CN102234250A

CN102234250A - Antitumor bioactive alkaloid compound in Agelas clathrodes

Info

Publication number: CN102234250A
Application number: CN2010101572122A
Authority: CN
Inventors: 林厚文; 杨帆; 姜波; 陈万生; 龚晓斌
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2010-04-27
Filing date: 2010-04-27
Publication date: 2011-11-09

Abstract

The invention relates to the technical field of medicine, specifically to an alkaloid compound in Agelas clathrodes, wherein the chemical structural formula of the compound is as shown in the figure. The chemical constitution and stereo configuration of the compound is determined by a plurality of modern spectrum analysis and especially by applying a plurality of advanced integrated parsing of two-dimension nuclear magnetic resonance. In vitro antitumor test shows that the alkaloid compound provided by the invention has an obvious effect of inhibiting tumor cells such as HepG2 human liver cancer cells and Caco-2 human intestinal cancer cells and thus can be used for preparing antitumor drugs. The invention provides a lead compound for developing new antitumor drugs and has an important value to develop and utilize Chinese ocean medicinal resources.

Description

A kind of alkaloid compound in the sponge of Xisha with anti-tumor activity

Technical field

The present invention relates to medical technical field, specifically is to resemble from the marine animal that originates from marine site, China Xisha Islands to separate a kind of new alkaloid compound C with anti-tumor activity that obtains the ear sponge Agelas clathrodes ₁₁H ₁₄O ₄N ₅ ⁺And the application in the preparation antitumor drug.

Background technology

Sponge is a kind of marine animal, and its living environment has characteristics such as low temperature, high pressure, high salt, and widely distributed in marine site, the world, all there is distribution in the Xisha Islands marine site of China from the Bohai Sea to the South Sea.In to marine site, China Xisha Islands Biological resources system thinking process, collect a kind of sponge and be orange, thick 1.5～10 centimetres of body wall inhabits in 10 meters the deep water.Through identifying that this kind sponge for resembling the ear sponge, belongs to Demospongiae, Agelasida order, Agelasidae section, Agelas genus.But do not see so far from then on to plant in the sponge (Agelasclathrodes) and be separated to alkaloid compound C ₁₁H ₁₄O ₄N ₅ ⁺Report.

Summary of the invention

The invention provides a kind of new alkaloid compound with anti-tumor activity that extraction separation obtains the ear sponge that resembles from marine site, China Xisha, its molecular formula is C ₁₁H ₁₄O ₄N ₅ ⁺, chemical structural formula is:

The preparation method of The compounds of this invention is as follows:

1. prepare total medicinal extract:

Use acetone, methylene dichloride and methanol solution supersound extraction routinely successively after ear sponge Agelas clathrodes cleans, shreds with fresh resembling, to extracting liquid colourless, united extraction liquid behind reclaim under reduced pressure acetone, methylene dichloride, the methyl alcohol, gets total medicinal extract;

2. prepare alkaloid compound C ₁₁H ₁₄O ₄N ₅ ⁺:

Above-mentioned total medicinal extract is dissolved in 85%～95% methyl alcohol, uses the 1000ml petroleum ether extraction, discard petroleum ether layer; Adjust methanol concentration to 60%～70% again, use dichloromethane extraction, with dichloromethane extraction liquid concentrating under reduced pressure, reclaim methylene dichloride, medicinal extract is concentrated into dried; With volume ratio is that 1: 1 methylene chloride mixed solvent dissolves medicinal extract, carry out Sephandex LH-20 gel filtration chromatography, it with volume ratio 1: 1 methylene chloride mixed solvent wash-out, monitoring result according to thin-layer chromatography, collection contains the flow point of alkaloid compound, reclaims solvent and is concentrated into dried alkaloid compound raw product; Thereafter carrying out reverse phase silica gel ODS column chromatography and separate, is that 1: 1～1: 0 methanol mixed solvent carries out gradient elution with volume ratio; Separate (the detection wavelength is 265nm for 10% methanol, flow velocity 1.5mL/min) through RPLC again, get pure alkaloid compound C ₁₁H ₁₄O ₄N ₅ ⁺

Activity test in vitro proves that it all has certain inhibition activity to these two kinds of different tumour cells of HepG2, Caco-2, therefore can be used for preparing antitumor drug.

The The compounds of this invention preparation method is simple, and anti-tumor activity is remarkable.The present invention provides new lead compound for researching and developing new antitumor drug, for development and use China ocean medicine resource provides scientific basis.

Embodiment

Now in conjunction with the embodiments, the present invention is described in detail, but protection domain is not limited to embodiment.

Embodiment 1. preparation The compounds of this invention

Get and resemble ear sponge 240g (weight in wet base), clean, shred, use 1500 milliliters of acetone, 1500 milliliters of methylene dichloride and each supersound extraction of 1500 ml methanol solution three times successively respectively, united extraction liquid, decompression and solvent recovery, total medicinal extract 3.5g; Total medicinal extract is dissolved in 1000 milliliter of 90% methyl alcohol,, discards petroleum ether layer, adjust methanol concentration to 60% with 1000 milliliters of petroleum ether extractions; With dichloromethane extraction three times, each 1000 milliliters, combining extraction liquid, concentrating under reduced pressure reclaims methylene dichloride, medicinal extract is concentrated into dried, 2.1g; Add 5 milliliters of dissolvings of 1: 1 methylene chloride mixed solvent of volume ratio, carry out Sephadex LH-20 gel filtration chromatography, it with the mobile phase volume ratio 1: 1 methylene chloride mixed solvent wash-out, according to the thin-layer chromatography monitoring result, collection contains the alkaloid compound flow point, reclaim solvent with the vacuum decompression Rotary Evaporators and be concentrated into driedly, obtain alkaloid compound raw product 0.67 gram; Thereafter carrying out reverse phase silica gel ODS column chromatography and separate, is 1: 1～1: 0 methanol mixed solvent gradient elution with volume ratio; Separate (the detection wavelength is 265nm for 10% methanol, flow velocity 1.5mL/min) through RPLC again, get pure alkaloid compound C ₁₁H ₁₄O ₄N ₅ ⁺3.0 milligram.Compound C ₁₁H ₁₄O ₄N ₅ ⁺Physico-chemical property and nuclear magnetic resonance spectrum data as follows:

The compounds of this invention C ₁₁H ₁₄O ₄N ₅ ⁺Be yellow solid, by high resolution mass spectrum (HR-API-MS ⁺) in the accurate molecular weight m/z 281.1213[M+1 that provides of quasi-molecular ion peak] ⁺, (Calcd.for C ₁₁H ₁₅O ₄N ₅ ⁺, 281.1202), provide the molecular formula C of this compound ₁₁H ₁₄O ₄N ₅ ⁺UV spectrum λ _Max(MeOH)=265.0nm, ¹H and ¹³The C nuclear magnetic resonance data sees Table 1.

Table 1. The compounds of this invention ¹H and ¹³C nmr spectrum data (DMSO-d ₆, 400MHz/100MHz)

The present invention is to Compound C ₁₁H ₁₄O ₄N ₅ ⁺Carried out anticancer experiment in vitro, HepG2 (human hepatoma cell strain), Caco-2 (strain of people's colon-cancer cell) that the tumor cell line that experiment is used provides as the biological company limited of Shanghai Tian Jia.Experimental result shows Compound C ₁₁H ₁₄O ₄N ₅ ⁺Therefore activity with obvious suppression tumour can be used to prepare antitumor drug.

HepG2 human liver cancer cell and Caco-2 people's colon-cancer cell cytotoxic activity are measured and adopted mtt assay: experiment divides blank group, positive controls and The compounds of this invention C ₁₁H ₁₄O ₄N ₅ ⁺Three groups.Positive control drug is a vincristine(VCR).The compounds of this invention and positive control drug are dissolved with DMSO respectively, be made into 100,50,25,12.5,6.25,3.125 (mcg/ml) 6 kinds of concentration; The HepG2 human liver cancer cell trysinization that will be in logarithmic phase becomes individual cells, with 1 * 10 ⁵The concentration 100 μ l/ holes of Cell/mL are inoculated in 96 well culture plates, and adherent growth 24h adds test unit sample solution 10 μ l/ holes, and each concentration is established 3 multiple holes, and what the blank group was used is the 0.1%DMSO nutrient solution.The HepG2 human liver cancer cell is put 5%CO ₂Incubator is inhaled after cultivating 48h under 37 ℃ and is removed nutrient solution, adds 10 μ l MTT solution (5mg/mL), puts CO again ₂Supernatant liquor was removed in suction after incubator was hatched 4h, added 100 μ l DMSO at 37 ℃ of shaking tables vibration 15min, and microplate reader is measured optical density (OD value) in the 570nm wavelength.

Calculate the inhibiting rate of analyte to growth of cancer cells, half effective inhibition concentration IC with following formula ₅₀Value adopts the Logit method to calculate:

The results are shown in Table 2.

Table 2 Compound C ₁₁H ₁₄O ₄N ₅ ⁺External antitumor activity

By table 2 as seen, Compound C ₁₁H ₁₄O ₄N ₅ ⁺To the restraining effect of two kinds of different tumor cell lines near in addition be better than the positive control drug vincristine(VCR), therefore can be used to prepare antitumor drug.

The present invention provides new lead compound for developing new antitumor drug, and is significant to the marine pharmaceutical organism resource of developing sharp China.

Claims

1. alkaloid compound C ₁₁H ₁₄O ₄N ₅ ⁺, chemical structural formula is as follows:

2. the application of the described compound of claim 1 in the preparation antitumor drug.

3. the preparation method of the described compound of claim 1, step is as follows:

1) prepare total medicinal extract:

Each supersound extraction is to extracting liquid colourless routinely to use acetone, methylene dichloride and methanol solution after ear sponge Agelas clathrodes cleans, shreds with fresh resembling successively, and united extraction liquid behind reclaim under reduced pressure acetone, methylene dichloride, the methyl alcohol, gets total medicinal extract;

2) preparation Compound C ₁₁H ₁₄O ₄N ₅ ⁺:

Above-mentioned total medicinal extract is dissolved in 85%～95% methyl alcohol, uses petroleum ether extraction, discard petroleum ether layer; Adjust methanol concentration to 60%～70% again, use dichloromethane extraction, with dichloromethane extraction liquid concentrating under reduced pressure, reclaim methylene dichloride, medicinal extract is concentrated into dried; With volume ratio is that 1: 1 methylene chloride mixed solvent dissolves medicinal extract, carry out Sephandex LH-20 gel filtration chromatography, it with volume ratio 1: 1 methylene chloride mixed solvent wash-out, monitoring result according to thin-layer chromatography, collection contains the flow point of alkaloid compound, reclaims solvent and is concentrated into dried alkaloid compound raw product; Thereafter carrying out reverse phase silica gel ODS column chromatography and separate, is that 1: 1～1: 0 methanol mixed solvent carries out gradient elution with volume ratio; Separate (the detection wavelength is 265nm for 10% methanol, flow velocity 1.5mL/min) through RPLC again, get alkaloid compound C ₁₁H ₁₄O ₄N ₅ ⁺