CN116554187A

CN116554187A - Novel framework type cytochalasin and application thereof in preparation of medicines with anti-tumor activity

Info

Publication number: CN116554187A
Application number: CN202310570234.9A
Authority: CN
Inventors: 石宝宝; �田淳; 吕晓; 艾洪莲; 李正辉
Original assignee: South Central University for Nationalities
Current assignee: South Central Minzu University
Priority date: 2023-05-19
Filing date: 2023-05-19
Publication date: 2023-08-08

Abstract

The invention relates to the technical field of medicinal chemistry, and in particular discloses endophytic fungi from fritillaria hupehensisBoeremia exiguaThe novel skeleton type cytochalasin compound of a 5/6/5/6/8 five-ring system obtained by separation is named as boerelasin B. The MTS method is adopted to evaluate the cytotoxic activity of the compound on 5 human tumor cells, and the biological activity evaluation shows that the compound has different degrees of cytotoxic activity on 5 human tumor cells, wherein the compound has obvious cytotoxic activity on SMMC-7721 (human liver cancer cells) and A549 (human lung cancer cells)，IC ₅₀ <10μM; cytotoxic activity on HL-60 (human leukemia cells) and MCF-7 (human breast cancer cells) is equivalent to that of cisplatin as a positive control drug; has better inhibitory activity on SW480 (human colon cancer cells).

Description

Novel framework type cytochalasin and application thereof in preparation of medicines with anti-tumor activity

Technical Field

The invention relates to the technical field of medicine and chemistry, in particular to a cytochalasin compound derived from Hubei fritillary endophytic fungi B.exigua and application thereof in preparing medicines with anti-tumor activity.

Background

Fritillary bulb is a perennial herb, flowers of which are for gardening and ornamental, and underground bulbs of which are for medical use. The name is obtained by the bulb shape, and the 'materia medica Jing Ji Zhi' is: the name fritillary bulb is similar to the one of the poly-belongings. Fritillaria Hubei is one of the original fritillaria Hubei in China, and is a genuine medicinal material in Hubei province. Has effects of eliminating phlegm, relieving cough, removing toxic substance, and resolving hard mass. The Chinese medicine is mainly used for treating exogenous wind-heat cough and phlegm-heat cough, is a daily necessary Chinese medicine in the families of common people, and is also an important economic crop in Miao nations of Enshi families in Hubei province.

Endophytic fungi survive in healthy plant tissues and organs in a reciprocal symbiotic equilibrium relationship with plants. The synthetic route of the endophyte organism itself is rich and varied, and the endophyte organism provides a series of novel compounds which are difficult to obtain by artificial chemical synthetic route and have complex structures, and the endophyte organism is an important source of novel structure active natural products and clinical medicines, and many pesticides, anticancer medicines and antibacterial medicines are directly or indirectly derived from the endophyte. In recent years, natural product researchers have separated different endophytes from bulbs of various fritillaries, and studied chemical components and biological activities of the separated endophytes. For example: all fritillary endophytic fungi fermentation filtrate belonging to fusarium show antioxidant activity; three novel heterozygous polyketides are separated from solid fermentation products of the fritillaria Hubei endophytic fungi, and the compounds are found to have obvious cytotoxic activity on the mouse lymphoma cells L5178Y through a biological activity experiment; 4 endophytes are separated and identified from fritillaria hupehensis, the antibacterial activity of the secondary metabolite of 3 endophytes is superior to that of bacterial Pseudomonas sp.BM-X-6, and the antibacterial spectrum of the secondary metabolite of Arthrinium sp.BM-Z-5 is the widest, and the research shows that fritillaria hupehensis endophytes have certain resistance to biological damage.

Based on endophytic fungi, the method has the advantages of abundant resources, various types, short culture period, high feasibility of gene manipulation and the like, and cytochalasin compounds have various biological activities, such as anti-tumor, antibacterial, anti-inflammatory, antioxidant and the like. The applicant has explored the antitumor activity of cytochalasin isolated from endophytic fungi of fritillaria hupehensis.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide cytochalasin and application thereof in preparing medicines with anti-tumor activity.

Cytochalasin with anti-tumor activity has the following structural formula:

the molecular formula: c (C) ₂₉ H ₃₅ NO ₄

The name is: boerelasin B

It is a further object of the invention to verify that the cytochalasin B has varying degrees of cytotoxic activity against 5 human tumor cells.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the invention discloses a novel skeleton type cytochalasin of 5/6/5/6/8 five-ring system from fritillaria Hubei endophytic fungi B.exigua.

2. The invention provides cytochalasin with anti-tumor activity, which has different degrees of cytotoxic activity on 5 kinds of human tumor cells.

3. The cytochalasin for protecting the pharmaceutical application can be obtained by extracting and purifying plant endophytic fungi, and has the advantages of short culture period, high operation feasibility, no chemical pollution, environment friendliness and the like.

Drawings

FIG. 1 shows a hydrogen spectrum (800 MHz, CDCl) of the compound obtained in example 1 in the specific embodiment ₃ )；

FIG. 2 is a schematic illustration of an embodiment ofCarbon spectra (201 MHz, CDCl) of the Compound prepared in example 1 ₃ ) And a DEPT diagram;

FIG. 3 is a HMQC chart of the compound prepared in example 1 of the specific embodiment;

FIG. 4 is a HMBC diagram of the compound prepared in example 1 in an embodiment;

FIG. 5 shows the compound obtained in example 1 of the specific embodiment ¹ H- ¹ H COSY pattern;

FIG. 6 is a ROESY chart of the compound prepared in example 1 in the specific embodiment;

FIG. 7 is a HR-ESI-MS plot of the compound prepared in example 1 of the present embodiments;

FIG. 8 is a graph showing measured ECD and calculated ECD of the compound prepared in example 1 of the present embodiment.

Detailed Description

For the purpose of making the object and summary of the present application more apparent, the applicant will make a clear and complete description of the technical solution of the present invention in connection with the specific embodiments below.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1:

the raw material Boeremia exigua (B.exigua) is separated from the root of a fresh plant of the Hubei fritillary bulb, the fritillary bulb is collected from a new pool and country double river community of Enshi soil family Miao nationality, hubei province, the strain is named as a shellfish 21, and the maximum similarity between the sequence and the Boeremia exigua is up to 100 percent and the accession number of a gene library is obtained by comparing the ITS sequence measurement result: MT154621.1. It was identified as Boeremia exigua and this strain was deposited in the university of south China national medical college microorganism strain library (published: lv Xiao, leaf, ma Xujun, etc. Boeremia exigua chemical composition of Hubei fritillary endophytic fungus and its anti-inflammatory activity research [ J ]. University of south China national university (Nature science edition), 2022, 41 (2): 174-179).

The specific separation process of the fritillary endophytic fungi fritillary bulb 21 comprises the following steps: taking fritillaria Hubei (root of fresh plant), washing with tap water, cutting into small blocks, rinsing with 75% V/V ethanol for 60s, rinsing with sterile water for 3 times, soaking and sterilizing with 0.1% mercuric chloride (1 g mercuric chloride is dissolved in 1000g distilled water) for 40s, rinsing with sterile water for 3 times again, absorbing water with high-pressure sterilized filter paper, cutting off the cuts at two ends of fritillaria Hubei root, separating from the middle, inoculating the small blocks onto Potato Dextrose Agar (PDA) culture medium containing a chlortetracycline mixture under aseptic condition, and culturing in a constant-temperature incubator at 25 ℃. After the fungus hypha grows out, the hypha is selected for purification culture to obtain a single strain, and the single strain is transferred into a test tube filled with PDA culture medium and is placed in a refrigerator at 4 ℃ for preservation.

The strain shellfish 21 is subjected to expansion fermentation by adopting oat solid fermentation: activating strain, taking out the test tube from the refrigerator, placing for 1 hour in a sterile normal temperature environment, taking out bacterial blocks with the diameter of about 5mm from the test tube under a sterile operation table, inoculating the bacterial blocks onto a flat-plate PDA culture medium, and growing the bacterial blocks for about 7 days, wherein bacterial colonies are full of PDA for standby. Oat culture medium ratio: oat 50 g/bottle, distilled water 50 mL/bottle, put in 500mL culture bottle, sterilized at 120 ℃ for 20 minutes, cooled, and inoculated on oat culture medium by picking fungus blocks with the diameter of 5mm from a flat-plate PDA culture medium in a sterile environment, 272 bottles are taken in total, and the culture conditions are as follows: dark culture was performed at 25℃for 35d.

Isolation and purification of cytochalasin from the compounds described in the claims and specification summary: the above fermented oat solid medium (total 15 kg) was triturated and soaked 5 times with organic mixed reagent (dichloromethane-methanol=1:1, v/v), 24h each time. And (3) centrifuging after each soaking, combining the extracting solutions after 5 times of centrifugation, concentrating under reduced pressure, evaporating to dryness, adding a small amount of water for dissolving, extracting with ethyl acetate for 5 times, combining the ethyl acetate parts, and concentrating under reduced pressure to obtain 418.77g of crude extract. Chromatography on 80-100 mesh normal phase silica gel Column (CH) ₂ Cl ₂ MeOH:100:0,50:1,20:1,10:1,5:1, v/v) and eluting with thin layer chromatography (chloroform as developing solvent: methanol=10:1, v/v;thin layer chromatography silica gel plate, qingdao ocean chemical plant) to detect and develop color, and rough segmentation to obtain five components A-E.

The C fraction (eluted fraction with a volume ratio of dichloromethane/methanol of 20:1, eluent amount of 5 column volumes, 15.5 g) was subjected to high performance medium pressure liquid chromatography (Biotage SP1, reversed phase packing material: RP-18, 20-45 μm, fuji Silysia Chemical ltd., japan) with a methanol water system (methanol/water=50:50, 60:40,70:30,80:20,100:0, v/v), flow rate of 20mL/min, gradient elution was performed, and the elution time of each ratio was 60min. Detecting and developing by thin layer chromatography (chloroform: methanol=20:1, v/v; thin layer chromatography silica gel plate, qingdao ocean chemical plant), combining the same or similar components, and finally combining into eight subfractions, and marking as C1-C8 from small to large in sequence according to polarity. The C6 fraction (eluted fraction at a methanol/water volume ratio of 60:40, 3.6 g) was subjected to normal phase silica gel column (200-300 mesh), dichloromethane methanol system (dichloromethane/methanol=20:1, 10:1,1:1, v/v) was eluted in gradient, and detected and developed by thin layer chromatography (developing solvent chloroform: methanol=10:1, v/v; thin layer chromatography silica gel plate, qingdao marine chemical factory), and the same or similar fractions were combined to obtain 5 subfractions, which were labeled as C6-1 to C6-5 in order from small to large in polarity. C6-2 (component eluted at a volume ratio of methylene chloride/methanol of 10:1, eluent amount of 5 column volumes, 68 mg) was subjected to Sephadex LH-20 gel column chromatography (Pharmacia Fine Chemical Co., ltd., sweden) eluting with methanol (methanol amount of 10 column volumes), and then 24.7mg of the obtained product was subjected to high performance liquid chromatography (Agilent 1260; column: agilent Zorbax SB-C18, specification: 9.4 mm. Times.150 mm,5 μm; acetonitrile-water: 45:55-55:45, v/v; flow rate: 4 mL/min) gradient elution for 25min, to obtain 1.5mg of the compound of the present invention (retention time: 16.6 min).

Structural identification of the compound: the cytochalasin compound prepared in example 1 was dissolved in 0.5mL of deuterated chloroform, transferred to a nuclear magnetic resonance tube by a 200. Mu.L pipette, and the relative configuration was determined by detecting hydrogen spectrum, carbon spectrum and two-dimensional spectrum (as shown in FIGS. 1 to 6) on a nuclear magnetic resonance apparatus (Brookfield Avance III 600MHz, germany), and the three-dimensional configuration of the compound was determined by calculating ECD (as shown in FIG. 8). The structure of the compound is obtained by combining the physicochemical data and is named as boerelasin B.

Nuclear magnetic resonance data of the obtained cytochalasin:

other physicochemical data:

appearance: white amorphous powder according to high resolution mass spectrum hresis ([ m+h)] ⁺ ，m/z 462.2640)。

From the results of the above detection, it was confirmed that the structural formula of the compound obtained in this example was:

the molecular formula: c (C) ₂₉ H ₃₅ NO ₄

Example 2: MTS detection of cell viability

Principle of cell Activity detection by MTS method:

MTS is a novel MTT analogue, which is totally called 3- (4, 5-dimethylazol-2-yl) -5 (3-carboxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, and is a yellow dye. Succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTS to generate soluble Formazan (Formazan) compounds, and the content of the Formazan can be measured at 490nm by an enzyme-labeled instrument. In general, the formazan production amount is proportional to the number of living cells, and thus the number of living cells can be estimated from the optical density OD value.

The experimental method comprises the following steps:

1. inoculating cells: five kinds of tumor cells (Kunming plant institute of China academy of sciences) were inoculated into 96-well plates with about 5000 cells per well in a single cell suspension prepared by using DMEM medium containing 10% fetal bovine serum, and the cell volume per well was 100. Mu.L, and the cells were inoculated and cultured 12-24 hours in advance.

2. Adding a solution of a compound to be tested: the compound obtained in example 1, cisplatin and taxol were dissolved in DMSO, and the compound was screened again at concentrations of 40. Mu.M, 20. Mu.M, 10. Mu.M, 5. Mu.M, 2.5. Mu.M, 1.25. Mu.M, 0.62. Mu.M, 0.31. Mu.M, 0.16. Mu.M and 0.08. Mu.M, respectively, with a final volume of 200. Mu.L per well, and 3 multiplex wells were used for each treatment.

3. Color development: after culturing at 37℃for 48 hours, all the culture solution in the wells was discarded from the adherent cells (A549, SMMC-7721, MCF-7, SW 480), and 20. Mu.L of MTS solution and 100. Mu.L of culture solution (DMEM medium containing 10% fetal bovine serum) were added to each well; suspension cells (HL-60) were discarded with 100. Mu.L of culture supernatant and 20. Mu.L of MTS solution was added to each well; 3 blank wells (mixed solution of MTS solution 20. Mu.L and culture solution 100. Mu.L) were used, and incubation was continued for 2 to 4 hours to allow the reaction to proceed sufficiently, and then the light absorption value was measured.

4. Colorimetric: selecting 490nm wavelength, reading light absorption value of each hole by a multifunctional enzyme labeling instrument (MULTISKAN FC), recording the result, drawing a cell growth curve by taking the concentration as an abscissa and the cell survival rate as an ordinate after data processing, and calculating the IC of the compound by using a two-point method (Reed and Muench method) ₅₀ Values.

5. Positive control compound: each experiment is performed by comparing two positive compounds of Cisplatin (Cisplatin) and Paclitaxel (Paclitaxel), drawing cell growth curve with concentration as abscissa and cell survival rate as ordinate, and calculating IC of the compound by two-point method (Reed and Muench method) ₅₀ Values. The cell viability was calculated as: cell viability% = drug/blank x 100%.

Experimental results:

the results show that the compound boerelasin B of the invention has obvious cytotoxic activity on SMMC-7721 (human liver cancer cell) and A549 (human lung cancer cell), and IC ₅₀ <10 mu M, has obvious inhibitory activity on HL-60 (human leukemia cells) and MCF-7 (human breast cancer cells), and IC ₅₀ The values are 14.16 mu M and 19.21 mu M, which are equivalent to the positive control drug cisplatin; has good inhibitory activity on SW480 (human colon cancer cells), and IC ₅₀ The value was 30.56. Mu.M.

Table 1: IC of Boerelasin B, cisplatin and paclitaxel on five tumor cells ₅₀ Value of (. Mu.M)

Claims

1. A novel framework type cytochalasin compound, which has the structural formula:

2. the cytochalasin compound as claimed in claim 1 wherein the compound has the formula:

3. a process for the preparation of a compound as claimed in claim 1 or 2, comprising the steps of:

1) Performing amplification culture on the Hubei fritillary endophytic fungi B.exigua by adopting oat solid fermentation, mashing a fermented oat solid culture medium, soaking and extracting by using an organic mixed reagent, concentrating an extracting solution to be dry, adding water for dissolution, and extracting by using ethyl acetate;

2) Separating ethyl acetate extract by silica gel column to obtain five components A-E;

3) Performing medium-pressure liquid phase preparation chromatographic separation on the component C to obtain eight sub-components C1-C8;

4) Separating the C6 component by a silica gel column to obtain five sub-components of C6-1 to C6-5;

5) Separating C6-2 component by gel column chromatography, and purifying the obtained product by high performance liquid chromatography to obtain the compound.

4. The method according to claim 3, wherein the organic mixed reagent in the step 1) is a dichloromethane-methanol mixed solvent in a volume ratio of 1:1;

oat culture medium ratio: the solid-to-liquid ratio of oat to distilled water is 1g to 1mL, and the oat is sterilized at the high temperature of 120 ℃ for standby;

conditions for the expansion culture: dark culture was performed at 25℃for 35d.

5. The method according to claim 3, wherein the conditions for the separation of the silica gel column in step 2) comprise: eluting with dichloromethane-methanol as eluent in the volume ratio of 100:0,50:1,20:1,10:1 and 5:1 sequentially to obtain five eluting components by TLC detection, wherein the developing agent is chloroform: methanol=10:1, v/v.

6. The method according to claim 3, wherein the conditions for the medium pressure liquid preparative chromatography in step 3) include: the RP-18 chromatographic column is eluted sequentially by taking methanol-water as eluent according to the volume ratio of 50:50,60:40,70:30,80:20 and 100:0, the flow rate is 20mL/min, the elution time of each proportion is 60min, and the TLC detection is carried out to obtain eight elution subfractions, wherein the developing agent is chloroform: methanol=20:1, v/v.

7. The method according to claim 3, wherein the conditions for the separation of the silica gel column in the step 4) comprise: eluting sequentially with dichloromethane-methanol as eluent at volume ratio of 20:1,10:1 and 1:1 to obtain five eluting components by TLC detection, wherein the developing agent is chloroform: methanol=10:1, v/v.

8. The method according to claim 3, wherein the gel column chromatography in step 5) uses methanol as an eluent;

the conditions of the high performance liquid chromatography in step 5) include: and C18, performing gradient elution for 25min by using a mixed solvent of acetonitrile and water as an eluent according to an initial volume ratio of acetonitrile to water of 45:55 and a final volume ratio of 55:45, wherein the flow rate is 4mL/min.

9. Use of a compound according to claim 1 or 2 for the preparation of an antitumor drug.

10. Use of a compound according to claim 1 or 2 for the manufacture of a medicament for inhibiting the growth of tumour cells SMMC-7721, a549, HL-60, MCF-7 and/or SW 480.