阅读说明：本技术 一种高效抗肿瘤活性多肽Tubulysin M的制备方法 (Preparation method of efficient anti-tumor active polypeptide Tubulysin M ) 是由 吴正治 龙伯华 于 2020-05-22 设计创作，主要内容包括：本发明提供了一种高效抗肿瘤活性多肽Tubulysin M的制备方法,包括步骤S1,提供化合物2；步骤S2,以化合物2作为关键原料制备活性多肽Tubulysin M。本发明的制备方法采用了创新性的路线,优化了活性多肽Tubulysin M的全合成过程,总产率较高,立体选择性好。(The invention provides a preparation method of a high-efficiency anti-tumor active polypeptide Tubulysin M, which comprises the steps of S1, providing a compound 2; step S2, preparing the active polypeptide Tubulysin M by using the compound 2 as a key raw material. The preparation method provided by the invention adopts an innovative route, optimizes the whole synthesis process of the active polypeptide Tubulysin M, and has the advantages of high total yield and good stereoselectivity.)

1. A preparation method of a high-efficiency anti-tumor active polypeptide Tubulysin M is characterized by comprising the following steps:

step S1, providing compound 2;

step S2, preparing an active polypeptide Tubulysin M by using the compound 2 as a key raw material;

wherein, the chemical formula of the compound 2 is as follows:

the chemical formula of the active polypeptide TubulysinM is as follows:

2. the method according to claim 1, wherein the step S2 is synthesized by the following steps:

wherein, the chemical formulas of the compound 3, the compound 7 and the compound 9 used in the reaction formula are as follows:

the method comprises the following steps:

(1) heating and refluxing the compound 2 in trifluoroacetic acid until the reaction is finished, concentrating under reduced pressure, and drying in vacuum to obtain an intermediate;

dissolving the intermediate in dichloromethane, adding a compound 3 and HATU, uniformly stirring, adding diisopropylethylamine, stirring at room temperature for reaction, adding water for quenching reaction, extracting, then combining organic phases, sequentially washing with water and saturated salt solution, separating liquid, adding anhydrous sodium sulfate to the organic phases for drying, concentrating under reduced pressure to obtain a crude product, and performing column chromatography separation by using a mixed solution of petroleum ether and ethyl acetate as an eluent to obtain a compound 4;

(2) dissolving the compound 4 in dichloromethane, adding 2, 6-dimethylpyridine and tert-butyldimethylsilyl trifluoromethanesulfonate under cooling in an ice-water bath, heating to room temperature, stirring for reaction, adding water for quenching reaction, extracting and combining organic phases, washing with dilute hydrochloric acid and saturated salt water in sequence, separating liquid, adding the organic phases, drying with anhydrous sodium sulfate, concentrating under reduced pressure to obtain a crude product, and performing column chromatography separation by using a mixed solution of petroleum ether and ethyl acetate as an eluent to obtain a compound 5;

(3) dissolving the compound 5 in DMF, adding 60% sodium hydride solid under cooling in an ice water bath, then adding methyl iodide, slowly heating to room temperature, stirring for reaction, cooling in the ice water bath, adding a saturated ammonium chloride aqueous solution for quenching reaction, extracting and combining organic phases, washing with water and saturated salt solution, separating liquid, adding the organic phase and anhydrous sodium sulfate for drying, concentrating under reduced pressure to obtain a crude product, performing column chromatography by using a mixed solution of petroleum ether and ethyl acetate as an eluent, and separating to obtain a compound 6;

(4) dissolving the compound 6 in a tetrahydrofuran/water mixed solvent, adding a sodium hydroxide solid under the cooling of an ice water bath, then heating to room temperature, stirring for reaction, cooling the reaction product in the ice water bath, acidifying the solution until the pH value is 2, extracting and combining organic phases, washing with water and saturated saline solution in sequence, separating liquid, adding the organic phase and anhydrous sodium sulfate for drying, and concentrating under reduced pressure to obtain an intermediate acid;

dissolving the intermediate in dichloromethane, adding a compound 7 and HATU, uniformly stirring, adding diisopropylethylamine, stirring at room temperature for reaction, adding water for quenching reaction, extracting and combining organic phases, sequentially washing with water and saturated salt solution, separating liquid, adding anhydrous sodium sulfate to the organic phase for drying, concentrating under reduced pressure to obtain a crude product, performing column chromatography by using a mixed solution of petroleum ether and ethyl acetate as an eluent, and separating to obtain a compound 8;

(5) dissolving the compound 8 in a tetrahydrofuran/water mixed solvent, adding triphenylphosphine, heating and refluxing to react until the reaction is finished, concentrating under reduced pressure, removing water by using toluene, and drying in vacuum to obtain an intermediate amine;

dissolving the intermediate amine in dichloromethane, adding a compound 9 and HATU, uniformly stirring, adding diisopropylethylamine, stirring at room temperature for reaction, adding water for quenching reaction, extracting and combining organic phases, sequentially washing with water and saturated salt solution, separating liquid, adding an organic phase, drying with anhydrous sodium sulfate, concentrating under reduced pressure to obtain a crude product, performing column chromatography by using a mixed solution of petroleum ether and ethyl acetate as an eluent, and separating to obtain a compound 10;

(6) dissolving the compound 10 in methanol, adding ammonium fluoride solid, heating and refluxing for reaction, concentrating under reduced pressure, diluting with water, extracting and combining organic phases, washing with saturated saline solution, separating liquid, adding the organic phases, drying with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a first intermediate;

dissolving the first intermediate in a tetrahydrofuran/water mixed solvent, adding a sodium hydroxide solid, heating and refluxing for reaction, cooling in an ice water bath, acidifying until the pH value of the solution is 2, extracting and combining organic phases, washing with water and saturated saline solution in sequence, separating the liquid, adding organic phase and anhydrous sodium sulfate, drying, and concentrating under reduced pressure to obtain a second intermediate;

dissolving the second intermediate in pyridine, adding acetic anhydride, stirring at room temperature for reaction, adding water for quenching reaction, cooling in an ice water bath, acidifying until the pH value of the solution is 2, extracting and combining organic phases, washing with water and saturated salt solution in sequence, separating liquid, adding organic phase and anhydrous sodium sulfate for drying, and concentrating under reduced pressure to obtain a third intermediate;

dissolving the third intermediate in dichloromethane, adding trifluoroacetic acid, stirring at room temperature for reaction, concentrating under reduced pressure, and drying to obtain a fourth intermediate;

dissolving the fourth intermediate in a mixed solvent of acetonitrile and methanol, adding a formaldehyde aqueous solution, stirring at room temperature for reaction, adding sodium cyanoborohydride, stirring for reaction till the reaction is finished, dropwise adding glacial acetic acid till the pH value of the solution is 5, then stirring at room temperature for reaction till the reaction is finished, concentrating under reduced pressure, separating and purifying by silica gel column chromatography, and pulping and purifying by isopropyl ether to obtain the compound 1.

3. The production method according to claim 2, wherein in the step (1), the ratio of compound 2: compound 3: HATU: the molar ratio of diisopropylethylamine is 1: 1.1-1.5: 2-3: 4-6, preferably 1: 1.2: 3: 6;

preferably, in the mixed liquid of petroleum ether and ethyl acetate, the ratio of petroleum ether: the volume ratio of ethyl acetate is 4: 1;

preferably, the solvent used for the extraction is dichloromethane.

4. The production method according to claim 2, wherein in the step (2), the ratio of compound 4: 2, 6-lutidine: the molar ratio of tert-butyldimethylsilyl trifluoromethanesulfonate is 1: 3-5: 1.5-2, preferably 1: 3: 1.5;

preferably, 2, 6-dimethylpyridine and tert-butyldimethylsilyl trifluoromethanesulfonate are added under the cooling of an ice water bath, the temperature is raised to the room temperature after 20-30 min, stirring is carried out for reaction for 3-4 h, and more preferably, the temperature is raised to the room temperature after 30min, stirring is carried out for reaction for 4 h;

preferably, the dilute hydrochloric acid is 1M dilute hydrochloric acid;

preferably, in the mixed liquid of petroleum ether and ethyl acetate, the ratio of petroleum ether: the volume ratio of ethyl acetate is 8: 1.

5. the production method according to claim 2, wherein in the step (3), the ratio of compound 5: sodium hydride: the molar ratio of methyl iodide is 1: 3-5: 1.5-2, preferably 1: 5: 1.5;

preferably, the solvent used for the extraction is ethyl acetate;

preferably, in the mixed liquid of petroleum ether and ethyl acetate, the ratio of petroleum ether: the volume ratio of ethyl acetate is 10: 1.

6. the method according to claim 2, wherein in the step (4), the ratio of compound 6: sodium hydroxide: compound 7: HATU: the molar ratio of diisopropylethylamine is 1: 10-15: 1.1-1.5: 2-3: 4-6, preferably 1: 10: 1.2: 3: 6;

preferably, adding sodium hydroxide solid under cooling in an ice water bath, heating to room temperature for 20-30 min, stirring and reacting for 2-3 h, more preferably heating to room temperature for 30min, and stirring and reacting for 2 h;

preferably, the ratio of tetrahydrofuran in the tetrahydrofuran/water mixed solvent is: the volume ratio of water is 1: 1;

preferably, in the mixed liquid of petroleum ether and ethyl acetate, the ratio of petroleum ether: the volume ratio of ethyl acetate is 8: 1.

7. the production method according to claim 2, wherein in the step (5), the ratio of compound 8: triphenylphosphine: compound 9: HATU: the molar ratio of diisopropylethylamine is 1: 10-15: 1.1-1.5: 2-3: 4-6, preferably 1: 10: 1.2: 3: 6;

preferably, the ratio of tetrahydrofuran in the tetrahydrofuran/water mixed solvent is: the volume ratio of water is 20: 1;

preferably, in the mixed solution of petroleum ether and ethyl acetate, the ratio of petroleum ether: the volume ratio of ethyl acetate is 4: 1.

8. the method according to claim 2, wherein in the step (6), the ratio of compound 10: ammonium fluoride: sodium hydroxide: acetic anhydride: trifluoroacetic acid: formaldehyde: molar ratio of sodium cyanoborohydride 1: 50-100: 10-15: 20-50: 20-30: 10-20: 10-20, preferably 1: 100: 10: 30: 20: 20: 20;

preferably, the ratio of tetrahydrofuran in the tetrahydrofuran/water mixed solvent is: the volume ratio of water is 1: 1;

preferably, the ratio of acetonitrile in the mixed solvent of acetonitrile and methanol is: the volume ratio of methanol is 1: 1;

preferably, the mass fraction of the formaldehyde aqueous solution is 37%;

preferably, the mobile phase used in the silica gel column chromatography separation and purification is methanol: volume ratio of dichloromethane is 1: 10 of the mixture.

9. The method according to claim 1, wherein in step S1, compound 2 is synthesized by the following scheme:

the method comprises the following steps:

step 11, dissolving a starting material L-valinol 2-1 in a tetrahydrofuran/water mixed solvent, adding a sodium bicarbonate solid and benzyl chloroformate, and reacting at room temperature overnight to obtain a compound 2-2;

step 12, dissolving the compound 2-2 obtained in the step 1 in acetonitrile, adding 2-iodoxybenzoic acid, and carrying out heating reflux reaction to obtain an intermediate aldehyde 2-3;

dissolving the intermediate aldehyde 2-3 in dichloromethane, adding a Wittig reagent 2-4 and tetramethylguanidine, and carrying out heating reflux reaction to obtain a compound 2-5;

step 13, dissolving the compound 2-5 in a tetrahydrofuran/water mixed solvent, adding a sodium hydroxide solid, and heating and refluxing for reaction to obtain a compound 2-6;

taking the compound 2-6 as a reaction substrate, adding a coupling reagent and triphenylphosphine, reacting with the compound 2-7 to prepare a thiazoline intermediate product, adding an oxidation reagent, and synthesizing the compound 2-8 by a one-pot method;

step 14, dissolving the compound 2-8 in tetrahydrofuran, adding concentrated hydrochloric acid, and reacting at room temperature to obtain a compound 2-9;

and step 15, under the protection of inert gas, dissolving the compound 2-9 in anhydrous tetrahydrofuran, adding (S) -Me-CBS and borane dimethyl sulfide complex under the cooling of ice-water bath, and heating to room temperature for reaction to obtain the compound 2.

10. The method according to claim 9, wherein in step 11, the ratio of L-valinol 2-1: sodium bicarbonate: the molar ratio of benzyl chloroformate is 1: 3-5: 1-1.02, preferably 1: 3: 1;

preferably, the reaction time of the step 11 is 10-15 hours, more preferably 12 hours, after the reaction is finished, the mixture is concentrated under reduced pressure, extracted by ethyl acetate, the organic phases are combined and washed by saturated saline solution, the organic phase is collected by liquid separation, then anhydrous sodium sulfate is added into the organic phase for drying, and the compound 2-2 is obtained by filtering, concentrating and draining;

preferably, in step 12, compound 2-2: 2-iodoxybenzoic acid: the molar ratio of the Wittig reagent 2-4 is 1: 2-3: 1.2-1.5, preferably 1: 2: 1.5.

Technical Field

The invention relates to the technical field of medicines, and in particular relates to a preparation method of a high-efficiency anti-tumor active polypeptide TubulysinM.

Background

The tetrapeptide compound Tubulisins was extracted from myxobacteria by Hofle et al in 2000, but its crystal structure was not determined until 2004. Tubulysin A-Z is a family of compounds, as shown in Table 1, which are natural products.

TABLE 1 structural formulas of Tubulisins family compounds

The active polypeptide Tubulysin M is obtained by structural modification of chemists, namely, sensitive acetal is replaced by stable methyl. Researches show that the active polypeptide Tubulysin M has extremely strong cytotoxic activity and IC (Integrated Circuit) on human cancer cells₅₀Values in the lower nanomolar range are shown in Table 2 (ref J.Am.chem.Soc.,2016,138(5): 1698-1708). The active polypeptide Tubulysin M is a cytotoxic Tubulysin which inhibits tubulin polymerization and leads to cell cycle arrest and apoptosis. In addition, researches show that the active polypeptide Tubulysin M also has the advantages of angiogenesis inhibition and multi-drug resistance. Compared with other anticancer drugs, the active polypeptide Tubulysin M has obvious advantages in the aspect of water solubility, and shows super-strong anticancer activity on specific cancer cells, so that the active polypeptide Tubulysin M becomes one of the well-concerned target molecules in the research and development of novel anticancer drugs.

TABLE 2 in vitro cytotoxic Activity of the active polypeptide Tubulysin M on human cancer cells

From the development process of the active polypeptide Tubulysin M for a short period of time, the active polypeptide Tubulysin M is receiving more and more attention due to the unique biological activity thereof and has become a star molecule in anti-tumor drugs. However, the synthesis is not ideal, most of the synthetic routes reported in the literature on the chemical synthesis of the active polypeptide Tubulysin M and analogues thereof up to now are not perfect, and a great space for further exploration exists.

Therefore, how to provide a simple and rapid route which can be synthesized in large quantity has important significance for further biological research and clinical application realization of the active polypeptide Tubulysin M.

In view of this, the present application is specifically made.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of the active polypeptide Tubulysin M, which adopts an innovative route, optimizes the whole synthesis process of the active polypeptide Tubulysin M, and has higher total yield and good stereoselectivity.

In order to achieve the technical effects, the basic idea of the technical scheme adopted by the invention is as follows:

a preparation method of a high-efficiency anti-tumor active polypeptide Tubulysin M, which comprises the following steps:

step S1, providing compound 2;

step S2, preparing an active polypeptide Tubulysin M by using the compound 2 as a key raw material;

wherein, the chemical formula of the compound 2 is as follows:

the chemical formula of the active polypeptide Tubulysin M is as follows:

as an embodiment, the step S2 adopts the following synthetic route:

wherein, the chemical formulas of the compound 3, the compound 7 and the compound 9 used in the reaction formula are as follows:

the method comprises the following steps:

(1) heating and refluxing the compound 2 in trifluoroacetic acid until the reaction is finished, concentrating under reduced pressure, and drying in vacuum to obtain an intermediate;

dissolving the intermediate in dichloromethane, adding a compound 3 and HATU, uniformly stirring, adding diisopropylethylamine, stirring at room temperature for reaction, adding water for quenching reaction, extracting, then combining organic phases, sequentially washing with water and saturated salt solution, separating liquid, adding anhydrous sodium sulfate to the organic phases for drying, concentrating under reduced pressure to obtain a crude product, and performing column chromatography separation by using a mixed solution of petroleum ether and ethyl acetate as an eluent to obtain a compound 4;

(2) dissolving the compound 4 in dichloromethane, adding 2, 6-dimethylpyridine and tert-butyldimethylsilyl trifluoromethanesulfonate under cooling in an ice-water bath, heating to room temperature, stirring for reaction, adding water for quenching reaction, extracting and combining organic phases, washing with dilute hydrochloric acid and saturated salt water in sequence, separating liquid, adding the organic phases, drying with anhydrous sodium sulfate, concentrating under reduced pressure to obtain a crude product, and performing column chromatography separation by using a mixed solution of petroleum ether and ethyl acetate as an eluent to obtain a compound 5;

(3) dissolving the compound 5 in DMF, adding 60% sodium hydride solid under cooling in an ice water bath, then adding methyl iodide, slowly heating to room temperature, stirring for reaction, cooling in the ice water bath, adding a saturated ammonium chloride aqueous solution for quenching reaction, extracting and combining organic phases, washing with water and saturated salt solution, separating liquid, adding the organic phase and anhydrous sodium sulfate for drying, concentrating under reduced pressure to obtain a crude product, performing column chromatography by using a mixed solution of petroleum ether and ethyl acetate as an eluent, and separating to obtain a compound 6;

(4) dissolving the compound 6 in a tetrahydrofuran/water mixed solvent, adding a sodium hydroxide solid under the cooling of an ice water bath, then heating to room temperature, stirring for reaction, cooling the reaction product in the ice water bath, acidifying the solution until the pH value is 2, extracting and combining organic phases, washing with water and saturated saline solution in sequence, separating liquid, adding the organic phase and anhydrous sodium sulfate for drying, and concentrating under reduced pressure to obtain an intermediate acid;

dissolving the intermediate in dichloromethane, adding a compound 7 and HATU, uniformly stirring, adding diisopropylethylamine, stirring at room temperature for reaction, adding water for quenching reaction, extracting and combining organic phases, sequentially washing with water and saturated salt solution, separating liquid, adding anhydrous sodium sulfate to the organic phase for drying, concentrating under reduced pressure to obtain a crude product, performing column chromatography by using a mixed solution of petroleum ether and ethyl acetate as an eluent, and separating to obtain a compound 8;

(5) dissolving the compound 8 in a tetrahydrofuran/water mixed solvent, adding triphenylphosphine, heating and refluxing to react until the reaction is finished, concentrating under reduced pressure, removing water by using toluene, and drying in vacuum to obtain an intermediate amine;

dissolving the intermediate amine in dichloromethane, adding a compound 9 and HATU, uniformly stirring, adding diisopropylethylamine, stirring at room temperature for reaction, adding water for quenching reaction, extracting and combining organic phases, sequentially washing with water and saturated salt solution, separating liquid, adding an organic phase, drying with anhydrous sodium sulfate, concentrating under reduced pressure to obtain a crude product, performing column chromatography by using a mixed solution of petroleum ether and ethyl acetate as an eluent, and separating to obtain a compound 10;

(6) dissolving the compound 10 in methanol, adding ammonium fluoride solid, heating and refluxing for reaction, concentrating under reduced pressure, diluting with water, extracting and combining organic phases, washing with saturated saline solution, separating liquid, adding the organic phases, drying with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a first intermediate;

dissolving the first intermediate in a tetrahydrofuran/water mixed solvent, adding a sodium hydroxide solid, heating and refluxing for reaction, cooling in an ice water bath, acidifying until the pH value of the solution is 2, extracting and combining organic phases, washing with water and saturated saline solution in sequence, separating the liquid, adding organic phase and anhydrous sodium sulfate, drying, and concentrating under reduced pressure to obtain a second intermediate;

dissolving the second intermediate in pyridine, adding acetic anhydride, stirring at room temperature for reaction, adding water for quenching reaction, cooling in an ice water bath, acidifying until the pH value of the solution is 2, extracting and combining organic phases, washing with water and saturated salt solution in sequence, separating liquid, adding organic phase and anhydrous sodium sulfate for drying, and concentrating under reduced pressure to obtain a third intermediate;

dissolving the third intermediate in dichloromethane, adding trifluoroacetic acid, stirring at room temperature for reaction, concentrating under reduced pressure, and drying to obtain a fourth intermediate;

dissolving the fourth intermediate in a mixed solvent of acetonitrile and methanol, adding a formaldehyde aqueous solution, stirring at room temperature for reaction, adding sodium cyanoborohydride, stirring for reaction till the reaction is finished, dropwise adding glacial acetic acid till the pH value of the solution is 5, then stirring at room temperature for reaction till the reaction is finished, concentrating under reduced pressure, separating and purifying by silica gel column chromatography, and pulping and purifying by isopropyl ether to obtain the compound 1.

As an embodiment, in the step (1), compound 2: compound 3: HATU: the molar ratio of diisopropylethylamine is 1: 1.1-1.5: 2-3: 4-6, preferably 1: 1.2: 3: 6.

preferably, in the step (1), the ratio of petroleum ether in the mixed solution of petroleum ether and ethyl acetate is: the volume ratio of ethyl acetate is 4: 1.

preferably, in the step (1), the solvent used for the extraction is dichloromethane.

As an embodiment, in the step (2), compound 4: 2, 6-lutidine: the molar ratio of tert-butyldimethylsilyl trifluoromethanesulfonate is 1: 3-5: 1.5-2, preferably 1: 3: 1.5.

preferably, in the step (2), 2, 6-lutidine and tert-butyldimethylsilyl trifluoromethanesulfonate are added under cooling of an ice-water bath, and the mixture is heated to room temperature after 20-30 min and stirred for reaction for 3-4 h, more preferably, the mixture is heated to room temperature after 30min and stirred for reaction for 4 h.

Preferably, in the step (2), the diluted hydrochloric acid is 1M diluted hydrochloric acid.

Preferably, in the step (2), the ratio of petroleum ether in the mixed solution of petroleum ether and ethyl acetate is: the volume ratio of ethyl acetate is 8: 1.

as an embodiment, in the step (3), compound 5: sodium hydride: the molar ratio of methyl iodide is 1: 3-5: 1.5-2, preferably 1: 5: 1.5.

preferably, in the step (3), the solvent used for the extraction is ethyl acetate.

Preferably, in the step (3), the ratio of petroleum ether in the mixture of petroleum ether and ethyl acetate is: the volume ratio of ethyl acetate is 10: 1.

as an embodiment, in the step (4), compound 6: sodium hydroxide: compound 7: HATU: the molar ratio of diisopropylethylamine is 1: 10-15: 1.1-1.5: 2-3: 4-6, preferably 1: 10: 1.2: 3: 6.

preferably, in the step (4), sodium hydroxide solid is added under cooling of an ice water bath, and the mixture is heated to room temperature for 20-30 min and stirred for reaction for 2-3 h, more preferably 30min and the mixture is heated to room temperature and stirred for reaction for 2 h.

Preferably, in the step (4), the ratio of tetrahydrofuran in the tetrahydrofuran/water mixed solvent is: the volume ratio of water is 1: 1.

Preferably, in the step (4), the ratio of petroleum ether in the mixed solution of petroleum ether and ethyl acetate is: the volume ratio of ethyl acetate is 8: 1.

as an embodiment, in the step (5), compound 8: triphenylphosphine: compound 9: HATU: the molar ratio of diisopropylethylamine is 1: 10-15: 1.1-1.5: 2-3: 4-6, preferably 1: 10: 1.2: 3: 6;

preferably, in the step (5), the ratio of tetrahydrofuran in the tetrahydrofuran/water mixed solvent is: the volume ratio of water is 20: 1.

Preferably, in the step (5), the ratio of petroleum ether in the mixed solution of petroleum ether and ethyl acetate is: the volume ratio of ethyl acetate is 4: 1.

as an embodiment, in the step (6), compound 10: ammonium fluoride: sodium hydroxide: acetic anhydride: trifluoroacetic acid: formaldehyde: molar ratio of sodium cyanoborohydride 1: 50-100: 10-15: 20-50: 20-30: 10-20: 10-20, preferably 1: 100: 10: 30: 20: 20: 20.

preferably, in the step (6), the ratio of tetrahydrofuran in the tetrahydrofuran/water mixed solvent is: the volume ratio of water is 1: 1.

Preferably, in the step (6), the ratio of acetonitrile in the mixed solvent of acetonitrile and methanol is: the volume ratio of methanol was 1: 1.

Preferably, in the step (6), the mass fraction of the aqueous formaldehyde solution is 37%.

Preferably, in the step (6), the mobile phase used in the silica gel column chromatography separation and purification is methanol: volume ratio of dichloromethane is 1: 10 of the mixture.

As an embodiment, in step S1, compound 2 is synthesized by the following scheme:

the method comprises the following steps:

step 11, dissolving a starting material L-valinol 2-1 in a tetrahydrofuran/water mixed solvent, adding a sodium bicarbonate solid and benzyl chloroformate, and reacting at room temperature overnight to obtain a compound 2-2;

step 12, dissolving the compound 2-2 obtained in the step 1 in acetonitrile, adding 2-iodoxybenzoic acid, and carrying out heating reflux reaction to obtain an intermediate aldehyde 2-3;

dissolving the intermediate aldehyde 2-3 in dichloromethane, adding a Wittig reagent 2-4 and tetramethylguanidine, and carrying out heating reflux reaction to obtain a compound 2-5;

step 13, dissolving the compound 2-5 in a tetrahydrofuran/water mixed solvent, adding a sodium hydroxide solid, and heating and refluxing for reaction to obtain a compound 2-6;

taking the compound 2-6 as a reaction substrate, adding a coupling reagent and triphenylphosphine, reacting with the compound 2-7 to prepare a thiazoline intermediate product, adding an oxidation reagent, and synthesizing the compound 2-8 by a one-pot method;

step 14, dissolving the compound 2-8 in tetrahydrofuran, adding concentrated hydrochloric acid, and reacting at room temperature to obtain a compound 2-9;

and step 15, under the protection of inert gas, dissolving the compound 2-9 in anhydrous tetrahydrofuran, adding (S) -Me-CBS and borane dimethyl sulfide complex under the cooling of ice-water bath, and heating to room temperature for reaction to obtain the compound 2.

In one embodiment, in step 11, the ratio of L-valinol 2-1: sodium bicarbonate: the molar ratio of benzyl chloroformate is 1: 3-5: 1-1.02, preferably 1: 3: 1.

preferably, the reaction time of the step 11 is 10-15h, more preferably 12h, after the reaction is finished, the reaction solution is concentrated under reduced pressure, extracted by ethyl acetate, the organic phases are combined and washed by saturated saline solution, the organic phases are collected by liquid separation, then anhydrous sodium sulfate is added into the organic phases for drying, and the compound 2-2 is obtained by filtering, concentrating and draining.

Preferably, in step 12, compound 2-2: 2-iodoxybenzoic acid: the molar ratio of the Wittig reagent 2-4 is 1: 2-3: 1.2-1.5, preferably 1: 2: 1.5.

compared with the prior art, the invention has the following beneficial effects:

1. the preparation method provided by the invention adopts an innovative route, optimizes the whole synthesis process of the active polypeptide Tubulysin M, and has the advantages of high total yield and good stereoselectivity.

2. The synthesis route adopted by the invention utilizes renewable resources, can be recovered, uses reagents with low toxicity, has little environmental pollution after reaction treatment, and meets the green chemical standard.

3. The preparation method has the advantages of convenient experimental operation, mild reaction conditions, simple separation and purification, suitability for mass preparation and convenience for realizing industrialization.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention are clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.

In the invention, HATU: 2- (7-azabenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate; DIPEA: diisopropylethylamine; DCM: dichloromethane; TBSOTf: tert-butyldimethylsilyl trifluoromethanesulfonate. English abbreviations for other used chemical agents are understood as being conventional in the art.