purified by 200-300 mesh silica gel column chromatography (volume ratio of methanol / dichloromethane = 4%-6%) to obtain white solid compound 13a, 13.56 g, yield 61%. 1HNMR(600MHz,DMSO)δ11.08(d,J=42.1Hz,1H),8.95(s,1H),8.57(s,1H),8.33(d,J=12.6Hz,1H),8.05(d,J=11.8Hz,2H),8.00-7.93(m,1H),7.92-7.83(m,2H),7.52(d,J=7.1Hz,1H),7.46(t,J=7.2Hz,1H),7.40(t,J=7.4Hz,2H),7.33-7.31(m,2H),7.16(dd,J=17.6,8.8Hz,2H),6.82-6.76(m,1H),5.35(s,1H),5.15-5.07(m,3H),4.92-4.81(m,2H),4.75(s,1H),4.57(d,J=9.8Hz,1H),4.36-4.26(m,2H),4.24-4.17(m,1H),4.13-4.01(m,43H),3.89(dd,J=17.0,6.7Hz,1H),3.72(s,1H),3.52-3.42(m,2H),3.21-3.16(m,2H),3.05-2.91(m,3H),2.65-2.59(m,1H),2.26(s,1H),2.11(t,J=11.0Hz,1H),2.02(s,3H),1.99(s,3H),1.95(s,3H),1.56(s,2H),1.13-1.06(m,2H),0.95(dd,J=20.7,9.5Hz,3H),0.83(t,J=6.9Hz,3H),0.78(d,J=6.0Hz,3H). 13 C NMR(151MHz,DMSO)δ171.9,171.9,171.8,171.1,171.1,170.6,170.2,169.9,169.8,169.8,169.7,169.6,168.1,167.5,162.2,158.7,155.3,151.9,137.2,128.4,127.8,127.4,124.9,123.6,122.5,121.7,116.4,109.9,72.3,71.0,70.2,69.5,62.9,61.8,59.1,54.9,54.3,53.0,52.8,52.5,50.5,39.5,35.7,34.4,30.8,28.9,25.2,20.9,20.7,20.6,14.8,11.2,10.6.HRMS(ESI+)calcd.for C 52 H 66 N10 O 16 NaS + (M+Na) + :1141.4271; found:1141.4280.

[0222] Referring to the synthesis method of compound 13a, a white solid compound 13b was obtained, 6.25 g, with a yield of 62%. 1 H NMR (600MHz, DMSO) δ11.25(s,1H),8.97(s,1H),8.56(s,1H),8.35(s,1H),8.06(d,J=14.9Hz,2H),7.98( d,J=9.9Hz,1H),7.91(d,J=7.0Hz,2H),7.58(d,J=7.7Hz,1H),7.32(s,1H),7.26(d,J=7.9Hz,1H),7.12( t,J=7.3Hz,1H),7.05-6.96(m,1H),5.36(s,1H),5.18(d,J=13.7Hz,1H),4.91(s,1H),4.86(d,J=7.7Hz, 1H),4.77(d,J=8.0Hz,1H),4.59(t,J=9.2Hz,1H),4.37-4.28(m,2H),4.19(dd,J=18.4,7.9Hz,1H),4.06 (dd,J=14.9,9.6Hz,3H),3.90(dd,J=17.1,6.9Hz,1H),3.73(s,1H),3.46(d,J=17.8Hz,2H),3.29-3.19( m,1H),3.17(s,1H),3.09-2.98(m,2H),2.90(dd,J=14.0,7.3Hz,2H),2.77(d,J=8.1Hz,1H),2.73(s,1H) ,2.62(s,1H),2.27(d,J=6.4Hz,1H),2.20-2.07(m,1H),2.03(s,3H),2.00(s,3H),1.95(s,3H),1.56(d, J=6.9Hz,2H),1.10(d,J=6.4Hz,2H),0.95(d,J=6.5Hz,3H),0.83(d,J=6.8Hz,3H),0.79(d,J=6.0Hz,3H). 13C NMR(151MHz,DMSO)δ172.0,171.9,171.1,170.7,170.2,169.9,169.8,169.7,169.6, 168.2,167.5,136.5,127.0,124.3,122.3,120.1,118.7,116.1,111.2,72.3,71.2,6 2.9,61.9,59.1,54.9,53.0,52.9,52.5,50.5,42.3,41.4,38.3,34.9,34.4,34.3,33 .6,30.6,29.0,25.2,21.0,20.7,20.6,14.8,11.2,11.0,10.6.HRMS(ESI+)calcd.for C 45 H 60 N 10 NaO 15 S + (M+Na) + :1035.3853; found:1035.3869.

[0223] Example 12: Synthesis of Compound 14, the principle is shown in the following formula:

[0224]

[0225] Take a 2500mL round-bottom flask and put in a magnetic stirrer. Add compound 13a (5.60g, 0.005mol) to a 2500mL round-bottom flask, add 300mL of a mixed solution of isopropanol and ethanol (volume ratio of isopropanol and ethanol = 2:1), and stir in an ice-water bath for 10min. A mixed solution of isopropanol and ethanol of m-chloroperbenzoic acid is added dropwise to the reaction solution and reacted at zero temperature for 30min. After the reaction is completed, the mixture of isopropanol and ethanol is removed under reduced pressure to obtain a crude product. The crude product is purified by 200-300 mesh silica gel column chromatography (volume ratio of methanol / dichloromethane = 5%-6%) to obtain a white solid compound 14a, 3.90g, with a yield of 70%.

[0226] Referring to the synthesis method of compound 14a, a white solid compound 14b was obtained, 3.00 g, with a yield of 73%. 1H NMR(600MHz,DMSO)δ11.67(s,1H),8.88(d,J=5.8Hz,1H),8.55(s,1H),8.30(s,1H),8.26(d,J=10.5Hz,2H),7.89(d,J=8.8Hz,1H),7.85(d,J=10.4Hz,1H),7.81(d,J=9.0Hz,1H),7.65(d,J=8.1Hz,1H),7.44-7.38(m,2H),7.19(t,J=7.6Hz,1H),7.09(t,J=7.4Hz,1H),5.33(s,1H),5.21(dd,J=19.6,8.7Hz,1H),5.04-4.99(m,1H),4.91(s,1H),4.85(dd,J=10.4,3.3Hz,1H),4.76(d,J=8.4Hz,1H),4.32-4.24(m,3H),4.07-4.00(m,3H),3.89(dd,J=17.3,7.1Hz,1H),3.70(d,J=4.0Hz,1H),3.48(d,J=17.0Hz,2H),3.41(d,J=18.1Hz,1H),3.25(dd,J=15.1,6.8Hz,2H),3.10(t,J=13.3Hz,1H),2.93(dd,J=10.1,6.6Hz,2H),2.79-2.69(m,1H),2.57-2.51(m,1H),2.24(dd,J=13.6,6.7Hz,1H),2.12(d,J=12.8Hz,1H),2.02(s,3H),1.98(s,3H),1.95(s,3H),1.58-1.50(m,2H),1.12-1.03(m,2H),0.92(d,J=7.0Hz,3H),0.82(t,J=7.2Hz,3H),0.79(d,J=6.5Hz,3H). 13C NMR(151MHz,DMSO)δ172.0,171.7,171.2,170.5,170.3,170.2,170.1,169.8,169.7, 169.6,167.9,166.9,137.4,132.3,127.2,123.2,121.3,119.5,112.3,110.8,72.3,7 1.1,62.9,61.9,59.1,52.9,52.6,52.4,50.7,49.9,42.3,41.1,39.5,36.8,34.7,34 .6,34.5,32.9,29.2,25.2,20.9,20.7,20.5,14.8,11.1,10.8.HRMS(ESI+)calcd.for C 45 H 60 N 10 O 16 NaS + (M+Na) + :1051.3802; found:1051.3816.

[0227] Example 13: Synthesis of compound 1a, the principle is shown in the following formula:

[0228]

[0229] Take a 2500mL round-bottom flask and put in a magnetic stirrer. Add compound 14a (4.00g, 0.0045mol) to the 2500mL round-bottom flask, add ethanethiol solution, and then add boron trifluoride etherate. After reacting for 2 hours, remove the solvent under reduced pressure to obtain a crude product from which the benzyl group has been removed. Then add 7N ammonia methanol solution to the crude product and stir at room temperature for 2 hours. Monitor the reaction by TLC until the raw material is exhausted, remove the ammonia methanol solution under reduced pressure to obtain the product α-Amanitin 1a 1.883g, with a yield of 50%.

[0230] 1H NMR (600MHz, DMSO) δ11.25(s,1H),9.21(s,1H),8.77(t,J=5.5Hz,1H),8.56(s,1H),8.37(s,1H),8 .24(d,J=10.4Hz,1H),8.21(s,1H),7.92(d,J=8.9Hz,1H),7.84(d,J=8.6Hz,1H),7.67(d,J=9.8Hz, 1H),7.42(d,J=8.7Hz,1H),7.37(s,1H),6.75(s,1H),6.61(d,J=8.6Hz,1H),5.18(s,1H),5.09(dd ,J=19.7,8.4Hz,1H),4.98-4.92(m,1H),4.84(dd,J=9.8,4.2Hz,1H),4.65(s,1H),4.41-4.36(m,3H ),4.25(ddd,J=27.6,14.9,8.0Hz,2H),3.90(dd,J=17.3,7.3Hz,1H),3.81(d,J=9.3Hz,1H),3.73( d,J=11.1Hz,1H),3.68(d,J=4.4Hz,1H),3.45-3.44(m,3H),3.41(s,1H),3.32-3.27(m,1H),3.23-3 .18(m,1H),3.17-3.15(m,1H),3.07(t,J=13.2Hz,1H),2.98-2.91(m,2H),2.74(t,J=13.8Hz,1H),2 .22-2.15(m,2H),1.86(t,J=11.0Hz,1H),1.59-1.50(m,2H),1.15-1.06(m,1H),0.85-0.77(m,9H). 13 C NMR (151MHz, DMSO) δ171.9,171.7,171.1,171.0,170.8,170.2,169.9,167.8,167.0,154.6,138.8,129.7,122.0,120.7,111.5,110.7,96. 7,71.9,68.6,63.5,62.1,59.2,55.8,54.9,53.9,52.7,50.8,50.0,4 8.6,42.4,41.3,37.8,37.3,34.6,33.4,29.2,25.2,14.7,11.4,10.7.

[0231] The synthesis principle of compound 1b is shown in the following formula:

[0232]

[0233] Take a 2500mL round-bottom flask and put in a magnetic stirrer. Add compound 14b (3.00g, 0.003mol) to the 2500mL round-bottom flask, add 7N ammonia methanol solution, and stir at room temperature for 2h. Monitor the reaction by TLC until the raw material is exhausted, and remove the ammonia methanol solution under reduced pressure to obtain a crude product. The crude product is purified by 200-300 mesh silica gel column chromatography (volume ratio methanol / dichloromethane = 10%-15%) to obtain a white solid compound Amaninamide 1b, 1.92g, yield 74%. 1 H NMR (600MHz, DMSO) δ11.80-11.03(m,1H),8.82(s,1H),8.57(s,1H),8.40(s,1H),8.29(d,J=10.3 Hz,1H),8.22(d,J=23.3Hz,1H),7.94(t,J=12.5Hz,1H),7.86(dd,J=26.5,7.9Hz,1H),7.69-7.62 (m,2H),7.40(d,J=7.7Hz,2H),7.18(dd,J=19.6,11.9Hz,1H),7.08(t,J=7.5Hz,1H),5.33-5.22( m,1H),5.16(dd,J=19.6,8.3Hz,1H),5.04-4.96(m,1H),4.85(dd,J=9.7,4.2Hz,1H),4.66(s,1H) ,4.44-4.369(m,3H),4.30-4.18(m,2H),3.90(dd,J=17.3,7.3Hz,1H),3.79(dd,J=29.6,10.1Hz, 2H),3.68(d,J=4.3Hz,1H),3.50-3.35(m,8H),3.31(dd,J=10.9,5.7Hz,8H),3.28-3.19(m,11H), 3.13(d,J=13.2Hz,1H),2.95(t,J=10.5Hz,2H),2.79(dd,J=27.3,10.7Hz,2H),2.23-2.16(m,2H) ,1.86(t,J=10.9Hz,1H),1.55(d,J=6.1Hz,2H),1.13-1.04(m,2H),0.80(dd,J=22.1,5.0Hz,9H). 13C NMR(151MHz,DMSO)δ171.9,171.7,171.21,171.0,170.8,170.2,169.9,167 .9,167.0,137.4,132.4,127.2,123.1,121.5,119.4,112.3,110.9,71.9,68 .7,63.5,62.10,59.2,58.9,55.8,54.0,52.6,50.9,49.9,42.4,41.7,41.0, 39.5,37.3,34.6,33.4,29.2,25.2,14.8,11.4,10.7.HRMS(ESI+)calcd.for C 39 H 54 N 10 NaO 13 S + (M+Na) + :925.3482; found:925.3489.

[0234] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. An intermediate for preparing cyclic peptide toxin compounds α-Amanitin and / or Amaninamide, the structure of which is shown in any one of compounds 4 to 8:

2. The method for preparing the intermediate according to claim 1, characterized in that: The preparation method of compound 4 is specifically the following steps: Step 1: Compound 2, pyridine, acetyl chloride and dichloromethane are mixed for acetylation reaction, and the obtained acetylation product is mixed with N,N-dimethylpyridine and di-tert-butyl dicarbonate for esterification reaction to obtain compound 3; Step 2: Mix the compound 3, N-Fmoc-asparagine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and dichloromethane for condensation reaction and deprotection reaction to obtain the compound 4; The preparation method of the compound 5 is specifically: mix the compound 4, N-Fmoc-cysteine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and dichloromethane for condensation reaction and deprotection reaction to obtain the compound 5; The preparation method of the compound 6 is specifically as follows: the compound 5, N-Fmoc-glycine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and dichloromethane are mixed to carry out condensation reaction and deprotection reaction to obtain the compound 6; The preparation method of the compound 7 is specifically as follows: the compound 6, N-Fmoc-isoleucine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and dichloromethane are mixed to carry out condensation reaction and deprotection reaction to obtain the compound 7; The preparation method of the compound 8 is specifically as follows: the compound 7, N-Fmoc-glycine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and dichloromethane are mixed to carry out condensation reaction and deprotection reaction to obtain the compound 8; The structures of compound 2 and compound 3 are shown below:

3. The preparation method according to claim 2, characterized in that: In the step 1, the molar ratio of compound 2, acetyl chloride and pyridine is 1:1-3:1-5; the molar ratio of compound 2, N,N-dimethylpyridine and di-tert-butyl dicarbonate is 1:0.1-1:1-5.

4. The preparation method according to claim 2, characterized in that: In the step 2, the molar ratio of compound 3, N-Fmoc-asparagine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1:1.

1.

5. The preparation method according to claim 2, characterized in that: In the step 3, the molar ratio of compound 4, N-Fmoc-cysteine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1:1.

1.

6. The preparation method according to claim 2, characterized in that: In step 4, the molar ratio of compound 5, N-Fmoc-glycine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1:1.

1.

7. The preparation method according to claim 2, characterized in that: In the step 5, the molar ratio of compound 6, N-Fmoc-isoleucine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1:1.

1.

8. The preparation method according to claim 2, characterized in that: In step 6, the molar ratio of compound 7, N-Fmoc-glycine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1.1:1.1:1.

1.

9. Use of the intermediate according to claim 1 in the preparation of cyclopeptide toxin compounds.

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