阅读说明：本技术 海绵来源杂萜类化合物etherianones及其制备方法与应用 (Sponge source heteraianones and preparation method and application thereof ) 是由 焦伟华 林厚文 于 2019-10-24 设计创作，主要内容包括：本发明涉及医药技术领域,公开了两种海绵来源杂萜类化合物etherianone A和etherianone B,化学结构式如下：本发明还公开了制备上述两种化合物的方法。本发明化合物etherianone A和etherianone B能够在体内抑制转基因斑马鱼的血管生成,etherianone A还对多种肿瘤细胞株具有很强的细胞毒性。这两个化合物可用于抗炎药物和抗肿瘤药物的开发。(The invention relates to the technical field of medicines, and discloses two sponge source diterpenoid compounds, namely, etherianone A and etherianone B, which have the following chemical structural formulas:)

1. A sponge source heteroterpenoid compound etherianone has a chemical structural formula as follows:

2. the process for the preparation of the sponge-derived terpenoid etherianone according to claim 1, characterized in that it comprises the steps of:

(a) cold soaking sponge with methanol, and concentrating the extractive solution to obtain extract;

(b) suspending the extract with water, extracting with dichloromethane or chloroform, concentrating the extractive solution under reduced pressure, sequentially passing through:

performing vacuum silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate, and mixing fractions containing sesquiterpene quinone compounds;

performing reversed-phase medium-pressure ODS column chromatography, and performing gradient elution by adopting a methanol-water system to obtain fine fractions containing the sesquiterpene quinone compounds;

separating by high performance liquid chromatography, and eluting by using 95% acetonitrile water to obtain the compound etherianone.

3. The method of claim 2, wherein in step (a), the sponge is frozen and chopped prior to being soaked.

4. The method of claim 2, wherein in step (b), the reversed-phase medium-pressure ODS column is performed with a methanol-water system gradient of 50:50 to 100: 0.

5. The method of claim 2, wherein the HPLC separation is performed at a flow rate of 2.0mL/min and a detection wavelength of 240 nm.

6. Use of the sponge-derived terpenoid etherianone according to claim 1 for the preparation of an anti-inflammatory or anti-tumor medicament.

7. Use according to claim 6, characterized in that it uses the inhibitory activity of etherianone A and/or etherianone B on angiogenesis.

8. Use according to claim 6, characterized in that it uses the cytotoxicity of etherianone A against tumor cells.

9. The use as claimed in claim 6 wherein the antineoplastic drug is one or more of an anti-lung cancer drug, an anti-cervical cancer drug and an anti-colon cancer drug.

Technical Field

The invention relates to the technical field of medicines, in particular to sponge source diterpenoid compounds etherianones and a preparation method and application thereof.

Background

Stubborn sponge (Dysidea ethria) is the sponge of Demospongiae (Demospongiae) Demospongiae (Dictyocetida) Despongiaceae (Dysideidae). For nearly half a century, scholars at home and abroad have systematically studied the chemical composition and biological activity of the characteristic sesquiterpene quinone heteroterpenoid contained in the sponge, and the compound contains C₁₅Sesquiterpene fragments of the Unit and a C₆Units of p-benzoquinone or hydroquinone boundTherefore, the heteroterpene skeleton containing 21 carbons is the most important metabolite of the catarrhal sponge, and the changeable rephoto skeleton and the abundant and diverse biological activities of the heteroterpene skeleton attract the continuous and wide attention of scholars at home and abroad. These heteroterpenoids have a wide range of biological activities, including antibacterial activity¹And an antifungal agent²And an antitumor agent³And oxidation resistance⁴Anti-inflammatory agent⁵Anti-allergic activity⁶And PTP1B enzyme inhibitory Activity⁷. The wide range of biological activities and diverse structures of this class of compounds has attracted continuing attention by chemists and biologists.

Angiogenesis refers to a process of forming a new blood capillary-dominated vascular system from an existing blood vessel by migration and proliferation of vascular endothelial cells based on the existing blood capillary⁸. Angiogenesis is a balanced process in which many molecules that promote or inhibit angiogenesis participate in regulation. A great deal of research shows that angiogenesis plays an important role in the growth and metastasis of malignant tumors, and the inhibition of tumor angiogenesis is an important research direction of biological targeted therapy of tumors⁹. Natural products are becoming an important source of new angiogenesis inhibitors.

Reference documents:

[1](a)Urban,S.；Capon,R.J.5-epi-Isospongiaquinone,a New Sesquiterpene/Quinone Antibiotic from an Australian Marine Sponge,Spongia hispida.J.Nat.Prod.1992,55,1638–1642.(b)Schmalzbauer,B.；Herrmann,J.；Müller,R.；Menche,D.Total Synthesis and Antibacterial Activity of Dysidavarone A.Org.Lett.2013,15,964-967.

[2]Ciavatta,M.L.；Lopez Gresa,M.P.；Gavagnin,M.；Romero,V.；Melck,D.；Manzo,E.；Guo,Y.W.；Van Soest,R.；Cimino,G.Studies on puupehenone-metabolites of a Dysidea sp.:structure and biological activity.Tetrahedron 2007,63,1380–1384.

[3](a)Daletos,G.；de Voogd,N.J.；Müller,W.E.G.；Wray,V.；Lin,W.；Feger,D.；Kubbutat,M.；Aly,A.H.；Proksch,P.Cytotoxic and Protein Kinase Inhibiting Nakijiquinones and Nakijiquinols from the Sponge Dactylospongia metachromia.J.Nat.Prod.2014,77,218-226.(b)Hwang,I.H.；Oh,J.；Zhou,W.；Park,S.；Kim,J.H.；Chittiboyina,A.G.；Ferreira,D.；Song,G.Y.；Oh,S.；Na,M.；Hamann,M.T.Cytotoxic Activity of Rearranged Drimane Meroterpenoids against Colon Cancer Cells via Down-Regulation ofβ-Catenin Expression.J.Nat.Prod.2015,78,453-461.

[4]Utkina,N.K.；Denisenko,V.A.；Krasokhin,V.B.Sesquiterpenoid Aminoquinones from the Marine Sponge Dysidea sp.J.Nat.Prod.2010,73,788–791.

[5]McNamara,C.E.；Larsen,L.；Perry,N.B.；Harper,J.L.；Berridge,M.V.；Chia,E.W.；Kelly,M.；Webb,V.L.Anti-inflammatory Sesquiterpene-quinones from the New Zealand Sponge Dysidea cf.cristagalli.J.Nat.Prod.2005,68,1431–1433.

[6]Jiao,W.H.；Cheng,B.H.；Shi,G.H.；Chen,G.D.；Gu,B.B.；Zhou,Y.J.；Hong,L.L.；Yang,F.；Liu,Z.Q.；Qiu,S.Q.；Liu,Z.G.；Yang,P.C.；Lin,H.W.Dysivillosins A–D,Unusual Antiallergic Meroterpenoids from the Marine Sponge Dysidea villosa.Sci.Rep.2017,7,8947.

[7]Jiao,W.H.；Li,J.；Zhang,M.M.；Cui,J.；Gui,Y.H.；Zhang,Y.；Li,J.Y.；Liu,K.C.；Lin,H.W.Frondoplysins A and B,Unprecedented Terpene-Alkaloid Bioconjugates from Dysidea frondosa.Org.Lett.2019,21,6190-6193.

[8]Carmeliet,P.；Jain,R.K.Angiogenesis in cancer and other diseases.Nature 2000,407,249-257.

[9]Kodera,Y.；Katanasaka,Y.；Kitamura,Y.；et al.Sunitinib inhibits lymphatic endothelial cell functions and lymphnode metastasis in a breast cancer model through inhibition of vascular endothelial growth factor receptor 3.Breast Cancer Res 2011,13,R66.

disclosure of Invention

The invention discloses a sponge source terpenoid compound etherianone, which has the following chemical structural formula:

the second purpose of the invention is to disclose the preparation method of the sponge source diterpenoid compound etherianone, which comprises the following steps:

(a) cold soaking sponge with methanol, and concentrating the extractive solution to obtain extract;

(b) suspending the extract with water, extracting with dichloromethane or chloroform, concentrating the extractive solution under reduced pressure, sequentially passing through:

performing vacuum silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate, and mixing fractions containing sesquiterpene quinone compounds;

performing reversed-phase medium-pressure ODS column chromatography, and performing gradient elution by adopting a methanol-water system to obtain fine fractions containing the sesquiterpene compounds;

separating by high performance liquid chromatography, and eluting by using 95% acetonitrile water to obtain the compound etherianone.

Preferably, in step (a), the sponge sample is soaked in the solution and frozen and cut up.

Preferably, in the step (b), the gradient of the methanol-water system adopted by the reversed-phase medium-pressure ODS column is 50: 50-100: 0. Further, the methanol-water system gradient changes were 50:50, 70:30, 80:20, 90:10, and 100: 0.

Preferably, the high performance liquid chromatography has a flow rate of 2.0mL/min and a detection wavelength of 240 nm.

The third purpose of the invention is to disclose the application of sponge source diterpenoid compound etherianone, in particular to the application in preparing anti-inflammatory drugs and antitumor drugs.

Activity tests prove that the compound of the invention, namely the etherianone A, shows strong cytotoxicity to 4 human tumor cells in vitro, and the etherianone A and the etherianone B have strong inhibitory activity to angiogenesis of a zebra fish model in vivo, so that the compound of the invention can be used for developing antitumor drugs.

The application of the sponge-derived heteroterpenoid compounds etherianone A and etherianone B is characterized in that the anti-tumor drug is one or more of drugs directly used for preventing, diagnosing, detecting, protecting, treating and researching tumors and directly related diseases thereof, and is one or more of anti-lung cancer drugs, anti-cervical cancer drugs and anti-colon cancer drugs; the tumor is one or more of lung cancer, cervical cancer or colon cancer.

The anti-lung cancer drug is one or more of drugs directly used for preventing, diagnosing, detecting, protecting, treating and researching lung cancer and directly related diseases thereof, the lung cancer is one or more of non-small cell lung cancer and small cell lung cancer, and the non-small cell lung cancer is one or more of phosphorus cancer, adenocarcinoma or large cell lung cancer.

The anti-cervical cancer drug is one or more of drugs directly used for preventing, diagnosing, detecting, protecting, treating and researching cervical cancer and directly related diseases thereof, and the cervical cancer is one or more of atypical hyperplasia, in-situ cancer, early stage infiltration cancer under the microscope or infiltration cancer.

The colon cancer resisting medicine is one or more of medicines directly used for preventing, diagnosing, detecting, protecting, treating and researching colon cancer and directly related diseases thereof, and the colon cancer is one or more of left half colon cancer, right half colon cancer or rectal cancer.

The invention has the beneficial effects that: the preparation method is simple, the compounds of the etherianone A and the etherianone B prepared by the method have obvious inhibitory activity to the generation of transgenic zebra fish blood vessels in vivo, and the etherianone A has strong cytotoxicity to various human tumor strains. The invention provides a new lead compound for researching and developing new anti-tumor drugs and provides scientific basis for developing and utilizing marine medicinal resources in China.

Drawings

FIG. 1 is a schematic representation of the key correlation of two-dimensional nuclear magnetic data of etherianone A;

FIG. 2 is a correlation plot of measured carbon spectrum data and calculated carbon spectrum data for etherianone A;

FIG. 3 is a comparison graph of an actually measured ECD spectrum of etherianone A and a calculated ECD spectrum of two configurations;

FIG. 4 is a fluorescent microscope photograph of etherianone A and PTK787 experiments in inhibiting angiogenesis in transgenic zebrafish;

FIG. 5 shows the results of quantitative analysis of ISV of the experiment of inhibiting angiogenesis in transgenic zebra fish by etherianone A and PTK 787.

Detailed Description

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

EXAMPLE 1 preparation of the Compounds Etherianone A and Etherianone B

The preparation method comprises the steps of taking catadroma sponge (wet weight 514g) which is frozen and chopped in advance, respectively carrying out cold extraction on the catadroma sponge by using methanol (500mL) for 5 times, combining extracting solutions, carrying out reduced pressure concentration on the extracting solutions to obtain a total extract, suspending the total extract into 60% methanol water, carrying out extraction on the total extract by using dichloromethane with the same volume for 5 times, combining the extracting solutions, and carrying out reduced pressure concentration to obtain a dichloromethane extract (2.6g), wherein the existence of etherianone A and etherianone B is detected in the dichloromethane extract by LC-DAD/MS.

Separating the extract with reduced pressure silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate, obtaining fraction containing the compound according to TLC analysis result, subjecting the fraction to reversed phase medium pressure ODS column chromatography, gradient eluting with methanol-water system (50:50, 70:30, 80:20, 90:10 and 100:0), and separating the obtained fine fraction containing sesquiterpene compound with high performance liquid chromatography (95% acetonitrile water, flow rate of 2.0mL/min, detection wavelength of 240nm) to obtain compound etherianone A (C: 95% acetonitrile water, flow rate of 2.0mL/min, detection wavelength of 240nm), and collecting extract₂₅H₃₀O₂) And etherianone B (C)₂₅H₃₀O₂). The physicochemical properties and nmr data for the two compounds are as follows:

etherianone A colorless oil; [ alpha ] to]_D ²⁰-2.0(c 0.05,MeOH)；UV(MeOH)λ_max(logε)224(3.05),252(2.54)nm；CD(MeOH)λ(Δε)236(-3.00),266(-0.38),289(-0.89),338(+0.24)nm；DART-MS:m/z 363.2[M+H]⁺；DART-HRMS:m/z 363.2316[M+H]⁺(calcd for C₂₅H₃₁O₂,363.2319)，¹H NMR(600MHz,CDCl₃)and ¹³C NMR(150MHz,CDCl₃) See table 1;

etherianone B, colorless oil; [ alpha ] to]_D ²⁰-4.0(c 0.06,MeOH)；UV(MeOH)λ_max(logε)223(3.19),249(2.66)nm；CD(MeOH)λ(Δε)228(-4.21),265(+0.70),287(-0.73),337(+1.16)nm；DART-MS:m/z 363.2[M+H]⁺；DART-HRMS:m/z 363.2314[M+H]⁺(calcd for C₂₅H₃₁O₂,363.2319)，¹H NMR(600MHz,CDCl₃)and ¹³C NMR(150MHz,CDCl₃) See table 1.

TABLE 1 NMR spectra data (CDCl) of the compounds etherianone A and etherianone B₃)

The structure of the key correlation diagram of the two-dimensional nuclear magnetic data of the compound etherianone a shown in fig. 1 is as follows:

FIG. 2 shows the correlation of the measured carbon spectrum data and the calculated carbon spectrum data of etherianone A, the correlation coefficient r²0.9973, suggesting that the calculated carbon spectrum value is highly consistent with the test; FIG. 3 shows the measured ECD (electronic circular dichroism) curve (1) and the calculated ECD curves (1a and 1b) for compound etherianone A, which determined the absolute configuration of the compound as 1S,5S,8S,9R,10S,16S, and has the following structural formula:

example 2 experiment of inhibiting angiogenesis in transgenic Zebra fish

Transgenic zebrafish in vivo angiogenesis inhibition experiments were performed on the compounds of the invention, etherianone a and etherianone B, and parallel experiments were performed with Vatalanib (Vatalanib, PTK787) as a positive control. The samples were dissolved in DMSO and stored at low temperature, and the concentration of DMSO in the final system was controlled within a range that did not affect the assay activity (final concentration < 0.1%). Each sample was set with 5 replicate wells in the test.

The specific experimental steps are as follows:

obtaining and culturing zebra fish embryos: the male and female zebra fish are separately fed with artificial granular bait and newly hatched Artemia nauplii (Artemia nauplii) by alternately lighting for 14 h/darkness for 10 h. When eggs are collected, healthy and mature zebra fish is taken and placed into a mating jar according to the ratio of male to female being 1:1 or 1:2, and fertilized eggs are obtained 9-10 days later. Sterilizing and cleaning fertilized egg, transferring into water (containing 5mmol/L NaCl, 0.17mmol/L KCl, 0.4mmol/L CaCl) for culturing zebra fish embryo₂，0.16mmol/L MgSO₄) Culturing at 28 deg.C under controlled light.

Experiment of angiogenesis inhibiting activity: after fertilized eggs developed for 24h, the egg membranes were removed using 1g/L Pronase E solution (Sigma, US). Normal zebrafish embryos were picked under a stereomicroscope and transferred to 24-well plates, 8 per well. Two compounds, i.e., etheriane a and etheriane B, were made into a mother solution with DMSO, diluted with distilled water, and a volume thereof was added to each well, and then the volume was increased to 2mL with culture water so that the concentrations of the two compounds were 1.25, 2.5, and 5.0 μ M/L, respectively, in a control group (control) with 1% DMSO, and the positive drug was PTK 787. The embryos were then placed in a light incubator at 28 ℃ with a lid on top to allow the embryos to continue to develop. At 48hpf (hours post fertilization), zebrafish internode blood vessels (ISVs) were observed and counted by fluorescence microscopy, inhibition rates were calculated, and embryo death with malformation was observed.

Inhibition rate/% (number of blood vessels in normal control group-number of blood vessels in administration group)/number of blood vessels in normal control group × 100%.

And calculating the median inhibitory concentration IC of the two compounds on the inhibitory activity of the new blood vessels₅₀The value is obtained.

The results are as follows:

the anti-angiogenic activity of compound etherianone a in the transgenic zebrafish Tg (VEGFR2: GFP) embryo model is shown in fig. 4 and 5 (n ═ 5). Specifically, fig. 4 shows a graph showing the change of the interganglionic vessel ISV after 24 hours of adding different concentrations of etiianone a by observing the side surface of the trunk of the zebra fish fry; vartanib (PTK787) was used as a positive control; white arrows indicate ISVs, white asterisks indicate disappearance of neovascular ISVs. Figure 5 shows the quantitative analysis of ISV after addition of the compound etherianone a in the zebrafish model. Data were derived from 5 separate experiments, expressed as standard deviations, # p <0.05, # p <0.01 compared to the control group.

FIGS. 4 and 5 show that compound etherianone A has strong inhibitory activity, IC, on inhibiting angiogenesis in transgenic zebrafish₅₀The values are respectively 2.4 mu M, and the dose dependence relationship is good; the IC of etherianone B for inhibiting angiogenesis activity was tested in the same manner₅₀The value was 8.7. mu.M.

Example 3 in vitro cytotoxicity assay

The MTT method is adopted to test the compound etherianone A for in vitro cytotoxic activity. The compound was dissolved in DMSO and stored at low temperature, and the concentration of DMSO in the final system was controlled within a range that did not affect the assay activity. Each sample was set with 3 duplicate wells in the test.

The specific experimental steps are as follows:

selecting 4 human tumor cell strains, myeloma NCI-H929 cell, lung cancer A549 cell, liver cancer HepG2 cell and ovarian cancer SK-OV-3 cell (Shanghai Life sciences research institute of Chinese academy of sciences), culturing in RPMI 1640 culture solution with 5% CO₂And 95% air at 37 deg.C, adding 10% (V/V) fetal calf serum and 80 U.mL^-1Penicillin/streptomycin. Cells were added to 96-well plates and incubated for 48h with 3 concentrations (1.25, 5, 20. mu.M) of etherianone A. Selecting 5-fluorouracil, testing absorbance OD value in MAX 340 spectrophotometer under 490nm condition, and calculating inhibition rate and IC₅₀The value is obtained.

The results are as follows:

the compound etilianone A shows strong cytotoxicity and IC to myeloma NCI-H929 cells, lung cancer A549 cells, liver cancer HepG2 cells and ovarian cancer SK-OV-3 cells₅₀The values were 8.3, 12.2, 7.4 and 9.4. mu.M, respectively.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.