CN100360529C - Sesquiterpene subergorgia suberosa ketone and its preparing process and application - Google Patents
Sesquiterpene subergorgia suberosa ketone and its preparing process and application Download PDFInfo
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- CN100360529C CN100360529C CNB200510033704XA CN200510033704A CN100360529C CN 100360529 C CN100360529 C CN 100360529C CN B200510033704X A CNB200510033704X A CN B200510033704XA CN 200510033704 A CN200510033704 A CN 200510033704A CN 100360529 C CN100360529 C CN 100360529C
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Abstract
The present invention relates to a new formula (1) sesquiterpene subergorgia suberosa ketone which is obtain by the separation of subergorgia suberosa at south China sea. The present invention also relates to a preparing process of the compound and the application of the compound in treating a breast cancer, leukemia, an oral cancer, a liver cancer and a lung cancer. An experiment verifies that the compound of the present invention has an obvious inhibiting function on the growth of the cancer cells.
Description
Technical field
The present invention relates to a kind of new sesquiterpene subergorgia suberosa ketone, also relate to its preparation method and its application aspect the anticancer growth.
Background technology
Be rich in sesquiterpene among the subergorgia suberosa Subergorgia suberosa, up to the present domestic and international according to the literature researcher is separated to 12 sesquiterpenes and 5 sterols from the subergorgia suberosa in different waters, the world, and carried out a series of Cytotoxic screenings, find that wherein the part sesquiterpene shows the potential cytotoxicity to Lu-csf-1 P-388, A549, HT-29 cell strain and cervical cancer HeLa cell strain.
Summary of the invention
The objective of the invention is to from subergorgia suberosa, to isolate one and new cancer cells is had strong inhibiting sesquiterpene, another purpose provides the preparation method of this sesquiterpene, and further purpose provides the application of this sesquiterpene in the preparation cancer therapy drug.
The present invention is by multiple normal pressure column chromatography and one dimension, two dimensional NMR wave spectrum, from the subergorgia suberosa of the South Sea, separate and obtain a new sesquiterpene subergorgia suberosa ketone, this sesquiterpene all has in various degree restraining effect to human breast cancer cell strain, lung cancer and hepatoma cell strain etc., thereby has realized purpose of the present invention.
Sesquiterpene subergorgia suberosa ketone of the present invention is represented by formula (1):
Structure is identified:
The compound of general formula (1), its ESIMS spectrum is at m/z 369[M-H]
-The place shows quasi-molecular ion peak, in conjunction with high resolution HRESIMS and
13C NMR (DEPT) spectrum, releasing its molecular formula is C
20H
26N
4O
3Its
13CNMR (DEPT) spectrum shows 20 carbon signals, comprises 15 basic framework carbon [3 methyl (δ
c16.7,27.0,34.4), 5 methylene radical (δ
c40.8,41.3,27.0,27.9,48.5), 4 methyne (δ
c44.0,52.4,36.6,49.7), 2 quaternary carbon (δ
c56.7,39.7) and a ketone group (δ
c216.5, s)] and 5 low carbon [δ
c108.1 (s), 140.6 (d), 150.2 (s), 151.9 (s), 155.8 (s)].
1H NMR spectrum shows 3 methyl [δ
H0.80 (3H, d, J=7.0Hz), 1.14 (3H, s), 1.16 (3H, s)] and an alkene hydrogen [δ
H7.91 (1H, s)].The compound suberosenone and the suberosanone (reference: 1.Bokesch H R of these NMR data and reference report, McKee T C, Cardellina J H, BoydM R.Suberosenone, a new cytotoxin from Subergorgia suberosa.Tetrahedron Letters, 1996,37:3259-3262; 2.Sheu J H, Hung K C, Wang G H, Duh C Y.New cytotoxicsesquiterpenes from the gorgonian Isis hippuris.J Nat Prod, 2000,63:1603-1607.) very similar, just have more 5 low carbon.Above data declaration sesquiterpene subergorgia suberosa ketone of the present invention is a sesquiterpene that is similar to suberosenone, is connected with one five carbon aromatic nitrogen-contg heterogeneous ring compound simultaneously as side chain.
According to its HMBC spectrum, degree of unsaturation, molecular weight and with document (Yu Dequan, Yang Junshan. analytical chemistry handbook: the 7th fascicle. Beijing: Chemical Industry Press, 1999.) in NMR data contrasts, infer this five carbon aromatic nitrogen-contg heterogeneous ring compound should be 6 ', 8 '-xanthine.In the HMBC spectrum, δ
H4.46 (1H, dd, J=4.6,14.3Hz, H-6a), 4.25 (1H, dd, J=9.0,14.1Hz, H-6b) and δ
c(150.2 s, C-4 '), 151.9 (s, C-8 ') are relevant, this explanation 6 ', 8 '-xanthine is by the C-6[δ on C-N key and the suberosenone part
c41.3 (t)] link to each other.In NOESY spectrum, H-5 is relevant with H-2 and Me-7, but H-11 discord H-12 is correlated with, and this shows H-2, and Me-7 and H-11 be in the same one side of six membered ring, and is the a-configuration, because the C-12 methylene radical is on the beta-position of C-1.In sum, the structure of sesquiterpene subergorgia suberosa ketone of the present invention is represented by formula (1).
The preparation method of sesquiterpene subergorgia suberosa ketone of the present invention, it is characterized in that the subergorgia suberosa organic solvent extraction, described organic solvent can be a methyl alcohol, ethanol, methylene dichloride, methyl alcohol-methylene dichloride or ethanol-methylene dichloride etc., the extracting solution concentrating under reduced pressure is got crude extract, crude extract is outstanding water-soluble, with chloroform or ethyl acetate extraction, concentrating under reduced pressure gets chloroform layer or ethyl acetate layer, chloroform layer or ethyl acetate layer are carried out the normal pressure silica gel column chromatography, with the mixing system of two kinds of organic solvents chloroform-acetone for example, chloroform-ethyl acetate, sherwood oil-acetone or petroleum ether-ethyl acetate equal solvent system are that eluent carries out gradient elution, and thin-layer chromatography TLC follows the trail of and merges component; To on TLC, can wash with chloroform-methanol or chloroform-acetone solvent system gradient with 9: 1 chloroform-methanol solvent systems unfolded component of volume ratio, get the crude product of formula (1) compound, this purifying crude is got pure formula (1) compound, purification process adopts usual method, as recrystallization, mistake post etc.This compound has uv-absorbing under ultraviolet lamp 254nm wavelength, be 9: 1 solvent systems R when launching with the chloroform-methanol volume ratio
fValue is about 0.65.
The application of sesquiterpene subergorgia suberosa ketone of the present invention in the cancer therapy drug of preparation treatment mammary cancer, leukemia, oral carcinoma, liver cancer and lung cancer.
By the external activity quantitative screening, find that sesquiterpene subergorgia suberosa ketone of the present invention has strong restraining effect to the growth of human breast cancer cell strain MDA-MB-231, its half inhibiting rate IC50 is 8.87 μ g/mL, by the qualitative screening of external activity, find that sesquiterpene subergorgia suberosa ketone also has the obvious suppression effect to human breast cancer cell strain MCF-7 under 100 μ mol/L and 50 μ mol/L concentration; In addition, sesquiterpene subergorgia suberosa ketone is also distinguished the potential restraining effect of showed different to mouse lymphocyte leukemia L1210, human oral cancer KB clone, lung cancer and hepatoma cell strain etc.
Description of drawings
Fig. 1 sesquiterpene subergorgia suberosa ketone is to the restraining effect of human breast cancer cell strain MDA-mB-231, and wherein Qi4 represents subergorgia suberosa ketone.
Embodiment
Following embodiment further specifies of the present invention, but the invention is not restricted to following embodiment.
Embodiment 1: the preparation of sesquiterpene subergorgia suberosa ketone
Subergorgia suberosa with weight in wet base 3Kg is a raw material, extract 3 times with the mixed organic solvents (about 20 liters) of ethanol and methylene dichloride (volume ratio 2: 1) lyophilize chopping back, the extracting solution concentrating under reduced pressure is got crude extract, crude extract is outstanding water-soluble, use chloroform extraction successively 3 times (each 600mL), concentrating under reduced pressure gets chloroform layer 50 grams.Mixed solvent with chloroform and methyl alcohol is mixed silica gel with the chloroform layer dissolving, mix about 900 gram silica gel with about 2500 milliliters of chloroforms and carry out wet method dress post, with chloroform-acetone (the 2000mL volume ratio changed from 100: 0 to 0: 100) solvent systems gradient elution, thin-layer chromatography TLC (GF
254) follow the trail of merge 8 components.Will be at TLC (GF
254) go up and can wash with chloroform-methanol (the 800mL volume ratio changed from 12: 1 to 10: 2) solvent systems gradient with 9: 1 solvent systems unfolded of chloroform-methanol volume ratio component, get the crude product of formula (1) compound, with the mixed solvent recrystallization of this crude product with chloroform-methanol, get pure formula (1) compound, this compound has uv-absorbing under ultraviolet lamp 254nm wavelength, R when launching with 9: 1 solvent systems of chloroform-methanol volume ratio
fValue is about 0.65.Its spectroscopic data is as follows:
1H NMR(500MHz，CDCl
3)δ
H：2.40(1H，m，H-2)，2.50(2H，m，H-3)，3.32(1H，dd，J＝4.4，8.3Hz，H-5)，4.46(1H，dd，J＝4.6Hz，14.3，H-6a)，4.25(1H，dd，J＝9.0，14.1Hz，H-6b)，0.80(3H，d，J＝7.0Hz，H-7)，1.54(1H，m，H-8)，1.87，1.19(each1H，m，H-9)，1.59(2H，m，H-10)，1.77(1H，m，H-11)，1.69，1.65(each 1H，d，J＝14.7Hz，H-12)，1.14(3H，s，Me-14)，1.16(3H，s，Me-15)，7.91(1H，s，H-2’).
13C NMR(125MHz，CDCl
3)δ
c：56.7(s，C-1)，44.0(d，C-2)，40.8(t，C-3)，216.5(s，C-4)，52.4(d，C-5)，41.3 (t，C-6)，16.7(q，C-7)，36.6(d，C-8)，27.0(t，C-9)，27.9(t，C-10)，49.7(d，C-11)，48.5(t，C-12)，39.7(s，C-13)，27.0(q，C-14)，34.4(q，C-15)，140.6(d，C-2’)，150.2(s，C-4’)，108.1(s，C-5’)，155.8(s，C-6’)，151.9(s，C-8’).Negative-ion ESIMS m/z：369[M-1]
-.
Embodiment 2: the qualitative screening experiment of external activity
The two male human breast cancer cell strain MCF-7 of the human breast cancer cell strain MDA-mB-231 of ER and PR jack to jack adapter and ER and PR are available from U.S. ATCC.Human breast cancer cell strain MDA-mB-231 cell cultures is being contained 10% foetal calf serum, 1 * 10
5U/L penicillin, 100mg/L Streptomycin sulphate, 2g NaHCO
3In the RPMI1640 nutrient solution of 0.3g glutamine, human breast cancer cell strain MCF-7 cell cultures is containing 10% foetal calf serum, 1 * 10
5U/L penicillin, 100mg/L Streptomycin sulphate, 2g NaHCO
3With (DMEM in the Eagle nutrient solution of the Dulbecco of 0.3g glutamine improvement; Gibeo BRL Grand Island, NY), saturated humidity, 37 ℃ and 5%CO
2After being cultured to logarithmic phase and 80% degrees of fusion under the concentration,, make single cell suspension with the corresponding nutrient solution that contains 10% new-born calf serum (Gibeo BRL) with 0.25% pancreatin solution digestion, carry out cell counting after, cell concn is adjusted into 5 * 10
7Individual cell/L is inoculated in 96 orifice plates with every hole 100 μ L, cultivates 24h.With adherent cell grouping, every group of 3 parallel holes.One group is not dosing group, all the other groups add 100.000 μ mol/L and 50 μ mol/L for the reagent thing, blank is made in the hole that with inoculating cell not, only adds 100 μ L nutrient solutions simultaneously.Then cell is put CO
2Continue to cultivate 48～72h in the incubator.Dilution back microscopically is observed the dosing group tangible cell growth inhibition.
Embodiment 3: the experiment of external activity quantitative screening
Human breast cancer cell strain MDA-mB-231 cell cultures is being contained 10% foetal calf serum, 1 * 10
5U/L penicillin, 100mg/L Streptomycin sulphate, 2g NaHCO
3In the RPMI1640 nutrient solution of 0.3g glutamine, human breast cancer cell strain MCF-7 cell cultures is containing 10% foetal calf serum, 1 * 10
5U/L penicillin, 100mg/L Streptomycin sulphate, 2g NaHCO
3With (DMEM in the Eagle nutrient solution of the Dulbecco of 0.3g glutamine improvement; Gibeo BRL Grand Island, NY), saturated humidity, 37 ℃ and 5%CO
2After being cultured to logarithmic phase and 80% degrees of fusion under the concentration,, make single cell suspension with the corresponding nutrient solution that contains 10% new-born calf serum (Gibeo BRL) with 0.25% pancreatin solution digestion, carry out cell counting after, cell concn is adjusted into 5 * 10
4Individual cell/mL is inoculated in 96 orifice plates with every hole 100 μ L, cultivates 24h.With adherent cell grouping, every group of 2 parallel holes.One group is not dosing group, all the other groups add the sesquiterpene subergorgia suberosa ketone of 100.000 μ mol/L, 33.333 μ mol/L, 11.111 μ mol/L, 3.704 μ mol/L, 1.235 μ mol/L, 0.412 μ mol/L, 0.137 μ mol/L, 0.046 μ mol/L respectively, and blank is made in the hole that with inoculating cell not, only adds 100 μ L nutrient solutions simultaneously.Then cell is put CO
2Continue to cultivate 48-72h in the incubator.Every hole adds 20 μ L 5mg/mL tetrazole (MTT) serum-free mediums, 37 ℃ are removed supernatant liquor behind the cultivation 4h down, add 100 μ LDMSO, at automatic microplate reader (DYNEX Technologies, Chantilly, VA) go up 570nm wavelength place and measure absorbance value (OD), and be calculated as follows cell growth rate: cell growth rate=(experimental group OD ÷ control group OD) * 100%.Experimental result sees Table 1 and Fig. 1.
Table 1 sesquiterpene subergorgia suberosa ketone is for the restraining effect of human breast cancer cell strain MDA-MB-231 growth
| The concentration of sesquiterpene subergorgia suberosa ketone (μ mol/L) | Cell growth rate (%) | |
| |
|
|
| 100.000 | 13.388 | 16.272 |
| 33.333 | 36.457 | 35.427 |
| 11.111 | 53.347 | 72.503 |
| 3.704 | 74.356 | 83.419 |
| 1.235 | 92.276 | 100.309 |
| 0.412 | 80.330 | 91.040 |
| 0.137 | 96.601 | 102.781 |
| 0.046 | 97.837 | 101.133 |
Claims (3)
1. the compound of following formula (1)
Formula (1)
2. the preparation method of the compound of claim 1, it is characterized in that subergorgia suberosa extracts with the mixed organic solvents of 2: 1 ethanol of volume ratio and methylene dichloride, the extracting solution concentrating under reduced pressure is got crude extract, crude extract is outstanding water-soluble, with chloroform or ethyl acetate extraction, concentrating under reduced pressure must extract organic solvent layer, organic solvent layer is carried out the normal pressure silica gel column chromatography, chloroform-acetone solvent the system that changed from 100: 0 to 0: 100 with volume ratio is that eluent carries out gradient elution, and thin-layer chromatography is followed the trail of and merged component; To on thin-layer chromatography, can wash with the chloroform-methanol solvent systems gradient that volume ratio changed from 12: 1 to 10: 2 with 9: 1 chloroform-methanol solvent systems unfolded component of volume ratio, get the crude product of formula (1) compound, this purifying crude is got pure formula (1) compound.
3. the application of the compound of claim 1 in the cancer therapy drug of preparation treatment mammary cancer.
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| CN100528877C (en) * | 2007-11-13 | 2009-08-19 | 中国科学院南海海洋研究所 | New purine alkaloid and preparation method and application thereof |
| CN101343274B (en) * | 2008-08-18 | 2010-11-03 | 中国科学院南海海洋研究所 | Carbazoles alkaloid, preparation and application thereof |
| CN102775337B (en) * | 2011-12-03 | 2016-01-20 | 中国海洋大学 | A kind of 9,10-open loop steroid compound and preparation method thereof and application |
| CN112876439B (en) * | 2021-01-15 | 2022-06-21 | 宁波大学 | Nano-docetaxel type sesquiterpenoids, preparation method and application thereof |
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Non-Patent Citations (2)
| Title |
|---|
| Suberosenone, a New Cytotoxin from Subergorgia suberosa. Heidi R. Bokesch et al.Tetrahedron Letters,Vol.37 No.19. 1996 * |
| 海洋生物柳珊瑚的化学成分及生物活性研究进展. 张文，郭跃伟.中国天然药物，第1卷第2期. 2003 * |
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