CN103012099B - Fluorenone compounds and preparation method and application thereof - Google Patents
Fluorenone compounds and preparation method and application thereof Download PDFInfo
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- CN103012099B CN103012099B CN201310014162.6A CN201310014162A CN103012099B CN 103012099 B CN103012099 B CN 103012099B CN 201310014162 A CN201310014162 A CN 201310014162A CN 103012099 B CN103012099 B CN 103012099B
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Abstract
The invention discloses fluorenone compounds and a preparation method and application thereof. According to the structural formulas of the fluorenone compounds, R1 is -H, R2 is -OH, a molecular formula is C19H18O6, and one compound is named as gramniphenol D; and R1 is -OH, R2 is -H, a molecular formula is C19H18O6, and the other compound is named as gramniphenol E. According to the preparation method, the fluorenone compounds are prepared from the raw materials including dried branches, leaves or fruits of arundina graminifolia. The preparation method comprises the steps of extract extraction, organic solvent extraction, silica column chromatography and high pressure liquid chromatography separation of the raw materials. The invention further discloses the application of the fluorenone compounds to preparation of anti-AIDS medicines. The gramniphenol D and the gramniphenol E are first detected natural fluorenone replaced by 2-hydroxy-3-methylbut-3-enyloxy. According to cytotoxicity detection on C8166 host cells and inhibition tests on an HIV-1IIIB-induced cytopathic effect (CPE) of the C8166, the two fluorenone compounds have good anti-HIV-1 activity, the EC50 values of the two fluorenone compounds are respectively 1.460.15 mug/mL and 1.580.20 mug/mL, and the therapeutic index (IT) values of the two fluorenone compounds are respectively 137 and 126.6. The fluorenone compounds have novel structures and high activity, and can serve as guiding compounds of the anti-AIDS medicines.
Description
Technical field
The invention belongs to effective ingredients in plant extractive technique field, be specifically related to a kind of Fluorenone compounds and its preparation method and application.
Background technology
The orchid family (Orchidaceae) is monocotyledons, and leaf of bamboo Cymbidium (Arundina) is a genus of the orchid family, is Lu Shenglan.Total approximately 5 kinds of this genus, is distributed in Tropical Asian to more Oceanian island.Wherein 2 kinds of Purpleback Murdannia A. graminifolia and narrow leaf Purpleback Murdannia A. stenopetala etc. also produce south China.Purpleback Murdannia, the high 40 ~ 80cm of plant, more than sometimes can reaching 1m; Underground root stock is everlasting and is connected basal part of stem place and be ovoid and expand, pseudobulb seemingly, stem is upright, constant is grown thickly or growth in flakes, produces Zhejiang, Jiangxi, Fujian, Taiwan, Southern Hunan, Guangdong, Hainan, Guangxi, South Sichuan (Miyi), Guizhou (Rongjiang, Xingyi), Yunnan (Deng Chuan, Fengqing, Jinghong, Xichou, screen limit etc.) and Southeastern Tibet (Motuo).Be born in by Cao Po, trench, under shrubbery or in woods 400 ~ 2800 meters of height above sea level.Also there is distribution on Nepal, Sillim, Bhutan, India, Sri Lanka, Burma, Vietnam, Laos, Cambodia, Thailand, Malaysia, Indonesia, the Ryukyu Islands and Tahiti and other places.Purpleback Murdannia medicinal part is rhizome and cauline leaf, and its property hardship is flat.Clearing heat and detoxicating, dispel rheumatism, pain relieving, diuresis.Be used for the treatment of jaundice, heat is drenched, beriberi oedema, hernia stomachache, rheumatic arthralgia, stomachache, urinary tract infections, venomous snake bite, sore and toxic, wound etc.Purpleback Murdannia is the conventional plant amedicas of the people of In Xishuangbanna the Dai nationality, and the local compatriot of the Dai nationality is called " agriculture still " the Purpleback Murdannia of opening beautiful flower, is a kind of removing toxic substances good medicine common to all.
Fluorenone cumarone is because plant Fluorenone constituent structure type is many, and stereochemistry complexity, has multiple biological activity, very active to the research in this field both at home and abroad, no matter be naturally occurring, or the Fluorenone compounds that obtains of synthetic, chemist's extensive concern all caused.
Summary of the invention
The first object of the present invention is to provide a kind of Fluorenone compounds; The second object is to provide the preparation method of described Fluorenone compounds; The 3rd object is to provide the application of described Fluorenone compounds in the medicine of the anti-AIDS of preparation.
The first object of the present invention is achieved in that described Fluorenone compounds is taking the Purpleback Murdannia branch, leaf or the fruit that are dried as raw material, separates and obtains through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography, and its structural formula is:
。
Described R
1for-H, R
2for-OH, its molecular formula is C
19h
18o
6, this compound called after gramniphenol D.
Described R
1for-OH, R
2for-H, its molecular formula is C
19h
18o
6, this compound called after gramniphenol E.
The second object of the present invention is achieved in that the Purpleback Murdannia branch, leaf or the fruit that are dried are raw material, obtains through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separation, is specially:
A, medicinal extract extract: by Purpleback Murdannia branch, leaf or fruit coarse reduction to 20 ~ 40 order, use organic solvent supersound extraction 2 ~ 4 times, and each 30 ~ 60min, extracting solution merges; Extracting liquid filtering, when the volume of concentrating under reduced pressure extracting solution to 1/4 ~ 1/2, leaves standstill, and filtering throw out, is condensed into medicinal extract a;
B, organic solvent extraction: in medicinal extract a, add the water of 1 ~ 2 times of amount of weight ratio, use and the isopyknic organic solvent extraction of water 3 ~ 5 times, merge organic solvent extraction phase, concentrating under reduced pressure becomes medicinal extract b;
C, silica gel column chromatography: the acetone solution by medicinal extract b by 1.5 ~ 3 times of amounts of weight ratio, then weigh 80 ~ 100 order silica gel silica gel mixed samples of 0.8 ~ 1.2 times with medicinal extract, then go up silica gel column chromatography, dress post silica gel is 160 ~ 200 orders, consumption is 6 ~ 8 times of amounts of medicinal extract b weight; The organic solvent solution gradient elution that is 1:0 ~ 0:1 by volume ratio, collects gradient eluent, concentrated, through TLC monitoring, merges identical part;
D, reversed phase column chromatography: by reversed phase column chromatography in the 9:1 part of C step elutriant, reversed-phase column is with reversed material C-18 dress post; The methanol aqueous solution that is 20 ~ 100% with volume content carries out gradient elution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part;
E, high performance liquid chromatography separate: 45% ~ 60% methanol aqueous solution wash-out part of D step elutriant, through high performance liquid chromatography separation and purification, is obtained to described Fluorenone compounds;
High performance liquid chromatography separation and purification described in F, E step is taking 40 ~ 60% methyl alcohol as moving phase, flow velocity 10 ~ 14ml/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, the chromatographic peak of collection 10 ~ 25min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol D.
High performance liquid chromatography separation and purification described in G, E step is taking 40 ~ 60% methyl alcohol as moving phase, flow velocity 10 ~ 14ml/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, the chromatographic peak of collection 25 ~ 40min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol E.
The 3rd object of the present invention is achieved in that described Fluorenone compounds is in the application of preparing in anti-AIDS drug.
Fluorenone compounds of the present invention is separated first, has determined for Fluorenone compounds, and characterized its concrete structure and be by nucleus magnetic resonance and measuring method of mass spectrum:
。
Its isomeric compound gramniphenol D, gramniphenol E can separate by method of the present invention.Gramniphenols D and gramniphenol E are the natural Fluorenones that has 2-hydroxy-3-methylbut-3-enyloxy to replace of finding first.Taking gramniphenol D, gramniphenol E as raw material, through the cytotoxicity of C8166 host cell is detected, and inhibition test to HIV-1IIIB induction C8166 cytopathy (CPE), two kinds of Fluorenone compounds have good anti-HIV-1 activity, EC
50value is respectively 1.46 ± 0.15 μ g/mL and 1.58 ± 0.20 μ g/mL, and therapeutic index (TI) values is 137 and 126.6.It is good that above result has disclosed the compounds of this invention novel structure activity, can be used as the guiding compound of anti-AIDS medicine, in the medicine of the anti-AIDS of preparation, has good application prospect.
Brief description of the drawings
Fig. 1 be compound gramniphenol D carbon-13 nmr spectra (
13c NMR);
Fig. 2 be compound gramniphenol D proton nmr spectra (
1h NMR);
Fig. 3 be compound gramniphenol E carbon-13 nmr spectra (
13c NMR);
Fig. 4 be compound gramniphenol E proton nmr spectra (
1h NMR);
Fig. 5 is the main HMBC(of compound gramniphenol D
→) and
1h-
1h COSY(
–) relevant.
Embodiment
Below in conjunction with accompanying drawing, the present invention is further illustrated, but never in any form the present invention is limited, and any conversion or the improvement done based on training centre of the present invention, all fall into protection scope of the present invention.
Fluorenone compounds of the present invention is taking the Purpleback Murdannia branch, leaf or the fruit that are dried as raw material, separates and obtains through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography, and its structural formula is:
。
Described R
1for-H, R
2for-OH, its molecular formula is C
19h
18o
6, this compound called after gramniphenol D.
Described R
1for-OH, R
2for-H, its molecular formula is C
19h
18o
6, this compound called after gramniphenol E.
Fluorenone compounds preparation method of the present invention, taking the Purpleback Murdannia branch, leaf or the fruit that are dried as raw material, separates and obtains through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography, is specially:
A, medicinal extract extract: by Purpleback Murdannia branch, leaf or fruit coarse reduction to 20 ~ 40 order, use organic solvent supersound extraction 2 ~ 4 times, and each 30 ~ 60min, extracting solution merges; Extracting liquid filtering, when the volume of concentrating under reduced pressure extracting solution to 1/4 ~ 1/2, leaves standstill, and filtering throw out, is condensed into medicinal extract a;
B, organic solvent extraction: in medicinal extract a, add the water of 1 ~ 2 times of amount of weight ratio, use and the isopyknic organic solvent extraction of water 3 ~ 5 times, merge organic solvent extraction phase, concentrating under reduced pressure becomes medicinal extract b;
C, silica gel column chromatography: the acetone solution by medicinal extract b by 1.5 ~ 3 times of amounts of weight ratio, then weigh 80 ~ 100 order silica gel silica gel mixed samples of 0.8 ~ 1.2 times with medicinal extract, then go up silica gel column chromatography, dress post silica gel is 160 ~ 200 orders, consumption is 6 ~ 8 times of amounts of medicinal extract b weight; The organic solvent solution gradient elution that is 1:0 ~ 0:1 by volume ratio, collects gradient eluent, concentrated, through TLC monitoring, merges identical part;
D, reversed phase column chromatography: by reversed phase column chromatography in the 9:1 part of C step elutriant, reversed-phase column is with reversed material C-18 dress post; The methanol aqueous solution that is 20 ~ 100% with volume content carries out gradient elution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part;
E, high performance liquid chromatography separate: 45% ~ 60% methanol aqueous solution wash-out part of D step elutriant, through high performance liquid chromatography separation and purification, is obtained to described Fluorenone compounds;
High performance liquid chromatography separation and purification described in F, E step is taking 40 ~ 60% methyl alcohol as moving phase, flow velocity 10 ~ 14ml/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, the chromatographic peak of collection 10 ~ 25min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol D.
High performance liquid chromatography separation and purification described in G, E step is taking 40 ~ 60% methyl alcohol as moving phase, flow velocity 10 ~ 14ml/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, the chromatographic peak of collection 25 ~ 40min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol E.
Organic solvent described in A step is the one in 70 ~ 100% acetone, ethanol or methyl alcohol.
Organic solvent described in B step is the one in ethyl acetate, chloroform, ether, sherwood oil or benzene.
Organic solvent solution described in C step is the one in normal hexane-acetone, chloroform-acetone, chloroform-methanol, sherwood oil-acetone or petroleum ether-ethyl acetate.
The volume proportion of the organic solvent solution described in C step is 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1.
High performance liquid chromatography separation and purification described in E step is taking 40 ~ 60% methyl alcohol as moving phase, flow velocity 10 ~ 14ml/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, the chromatographic peak of collection 10 ~ 40min, repeatedly cumulative rear evaporate to dryness.
Fluorenone compounds of the present invention is in the application of preparing in anti-AIDS drug.
Purpleback Murdannia of the present invention is not limited by area and kind, all can realize the present invention.
Embodiment 1
Get dry Purpleback Murdannia branch, leaf and/or fruit 5.8kg, coarse reduction to 40 order, the acetone supersound extraction with 70% 4 times, each 60min, extracting solution merges; Extracting liquid filtering, is evaporated to 1/4 of volume; Leave standstill, filtering throw out, is condensed into 674g medicinal extract a; In medicinal extract a, add 630g water, use and the isopyknic ethyl acetate extraction of water 5 times, merge extraction phase, concentrating under reduced pressure becomes 496g medicinal extract b; With 200 order silica gel 1900g dress posts, in medicinal extract b, add the acetone solution of 478.5g, then add 100 order silica gel 496g to mix sample, mix upper prop after sample; Be respectively the chloroform-methanol organic solvent solution gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 by volume ratio, collect gradient eluent, concentrated, monitor through TLC, merge identical part, obtain 6 parts, the elutriant c of the chloroform-methanol organic solvent solution of volume ratio 9:1 is 81.2g; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c, carries out gradient elution taking volume content as 20 ~ 100% methanol aqueous solution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part; Get the elutriant obtaining with volume content 45 ~ 60% methanol aqueous solution wash-outs, again taking 55% methyl alcohol as moving phase, flow velocity 10ml/min, 21.2 ' 250mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 50mL, collect the chromatographic peak of 19min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol D; Collect the chromatographic peak of 28min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol E.
Embodiment 2
Get dry Purpleback Murdannia branch, leaf and/or fruit
5kg, coarse reduction to 20 order, the ethanol ultrasonic extraction with 100% 2 times, each 50min, extracting solution merges; Extracting liquid filtering, is evaporated to 1/3 of volume; Leave standstill, filtering throw out, is condensed into 590g medicinal extract a; In medicinal extract a, add the water of 590g, use and the isopyknic chloroform extraction of water 3 times, merge extraction phase, concentrating under reduced pressure becomes 438g medicinal extract b; With 160 order silica gel 3504g dress posts, in medicinal extract b, add the acetone solution of 1314g, then add 80 order silica gel 350.4g to mix sample, mix upper prop after sample; Be respectively normal hexane-acetone organic solvent solution gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 by volume ratio, collect gradient eluent, concentrated, through TLC monitoring, merge identical part; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c, carries out gradient elution taking volume content as 20 ~ 100% methanol aqueous solution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part; Get the elutriant obtaining with volume content 45 ~ 60% methanol aqueous solution wash-outs, again taking 45% methyl alcohol as moving phase, flow velocity 14ml/min, 21.2 ' 250mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 55mL, collect the chromatographic peak of 22min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol D; Collect the chromatographic peak of 37min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol E.
Embodiment 3
Get dry Purpleback Murdannia branch, leaf and/or fruit 6kg, coarse reduction to 30 order, the methyl alcohol supersound extraction with 80% 4 times, each 30min, extracting solution merges; Extracting liquid filtering, is evaporated to 1/2 of volume; Leave standstill, filtering throw out, is condensed into 700g medicinal extract a; In medicinal extract a, add the water of 1400g, use and the isopyknic extracted with diethyl ether of water 4 times, merge extraction phase, concentrating under reduced pressure becomes 520g medicinal extract b; With 180 order silica gel 3638g dress posts, in medicinal extract b, add the acetone solution of 1040g, then add 90 order silica gel 624g to mix sample, mix upper prop after sample; Be respectively chloroform-acetone organic solvent solution gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 by volume ratio, collect gradient eluent, concentrated, through TLC monitoring, merge identical part; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c, carries out gradient elution taking volume content as 20 ~ 100% methanol aqueous solution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part; Get the elutriant obtaining with volume content 45 ~ 60% methanol aqueous solution wash-outs, again taking 60% methyl alcohol as moving phase, flow velocity 12ml/min, 21.2 ' 250mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254nm that UV-detector detects wavelength, each sample introduction 60mL, collect the chromatographic peak of 10min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol D; Collect the chromatographic peak of 26min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol E.
Embodiment 4
Get dry Purpleback Murdannia branch, leaf and/or fruit 5.5kg, coarse reduction to 40 order, the ethanol ultrasonic extraction with 90% 3 times, each 45min, extracting solution merges; Extracting liquid filtering, is evaporated to 1/4 of volume; Leave standstill, filtering throw out, is condensed into 640g medicinal extract a; In medicinal extract a, add the water of 960g, use and the isopyknic petroleum ether extraction of water 4 times, merge extraction phase, concentrating under reduced pressure becomes 475g medicinal extract b; With 160 order silica gel 2850g dress posts, in medicinal extract b, add the acetone solution of 712.5g, then add 80 order silica gel 475g to mix sample, mix upper prop after sample; Be respectively sherwood oil-acetone organic solvent solution gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 by volume ratio, collect gradient eluent, concentrated, through TLC monitoring, merge identical part; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c, carries out gradient elution taking volume content as 20 ~ 100% methanol aqueous solution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part; Get the elutriant obtaining with volume content 45 ~ 60% methanol aqueous solution wash-outs, again taking 50% methyl alcohol as moving phase, flow velocity 10ml/min, 21.2 ' 250mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, and it is 254nm that UV-detector detects wavelength, collects the chromatographic peak of 21min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol D; Collect the chromatographic peak of 35min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol E.
Embodiment 5
Get dry Purpleback Murdannia branch, leaf and/or fruit 5kg, coarse reduction to 20 order, the methyl alcohol supersound extraction with 70% 4 times, each 35min, extracting solution merges; Extracting liquid filtering, is evaporated to 1/2 of volume; Leave standstill, filtering throw out, is condensed into 580g medicinal extract a; In medicinal extract a, add the water of 1160g, use and the isopyknic benzene extraction of water 5 times, merge extraction phase, concentrating under reduced pressure becomes 430g medicinal extract b; With 200 order silica gel 3010
gdress post adds the acetone solution of 1290g in medicinal extract b, then adds 100 order silica gel 344g to mix sample, mixes upper prop after sample; Be respectively the petroleum ether-ethyl acetate organic solvent solution gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 by volume ratio, collect gradient eluent, concentrated, through TLC monitoring, merge identical part; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c, carries out gradient elution taking volume content as 20 ~ 100% methanol aqueous solution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part; Get the elutriant obtaining with volume content 45 ~ 60% methanol aqueous solution wash-outs, again taking 40% methyl alcohol as moving phase, flow velocity 12ml/min, 21.2 ' 250mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, and it is 254nm that UV-detector detects wavelength, collects the chromatographic peak of 25min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol D; Collect the chromatographic peak of 40min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol E.
Embodiment 6
Getting compound gramniphenol D prepared by embodiment 1, is red jelly; Optical value [a] 24.8 D-16.8 (solvent is methyl alcohol c 0.25); Measuring method is: with nucleus magnetic resonance, in conjunction with other spectroscopic technique qualification structure.
(1) UV spectrum (solvent is methyl alcohol),
λ max(log
e): 210(3.86), 268(3.82), 320(2.58), 346(2.89) and nm;
(2) infrared spectra (pressing potassium bromide troche),
n max3328,2972,2895,1698,1610,1548,1456,1187,1114 cm
-1;
(3) HRESIMS shows the compounds of this invention quasi-molecular ion peak
m/z[365.1002 M+H]
+(calculated value is 365.1001), in conjunction with
13c and
1h NMR spectrum (Fig. 1 and Fig. 2, carbon spectrum hydrogen spectrum attribution data is in table 1) provides its molecular formula C
19h
18o
6.
1h NMR(C
5d
5n, 500 MHz) and
13c NMR(C
5d
5n, 125 MHz) data, in table 1.
The infrared absorption of Gramniphenol D illustrates hydroxyl (3328 cm in its structure
-1), carbonyl (1698 cm
-1) and aromatic ring (1610,1548,1456 cm
-1).Ultra-violet absorption spectrum is 268,320, and 346 nm also illustrate the existence of aromatic ring.
1h and
13c NMR spectrum shows 19 carbon signals and 18 hydrogen signals, shows Isosorbide-5-Nitrae, the Fluorenone structure (H-2, H-3, H-6, H-8, and C-1-C-9a) of 5,7-oxidation, 1 methoxyl group (
d c57.0,
d h3.91), 2 phenolic hydroxyl group protons (
δ h10.50,11.02), also has 1 2-hydroxy-3-methylbut-3-enyloxy unit[-OCH
2cH(OH) C(CH
2) (CH
3)] (H-1 ￠, H-2 ￠, H-4 ￠, H-5 ￠, and C-1 ￠-C-5 ￠).Methoxyl group proton in HMBC spectrum (
δ h3.91) and C-5(
δ c159.5) related description methoxyl group is in C-5 position.In addition, H-1 ￠ (
d h3.95 and 4.12) and C-7(
d c152.0) HMBC related description 2-hydroxy-3-methylbut-3-enyloxy group is substituted in C-7 position.Finally, hydroxyl signal (
d h11.02) and C-1(
d c150.5), C-2(
d c118.3), and C-9a (
d c116.7) HMBC is relevant, and another 1 hydroxyl signal (
d h10.50) with C-3 (
d c129.1), C-4(
d c145.0), and C-4a(
d c125.1) be correlated with, is positioned 2 phenolic hydroxyl group protons for respectively C-1 and C-4.By contrasting with NMR spectrum and the optical value of known compound stachylines A, C-2' is configured as
s.So far the structure of this compound is determined.
Embodiment 7
Getting compound gramniphenol E prepared by embodiment 1 is red jelly; Optical value [a] 25.0 D-14.6 (solvent is methyl alcohol c 0.25); Measuring method is: with nucleus magnetic resonance, in conjunction with other spectroscopic technique qualification structure.
(1) UV spectrum (solvent is methyl alcohol),
λ max(log
e): 210(3.79), 266(3.86), 320(2.52), 346(2.73) and nm;
(2) infrared spectra (pressing potassium bromide troche),
n max3325,2967,2897,1701,1611,1546,1452,1182,1120 cm
-1;
(3) HRESIMS shows the compounds of this invention quasi-molecular ion peak
m/z[365.1009 M+H]
+(calculated value is 365.1001), in conjunction with
13c and
1h NMR spectrum (Fig. 3 and Fig. 4, carbon spectrum attribution data is in table 1) provides its molecular formula C
19h
18o
6.
1h NMR(C
5d
5n, 500 MHz) and
13c NMR(C
5d
5n, 125 MHz) data, in table 1.
The molecular formula C of Gramniphenol E
19h
18o
6identical with gramniphenol D,
1h and
13also closely similar with Gramniphenol D of C NMR spectrum.Find that by contrast the difference of these two compounds is the replacement difference on Fluorenone ring.
d h6.57(d,
j=1.9 Hz), 6.28(d,
j=1.9Hz), 6.67 (d,
j=1.7Hz), and 6.83(d,
j=1.7 Hz)
1h NMR signal instruction gramniphenol E is the Fluorenone that 2,4,5,7-oxidation replaces.Methoxyl group signal in HMBC spectrum (
δ h3.85) and C-5(
δ c153.3) illustrate that methoxyl group is in C-5 position.In addition, H-1 ￠ (
d h3.93 and 4.22) and C-7(
δ chMBC related description 157.3) (
s)-2-hydroxy-3-methylbut-3-enyloxy is in C-7 position.Hydroxyl signal in HMBC spectrum (
d h11.11) and C-1(
d c105.9), C-2(
d c159.5), and C-3(
d c110.6) relevant, another 1 hydroxyl (
d h10.83) and C-3(
d c110.6), C-4(
d c154.3), and C-4a(
d c124.1) relevant, two hydroxyls of this explanation replace respectively in C-2 and C-4 position.So far the structure of this compound is determined.
Table 1 compound
1h and
13(solvent is C to C NMR data
5d
5n)
Embodiment 8
Get compound gramniphenol D, gramniphenol E prepared by embodiment 2 and carry out structure determination by the method in embodiment 6,7 respectively, result is: its structure is with embodiment 6,7, and molecular formula is respectively C
19h
18o
6and C
19h
18o
6.
Embodiment 9
Get compound gramniphenol D, gramniphenol E prepared by embodiment 3 and carry out structure determination by the method in embodiment 6,7 respectively, result is: its structure is with embodiment 6,7, and molecular formula is respectively C
19h
18o
6and C
19h
18o
6.
Embodiment 10
Get compound gramniphenol D, gramniphenol E prepared by embodiment 4 and carry out structure determination by the method in embodiment 6,7 respectively, result is: its structure is with embodiment 6,7, and molecular formula is respectively C
19h
18o
6and C
19h
18o
6.
Embodiment 11
Get compound gramniphenol D, gramniphenol E prepared by embodiment 5 and carry out structure determination by the method in embodiment 6,7 respectively, result is: its structure is with embodiment 6,7, and molecular formula is respectively C
19h
18o
6and C
19h
18o
6.
Embodiment 12
Get Fluorenone compounds (gramniphenol D) prepared by embodiment 1 and carry out anti-HIV-1 activity test, as follows:
Measure medicine and compound: testing sample dissolves with DMSO, positive control compound Zidovodine (AZT, 3 '-Azido-3 '-deoxythymidine), purchased from Sigma company, is dissolved in RPMI-1640 perfect medium, 0.22 μ m membrane filtration degerming ,-20 DEG C of preservations after packing.
Reagent: HEPES(N-2(2-Hydroxyothyl) piperazine-N'-(2-ethanesufonic acid), MTT(3-(4; 5-dimethylthiazol-2-yl)-2; 5-diphenyl tetrazolium bromide), DMF(N, N '-Dimethyl formamine), penicillin (Penicillin), Vetstrep (Streptomycin sulfate), glutamine (Glutamine) is all purchased from Sigma company; DMSO, 2 mercapto ethanol (2-Me, 2-Mercaptoethanol) are Bio-Rad company product.RPMI-1640 and newborn calf serum are Gibco company product.
Substratum: RPMI-1640 perfect medium, contains 10 % newborn calf serums, 2 mM L-glutaminate, 10 mM HEPES, 50 μ M 2 mercapto ethanols, 100,000 IU penicillin, 100 μ g/mL Streptomycin sulphates.
Cell and virus: human T lymphocyte is C8166, MT4 and HIV-1 experiment strain HIV-1IIIB all come from Britain Medical Research Council, AIDS Reagent Project.All cells and virus are all cultivated with the RPMI-1640 perfect medium containing 10% calf serum.Prepare according to a conventional method HIV-1IIIB, titration also calculates viral TCID
50.After the packing of virus stock solution, put-70 DEG C of preservations.Cell and virus freezing and thawing according to a conventional method.
HIV-1 infectious titration: carry out titration by method improvement described in Johnson & Byington, be summarized as follows: HIV-1IIIB stock solution is done on 96 orifice plates to 4 times of dilutions, 10 gradients, 6 repeating holes of every gradient arrange control wells 6 holes simultaneously.Every hole adds C8166 cell 50 μ L(4 × 10
5/ mL), every hole final volume is 200 μ L.37 DEG C, 5% CO
2cultivate.Within the 3rd day, add fresh RPMI-1640 perfect medium 100 μ L, within the 7th day, under inverted microscope, observe cytopathic effect (the Cytopathic Effect of HIV-1 induction in every hole, CPE), whether there is the formation of synplasm (Syncytium) to determine with every hole; Calculate viral TCID by Reed & Muench method
50(50% Tissue Culture Infection Dose).
Sample detects the cytotoxicity of C8166 host cell: 4 × 10
5/ mL C8166 cell suspension 100
μl mixes from different testing compound solutions, establishes three repeating holes.The not control wells containing compound is set simultaneously, 37 DEG C, 5% CO
2cultivate 3 days, adopt MTT colorimetric determination cytotoxicity.ELx800 ELISA instrument is measured OD value, and measuring wavelength is 595 nm, and reference wavelength is 630 nm.Calculate CC
50value (50% Cytotoxic Concentration), the compound concentration while 50% normal T lymphocyte series C8166 being produced to toxicity.
The inhibition test of sample to HIV-1IIIB induction C8166 cytopathy (CPE): by 8 × 10
5/ mL C8166 cell 50 μ L/ holes are inoculated on the 96 porocyte culture plates that contain 100 μ L/ hole doubling dilution compounds, then add the HIV-1IIIB dilution supernatant (M.O.I.0.0016) of 50 μ L.If 3 repeating holes.The not normal cell control wells containing compound is set simultaneously, 37 DEG C, 5% CO
2cultivate 3 days, under inverted microscope, (100 ×) count plasmodial formation.EC
50(50% Effective Concentration) is the compound concentration while suppressing Syncytium formation 50%.
Calculation formula: draw dose response curve according to experimental result, calculate by Reed & Muench method the 50% effective concentration (EC that compound suppresses virus
50), 50% cell growth inhibiting concentration (CC
50) and the therapeutic index TI value (Therapeutic index) of Anti-HIV-1 Active be: TI=CC
50/ EC
50.
Test-results: through the cytotoxicity of C8166 host cell is detected, and HIV-1IIIB is induced to the inhibition test of C8166 cytopathy (CPE), Fluorenone compounds (gramniphenol D) has good anti-HIV-1 activity, EC
50value is 1.46 ± 0.15 μ g/mL, and therapeutic index (TI) values is that 137(is in table 2).It is good that above result has disclosed the compounds of this invention novel structure activity, can be used as the guiding compound of anti-AIDS medicine, in the medicine of the anti-AIDS of preparation, has good application prospect.
Embodiment 13
Get Fluorenone compounds (gramniphenol E) prepared by embodiment 1 and carry out anti-HIV-1 activity test by the identical method of embodiment 12, test-results: through the cytotoxicity of C8166 host cell is detected, and inhibition test to HIV-1IIIB induction C8166 cytopathy (CPE), Fluorenone compounds (gramniphenol E) has good anti-HIV-1 activity, EC
50value is 1.58 ± 0.2 μ g/mL, and therapeutic index (TI) values is that 137(is in table 2).It is good that above result has disclosed the compounds of this invention novel structure activity, can be used as the guiding compound of anti-AIDS medicine, in the medicine of the anti-AIDS of preparation, has good application prospect.
The Anti-HIV activity of table 2 compound
Claims (7)
1. a Fluorenone compounds, is characterized in that: described Fluorenone compounds is taking the Purpleback Murdannia branch, leaf or the fruit that are dried as raw material, separates and obtains through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography, and its structural formula is:
, C-2' is configured as S; R
1for-H, R
2for-OH, its molecular formula is C
19h
18o
6, this compound called after gramniphenol D; Or R
1for-OH, R
2for-H, its molecular formula is C
19h
18o
6, this compound called after gramniphenol E.
2. a Fluorenone compounds preparation method claimed in claim 1, Purpleback Murdannia branch, leaf or the fruit that it is characterized in that being dried is raw material, obtains through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separation, is specially:
A, medicinal extract extract: by Purpleback Murdannia branch, leaf or fruit coarse reduction to 20 ~ 40 order, use organic solvent supersound extraction 2 ~ 4 times, and each 30 ~ 60min, extracting solution merges; Extracting liquid filtering, when the volume of concentrating under reduced pressure extracting solution to 1/4 ~ 1/2, leaves standstill, and filtering throw out, is condensed into medicinal extract a;
B, organic solvent extraction: in medicinal extract a, add the water of 1 ~ 2 times of amount of weight ratio, use and the isopyknic organic solvent extraction of water 3 ~ 5 times, merge organic solvent extraction phase, concentrating under reduced pressure becomes medicinal extract b;
C, silica gel column chromatography: the acetone solution by medicinal extract b by 1.5 ~ 3 times of amounts of weight ratio, then weigh 80 ~ 100 order silica gel silica gel mixed samples of 0.8 ~ 1.2 times with medicinal extract, then go up silica gel column chromatography, dress post silica gel is 160 ~ 200 orders, consumption is 6 ~ 8 times of amounts of medicinal extract b weight; The organic solvent solution gradient elution that is 1:0 ~ 0:1 by volume ratio, collects gradient eluent, concentrated, through TLC monitoring, merges identical part;
D, reversed phase column chromatography: by reversed phase column chromatography in the 9:1 part of C step elutriant, reversed-phase column is with reversed material C-18 dress post; The methanol aqueous solution that is 20 ~ 100% with volume content carries out gradient elution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part;
E, high performance liquid chromatography separate: the elutriant that will obtain with volume content 45 ~ 60% methanol aqueous solution wash-outs, through high performance liquid chromatography separation and purification, obtains described Fluorenone compounds;
High performance liquid chromatography separation and purification described in F, E step is taking 40 ~ 60% methyl alcohol as moving phase, flow velocity 10 ~ 14ml/min, 21.2 × 250mm, the anti-phase preparative column of Zorbax PrepHT GF of 5 μ m is stationary phase, it is 254nm that UV-detector detects wavelength, each sample introduction 50 ~ 60 μ L, the chromatographic peak of collection 10 ~ 25min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol D;
High performance liquid chromatography separation and purification described in G, E step is taking 40 ~ 60% methyl alcohol as moving phase, flow velocity 10 ~ 14ml/min, 21.2 × 250mm, the anti-phase preparative column of Zorbax PrepHT GF of 5 μ m is stationary phase, it is 254nm that UV-detector detects wavelength, each sample introduction 50 ~ 60 μ L, the chromatographic peak of collection 25 ~ 40min, repeatedly cumulative rear evaporate to dryness, obtains described Fluorenone compounds gramniphenol E.
3. the preparation method of Fluorenone compounds according to claim 2, is characterized in that: the one in acetone, ethanol or methyl alcohol that the organic solvent described in A step is 70 ~ 100%.
4. the preparation method of Fluorenone compounds according to claim 2, is characterized in that: the organic solvent described in B step is the one in ethyl acetate, chloroform, ether, sherwood oil or benzene.
5. the preparation method of Fluorenone compounds according to claim 2, is characterized in that: the organic solvent solution described in C step is the one in normal hexane-acetone, chloroform-acetone, chloroform-methanol, sherwood oil-acetone or petroleum ether-ethyl acetate.
6. the preparation method of Fluorenone compounds according to claim 2, is characterized in that: the volume proportion of the organic solvent solution described in C step is 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1.
7. a Fluorenone compounds claimed in claim 1 is in the application of preparing in anti-AIDS drug.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4011266A (en) * | 1973-10-31 | 1977-03-08 | Xerox Corporation | 2-vinyl-fluorenone and derivatives thereof |
| CN1110875A (en) * | 1993-06-18 | 1995-10-25 | 大塚制药株式会社 | Fluorenone derivatives, process for preparing the same and central or peripheral nerve degeneration repair and protective agent |
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2013
- 2013-01-15 CN CN201310014162.6A patent/CN103012099B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4011266A (en) * | 1973-10-31 | 1977-03-08 | Xerox Corporation | 2-vinyl-fluorenone and derivatives thereof |
| CN1110875A (en) * | 1993-06-18 | 1995-10-25 | 大塚制药株式会社 | Fluorenone derivatives, process for preparing the same and central or peripheral nerve degeneration repair and protective agent |
Non-Patent Citations (2)
| Title |
|---|
| 朱慧.鼓槌石斛及竹叶兰植物化学成分的研究.《中国优秀硕士学位论文全文数据库农业科技辑》.2007,(第6期),D047-438. |
| 鼓槌石斛及竹叶兰植物化学成分的研究;朱慧;《中国优秀硕士学位论文全文数据库农业科技辑》;20071215(第6期);D047-438 * |
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