CN104805147B - A kind of preparation method of dioxin-like compound, purification process and application - Google Patents

Abstract

The invention discloses a preparation method, purification method and application of a dioxin compound. The preparation method of the compound shown in formula I disclosed by the present invention comprises the following steps: (1) inoculating the Aspergillus terreus spore powder solution into the seed medium for cultivation to obtain the seed liquid; wherein, the Aspergillus terreus spore powder The amount of the solution added is 0.5% to 1.0%; (2) inoculating the seed liquid into the fermentation medium and culturing for 7 to 10 days to obtain a fermentation liquid containing the compound shown in formula I; wherein, the The volume ratio of the seed liquid to the fermentation medium is 1:120～1:180. The raw materials in the preparation method of the present invention are easy to obtain, the preparation cycle is short, the process is simple, and it is more environmentally friendly; and the target compound prepared by the present invention has a high yield and high purity; at the same time, the compound has a strong effect on neuraminidase inhibitory effect.

Description

Preparation method, purification method and application of a dioxin-like compound

technical field

The invention relates to a preparation method, purification method and application of a dioxin compound.

Background technique

Neuraminidase (also known as sialidase, neuraminidase, NA) is a glycoprotein distributed on the envelope of influenza A and B viruses. It can cleave the sialic acid residue at the end of the influenza virus receptor on the cell surface, release progeny virus particles from the infected cell membrane, and promote the spread of influenza virus in the respiratory tract. Therefore, neuraminidase is an important target for the development of anti-influenza virus drugs . The neuraminidase inhibitors currently in clinical use mainly include zanamivir, oseltamivir, and peramivir. The oral utilization of zanamivir is low, the volume of distribution in the body is small, and the renal clearance is fast, so it can only be used as a local drug; during the use of oseltamivir, a large number of drug-resistant virus strains appeared clinically; Approved for listing in a few countries around the world. In summary, designing and synthesizing or screening new and effective neuraminidase inhibitors from compound libraries has become a hot and difficult point in current research.

At present, the preparation method of 4H-1,3-dioxin-2,4-dione is mainly obtained by extracting from the fruiting body of ferulus mushroom (Pleurotuseryngii var.ferulae), and it has been reported that the compound has the ability to inhibit Action of neutrophil elastase. However, in the above-mentioned preparation method, the source of ferulic mushroom is relatively limited, and it is only produced in Xinjiang region. At the same time, the yield of 4H-1,3-dioxin-2,4-dione produced in this preparation method is relatively low, and only 5 mg of the compound can be prepared from 4 kg of mushroom fruiting bodies.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects of limited raw material source and low yield of target compound in the existing 4H-1,3-dioxin-2,4-dione preparation method, and provide A preparation method, purification method and application of dioxin-like compounds are provided. The raw materials in the preparation method of the present invention are easy to obtain, the preparation period is short, the process is simple, and it is more environmentally friendly; and the target compound prepared by the present invention has a high yield and high purity; at the same time, the compound has a strong effect on neuraminidase It has certain guiding significance for the design and research of new neuraminidase inhibitor anti-influenza drugs.

The present invention provides a kind of preparation method of the compound shown in formula I, it comprises the following steps:

(1) Inoculate the Aspergillus terreus spore powder solution into the seed medium and cultivate it to obtain the seed liquid; wherein, the added amount of the Aspergillus terreus spore powder solution is 0.5% to 1.0%, and the percentage is Refers to the percentage of the volume of Aspergillus terreus spore powder solution accounting for the seed culture medium volume;

(2) Inoculate the seed liquid into the fermentation medium and cultivate it for 7-10 days to obtain a fermentation liquid containing the compound shown in formula I; wherein, the volume ratio of the seed liquid to the fermentation medium is 1 :120～1:180.

In step (1), the Aspergillus terreus can be conventional Aspergillus terreus or its variants in the art, as long as the compound shown in formula I can be produced during fermentation. The preferred strain of the present invention is Aspergillus terreus (Aspergillus terreus) PF26 strain. The Aspergillus terreus strain has been submitted to the General Microorganism Center of China Microbiological Culture Collection Management Committee for preservation on August 31, 2011. The preservation number is CGMCC NO.5208. This strain has been registered in CN102827777A published in. The described Aspergillus terreus spore powder solution is preferably an aqueous glycerol solution of Aspergillus terreus spore powder, wherein the glycerol aqueous solution is preferably a 40% aqueous glycerol solution by mass percentage, and the percentage refers to that the mass percentage of glycerol accounts for Percentage of total mass of glycerin and water. In the described Aspergillus terreus spore powder solution, the volume mass of the glycerol aqueous solution and the Aspergillus terreus spore powder is preferably 40 mg/mL.

In step (1), the cultivation method and conditions can be conventional methods and conditions in the field, preferably: at 25°C to 37°C, shaking in a shaker with a rotation speed of 150rpm to 250rpm Cultivate for 18 to 30 hours. The culture time is preferably 12-24 hours. Described seed culture medium is the seed culture medium of Aspergillus terreus routinely used in this field. The seed culture medium preferably comprises: 0.2% to 1.0% of yeast extract, 1.0% to 3.0% of glucose, 0.5% to 1.5% of peptone, and the rest is water; the pH of the seed culture medium The value is 5.0 to 6.0.

In step (2), the culture method and conditions can be conventional methods and conditions in the field, preferably, at 28°C-37°C, in a shaker with a rotation speed of 150rpm-250rpm 7-10 days. The fermentation medium is a conventional fermentation medium of Aspergillus terreus seed liquid in the art. The fermentation medium preferably includes: 2.0%-4.0% glucose, 1.0%-3.0% ammonium sulfate, 0.5%-1.5% yeast extract, 0.1%-0.2% magnesium sulfate, 0.005%- 0.01% ferrous sulfate, and the rest is water; the pH value in the fermentation medium is 5.0-6.0.

In step (1) and step (2), the water is generally distilled water.

The fermentation broth containing the compound represented by formula I, preferably, further includes a purification step. The method and condition of the step of described purification can be conventional method and condition in the art, and the present invention preferably comprises the following steps:

a. The fermented liquid containing the compound shown in formula I is subjected to solid-liquid separation, and then the liquid is collected; concentrated after extraction with an organic solvent, to obtain concentrate A;

b. Using preparative liquid chromatography to separate and purify the concentrate A, collect the eluent with a methanol concentration of 0 to 30%, and concentrate to obtain the concentrate B; wherein, the eluent during separation and purification is methanol and water The mixed solution, the elution method is gradient elution, and the gradient elution is that the concentration of methanol rises linearly to 100% within 30min, and the concentration is the volume ratio of the volume of methanol to the mixed solution; The flow rate of liquid removal is 3mL/min～5mL/min;

c. Using silica gel column chromatography to separate and purify the concentrate B; obtain the compound shown in formula I; wherein, the eluent is a mixture of chloroform and methanol; in the eluent, chloroform and The volume ratio of methanol is 5:1～60:1.

In step a, the method and conditions for solid-liquid separation can be conventional methods and conditions for solid-liquid separation in the art, preferably by centrifugation. Wherein, the speed of the centrifugation is preferably 3000rpm-5000rpm, and the time of the centrifugation is preferably 0.5-1 hour. The organic solvent can be a conventionally used solvent for extraction in this field, preferably one or more of chloroform, ethyl acetate and n-butanol, more preferably ethyl acetate. The method and conditions for concentration can be conventional methods and conditions for concentration in the art, preferably concentration under reduced pressure. The temperature of the concentrated under reduced pressure is preferably 40°C-45°C. The vacuum degree of the vacuum concentration is preferably 700mmHg-800mmHg.

In step b, the concentrate A is preferably dissolved with experimental water, and then the operation of preparative liquid phase separation is carried out. The amount of water used in the experiment is not specifically limited, and it is generally sufficient to dissolve the concentrate A. The experimental water is generally distilled water. Described preparative liquid phase is the conventional preparative liquid phase of this area, preferably the model of Shimadzu Corporation is the high-performance liquid chromatograph of SHIMADZULC-20AP, and the specification of post in this high-performance liquid chromatograph is preferably 10 Asahi's model is Ultimate AQ C18 analytical chromatographic column (10×250mm, 5μm). The volume and mass of the eluent and the concentrate A are preferably 4L/g-5L/g. The gradient elution is preferably a wash with methanol concentration from 0→10%→20%→30%→40%→50%→60%→70%→80%→90%→100% within 30min. Eluent for elution. The flow rate of the eluent is preferably 4mL/min. The method and conditions for concentration can be conventional methods and conditions for concentration in the art, preferably concentration under reduced pressure. The temperature of the concentrated under reduced pressure is preferably 40°C-45°C. The vacuum degree of the vacuum concentration is preferably 700mmHg-800mmHg.

In step c, the concentrate B is preferably dissolved with experimental water, and then the operation of preparative liquid phase separation is carried out. The amount of water used in the experiment is not specifically limited, and it is generally sufficient to dissolve the concentrate B. The experimental water is generally distilled water. The silica gel used in the silica gel column chromatography is conventional silica gel in the field, preferably 200-300 mesh silica gel. Wherein, the silica gel chromatography column used in the silica gel column chromatography is a silica gel chromatography column routinely used in the art, preferably a normal phase silica gel chromatography column or a reverse phase silica gel chromatography column, more preferably a normal phase silica gel chromatography column Column. The mass ratio of the filling amount of the silica gel to the concentrate B is preferably 40:1˜5:1. The specifications of the silica gel chromatography column can be selected according to common knowledge in the field. The volume ratio of chloroform and methanol in the eluent is preferably 30:1-10:1, more preferably 20:1. The flow rate of the eluent is preferably 5 mL/min˜15 mL/min.

The purification method may further include the following steps: using gel column chromatography to further separate and purify the compound shown in formula I to obtain the compound product shown in formula I; wherein, the elution The solution is a mixture of methanol and water; the gel in the gel column chromatography is hydroxypropyl dextran gel (Sephadex LH-20);

Wherein, the gel chromatography column used in the gel column chromatography is a gel chromatography column commonly used in the field, preferably, the particle size of the gel in the gel chromatography column is 18 μm to 111 μm , more preferably 50 μm to 100 μm. The specifications of the silica gel chromatography column can be selected according to common knowledge in the field. The mass ratio of the loading amount of the gel to the compound represented by formula I is preferably 80:1-240:1. The volume ratio of methanol and water in the eluent is preferably 0:100-10:90, more preferably 5:95-20:80. The flow rate of the eluent is preferably 1 mL/min-2 mL/min.

The present invention also provides a purification method of Aspergillus terreus fermented liquid containing the compound shown in formula I, and described purification method comprises the following steps:

a. After the fermentation liquid containing the compound shown in formula I is subjected to solid-liquid separation, the liquid is collected; after extraction with an organic solvent, it is concentrated to obtain concentrate A;

b. Using preparative liquid chromatography to separate and purify the concentrate A, collect the eluent with a methanol concentration of 0 to 30%, and concentrate to obtain the concentrate B; wherein, the eluent during separation and purification is methanol and water The mixed solution, the elution method is gradient elution, and the gradient elution is that the concentration of methanol rises linearly to 100% within 30min, and the concentration is the volume ratio of the volume of methanol to the mixed solution; The flow rate of liquid removal is 3mL/min～5mL/min;

c. Using silica gel column chromatography to separate and purify the concentrate B; obtain the compound shown in formula I; wherein, the eluent is a mixture of chloroform and methanol; in the eluent, chloroform and The volume ratio of methanol is 5:1～60:1.

In step a, the method and conditions for solid-liquid separation can be conventional methods and conditions for solid-liquid separation in the art, preferably by centrifugation. Wherein, the speed of the centrifugation is preferably 3000rpm-5000rpm, and the time of the centrifugation is preferably 0.5-1 hour. The organic solvent can be a conventionally used solvent for extraction in this field, preferably one or more of chloroform, ethyl acetate and n-butanol, more preferably ethyl acetate. The method and conditions for concentration can be conventional methods and conditions for concentration in the art, preferably concentration under reduced pressure. The temperature of the concentrated under reduced pressure is preferably 40°C-45°C. The vacuum degree of the vacuum concentration is preferably 700mmHg-800mmHg.

In step b, the concentrate A is preferably dissolved with experimental water, and then the operation of preparative liquid phase separation is carried out. The amount of water used in the experiment is not specifically limited, and it is generally sufficient to dissolve the concentrate A. The experimental water is generally distilled water. Described preparative liquid phase is the conventional preparative liquid phase of this area, preferably the model of Shimadzu Corporation is the high-performance liquid chromatograph of SHIMADZULC-20AP, and the specification of post in this high-performance liquid chromatograph is preferably 10 Asahi's model is Ultimate AQ C18 analytical chromatographic column (10×250mm, 5μm). The volume and mass of the eluent and the concentrate A are preferably 4L/g-5L/g. The gradient elution is preferably a wash with methanol concentration from 0→10%→20%→30%→40%→50%→60%→70%→80%→90%→100% within 30min. Eluent for elution. The flow rate of the eluent is preferably 4mL/min. The method and conditions for concentration can be conventional methods and conditions for concentration in the art, preferably concentration under reduced pressure. The temperature of the concentrated under reduced pressure is preferably 40°C-45°C. The vacuum degree of the vacuum concentration is preferably 700mmHg-800mmHg.

In step c, the concentrate B is preferably dissolved with experimental water, and then the operation of preparative liquid phase separation is carried out. The amount of water used in the experiment is not specifically limited, and it is generally sufficient to dissolve the concentrate B. The experimental water is generally distilled water. The silica gel used in the silica gel column chromatography is conventional silica gel in the field, preferably 200-300 mesh silica gel. Wherein, the silica gel chromatography column used in the silica gel column chromatography is a silica gel chromatography column routinely used in the art, preferably a normal phase silica gel chromatography column or a reverse phase silica gel chromatography column, more preferably a normal phase silica gel chromatography column Column. The mass ratio of the filling amount of the silica gel to the concentrate B is preferably 40:1˜5:1. The specifications of the silica gel chromatography column can be selected according to common knowledge in the field. The volume ratio of chloroform and methanol in the eluent is preferably 30:1-10:1, more preferably 20:1. The flow rate of the eluent is preferably 5 mL/min˜15 mL/min.

The purification method may further include the following steps: using gel column chromatography to further separate and purify the compound shown in formula I to obtain the compound product shown in formula I; wherein, the elution The solution is a mixture of methanol and water; the gel in the gel column chromatography is hydroxypropyl dextran gel (Sephadex LH-20);

Wherein, the gel chromatography column used in the gel column chromatography is a gel chromatography column commonly used in the field, preferably, the particle size of the gel in the gel chromatography column is 18 μm to 111 μm , more preferably 50 μm to 100 μm. The specifications of the silica gel chromatography column can be selected according to common knowledge in the field. The mass ratio of the loading amount of the gel to the compound represented by formula I is preferably 80:1-240:1. The volume ratio of methanol and water in the eluent is preferably 0:100-10:90, more preferably 5:95-20:80. The flow rate of the eluent is preferably 1 mL/min-2 mL/min.

The present invention also provides an application of a compound as shown in formula I in the preparation of neuraminidase inhibitors;

The present invention also provides an application of the compound shown in formula I in the preparation of anti-influenza virus drugs;

The influenza virus can be a conventional influenza virus in the art, preferably an H1N1 subtype influenza virus.

On the basis of not violating common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive progress effect of the present invention is:

The method for separating and purifying dioxin-like compounds in the present invention has convenient material source, short preparation cycle, environmental protection and simple process. At the same time, the compound has a strong inhibitory effect on neuraminidase, which has certain guiding significance for the design and research of new neuraminidase inhibitor anti-influenza drugs.

detailed description

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

The source of each raw material in following embodiment is as follows:

The preparation liquid phase in the step b of following embodiment is the model of Shimadzu Company is the high performance liquid chromatography of SHIMADZULC-20AP, and the specification of column in this high performance liquid chromatography is the analysis of the model of Yuexu company is Ultimate AQ C18 Chromatography column (10×250mm, 5μm).

The compound product shown in formula I in step d of the following examples refers to the compound product shown in formula I obtained after separation by gel column chromatography.

The Aspergillus terreus in the following examples is the Aspergillus terreus PF26 strain, and the Aspergillus terreus strain was submitted to the General Microorganism Center of the China Microbiological Culture Collection Management Committee for preservation on August 31, 2011, and the preservation number is CGMCC NO.5208. The strain has been disclosed in CN102827777A.

The preparation of the fermented liquid of embodiment 1 Aspergillus terreus (Aspergillus terreus)

(1) Inoculate 0.15mL of Aspergillus terreus spore powder solution (80mg of Aspergillus terreus spore powder dissolved in 2mL of 40% glycerol aqueous solution) into a 250mL triangular shaker flask containing 30mL of seed medium, at 25°C at 220rpm Vibrate and cultivate in a shaker for 12 hours to obtain seed liquid. Wherein, the seed medium contains: 0.5% yeast extract, 2% glucose, 1% peptone, and the rest is experimental water. The pH value of the seed culture medium is 5.5;

(2) Inoculate 1mL of seed solution into a 750mL Erlenmeyer shaker flask containing 150mL of fermentation medium, and culture in a shaker at 220rpm for 168 hours (7 days) at 28°C. A total of 4L of Aspergillus terreus fermentation liquid containing the compound represented by formula I was enriched. The fermentation medium contains: 3% glucose, 2% ammonium sulfate, 1% yeast extract, 0.1% magnesium sulfate, 0.005% ferrous sulfate, and the rest is experimental water. The pH value in the fermentation medium is 5.5.

The preparation of the fermented liquid of embodiment 2 Aspergillus terreus (Aspergillus terreus)

(1) Inoculate 0.15mL of Aspergillus terreus spore powder solution (80mg of Aspergillus terreus spore powder dissolved in 2mL of 40% glycerol aqueous solution) into a 250mL Erlenmeyer shaker flask containing 30mL of seed medium, at 37°C at 150rpm Shake culture in a shaker for 24 hours to obtain seed liquid. Wherein, the seed medium contains: 0.2% yeast extract, 1% glucose, 0.5% peptone, and the rest is experimental water. The pH value in the seed culture medium is 5.0;

(2) Inoculate 0.8mL of seed liquid into a 750mL Erlenmeyer shaker flask containing 150mL of fermentation medium, and culture in a shaker at 220rpm at 37°C for 168 hours (7 days). A total of 4L of Aspergillus terreus fermentation liquid containing the compound represented by formula I was enriched. The fermentation medium comprises: 2% glucose, 1% ammonium sulfate, 0.5% yeast extract, 0.1% magnesium sulfate, 0.005% ferrous sulfate, and the rest is experimental water. The pH value in the fermentation medium is 5.0.

The preparation of the fermented liquid of embodiment 3 Aspergillus terreus (Aspergillus terreus)

(1) Inoculate 0.3mL of Aspergillus terreus spore powder solution (80mg of Aspergillus terreus spore powder dissolved in 2mL of 40% glycerol aqueous solution) into a 250mL triangular shaker flask containing 30mL of seed medium, at 30°C at 220rpm Shake culture in a shaker for 24 hours to obtain seed liquid. Wherein, the seed medium contains: 1% yeast extract, 3% glucose, 1.5% peptone, and the rest is experimental water. The pH value of the seed medium is 6.0;

(2) Inoculate 1.2mL of seed liquid into a 750mL Erlenmeyer shaker flask containing 150mL of fermentation medium, and culture in a shaker at 220rpm at 30°C for 240 hours (ie 10 days). A total of 4L of Aspergillus terreus fermentation liquid containing the compound represented by formula I was enriched. The fermentation medium comprises: 4% glucose, 3% ammonium sulfate, 1.5% yeast extract, 0.2% magnesium sulfate, 0.01% ferrous sulfate, and the rest is experimental water. The pH value in the fermentation medium is 6.0.

The preparation of the fermented liquid of embodiment 4 Aspergillus terreus (Aspergillus terreus)

The culture time of step (2) in Example 1 was replaced by 8 days from 7 days, and other conditions were the same as in Example 1.

The preparation of the fermented liquid of embodiment 5 Aspergillus terreus (Aspergillus terreus)

The culture time of step (2) in Example 1 was replaced by 9 days from 7 days, and other conditions were the same as in Example 1.

Example 64H-1,3-dioxin-2,4-diketone purification

a. the Aspergillus terreus fermented liquid containing the compound shown in formula I by 4L enriched among the embodiment 1, centrifugal 30 minutes in the centrifuge of 3000rpm, obtain supernatant; Add 4L ethyl acetate to extract supernatant, The collected organic phase was evaporated and concentrated to dryness under reduced pressure to obtain Concentrate A, a total of 160mg;

b. Dissolve the concentrate A with 2mL of experimental water, and use the preparative liquid phase for separation. A mixture of methanol and water is used as the eluent for gradient elution; wherein, the concentration of methanol rises from 0% to 100% within 30 minutes. Specifically, the elution method is to increase the concentration of methanol from 0 to 10 within 30 minutes. %→20%→30%→40%→50%→60%→70%→80%→90%→100% gradient elution, collect eluent with methanol concentration of 0%～30%, flow rate is 3mL/ min, the injection volume is 800 μL (single injection volume); the collected 0-30% eluate is then concentrated to dryness under reduced pressure to obtain 8.0 g of concentrate B;

c. Dissolve the concentrate B with 3mL of experimental water, mix the sample with 800mg of normal phase silica gel (200-300 mesh) and load it into the column. The mass ratio of the silica gel loading to the concentrate B is 40:1. (volume ratio 10:1) mixed solution was eluted at a flow rate of 5mL/min; it was collected in a test tube and combined for TLC (thin layer chromatography) detection. The developer system used for detection was chloroform-methanol (volume ratio 10:1). The eluate with Rf=0.18 was collected, combined and concentrated to dryness under reduced pressure to obtain 7.0 g of the compound represented by formula I; the yield was 87.5%, and the HPLC purity was 98.3%.

The compound shown in formula I obtained in step c can also be further separated and purified:

The compound shown in formula I prepared in step c was dissolved with 1 mL of experimental water and separated by Sephadex LH-20 gel column chromatography, and eluted with 500 mL of 5% aqueous methanol; test tube collection, TLC (thin layer chromatography method) detection combined, the developer system used for detection is chloroform-methanol (volume ratio 10:1), collect the eluate with Rf=0.18, combine the eluate and distill under reduced pressure to obtain 4.3mg of the compound shown in formula I The compound product (4H-1,3-dioxin-2,4-dione) has a total yield of 2.69% and an HPLC purity of 99.5%.

Its physical and chemical properties and NMR spectrum data are as follows:

White amorphous powder, easily soluble in DMSO, soluble in water, insoluble in methanol, ethanol, etc.

Its methanol solution has characteristic UV absorption at 227nm and 258nm.

EI-MS: m/z112[M-2H] ⁺ (100), 69(56), 68(21), 44(11), 43(52), 42(33);

¹ H-NMR (400MHz, DMSO-d ₆ ): 5.44 (d, J=7.6Hz, H-5), 7.37 (d, J=7.6Hz, H-6).

¹³ C-NMR: 155.7(C-2), 164.4(C-4), 100.3(C-5), 142.4(C-6).

Example 74H-1,3-dioxin-2,4-diketone purification

a. the Aspergillus terreus fermented liquid containing the compound shown in formula I by the 4L enriched in embodiment 2, in the centrifuge of 5000rpm, centrifuge 60 minutes, get supernatant; Add 4L chloroform to extract supernatant, collect organic The phase was evaporated and concentrated to dryness under reduced pressure to obtain Concentrate A, a total of 175 mg.

b. Dissolve the concentrate A with 2mL of experimental water, and use the preparative liquid phase for separation; use the mixture of methanol and water as the eluent for gradient elution; wherein, the concentration of methanol rises from 0% to 100% within 30 minutes %, collect the eluent with a methanol concentration of 0% to 30%, the flow rate is 5mL/min, and the injection volume is 800 μL (single injection volume); , to obtain 8.1mg concentrate B;

c. Dissolve the concentrate B with 1 mL of experimental water, mix the sample with 800 mg of normal phase silica gel (200-300 mesh) and load it into the column. The mass ratio of the silica gel loading to the concentrate B is 5:1. (volume ratio 30:1) mixed solution was eluted at a flow rate of 15mL/min; it was collected in a test tube and combined for TLC (thin layer chromatography) detection. The developer system used for detection was chloroform-methanol (volume ratio 10:1). The eluents with Rf=0.18 were collected, combined and concentrated to dryness under reduced pressure to obtain 7.1 mg of the compound represented by formula I; the yield was 87.4%, and the HPLC purity was 98.3%.

The compound shown in formula I obtained in step c can also be further separated and purified:

The compound shown in formula I prepared in step c was dissolved with 1 mL of experimental water and separated by Sephadex LH-20 gel column chromatography, and eluted with 500 mL of 5% aqueous methanol; test tube collection, TLC (thin layer chromatography method) detection combined, the developer system used in the detection is chloroform-methanol (volume ratio 10:1), the eluate with Rf=0.18 was collected, the collected eluate was combined and then distilled under reduced pressure to obtain 4.5 mg of the target compound (4H -1,3-dioxin-2,4-dione), the total yield is 2.69%, and the HPLC purity is 99.5%; its physical and chemical properties and nuclear magnetic resonance spectrum data are the same as in Example 6.

Purification of Example 84H-1,3-dioxin-2,4-dione

Dissolve the concentrate B in step c of Example 6 with 5 mL of experimental water, mix the sample with 800 mg of normal phase silica gel (200-300 mesh) and load it into the column, and use 500 mL of chloroform and methanol (the volume ratio of chloroform and methanol is 40:1) Elution is performed as an eluent. The remaining conditions are the same as steps a to c in Example 6, and finally the yield of the compound shown in formula I is 74.5%, and the product purity is 93.7%. Its physical and chemical properties and nuclear magnetic resonance spectrum data are with embodiment 6.

Purification of Example 94H-1,3-dioxin-2,4-dione

The Aspergillus terreus fermented liquid containing the compound shown in formula I prepared in Example 4 is separated and purified according to the same purification method as in Example 6, and 500 mL of chloroform and methanol are used in step c (the volume ratio of chloroform and methanol is 5 : 1) Elution was carried out as an eluent to obtain 3.1 mg of the compound product represented by formula I, the total yield was 1.9%, and the HPLC purity was 98.1%.

Example 104H-1,3-dioxin-2,4-diketone purification

The Aspergillus terreus fermented liquid containing the compound shown in formula I prepared in Example 5 is separated and purified according to the same purification method as in Example 6. In step c, use 500mL chloroform and methanol (the volume ratio of chloroform and methanol is 60 : 1) Elution was carried out as an eluent to obtain 3 mg of the compound product represented by formula I, with a total yield of 1.8% and an HPLC purity of 98.7%.

Comparative Example 1

The culture time of step (2) in Example 1 was replaced from 7 days to 5 days, and the remaining conditions were the same as in Example 1, and the same purification method as in Example 2 was used for separation and purification to obtain 2.2 mg of the compound represented by formula I Product, the total yield is 1.3%, and the HPLC purity is 93.7%.

Comparative Example 2

The culture time of step (2) in Example 1 was replaced by 7 days to 6 days, and the remaining conditions were the same as in Example 1, and the same purification method as in Example 2 was used for separation and purification to obtain 2.5 mg of the compound shown in formula I, The overall yield was 1.5%, and the HPLC purity was 96.3%.

Comparing Examples 1 and 2 shows that the cultivation time has a great influence on the final yield of the product, and the cultivation time of 7 to 10 days is beneficial to the improvement of the yield of the product.

Effect Example 1

The method of neuraminidase inhibitor screening model (content described in Example 3 on page 7 of CN102586295 specification) was used to detect its neuraminidase inhibitory activity. Add neuraminidase, substrate MUNANA (methylumbelliferyl-N-acetyl-α-D-neuralminic acid), MES buffer (2-(N-morpholino)ethanesulfonic acid aqueous solution, pH value is 6.5 to the reaction system ) and the inhibitor to be screened, and detect the change of fluorescence intensity under the excitation light of 360nm and the emission light of 460nm after incubation.

Wherein, the reaction system is: 1-3 μL of the inhibitor to be tested, 80-90 μL of NA enzyme solution, 6-10 μL of 100 μL MUNANA solution, and 70-80 μL of 32.5 mM MES buffer solution.

Alternatively, the reaction system is: 2 μL of the inhibitor to be tested, 70 μL of enzyme solution, 8 μL of 100 μM MUNANA solution, and 72 μL of 32.5 mM MES buffer.

Take the target compound obtained in Example 2, that is, 4H-1,3-dioxin-2,4-dione, and prepare it into a 1mM stock solution, and then double-dilution it into 100μM, 10μM, 1μM, 100nM, 10nM and 1nM concentration.

The reaction system for activity screening is: take 2 μL of the test sample, add 70 μL of NA enzyme solution (made in the laboratory), 8 μL of 1M MUNANA solution and 72 μL of 32.5 mM MES buffer, mix well, react at 37 °C for 30 min, and excite at 360 nm Fluorescence intensity change F was detected under light and 460nm emitted light.

Experimental water was selected as the blank control.

Inhibition rate (%) = (F control - F sample) / F control × 100%

F control refers to the change value of the fluorescence intensity detected under the excitation light of 360nm and the emission light of 460nm by the experimental water;

F sample is the change value of the fluorescence intensity detected under the excitation light of 360nm and the emission light of 460nm of the sample to be tested.

Table 1: Neuraminidase inhibition rate/% of compound 4H-1,3-dioxin-2,4-dione (C1)

The results of the inhibition experiment are shown in Table 1: the _IC50 value of 4H-1,3-dioxin-2,4-dione on neuraminidase is 212.5 μM, indicating that the compound has certain neuraminidase inhibitory activity .

Claims (19)

1. a preparation method of the compound shown in formula I, is characterized in that comprising the following steps: (1) Inoculate the Aspergillus terreus spore powder solution into the seed culture medium and cultivate to obtain the seed liquid; wherein, the addition amount of the Aspergillus terreus spore powder solution is 0.5% to 1.0%, and the percentage is Refers to the percentage of the volume of Aspergillus terreus spore powder solution accounting for the seed culture medium volume; (2) Inoculate the seed liquid into the fermentation medium and cultivate it for 7-10 days to obtain the fermentation liquid containing the compound shown in formula I; wherein, the volume ratio of the seed liquid to the fermentation medium is 1:120～1:180. 2. preparation method as claimed in claim 1, is characterized in that, in step (1), described Aspergillus terreus spore powder solution is the glycerin aqueous solution of Aspergillus terreus spore powder, and wherein, described glycerol aqueous solution is that mass percentage is 40% glycerin aqueous solution; in the described Aspergillus terreus spore powder solution, the mass-to-volume ratio of glycerol aqueous solution to Aspergillus terreus spore powder is 40 mg/mL. 3. The preparation method according to claim 1, characterized in that, in step (1), the culture method and conditions are as follows: under the condition of 25° C. to 37° C., at a speed of 150 to 250 rpm shake culture in a shaker for 18-30 hours; the seed medium contains: 0.2%-1.0% yeast extract, 1.0%-3.0% glucose, 0.5%-1.5% peptone, and the rest is water; The pH value of the seed culture medium is 5.0-6.0. 4. The preparation method according to claim 1, characterized in that, in step (2), the culture method and conditions are: under the condition of 28°C-37°C, in a shaker with a rotation speed of 150rpm-250rpm Shaking culture in bed for 7-10 days; the fermentation medium contains: 2.0%-4.0% glucose, 1.0%-3.0% ammonium sulfate, 0.5%-1.5% yeast extract, 0.1%-0.2% Magnesium sulfate, 0.005% to 0.01% of ferrous sulfate, and the balance is water; the pH value in the fermentation medium is 5.0 to 6.0. 5. The preparation method according to claim 1, wherein the fermented liquid containing the compound shown in formula I further comprises a purification step; the purification step comprises the following steps: a. The fermented liquid containing the compound shown in formula I is subjected to solid-liquid separation, and then the liquid is collected; concentrated after extraction with an organic solvent, to obtain concentrate A; b. Using preparative liquid chromatography to separate and purify the concentrate A, collect the eluent whose methanol concentration is 0-30%, and concentrate to obtain the concentrate B; wherein, the eluent during separation and purification is methanol and water The mixed solution, the elution method is gradient elution, and the gradient elution is that the concentration of methanol rises linearly to 100% within 30min, and the concentration is the volume ratio of the volume of methanol to the mixed solution; The flow rate of liquid removal is 3mL/min～5mL/min; c. The concentrate B is separated and purified by silica gel column chromatography; the compound shown in formula I is obtained; wherein, the eluent is a mixture of chloroform and methanol; in the eluent, chloroform and The volume ratio of methanol is 5:1～60:1. 6. The preparation method according to claim 5, characterized in that, in step a, the method of solid-liquid separation is separation by centrifugation; wherein, the speed of centrifugation is 3000rpm～5000rpm, the The time of described centrifugation is 0.5～1 hour; Described organic solvent is one or more in chloroform, ethyl acetate and n-butanol; Described concentration is decompression concentration; The temperature of described decompression concentration 40°C to 45°C; the vacuum degree of the vacuum concentration is 700mmHg to 800mmHg. 7. The preparation method according to claim 5, wherein in step b, the volume-to-mass ratio of the mixed solution to the concentrate A is 4L/g to 5L/g; the gradient wash The eluent is eluted with methanol concentration from 0→10%→20%→30%→40%→50%→60%→70%→80%→90%→100% within 30min; The flow rate of the eluent is 4mL/min; the concentration is reduced pressure concentration; the temperature of the reduced pressure concentration is 40°C to 45°C; the vacuum degree of the reduced pressure concentration is 700mmHg to 800mmHg. 8. the preparation method as claimed in claim 5 is characterized in that, in step c, the silica gel chromatography column used in silica gel column chromatography is normal phase silica gel chromatography column or reverse phase silica gel chromatography column; Described The mass ratio of the filling amount of silica gel to the concentrate B is 40:1 to 5:1; the volume ratio of chloroform to methanol in the eluent is 30:1 to 10:1; the elution The flow rate of liquid is 5mL/min～15mL/min. 9. The preparation method according to any one of claims 5 to 8, characterized in that, the step of purifying further comprises the following steps: using gel column chromatography to further process the compound shown in formula I Separation and purification to obtain the compound product shown in formula I; wherein, the eluent is a mixture of methanol and water; the gel in the gel column chromatography is hydroxypropyl dextran gel; 10. The preparation method according to claim 9, characterized in that, the particle size of the gel in the gel chromatography column used in the gel column chromatography is 18 μm to 111 μm; the packing of the gel The mass ratio of the amount to the compound shown in formula I is 80:1～240:1; the volume ratio of methanol and water in the eluent is 0:100～10:90; the eluent’s The flow rate is 1mL/min～2mL/min. 11. a kind of purification method containing the Aspergillus terreus fermented liquid of the compound shown in formula I, described purification method comprises the following steps: a. After the fermentation liquid containing the compound shown in formula I is subjected to solid-liquid separation, the liquid is collected; after extraction with an organic solvent, it is concentrated to obtain concentrate A; b. Using preparative liquid chromatography to separate and purify the concentrate A, collect the eluent whose methanol concentration is 0-30%, and concentrate to obtain the concentrate B; wherein, the eluent during separation and purification is methanol and water The mixed solution, the elution method is gradient elution, and the gradient elution is that the concentration of methanol rises linearly to 100% within 30min, and the concentration is the volume ratio of the volume of methanol to the mixed solution; The flow rate of liquid removal is 3mL/min～5mL/min; c. Using silica gel column chromatography to separate and purify the concentrate B; obtain the compound shown in formula I; wherein, the eluent is a mixture of chloroform and methanol; in the eluent, chloroform and The volume ratio of methanol is 5:1～60:1. 12. The preparation method according to claim 11, characterized in that, in step a, the method of solid-liquid separation is separation by centrifugation; wherein, the speed of centrifugation is 3000rpm-5000rpm, the The time of described centrifugation is 0.5～1 hour; Described organic solvent is one or more in chloroform, ethyl acetate and n-butanol; Described concentration is decompression concentration; The temperature of described decompression concentration 40°C to 45°C; the vacuum degree of the vacuum concentration is 700mmHg to 800mmHg. 13. The preparation method according to claim 11, characterized in that, in step b, the volume-to-mass ratio of the mixed solution to the concentrate A is 4L/g to 5L/g; the gradient wash The eluent is eluted with methanol concentration from 0→10%→20%→30%→40%→50%→60%→70%→80%→90%→100% within 30min; The flow rate of the eluent is 4mL/min; the concentration is reduced pressure concentration; the temperature of the reduced pressure concentration is 40°C to 45°C; the vacuum degree of the reduced pressure concentration is 700mmHg to 800mmHg. 14. the preparation method as claimed in claim 11 is characterized in that, in step c, the silica gel chromatography column used in silica gel column chromatography is normal phase silica gel chromatography column or reverse phase silica gel chromatography column; Described The mass ratio of the filling amount of silica gel to the concentrate B is 40:1 to 5:1; the volume ratio of chloroform to methanol in the eluent is 30:1 to 10:1; the elution The flow rate of liquid is 5mL/min～15mL/min. 15. The preparation method according to any one of claims 11 to 14, characterized in that, the step of purifying further comprises the following steps: subjecting the compound shown in formula I to further purification by gel column chromatography Separation and purification to obtain the compound product shown in formula I; wherein, the eluent is a mixture of methanol and water; the gel in the gel column chromatography is hydroxypropyl dextran gel; 16. The preparation method according to claim 15, characterized in that, the particle size of the gel in the gel chromatography column used in the gel column chromatography is 18 μm to 111 μm; the packing of the gel The mass ratio of the amount to the compound shown in formula I is 80:1～240:1; the volume ratio of methanol and water in the eluent is 0:100～10:90; the eluent’s The flow rate is 1mL/min～2mL/min. 17. Application of a compound as shown in formula I in the preparation of neuraminidase inhibitors; 18. Application of a compound as shown in formula I in the preparation of anti-influenza virus drugs; 19. The use of the compound represented by formula I as claimed in claim 18 in the preparation of anti-influenza virus drugs, characterized in that the influenza virus is H1N1 subtype influenza virus.