CN100404537C - Quinazolin allkaloids compound, prepn. method and use thereof - Google Patents

Quinazolin allkaloids compound, prepn. method and use thereof Download PDF

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CN100404537C
CN100404537C CNB2005100798879A CN200510079887A CN100404537C CN 100404537 C CN100404537 C CN 100404537C CN B2005100798879 A CNB2005100798879 A CN B2005100798879A CN 200510079887 A CN200510079887 A CN 200510079887A CN 100404537 C CN100404537 C CN 100404537C
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formula
preparation
methanol
chloroform
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顾谦群
辛志宏
朱伟明
方玉春
朱天骄
刘红兵
段琳
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Ocean University of China
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Abstract

The present invention relates to a Quinazolin allkaloids compound, a preparation method and use thereof. The present invention uses Penicillium auratiogriseum SP-19 separated from a sea sample to produce the allkaloids compound with a novel structure. An experiment indicates that the allkaloids compound can serve as a cell proliferation inhibitor or an antineoplastic agent.

Description

Quinazolin allkaloids compound and its production and use
Technical field:
The present invention relates to that (deposit number is: the method that CCTCC M205048) prepares quinazolin allkaloids compound with yellow grey mould fungi SP-19 (Penicillium auratiogriseum SP-19); The invention still further relates to the purposes of this compounds in preparation inhibition of cell proliferation or antineoplastic agent.
Background technology:
The research report of relevant quinazolin allkaloids compound has only 3 pieces, this compounds is also to be to separate from the grey mould fungi of Huang (Penicillium auratiogriseum) to obtain Anacine by the Englishman first, but at that time structure fixed not to (Boyes-Korkis, J.M.Gurney, K.A.Penn, J; Et al. Anacine, a new benzodiazepine metabolite of Penicilliumaurantiogriseum produced with other alkaloids in submerged fermentation.J.Nat.Prod.1993,56 (10), 1707-1717); Then revised the structure of Anacine by Larsen etc., (Larsen, T.O; Franzyk, H; Jensen, S.R.UV-guided isolation of verrucines A and B, novel quinazolines fromPenicillium verrucosum structurally related to anacine from Penicillium aurantiogriseum.J.Nat.Prod.1999,62 (11), 1578-1580); Synthesize this compound by people such as Wang at last, thereby further confirmed correct (Wang, the H of its structure; Sim, M.M.Total Solid Phase Syntheses of the Quinazoline Alkaloids:Verrucines A and B and Anacine.J.Nat.Prod.2001,64 (12), 1497-1501); Do not see any report about the activity of this compound.The inventor discovers that (deposit number is: CCTCC M205048) crude extract of liquid fermentation production after ultrasonication has the good cell cycle to suppress active to yellow grey mould fungi SP-19 (Penicillium auratiogriseum SP-19), then its activeconstituents is studied, found that 3 new quinazolin allkaloids compounds have anti-tumor activity, have not yet to see any chemical structure and active report of cell inhibitory effect, so do not see as yet also on the market that relevant therewith medicine is arranged these 3 compounds.
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 the quinazolin allkaloids compound of novel structure first from the grey mould fungi of Huang SP-19 (Penicillium auratiogriseum SP-19) fermented product, shown in I, formula II:
Figure C20051007988700041
Its constitutional features is: the basic framework of formula I, formula II compound is quinazoline, wherein R 1Be hydrogen, alkyl, hydroxyl or acyl group, R 2, R 3Be hydrogen, amino, hydroxyl, alkoxyl group, acyloxy or amido.
Preferred The compounds of this invention is the R of described formula I compound 1 and compound 2 1, R 2Be hydrogen, the R of compound 1 3Be hydroxyl, the R of compound 2 3Be methoxyl group; The R of formula II compound 3 1, R 2Be hydrogen.
In the foregoing invention most preferred be liquid fermentate by the yellow grey mould fungi SP-19 (Penicilliumauratiogriseum SP-19) of marine source through silica gel column chromatography, be that solvent carries out gradient elution with sherwood oil, sherwood oil-chloroform, chloroform-methanol.95: 5 eluate of chloroform-methanol again through Sephadex LH-20 (chloroform-methanol 1: 1), through preparation HPLC, gets formula I, formula II compound with 70: 30 wash-outs of methanol-water again.
The present invention adopts mtt assay to test formula I, the formula II compound anti-tumor activity to P388, HL60, BEL-7402 and A549 cell strain.Experiment confirm, formula I, formula II compound all have inhibited proliferation to these four 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, when using as probe, formula I, formula II compound dissolve in methyl alcohol, water or the aqueous methanol, also dissolve in the aqueous solution of 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.
Yellow grey mould fungi SP-19 (Penicillium auratiogriseum SP-19) strain obtains by separating from coastal waters, Qingdao Mycale plumose [Mycale plumosa (CarTer)], and is accredited as yellow grey mould fungi SP-19 (Penicillium auratiogriseum SP-19) through means of taxonomic research.This bacterial strain has been deposited in Chinese typical culture collection center (deposit number: CCTCC M205048) on May 26th, 2005.This bacterial strain has following microorganism mycology feature:
Bacterium colony on the Cha Shi substratum, grow the appropriateness, 25-28 ℃ 12 days, diameter 37-40 millimeter, quality is velvet-like to particulate state, edge greyish-green, middle part tawny, reverse side pistac.Conidiophore betides matrix mycelia and coremium, falx stem 12-500 * 3.0-4.0 micron, and wall is coarse; Penicillus three is verticillate, and each penicillus has accessory branch 2-3,14-25 * 3.0-4.0 micron, and wall is coarse; Every 4-6 (8) that takes turns is individual for metulae, 8-12 * 2.5-3.0 micron; Conidium is spherical or subsphaeroidal, the 3.0-4.0 micron, and smooth, conidia chain is long column shape.
Of particular note, the method of producing formula I compound of the present invention through organism of fermentation can adopt other any microorganism that can produce quinazolin allkaloids compound, all can be used as and produces plain bacterium and be used for preparation I compound as long as can produce the microorganism of this compounds.
Embodiment:
The chemical structure of the compound of indication is in following embodiment: (Arabic numerals in the structural formula are marks of carbon atom in the chemical structure): formula I, formula II compound, wherein R 1Be hydrogen, alkyl, hydroxyl or acyl group, R 2, R 3Be hydrogen, amino, hydroxyl, alkoxyl group, acyloxy or amido.
Figure C20051007988700051
The R of its Chinese style I compound 1 and compound 2 1, R 2Be hydrogen, the R of compound 1 3Be hydroxyl, the R of compound 2 3Be methoxyl group; The R of formula II compound 3 1, R 2Be hydrogen.
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, (deposit number is: CCTCC M205048) an amount of to get yellow grey mould fungi SP-19 (Penicilliumauratiogriseum SP-19), be inoculated on the PDA slant medium, in 28 degrees centigrade of incubators, cultivated 5 days.
It is an amount of to get 5 days yellow grey mould fungi SP-19 (Penicillium auratiogriseum SP-19) of slant culture, is inoculated into 150 mL nutrient solutions [substratum composition (%): Sorbitol 2%, maltose 2%, glutamine 1%, KH are housed 2PO 40.05%, MgSO 47H 2O 0.03%, tryptophan 0.05%, Yeast ext.0.3%, pH 6.5] the 500mL Erlenmeyer flask in, shaking table was cultivated 48 hours under 28 ℃, 120 rev/mins conditions, obtained seed culture fluid.Be inoculated in this seed culture fluid in the 500mL Erlenmeyer flask that 200 milliliters of production nutrient solutions (substratum is formed the same) are housed respectively by 5% inoculum size, be loaded on 28 ℃, the 120 rev/mins shaking tables, carry out 9 days by a definite date production fermentation, obtain mycelium and fermented liquid.
The acquisition of 2 medicinal extract
With cotton mycelium is separated with fermented liquid.Mycelium with acetone extraction 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 20 grams.
The separation and purification of 3 compounds
Behind medicinal extract (20 gram) dissolve with methanol, add 60 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, sherwood oil-chloroform, chloroform-methanol is that solvent carries out gradient elution, is divided into 10 stream parts.Component 4 (1.5 grams, 95: 5 eluates of chloroform-methanol) through Sephadex LH-20 (chloroform-methanol 1: 1), again through HPLC, gets compound 1 (22mg), 2 (16mg) and 3 (9mg) with 70: 30 wash-outs of methanol-water.
Compound 1 white unformed powder, [α] D 17+ 172 ° of (c1.8, CH 3OH); HRESIMS:397.1267[M+K] +, UV λ Max(CH 3OH) nm (log ε) 280 (4.60), 311 (4.28); IRv (KBr, cm -1): 3217,2958,1632,1606,1471,1388,1162,773,674; 1H reaches 13C NMR data see Table 1.
Figure C20051007988700061
Figure C20051007988700071
Compound 2 white unformed powder, [α] D 17+ 718 ° of (c13.9, CHCl 3); HRESIMS:373.1862[M+H] +, UV λ Max(CH 3OH) nm (log ε) 282 (3.90), 311 (3.71); IRv (KBr, cm -1): 3196,2958,1633,1605,1471,1387,1321,1155,1043,771,673; 1H reaches 13C NMR data see Table 2.
Compound 3 white unformed powder, white unformed powder, [α] D 17+ 44 ° of (c1.6, CHCl 3); HRESIMS:341.1617[M+H] +, UV λ Max(CH 3OH) nm (log ε) 314 (4.48); IRv (KBr, cm -1): 3197,2959,2919,2859,1634,1607,1582,1561,1460,1392,1321,764,665; 1H reaches 13C NMR data see Table 2.
Figure C20051007988700081
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 1,2,3 of separation and purification in the foregoing description 1.Accurately take by weighing an amount of sample, be mixed with the solution of desired concn, for active testing with methyl alcohol.
The succeeding transfer culture of clone and cell: active testing adopts P388, HL60, BEL-7402 and A549 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 (mtt assay)
The present invention adopts mtt assay, test evaluation the inhibition activity of tested sample to cancer cell multiplication.In the viable cell plastosome desaturase can metabolism reduction xanchromatic bromination 3-(4, the 5-dimethylthiazole)-2,5-phenylbenzene tetrazole is hepatic water-fast formazan, 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 P388 in the vegetative period of taking the logarithm, HL60, BEL-7402 and A549 cell, 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 L DMSO 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 1,2,3 is shown in table 4,5,6 respectively:
Figure C20051007988700091
Figure C20051007988700092
Figure C20051007988700093
3 conclusions
1,2,3 pairs of compounds comprise that the cancer cells in people's Mammals source 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 (8)

1. formula I compound, wherein R 1, R 2Be hydrogen, R 3Be hydroxyl or methoxyl group; Or formula II compound, wherein R 1, R 2Be hydrogen.
Figure C2005100798870002C1
Formula I formula II
2. the preparation method of described formula I of claim 1 or formula II compound, it is characterized in that the yellow grey mould fungi Penicillium auratiogriseum SP-19 of fermentation culture, obtain the fermented product that contains above-mentioned formula I, II compound, separation and purification goes out formula I and formula II compound 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, sherwood oil-chloroform, chloroform-methanol is that solvent carries out gradient elution, 95: 5 eluate of chloroform-methanol, through Sephadex LH-20 column chromatography, 1: 1 eluted product of chloroform-methanol obtains formula I compound and formula II compound again through preparation HPLC separation and purification respectively with 70: 30 wash-outs of methanol-water again.
4. the preparation method of the described formula I of claim 3, II compound, the production bacterium of wherein said quinazolin allkaloids compound is yellow grey mould fungi Penicillium auratiogriseum SP-19, CCTCCM205048.
5. described formula I of claim 1 or the II compound purposes in preparation inhibition of cell proliferation or tumor cytotoxicity agent.
6. described formula I of claim 1 or II compound are in the purposes of preparation in the antitumor drug.
7. yellow grey mould fungi Penicillium auratiogriseum SP-19, its preserving number is: CCTCCM205048.
8. the described bacterial strain of claim 7 is used for the purposes of described formula I of production claim 1 or II compound.
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CN101947225B (en) * 2010-09-29 2012-10-10 厦门大学 Anticancer application of alkaloid
CN102276613B (en) * 2011-06-01 2013-10-23 中国海洋大学 Sesquiterpene alkaloid compound as well as preparation method and application thereof
CN109810055A (en) * 2019-01-17 2019-05-28 广东轻工职业技术学院 A kind of new bio alkali cpd in marine fungi source and its preparing the application in anti-lung-cancer medicament

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
Anacine, a new benzodiazepine metabolite ofPenicilliumAurantiogriseum produced with other alkaloids insubmergedfermentation.. Boyes-Korkis ET AL.J. Nat. Prod,Vol.56 No.10. 1993
Anacine, a new benzodiazepine metabolite ofPenicilliumAurantiogriseum produced with other alkaloids insubmergedfermentation.. Boyes-Korkis ET AL.J. Nat. Prod,Vol.56 No.10. 1993 *
Total Solid Phase Syntheses of the QuinazolineAlkaloids:Verrucines A and B and Anacine.. Wang, Haishan, Sim, Mui Mui.Journal of Natural Products,Vol.64 No.12. 2001
Total Solid Phase Syntheses of the QuinazolineAlkaloids:Verrucines A and B and Anacine.. Wang, Haishan, Sim, Mui Mui.Journal of Natural Products,Vol.64 No.12. 2001 *
UV-guided isolation of verrucines A and B, novel quinazolinesfrom Penicillium verrucosum structurally related to anacinefrom Penicillium aurantiogriseum.. Larsen, Thomas Ostenfeld, Franzyk, Henrik, Jensen, SorenRosendal.Journal of Natural Products,Vol.62 No.11. 1999
UV-guided isolation of verrucines A and B, novel quinazolinesfrom Penicillium verrucosum structurally related to anacinefrom Penicillium aurantiogriseum.. Larsen, Thomas Ostenfeld, Franzyk, Henrik, Jensen, SorenRosendal.Journal of Natural Products,Vol.62 No.11. 1999 *
文章题目Quinocitrinines A and B, new quinoline alkaloids fromPenicillium citrinum thom 1910, a permafrost fungus.. Kozlovsky, A. G. ET AL.Journal of Antibiotics,Vol.56 No.5. 2003
文章题目Quinocitrinines A and B, new quinoline alkaloids fromPenicillium citrinum thom 1910, a permafrost fungus.. Kozlovsky, A. G. ET AL.Journal of Antibiotics,Vol.56 No.5. 2003 *

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