CN101519435B

CN101519435B - Cyclic nonapeptide compound in Hsisha sponge and application thereof

Info

Publication number: CN101519435B
Application number: CN2009100489873A
Authority: CN
Inventors: 张红军; 杨帆; 林厚文; 董涛; 曹桂东; 陈万生
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2009-04-09
Filing date: 2009-04-09
Publication date: 2011-09-14
Anticipated expiration: 2029-04-09
Also published as: CN101519435A

Abstract

The invention relates to the technical field of medicaments, which is a novel cyclic nonapeptide compound with antineoplastic activity produced from a marine animal Phakellia fusca in the territorialwaters near Hsisha Islands. The chemical structural formula of the novel cyclic nonapeptide compound is shown as below. The in vitro activity test proves that the compound has remarkable inhibiting activity for various tumour cells, wherein the IC50 value of a mouse P388 is only 5.6 mu g/ml and is obviously superior to a positive control medicament vincristine. Therefore, the novel cyclic nonapeptide compound can be used for preparing antitumor medicaments. The invention provides a lead compound for developing a novel antitumor medicament.

Description

A kind of ring nonapeptide compounds and uses thereof in the sponge of Xisha

Technical field

The present invention relates to medical technical field, is a kind of new ring nonapeptide compounds with anti-tumor activity that originates among the brown flat sponge Phakellia fusca of marine animal of surrounding waters, the Xisha Islands.

Background technology

Sponge is a kind of multicellular animals of low grade, and it is widely distributed but often contain the rare secondary metabolite with antitumour activity of a lot of structures, is the first-selected marine organisms research object of marine natural product chemistry man therefore always.The little axle of the medicine source material Phakellia ordinary guiding principle of fusca Sponge of the present invention (Demospongiae) Halichondrida (Halichondrida) Spongiidae (Axinellidae) sponge.2002,1 new cyclic peptide phakelliastatin 12 and patent applied for (application number: 01113389.9) once from the P.fusca sponge that gather on island, Yongxing, Xisha, China South Sea, had been found.All Phakellistatins class cyclic peptide compounds are before all belonged to the sponge from Phakellia by Pettit group of the upright university of State of Arizona, US to be found, but does not see relevant report with new ring nonapeptide compounds of anti-tumor activity so far.

Summary of the invention

The invention provides a kind of new ring nonapeptide compounds that is separated to from the brown flat sponge of surrounding waters, the Chinese Xisha Islands, its chemical structural formula is as follows:

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

1. extract the preparation total extract:

After the brown flat sponge of exsiccant (P.fusca) pulverized, extract with 95% ethanol cold soaking routinely, extracting solution, with extracting solution concentrate medicinal extract, add 50% the ethyl acetate aqueous solution and extract, get the ethyl acetate total extract.

2. separation and purification:

1) preparation crude extract: the ethyl acetate total extract with 90% methyl alcohol suspendible, behind petroleum ether degreasing, with methyl alcohol water layer n-butanol extraction, is got crude extract routinely;

2) separation and purification cyclic peptide compound: with Sephadex LH-20 gel column chromatography on the crude extract, with pure methyl alcohol is eluent, the result is known in inspection according to thin-layer chromatography, collection contains the elutriant part of cyclic peptide compound, carries out reversed-phase silica gel column chromatography again, is 1 with volume ratio: 1-1: 0 methanol mixed solvent gradient elution, again with reversed phase high efficiency liquid phase (55% methanol, flow velocity 1.5ml/min, the detection wavelength is 280nm) preparation, get The compounds of this invention C ₄₈H ₆₈N ₁₂O ₁₄

Activity test in vitro proves that The compounds of this invention all has certain inhibition activity to multiple different tumour cells such as P388, BEL-7402, SPC-A-1, HAC and Colo-26.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.

Embodiment 1. preparation The compounds of this invention

Get the brown flat sponge 15kg after the drying and crushing, extract 8 times with 95% ethanol 50L cold soaking respectively, each week, united extraction liquid, extracting solution gets medicinal extract through concentrating under reduced pressure, and medicinal extract extracts with 50% the ethyl acetate aqueous solution, gets the ethyl acetate total extract, the ethyl acetate total extract is used petroleum ether degreasing with 90% methyl alcohol suspendible; With pure water layer n-butanol extraction, get crude extract 50g;

Above-mentioned crude extract 50g is carried out Sephadex LH-20 gel column chromatography, with pure methyl alcohol is eluent, the result is known in inspection according to thin-layer chromatography, collection contains the elutriant part of cyclic peptide compound, carries out reversed-phase silica gel column chromatography again, is 1 with volume ratio: 1-1: 0 methanol mixed solvent gradient elution, again with reversed phase high efficiency liquid phase (55% methanol, flow velocity 1.5ml/min, the detection wavelength is 280nm) preparation, get Compound C ₄₈H ₆₈N ₁₂O ₁₄, its physico-chemical property and nuclear magnetic resonance spectrum data are as follows:

Compound C ₄₈H ₆₈N ₁₂O ₁₄: the white glass sprills.Show [M+H] among the ESI-MS ⁺Peak 1037.54, [M+Na] ⁺Peak 1059.55 determines that its molecular weight is 1036.The nuclear magnetic resonance spectrum data see Table 1.

Table 1 Compound C ₄₈H ₆₈N ₁₂O ₁₄Nuclear magnetic resonance spectrum data (DMSO-d ₆, 600MHz/150MHz)

The anti tumor activity in vitro experiment:

P388 (mouse leukemia), BEL-7402 (people's liver cancer), SPC-A-1 (people's lung cancer), HAC (rat liver cancer ascitic tumor), Colo-26 (mouse junction cancer) that the tumor cell line that experiment is used provides as Shanghai Tian Jia company.

Experimental technique adopts conventional tetramethyl-azo azoles salt (MTT) colorimetry, and platform is expected blue staining.Experiment divides 3 groups: blank group, positive controls and The compounds of this invention group.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 kinds of concentration of 6.25,3.125 (mcg/ml), cell is cultivated with 10% calf serum earlier routinely, attached cell makes cell be in logarithmic phase with the digestion of 0.2% trysinization liquid when going down to posterity, and cell inoculation is in 96 well culture plates during experiment, 180 microlitres are inoculated in every hole, and concentration is 5 * 10 ⁴The cell suspension of individual/milliliter.Put 37 ℃ CO ₂After the pre-overnight incubation of incubator, every hole adds 20 microlitre drug solutions, and the blank group adds 20 microlitre DMSO, and every kind of concentration repeats 5 holes.Drug effect carries out mtt assay in cell after 3 days measures, and adding concentration in the every hole of 96 well culture plates is MTT working fluid 10 microlitres of 5 mg/ml, hatches 4 hours for 37 ℃, abandons supernatant liquor, adds 100 microlitre DMSO, measures the OD value with microplate reader under 570 nano wave lengths.Platform expects that blue row dyes method, adds platform and expects blue working fluid, living cell counting.Calculate the inhibiting rate of analyte by following formula to growth of cancer cells:

Half effective inhibition concentration IC ₅₀Value adopts the Logit method to calculate, and the results are shown in Table 2:

Table 2 Compound C ₄₈H ₆₈N ₁₂O ₁₄Half effective inhibition concentration (μ g/ml) to tumour cell

By table 2 as seen, Compound C ₄₈H ₆₈N ₁₂O ₁₄Multiple different tumor cell line is shown that all certain restraining effect is arranged, wherein to the IC of mouse P388 ₅₀Value only is 5.6 μ g/ml, and obviously is better than contrasting the medicine vincristine(VCR), so can be used to prepare antitumor drug.The present invention provides new lead compound for developing new antitumour drug.

Claims

1. one kind is encircled the nonapeptide compounds, it is characterized in that chemical structural formula is as follows:

2. the application of the described ring nonapeptide of claim 1 compounds in the preparation antitumor drug.