Synthesis method of amanitine intermediate

Abstract

The invention belongs to the technical field of medicines and chemistry, and particularly relates to a synthesis method of an amanitine intermediate, which comprises the following steps: step 1, carrying out condensation reaction on a compound in a formula 1 in an organic solvent in the presence of a peptide coupling agent and organic alkali to obtain a compound 2; the preparation method of the intermediate compound 2 is mild in condition, easy to control, simple and convenient to operate, high in conversion rate, high in selectivity, capable of obtaining a high-purity product, capable of solving the amplification problem, capable of reducing the cost and beneficial to industrialization.

Description

Technical Field

[0001] This invention relates to a method for synthesizing muscarine intermediates, belonging to the fields of pharmaceuticals and chemical technology. Background Technology

[0002] Amanitin, a natural and potent RNApol II inhibitor, is derived from the fungus *Amanita muscaria*. α-Amanitin and β-Amanitin, along with seven other macrocyclic derivatives (γ-amanitin, ε-amanitin, amaniin, amanitinamide, amanullin, amanullinic acid, and proamanullin), constitute the amannitin family. Amanita toxins are biochemical reagents with wide applications in molecular biology, developmental biology, genetics, biochemistry, medicine, and biocontrol. Although widely used in the laboratory to explore transcriptional mechanisms, α-Amanitin has proven too toxic, particularly to the liver, to be suitable for further development as an anticancer agent. However, this molecule presents many advantages as a potential ADC payload, including an intracellular target, favorable physicochemical properties (including hydrophilicity), insensitivity to efflux pumps, and the ability to induce cytotoxicity in quiescent cancer cells. β-Amanitin was first conjugated with albumin in 1973, demonstrating selective killing of macrophages. This derivative was later conjugated with anti-MUC1 and anti-PSMA antibodies and showed strong selective cytotoxicity in preclinical models.

[0003] Compound 2 is an important intermediate in the synthesis of amatoxins, and its chemical structure is shown below:

[0004]

[0005] In patent CN111051310B, the specification

[0235] to

[0240] discloses the synthesis of a similar intermediate using DPPA and DIEA as condensation conditions. The reaction yield is only 41%, which requires 10 molar equivalents of peptide coupling agent and base relative to the substrate, respectively. The amount used is relatively high, and the solvent is 120V, which further increases the production cost and is not conducive to industrial production.

[0006]

[0007] In patent CN108495840A, the specification

[0283] to

[0286] discloses the synthesis of the following similar intermediates, using PyBOP, HOBt and DIEA as condensation conditions, with a reaction yield of only 35%, a solvent of 110V, and PyBOP has the potential to be explosive, which is not conducive to industrial scale-up.

[0008]

[0009] In patent KR102092817B1, the specification

[0390] to

[0392] discloses the synthesis of the following similar intermediates, using PyAOP, HOAt and DIEA as condensation conditions, with a reaction yield of 40% and a solvent of 150V, which is not conducive to industrial scale-up.

[0010]

[0011] The conditions for self-condensation cyclization of tryptophan amino groups with L-isoleucine carboxylic acid to form peptide bonds, as described above, currently disclosed in the prior art, yield only 35-41%. During repeated verification, the inventors found that the above condensation conditions not only used excessively high equivalents of peptide coupling agents or bases, but some peptide coupling agents also exhibited potential explosiveness. Furthermore, because the raw materials have multiple sites that can undergo condensation cyclization with carboxyl groups, the reaction typically requires extremely dilute solvents, resulting in huge solvent consumption. The peptide coupling agent and substrate usually need to be added in multiple batches, and yields close to those of the prior art can only be repeatedly obtained in milligram-level experiments. Reducing the reaction solvent ratio or increasing the feed volume leads to a sharp increase in intramolecular or intermolecular condensation byproducts, making scale-up production impractical. Therefore, based on the shortcomings of the prior art, there is a need in the art for a better method for synthesizing muscarinic intermediate 2. Summary of the Invention

[0012] The first aspect of the present invention relates to a method for preparing the compound of formula 2, wherein the reaction formula is as follows:

[0013]

[0014] Includes the following steps:

[0015] Step 1: Compound 1 is subjected to a condensation reaction in an organic solvent in the presence of a peptide coupling agent and an organic base to obtain compound 2;

[0016] R1 is independently selected from hydrogen, C 1-6 Alkyl, with one or more R d Replacement C 1-6 Alkyl or -C(O)-R e ;

[0017] R a R b and R c Each is independently selected from C 1-6 Alkyl or phenyl;

[0018] R d Selected independently from C 1-6 alkoxy or phenyl;

[0019] R e Selected from C 1-6 Alkyl, C 1-6 Halogenated alkyl or phenyl;

[0020] R2 is selected from NH2, -Z-R3, OH, OR4, and NHR5;

[0021] PEG 1-8 Chain representation

[0022] Z is selected from -C1-C5 alkylene-, -NH-, -C1-C8 alkylene-PEG. 1-8 -,-PEG 1-8 -or any combination thereof;

[0023] R3 is selected from:

[0024]

[0025] R4 is C 1-6 Alkyl or vinyl-substituted C 1-6 alkyl;

[0026] R5 is selected from benzyloxycarbonyl, 9-fluorenyloxycarbonyl, tert-butyloxycarbonyl, tert-valeryl, acetyl, and triphenylmethyl.

[0027] R6 is selected from H, -OR f or n is 1, 2, 3, 4, 5, 6, 7;

[0028] R f C 1-6 Alkyl, benzyl, or with one or more R f-1 Substituted benzyl;

[0029] R f-1 Independently selected from halogens, C 1-6 Alkyl, nitro, or cyano groups.

[0030] In some schemes, R1 is independently selected C 1-6 Alkyl, with one or more R d Replacement C 1-6 Alkyl or -C(O)-R e ;R a R b and R c Each is independently selected from C 1-6 Alkyl or phenyl; R d Selected independently from C 1-6 alkoxy or phenyl; R e Selected from C 1-6Alkyl, C 1-6 Halogenated alkyl or phenyl;

[0031] In some schemes, R2 is selected from NH2, OH, OR4, and NHR5; R4 is C 1-6 Alkyl or vinyl-substituted C 1-6 Alkyl group; R5 is selected from benzyloxycarbonyl, 9-fluorenyloxycarbonyl, tert-butoxycarbonyl, pivaloyl, acetyl, and triphenylmethyl.

[0032] In some schemes, R6 is selected from H or -OR f ;R f C 1-6 Alkyl, benzyl, or with one or more R f-1 Substituted benzyl; R f-1 Independently selected from halogens, C 1-6 Alkyl, nitro, or cyano groups.

[0033] In some schemes, R1 is independently selected C 1-6 Alkyl or -C(O)-R e ;R a R b and R c Each is independently selected from C 1-6 Alkyl or phenyl; R e Selected from C 1-6 Alkyl, C 1-6 Halogenated alkyl or phenyl;

[0034] In some schemes, R2 is selected from NH2, OR4, and NHR5; R4 is C 1-6 Alkyl or vinyl-substituted C 1-6 Alkyl group; R5 is selected from benzyloxycarbonyl, 9-fluorenyloxycarbonyl, tert-butoxycarbonyl, pivaloyl, acetyl, and triphenylmethyl.

[0035] In some schemes, R6 is -OR f ;R f C 1-6 Alkyl, benzyl, or with one or more R f-1 Substituted benzyl; R f-1 Independently selected from halogens, C 1-6 Alkyl, nitro, or cyano groups.

[0036] In some schemes, R1 is independently selected or -C(O)-R e ;R a R b and R c Each is independently selected from C 1-6 Alkyl or phenyl; R e Selected from C1-6 Alkyl, C 1-6 Halogenated alkyl or phenyl;

[0037] In some schemes, R2 is selected from NH2 or OR4; R4 is C 1-6 Alkyl or vinyl-substituted C 1-6 alkyl;

[0038] In some schemes, R6 is -OR f ;R f C 1-6 Alkyl, benzyl, and one or more R f-1 Substituted benzyl; R f-1 Selected independently from C 1-6 Alkyl, nitro, or cyano groups.

[0039] In some schemes, R1 is selected from or -C(O)-R e ;R a R b and R c Each is independently selected from C 1-6 Alkyl; R e Selected from C 1-6 Alkyl, C 1-6 Halogenated alkyl or phenyl;

[0040] In some schemes, R2 is NH2 or OR4; R4 is C 1-6 alkyl;

[0041] In some schemes, R6 is -OR f ;R f C 1-6 Alkyl, benzyl, and one or more R f-1 Substituted benzyl; R f-1 Selected independently from C 1-6 Alkyl or nitro groups.

[0042] In some schemes, R1 is selected from or -C(O)-R e ;R a R b and R c Each is independently selected from C 1-6 Alkyl; R e Selected from C 1-6 alkyl;

[0043] In some schemes, R2 is NH2 or OR4, and R4 is C. 1-3 alkyl;

[0044] In some schemes, R6 is -OR f ;R fC 1-3 Alkyl, benzyl, and one or more R f-1 Substituted benzyl; R f-1 Selected independently from C 1-6 Alkyl group.

[0045] In some schemes, R1 is selected from or -C(O)-R e ;R a R b and R c Each is independently selected from C 1-4 Alkyl; R e It is methyl;

[0046] In some schemes, R2 is NH2 or OR4; R4 is methyl;

[0047] In some schemes, R6 is -OR f ;R f It can be methyl or benzyl.

[0048] In some schemes, R1 is acetyl; R2 is NH2; and R6 is methoxy.

[0049] In some schemes, R1 is acetyl; R2 is NH2; and R6 is benzyloxy.

[0050] In some schemes, R1 is acetyl; R2 is OCH3; and R6 is methoxy.

[0051] In some schemes, R1 is tert-butyldimethylsilyl; R2 is NH2; and R6 is benzyloxy.

[0052] As a further improvement of the present invention, including but not limited to, the condensation reaction includes the following steps: reacting the compound of formula 1 in an organic solvent in the presence of a peptide coupling agent, selectively by adding an organic base at low temperature, to obtain compound 2.

[0053] As a further improvement of the present invention, the selective low-temperature reaction temperature is selected from -10 to 10°C, preferably -5 to 5°C.

[0054] As a further improvement to the present invention, the inventors have conducted in-depth research and discovered that low-temperature feeding reduces the occurrence of side reactions, thereby reducing the generation of impurities and improving the reaction yield.

[0055] As a further improvement of the present invention, including but not limited to, in the reaction of step 1, the peptide coupling agent is selected from one or any combination of TsTU and TsTU analogs, and the TsTU analogs are selected from O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N′,N′-tetramethylurea tetrafluoroborate (TOTU), O-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N′-N′-tetramethylurea tetrafluoroborate (TPTU) or 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea tetrafluoroborate (TATU), and the peptide coupling agent is preferably N,N,N′,N′-tetramethyl-O-(N-succinimide)urea tetrafluoroborate (TsTU).

[0056] As a further improvement of the present invention, including but not limited to, in the reaction of step 1, the organic base is selected from N-methylmorpholine (NMM), N,N-dimethylpiperazine, N-methylpiperidine, 4-dimethylaminopyridine, morpholine, pyridine, preferably N-methylmorpholine (NMM).

[0057] As a further improvement of the present invention, the inventors, through creative labor, finally determined suitable peptide coupling agents and bases. For example, as can be seen from sequences 1 to 14 in Table 1, when using peptide coupling agents other than TsTU and TsTU analogs, more intramolecular or intermolecular byproducts are generated, and the yield decreases with the increase of feed amount. When using TsTU and NMM in combination, not only can the solvent volume ratio be greatly reduced, but the peptide coupling agent and substrate can be added at one time, and the yield can also be increased to 83%. The combination of TsTU and TsTU analogs and NMM in the present invention not only has good selectivity, but also greatly reduces the byproducts generated when using other peptide coupling agents and bases. It also has the advantages of simple post-processing, good reproducibility, and high conversion rate, making it suitable for industrial production.

[0058] As a further improvement of the present invention, including but not limited to, the organic solvent being one or more of ether solvents, haloalkane solvents, ketone solvents, aromatic hydrocarbon solvents, nitrile solvents, ester solvents, sulfone solvents, and amide solvents, preferably one or two of sulfone solvents and amide solvents; the sulfone solvent is preferably dimethyl sulfoxide; the amide solvent may be one or more of N,N-dimethylformamide, N-methylpyrrolidone, and N,N-dimethylacetamide, preferably N,N-dimethylformamide; the ether solvent may be one or more of tetrahydrofuran, methyltetrahydrofuran, and dioxane, preferably tetrahydrofuran or dioxane; the haloalkane solvent may be one or more of dichloromethane, dichloroethane, and chloroform, preferably dichloromethane; the ketone solvent may be acetone; the aromatic hydrocarbon solvent may be one or two of toluene and pyridine, preferably toluene; the nitrile solvent is preferably acetonitrile; the ester solvent may be one or more of ethyl acetate, ethyl formate, and isopropyl acetate, preferably ethyl acetate;

[0059] As a further improvement of the present invention, including but not limited to, the present invention preferably uses an organic solvent, mainly selected from the viewpoints of reactivity, selectivity and ease of acquisition; the organic solvent in step 1 is selected from tetrahydrofuran, dioxane, dichloromethane, chloroform, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, ethyl acetate, toluene, acetone, acetonitrile, dimethyl sulfoxide, preferably N,N-dimethylformamide.

[0060] As a further improvement of the present invention, including but not limited to, in the reaction of step 1, the molar ratio of the compound of formula 1 to the peptide coupling agent is 1:(1 to 3), preferably 1:(1.5 to 2.8), for example 1:2, 1:2.5.

[0061] As a further improvement of the present invention, including but not limited to, in the reaction of step 1, the molar ratio of the compound of formula 1 to the organic base is 1:(1.5 to 5), preferably 1:(2 to 3.2), for example 1:2.5, 1:3.

[0062] As a further improvement of the present invention, including but not limited to, in the reaction of step 1, the volume of organic solvent used (mL) is 6 to 50 times the mass of compound of formula 1 (g), preferably 25 to 45 times, for example 30 times, 35 times, 36 times, 37 times, or 38 times.

[0063] As a further improvement of the present invention, including but not limited to, the reaction temperature in step 1 is 15-35°C, preferably 20-30°C.

[0064] As a further improvement of the present invention, including but not limited to, the reaction time in step 1 is 10 to 48 hours, preferably 10 to 36 hours, for example 24 hours, 28 hours, or 36 hours.

[0065] As a further improvement of the present invention, including but not limited to, after the condensation reaction in step 1 is completed, the solvent is evaporated and purified (e.g., by reverse phase preparation) to obtain compound 2.

[0066] The second aspect of the present invention also provides a method for preparing intermediate compound 2, which can be further used in the preparation of amatoxins or in the preparation of ADC conjugate toxin linkers.

[0067] Beneficial technical effects of the present invention:

[0068] 1. The present invention overcomes the shortcomings of the prior art and provides a method for synthesizing amanita kinase intermediate compound 2 based on the structural characteristics of amanita kinase itself.

[0069] 2. Patent CN111051310B uses DPPA as a peptide coupling agent, achieving a yield of 41%. This requires 10 molar equivalents of peptide coupling agent and base relative to the substrate, with a solvent volume of 120V, resulting in high reagent consumption and increased byproducts during scale-up, leading to a sharp decrease in yield. Through inventive effort, the inventors of this application unexpectedly discovered suitable peptide coupling agents and bases, significantly reducing the amount of peptide coupling agent and base used, lowering production costs, and also significantly increasing the yield by 42%.

[0070] 3. Patent CN108495840A uses PyBOP, HOBt, and DIPEA as condensation conditions, with a yield of only 35% and a solvent volume of 110V. Furthermore, PyBOP has the potential to explode, and the amount of byproducts increases during scale-up, resulting in a sharp decrease in yield, which is not conducive to industrial scale-up. This application selects a preparation method suitable for peptide coupling agents and bases, with mild conditions, easy control, good reproducibility, and a significantly improved yield compared to the prior art.

[0071] 4. Through experimental screening of different condensation conditions, the inventors found that the combination of TsTU and TsTU analogs with NMM was significantly more effective than other combinations, with stable reaction and high yield. During the experiment, it was found that when using peptide coupling agents other than TsTU and TsTU analogs, more intramolecular or intermolecular byproducts were generated, and the yield decreased with the increase of feed amount, and the subsequent products could not meet the ICH quality requirements.

[0072] 5. The present invention provides a method for preparing intermediate compound 2, which uses new compound 1 as raw material and performs a condensation reaction in the presence of a peptide coupling agent and an organic base to form peptide bonds, thereby obtaining new compound 2. The method is simple to operate, has a high conversion rate and high selectivity, and can obtain high-purity products with a purity of over 99%. It can also solve the scale-up problem and is significantly superior to the condensation conditions for forming peptide bonds reported in the prior art.

[0073] 6. The conditions of this invention not only solve the problem of using large amounts of solvent in existing technologies, but also the problem of batch feeding, further solving the problem that existing technologies can only produce in small batches. Since the raw materials have multiple sites that can undergo condensation and cyclization with carboxyl groups, the reactions disclosed in existing technologies usually need to be carried out in extremely dilute solvents, requiring huge amounts of solvent. The peptide coupling agent and substrate usually need to be added in multiple batches, and existing technologies can only achieve yields close to those reported in existing technologies at the milligram level. When existing technologies reduce the proportion of the reaction solvent or scale up the feeding, it leads to a sharp increase in intramolecular or intermolecular condensation byproducts, making large-scale production infeasible. Attached Figure Description

[0074] Figure 1 The LCMS spectrum of the product obtained in Example 5 of this invention;

[0075] Figure 2 The product obtained in Embodiment 5 of the present invention 1 H-NMR spectrum. Detailed Implementation

[0076] The preparation method of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0077] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available. Specific implementation examples:

[0079] Example 1

[0080]

[0081] Compound 1 (7 g, 6.87 mmol) and TsTU (4.14 g, 13.74 mmol) were dissolved in DMF (250 mL). The solution was cooled to 0 °C, and NMM (1.74 g, 17.17 mmol) was added with stirring. After the addition was complete, the mixture was allowed to warm to room temperature and reacted for 36 h. LCMS analysis showed that the reaction was complete. The solvent was evaporated, and the mixture was prepared by reverse phase reaction (ACN / H2O + TFA). The solution was then lyophilized to give a white solid (5.35 g, yield 77.8%).

[0082] Example 2

[0083]

[0084] Compound 1 (7 g, 6.39 mmol) and TsTU (4.81 g, 15.98 mmol) were dissolved in DMF (260 mL). The solution was cooled to 0 °C, and NMM (1.94 g, 19.17 mmol) was added with stirring. After the addition was complete, the mixture was allowed to warm to room temperature and reacted for 36 h. LCMS analysis showed that the reaction was complete. The solvent was evaporated, and the mixture was prepared by reverse phase reaction (ACN / H2O + TFA). The solution was then lyophilized to give a white solid (5.7 g, yield 82.8%).

[0085] Example 3

[0086]

[0087] Compound 1 (7 g, 6.77 mmol) and TsTU (4.08 g, 13.54 mmol) were dissolved in DMF (250 mL). The solution was cooled to 0 °C, and NMM (1.72 g, 16.99 mmol) was added with stirring. After the addition was complete, the mixture was allowed to warm to room temperature and reacted for 36 h. LCMS analysis showed that the reaction was complete. The solvent was evaporated, and the mixture was prepared by reverse phase reaction (ACN / H2O + TFA). The solution was then lyophilized to give a white solid (5.40 g, yield 78.5%).

[0088] Example 4

[0089]

[0090] Compound 1 (5 g, 4.03 mmol) and TsTU (2.43 g, 8.07 mmol) were dissolved in 150 mL of DMF. The solution was cooled to 0 °C, and NMM (1.02 g, 10.08 mmol) was added with stirring. After the addition was complete, the mixture was allowed to warm to room temperature and reacted for 28 h. LCMS analysis showed that the reaction was complete. The solvent was evaporated, and the mixture was prepared by reverse phase reaction (ACN / H2O + TFA). The solution was then lyophilized to give a white solid (3.73 g, yield 75.7%).

[0091] Example 5

[0092]

[0093] Compound 1 (21 g, 19.17 mmol) and TsTU (11.55 g, 38.35 mmol) were dissolved in DMF (800 mL). The solution was cooled to 0 °C, and NMM (4.85 g, 47.94 mmol) was added with stirring. After the addition was complete, the mixture was allowed to warm to room temperature and reacted for 24 h. LCMS analysis showed that the reaction proceeded completely. The solvent was evaporated, and the mixture was prepared by reverse phase reaction (ACN / H2O + TFA). The solution was lyophilized to give a white solid (17.12 g, yield 82.9%, LCMS purity 99.17%, retention time RT = 1.590 min). The LCMS spectrum is shown below. Figure 1 As shown, 1 H-NMR spectrum as shown Figure 2 As shown.

[0094] Example 6

[0095]

[0096] Referring to the method in Example 1, compound 1 (1 eq) and peptide coupling agent (2 eq) were dissolved in a solution of DMF, cooled to 0°C, and NMM was added with stirring. After the addition was complete, the mixture was naturally heated to room temperature and reacted for 24 h. The reaction was detected by LCMS. The solvent was evaporated by an oil pump, and the mixture was prepared by reverse phase (ACN / H2O+TFA). The compound of formula 2 was obtained by freeze drying. The detection results are shown in Table 1.

[0097] Table 1

[0098]

[0099]

[0100] Through inventive effort, the inventors have identified suitable peptide coupling agents and bases. For example, as shown in Tables 1 (Sequences 1-14), when using peptide coupling agents other than TsTU and its analogues, more intramolecular or intermolecular byproducts are generated, and the yield decreases with increasing feed amount. Only when using TsTU and its analogues in combination with NMM can the yield reach 83%, which is 42-48% higher than the yields of existing technologies CN111051310B, CN108495840A, and KR102092817B1. The combination of TsTU and NMM in this invention not only offers good selectivity but also avoids the byproducts generated when using other peptide coupling agents and bases. It also boasts advantages such as simple post-processing, good reproducibility, and high conversion rate, making it suitable for industrial production.

[0101] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing a compound of formula 2, wherein the reaction formula is as follows: Includes the following steps: Step 1: Compound 1 is subjected to a condensation reaction in an organic solvent in the presence of a peptide coupling agent and an organic base to obtain compound 2; Among them, R1 is independently selected from hydrogen, C 1-6 Alkyl, with one or more R d Replacement C 1-6 Alkyl or -C(O)-R e ;R a R b and R c Each is independently selected from C 1-6 Alkyl or phenyl; R d Selected independently from C 1-6 alkoxy or phenyl; R e Selected from C 1-6 Alkyl, C 1-6 Halogenated alkyl or phenyl; R2 is selected from NH2, -Z-R3, OH, OR4, and NHR5; PEG 1-8 Chain representation Z is selected from -C1-C5 alkylene-, -NH-, -C1-C8 alkylene-PEG. 1-8 -,-PEG 1-8 -or any combination thereof; R3 is selected from: R4 is C 1-6 Alkyl or vinyl-substituted C 1-6 alkyl; R5 is selected from benzyloxycarbonyl, 9-fluorenyloxycarbonyl, tert-butoxycarbonyl, tert-valeryl, acetyl, triphenylmethyl, preferably triphenylmethyl; R6 is selected from H, -OR f or n is 1, 2, 3, 4, 5, 6, 7; R f benzyl or formed by one or more R f-1 Substituted benzyl; R f-1 Independently selected from halogens, C 1-6 Alkyl, nitro, or cyano groups.

2. The preparation method according to claim 1, characterized in that, R1 is or -C(O)-R e R a R b and R c Each is independently selected from C 1-6 Alkyl, R e C 1-6 Alkyl; preferably R1 is or -C(O)-R e R a R b and R c Each is independently selected from C 1-4 Alkyl, R e It is methyl; And / or, R2 is NH2, OH, or OR4, and R4 is C 1-6 Alkyl or vinyl-substituted C 1-6 Alkyl group; preferably R2 is NH2, OH or OR4, and R4 is C 1-6 Alkyl; further preferably R2 is NH2 or OR4; R4 is methyl; And / or, R6 is -OR f ;R f benzyl or formed by one or more R f-1 Substituted benzyl; R f-1 Independently selected from halogens, C 1-6 Alkyl, nitro, or cyano; preferably R6 is -OR f R f For benzyl groups are one or more R groups f-1 Substituted benzyl, R f-1 Selected independently from C 1-6 Alkyl group; further preferably R6 is -OR f R f It can be methyl or benzyl.

3. The preparation method according to claim 1 or 2, characterized in that, The condensation reaction comprises the following steps: reacting compound 1 in an organic solvent in the presence of a peptide coupling agent, selectively with the addition of an organic base at low temperature, to obtain compound 2.

4. The preparation method according to claim 3, characterized in that, The low-temperature reaction temperature is selected from -10 to 10℃, preferably -5 to 5℃.

5. The preparation method according to claim 1 or 2, characterized in that, In step 1, the peptide coupling agent is selected from one or any combination of N,N,N′,N′-tetramethyl-O-(N-succinimide)urea tetrafluoroborate (TsTU) and TsTU analogs.

6. The preparation method according to claim 5, characterized in that, In step 1, the TsTU analogue is selected from O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N′,N′-tetramethylurea tetrafluoroborate, O-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N′-N′-tetramethylurea tetrafluoroborate, or 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea tetrafluoroborate.

7. The preparation method according to claim 1 or 2, characterized in that, In step 1, the organic base is selected from N-methylmorpholine, N,N-dimethylpiperazine, N-methylpiperidine, 4-dimethylaminopyridine, morpholine, pyridine, and preferably N-methylmorpholine.

8. The preparation method according to claim 1 or 2, characterized in that, In step 1, the organic solvent is one or more of ether solvents, haloalkane solvents, ketone solvents, aromatic hydrocarbon solvents, nitrile solvents, ester solvents, sulfone solvents, and amide solvents, preferably one or two of sulfone solvents and amide solvents; the sulfone solvent is preferably dimethyl sulfoxide; the amide solvent can be one or more of N,N-dimethylformamide, N-methylpyrrolidone, and N,N-dimethylacetamide, preferably N,N-dimethylformamide; the ether solvent can be one or more of tetrahydrofuran, methyltetrahydrofuran, and dioxane, preferably tetrahydrofuran or dioxane; the haloalkane solvent can be one or more of dichloromethane, dichloroethane, and chloroform, preferably dichloromethane; the ketone solvent can be acetone; the aromatic hydrocarbon solvent can be one or two of toluene and pyridine, preferably toluene; the nitrile solvent is preferably acetonitrile; the ester solvent can be one or more of ethyl acetate, ethyl formate, and isopropyl acetate, preferably ethyl acetate; And / or, preferably, the organic solvent in step 1 is selected from tetrahydrofuran, dioxane, dichloromethane, chloroform, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, ethyl acetate, toluene, acetone, acetonitrile, dimethyl sulfoxide, and preferably N,N-dimethylformamide.

9. The preparation method according to claim 1 or 2, characterized in that, The preparation method of the compound of Formula 2 satisfies one or more of the following conditions: 1) In the reaction of step 1, the molar ratio of compound of formula 1 to peptide coupling agent is 1:(1-3), preferably 1:(1.5-2.8); 2) In the reaction of step 1, the molar ratio of compound of formula 1 to organic base is 1:(1.5-5), preferably 1:(2-3.2); 3) In step 1, the volume of organic solvent used (mL) is 6 to 50 times the mass of compound 1 (g), preferably 30 to 45 times. 4) In step 1, the reaction temperature is 15–35°C, preferably 20–30°C; 5) In step 1, the reaction time is 5 to 48 hours, preferably 10 to 36 hours; 6) The condensation reaction in step 1 includes the following steps: dissolving compound 1, TsTU and TsTU analogues in an organic solvent, cooling to -5 to 5°C, adding NMM with stirring, and then naturally heating to 20 to 30°C for 10 to 36 hours. 7) The condensation reaction in step 1 includes the following steps: dissolving compound 1, TsTU and TsTU analogues in DMF, cooling to -5 to 5°C, adding NMM with stirring, and then naturally heating to 20 to 30°C for 10 to 36 hours. 8) After the condensation reaction in step 1 is completed, the solvent is evaporated and purified to obtain compound 2.

10. A method for preparing amatoxins, comprising the method for preparing the compound of formula 2 as described in any one of claims 1 to 9; And / or, a method for preparing a compound of formula 2 as described in any one of claims 1 to 9 may be further used for the preparation of amatoxins or for the preparation of ADC conjugate toxin linkers.

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