阅读说明：本技术 一种星孢菌素提取方法 (Method for extracting staurosporine ) 是由 付光明 戴绪霖 霍育生 张文凯 于 2019-11-18 设计创作，主要内容包括：本发明公开了一种星孢菌素的提取方法,包含以下步骤：1)板框过滤；2)丙酮提取；3)萃取；4)结晶及真空干燥。本发明所揭示的星孢菌素提取方法生产成本低,提取周期短,操作简单,方法稳定,易于放大,在工业化生产中具有极高的实用价值。(The invention discloses a method for extracting staurosporine, which comprises the following steps: 1) filtering by a plate frame; 2) extracting with acetone; 3) extracting; 4) crystallizing and vacuum drying. The method for extracting the staurosporine disclosed by the invention has the advantages of low production cost, short extraction period, simplicity in operation, stability, easiness in amplification and extremely high practical value in industrial production.)

1. A method for extracting staurosporine is characterized by comprising the following steps:

step 1: adding a filter aid into the staurosporine fermentation liquor, and filtering through a plate frame to obtain staurosporine fungus residues;

step 2: extracting the staurosporine fungi residues in the step 1 by using a solvent to obtain an extraction concentrated solution;

and step 3: adding an organic solvent A, an aqueous solution and an extraction aid into the extraction concentrated solution obtained in the step 2, extracting and layering, drying and filtering an organic phase, concentrating, adding an organic solvent B, crystallizing and filtering to obtain a crude product of the staurosporine;

and 4, step 4: dissolving the crude staurosporine in the step 3 by using a proper amount of mixed solvent, filtering by using a filter membrane, concentrating under reduced pressure, adding an organic solvent B for crystallization, and filtering to obtain a staurosporine crystal;

and 5: and (4) drying the staurosporine crystal product in the step (4) to obtain a staurosporine finished product.

2. The method according to claim 1, wherein the solvent of step 2 is one of isopropanol, ethanol, acetone and methanol.

3. The method according to claim 1, wherein the organic solvent A in step 3 is one of butyl acetate, ethyl acetate, dichloromethane and chloroform.

4. The method according to claim 1, wherein the aqueous solution in step 3 is an aqueous solution formed by one of potassium carbonate, sodium hydroxide, potassium hydroxide and ammonia, and the concentration of the aqueous solution is preferably 0.1-3.0% by mass.

5. The method of claim 1, wherein the extraction aid of step 3 is one of methanol, ethanol, and acetone.

6. The method according to claim 1, wherein the organic solvent B in step 3 and step 4 is one of methanol, acetone, diethyl ether and isopropyl ether.

7. The method according to claim 1, wherein the mixed solvent in the step 4 is one of ethyl acetate, chloroform and dichloromethane and one of acetone, methanol and ethanol, the volume ratio of the two solvents is 88: 12-94: 6, and the reduced pressure concentration temperature is 25-35 ℃.

8. The method of claim 1, wherein the filter of step 4 is a 0.22 μm filter.

9. The method as claimed in claim 1, wherein the crystalline staurosporine of step 5 is dried under vacuum at 60 ± 5 ℃ for 16 ± 2 h.

Technical Field

The invention belongs to the field of separation and purification of chemicals, and particularly relates to a method for extracting staurosporine.

Background

Staurosporine (STA, structural formula (1)) is a natural product with good pharmaceutical and biological activities. The compound was isolated from Agrobacterium in 1997 by Omura as the first indolocarbazole alkaloid. The analysis of X single crystal diffraction experiment shows that the center is indole carbazole ring, the N below the ring is connected with micromolecule sugar, and the upper part is connected with lactam ring. STA is a strong PKC inhibitor (IC50 ═ 2.7nM), acting mainly on various kinases, topoisomerases, and cell cycle regulators in cell signaling pathways.

The staurosporine derivative midostaurin (structural formula (2)) obtained by structural modification is approved in 2017 to treat adult patients with acute myelogenous leukemia which are positive to the latest diagnosed FLT3 mutation (detected by an FDA approved detection method) and suitable for standard induction and consolidation chemotherapy.

Staurosporine is a key intermediate of midostaurin, and the production process of staurosporine directly affects the production cost and quality of midostaurin. Therefore, the development of a method for extracting and purifying staurosporine is of great significance.

US4107297 discloses a method for extracting and purifying staurosporine. After fermentation, adjusting the pH value of a culture medium to 10 by using ammonia water, and adding n-butyl acetate for extraction at room temperature; adding HCl aqueous solution, and transferring the product into the aqueous solution; adjusting the pH value to 10 by using ammonia water, extracting by using ethyl acetate, and concentrating to obtain a brown coagulant; washing with diethyl ether to remove fat; performing silica gel chromatography, eluting with chloroform-methanol, controlling with thin layer, concentrating, and drying to obtain pure product. The purity reaches more than 99 percent.

US4973552 discloses a method for extracting staurosporine, which comprises filtering zymocyte liquid, extracting the residue with tetrahydrofuran under stirring for 1 hr, filtering, washing the mycelium layer with acetone, concentrating the filtrate to obtain water solution, and extracting with chloroform to obtain crude extract; the crude extract is chromatographed by silica gel, and the eluent is n-hexane-tetrahydrofuran gradient; and preparing the mixture by using sephadex LH-20 again to obtain a finished product.

US7608420, discloses a process wherein the product is extracted from the fermentation broth using methanol, acetone and tetrahydrofuran (preferably tetrahydrofuran), and then ultrafiltered using a ceramic membrane, a polymer membrane or a metal membrane, etc., with a molecular weight cut-off of 10kDa or less; adjusting the pH value of the solution to 10-13, and centrifuging or filtering; then tetrahydrofuran-methanol is adopted for crystallization to obtain the staurosporine with the content of 89 percent and the total yield of 64 percent.

In the methods disclosed in the above patents, the extraction methods of US4107297 and US4973552 both adopt silica gel column chromatography, so the product quality is good, the purity reaches more than 99%, but the production cost is high and the yield is low. The method of US7608420 needs ultrafiltration technology to remove biomacromolecule impurities, has high extraction yield reaching 64%, but the obtained staurosporine has poor quality and the content is only 89%.

Methods for obtaining staurosporine fermentation broth are mentioned in patent documents CN 201810572019.1, US4107297, US4973552, US7608420, US20060194294, etc.

Therefore, it is needed to provide a process which is simple, low in cost, easy to scale up, short in production period, high in yield (more than 48% in total), and good in quality of obtained staurosporine (the content is more than 99.5%, and the maximum single impurity is not more than 0.10%). Therefore, the method for purifying staurosporine disclosed by the invention has great application value.

Disclosure of Invention

The invention provides a method for extracting and purifying staurosporine. The method comprises the working procedures of plate-and-frame filtration, extraction, concentration, crystallization and the like, and has the advantages of low cost, short production period, high efficiency and good product quality, and is more suitable for practical production and application. The method comprises the following specific steps:

step 1: adding a filter aid into the staurosporine fermentation liquor, and filtering through a plate frame to obtain staurosporine fungus residues;

step 2: extracting the staurosporine fungi residues in the step 1 by using a solvent to obtain an extraction concentrated solution;

and step 3: adding an organic solvent A, an aqueous solution and an extraction aid into the extraction concentrated solution obtained in the step 2, extracting and layering, drying and filtering an organic phase, concentrating, adding an organic solvent B, crystallizing and filtering to obtain a crude product of the staurosporine;

and 4, step 4: dissolving the crude staurosporine in the step 3 by using a proper amount of mixed solvent, filtering by using a filter membrane, concentrating under reduced pressure, adding an organic solvent B for crystallization, and filtering to obtain a staurosporine crystal;

and 5: and (4) drying the staurosporine crystal product in the step (4) to obtain a staurosporine finished product.

And in addition, the staurosporine fermentation liquid in the step 1 is marine streptomycete fermentation liquid.

And the solvent in the step 2 is one of isopropanol, ethanol, acetone and methanol.

And the organic solvent A in the step 3 is one of butyl acetate, ethyl acetate, dichloromethane and trichloromethane.

And the aqueous solution in the step 3 is formed by one of potassium carbonate, sodium hydroxide, potassium hydroxide and ammonia, and the mass percentage concentration is 0.1-3.0%.

And the extraction auxiliary agent in the step 3 is one of methanol, ethanol and acetone.

And the organic solvent B in the step 3 and the step 4 is one of methanol, acetone, diethyl ether and isopropyl ether.

And the mixed solvent in the step 4 is one of ethyl acetate, trichloromethane and dichloromethane and one of acetone, methanol and ethanol respectively, the volume ratio of the two solvents is 88: 12-94: 6, and the reduced pressure concentration temperature is 25-35 ℃.

Furthermore, the filter of step 4 is a 0.22 μm filter.

And the staurosporine crystal product of the step 5 is dried under vacuum for 16 plus or minus 2 hours at the temperature of 60 plus or minus 5 ℃.

The method for extracting and purifying the staurosporine does not need silica gel column chromatography, only needs to filter the mycelium when the fermentation mycelium does not start autolysis, adopts proper solvent to extract the staurosporine, and avoids release of a large amount of intracellular impurities caused by bacteriolysis. Concentrating the extractive solution, extracting, and removing biomacromolecule impurities of protein and nucleic acid in the extractive solution with alkaline water solution. Finally, removing the rest organic impurities through crystallization. The purification method has the advantages of simple process, easy amplification, short production period, high yield (more than 48 percent in total), and good quality of the obtained staurosporine (the content is more than 99.5 percent, and the maximum single impurity is not more than 0.10 percent). Therefore, the method for purifying staurosporine disclosed by the invention has great application value.

Drawings

FIG. 1 shows staurosporine prepared according to the present invention¹H-NMR chart;

FIG. 2 shows the preparation of the present inventionStaurosporine of¹³C-NMR chart;

FIG. 3 is a mass spectrum of staurosporine prepared according to the present invention;

FIG. 4 is an infrared spectrum of staurosporine prepared according to the present invention;

FIG. 5 is a typical HPLC chart of staurosporine fermentation broth according to the present invention;

FIG. 6 is a typical HPLC chart of a staurosporine crystal prepared according to the present invention;

Detailed Description

The present invention will be described with reference to examples.