CN101591314B

CN101591314B - Ophiobolin di-sesquiterpene compound and preparation method and application thereof

Info

Publication number: CN101591314B
Application number: CN2008101729460A
Authority: CN
Inventors: 朱伟明; 卢圳域; 洪葵; 缪承杜
Original assignee: Ocean University of China; Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
Current assignee: Ocean University of China; Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
Priority date: 2008-10-24
Filing date: 2008-10-24
Publication date: 2011-08-10
Anticipated expiration: 2028-10-24
Also published as: CN101591314A

Abstract

The invention relates to an ophiobolin di-sesquiterpene compound and a preparation method and application thereof. The preparation method produces the compound of novel structure by using Aspergillus ustus 094102 (culture collection number: CCTCC M 208153) separated from soil samples of the root system of mangrove plant Bruguiera gymnorrhiza in Touyuan town, Wenchang. Experiments show that the ophiobolin di-sesquiterpene compound can be used as cell proliferation restraining agent or antitumor agent.

Description

Ophiobolin (ophiobolin) class sesterterpene ene compound and its production and use

Technical field:

The present invention relates to prepare the method for ophiobolin (ophiobolin) class sesterterpene ene compound with Aspergillus ustus 094102 (Aspergillus ustus094102); The invention still further relates to the purposes of this compounds in preparation inhibition of cell proliferation or antineoplastic agent.

Background technology:

Ophiobolin (ophiobolin) class sesterterpene ene compound has some bibliographical informations, such compound exhibits broad-spectrum antibacterial activity, as nematicide, antimycotic and antibacterium etc., in addition kinds of tumor cells is had remarkable cytotoxic activity.It is reported that the ophiobolinA of this family can suppress the active of calcium ion activatory cyclic nucleotide phosphodiesterase and cause the apoptosis of mouse leukemia cell L1210 cell.

Up to the present found kind surplus this compounds 30, the constitutional features of this compounds is all to have a tricyclic 5-8-5 ring structure.In addition, there are 14,17 to form epoxy constructions, as (Antonio, E. such as ophiobolinA, ophiobolin J, Anna, A., Alessio C., et al.Ophiobolin E and 8-epi-ophiobolin J produced by Drechsleragigantean, a potential mycoherbicide of weedy grasses.Phytochemistry, 2006,67,2281-2287); There are 16,17 two keys to be hydrogenated and saturated, as (Athanasios T. such as ophiobolins B-D, ophiobolin F, ophiobolin M, Akinlolu A.A., Jan S.T., et al.Ophiobolin M and Analogues, NoncompetitiveInhibitors of Ivermectin Binding with Nematocidal Activity.Bioorg.Med.Chem., 1996,4,531-536); Have between 5,21 and to form lactonic ring or hemiketal, as (Hong W. such as ophiobolin A lactone, ophiobolin H, ophiobolin L, Takuya I., Masahiro K., et al.Cytotoxic sesterterpenes, 6-epi-ophiobolin G and6-epi-ophiobolin N, from marine derived fungus Emericella vaiecolor GF10.Tetrahedron, 2004,60,6015-6019).The inventor discovers that the crude extract of Aspergillus ustus 094102 (Aspergillus ustus094102) liquid fermentation production after ultrasonication has good necrocytosis activity, then its activeconstituents is studied, found that three ophiobolins (ophiobolins) class sesterterpene ene compound has anti-tumor activity, have not yet to see chemical structure and the active report of cell inhibitory effect, so do not see as yet also on the market that relevant therewith medicine is arranged this compound.

Summary of the invention:

The present invention aims to provide a kind of new compound with anti-tumor activities such as cell inhibitory effect and direct killing cancer cells of structure uniqueness.

The present invention also aims to provide the preparation method and the purposes of new compound in preparation tumor cell proliferation inhibitor or antineoplastic agent thereof of a class new compound.

The present invention has found ophiobolin (ophiobolins) the class sesterterpene ene compound of novel structure first from Aspergillus ustus 094102 (Aspergillus ustus094102) fermented product, shown in I, formula II:

Formula I formula II

Its constitutional features is: R among the formula I ₁, R ₂Be hydrogen, alkyl, acyl group, R among the formula II ₁Be hydrogen, alkyl.

Formula I of the present invention, formula II compound be R in the compound 1 wherein preferably ₁R ₂Be methyl, 21 methoxyl group is a beta comfiguration; With R in the compound 2 ₁R ₂Be methyl, 21 methoxyl group is the α configuration.R in the described formula II compound 3 ₁Be methyl.

Most preferred is that liquid fermentate by the Aspergillus ustus 094102 (Aspergillus ustus094102) in mangrove forest source is through silica gel column chromatography in the foregoing invention, with sherwood oil-acetone is that solvent carries out gradient elution, the eluate of sherwood oil-acetone 3:1, be eluent through gel filtration chromatography with chloroform-methanol 1:1 again, eluted product is again through reversed phase column chromatography, with the methanol-water is that solvent carries out gradient elution, methanol-water 85:15 eluted product gets formula I1 and 2, formula II compound 3 through preparation HPLC separation and purification with methanol-water 90:10 wash-out.

The present invention adopts srb assay to test compound 1,2, and the anti-tumor activity of 3 pairs of A549 human lung carcinoma cell lines adopts mtt assay to test compound 1,2, the anti-tumor activity of 3 pairs of HL-60 human leukemia cell lines.Experiment confirm, these 3 compounds all have inhibited proliferation to these two kinds of tumour cells.

Therefore formula I of the present invention, formula II compound useful as inhibitors of cell proliferation or tumor cytotoxicity agent.

Formula I, formula II compound and various medicine acceptable carrier, vehicle or supplementary product compatibility can be made into antitumor drug, are used for tumor treatment.

Formula I, formula II compound also can be used as the low molecular biosciences probe that suppresses cell proliferation and are used for life science, and when using as probe, formula I, formula II compound dissolve in the methyl alcohol, also dissolve in the dimethyl sulfoxide (DMSO) to be applied.

Formula I of the present invention, formula II compound can be cultivated by microbial fermentation, separation and purification and obtaining from fermented product then; Also can be by above-mentioned preferred compound through the synthetic acquisition of chemical modification method well known to those skilled in the art.

Of particular note, the method of producing formula I of the present invention, formula II compound through organism of fermentation can adopt other any microorganism that can produce this compounds, all can be used as and produces bacterium and be used for preparation formula I, formula II compound as long as can produce the microorganism of this compounds.

Enumerated in the embodiments of the invention and utilized Aspergillus ustus 094102 strain to prepare preferred formula I of the present invention, formula II examples for compounds.

This fungi 094102 strain is by separating from the garden mangrove forest Bruguiera conjugata soil of Wenchang, and be accredited as Aspergillus ustus Aspergillus ustus through means of taxonomic research and molecular biology research, and by China's typical culture collection center preservation (numbering: CCTCC M208153, date: on October 10th, 2008, place: Chinese Wuhan, Wuhan University).

This Aspergillus ustus 094102 (Aspergillus ustus 094102) strain has following microorganism mycology feature:

Bacterium colony is cultivated diameter 45～60mm of 15 days at 28 ℃ of diameter 25～35mm that cultivate 7 days of Cha Shi substratum; Bacterium colony is beige, adularescent edge, quality flocculence, no transudate; The uneven tawny of bacterium colony reverse side.Conidiophore betides aerial hyphae, and the conidial head children time be spherical, becomes radiation shape after always.Falx stem 400～800 μ m * 10～16 μ m, the tool brown, wall is smooth.Top capsule sphere or semisphere, 6～13 μ m, most surfaces can be educated; The conidial fructification bilayer, metulae 4～9 μ m * 3～3.5 μ m, bottle stalk 4～10 μ m * 2～3 μ m; The conidium sphere, 3～5 μ m, obviously coarse, the tool pimple.

This Aspergillus ustus 094102 (Aspergillus ustus 094102) strain has following molecular biological characteristics:

The ITS sequence information:

GGGGTTCGAGTGAGACTCTGGGTCACCTCCCACCCGTGTCTATCGTACCTTGTTGCTTC

GGCGGGCCCGCCGTTTCGACGGCCGCCGGGGAGGCCTTGCGCCCCCGGGCCCGCGCC

CGCCGAAGACCCCAACATGAACGCTGTTCTGAAAGTATGCAGTCTGAGTTGATTATCG

TAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCA

GCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACG

CACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTC

AAGCACGGCTTGTGTGTTGGGCCCCCGTCCCCCTCTCCGGGGGACGGGCCCGAAAGG

CAGCGGCGGACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCTGCTCTGTAGG

CCCGGCCGGCGCCAGCCGACACCCAACTTTATTTTTCTAAGGTTGACCTCGGATCAGG

TAGGGATACCCGCTGAACTTAAGCATATCAATAA

The 18S sequence information:

GCCGGCTTGTCTCAGATTAGCCATGCATGTCTAAGTATAAGCAATCTATACTGTGAAAC

TGCGAATGGCTCATTAAATCAGTTATCGTTTATTTGATAGTACCTTACTACATGGATACCT

GTGGTAATTCTAGAGCTAATACATGCTAAAAACCCCGACTTCGGGAGGGGTGTATTTAT

TAGATAAAAAACCAATGCCCCTCGGGGCTCCTTGGTGATTCATAATAACTTAACGAATC

GCATGGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTGCCCTATCAACTTTCGATGGT

AGGATAGTGGCCTACCATGGTGGCAACGGGTAACGGGGAATTAGGGTTCGATTCCGGA

GAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTAC

CCAATCCCGACACGGGGAGGTAGTGACAATAAATACTGATACGGGGCTCTTTTGGGTC

TCGTAATTGGAATGAGTACAATCTAAACCCCTTAACGAGGAACAATTGGAGGGCAAGT

CTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGT

TAAAAAGCTCGTAGTTGAACCTTGGGTCTGGCTGGCCGGTCCGCCTCACCGCGAGTAC

TGGTCCGGCTGGACCTTTCCTTCTGGGGAAGCCCATGGCCTTCACTGGCTGTGGGGGG

AACCAGGACTTTTACTGTGAAAAAATTAGAGTGTTCAAAGCAGGCCTTTGCTCGGATA

CATTAGCATGGAATAATAGAATAGGACGTGCGGTTCTATTTTGTTGGTTTCTAGGACCG

CCGTAATGATTAATAGGGATAGTCGGGGGCGTCAGTATTCAGCTGTCAGAGGTGAAATT

CTTGGATTTGCTGAAGACTAACTACTGCGAAAGCATTCGCCAAGGATGTTTTCATTAAT

CAGGGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCGTCGTAGTCTTAACCATA

AACTATGCCGACTAGGGATCGGGCGGCGTTTCTATGATGACCCGCTCGGCACCTTACG

AGAAATCAAAGTTTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAG

AAATTGACGGAAGGGCACCACAAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACAC

GGGGAAACTCACCAGGTCCAGACAAAATAAGGATTGACAGATTGAGAGCTCTTTCTTG

ATCTTTTGGATGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATT

GCGATAACGAACGAGACCTCGGCCCTTAAATAGCCCGGTCCGCGTCTGCGGGCCGCTG

GCTTCTTAGGGGGACTATCGGCTCAAGCCGATGGAAGTGCGCGGCAATAACAGGTCTG

TGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGACAGGGCCAGCGAGTAC

ATCACCTTGGCCGAGAGGCCTGGGTAATCTTGTTAAACCCTGTCGTGCTGGGGATAGA

GCATTGCAATTATTGCTCTTCAACGAGGAATGCCTAGTAGGCACGAGTCATCAGCTCGG

CCGAAAGGGGGTCGAGGGAACA

Of particular note, the method of producing formula I of the present invention, formula II compound through organism of fermentation can adopt other any microorganism that can produce ophiobolin (ophiobolin) class sesterterpene ene compound, all can be used as and produces plain bacterium and be used for preparation formula I, formula II compound as long as can produce the microorganism of this compounds.

Embodiment:

The chemical structure of the compound 1,2,3 of indication is respectively in following embodiment: (Arabic numerals in the structural formula are marks of carbon atom in the chemical structure)

Formula I formula II

The R of compound 1 among the formula I ₁R ₂Be methyl, 21 methoxyl group is a beta comfiguration; The R of compound 2 ₁R ₂Be methyl, 21 methoxyl groups are the α configuration.The R of compound 3 among the formula II ₁Be methyl.

The fermentative production and the separation and purification of embodiment 1 compound 1,2,3

1 fermentative production

Produce the fermentation culture of bacterium: by the ordinary method of culturing micro-organisms, it is an amount of to get Aspergillus ustus 094102 (Aspergillus ustus094102), is inoculated on the PDA slant medium, cultivates 4 days in 28 degrees centigrade of incubators.

It is an amount of to get 4 days Aspergillus ustus of slant culture 094102 (Aspergillus ustus 094102), be inoculated into 120mL nutrient solution [substratum composition (grams per liter): N.F,USP MANNITOL 20.0 is housed, maltose 20.0, glucose 10.0, monosodium glutamate 10.0, yeast extract paste 3.0, corn steep liquor 1.0, potassium primary phosphate 0.5, sal epsom 0.3, pH6.5] the 500mL Erlenmeyer flask in, shaking table was cultivated 48 hours under 28 ℃, 120 rev/mins conditions, obtained the seed culture fluid of Aspergillus ustus 094102 (Aspergillus ustus094102).This seed culture fluid is inoculated in respectively by 10% inoculum size is equipped with 300 milliliters and produces nutrient solutions [substratum is formed (grams per liter): N.F,USP MANNITOL 20.0, maltose 20.0, glucose 10.0, monosodium glutamate 10.0, yeast extract paste 3.0, corn steep liquor 1.0, potassium primary phosphate 0.5, sal epsom 0.3, pH6.5] the 1000mL Erlenmeyer flask in, carry out 24 ℃ the production of leaving standstill in 30 days by a definite date and ferment, obtain mycelium and fermented liquid.

The acquisition of 2 medicinal extract

With silk mycelium is separated with fermented liquid.Mycelium with the broken lixiviate of acetone three times, is evaporated to and does not contain acetone, and the gained water layer is with equal-volume ethyl acetate extraction three times, combined ethyl acetate extraction liquid concentrating under reduced pressure, crude extract.After the fermented liquid concentrating under reduced pressure is 1/4th volumes, use ethyl acetate extraction three times, merge the medicinal extract of mycelium and fermented liquid, totally 15.0 grams.

The separation and purification of 3 compounds

Medicinal extract (15.0 gram) is with after the dissolving of chloroform-methanol (9:1) mixed solvent, add 70 gram 200～300 order silica gel Hs (Qingdao Haiyang Chemical Industry Group Corp.'s product) and mix sample, after the removal of solvent under reduced pressure, use silica gel column chromatography, with sherwood oil-acetone is that solvent carries out gradient elution, is divided into 10 stream parts.Fr-6 (1.8 grams, sherwood oil-acetone 9:3 eluate), be eluent through gel filtration chromatography with chloroform-methanol 1:1 again, eluted product is again through reversed phase column chromatography, with the methanol-water is that solvent carries out gradient elution, wherein methanol-water 85:15 eluted product gets formula I, formula II compound 1 (16mg), 2 (14mg), 3 (3mg) through preparation HPLC separation and purification with methanol-water 90:10 wash-out.

Compound 1, white unformed powder, [α] _D ²⁰+ 34.7 ° (c0.1, MeOH), molecular formula C ₂₇H ₄₂O ₄UV λ _MaxNmin MeOH:238; IR v _MaxCm ^-1(KBr): 3400,2966,2864,1728,1643,1275,1088; ¹H reaches ¹³C NMR data see Table 1 and table 2.

Compound 2, white unformed powder, [α] _D ²⁰+ 72.6 ° (c0.1, MeOH), molecular formula C ₂₇H ₄₂O ₄UV λ _MaxNmin MeOH:238; IR v _MaxCm ^-1(KBr): 3420,2958,2813,1710,1655,1290,1088; ¹H reaches ¹³C NMR data see Table 1 and table 2.

Compound 3, white unformed powder, [α] _D ²⁰+ 58.2 ° (c0.1, MeOH), molecular formula C ₂₅H ₃₈O ₂UV λ _MaxNmin MeOH:238; IR v _MaxCm ^-1(KBr): 3435,1670,1659,1376,1230,1154; ¹H reaches ¹³C NMR data see Table 1 and table 2.

Table 1 compound 1～3 ¹H (600MHz in CDCl ₃)

Table 2 compound 1～3 ¹³C NMR data (150MHz in CDCl ₃)

The test of embodiment 2 anti-tumor activities

1 laboratory sample and experimental technique

The preparation of sample solution: specimen is the pure product compound 1～3 of separation and purification in the foregoing description 1.Accurately take by weighing an amount of sample, the solution with DMSO is mixed with desired concn supplies active testing.

The succeeding transfer culture of clone and cell: active testing adopts A549 and HL-60 clone.Various cells are all with the RPMI-1640 substratum that contains 10%FBS, succeeding transfer culture in 37 ℃ of incubators that feed 5% carbonic acid gas.

Cell inhibitory effect activity test method (srb assay and mtt assay)

The present invention adopts srb assay and mtt assay, test evaluation tested sample to cancer cells A549 and HL-60 inhibition of proliferation activity.In the viable cell plastosome desaturase can metabolism reduction xanchromatic bromination 3-(4, the 5-dimethylthiazole)-2,5-phenylbenzene tetrazole is the hepatic water-fast first moon for (formazan), and what of formazan can be measured its optical density by microplate reader and try to achieve.Because the amount of formazan is directly proportional with viable count, so can obtain the number of viable cell according to optical density, medicine suppresses or the ability of killing tumor cell thereby understand.

During active testing, the HL-60 cell in the vegetative period of taking the logarithm, being mixed with density with fresh RPMI-1640 substratum is every milliliter 5 * 10 ⁴The cell suspension of individual cell is inoculated in 96 orifice plates by every hole 200 microlitres, and cultivation is after 24 hours down at 37 ℃, and every hole adds the sample solution of 2 microlitre different concns, continues to cultivate 72 hours.Add 20 μ L then and contain the IPMI-1640 solution (5mg/L) of MTT, cultivated again 4 hours, add 150 μ LDMSO dissolving formazan after shifting out 150 μ L nutrient solutions, in 540nm place its optical density of mensuration.According to IR%=(OD _Blank-OD _Sample)/OD _Blank* 100% formula is calculated the cell proliferation inhibition rate (IR%) under each concentration.

2 experimental results

The cell inhibitory effect activity of compound 1～3:

1～3 couple of A549 of table 3. compound and HL-60 cell inhibiting rate

3 conclusions

1～3 pair of human cancer cell of compound has antitumor action.Therefore, formula I of the present invention, formula II compound can be used as antineoplastic agent (being antitumor drug) and are used for tumor treatment, and the low molecular biosciences probe that also can be used as cell inhibitory effect is used for exploring the Life Science Experiment research of biological phenomena essence.

Claims

1. formula I or formula II compound, its Chinese style I compound is compound 1 and compound 2, wherein R in the compound 1 ₁, R ₂Be methyl, the methoxyl group of 21-position is a beta configuration; R in the compound 2 ₁, R ₂Be methyl, the methoxyl group of 21-position is α-configuration; Formula II compound is compound 3, wherein R ₁Be methyl

Formula I formula II.

2. the preparation method of described formula I of claim 1 or formula II compound, it produces bacterium is Aspergillus ustus 094102 (Aspergillus ustus 094102), deposit number: CCTCC M 208153, preservation date: on October 10th, 2008, preservation place: Chinese Wuhan, Wuhan University, obtain the fermented product that contains above-mentioned formula I, formula II compound by fermentation culture Aspergillus ustus 094102, separation and purification goes out formula I compound 1 and compound 2, formula II compound 3 from fermented product then.

3. the described preparation method of claim 2, wherein with described fermented product through silica gel column chromatography, with sherwood oil-acetone is that solvent carries out gradient elution, 3: 1 eluate of sherwood oil-acetone, be at 1: 1 eluent through gel filtration chromatography with chloroform-methanol again, eluted product is that solvent carries out gradient elution again through reversed phase column chromatography with the methanol-water, 85: 15 eluted product of methanol-water get formula I compound 1 and 2, formula II compound 3 through preparation HPLC separation and purification with 90: 10 wash-outs of methanol-water.

4. described formula I of claim 1 or the formula II compound purposes in preparation inhibition of cell proliferation or tumor cytotoxicity agent.

5. described formula I of claim 1 or formula II compound are preparing the purposes that suppresses in the tumor cell proliferation medicine.

6. described formula I of claim 1 or formula II compound are in the purposes of preparation in the antitumor drug.