CN104478688A

CN104478688A - New sesquiterpene quinine compound in dysidea avara and application thereof

Info

Publication number: CN104478688A
Application number: CN201410678842.2A
Authority: CN
Inventors: 周同永; 周国泰
Original assignee: XINING YIGE INTELLECTUAL PROPERTY ADVISORY SERVICES Co Ltd
Current assignee: XINING YIGE INTELLECTUAL PROPERTY ADVISORY SERVICES Co Ltd
Priority date: 2014-11-24
Filing date: 2014-11-24
Publication date: 2015-04-01

Abstract

The invention relates to the marine natural pharmaceutical chemistry field and in particular relates to a new sesquiterpene quinine compound Dysideanone D and application thereof. The study on pharmacological activity of Dysideanone shows that Dysideanone has the effect of inhibiting various tumors and has a strongest inhibition effect on cervical cancer cells; the inhibition effect is superior to the positive medicine; the Dysideanone has a poor inhibition effect on normal cells. Consequently, Dysideanone can be used for preparing anti-tumor medicine, especially anti-cervical cancer medicine.

Description

A kind of new sesquiterpene naphtoquinone compounds and uses thereof in greedy stubborn sponge

Technical field

The present invention relates to marine natural medicinal chemistry art, be specifically related to a kind of new sesquiterpene naphtoquinone compounds and uses thereof in greedy stubborn sponge.

Background technology

Marine organisms contain different from terrestrial life, that chemical structure is special active substance.For finding the difficult diseases active substances such as treatment cardiovascular and cerebrovascular diseases, tumour, acquired immune deficiency syndrome (AIDS), senile dementia, people turn one's attention to ocean one after another.China's marine drug research and development, through making great efforts for many years, achieve gratifying effect, have found a collection of novel anti-AIDS, antitumor and antiatherogenic medicine.

Research finds, sponge is rich in multiple secondary metabolite, novel structure, and pharmacologically active is extensive.Sesquiterpene quinone and sesquiterpene quinhydrones are wherein typical compounds.With from the stubborn sponge of greediness dysidea avarathe sesquiterpene quinhydrones Avarol that middle separation obtains and derivative Avarone thereof is example, and the two anti-tumor activity is very remarkable, and result for the treatment of is similar to endoxan, daunorubicin and methotrexate; HIV (human immunodeficiency virus)-resistant activity is similar to AZT; And aldose reductase inhibition activity and Sorbinil close.

Jiao WH etc. are to the greedy stubborn sponge (Sponge in the South Sea dysidea avara) new separation obtains the special sesquiterpene naphtoquinone compounds of three kinds of structures in chemical constitution study, called after Dysideanone A, B and C(Dysideanones A C, Unusual Sesquiterpene Quinones from the South China Sea Sponge dysidea avara, j. Nat. Prod. 2014,77,346 350).To these three compound activity the selection result displays, Dysideanone B has significant restraining effect to HeLa tumor cell line and HepG2 tumor cell line, and IC50 is respectively 7.1 and 9.4 μMs.Dysideanone A, B and C-structure formula as follows:

Contriver is to the stubborn sponge of greediness dysidea avarain chemical constitution study process, the sesquiterpene quinones that isolation identification one is new from dichloromethane extract chemical structure is Dysideanone A 1,17 dehydrations and 3,4 carbon-carbon double bonds displacements are 4 and 11.This sesquiterpene naphtoquinone compounds is reported first, called after Dysideanone D.Cytotoxicity test shows, this compound on tumor cell has significant cytotoxicity, more weak to normal cell toxic action, can as the activeconstituents preparing antitumor drug.

Summary of the invention

The object of this invention is to provide a kind of from the stubborn sponge of greediness dysidea avaraa kind of new sesquiterpene naphtoquinone compounds Dysideanone D that middle separation obtains.

Another object of the present invention is to provide Dysideanone D preparing the application in antitumor drug.

According to an aspect of the present invention, the Compound D ysideanone D with the following chemical structure formula is provided:

According to a further aspect in the invention, the compounds of this invention Dysideanone D is provided to prepare the application in antitumor drug.For realizing this object, the present invention adopts mtt assay to screen the inhibit activities of this compound to kinds of tumor cells, comprising: human cervical carcinoma HeLa, people liver cancer HepG2, people lung cancer A549 and human colon carcinoma HCT15.Research finds, Dysideanone D has significant restraining effect (effect is better than positive drug) to human cervical carcinoma HeLa tumor cell line, relatively weak to other three kinds of tumor cell line inhibit activities; To human normal cell line without obvious cytotoxicity.Further cytologic experiments shows, Dysideanone D may with apoptosis-related to the restraining effect of human cervical carcinoma HeLa tumour.Based on standard pharmacological trials, thus the compounds of this invention Dysideanone D may be used for antitumor field, preferred cervical cancer.

Be MTT test and result below.

The determination of activity of In Vitro Anti human cervical carcinoma HeLa, people liver cancer HepG2, people lung cancer A549, human colon carcinoma HCT15 and Human umbilical vein endothelial cells (VEC):

Beating holes method measures KB and cell strain inhibit activities, test method is as follows: take the logarithm vegetative period cell cultures in 96 well culture plates, every hole 100 μ L (containing 1000-1200 tumour cell), next day, administration group adds containing different concns compound, 4 dosage groups established by every medicine, and often group establishes 3 parallel plates.Control group adds isopyknic solvent with compound, puts 5%CO ₂in 37 DEG C of cultivations in incubator, nutrient solution is discarded after 4d, every hole adds 200 μ L 0.2% MTT solution, 37 DEG C of insulation 4h, abandoning supernatant, every hole adds DMSO 150 μ L and dissolves first hairpin particle, after slight concussion, by microplate reader, at reference wavelength 450nm, under determined wavelength 570nm condition, measure optical density(OD) (OD).With the cell strain of solvent control process for control group, using taxol as positive control medicine.

LOGIT method and then computerized compound IC is used by gained cell inhibitory rate ₅₀numerical value.

Test-results shows, the compounds of this invention Dysideanone D has significant restraining effect (effect is better than positive drug) to human cervical carcinoma HeLa tumor cell line, relatively weak to other three kinds of tumor cell line inhibit activities; On the other hand, the restraining effect of Compound D ysideanone D to normal cell (Human umbilical vein endothelial cells VEC) is less, shows that the compounds of this invention Dysideanone D has antitumor action, and has certain selectivity.Test-results is in table 1.

Table 1 the compounds of this invention I is to tumour cell and Normocellular restraining effect (IC ₅₀, μM)

Accompanying drawing explanation

Fig. 1 is that Dysideanone D calculates ECD and test ECD figure compares

Fig. 2 is that Dysideanone D acts on 24 hours HeLa morphocytologies change (control group and treatment group).

Embodiment

Embodiment 1:Dysideanone D preparation method

Dry sponge 6kg, 95% alcohol heat reflux extracts 3 times, each 20L.Extracting solution concentrating under reduced pressure obtains medicinal extract 320g.Medicinal extract is dispersed in 3L water, with dichloromethane extraction 3 times, each 3L.Combined dichloromethane extraction liquid, concentrating under reduced pressure obtains medicinal extract 118g.Medicinal extract 100ml methylene dichloride and acetone mixed solvent (3:1, V/V) are dissolved, and mix and mix sample (200g, 200-300 order), volatilize solvent with silica gel.Adopt 200-300 order silica gel (1.5kg) to fill post to be separated.With dichloromethane-acetone gradient elution (10:1,5:1,2:1,1:1, V/V).Collect dichloromethane-acetone (2:1) elution fraction, after decompression and solvent recovery, use methyl chloride-acetone gradient elution (4:1,3:1,2:1, V/V) further, collect dichloromethane-acetone (2:1) elution fraction.This part preparative high performance liquid chromatography is separated, water-methanol gradient elution, obtains pure Compound D ysideanone D(11.3mg from water-methanol (volume ratio 15:85) wash-out position).

Structural identification: pale yellow powder; HR-ESIMS shows [M+H] ⁺for m/z 295.2542; ¹h NMR (CDCl ₃, δ ppm, jin Hz, 600 MHz) and ¹³c NMR (CDCl ₃, δ ppm, 150 MHz) and data are in table 2.In conjunction with DEPT, HSQC, HMBC, NOESY spectrum and data in literature (Dysideanones A C, Unusual Sesquiterpene Quinones from the South China Sea Sponge dysidea avara, j. Nat. Prod. 2014,77,346 350) its two dimensional structure and relative configuration can be determined.Absolute configuration is confirmed by ECD, and ECD figure is shown in accompanying drawing 1.

Table 2 ¹h NMR and ¹³c NMR signals assignment

Embodiment 2: morphological observation and AO(acridine orange) dyeing

HeLa tumour cell establishes not dosing group (control group) and dosing group (5 μMs, treatment group).Cell is after effect different time (0,2,4,8,12,24 hours) after 4% paraformaldehyde fixes 30min, and the PBS washing of 0.01M 2-3 time, AO dyes 10min, observation of cell nuclear morphology under fluorescent microscope.

Find that the cellular form of negative treatment group is normal, complete through staining reagent stained cells core, the many cellular fories after Dysideanone D process become circle or shrinkage, under fluorescent microscope, excite discovery karyopyknosis with blue light.

After AO fluorescent dye, the fine and close dense dye of typical apoptotic cell karyon, in block or particulate state yellow-green fluorescence; The even light dye of normal cell; Dead cell does not dye.Experimental result shows: the even light dye of negative control group cell core, in yellow-green fluorescence, has several apoptotic cell being dyed to fine and close dense dye because natural apoptosis causes once in a while; Dysideanone D treatment group is with extended durations of action, be dyed to and obviously increase in cell number that is block or the fine and close dense dye of particulate state, result shows the cellular portions apoptosis after compound treatment, illustrates that Dysideanone D inhibition tumor cell propagation may be correlated with apoptosis.

Accompanying drawing 2 changes (control group and treatment group) for Dysideanone D acts on 24 hours HeLa morphocytologies.

Claims

1. a sesquiterpene naphtoquinone compounds, its chemical structural formula is as follows:

。

2. the compound described in claim 1 is preparing the application in antitumor drug, and described tumour is cervical cancer, liver cancer, lung cancer and colorectal carcinoma.

3. application according to claim 2, is characterized in that described tumour is cervical cancer.