CN109879885B - Novel compound and preparation method and application thereof - Google Patents

Abstract

The invention discloses a natural pyran naphthoquinone compound shown as a formula I or pharmaceutically acceptable salt thereof, and a preparation method and application thereof. The compound is obtained by first separating from a solid fermentation extract of erythromyceliophthora pseudopekinensis (Nectria pseudopekinensis) Z8-6(7761) (with the preservation number of CGMCC No. 17473). The compound has the activity of inhibiting the reproduction and growth of tumor cells, and can be used for preparing a medicament for inhibiting the proliferation of eukaryotic tumor cells or preparing a medicament for preventing and/or treating tumors.

Description

Novel compound and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a naphthoquinone pyrane compound in rice fermentation products of Nectriapeudeuterochia fungi, and a preparation method and application thereof.

Background

Polyketides are of a rich structural type and diverse biological activities, such natural products are produced by successive condensation reactions of lower carboxylic acid units, have the same biosynthetic pathway, are widely distributed in plants and fungi, are important sources of drugs, and important polyketides derived from fungi include: lovastatin (a hypolipidemic drug), griseofulvin (an antifungal drug), mycophenolic acid (mycophenolic acid) and the like.

Aromatic polyketones belong to an important class of polyketones, and various aromatic polyketones compounds are separated from fungi, wherein the aromatic polyketones comprise naphthopyr and naphthoquinone, and rearrangement, ring opening and dimerization derivatives thereof, and the compounds have various biological activities such as antibacterial activity, antifungal activity, antitumor activity and the like. Many quinone natural products exhibit strong antitumor activity, and thus their antitumor effects are widely regarded in studies at various stages in vitro, in vivo and clinically, and notable examples include: mitomycin (mitomycin), which is an antitumor drug currently used in clinical practice, and anthracyclines such as daunorubicin (daunorubicin), doxorubicin (doxorubicin), and aclarubicin (aclarubicin), etc.; the in vitro anti-tumor effect of vitamin K and its derivative menadione has also been reported.

Fungi of the genus Nectria are capable of producing a range of naphthoquinones in dark colors, and are structurally classified mainly into nitrogen atom-free fusarium (fusabin) derivatives and nitrogen atom-containing fusarium botryoides (bostrycoidin) derivatives. The fungus of the genus Nectria (Nectria pseudotrichia) is generally recognized as a phytopathogen and its chemical composition is reported to include naphthoquinones, isocoumarins and terpenoids. The anti-tumor activity and the application of the novel naphthopyran quinone compound separated and prepared from rice leavening of the fungi of the genus Nectria related to the invention are not reported so far.

Disclosure of Invention

The invention aims to provide a natural naphthopyran quinone compound, a preparation method thereof and application thereof in preparing an anti-tumor medicament.

In a first aspect of the invention there is provided a compound of formula I or a pharmaceutically acceptable salt thereof:

the second aspect of the invention provides a pseudoplexus erythraea (Nectria pseudotrichia) Z8-6(7761), which is isolated from twigs of trees with altitude of 900 m of Tiantang Zi of Anhui, and is preserved in the general microorganism center of China Committee for culture Collection of microorganisms (CGMCC for short, the address: No. 3 of West Lu 1 of the North Chen West Lu of the Korean district of Beijing, institute of microbiology, China academy of sciences, zip code 100101) in 26 days of 2019, the preservation number is CGMCC No.17473, the classification name is pseudoplexus erythraea (Nectria pseudotrichia) and the strain name is Z8-6 (7761).

In a third aspect of the invention, there is provided a fermentation broth or extract of the strain of the invention; specifically, the fermentation liquor or the extract contains the compound shown in the formula I.

The fourth aspect of the invention provides the application of the strain in preparing the compound shown in the formula I or the pharmaceutically acceptable salt thereof.

The fifth aspect of the present invention provides a process for producing the compound represented by the above formula I, which comprises using a strain having a collection number of CGMCC No.17473, and being classified and named as Haematococcus pseudopekinensis (Nectria pseudotrichia) and named as Z8-6(7761) in the process.

In a preferred embodiment of the present invention, the method comprises the steps of:

a) performing solid fermentation on hypocrella pseudopekinensis (Nectria pseudotrichia) Z8-6(7761) to obtain a fermentation culture;

b) extracting the fermentation culture obtained in the step a) by using a proper organic solvent for proper times, combining extracting solutions, and separating the organic solvent from the extracting solutions to obtain a crude extract;

c) subjecting the crude extract obtained in step b) to chromatographic separation to obtain an active ingredient solution, and subjecting the active ingredient solution to high performance liquid chromatography to obtain the compound.

In the preparation method, in the step a), the culture medium used for fermentation is a rice culture medium; the rice culture medium can be prepared by mixing rice and water according to the proportion of 1.6Kg to 2400 mL.

The fermentation temperature is 20-30 ℃, preferably 22-28 ℃, and more preferably 25 ℃;

the fermentation time is 35-45 days, preferably 38-42 days, and more preferably 40 days.

In the preparation method, the extraction mode in the step b) is soaking extraction;

the extraction method is to soak the fermentation culture obtained in the step a) in the organic solvent for a certain time (the soaking time is at least 12 hours, the preferred soaking time is 0.5-2 days, and the more preferred soaking time is 1 day);

the extraction times are 1-5 times, the preferred extraction times are 2-4 times, and the more preferred extraction times are 3 times;

the organic solvent is methanol, acetone or ethyl acetate, the preferable organic solvent is acetone or ethyl acetate, and the more preferable organic solvent is ethyl acetate;

the organic solvent is separated in step b) by distillation, and a more preferred method for separating the organic solvent is distillation under reduced pressure.

In the preparation method, in the step c), the chromatographic separation is silica gel column chromatographic separation and/or gel chromatographic separation, wherein the silica gel column chromatographic separation is preferably reduced pressure silica gel column chromatographic separation;

preferably, the crude extract obtained in step b) is subjected to silica gel column chromatography and gel chromatography in sequence;

preferably, in the silica gel column chromatography separation step, the mobile phase is a mixture of petroleum ether and ethyl acetate; elution was performed according to the following elution procedure: gradient elution is sequentially carried out by adopting 9 mobile phases, petroleum ether and column volume of which the volume ratio is 100:0,98:2,96:4,92:8,90:10,88:12,85:15,80:20 and 75:25 in the 9 mobile phases are respectively eluted in a gradient manner by 2-3 times, and eluent obtained by eluting the petroleum ether and ethyl acetate by using the volume ratio of 75:25 as the mobile phases is collected, namely the active component solution.

Preferably, in the gel chromatography separation step, the mobile phase is a solution of chloroform to methanol in a volume ratio of 1: 1.

Specifically, the method comprises the following steps: the active fraction solution was separated by gel chromatography using Sephadex LH-20 (from GEHealthcare Bio-Sciences AB) as a gel packing of 60cm × 2.5cm (from New Ville glass instruments), 60g of gel was packed in a column, the mobile phase was a 1:1 volume chloroform/methanol solution, which was received in the flow order of the mobile phase, and each 5mL of the collected subfractions, and the 16 th subfraction (designated as subfraction 1-16) was found to have inhibitory activity under the guidance of activity tracking [ the inhibitory activity of 20 fractions against breast cancer cells (MCF-7) and cervical cancer cells (HeLa), and the specific test method was as described in example 3, and an inhibitory rate of more than 50% was regarded as having inhibitory activity ].

In the preparation method, in the step c), in the high performance liquid chromatography separation step, the mobile phase is acetonitrile-water solution; preferably, the volume ratio of acetonitrile to water is 42: 58.

Preferably, the conditions for the high performance liquid chromatography separation are: the elution was carried out isocratically using a C18 reverse phase column and a mixture of acetonitrile and water at a volume ratio of 42:58 as a mobile phase.

In one embodiment of the invention, the chromatograph employed is specifically an Agilent model 1260 semi-preparative high pressure liquid chromatograph. The C18 reversed phase chromatographic column is specifically an Agilent C18 reversed phase semi-preparative chromatographic column (5 μm; 9.4X 250 mm). The flow rate of the isocratic elution was 2.0mL/min, and the elution time was 50 min.

Purifying by high performance liquid chromatography under the above conditions, and collecting the elution peak with retention time of 38.0min to obtain the compound shown in formula I.

In a sixth aspect, the present invention provides a pharmaceutical composition, which contains the compound represented by formula I provided in the first aspect of the present invention or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

The pharmaceutical composition can be prepared into various forms such as injection, tablets, powder, granules, capsules, oral liquid, ointment, cream and the like. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field. The carrier includes diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, etc. which are conventional in the pharmaceutical field.

The pharmaceutical composition can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical mediated method; or mixed or coated with other materials and introduced into body.

The seventh aspect of the invention provides the use of the compound represented by formula I or a pharmaceutically acceptable salt thereof according to the first aspect of the invention, or the pharmaceutical composition according to the sixth aspect of the invention, in the preparation of a medicament for inhibiting the proliferation of eukaryotic tumor cells, or in the preparation of a medicament for preventing and/or treating tumors;

preferably, the eukaryote is a mammal;

preferably, the tumor cell is a cancer cell; further preferably, the cancer cell is a breast cancer cell, a kidney cancer cell, a liver cancer cell, a glioma cell, a cervical cancer cell and a colon cancer cell; more preferably, the breast cancer cell is a breast cancer cell MCF-7, the kidney cancer cell is a kidney cancer cell ACHN and a kidney clear cell adenocarcinoma cell 786-O, the liver cancer cell is a liver cancer cell HepG2, the glioma cell is gliomas SHSY5Y, H4 and U251, the cervical cancer cell is a cervical cancer cell HeLa, and the colon cancer cell is a colon cancer cell SW480 and SW 620.

Preferably, the tumor is a carcinoma; further preferably, the cancer is breast cancer, kidney cancer, liver cancer, glioma, cervical cancer and colon cancer.

The compound shown in the formula I provided by the invention is a novel compound which is obtained and named after being separated from a microorganism solid fermentation extract for the first time, and the anti-tumor activity of the compound is reported for the first time.

The invention identifies whether the compound has the function of inhibiting the tumorigenesis and development by constructing a cell screening model. The constructed cell screening model comprises breast cancer cells (MCF-7), kidney cancer cells (ACHN), clear cell adenocarcinoma cells (786-O), liver cancer cells (HepG2), glioma cells (SHSY5Y, H4 and U251), cervical cancer cells (HeLa) and colon cancer cells (SW480 and SW 620).

Drawings

FIG. 1 shows NMR of a compound of formula I¹H-NMR spectrum;

FIG. 2 shows NMR of a compound of formula I¹³A C-NMR spectrum;

FIG. 3 is a nuclear magnetic resonance HSQC spectrum of the compound of formula I;

FIG. 4 is a nuclear magnetic resonance HMBC spectrum of the compound shown in formula I;

FIG. 5 is the measured CD spectrum and the calculated ECD spectrum of the compound shown in formula I;

FIG. 6 is a graph showing the dose effect of tumor cells including breast cancer cells (MCF-7), kidney cancer cells (ACHN), renal clear cell adenocarcinoma cells (786-O), liver cancer cells (HepG2), glioma cells (SHSY5Y, H4 and U251), cervical cancer cells (HeLa) and colon cancer cells (SW480 and SW620) on the compound of formula I;

FIG. 7 shows the results of the compounds of formula I on tumor cells [ breast cancer cell (MCF-7), renal cancer cell (ACHN), renal clear cell adenocarcinoma cell (786-O), liver cancer cell (HepG2), glioma cell (SHSY5Y, H4 and U251), cervical cancer cell (HeLa) and colon cancer cell (SW480 and SW620) ]]IC of₅₀A value;

FIG. 8 shows the positive control drug cisplatin (DDP) on tumor cells [ breast cancer cell (MCF-7), renal cancer cell (ACHN), renal clear cell adenocarcinoma cell (786-O), liver cancer cell (HepG2), glioma cell (SHSY5Y, H4 and U251), cervical cancer cell (HeLa) and colon cancer cell (SW480 and SW620) ]]IC of₅₀The value is obtained.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

The strain N used in the embodiment is a strain N which is deposited in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of West Luo No.1 of the sunward area of Beijing, China institute of microbiology, China academy of sciences, postal code 100101) in 26.3.26.2019, the preservation number is CGMCC No.17473, the strain is classified and named as Chitosa pseudochaeli (Nectria pseudolari), and the strain name is Z8-6 (7761).

Example 1 preparation of Compounds of formula I

a. Solid fermentation of Strain Z8-6(7761)

Activation of Strain Z8-6 (7761). PDA culture medium: 200g of potato, 20g of glucose, 15g of agar and 1000mL of purified water are sterilized by high-pressure steam at 121 ℃ for 30 minutes to prepare a test tube inclined plane, and mycelium is selected to be inoculated on the test tube inclined plane and cultured for 10 days at 25 ℃.

Solid fermentation of strain Z8-6 (7761). Preparing a rice culture medium (1.6 Kg of rice and 2400mL of purified water are equally added into 20 triangular bottles of 500mL, soaked overnight, sterilized by high-pressure steam at 121 ℃ for 30 minutes, and cooled for standby); selecting mycelium from test tube slant, placing in test tube containing sterile water to obtain bacterial suspension, and mixing the prepared bacterial suspension (spore concentration is 1 × 10)⁶one/mL) of the cells were inoculated onto a rice medium in an amount of 5mL, and subjected to fermentation culture at 25 ℃ for 40 days while being aseptically cultured to obtain a fermentation culture.

b. Extraction of crude extract

B, mashing the fermentation culture obtained in the step a by using a glass rod (0.8cm multiplied by 30cm), adding 4.0L of ethyl acetate into a 500mL triangular flask (20 bottles in total and 200mL in each bottle), standing and soaking for 1 day, filtering at room temperature and normal pressure by using medium-speed qualitative filter paper (purchased from special paper industries, Ltd.) with the diameter of 12.5cm to obtain an organic phase and residues, recording the organic phase as a crude extract I, and recording the residues as residues I;

adding 4.0L ethyl acetate into residue I (20 bottles in total, 200mL in each bottle), standing and soaking for 1 day, filtering to obtain an organic phase and residue, recording the organic phase as a crude extract II, and recording the residue as residue II;

adding 4.0L ethyl acetate into residue II (20 bottles in total, 200mL in each bottle), standing and soaking for 1 day, filtering to obtain an organic phase and residue, recording the organic phase as a crude extract III, and recording the residue as residue III;

and combining the crude extract I, the crude extract II and the crude extract III to be recorded as a crude extract.

The crude extract was distilled under reduced pressure to dryness to obtain 8.6g of a crude extract.

c. Separation and purification of crude extract

Loading the crude extract into reduced pressure chromatography silica gel column (40cm × 8.5cm) from Beijing glass instruments; the silica gel used was thin layer chromatography silica gel H, purchased from Qingdao ocean chemical Co., Ltd. Packing 120g silica gel in a column, and performing reduced pressure silica gel column chromatography by using a petroleum ether-ethyl acetate-methanol system, wherein the mobile phase is as follows: petroleum ether ethyl acetate (v/v) 100:0,98:2,96:4,92:8,90:10,88:12,85:15,80:20,75:25,70:30,65:35,60:40,55:45,50:50,45:55,40:60,35:65,30:70,25:75,20:80,10:90,0: 100; ethyl acetate methanol (v/v) 98:2,95:5,90:10,0: 100; the total of 26 gradients, the elution volume of each gradient is 400mL, the eluent of each gradient is distilled under reduced pressure, the crude extract is divided into 26 components according to the order of polarity from small to large, under the guidance of activity tracking [ the inhibitory activity of 26 components on breast cancer cells (MCF-7) and cervical cancer cells (HeLa) is tested, the specific test method is described in example 3, the inhibitory activity is considered to be present when the inhibitory rate is more than 50% ], the component (named as component 1) eluted by using ethyl acetate 75:25(v/v) as a mobile phase in petroleum ether has the inhibitory activity, and the active component 1(110mg) is collected.

Separating active component 1 by gel chromatography, wherein the specification of gel chromatography column is 60cm × 2.5cm (from New Vill glass instruments Co.), the used gel filler is Sephadex LH-20 (from GE Healthcare Bio-Sciences AB Co.), 60g gel is loaded in the column, the mobile phase is a solution with the volume ratio of chloroform to methanol of 1:1, the solution is received according to the flowing sequence of the mobile phase, every 5mL is a subfraction, 20 subfractions are collected in total, then, under the guidance of activity tracking [ test 20 fractions for inhibitory activity against breast cancer cells (MCF-7) and cervical cancer cells (HeLa), and the specific test method described in example 3, the inhibitory rate of more than 50% was regarded as inhibitory activity ], 16 th sub-fraction (named sub-fractions 1 to 16) was found to have inhibitory activity, and active sub-fractions 1 to 16(26mg) were collected.

Purification of the active subfractions 1-16 by HPLC affords 1.6mg of the compound of formula I, wherein the HPLC conditions are: adopting Agilent1260 type semi-preparative high pressure liquid chromatograph, Agilent C₁₈Reverse phase semi preparative chromatography column (5 μm, 9.4X 250mm), flow rate 2.0mL/min, with acetonitrile and water as mobile phase, conditions were prepared: acetonitrile/water (42/58,v/v) isocratic elution for 50 minutes to obtain the compound shown in the formula I, and the retention time t of the compound_R38.0 min.

In summary, 8.6g of crude extract was isolated and purified to give 1.6mg of the compound of formula I with a yield of 0.02%.

Example 2 structural characterization of Compounds of formula I

The compound of formula I prepared in example 1 was analyzed by high resolution mass spectrometry (HRESIMS), infrared spectroscopy (IR), ultraviolet spectroscopy (UV), nuclear magnetic resonance spectroscopy (NMR), circular dichroism spectroscopy (CD) and optical rotation spectroscopy (ORD), wherein the high-resolution Mass spectrum is tested by a national institute of medicine and metabolite analysis research center of the institute of medicine of Chinese medical science by a test provided by the institute of poisonous drug of military medical institute (the model of a test instrument is Agilent Accurate-Mass-Q-TOF LC/MS G6550), a nuclear magnetic resonance spectrum (the model of a test instrument is Bruker Avance III-600) and a circular dichroism spectrum (the model of a test instrument is JASCO J-815), and an infrared spectrum (the model of a test instrument is Nicolet Magna-IR 750), an ultraviolet spectrum (Shimadzu Biospec-1601) and an optical spectrum (the model of a test instrument is Autopol IV) are tested by the institute of microbiology of Chinese medical science institute.

The physicochemical data of the compound of formula I prepared in example 1, analyzed by the above tests, are: a yellow powder; the molecular formula is as follows: c₂₀H₂₂O₇(ii) a Molecular weight: 374; high resolution mass spectrometry HRESIMS: m/z 375.1441[ M + H ]]⁺(calculated C)₂₀H₂₃O₇375.1438); hydrogen spectrum (¹H-NMR) and carbon Spectroscopy (¹³C-NMR) data are shown in Table 1:

TABLE 1 hydrogen spectra of compounds of formula I: (¹H-NMR) and carbon Spectroscopy (¹³C-NMR) data

^aCDCl at 600MHz₃Is a solvent test.

^bCDCl at 150MHz₃Is a solvent test.

The chemical formula of the compound is confirmed to be shown in the formula I through the nuclear magnetic resonance hydrogen spectrum (shown in the figure 1), the carbon spectrum (shown in the figure 2) and the mass spectrum of the compound shown in the formula I, the two-dimensional nuclear magnetic resonance spectrum HSQC (shown in the figure 3) and the HMBC (shown in the figure 4) spectrum of the compound, actually measured CD and calculated ECD spectrogram (shown in the figure 5):

in summary, the natural compound shown in the formula I is extracted and separated from the erythromyceliophthora pseudopilea (Nectria pseudotrichia) Z8-6(7761), and the structural characteristics of the compound are identified through further purification and combination of high-resolution mass spectrometry, nuclear magnetic resonance technology and circular dichroism chromatogram data analysis.

Example 3 Effect of Compounds of formula I on reproductive growth of tumor cells

Constructing a cell screening model: breast cancer cells (MCF-7), kidney cancer cells (ACHN), renal clear cell adenocarcinoma cells (786-O), liver cancer cells (HepG2), glioma cells (SHSY5Y, H4 and U251), cervical cancer cells (HeLa) and colon cancer cells (SW480 and SW 620).

The effect of the compound of formula I prepared in example 1 on the reproductive growth of the above 5 tumor cells was examined by MTS method (MTS colorimetric method). To verify the dose effect of several tumor cells on the compound, the compound of formula I prepared in example 1 and cisplatin, a positive control, were prepared into test solutions with concentrations of 50.00. mu.M, 25.00. mu.M, 12.50. mu.M, 6.25. mu.M and 3.13. mu.M, respectively, using DMSO (Dimethyl sulfoxide, DMSO) as a solvent, and three replicates were set for each treatment concentration.

The specific experimental method is as follows:

1. tumor cell suspension was inoculated into 96-well plates at 100. mu.L (concentration 1.0X 10)⁴pieces/mL), DMEM medium (purchased from Invitrogen, and sterilized with liquid medium by adding an appropriate volume of water as described before use) 100 μ L.

2. Tumor cells were incubated at 37 ℃ with 5% CO₂After 24 hours, the culture medium was poured out of each well and washed 1 time with PBS, and the experimental groups were added with different concentrations of the compound of formula I prepared as described aboveInoculating 3 holes with each concentration of the solution to be detected, and adding 200 mu L of the solution into each hole; cisplatin solutions with different concentrations are added into the positive control group, 3 holes are inoculated at each concentration, and 200 mu L of cisplatin solution is added into each hole; adding 200 mu L of liquid DMEM culture solution into the negative control group; it was placed at 37 ℃ in 5% CO₂The culture chamber of (2) was incubated for 4 hours.

3. After the culture is finished, the compound solution shown in the formula I, the positive control solution and the culture solution of the negative control group are replaced by fresh DMEM liquid culture medium in the same volume, and then the culture solution is cultured at 37 ℃ and 5% CO₂The culture was performed in the incubator of (1) for 48 hours.

4. The tumor cells were treated with 20. mu.L (concentration 0.5mg/mL) of MTS solution per well and then placed at 37 ℃ in 5% CO₂The culture was performed in the incubator of (1) for 1.5 hours.

5. The medium and MTS solution were removed by centrifugation (1500r/min,5min), 200. mu.L of DMSO solution was added to each well, and the mixture was allowed to stand at 37 ℃ for 1 hour.

6. Measuring the optical density OD value of the mixture cultured in the above steps at 490nm wavelength by use of enzyme-linked immunosorbent assay (SUNRISE-basic TECAN), and calculating the tumor cell growth inhibition rate according to a formula. When the OD value was measured, a blank containing 200. mu.L of DMSO solution was set.

In the experiment, the OD value of the negative control group, the OD value of the positive control group and the OD value of the experimental group are the values of blank group OD deduction.

The results of the compounds of formula I on the inhibition of 10 tumor cells are shown in FIG. 6.

The IC of the natural compound shown in the formula I on 10 tumor cells is deduced according to the obtained inhibition rate curve graph (shown in figure 6) of the compound shown in the formula I at each treatment concentration₅₀The value is obtained.

Wherein the IC₅₀Represents the drug concentration that results in 50% inhibition of cell proliferation.

IC of Compounds of formula I on 10 tumor cells tested₅₀The values are shown in FIG. 7.

The IC of the positive control drug cisplatin (DDP) on the 10 tumor cells tested was obtained according to the above method₅₀The values are shown in FIG. 8.

In conclusion, the compound shown in the formula I has obvious inhibitory activity on breast cancer cells (MCF-7), kidney cancer cells (ACHN), renal clear cell adenocarcinoma cells (786-O), liver cancer cells (HepG2), glioma cells (SHSY5Y, H4 and U251), cervical cancer cells (HeLa) and colon cancer cells (SW480 and SW 620). Therefore, the invention provides candidate compounds for researching and developing new anti-tumor (breast cancer, kidney cancer, liver cancer, glioma, cervical cancer and colon cancer) medicaments, and also provides scientific basis for developing and utilizing natural active substances from microorganisms.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (14)

1. A compound of formula I, or a pharmaceutically acceptable salt thereof:

(formula I).

2. A process for preparing a compound of claim 1 comprising: fermentation of Haemaria pseudonarum (A. pseudonarum:)Nectriapseudotrichia) A strain Z8-6(7761) which gives a compound according to claim 1;

the said Chitosan pseudoplex (C.), (Nectriapseudotrichia) Z8-6(7761), which has a preservation number of CGMCC 17473 in China general microbiological culture Collection center (CGMCC).

3. The method of claim 2, wherein: the preparation method comprises the following steps:

a) to the said Chitosan pseudopeica (C.), (Nectriapseudotrichia) Z8-6(7761) performing solid fermentation to obtain fermentation culture;

b) extracting the fermentation culture obtained in the step a) by using an organic solvent, collecting an extracting solution, and separating the organic solvent to obtain a crude extract;

c) subjecting the crude extract obtained in step b) to chromatographic separation to obtain an active ingredient solution, and subjecting the active ingredient solution to high performance liquid chromatography to obtain the compound.

4. The production method according to claim 3, characterized in that: in the step a), the culture medium used for fermentation is a rice culture medium; the fermentation temperature is 20-30 ℃; the fermentation time is 35-45 days.

5. The method of claim 4, wherein:

the fermentation temperature is 22-28 ℃; the fermentation time is 38-42 days.

6. The production method according to claim 3, characterized in that: the extraction mode in the step b) is soaking extraction;

the extraction method comprises the following steps: soaking the fermentation culture obtained in step a) in the organic solvent for at least 12 hours;

the extraction times are 1-5 times;

the organic solvent is methanol, acetone or ethyl acetate;

the way of separating the organic solvent in step b) is distillation.

7. The method of claim 6, wherein:

the extraction method comprises the following steps: soaking the fermentation culture obtained in the step a) in the organic solvent for 0.5-2 days;

the extraction times are 2-4 times;

the organic solvent is acetone or ethyl acetate;

the organic solvent is separated in the step b) by vacuum distillation.

8. The production method according to claim 3, characterized in that: in the step c), the chromatographic separation is silica gel column chromatographic separation and/or gel chromatographic separation;

in the step of silica gel column chromatography separation, the mobile phase is a mixture of petroleum ether and ethyl acetate; elution was performed according to the following elution procedure: sequentially adopting 9 mobile phases for gradient elution, wherein the volume ratio of petroleum ether to ethyl acetate in the 9 mobile phases is 100:0,98:2,96:4,92:8,90:10,88:12,85:15,80:20 and 75:25 respectively, performing gradient elution for 2-3 times of column volume, and collecting eluent obtained by eluting the mobile phases with the volume ratio of petroleum ether to ethyl acetate of 75: 25;

in the gel chromatography separation step, the mobile phase is a solution with the volume ratio of chloroform to methanol being 1:1, and in the high performance liquid chromatography separation step, the mobile phase is a mixed solution of acetonitrile-water.

9. The method of claim 8, wherein: the silica gel column chromatography separation is decompression silica gel column chromatography separation;

sequentially carrying out silica gel column chromatography and gel chromatography separation on the crude extract obtained in the step b);

the conditions of the high performance liquid chromatography separation are as follows: the elution was carried out isocratically using a C18 reverse phase column and a 42:58 volume ratio acetonitrile-water mixture as the mobile phase.

10. A pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable excipients.

11. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 10, in the manufacture of a medicament for inhibiting the proliferation of eukaryotic tumor cells;

the eukaryote is a mammal; the tumor cell is a cancer cell.

12. Use according to claim 11, characterized in that: the cancer cell is selected from any one of the following: is breast cancer cell, renal cancer cell, liver cancer cell, glioma cell, cervical cancer cell and colon cancer cell.

13. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of claim 10 for the manufacture of a medicament for the prevention and/or treatment of tumors; the tumor is cancer.

14. Use according to claim 13, characterized in that: the cancer is selected from any one of the following: breast cancer, kidney cancer, liver cancer, glioma, cervical cancer and colon cancer.

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