CN109956951B

CN109956951B - Cervidomycin (Cervinomycin) C1, C2, C3 and C4, and its preparation method and application

Info

Publication number: CN109956951B
Application number: CN201910288024.4A
Authority: CN
Inventors: 武临专; 胡笑文; 孙伟; 江冰娅; 李书芬; 刘红宇; 余利岩; 山广志
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2019-04-11
Filing date: 2019-04-11
Publication date: 2021-01-01
Anticipated expiration: 2039-04-11
Also published as: CN109956951A

Abstract

The invention relates to Cervidomycin (cervinomycin) C1, C2, C3 and C4, and a production method and application thereof. The deer-color mycin C component is deer-color mycin C1, C2, C3 and C4, and the chemical structures thereof are shown in formulas (1), (2), (3) and (4). Through fermentation culture of streptomycete with preservation number of CGMCC NO.16425, the fermentation culture is harvested, extracted, separated and purified to obtain 4 deer chroman C components. The deer-color mycin C component is expected to be used as a lead compound for researching and developing antitumor drugs.

Description

Cervidomycin (Cervinomycin) C1, C2, C3 and C4, and its preparation method and application

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to cerhromycin (cervinomycin) C1, C2, C3 and C4, and a production method and application thereof.

Background

With the rapid growth and aging of the global population, the incidence and mortality of cancer also rapidly increase worldwide. According to the statistics of world health organization 2015, cancer is the first or second cause of death in the population under 70 years of age in 91 countries and the third or fourth cause of death in the population under 70 years of age in another 22 countries. Since 2010, cancer has been the leading cause of death in the Chinese population and a major public health problem.

Among many cancers, pancreatic cancer is a malignant disease with poor prognosis. Early diagnosis of pancreatic cancer is very difficult and there is also no particularly effective treatment regimen; the only possible treatment for pancreatic cancer is surgical resection, but there is a recurrence of the disease after surgery. The above factors result in low cure rate and short survival time (average survival time is 3-6 months, 5-year survival rate is less than 5%) for pancreatic cancer patients. Therefore, the clinical need for drugs for treating pancreatic cancer is very urgent.

Cerhromycin (cervinomycin) is a polycyclic xanthone compound catalyzed by microorganism II type polyketide synthase and the like, and is separated, purified and chemically identified by Japanese village wisdom and the like for the first time. Polycyclic xanthones generally have strong antitumor cell activity, such as IC of arixanthomycin A on colorectal cancer HCT-116 cell line reported in 2014₅₀0.15. mu.M; IC of xanthoholipin on leukemia cell line was reported in 2002₅₀<0.3 μ M, cancer cell line IC against oral squamous cell₅₀<2 mu M; in 2009, the IC of simamicin alpha on tumor cell lines was reported₅₀At 0.3-19nM, additional researchers found that simamicin α was able to upregulate caspase-3 activity, fragmenting DNA, and ultimately leading to apoptosis. The deer color mycin has very strong anti-tumor cell activity as the polycyclic xanthone compounds; for example, in previous studies, the present inventors isolated 4 members (B) of the Cerhromycin B-series compounds from the secondary metabolites of 1 strain of Streptomyces (CGMCC NO.16425)_1-4) Their IC on tumor cells (pancreatic cancer cells SW1990, prostate cancer cells PC3, non-small cell lung cancer cells H1299)₅₀Is 0.8-106.6 nM.

The deer-chromomycin B component series compounds show stronger inhibitory activity to gram-positive bacteria such as staphylococcus aureus, the minimum inhibitory concentration MIC of the deer-chromomycin B component series compounds is 0.06-0.004 mu g/ml, and the deer-chromomycin B component series compounds are expected to become lead compounds for developing anti-gram-positive bacteria medicaments, so that the inventor has applied for related patents (Chinese invention patent, application number CN 201811341427.2).

The inventor carries out deep research on secondary metabolites generated by streptomyces CGMCC NO.16425 (see CN201811341427.2), separates 4 new components of the cerulomycins C1-4 from the strain, and determines the chemical structure of the cerulomycins C1-4 through spectroscopic data such as ultraviolet spectrum, high-resolution mass spectrum and nuclear magnetic resonance and X-ray diffraction analysis. Compared with the Cerhromycin B component, the Cerhromycin C component does not contain oxazolidine rings.

Disclosure of Invention

The invention firstly relates to a group of deer-color mycin C component compounds which are deer-color mycin C1, C2, C3 and C4 respectively. They are new polycyclic xanthenone compounds with chemical structures shown in formulas (1), (2), (3) and (4),

the deer-color mycin C1 component is a yellow amorphous powder with molecular formula of C28H23NO9 molecular weight 517, can be dissolved in organic solvents such as dimethyl sulfoxide and the like, and is insoluble in water;

the deer-color mycin C2 component is dark purple amorphous powder, has molecular formula of C28H21NO9 and molecular weight of 515, is slightly soluble in organic solvents such as dimethyl sulfoxide and the like, and is insoluble in water; it is similar to the structure of the C1 component of the deer-color mycin and belongs to the E-ring quinoid (oxidized) structure of the C1 component;

the deer-color mycin C3 component is golden yellow amorphous powder, has molecular formula of C26H19NO8 and molecular weight of 473, can be dissolved in organic solvents such as dimethyl sulfoxide and the like, and is insoluble in water;

the deer-color mycin C4 component is brick-red amorphous powder with molecular formula of C26H17NO8 and molecular weight of 471, is slightly soluble in organic solvents such as dimethyl sulfoxide and the like, and is insoluble in water; it is similar to the structure of the C3 component of deer-color mycin, belonging to the E-ring quinoid (oxidized) structure of the C3 component.

The invention also relates to a preparation method of the deer chromamycin C component compound, which comprises the following steps:

(1) fermenting and culturing streptomyces CGMCC NO.16425, and harvesting a fermentation product;

(2) extracting, separating and purifying the Cervidomycin (Cervinomycin) C component from the fermentation product.

The fermentation culture streptomyces CGMCC NO.16425 comprises the following steps:

1) seed culture

Firstly, a streptomycete CGMCC NO.16425 spore suspension frozen and preserved in a refrigerator at 70 ℃ below zero is melted and inoculated on a spore culture medium plate for culture.

The spore culture medium consists of (g/L): 20.0 of starch, 20.0 of soybean cake powder, 15.0 of agar powder and 7.0 of pH.

Each plate (9.0 cm diameter) was filled with about 20ml of spore medium, or each slant was filled with 20-30ml of spore medium.

The spore culture condition of the streptomycete CGMCC NO.16425 is 26-30 ℃ and 6-10 days. Washing spore with sterile water, and oscillating to obtain fresh spore suspension (containing about 10% of spore⁸Spores/ml) for inoculating fermentation medium plates; alternatively, the spores are plated or scooped up and inoculated into a liquid fermentation medium.

2) Solid state fermentation of said streptomyces

The solid state fermentation method comprises the following steps: inoculating the fresh spore suspension of streptomycete CGMCC NO.16425 to a fermentation medium plate.

The composition of the fermentation medium is (g/L): 10.0 parts of corn starch, 10.0 parts of cottonseed cake meal, 10.0 parts of threonine, 15.0 parts of agar powder and 7.0 parts of pH value.

Each fermentation medium plate (diameter 15.0cm) was filled with about 45 ml of fermentation medium in an inoculum size of about 10⁶And (4) spores.

And culturing the fermentation medium plate at 26-30 ℃ for 8-12 days, and harvesting the fermentation culture for separating and purifying the deer-tryptophin C component compound.

The extraction, separation and purification of the Cervidomycin (Cervinomycin) C component from the fermentation product comprises the following steps,

(1) extracting the fermentation culture with organic solvent such as ethyl acetate, and rotary-distilling the extractive solution under reduced pressure to obtain ethyl acetate crude extract;

(2) the ethyl acetate crude extract adopts reversed phase C₁₈Removing main impurities from the column, eluting with 50% methanol-water, 60% methanol-water and 70% methanol for 2-3 column volumes, collecting eluates, analyzing by TLC or HPLC, mixing eluates containing CeratomycinCollecting liquid of the component C; removing the eluting solvent by rotary evaporation under reduced pressure to obtain refined product containing Cerulomycin C1-2 component mixture and crude product containing Cerulomycin C3-4 component mixture;

(3) performing reverse phase preparation and semi-preparative HPLC purification on the refined product of the deer-tryptophycetin C1-2 component mixture to respectively obtain a deer-tryptophycetin C1 component and a deer-tryptophycetin C2 component, wherein a mobile phase used by the reverse phase HPLC is 50% acetonitrile-water, collecting an elution peak containing the deer-tryptophycetin C component, and performing reduced pressure rotary evaporation or freeze drying to obtain pure products of the deer-tryptophycetin C1 and C2 components;

(4) separating the crude product of the deer-tryptophycetin C3-4 component mixture by a normal phase silica gel chromatographic column, performing gradient elution by using methanol (0-15%) -dichloromethane, and combining eluates with the same components after HPLC analysis; removing the eluting solvent by rotary evaporation under reduced pressure to obtain refined product of Cerhromycin C3-4 component mixture;

(5) and (3) carrying out reverse phase preparation and semi-preparative HPLC purification on the refined product of the deer-tryptophycetin C3-4 component mixture to respectively obtain pure deer-tryptophycetin C3 component and pure deer-tryptophycetin C4 component, wherein a mobile phase used by the reverse phase HPLC is 45% acetonitrile-water, collecting an elution peak containing the deer-tryptophycetin C component, and carrying out reduced pressure rotary evaporation or freeze drying to obtain pure deer-tryptophycetin C3 and pure deer-C4 component.

About 13L of fermentation culture of streptomyces CGMCC NO.16425 can obtain 7.5mg of pure Cerromycin C1 component, 1.5mg of pure Cerromycin C2 component, 6.4mg of pure Cerromycin C3 component and 1.2mg of pure Cerromycin C4 component through the separation and purification steps. The separation and purification process of the Cerhromycin C component can be seen in FIG. 2.

The invention also relates to a method for obtaining Cervidomycin (cervinomycin) C2 and C4 by oxidation reaction by using Cervidomycin (cervinomycin) C1 and C3 as substrates, wherein the method comprises the following steps:

(1) dissolving Cerhromycin C1 (or C3) in dichloromethane-methanol (1:1) solution, adding weak oxidant (such as silver oxide) with the same mass as the component to be oxidized, stirring at room temperature, taking a little reaction supernatant every 1 hour after the oxidation reaction is finished (about 5 to 9 hours are needed), re-dissolving in acetonitrile after rotary evaporation, monitoring the reaction process by HPLC, wherein the HPLC monitors the type of a chromatographic column, namely Agilent zorbax SB-C8 column, 4.6 x 250mm, and the gradient elution is carried out by using 40-70% acetonitrile-water, the flow rate is 1.0ml/min, and filtering by using a filter membrane to remove the weak oxidant (silver oxide);

(2) separating and purifying the filtrate with silica gel column, gradient eluting with methanol (0-10%) -dichloromethane, and vacuum rotary evaporating to obtain semi-pure Ceratomycin C2 (or C4) component;

(3) and (3) performing preparative or semi-preparative reverse phase HPLC (high performance liquid chromatography) refining on the semi-pure product, wherein an eluent is acetonitrile (50% or 45%) -water, combining elution peaks containing a Cerromycin C2 (or C4) component, and performing freeze drying or reduced pressure rotary evaporation to obtain pure products of the target compounds, namely Cerromycin C2 and C4 components.

The invention also relates to application of the cerviolomycin (cervinomycin) C1, C2, C3 and C4 in preparation of antitumor drugs, preferably, the tumors are pancreatic cancer or colorectal cancer.

In order to improve the yield of the Cerhromycin C component of the streptomyces CGMCC NO.16425, the fermentation medium needs to contain rich carbon source and nitrogen source. The carbon source includes various monosaccharides (e.g., glucose and fructose), disaccharides (e.g., maltose and sucrose), polysaccharides (e.g., starch and dextrin), and the like; nitrogen sources, particularly organic nitrogen sources, include amino acids, polypeptides and peptones (of animal, plant or microbial origin), soybean meal, cottonseed meal, yeast meal, corn steep liquor, peanut meal and the like. These media components are used to provide the precursors or building blocks required for biosynthesis of the C component of Cerromycin, in addition to the nutrients and energy necessary for the growth of Streptomyces CGMCC NO. 16425. Thus, the fermentation medium of Streptomyces CGMCC NO.16425 includes, but is not limited to, the fermentation medium formulation described above.

The invention has the beneficial effects that: the deer chromacin C component related by the invention belongs to a group of polycyclic xanthone novel antibiotics, which have strong tumor cell growth inhibition activity, particularly show excellent inhibition activity on pancreatic cancer cells, and are expected to be used as lead compounds for research and development of antitumor drugs. The invention also provides a streptomyces CGMCC NO.16425 as a strain for producing the Cerromycin C component, a method for fermenting and culturing the strain and separating and purifying the Cerromycin C1, C2, C3 and C4 components from a fermentation culture, and a method for preparing the Cerromycin C2 component from the Cerromycin C1 component and preparing the Cerromycin C4 component from the Cerromycin C3 component by chemical oxidation reaction.

Drawings

FIG. 1 chemical structures of Cerromycin C1, C2, C3 and C4.

FIG. 2 is a flow chart of separation and purification of Cervidomycin C component.

FIG. 3 of Cervidomycin (cervinomycin) C1¹H-NMR nuclear magnetic spectrum.

FIG. 4A view of Cervidomycin (cervinomycin) C1¹³C-NMR nuclear magnetic spectrum.

FIG. 5 of Cervidomycin (cervinomycin) C1¹H-¹H COSY nuclear magnetic spectrum.

FIG. 6 HSQC nuclear magnetic spectrum of Cervidomycin (cervinomycin) C1.

FIG. 7 HMBC nuclear magnetic spectrum of Cervidomycin (cervinomycin) C1.

FIG. 8 NOESY nuclear magnetic spectrum of Cervidomycin (cervinomycin) C1.

FIG. 9 of Cervidomycin (cervinomycin) C2¹H-NMR nuclear magnetic spectrum.

FIG. 10 preparation of Cervidomycin (cervinomycin) C2¹³C-NMR nuclear magnetic spectrum.

FIG. 11 of Cervidomycin (cervinomycin) C2¹H-¹H COSY nuclear magnetic spectrum.

FIG. 12 HSQC nuclear magnetic spectrum of Cervidomycin (cervinomycin) C2.

FIG. 13 HMBC nuclear magnetic spectrum of Cervidomycin (cervinomycin) C2.

FIG. 14 NOESY nuclear magnetic spectrum of Cervidomycin (cervinomycin) C2.

FIG. 15 of Cervidomycin (cervinomycin) C3¹H-NMR nuclear magnetic spectrum.

FIG. 16 of Cervidomycin (cervinomycin) C3¹³C-NMR nuclear magnetic spectrum.

FIG. 17 of Cervidomycin (cervinomycin) C3¹H-¹H COSY nuclear magnetic spectrum.

FIG. 18 HSQC nuclear magnetic resonance spectrum of Cervidomycin (cervinomycin) C3.

FIG. 19 HMBC nuclear magnetic spectrum of Cervidomycin (cervinomycin) C3.

FIG. 20 NOESY nuclear magnetic spectrum of Cervidomycin (cervinomycin) C3.

FIG. 21 of Cervidomycin (cervinomycin) C4¹H-NMR nuclear magnetic spectrum.

FIG. 22 is a perspective view of a structure of Cervidomycin (Cervinomycin) C2 molecule.

FIG. 23 is a diagram of the unit cell packing along the a-axis of the Cerhromycin (cervinomycin) C2 molecule.

Detailed Description

Example 1: streptomyces CGMCC No.16425 solid state fermentation culture

(1) Preparation of fresh spore suspension: streptomyces CGMCC No.16425 spore suspension frozen and preserved in a refrigerator at 70 ℃ below zero is evenly coated and inoculated on 7-10 spore culture medium plates (the culture medium components are 20.0g/L of starch, 20.0g/L of soybean cake powder, 15.0g/L of agar powder, pH is 7.0, the diameter of each plate is 9.0cm, and each plate is filled with about 20ml of culture medium). When cultured at 28 ℃ for 8-9 days, a layer of gray spores grows on the surface of the plate medium. The spores were washed off each plate with 8.0-10.0ml of sterile water and shaken to obtain a concentration of about 1X 10⁸Spores/ml spore suspension used to inoculate fermentation medium plates.

(2) Solid state fermentation culture: the spore suspension was spread evenly onto fermentation medium plates (medium composition: corn starch 10.0g/L, cottonseed meal 10.0g/L, threonine 10.0g/L, agar meal 15.0g/L, pH 7.0; plate diameter 15.0cm, each plate filled with about 45 ml of medium) each coated with about 50. mu.l of spore suspension. Culturing at 28 deg.C for 8-9 days, and collecting culture (containing agar medium) of each fermentation plate for separation and purification of Cerhromycin C component.

Example 2: separation and purification of deer chromomycin C component

(1) Ethyl acetate extract:

streptomyces CGMCC NO.16425 solid fermentation culture (about 13L) is added with equal volume of ethyl acetate for extraction, and the extraction is carried out for 2 days each time and 2 times in total. The ethyl acetate extract was collected and rotary evaporated in vacuo to give a brown ethyl acetate extract (10 g).

(2) And (3) refining the deer-color mycin C component mixture (removing impurities by using a reverse phase silica gel chromatographic column):

the brown ethyl acetate extract was redissolved in an appropriate amount of methanol (about 150ml) and accelerated to dissolution with ultrasound. Approximately 15g of reverse phase (ODS) silica gel column packing was weighed in a ratio of 1:1.5, added to a methanol solution, mixed and then subjected to vacuum rotary evaporation to remove the methanol solvent, to obtain a sample-mixed ODS powder.

The ODS silica gel chromatography powder after sample mixing was loaded on a loading column and then connected to a separation column packed with about 120g of reverse phase silica gel column packing. Reverse phase chromatography column purification was performed to remove major impurities using a methanol-water system as the mobile phase. Eluting 3-4 column volumes with pure water, and eluting 2-3 column volumes with 50% methanol-water, 60% methanol-water and 70% methanol-water respectively at flow rate of 20 ml/min; collecting the eluates of each stage, and collecting about 70ml of each collecting tube; HPLC and TLC detect the target product in each collection tube, and the same constituent collection tubes are combined. After vacuum rotary evaporation, refined product (0.22g) containing Cerhromycin C1-2 component mixture and refined product (4.37g) containing Cerhromycin C3-4 component mixture are obtained.

(3) Semipure fractions of cervinomycin C1 and C2 (reverse phase preparative HPLC purification):

dissolving the refined product containing the deer-color mycin C1-2 component mixture with 10.0ml DMSO, filtering with 0.22 μm filter membrane, and separating and purifying by preparative HPLC, wherein the preparation column type is as follows: YMC-Pack ODS-A column, 20X 250 mm. Gradient elution is carried out by adopting a 50-100% acetonitrile-water system at the flow rate of 5.0ml/min, elution peaks containing the components of the Cereus C1 and the Cereus C2 are respectively collected, and a semi-pure product (16.4mg) containing the component of the Cereus C1 and a semi-pure product (3.6mg) containing the component of the Cereus C2 are obtained after the elution solvent is removed by vacuum rotary evaporation.

(4) Pure deer chromacin C1 and C2 fractions (reverse phase semi-preparative HPLC purification):

a refined product of the mixture containing the deer-color mycin C1 component is dissolved in a small amount of acetonitrile and purified by reverse phase preparative HPLC. The type of the chromatographic column: COSMOSIL packed column, 5C18-PAQ, 10 × 250 mm. Isocratic elution is carried out by adopting a 50% acetonitrile-water system, the flow rate is 2.0ml/min, and a chromatographic peak which appears near 17.9min under the wavelength of 254nm is collected to obtain an eluent containing the deer-color mycin C1 component. Removing acetonitrile in the eluent by vacuum rotary evaporation, and removing water by freeze drying to obtain 7.5mg of yellow Cerromycin C1 pure product with purity of over 99% by HPLC analysis.

A refined product of the mixture containing the deer-color mycin C2 component is dissolved in a small amount of acetonitrile and purified by reverse phase preparative HPLC. The column model, mobile phase parameters and detection parameters are the same as above, and chromatographic peak at wavelength of 254nm around 14.4min is collected to obtain eluate containing Cerhromycin C2 component. Removing acetonitrile in the eluent by vacuum rotary evaporation, and removing water by freeze drying to obtain purple pure product of the deer-color-mycin C2, wherein the purity of HPLC analysis is more than 99%.

(5) A refined product of the deer-color mycin C3-4 component mixture (normal phase silica gel column purification):

4.37g of a purified product containing the deer-color mycin C3-4 component was dissolved in about 300ml of methylene chloride, and then 7g of a silica gel column packing was added thereto, and after mixing, the solvent was removed in a vacuum rotary evaporator to obtain a stirred silica gel powder.

Loading the silica gel powder after sample mixing into a sample loading column, performing gradient elution for 200min by using 0-15% methanol-dichloromethane at the flow rate of 10.0ml/min, and collecting eluent, wherein each collecting pipe is about 70 ml; detecting compounds in each collecting tube by HPLC or TLC, and combining collecting liquid containing deer-color mycin C3-4 component mixture; the solvent was removed by rotary vacuum evaporation to obtain a purified product (0.38g) of a mixture containing Ceratomycin C3-4.

(6) Semipure fractions of cervinomycin C3 and C4 (reverse phase preparative HPLC purification):

dissolving the refined product containing the deer-color mycin C3-4 component mixture with 15.0ml DMSO, filtering with 0.22 μm filter membrane, and separating and purifying by preparative HPLC, wherein the preparation column type is as follows: YMC-Pack ODS-A column, 20X 250 mm. Gradient elution is carried out by adopting a 50-100% acetonitrile-water system at the flow rate of 5.0ml/min, elution peaks containing the components of the Cereus C3 and the Cereus C4 are respectively collected, and a semi-pure product (15.3mg) containing the component of the Cereus C3 and a semi-pure product (3.6mg) containing the component of the Cereus C4 are obtained after the elution solvent is removed by vacuum rotary evaporation.

(7) Pure deer chromacin C3 and C4 fractions (reverse phase semi-preparative HPLC purification):

dissolving the semi-pure product of the deer-color mycin C3 component in a small amount of acetonitrile respectively, and purifying by reversed-phase semi-preparative HPLC. The type of the chromatographic column: COSMOSIL packed column, 5C18-PAQ, 10 × 250 mm. Isocratic elution is carried out by adopting a 45% acetonitrile-water system, the flow rate is 2.0ml/min, and a chromatographic peak which appears near 27.6min under the wavelength of 254nm is collected to obtain an eluent containing the deer-color mycin C3 component. The eluent is subjected to vacuum rotary evaporation to remove acetonitrile, and then freeze drying is carried out to obtain yellow pure product 6.4mg of the deer-color mycin C3 component, and the purity of HPLC analysis is more than 99%.

Dissolving the semi-pure product of the deer-color mycin C4 component in a small amount of acetonitrile respectively, and purifying by reversed-phase semi-preparative HPLC. The column model, mobile phase parameters and detection parameters are as above, and chromatographic peak around 21.3min at 254nm wavelength is collected to obtain eluate containing Cerromycin C4 component. The eluent is subjected to vacuum rotary evaporation to remove acetonitrile, and then freeze drying is carried out to obtain a purple pure product of the deer-color mycin C4 component, wherein the purity of the HPLC analysis is more than 99%.

And C, analyzing the chemical structure of the deer-color mycin component: based on the ultraviolet-visible spectrum and high-resolution mass spectrum of the compound, and¹H-NMR、¹³C-NMR、DEPT、¹H-¹h correlation spectrum (¹H-¹H COSY)、¹H-¹³C-correlation spectroscopy (HSQC) and reverse detection remote¹H-¹³Analysis of the C heteronuclear multiple bond correlation spectroscopy (HMBC) confirmed the chemical structures of cervinomycin C1, C2, and C3, respectively. By ultraviolet-visible spectrum, high-resolution mass spectrum,¹H-NMR and the relation that the Cerromycin C3 is oxidized to generate the Cerromycin C4 determine the chemical structure of the Cerromycin C4 (figure 1). According to the result of chemical structure analysis, they belong to polycyclic xanthone antibiotics, and the molecular structure skeleton of the antibiotics is polycyclic (ABCDEF, six-ring) fusion. The Cerhromycin C component is the same as the Cerhromycin B component, and a carbon-carbon double bond in a horn ring in a polycyclic structure of the Cerhromycin C component is reduced into a single bond; in addition, the Cerhromycin C component lacks an oxazolidine ring and has a nitrogen-containing six-membered ring (lactam ring) with one more double bond than the Cerhromycin B component (which is a fused seven-ring). More particularly, the constituent Cercomycin C1 and C2 have a substituent (hydroxyethyl) on the N atom, whereas Cercomycin C₃And C₄The N atom of the component has no substituent (to which one H atom is attached). Nuclear magnetic spectra of deer chromans C1, C2, C3 and C4 are shown in fig. 3-21. NMR nuclear magnetic data for Cerromycin C1, C2, C3 and C4 are shown in tables 1-4. X-ray diffraction analysis shows that the Cerhromycin C2 contains 1 Cerhromycin C2 molecule and 2 solvent molecules of dimethyl sulfoxide in an asymmetric unit, as shown in FIGS. 22-23, the crystal belongs to a triclinic system, the space group is P-1, and the specific unit cell parameters are shown in Table 5.

TABLE 1 NMR Nuclear magnetic data for Cerromycin C1

TABLE 2 NMR Nuclear magnetic data for Cerromycin C2

TABLE 3 NMR Nuclear magnetic data for Cerromycin C3

TABLE 4 1H-NMR Nuclear magnetic data for Cerromycin C4

position	δ_H(J in Hz)
		3-OH	13.88,s
10	7.40,s
		11-OH	10.14,s
12-OCH3	3.97,s
		13	7.37,s
19	2.62,m
		20	2.79,t(7.2)
22	6.93,s
		24	6.53,s
26	2.27,s
		27-NH	11.95,s

TABLE 5 Luceromycin C2 unit cell parameters

Example 3: oxidative preparation of Ceratomycin C2 and C4 components

The separation and purification of the deer-color mycin C2 and C4 components in the example 2 have lower yield. Cerhromycin C2 and C4 fractions are oxidation products of Cerhromycin C1 and C3 fractions, respectively, and therefore Cerhromycin C2 and C4 fractions can be prepared by chemical oxidation methods using pure or semi-pure products of Cerhromycin C1 and C3 fractions obtained in example 2. The method comprises the following specific steps:

(1) ultrasonic dissolving 5.0mg (or 3.0mg) of Cerhromycin C1 (or C3) component in 25.0ml dichloromethane-methanol (volume ratio 1: 1); 5.0mg (or 3.0mg) of silver oxide was weighed out, added to the solution, and stirred at room temperature.

(2) Taking 100 mu l of reaction solution supernatant every 1h, and filtering by using a 0.22 mu m filter membrane to remove silver oxide; after removal of the solvent in the vacuum concentrator, the oxidation product was redissolved by adding 100. mu.l acetonitrile and the progress of the oxidation was monitored by HPLC analysis and after about 7h (or 5h) the oxidation reaction was complete. HPLC analysis monitoring conditions were 40% -70% acetonitrile-water system, gradient elution for 30min, flow rate 1.0 ml/min. After the reaction was completed, the reaction solution was filtered through a 0.22 μm filter to remove silver oxide, and the organic solvent was removed in a vacuum rotary evaporator to obtain a sample containing a component C2 (or C4) of Cerromycin.

(3) The sample containing the deer-color mycin C2 (or C4) component is dissolved in 5.0ml (or 3.0ml) of dichloromethane-methanol (volume ratio of 1:1) and subjected to preparative silica gel plate TLC separation and purification, and the sample development system is dichloromethane-methanol (volume ratio of 9: 1). After the silica gel plate after spreading is dried in the air, the Ceramicin C2 (or C4) component strip is scraped off, and dichloromethane solution containing a small amount of methanol is eluted; the solvent was evaporated off from the eluate on a vacuum rotary evaporator to obtain a semi-pure product of Cerhromycin C2 (or C4) fraction (3.9 mg or 2.4 mg).

(4) The above-mentioned semipure product of the deer-tryptophane C2 (or C4) fraction was dissolved in 2.0ml of DMSO and purified by preparative HPLC using the same column as in example 2. Isocratic elution is carried out by adopting a 50% (or 45%) acetonitrile-water system at the flow rate of 2.0ml/min, a chromatographic peak which appears near 14.4min (or 21.3min) under the wavelength of 254nm is collected, and the elution solvent is removed by vacuum rotary evaporation to obtain a pure product of 3.4mg (or 2.0mg) of purple deer-color-mycin C2 (or C4) component; the purity of HPLC analysis is above 99%.

Example 4: ceramicin C component with tumor cell growth inhibiting activity

(1) Preparing a sample:

weighing a proper amount of pure deer chromacin C1, C2, C3 and C4 components, dissolving the pure deer chromacin C1, C2, C3 and C4 components in DMSO, and preparing a sample mother solution to be detected with the concentration of 10 mM. BxPC-3(ATCC CRL-1687) and HCT116 cells (ATCC CCL-247) were seeded in 96-well plates at about (2-5). times.10 cells per well³Cells were cultured overnight at 37 ℃.

(2) And (3) determination:

BxPC-3 and HCT116 tumor cells were treated with BxPC-3 and HCT116 tumor cells by diluting the C1, C2, C3 and C4 sample stocks in culture medium to give final concentrations of 0.1, 1, 10, 100, 1000nM and 10. mu.M compound, setting triplicate wells per concentration, adding equal amounts of DMSO to the control group, and incubating at 37 ℃ in an incubator for 72 h. After 72h, MTT was added to the 96-well plate to a final concentration of 0.5mg/ml and incubation was continued for 3h in a 37 ℃ cell incubator.

(3) Half cell growth inhibitory Activity (IC)₅₀)：

Supernatant from the 96-well plate was discarded, crystals in the wells were dissolved by adding DMSO, and absorbance at 570nm was measured to calculate the concentration of the compound at which half of the cells were inhibited, i.e., IC 50. The deer-color mycin C1, C2, C3 and C4 components show strong inhibitory activity to the tested tumor cells. For example, IC of Cerromycin C3 component on BxPC-3₅₀754.7nM IC for HCT116₅₀The concentration of the adriamycin is 0.9nM, which is superior to that of the positive control drug adriamycin.

IC of Ceratomycin C1-C4 for BxPC-3 and HCT116₅₀The values are shown in Table 6 below.

TABLE 6 Cervidomycin C fraction up to half of inhibition of tumor cellConcentration at cell time (IC)₅₀)

Finally, it should be noted that the above examples are only used to help those skilled in the art understand the essence of the present invention, and should not be construed as limiting the scope of the present invention.

Claims

1. A group of deer-color-mycin C component compounds are deer-color-mycin C1, C3 and C4 which are polycyclic xanthone compounds with chemical structures shown in formulas (1), (3) and (4),

2. a method for preparing deer-color mycin C1, C2, C3 and C4, which comprises the following steps:

(2) extracting, separating and purifying the deer-chromamycin C component from the fermentation product;

wherein the content of the first and second substances,

the fermentation culture streptomyces CGMCC NO.16425 in the step (1) comprises the following steps:

1) the seed is cultured, and the seed is cultured,

the spore culture medium comprises the following components: 20.0g/L of starch, 20.0g/L of soybean cake powder, 15.0g/L of agar powder and 7.0 of pH;

the spore culture conditions are 26-30 ℃ and 6-10 days;

2) the streptomyces is subjected to solid-state fermentation,

the solid state fermentation method comprises the following steps: inoculating a fresh spore suspension of streptomycete CGMCC NO.16425 to a fermentation medium plate;

the fermentation medium comprises the following components: 10.0g/L of corn starch, 10.0g/L of cottonseed cake meal, 10.0g/L of threonine, 15.0g/L of agar powder and pH of 7.0;

culturing for 8-12 days at 26-30 ℃, and harvesting a fermentation culture;

the step (2) of extracting, separating and purifying the deer-color mycin C component from the fermentation product comprises the following steps,

1) extracting the fermentation culture with organic solvent, and rotary steaming the extractive solution under reduced pressure to obtain crude extract;

2) the crude extract adopts reversed phase C₁₈Removing main impurities by chromatographic column to obtain refined product containing Cerhromycin C1-C2 component mixture and crude product containing Cerhromycin C3-C4 component mixture;

3) reverse phase preparation and semi-preparative HPLC purification are carried out on the refined product of the deer-tryptophycetin C1-C2 component mixture to respectively obtain a deer-tryptophycetin C1 component and a deer-tryptophycetin C2 component, elution peaks containing the deer-tryptophycetin C component are collected and subjected to reduced pressure rotary evaporation or freeze drying to obtain pure products of the deer-tryptophycetin C1 and C2 components;

4) separating the crude mixture of ingredients C3-C4 of Ceratomycin by normal phase silica gel chromatography, mixing eluents with the same ingredients after HPLC analysis, and removing the eluting solvent by rotary evaporation under reduced pressure to obtain refined mixture of ingredients C3-C4 of Ceratomycin; performing reversed-phase preparation and semi-preparative HPLC purification on the refined product of the deer-tryptophacin C3-4 component mixture to respectively obtain pure products of a deer-tryptophacin C3 component and a deer-tryptophacin C4 component; the structural formula of the deer chromacin C1-C4 is shown in the following formulas (1) to (4)

3. A method for obtaining the deer-color mycins C2 and C4 by oxidation reaction by using the deer-color mycins C1 and C3 as substrates comprises the following steps:

(1) dissolving the deer-color mycin C1 or C3 in a dichloromethane-methanol solution, adding a weak oxidant with the same mass as that of a component to be oxidized, stirring at room temperature, and filtering with a filter membrane to remove the weak oxidant after the oxidation reaction is finished, wherein the weak oxidant is silver oxide;

(2) separating and purifying the filtrate with silica gel column, and vacuum rotary evaporating to obtain semi-pure product of Ceramicin C2 or C4;

(3) subjecting the semi-pure product to preparative or semi-preparative reverse phase HPLC refining, mixing elution peaks containing Cervus elaphus C2 or C4 component, and freeze drying or rotary steaming under reduced pressure to obtain pure product of target compound, i.e. Cervus elaphus C2 and C4 component; the structural formula of the deer chromacin C1-C4 is shown in the following formulas (1) to (4)

4. The use of the deer antler mycin C1, C3, C4 of claim 1 in the preparation of an anti-tumor medicament, wherein said tumor is pancreatic cancer or colorectal cancer.