WO2022247618A2 - Preparation of polyketone compound having anti-novel coronavirus activity, and use thereof - Google Patents

Description

Preparation and application of polyketide compounds with anti-new coronavirus activity technical field

The invention belongs to the field of compound synthesis, and in particular relates to the preparation and application of polyketide compounds.

Background technique

Endophytes in medicinal plants are characterized by diversity, and each endophyte has abundant secondary metabolites. Many biologically active alkaloids, polypeptides, and polyketides have been discovered from the secondary metabolites of endophytes. These secondary metabolites not only have the same or similar biological activities as the host plant, such as antibacterial, anti-inflammatory, anti-tumor, anti-hyperglycemic, anti-parasitic, etc., but also have many new biological activities. Therefore, the secondary metabolites of medicinal plant endophytes are a huge resource pool of potential medicinal substances and have good development prospects.

Contents of the invention

In order to make up for the gap in the prior art, the present invention aims to provide a polyketide compound and its active application. The present invention is a compound with a novel structure that has inhibitory activity against SARS-CoV-2 virus isolated from a solid fermentation product of a plant-derived endophyte and an application thereof. Specifically, a solid-state fermentation of the endophytic fungus Alternaria sp. was carried out, and a polyketide monomer compound with a novel structure was obtained from the fermentation product using a series of separation and purification methods. Using techniques such as mass spectrometry and nuclear magnetic resonance, the structure of the compound was determined, and the structure is:

The structure is shown in formula (I):

At the same time, the present invention claims protection for the preparation method of the above-mentioned polyketide compound.

(1) Strain fermentation: the endophytic fungus Alternaria sp. of the genus Alternaria was fermented in solid state with rice and purified water.

(2) Extraction and separation of metabolites: Use an equal volume of methanol to ultrasonically extract the solid fermentation product of the plant endophytic fungus Alternaria sp., filter through 8 layers of gauze, separate the extract from mycelium and rice, and concentrate the extract to dryness , the extract is subjected to medium-pressure column chromatography, with 300-400 mesh silica gel as the stationary phase, and chloroform/methanol as the mobile phase for gradient elution, by volume, the gradient is successively chloroform:methanol=100:0, 100:1, 100:2, 100:3, 100:5, 100:10, 0:100, the flow rate of the eluent is 30ml/min, collect the eluate (collect once every 500ml), hang the eluate to dry, The eluents were combined to obtain components 1-11; component 4 was passed through a silica gel column, with 200-300 mesh silica gel as the stationary phase, and the mobile phase was petroleum ether/ethyl acetate, and the gradient was petroleum ether in order by volume: Ethyl acetate=10:1, 10:3, 10:5, 10:6, 10:7, 10:9, the eluent flow rate is 30ml/min, collect the eluate (every 500ml once), the same as every Combine five eluents to obtain fractions 4-1 to 4-16; put fractions 4-11 on a Sephadex LH-20 gel column, the mobile phase is dichloromethane/methanol=1:1, and the flow rate of the eluent is 0.3ml/min, collect the eluate (collect once every 10ml), and combine every four parts of eluate to obtain components 4-11-1 to 4-11-6; Liquid chromatography, prepared with 48% methanol water, to obtain the monomeric compound with a retention time of 11 min.

(3) Structure identification: using deuterated dimethyl sulfoxide as solvent, Bruker Avance Ⅱ 500M nuclear magnetic resonance instrument was used to measure the nuclear magnetic spectrum of the isolated compound sample, and the molecular formula was deduced by mass spectrometry, and finally the structure of the product was characterized.

Further, the step (1) fermentation conditions: the bacterial strain Alternaria sp. was subjected to shaking culture with Fungi No. 4 culture medium, the culture condition was 28°C, 180r/min, and then the fermentation broth and mycelium of the shaking culture were inoculated in the container Static fermentation culture was carried out in conical flasks with rice solid medium for 40 days, and the culture temperature was 28°C.

Further, the No. 4 fungus culture medium is composed as follows in terms of mass percentage: 2% mannitol, 2% glucose, 0.5% yeast extract, 1% peptone, 0.05% KH ₂ PO ₄ , 0.03% MgSO ₄ ·7H ₂ O , 0.1% corn steep liquor, deionized water preparation.

Further, the rice solid medium is as follows: add 80 g of rice to every 110 ml of deionized water, and put it into an Erlenmeyer flask; each bottle of rice culture medium is inoculated with 20 ml of seed solution.

Another object of the present invention is to claim the application of the above-mentioned polyketide compound, which has inhibitory activity against SARS-CoV-2 virus and can be used to prepare drugs for inhibiting SARS-CoV-2 virus.

Beneficial effects: the present invention makes full use of plant endophyte resources, studies plant endophytes, finds new polyketide active compounds, and conducts anti-new coronavirus activity experiments on them, so as to obtain FDA-approved anti-SARS-CoV -2 The host-directed drug clofazimine for viral activity was used as a control group to explore the antiviral activity of the compound. The experimental results show that the SI index of clofazimine is 1.5, and the SI index of the isolated compound of the present invention is 3, the inhibition rate of the virus is higher than that of the control drug, and it has the activity of inhibiting the new coronavirus, and is looking for new lead compounds for drug development .

Description of drawings

Fig. 1 is the hydrogen NMR spectrogram of embodiment 1.

Fig. 2 is the carbon 13 nuclear magnetic resonance spectrogram of embodiment 1.

Fig. 3 is embodiment 1 HSQC spectrogram.

Fig. 4 is embodiment 1 HMBC spectrogram.

Detailed ways

The present invention is described in detail below through specific examples, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods used in the present invention are conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial sources. Alternaria sp. is a known bacterial species in the art. For illustration, in the following examples, the endophyte Alternaria sp. collected from Rhodiola tibetica blade parts

Example 1

(1) Fermentation of strains: the bacterial strain Alternaria sp. was cultured by shaking with fungus No. 4 medium, the culture condition was 28° C., 180 r/min; the composition of the fungus No. 4 medium: 2% mannitol, 2% glucose, 0.5 % yeast extract, 1% peptone, 0.05% KH ₂ PO ₄ , 0.03% MgSO ₄ ·7H ₂ O, 0.1% corn steep liquor, and deionized water.

Then the fermented liquid of shaking culture is inoculated in the Erlenmeyer flask that rice solid medium (rice solid medium formula: 80g rice, 110ml deionized water) is housed together with mycelia, every bottle of rice medium is inoculated with 20ml seed liquid, The static fermentation culture was carried out for 40 days, and the culture temperature was 28°C.

(2) Extraction and separation of metabolites: Use an equal volume of methanol to ultrasonically extract the solid fermentation product of the plant endophytic fungus Alternaria sp., filter through 8 layers of gauze, separate the extract from mycelium and rice, and concentrate the extract to dryness , the extract is subjected to medium-pressure column chromatography, with 300-400 mesh silica gel as the stationary phase, and chloroform/methanol as the mobile phase for gradient elution, by volume, the gradient is successively chloroform:methanol=100:0, 100:1, 100:2, 100:3, 100:5, 100:10, 0:100, the flow rate of the eluent is 30ml/min, and the eluate is collected (collected once every 500ml); The eluents were combined to obtain components 1-11; component 4 was passed through a silica gel column, with 200-300 mesh silica gel as the stationary phase, and the mobile phase was petroleum ether/ethyl acetate, and the gradient was petroleum ether in order by volume: Ethyl acetate=10:1, 10:3, 10:5, 10:6, 10:7, 10:9, the eluent flow rate is 30ml/min, collect the eluate (every 500ml once), the same as every Combine five eluents to obtain fractions 4-1 to 4-16; put fractions 4-11 on a Sephadex LH-20 gel column, the mobile phase is dichloromethane/methanol=1:1, and the flow rate of the eluent is 0.3ml/min, collect the eluate (collect once every 10ml), and combine every four parts of eluate to obtain components 4-11-1 to 4-11-6; Liquid chromatography, prepared with 48% methanol water, to obtain the monomeric compound with a retention time of 11 min.

(3) Structure identification: using deuterated dimethyl sulfoxide as solvent, Bruker Avance Ⅱ 500M nuclear magnetic resonance instrument was used to measure the nuclear magnetic spectrum of the isolated compound sample, and the molecular formula was deduced by mass spectrometry, and finally the structure of the product was characterized.

Its spectral data are as follows: ¹ H NMR (500Hz, DMSO-d ₆ ) δ: 9.42 (1H, brs, 3-OH), 6.68 (1H, d, J=8.0Hz, H-4), 7.04 (1H, t ,J=8.0Hz,H-5),6.65(1H,d,J=8.0Hz,H-6),2.57(1H,t,J=8.0Hz,H-2'),12.1(1H,brs, 3-OH'). ¹³ C NMR (125Hz, DMSO-d ₆ ) δ: 142.9 (C-1), 122.6 (C-2), 156.4 (C-3), 113.6 (C-4), 129.2 (C-5), 120.1 ( C-6), 64.9 (C-7), 57.7 (C-8), 27.9 (C-1'), 35.9 (C-2'), 174.5 (C-3').

Its HMBC correlation is as follows:

(2) The compound was dissolved, and the concentration was adjusted to 10 mg/mL. The initial gradient was 30-fold dilution, followed by 3-fold dilution, and 7 consecutive gradients. Culture in a 96-well cell plate, add 100 μl/well of DMEM complete medium to the wells of the virus control group, add 150 μl/well of DMEM complete medium to the wells of the cell control group, add 142.5 μl/well to the wells of the sample to be tested, and add culture to the remaining wells Base 100μl/well. Add 7.5 μl of the sample to be tested into the well of the sample to be tested. Gently blow and aspirate the liquid in the sample wells to be tested 6-8 times, then transfer 50 μl of the liquid to the corresponding wells listed below, and then all the wells are diluted 3-fold. Dilute SARS-CoV-2 pseudovirus (spike-D614G) to 1.3×10 ⁴ TCID 50/mL with DMEM complete medium, and add 50 μl to each well. The above 96-well plate was placed in a cell culture incubator (37°C, 5% CO ₂ ) and incubated for 1 hour. After 30 minutes of incubation, the Huh7 cells were digested and the cell concentration was diluted to 2×10 ⁵ cells/mL. After the incubation, add 100 μl of cells to each well to make the cells in each well 2×10 ⁴ cells, and put them in a 37°C, 5% CO ₂ cell incubator for 24 hours. After the incubation, discard 150 μl of supernatant and add 100 μl of Bright-GloTM luciferase detection reagent, react at room temperature in the dark for 2 minutes, pipette repeatedly, and transfer 150 μl of liquid to the white plate. Luminescence values (RLU) were read using a PerkinElmer EnSight Multi-Purpose Imaging Microplate Reader. Drug _EC50 values were calculated using luminescence values. The CC ₅₀ experiment process is the same as above, except that no pseudovirus is added to each well, so as to see the effect of the drug on the cell activity.

(3) Repeat the above method, and use the host-directed drug clofazimine with anti-SARS-CoV-2 virus activity approved by the FDA as a control group to compare and judge the inhibitory activity of the compound on the SARS-CoV-2 pseudovirus .

Table 1 activity test result:

the	Average EC 50 (ug/ml)	CC50 (ug/ml)	SI (SI=CC 50 /average(EC50)
Clofazimine	4	6	1.5
the compound	52	157	30

Note: SI>3 drugs are available

Experimental results show that the compound has inhibitory activity on the SARS-CoV-2 virus, especially the inhibitory rate on the virus is higher than that of clofazimine on the virus, and can be used to prepare drugs for inhibiting the SARS-CoV-2 virus.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto, any person familiar with the technical field within the technical scope of the disclosure of the present invention, according to the present invention Any equivalent replacement or change of the created technical solution and its inventive concept shall be covered within the scope of protection of the present invention.

Claims (7)

1. A polyketide compound with anti-new coronavirus activity, characterized in that it has the structure shown in formula (I):

   
2. The preparation method of polyketide compound as claimed in claim 1, is characterized in that, comprises the steps:

   (1) Strain fermentation: Alternaria sp. is fermented in solid state with rice and purified water;

   (2) Extraction and separation of metabolites: Use an equal volume of methanol to ultrasonically extract the solid fermentation product of the plant endophytic fungus Alternaria sp., filter through 8 layers of gauze, separate the extract from mycelium and rice, and concentrate the extract to dryness , the extract is subjected to medium-pressure column chromatography, with 300-400 mesh silica gel as the stationary phase, and chloroform/methanol as the mobile phase for gradient elution, by volume, the gradient is successively chloroform:methanol=100:0, 100:1, 100:2, 100:3, 100:5, 100:10, 0:100, the eluent flow rate is 30ml/min, collect the eluate once every 500ml, hang the eluate to dry, Combined to obtain components 1-11; component 4 was passed through a silica gel column, with 200-300 mesh silica gel as the stationary phase, and the mobile phase was petroleum ether/ethyl acetate, and the gradient was petroleum ether:ethyl acetate by volume. =10:1, 10:3, 10:5, 10:6, 10:7, 10:9, the flow rate of the eluent is 30ml/min, the eluate is collected every 500ml, and combined with every five eluates , to obtain components 4-1 to 4-16; put components 4-11 on Sephadex LH-20 gel column, mobile phase is dichloromethane/methanol 1:1, eluent flow rate is 0.3ml/min, every Collect the eluate once in 10ml, and combine every four eluents to obtain components 4-11-1 to 4-11-6; component 4-11-3 is prepared by high performance liquid chromatography and 48% methanol water , to obtain a monomeric compound with a retention time of 11 min.
3. The preparation method of polyketide compound as claimed in claim 2, is characterized in that, also comprises structural identification step (3):

   Using deuterated dimethyl sulfoxide as solvent, Bruker AvanceⅡ500M nuclear magnetic resonance instrument was used to measure the nuclear magnetic spectrum of the isolated compound sample, and the molecular formula was deduced by mass spectrometry, and finally the structure of the product was characterized.
4. The preparation method of polyketides as claimed in claim 2, characterized in that, the fermentation conditions in the step (1): the bacterial strain Alternaria sp. is subjected to shaking culture by using fungus No. 4 medium, and the culture conditions are 28°C, 180r /min, and then inoculate the fermented liquid and mycelia of the shaking culture in the Erlenmeyer flask equipped with rice solid medium for static fermentation for 40 days, and the culture temperature is 28°C.
5. The method for preparing polyketides according to claim 4, wherein the No. 4 culture medium for fungi is composed as follows in terms of mass percentage: 2% mannitol, 2% glucose, 0.5% yeast extract, 1% peptone , 0.05% KH2PO ₄ , 0.03% MgSO ₄ ·7H ₂ O, 0.1% corn steep liquor, deionized water preparation.
6. The preparation method of polyketide compound as claimed in claim 4, is characterized in that, described rice solid medium is: every 110ml deionized water adds 80g rice, packs in conical flask; Every bottle of rice culture medium inoculates 20ml seed liquid.
7. Use of the polyketide compound as claimed in claim 1 in the preparation of a drug for inhibiting the SARS-CoV-2 virus.

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