CN100410226C - Quinone compounds, and their preparing method and antitumour use - Google Patents

Quinone compounds, and their preparing method and antitumour use Download PDF

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CN100410226C
CN100410226C CNB200610035637XA CN200610035637A CN100410226C CN 100410226 C CN100410226 C CN 100410226C CN B200610035637X A CNB200610035637X A CN B200610035637XA CN 200610035637 A CN200610035637 A CN 200610035637A CN 100410226 C CN100410226 C CN 100410226C
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ethyl acetate
petroleum ether
quinones
nutrient solution
column chromatography
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CN1850765A (en
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佘志刚
林永成
夏雪奎
符立梧
梁永钜
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Sun Yat Sen University
National Sun Yat Sen University
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Abstract

The present invention relates to the field of compounds in quinone class, particularly to compounds in the quinone class having an anti-tumor activity and a preparation method and an application thereof. In the present invention, the red compound in the quinone class and an acetylation product thereof obtained by the extraction separation from a fermentation culture object of a marine fungus Halorosellinia sp. 1403 in South China Sea can effectively inhibit the growth of tumor cell strains. The present invention can be used for developing anti-tumor drugs and have a wide application prospect.

Description

Quinone compounds and preparation method thereof and antitumor application
Technical field
The present invention relates to the quinones field, be specifically related to quinones and preparation method thereof and application that a class has anti-tumor activity.
Background technology
Marine microorganism produces the active metabolite of novel structure with its unique pathways metabolism, and its meta-bolites becomes the abundant source of medicine, the meta-bolites of marine microorganism is pharmaceutical use widely also, as antitumor, treatment cardiovascular disorder, immunomodulator etc.Because the singularity of ocean environment, marine microorganism is of a great variety, wide material sources, screening pick-up rate height, according to the report of John professor, found that marine microorganism was one of main source in the marine natural product new compound in 2003 at 2005 " Natural Product Reports ".
Plant endogenesis epiphyte (Endophytic fungi) is lived in the tissue of higher plant, it is the natural moiety in the plant microecology system, set up harmonious symbiotic relationship with the host during evolution, endogenetic fungus is of a great variety at a conservative estimate, and nearly 1.5 * 10 6Kind because quantity is huge, and and other biological between ecological relationship closely, make this class fungi become potential and have the source that produces abundant secondary metabolite.
Mangrove forest is the plant that is grown in the torrid zone and tropical coastal tideland slob, and mangrove forest ecological system is one of the richest diversity, marine ecosystem that productivity is the highest in the world.Outer saline waters nutritive deficiency, but the mangrove swamp district is typical salty slightly waters band, compile the various inorganic salt and the organism that bring from upstream and ocean and add dropping of the dry branches and fallen leaves of mangrove plant body own, competent vegetable material is arranged, abundant planktonic algae and planktonic organism are arranged, and these conditions just are being well-suited for the breeding of microorganism (bacterium and fungi).In more than ten years in the past, the mangrove forest habitat is proved to be the abundant source of the new kind of fungi, and this has formed second largest ecological subclass of thalassiomycetes.
Summary of the invention
The object of the present invention is to provide a class to derive from the quinones with anti-tumor activity and the acetyl derivatives thereof of ocean mangrove fungi.
Another object of the present invention provides the preparation method of above-mentioned quinones.
Further purpose of the present invention provides the application of above-mentioned quinones in the preparation antitumor drug.
The present invention's extraction separation from the fermenting culture of South Sea thalassiomycetes Halorosellinia sp.1403 (hereinafter to be referred as fungi 1403) obtains a kind of quinones (1403C) of redness, and its structural formula is suc as formula (I): R wherein 1, R 2, R 3, R 4, R 5All be H.
Figure C20061003563700051
Quinones 1403C under the effect of catalyzer, obtains a series of ethanoyl through aceticanhydride/pyridine acetylize and replaces the acetyl quinones that replaces to penta-acetyl, and its structural formula is suc as formula (I), wherein R 1, R 2, R 3, R 4, R 5Be H or CH 3CO, R 1, R 2, R 3, R 4, R 5Be not H simultaneously.
The used fungi 1403 of the present invention has been preserved in Chinese typical culture collection center (CCTCC, Chinese Wuhan University are in the school), and preserving number is CCTCC NO:M201018, and preservation day is April 23 calendar year 2001.
Quinones 1403C of the present invention can obtain by extraction separation from the fermentation culture of fungi 1403, and preparation method's concrete steps are as follows:
(1) seed culture of fungi Halorosellinia sp.1403CCTCC NO:M 201018: substratum is by weight: glucose 0.5-1.5, yeast extract 0.05-0.15, peptone 0.1-0.3, agar 1-1.5, sodium-chlor 3-5, water 100 is made the test tube slant, the picking bacterial strain inserts the inclined-plane, cultivates 5-7 days for 30-35 ℃;
(2) fermentation culture of fungi Halorosellinia sp.1403CCTCC NO:M 201018: fermention medium is by weight: glucose 5-15, yeast extract 1-4, peptone 0.5-4, sodium-chlor 3-5, water 100, cultured bacterial strain in the inclined-plane is chosen into fermention medium, left standstill 1-2 month in room temperature 25-35 ℃;
(3) above-mentioned cultured filtering fermentation liquor is removed thalline and obtain nutrient solution;
(4) heating of fungi 1403 nutrient solutions concentrates (temperature is no more than 50 ℃) 1/20-1/5 to the stoste volume, with ethyl acetate extraction repeatedly, concentrating acetic acid ethyl acetate extract, carry out chromatographic separation in silicagel column, is the eluent gradient elution with petroleum ether-ethyl acetate-methyl alcohol;
(5) nutrient solution medicinal extract is collected the ethyl acetate/petroleum ether elutriant of 50%-100% through behind the column chromatography, and 70% ethyl acetate/petroleum ether is an elutriant, separates with polyamide column chromatography again, and recrystallization purifying promptly obtains red granules shape crystal 1403C.
The acetylize quinones is to obtain acetylate by 1403C through aceticanhydride/pyridine acetylize, and preparation method's concrete steps are as follows:
Get dried 1403C, under the effect of catalyzer, add exsiccant pyridine, add acetic anhydride again, under 10～80 ℃, stir 30min～15h, add water, chloroform extraction; Through silica gel column chromatography, petroleum ether-ethyl acetate is the eluent gradient elution, collects 5%-80% ethyl acetate/petroleum ether elutriant, recrystallization purifying, and a series of ethanoyl that obtain 1403C replace the acetylize quinones that replaces to penta-acetyl.
20～50% ethyl acetate/petroleum ether elutriants, the chloroform-methanol recrystallization obtains the product 1403C-3Ac (R of orange-yellow crystal triacetylization 1, R 2, R 4CH 3CO, R 3, R 5Be H).
Compared with prior art, the present invention has following beneficial effect: the present invention is through evidence, and quinones 1403-C and acetylate thereof all can effectively suppress the growth of tumor cell line, can be used for developing anti-tumor medicaments, have a extensive future.
Embodiment
The preparation of embodiment 1 compound 1403-C and acetylate thereof
(1) seed culture of fungi Halorosellinia sp.1403:
Substratum is by weight: glucose 0.5-1.5, and yeast extract 0.05-0.15, peptone 0.1-0.3, agar 1-1.5, sodium-chlor 3-5, water 100 is made the test tube slant, and the picking bacterial strain inserts the inclined-plane, cultivates 5-7 days for 30-35 ℃;
(2) fermentation culture of fungi Halorosellinia sp.1403:
Fermention medium is by weight: glucose 5-15, and yeast extract 1-4, peptone 0.5-4, sodium-chlor 3-5, water 100 chooses cultured bacterial strain in the inclined-plane into fermention medium, leaves standstill 1-2 month in room temperature 25-35 ℃;
(3) above-mentioned cultured filtering fermentation liquor is removed thalline and obtain nutrient solution;
(4) be no more than under 70 ℃ of conditions in temperature, nutrient solution is heated the 1/20-1/5 that is concentrated into the stoste volume, with ethyl acetate extraction repeatedly, concentrate acetic acid ethyl acetate extract, carrying out chromatographic separation in silicagel column, is the eluent gradient elution with petroleum ether-ethyl acetate-methyl alcohol.
(5) nutrient solution medicinal extract is collected the ethyl acetate/petroleum ether elutriant of 50%-100% through behind the column chromatography, and 70% ethyl acetate/petroleum ether is an elutriant, separates with polyamide column chromatography again, and recrystallization purifying promptly obtains red granules shape crystal 1403C.
Acetylate 1403C-3Ac is by 1403C, and at catalyzer, through the triacetyl product that aceticanhydride/pyridine acetylize obtains, preparation method's concrete steps are as follows:
Get exsiccant 1403C, add exsiccant pyridine and acetic anhydride, a spot of DMAP under the room temperature, stirs 3-6h, adds water, chloroform extraction.Through silica gel column chromatography, petroleum ether-ethyl acetate is the eluent gradient elution, collects 20～50% ethyl acetate/petroleum ether elutriants, and the chloroform-methanol recrystallization obtains orange-yellow crystal 1403-C-Ac-3.
The testing data of 1403C:
FABMS：337(M+1)，m/z：55.EA(w/％，)：C 56.56，H 4.617。Calculate straight (C 16H 16O 8): C 57.14, and H 4.76,232 ℃ of .IR v/cm of mp -1(KBr): 3514,3479,3374,3029,2987,2938,2896,1595,1475,1440,1398,1370,1300,1200,1208,1138,1082,997,948,864,821,632,554,491. 1HNMR (500Mz, CDCl 3, TMS): 13.35 (s, OH-9), 12.62 (s, OH-10), 6.46 (s, H-3), 4.77 (t, 4.5,4.5Hz, H-7, OH), 3.92 (s, CH 3-16), 3.54 (t, 4.5,4.5Hz, H-5), 2.74 (d, 18Hz, H-8a), 2.67 (d, 18Hz, H-8b), 1.24 (s, CH 3-15). 13CNMR (CDCl 3): 183.3 (C-4), 176.5 (C-1), 160.9 (C-10), 160.9 (C-2), 160.2 (C-9), 139.5 (C-11), 136.8 (C-12), 109.8 (C-14), 109.6 (C-3), 107.3 (C-13), 76.3 (C-7), 69.2 (C-6), 68.2 (C-5), 56.8 (C-16), 34.8 (C-8), 25.5 (C-15).
The testing data of 1403C-3Ac:
EI-MS：462，mp 259～260℃ 1HNMR(500Mz，CDCl 3，TMS)：6.026(H-3)，5.287(dd，)，3.98(d)，3.07(dd，15，15HZ)，2.9(dd，15，15HZ)，1.361(H-15)，2.12(CH3-22)，2.37(CH 3-21)，2.12(CH 3-20)，12.65(OH-9)，6.19(OH-7)； 13CNMR(CDCl 3)：184.8(C-4)，182.7(C-1)，170.5(C-18)，170.4(C-19)，169.9(C-17)，158.9(C-9)，158.9(C-2)，132.4(C-11)，132.1(C-12)，113.0(C-13)，112.0(C-14)，111.6(C-3)，73.6(C-6)，70.5(C-7)，69.4(C-8)，35.2(C-5)，29.7(C-15)，21.1(C-22)，20.8(C-21)，20.8(C-20).
Embodiment 2 MTT reduction method detection compound 1403C and 1403C-3Ac anti-tumor activities
Test
1, material:
1.1 four Cuo salt (MTT): (3-(4 for phosphate buffered saline buffer (PBS) the dissolving MTT of usefulness 0.01mol/L, 5-dimethythiazol-z-yl) 2,5-diphenyltetrazolium bromide, SIGMA) final concentration 5mg/mL, filtration sterilization, 4 ℃ keep in Dark Place after the packing.
1.2 the preparation of target cell (is example with the HepG2 cell): with the recovery and the cultivation of HepG2 cell:
A. the frozen pipe of taking-up human hepatoma cell strain HepG2 cell from liquid nitrogen container is inserted rapidly in 37 ℃ of water-baths, does not stop to shake to make it to dissolve rapidly, and aseptic technique moves in the centrifuge tube;
B. add full nutrient solution to 10mL, the centrifugal 5s of 1000rmp abandons supernatant;
C. repeat above operation once;
D. move in the culturing bottle 5%CO after making the cell mixing with full nutrient solution piping and druming 2, 37 ℃ of cultivations;
E. the observation of cell growing state is in time changed nutrient solution, divides bottle.
1.3 cell counting:
A. choose the logarithmic phase cell, trysinization, the full cultivation stops, and moves in the centrifuge tube, adds full the training to 10mL;
B. get and one after another drop ofly go in the tally one side groove, the total cellular score of microscopically counting four big lattice, divided by 4 takes advantage of 10 4, be every milliliter of contained cell count of nutrient solution;
C. adjust cell count to 1 * 10 5/ mL;
1.4 the preparation of compd E and F: get compd E and F respectively and join in the full training, adjusting concentration is 500 μ g/mL, ultrasonic emulsification, filtration sterilization, 4 ℃ of preservations.
2. test method (is example with the HepG2 cell)
A.96 each hole of orifice plate adds HepG2 cell 100 μ L (1 * 10 5/ mL), 5%CO 2, cultivate 4hr for 37 ℃.
B. add different concns study subject 100 μ L, contrast adds full training 100 μ L, continues to cultivate 48hr.
C. add each 10 μ L of MTT (5mg/mL), continue to cultivate 4hr.
D. remove nutrient solution.Every hole adds DMSO 100 μ L, and the 5-10min that vibrates gently makes the particle dissolving.
E. enzyme linked immunological instrument 570nm measures every hole OD value down.
F. calculate inhibiting rate
The average OD value that the average OD value that tumor cell destruction %=[(control group is measured-dosing group is measured)/the average OD value of control group mensuration] * 100%
G. with the logarithm mapping of inhibiting rate, try to achieve IC to drug level 50Value
With lg c is X-coordinate, and inhibiting rate is an ordinate zou, tries to achieve IC 50Value
Lung cancer cell line A549, hepatoma cell strain Hep-2, cancer cell of oral cavity strain KB, breast cancer cell strain MCF-7, the test method of the anti-medicine breast cancer cell of multiple medicines strain MCF-7/Adr is the same.
3. test-results
Test-results shows that compound 1403C and 1403C-3Ac all can effectively suppress lung cancer cell line A549, hepatoma cell strain Hep-2 and hepatoma cell strain Hep G2, cancer cell of oral cavity strain KB, breast cancer cell strain MCF-7, the growth of the anti-medicine breast cancer cell of multiple medicines strain MCF-7/Adr.1403C and 1403C-3Ac are to the IC of these JEG-3 50Value (μ g/ml) sees Table 1.
The cell in vitro poison test-results (IC of table 1,1403-C and 1403-C-Ac-3 50μ g/mL)
Cell strain 1403C 1403C-3Ac
Lung cancer cell line A549 2.44 0.69
Hepatoma cell strain Hep-2 3.15 1.00
Hepatoma cell strain Hep G2 4.41 0.85
Cancer cell of oral cavity strain KB 3.15 0.92
Breast cancer cell strain MCF-7 4.76 3.02
The anti-medicine breast cancer cell of multiple medicines strain MCF-7/Adr 5.45 2.97

Claims (3)

1. quinones, its structural formula is suc as formula (I)
Figure C2006100356370002C1
Wherein, R 1, R 2, R 4Be CH 3CO, R 3, R 5Be H.
2. the preparation method of the described quinones of claim 1 is characterized in that may further comprise the steps:
(1) seed culture of fungi Halorosellinia sp.1403: substratum is by weight: glucose 0.5-1.5, yeast extract 0.05-0.15, peptone 0.1-0.3, agar 1-1.5, sodium-chlor 3-5, water 100; Make the test tube slant, the picking bacterial strain inserts the inclined-plane, cultivates 5-7 days for 30-35 ℃;
(2) fermentation culture of fungi Halorosellinia sp.1403: fermention medium is by weight: glucose 5-15, yeast extract 1-4, peptone 0.5-4, sodium-chlor 3-5, water 100; Cultured bacterial strain in the inclined-plane is chosen into fermention medium, left standstill 1-2 month in room temperature 25-35 ℃;
(3) above-mentioned cultured filtering fermentation liquor is removed thalline and obtain nutrient solution;
(4) be no more than under 50 ℃ of conditions in temperature, nutrient solution is heated the 1/20-1/5 that is concentrated into the stoste volume, with ethyl acetate extraction repeatedly, concentrate acetic acid ethyl acetate extract, carrying out chromatographic separation in silicagel column, is the eluent gradient elution with petroleum ether-ethyl acetate-methyl alcohol;
(5) nutrient solution medicinal extract is collected the ethyl acetate/petroleum ether elutriant of 50%-100% through behind the column chromatography, and 70% ethyl acetate/petroleum ether is an elutriant, separates with polyamide column chromatography again, and recrystallization purifying promptly obtains red granules shape crystal 1403C; 1403C is meant that structural formula is suc as formula (I), R 1, R 2, R 3, R 4, R 5It all is the quinones of H;
(6) get dried 1403C, under the effect of catalyzer, add exsiccant pyridine, add acetic anhydride again, under 10～80 ℃, stir 30min～12h, add water, chloroform extraction; Through silica gel column chromatography, petroleum ether-ethyl acetate is the eluent gradient elution, collects 5%-80% ethyl acetate/petroleum ether elutriant, and recrystallization purifying obtains the acetylate of 1403C.
3. the application of the described quinones of claim 1 in the preparation antitumor drug.
CNB200610035637XA 2006-05-26 2006-05-26 Quinone compounds, and their preparing method and antitumour use Expired - Fee Related CN100410226C (en)

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CN102532092A (en) * 2012-01-16 2012-07-04 中山大学 Natural product Bostrycin derivative I as well as preparation method and application thereof
CN102558143B (en) * 2012-01-16 2014-04-23 中山大学 Derivant III of natural product Bostrycin and preparation method and purpose thereof
CN103304396B (en) * 2012-03-13 2015-06-24 上海医药工业研究院 Method for separating and purifying 1403C from fermentation liquid of 1403C

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CN1347865A (en) * 2001-07-12 2002-05-08 中山大学 Antineoplastic compound and its prepn and pharmaceutical use

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CN1347865A (en) * 2001-07-12 2002-05-08 中山大学 Antineoplastic compound and its prepn and pharmaceutical use

Non-Patent Citations (4)

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Title
Recent progress in bioactive metabolites of marinemicroorganisms. Liu，Xiao-hong等人.中国抗生素杂志，第vol.29卷第no.8期. 2004
Recent progress in bioactive metabolites of marinemicroorganisms. Liu，Xiao-hong等人.中国抗生素杂志，第vol.29卷第no.8期. 2004 *
中国南海红树内生真菌No.1403次级代谢物的研究. 姜广策等人.中山大学学报（自然科学版），第vol.39卷第no.6期. 2000
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