CN109652468B

CN109652468B - Preparation method and application of anti-tumor compound

Info

Publication number: CN109652468B
Application number: CN201910011187.8A
Authority: CN
Inventors: 洪葵; 田二丽; 邓子新
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2019-01-07
Filing date: 2019-01-07
Publication date: 2020-08-25
Anticipated expiration: 2039-01-07
Also published as: CN109652468A

Abstract

The invention discloses a preparation method and application of an anti-tumor compound. The compound belongs to a p-terphenyl type, and the p-terphenyl compound BTH-II0204-207: B is obtained by separating an active secondary metabolite from a mycelial part of Streptomyces himurium erythrophyllae 219807. The compound has strong effects of inhibiting breast cancer cell MCF-7, human liver cancer in vitro cell Bel7402 and human oral epidermoid cancer cell KB, and can be used for preparing antitumor drugs.

Description

Preparation method and application of anti-tumor compound

Technical Field

The invention belongs to the field of microbial medicines, and particularly relates to a preparation method and application of an active compound BTH-II0204-207: B.

Background

The structure of the terphenyl aromatic hydrocarbon compound contains 3 benzene rings, and the terphenyl aromatic hydrocarbon compound is divided into three isomers of ortho/meta/para according to the different positions of the connected benzene rings, and the terphenyl aromatic hydrocarbon compound has a changeable structure and remarkable activity. Of these, the para-terphenyl compounds are mostly isolated from fungi, and there are about 10 compounds derived from streptomyces. The compound has biological activities of cytotoxicity, antioxidation, antibiosis, anticoagulation, neuroprotection, immunosuppression, lipoxygenase inhibition and the like. The BTH-II0204-207: B belongs to a p-terphenyl compound, which is separated from a strain of B.pseudoalloleiK96243 (Biggins, J.B.; Liu, X.; Feng, Z.; et al. metals from the induced expression of crystalline single microorganisms found in the genome of Burkholderianum pseudoallolei.J.am.chem.Soc.2011, 133,1638-1641.) induced and expressed by a single operon for the first time as a natural product. In structural view, the compound is obtained from streptomyces for the first time, and the effect of inhibiting breast cancer cells MCF-7, human liver cancer in vitro cells Bel7402 and human oral epidermoid cancer cells KB which are the same as the effect of the compound in the invention are not related in the existing reports.

Disclosure of Invention

The invention aims to provide a preparation method of a p-terphenyl compound BTH-II0204-207: B from Streptomyces mangrove 219807.

The invention also aims to provide application of the p-terphenyl compound BTH-II0204-207: B in preparing antitumor drugs.

The purpose of the invention is realized by the following technical scheme:

the invention provides a method for preparing a p-terphenyl compound BTH-II0204-207: B by utilizing streptomyces mangrovei 219807, wherein the structural formula of the BTH-II0204-207: B is shown as a formula I, and an excimer ion peak M/z 293.1173 (a calculated value of [ M + H ] is 293.1173]⁺293.1099) of formula C₁₉H₁₆O₃，

The compound BTH-II0204-207: B is prepared from a fermentation culture of Streptomyces mangrovei 219807, the Streptomyces mangrovei 219807 is derived from the soil of the tri-mangrove in Hainan province of China, and the preservation number is CCTCCNO.M2015276.

Preferably, the method specifically comprises the following steps:

(1) activating the streptomyces mangrove 219807 by a solid culture medium, inoculating the activated streptomyces mangrove 219807 into an ISP2 liquid culture medium for culture, wherein the culture condition is that the culture is carried out for 3d at the temperature of 28 ℃ and at the speed of 220r/min, then inoculating the activated streptomyces mangrove 219807 into a DO culture medium at the inoculation amount of 10 percent, and culturing the activated streptomyces mangrove 219807 for 8d at the temperature of 28 ℃ and at the speed of 220 r/; the formula of the DO culture medium is as follows: 10g/L of glucose, 25g/L of dextrin, 20g/L of oat powder, 10g/L of cottonseed cake powder, 5g/L of fish meal, 2g/L of yeast extract and CaCO₃3g/L, and the pH value is 6.0;

(2) performing solid-liquid separation on the fermentation liquor obtained in the step (1) by using a centrifugal method, performing ultrasonic wall breaking on a mycelium part for three times by using acetone, concentrating the combined extracting solution under reduced pressure until the extracting solution does not contain acetone, extracting the extracting solution for three times by using ethyl acetate, and concentrating to obtain a crude extract of the mycelium part;

(3) and (3) performing gradient elution on the crude extract obtained in the step (2) in normal-phase silica gel column chromatography by using cyclohexane and acetone as eluent, wherein the ratio is cyclohexane: acetone is 9:1, 7:1, 5:1, 3:1, 7:3, 3:2, 1:1, 2:3, 1:3 and 1:5, the elution volume of each gradient is 1.5L, and ten elution components are obtained in total and are respectively concentrated under reduced pressure;

(4) the elution ratio obtained in the step (3) is cyclohexane: and (3) separating the concentrated extract of the part (7: 1) of acetone by normal phase silica gel column chromatography, wherein the eluent is petroleum ether: obtaining seven elution components in total by ethyl acetate 9:1, 8:2, 7:3, 6:4, 1:1, 4:6 and 1:9, and respectively concentrating under reduced pressure;

(5) and (3) taking the elution ratio obtained in the step (4) as petroleum ether: separating and purifying the concentrated extract with C18 reversed phase chromatographic column under isocratic CH (8: 2) condition₃CN:H₂O65: 35, flow rate 3.0mL/min, at 10.48min gave the monomeric compound of BTH-II0204-207: B.

The second purpose of the invention is to provide the application of the streptomyces mangrovei 219807 in the preparation of the p-terphenyl compound BTH-II0204-207: B.

The invention adopts MTT method to test 50% Inhibitory Concentration (IC) of B-terphenyl compound BTH-II0204-207 to breast cancer cell MCF-7, human liver cancer in vitro cell Bel7402 and human oral epidermoid cancer cell KB₅₀). The experimental result shows that BTH-II0204-207: B has very obvious resistance to the tested tumor cells, and the BTH-II0204-207: B can be used for preparing the antitumor drugs. The compound BTH-II0204-207: B is used as an active ingredient and is compatible with one or more pharmaceutically acceptable carriers, excipients or auxiliary materials to prepare an anti-tumor pharmaceutical composition. The medicine and the pharmaceutical composition can be used for treating tumor-related diseases. The compound can also be combined with known medicaments to form a compound preparation for treating tumor-related diseases.

The third purpose of the invention is to provide the application of the p-terphenyl compound BTH-II0204-207: B in preparing anti-tumor drugs.

Preferably, the anti-tumor drug is a drug for resisting breast cancer, liver cancer and oral epidermoid carcinoma.

The fourth purpose of the invention is to provide an anti-tumor pharmaceutical composition, wherein the p-terphenyl compound BTH-II0204-207: B is used as an effective component.

Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, excipients or auxiliary materials.

The fifth purpose of the invention is to provide the application of the anti-tumor pharmaceutical composition in preparing anti-tumor drugs.

Preferably, the anti-tumor medicine composition is a medicine for resisting breast cancer, liver cancer and oral epidermoid carcinoma.

The p-terphenyl compound BTH-II0204-207, which is prepared and separated from the fermentation culture of the mangrove streptomyces 219807, has good anti-tumor activity and can provide a chemical entity for the research and development of new drugs.

Drawings

FIG. 1 is a high resolution mass spectrum of BTH-II0204-207: B.

FIG. 2 is of BTH-II0204-207: B¹H-NMR spectrum.

FIG. 3 is of BTH-II0204-207: B¹³C-NMR spectrum.

Detailed Description

The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.

The p-terphenyl compound BTH-II0204-207: B of the present application was isolated from a fermentation culture of Streptomyces mangrovei 219807, the properties of which are disclosed in ZL 201510260295.0.

[ example 1 ] isolation and Structure confirmation of Compound

(1) Isolation of Compounds

And (3) loading the crude extract of the mycelium part obtained after fermentation treatment by adopting a dry method, performing gradient elution in silica gel of 300-400 meshes, wherein the eluent is cyclohexane and acetone, the ratio of the cyclohexane to the acetone is 9:1, 7:1, 5:1, 3:1, 7:3, 3:2, 1:1, 2:3, 1:3 and 1:5 in sequence, the elution volume of each gradient is 1.5L, decompressing and concentrating the eluent of each part, and mixing the eluent of 7:1 part of the concentrated extract is prepared by mixing normal phase silica gel with petroleum ether: ethyl acetate 9:1, 8:2, 7:3, 6:4, 1:1, 4:6, 19, obtaining seven elution components in total, and respectively concentrating under reduced pressure. And then 8:2, carrying out C18 semi-preparative liquid phase separation and purification on the components after partial decompression concentration, wherein the semi-preparative condition is equal CH₃CN:H₂O65: 35, flow rate 3.0mL/min, at 10.48min gave compound BTH-II0204-207: B.

(2) Structure validation

The structure of BTH-II0204-207: B was determined by MS and NMR. Wherein the mass spectrum, 1H-NMR spectrum and 13C-NMR spectrum of the compound BTH-II0204-207: B are shown in figure 1-3.

a) Structure identification of compound BTH-II0204-207: B

The compound is yellow powder, and HRESI-MS (+) obtains an excimer peak M/z 293.1173 (calculated value [ M + H ] +293.1099), and the molecular formula is C19H16O 3. In the 1H-NMR spectrum, 10 overlapped aromatic protons with coupling constants of 7-8 Hz, a single peak signal of [ H6.39(s) ] and a methoxyl hydrogen signal of [ H3.30(s) ] appear in 7.20-7.60 ppm of a low field region, so that the compound is supposed to exist more than one benzene ring. In 13C-NMR-DEPT spectra (CD3OD), a total of 19 carbon signals, including 18 aromatic carbon signals, 4 of which are very significant, presumably with a more symmetrical structure such that the signals overlap and one more methoxy carbon signal. These signals indicate that the compound may have three phenyl rings, two of which are symmetrically located and the other is pentasubstituted, two of which are phenyl rings and one of which is attached to a methoxy group, the remaining two substitutions most likely being hydroxy groups. The above conjecture is confirmed by consulting the literature (Biggins, J.B.; Liu, X.; Feng, Z.; et al, metals from the induced expression of crystalline microorganisms found in the genome of Burkholderia pseudo-allei.J.Am.chem.Soc.2011, 133,1638-1641.) and this is the first time this compound was obtained from a wild-type bacterium, and the nuclear magnetic data are shown in Table 1.

TABLE 1 of the Compound BTH-II0204-207: B¹H and¹³C-NMR data (CD)₃OD,in ppm)

The physicochemical properties and spectral data of BTH-II0204-207: B are as follows:

a yellow powder;

the molecular formula is as follows: c₁₉H₁₆O₃；

Molecular weight: 292.11, respectively;

HRESI-MS (m/z): 293.1173 (Calcd for [ M + H ] + 293.1099);

the maximum ultraviolet absorption is 217 nm and 264 nm;

¹h and¹³C-NMR data (500 and 125MHz, CD)₃OD) is shown in table 1.

[ example 2 ] BTH-II0204-207: B antitumor Activity assay

1. And (3) reagent sources: RPMI1640 medium was purchased from Hyclone, fetal bovine serum from Gibco, pancreatin from Gibco, and MTT from Biosharp.

2. Cell lines: breast cancer cell MCF-7, human liver cancer in vitro cell Bel7402 and human oral epidermoid cancer cell KB.

3. Methods for Activity evaluation test reference Mosmann, T.Rapid colorimetric assay for cellular growth and survival application to promotion and cytoxicity assays.J.Immunol.methods.1983,65(1-2),55-63. the optimization procedure was followed as follows:

(1) sample preparation: BTH-II0204-207: B1 mg was weighed precisely and dissolved into 20mg/mL stock by adding appropriate amount of DMSO. mu.L of the stock solution was taken, 104. mu.L of sterile medium was added, and a sample test mother liquor (4mg/mL) was obtained after dilution. And continuously performing gradient dilution by using a sterile culture medium, and respectively diluting to obtain sample solutions with the concentrations of 400, 40, 4, 0.4 and 0.04 mu g/mL for later use.

(2) Preparing a cell suspension: discarding the cell culture medium, washing twice with PBS, digesting with 0.25% pancreatin for a proper time, adding an equal amount of culture medium to stop digestion, blowing the solution into a suspension, centrifuging at 800rpm for 5min, discarding the supernatant, uniformly blowing the cells with 1mL of culture medium, diluting by a proper multiple, counting, and adjusting the concentration to 3-4 × 104/mL.

(3) The detection method comprises the following steps: and blowing and uniformly mixing the prepared cell suspension, adding the cell suspension into a sample adding groove, and sucking 190 uL/hole cell suspension to a 96-hole plate by using a row gun to obtain the cell to be detected with the density of 6000-8000/hole (the edge hole is filled with sterile PBS). Shaking the 96-well plate to uniformly distribute the cells in the holes, and then adhering the cells to the wall in an incubator for 24 hours. Samples to be tested are diluted into proper concentration by using culture medium, 10 mu L of the diluted samples are added into a 96-well plate inoculated with cells, the final concentration of the samples is 200, 20, 2, 0.2, 0.02 and 0.002 mu g/mL (each sample is provided with 3 parallel multiple wells), and the negative control group is DMEM culture medium without medicine. 5% CO₂Incubation is carried out for 72 hours at 37 ℃, and the effect of the drug is observed under an inverted microscope. MTT solution at a concentration of 5mg/mL was prepared with PBS, and 20. mu.L of the solution was added to each well after filtration through a 0.22 μm filter, and the culture was continued. The culture was terminated after 4h, the supernatant was removed, 100. mu.L of DMSO was added to each well, the mixture was placed on a shaker and shaken at low speed for 10min, after the crystals were fully dissolved, the absorbance of each well was measured at 550nm with a microplate reader, and the reference wavelength was set at 690 nm.

And (4) calculating a result: the cell growth survival rate was calculated according to the formula of (%) cell survival rate [ (compound-treated group-blank group)/(negative control group-blank group) ] × 100%, and finally the IC50 value was calculated.

4. Results of the experiment

The IC50 values for compound BTH-II0204-207: B on MCF-7, Bel7402 and KB cells are given in Table 2. The experimental result shows that BTH-II0204-207: B has obvious antitumor activity on tested tumor cells, and particularly for MCF-7 and KB cells, the IC50 value of the inhibitory activity reaches the nanogram level. The BTH-II 0204-207B can be used for preparing antitumor drugs, or can be used as an active ingredient to be compatible with one or more pharmaceutically acceptable carriers, excipients or auxiliary materials to prepare an antitumor drug composition.

TABLE 2 BTH-II0204-207 inhibitory Activity of B on MCF-7, Bel7402 and KB cells

Claims

1. A method for preparing a p-terphenyl compound BTH-II0204-207: B by using Streptomyces mangrove 219807 is disclosed, wherein the structural formula of the BTH-II0204-207: B is shown in formula I, and the calculated value of [ M + H ] peak M/z 293.1173]⁺293.1099) of formula C₁₉H₁₆O₃The compound BTH-II0204-207: B is prepared from a fermentation culture of Streptomyces mangrovei 219807, the Streptomyces mangrovei 219807 is derived from the soil of the third mangrove in Hainan province of China, the preservation number is CCTCC NO. M2015276,

2. the method according to claim 1, characterized in that it comprises in particular the steps of:

3. Use of Streptomyces mangrove 219807 in the process of claim 1 for the preparation of the p-terphenyl compound BTH-II0204-207: B.

4. The method of claim 1, wherein the p-terphenyl compound BTH-II 0204-207B is used for preparing antitumor drugs.

5. The use of claim 4, wherein the anti-tumor drug is a drug against breast cancer, liver cancer, oral epidermoid carcinoma.