CN101531644A - New daphnane diterpene compounds in Daphne genkwa as well as preparation method and application of same - Google Patents
New daphnane diterpene compounds in Daphne genkwa as well as preparation method and application of same Download PDFInfo
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- CN101531644A CN101531644A CN200910010388A CN200910010388A CN101531644A CN 101531644 A CN101531644 A CN 101531644A CN 200910010388 A CN200910010388 A CN 200910010388A CN 200910010388 A CN200910010388 A CN 200910010388A CN 101531644 A CN101531644 A CN 101531644A
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- 238000002360 preparation method Methods 0.000 title claims description 11
- -1 daphnane diterpene compounds Chemical class 0.000 title abstract description 19
- 150000001875 compounds Chemical class 0.000 claims abstract description 70
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- DZUKXCCSULKRJA-UHFFFAOYSA-N Isopratol Natural products C=1C(OC)=CC=C(C(C=2)=O)C=1OC=2C1=CC=C(O)C=C1 DZUKXCCSULKRJA-UHFFFAOYSA-N 0.000 claims abstract description 35
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- Saccharide Compounds (AREA)
Abstract
The invention belongs to the technical field of medicaments, and relates to a series of novel daphnane diterpene compounds, namely Genkwanine II, Genkwanin III, Genkwanin IV, Genkwanin V, Genkwanin VI, and Genkwanin VII which are extracted and separated from buds of Daphne genkwa, zucc. The compounds have the same daphnane diterpene parent nucleus and a chemical structural general formula shown in a formula (1). The invention also relates to new compounds, the antitumor physiological activity of medicaments prepared by the new compounds, and pharmaceutical application of the new compounds. The compounds can be combined with pharmaceutically acceptable carriers to prepare into clinically acceptable dosage forms to be used for the treatment of various cancers. A method for preparing the compounds has the advantages of simplicity, good reproducibility, and high extraction purity. The compounds have better antitumor activity effect.
Description
Technical field
The invention belongs to medical technical field, relate to daphane diterpene-kind compound new in the lilac daphne and preparation method thereof, the invention still further relates to the purposes of such new compound aspect anti-tumor biological.
Background technology
Thymelaeceae plant lilac daphne (Daphne genkwa sieb.et Zucc) is traditional removing water retention by purgation medicine.Aspect researchs such as antitumor to it at present, induced labor, kobadrin, desinsection, anti-inflammatory, immunologic function adjusting are more.Chinese scholars from the flower and alabastrum of thymelaeceae plant lilac daphne, root, separation obtained a series of daphane diterpene ortho-ester compounds with special construction.The daphane diterpene has another name called daphnetoxin compounds (Daphnetoxin) and has very strong biological activity, early 1970s, people isolate the mezereon element (Mezerein) and daphnetoxin (Daphnotoxin) with anti-tumor activity from daphne plant after, this compounds just becomes a research focus, comes into one's own owing to the daphane diterpene has unique anti-tumour phological activity.
Modern medicine study shows that cancer is a kind of cancer poison diffusion disease of especially severe, and the formation of cancer poison is the product of present mode of life, and more and more serious trend is arranged.Therefore searching is more effectively anticancer, antitumor drug is very urgent.
Summary of the invention:
Purpose of the present invention is intended to seek new anti-tumor prodrug from the Chinese medicine lilac daphne, extraction, the preparation method of such daphane diterpene-kind compound is provided, and studies their anti-tumor biological and medicinal use.Another object of the present invention is to provide the structure authentication method of this compounds.
(1) structural formula of a series of 6 new daphane diterpene-kind compounds of the present invention (Genkwanine II, Genkwanin III, Genkwanin IV, Genkwanin V, Genkwanin VI, Genkwanin VII) is as follows:
(2) new daphane diterpene-kind compound of the present invention (GenkwanineII, GenkwaninIII, GenkwaninIV, GenkwaninV, GenkwaninVI, GenkwaninVII) extraction, preparation method are as follows:
Selecting the dry flower 4000～6000g of thymelaeceae plant lilac daphne (Daphne genkwa.et zucc) for use is raw material, the volume ratio of 5～8 times of amounts is that cold soaking extracted 15～20 days under 60%～95% industrial alcohol room temperature, per 24～48h hour concentrating under reduced pressure once, the total medicinal extract 500～600g of accumulative total, total medicinal extract with 3～5L water suspendible after, use chloroform respectively, propyl carbinol extracts with the volume ratio of 1:1, extract 3～5 times, the chloroform layer dry extract 200～300g that obtains, n-butanol layer dry extract 200～300g, and concentrate back water liquid 10～15L;
Chloroform layer medicinal extract carries out the fast decompression column chromatography or the normal pressure column chromatography is separated, boiling range is 30～60 ℃, or 60～90 ℃ sherwood oil-acetone 50:1～2:1 (V/V) mixed solvent gradient elution, per 300～500ml volume is a cut, collect 40～50 cuts altogether, know through the silica gel thin-layer chromatography inspection, merge 6 cuts that obtain F1～F6
Cut F4 obtains 6 cuts of F4-1～F4-6 through chloroform-methanol 100:0～2:1 mixed solvent gradient elution, and F4-4 removes chlorophyll through MCI gel column chromatography methanol-water system gradient elution respectively, and silica gel column chromatography methylene chloride-methanol system wash-out separates, C
18Reversed-phase silica gel column chromatography and preparative high-performance liquid chromatographic methanol-water system are gradient elution separation, and Rotary Evaporators reclaims solvent and obtains white powder or transparence material, compound Genkwanin VI and Genkwanin VII;
Cut F6 is through chloroform-methanol 80:0～2:1 or methylene chloride-methanol 80:0～2:1 mixed solvent gradient elution, obtain 4 cuts of F6-1～F6-4, F6-2 removes chlorophyll through MCI gel column chromatography methanol-water system gradient elution respectively, silica gel column chromatography methylene chloride-methanol system wash-out separates, C
18Reversed-phase silica gel column chromatography and preparative high-performance liquid chromatographic methanol-water system are gradient elution separation, and Rotary Evaporators reclaims solvent and obtains white powder or transparence material, compound Genkwanin II, GenkwaninIII, GenkwaninIV, GenkwaninV.
The chloroform layer dry extract that obtains according to aforesaid extraction and extracting process obtains cut F1-F6 behind the sherwood oil-50:1～2:1 of acetone system (V/V) mixed solvent gradient elution silica gel column chromatography.
Cut F3 is through sherwood oil-acetone 80:0～2:1 or methylene chloride-methanol 100:0～2:1 mixed solvent gradient elution, obtain 7 cuts of F3-1～F3-7, cut F3-3 with methanol-water or alcohol-water system gradient elution, collects the cut of 20～50% wash-outs, through C through MCI gel column chromatography
18Reversed-phase silica gel column chromatography is collected the cut of 50～80% wash-outs with methanol-water system gradient elution, separates through anti-phase preparative high-performance liquid chromatographic, adopt methanol-water system gradient elution, behind the recovery solvent, obtain compound GenkwaninII altogether, GenkwaninIV, GenkwaninV.
The part that contains the daphane diterpene in the cut behind the wash-out is launched with the chloroform-methanol 30:1 of system～20:1, and its Rf value is 0.1～0.8; 254nm uviol lamp identification or developer differentiate that developer can be 10% sulfuric acid developer, or the inclined to one side alum acid of 10% sulfuric acid ammonium-developer, or 10% sulfuric acid-Vanillin developer.
(3) new daphane diterpene-kind compound of the present invention (Genkwanine II, GenkwaninIII, GenkwaninIV, GenkwaninV, GenkwaninVI GenkwaninVII) has better antitumor activity.
1. experiment material
1.1 be subjected to test product: compound GenkwanineII, GenkwaninIII, GenkwaninIV, GenkwaninV, GenkwaninVI, GenkwaninVII.
1.2 experimental cell strain and source HL-60: people's acute myeloid leukaemia cell
2. experimental technique
2.1 drug treating
The dissolving of medicine
Compound GenkwanineII, GenkwaninIII, GenkwaninIV, GenkwaninV, GenkwaninVI, GenkwaninVII is Powdered, uses the DMSO dissolving.Be made into the mother liquor that concentration is 100mmol/L, be stored in-20 ℃.Facing the time spent is 100 μ mol/L with corresponding nutrient solution with its dilution, 10 μ mol/L, and 1 μ mol/L experimentizes.When the sample of DMSO configuration experimentized, the final concentration of DMSO was 1 ‰.It is similar that bibliographical information yuanhuacine has anti-tumor activity and structure and testing compound, therefore chooses yuanhuacine as positive control drug, and concentration is 10 μ mol/L.
Administration is handled
The cell of taking the logarithm vegetative period is adjusted suitable cell density, is inoculated in 96 orifice plates, and 100 μ L/well are incubated at 37 ℃, 5%CO
2Incubator in.After cultivating 24h, be 100 μ mol/L with drug dilution, 10 μ mol/L, three concentration of 1 μ mol/L, 10 μ L/well, effect 48h.Set up blank group, administration group separately, establish 5 multiple holes for every group.
2.2 the measuring method of mtt assay
Behind the drug effect 48h, cell and 0.25mg/ml MTT are hatched 4h jointly under 37 ℃, every hole adds 100 μ l dimethyl sulfoxide (DMSO) (DMSO) behind the absorption nutrient solution, and the dissolving back uses microplate reader to measure its optical density(OD) OD value in 490nm fully.Be 100% with blank group OD value at last, calculate and respectively organize cell inhibitory rate.
2.3. statistical method
All data adopt the analysis of testing of SPSS (13.0) statistical packages.Each organize data with the mean value standard error (Mean ± S.E.) expression adopts One-Way ANOVA to estimate globality difference, and carry out Dunnett or Dunnett ' s T3 check organize between relatively.
3. experimental result
The inhibiting rate that table 1 compound survives tumour cell HL-60 (%) (M ± SE)
***P<0.001Vs?Control；
**P<0.01Vs?Control；
*P<0.05Vs?Control
The inhibiting rate (%) that table 2 compound YH-21 survives tumour cell HL-60 (M ± SE)
| control | 6.25μmol/L | 12.5μmol/L | 25μmol/L | 50μmol/L | IC50μmol/L | |
| YH-21 | 0.00±0.18 | 24.041±0.24 *** | 41.20±0.08 *** | 82.96±0.07 *** | 86.653±0.15 *** | 12.90 |
Can see that by above-mentioned experimental result after the related drug effect 48h of the present invention, 6 kinds of compounds all have certain lethal effect to the HL-60 cell strain.Wherein the effect of compound GenkwaninVI is the most obvious, secondly is GenkwaninVII.
Compound GenkwanineII, GenkwaninIII, GenkwaninIV is though GenkwaninV has certain lethal effect, DeGrain to the HL-60 cell strain.
The related daphane diterpenes of the present invention new compound, part has remarkable restraining effect to HL-60 people's acute myeloid leukaemia cell, and may have good inhibitory effect to other tumour cells, as A-549 people's lung cancer in non-cellule type cell, HeLa cervical cancer cell system etc., can be used for developing antitumor, anticancer class medicine, especially at clinical chemotherapy stage medicine.
Preparation method of the present invention is simple, favorable reproducibility, dna purity height.The compound that obtains has the better antitumor activity effect.
Description of drawings
Fig. 1 is Genkwanine II's
1H-NMR
Fig. 2 is Genkwanine II's
13C-NMR
Fig. 3 is the HSQC of Genkwanine II
Fig. 4 is the HMBC of Genkwanine II
Fig. 5 is GenkwanineIII's
1H-NMR
Fig. 6 is GenkwanineIII's
13C-NMR
Fig. 7 is the HSQC of GenkwanineIII
Fig. 8 is the HMBC of GenkwanineIII
Fig. 9 is GenkwaninIV's
1H-NMR
Figure 10 is GenkwaninIV's
13C-NMR
Figure 11 is the HMBC of GenkwaninIV
Figure 12 is GenkwaninV's
1H-NMR
Figure 13 is GenkwaninV's
13C-NMR
Figure 14 is the HSQC of GenkwaninV
Figure 15 is the HMBC of GenkwaninV
Figure 16 is GenkwaninVI's
1H-NMR
Figure 17 is GenkwaninVI's
13C-NMR
Figure 18 is the HMBC of GenkwaninVI
Figure 19 is GenkwaninVII's
1H-NMR
Figure 20 is GenkwaninVII's
13C-NMR
Figure 21 is the HMBC of GenkwaninVII
Embodiment
Selecting the dry flower 6000g of thymelaeceae plant lilac daphne (Daphne genkwa.et zucc) for use is raw material, the volume ratio of 6 times of amounts is that cold soaking extracted 20 days under the 95% industrial alcohol room temperature, every 48h hour concentrating under reduced pressure once adds up total medicinal extract 525g, after total medicinal extract is used 4L water suspendible, use chloroform respectively, propyl carbinol extracts with the volume ratio of 1:1, extracts the chloroform layer dry extract 245g that obtains 4 times, n-butanol layer dry extract 250g, and concentrate back water liquid 15L;
Chloroform layer medicinal extract carries out the fast decompression column chromatography or the normal pressure column chromatography is separated, boiling range is sherwood oil-acetone 30:1 of 30～60 ℃, 10:1,5:1,2:1 (V/V) mixed solvent gradient elution, every 500ml volume is a cut, collect 46 cuts altogether, know through the silica gel thin-layer chromatography inspection, merge 6 cuts that obtain F1～F6
Cut F4 is through chloroform-methanol 100:0,30:1,8:1,3:1,2:1 (V/V) mixed solvent gradient elution, obtain 6 cuts of F4-1～F4-6, F4-4 removes chlorophyll, the silica gel column chromatography methylene chloride-methanol 30:1 of system, 8:1 through MCI gel column chromatography methanol-water system gradient elution respectively, 2:1 (V/V) wash-out separates, C
18Reversed-phase silica gel column chromatography and preparative high-performance liquid chromatographic methanol-water system 70%, 80%, 90% are gradient elution separation, and Rotary Evaporators reclaims solvent and obtains white powder or transparence material, compound Genkwanin VI and Genkwanin VII;
Cut F6 is through chloroform-methanol 80:1,30:1,8:1,2:1 (V/V) mixed solvent gradient elution obtains 4 cuts of F6-1～F6-4, and F6-2 removes chlorophyll through MCI gel column chromatography methanol-water system gradient elution respectively, the silica gel column chromatography methylene chloride-methanol 30:1 of system, 10:1,2:1 (V/V) wash-out separates, C
18Reversed-phase silica gel column chromatography and preparative high-performance liquid chromatographic methanol-water system 60%, 80% are gradient elution separation, and Rotary Evaporators reclaims solvent and obtains white powder or transparence material, compound Genkwanin II, GenkwaninIII, GenkwaninIV, Genkwanin V.
Selecting the dry flower 6000g of thymelaeceae plant lilac daphne (Daphne genkwa.et zucc) for use is raw material, the volume ratio of 6 times of amounts is that cold soaking extracted 20 days under the 95% industrial alcohol room temperature, every 48h hour concentrating under reduced pressure once, the total medicinal extract 525g of accumulative total, after total medicinal extract is used 4L water suspendible, extract with the volume ratio of chloroform, extract 4 times, the chloroform layer dry extract 245g that obtains with 1:1.
Chloroform layer medicinal extract carries out the fast decompression column chromatography or the normal pressure column chromatography is separated, boiling range is sherwood oil-acetone 30:1 of 30～60 ℃, 10:1,5:1,2:1 (V/V) mixed solvent gradient elution, every 500ml volume is a cut, collect 46 cuts altogether, know through the silica gel thin-layer chromatography inspection, merge 6 cuts that obtain F1～F6
Cut F3 is through sherwood oil-acetone 80:0,50:1,10:1,5:1,2:1 (V/V) or methylene chloride-methanol 100:0,30:1,10:1,3:1,2:1 (V/V) mixed solvent gradient elution obtains 7 cuts of F3-1～F3-7, cut F3-3 through MCI gel column chromatography with methanol-water system gradient elution, collect the cut of 30% wash-out, through C
18Reversed-phase silica gel column chromatography is with methanol-water system gradient elution, collect the cut of 70% wash-out, separate through anti-phase preparative high-performance liquid chromatographic, adopt methanol-water system 55%, 65%, 80% gradient elution, after reclaiming solvent, obtain compound Genkwanin II altogether, GenkwaninIV, Genkwanin V.
New daphane diterpene-kind compound (Genkwanine II, GenkwaninIII, GenkwaninIV, Genkwanin V, GenkwaninVI, structure elucidation GenkwaninVII) of the present invention
Experimental example 1:
Genkwanine II, white amorphous powder (methyl alcohol) is soluble in chloroform, methyl alcohol.[α]
D 22-57.3 (c0.012, CHCl
3) high resolution mass spectrum provides quasi-molecular ion peak m/z 527.2258[M+Na]
+(calculated value is 527.2252), in conjunction with
1H,
13C NMR spectrum determines that molecular formula is C
27H
36O
9, show 10 degrees of unsaturation.
1H NMR (600MHz, CDCl
3) information provides the basic parent nucleus feature of daphane diterpene,
1δ in the H NMR spectrum
H8.12 (2H, m), 7.56 (1H, m) and 7.45 (prompting has a monosubstituted phenyl in the molecule for 2H, existence m); Three methyl feature proton signal δ
H1.87 (3H, s), 1.00 (3H, d, J=6.6), 0.96 (3H, d, J=7.2); A methylol hydrogen signal that is connected on the quaternary carbon, δ
H3.70 (1H, d, J=12) and 3.93 (1H, d, J=12); Four hydrogen signal δ on the oxygen carbon even
H4.13 (1H, d, J=10.2), 4.52 (1H, s), 3.24 (1H, bs), 5.95 (1H, s); And 2 feature hydrogen signal δ that are connected on the terminal double link
H5.17 (1H, s, H-16) and 5.14 (1H, s, H-16).
13Can see 27 carbon signals, three methyl carbon signal δ in the C NMR spectrum
C12.8,19.3,21.4; A pair of terminal double link carbon signal δ
C144.9 (C-15) and 114.0 (C-16), the carbon signal on one group of phenyl ring, wherein the feature quaternary carbon blackout of daphane diterpene ortho ester be the substitute is δ
C167.1 the ester carbonyl group carbon signal, illustrate the oxygen ring opening of ortho ester to form a benzoyloxy; δ
C67.3 connect the oxygen carbon signal with 64.5 feature, point out 6,7 to become the oxygen rings.
By hsqc spectrum all the hydrocarbon signals except that quaternary carbon have been carried out directly related, methyl hydrogen signal δ in HMBC spectrum
H1.87 (δ
C18.9) and δ
C144.9 (C-15), 114.0 (C-16), 73.7 have long-range being correlated with, and there is structure fragment 1 in prompting, and δ is described
H1.87 link to each other, be 17 methyl signals with terminal double link.δ
H1.00 (δ
C15.4) methyl hydrogen signal and δ
C33.9 36.1,73.1 have long-range being correlated with, and release structure fragment 2, δ
H0.96 (δ
C15.7) methyl hydrogen signal and δ
C35.1 31.4,75.5 have long-range being correlated with, its chemical environment is extremely similar to structure fragment 2, wouldn't belong to δ
H1.00 with 0.96 methyl signals be 18 or 19 methyl.H-14 (δ
H5.95) and δ
C167.1 long-range being correlated with arranged, prompting benzoyloxy and C-14 (δ
C77.2) be connected H-14 (δ
H5.95) go back and δ
C33.9 73.5,39.5,67.3 carbon signal has long-range relevant, releases structure fragment 3, illustrates and δ
C33.9 73.5 have relevant δ
H1.00 be 18 methyl hydrogen signals, δ
H0.96 be 19 methyl signals.δ
H4.13 (H-3) and δ
C15.7 (C-19), 35.1 (C-1), 31.4 (C-2), 79.8 (C-4), 76.7 (C-5) have long-range relevant, δ
H4.52 (H-5) and δ
C54.4 (C-10), 64.5 (C-6) have long-range relevant; δ
H3.24 (H-7) and δ
C68.0 (C-20), 64.5 (C-6), 39.5 (C-8), 77.2 (C-14) have long-range relevant; Can determine the structure of compound by above derivation, molecular formula is C
27H
36O
9, the molecular formula given with HR-ESIMS is consistent.Through sci finder literature search, do not appear in the newspapers, be a new compound, called after genkwanine II
Its
1H NMR spectrum,
13C NMR spectrum signal ownership and HMBC see Table 1, and relevant collection of illustrative plates is seen accompanying drawing 1-Fig. 4.
Table 1 gcnkwanin II's
1H NMR composes (600MHz, CDCl
3) and
13C NMR composes (150MHz, CDCl
3) data
Experimental example 2
GenkwanineIII, white crystals (methyl alcohol) is soluble in chloroform, methyl alcohol.[α]
D 22-39.3 (c0.019, CHCl
3) high resolution mass spectrum provides quasi-molecular ion peak m/z:527.2251[M+Na]
+(calculated value is 527.2252), in conjunction with
1H,
13C NMR spectrum determines that molecular formula is C
27H
36O
9, show 10 degrees of unsaturation, be the isomers of compound genkwanin II.
1H NMR (600MHz, CDCl
3) information provides the basic parent nucleus feature of daphane diterpene,
1δ in the H NMR spectrum
H8.00 (2H, m), 7.55 (1H, m) and 7.42 (prompting has a monosubstituted phenyl in the molecule for 2H, existence m); Three methyl feature hydrogen signal δ
H1.82 (3H, s), 0.82 (3H, d, J=7.2), 0.95 (3H, d, J=6.6); A methylol feature hydrogen signal δ who is connected on the quaternary carbon
H4.41 (1H, d, J=12) and 4.82 (1H, d J=12), shift to low than compound genkwanin II; Four hydrogen signal δ on the oxygen carbon even
H4.23 (1H, d, J=10.2), 5.02 (1H, s), 4.16 (1H, s), 4.10 (1H, s); And 2 feature hydrogen signal δ that are connected on the terminal double link
H5.12 (1H, s, H-16) and 5.11 (1H, s, H-16).
13Can see 27 carbon signals, three methyl carbon signal δ in the C NMR spectrum
C15.9,13.8,19.1; A pair of terminal double link carbon signal δ
C144.7 (C-15) and 114.8 (C-16), the carbon signal (δ on one group of phenyl ring
C129.9,129.6 * 2,128.5,133.3,128.5); Wherein the feature quaternary carbon blackout of daphane diterpene ortho ester be the substitute is δ
C167.2 the ester carbonyl group carbon signal, illustrate the oxygen ring opening of ortho ester to form a benzoyloxy, δ
C81.0 shift to low with company's oxygen carbon signal of 83.0 than genkwanin II, point out 6,7 oxygen ring openings.
Hsqc spectrum has carried out directly related to all the hydrocarbon signals except that quaternary carbon, methyl hydrogen signal δ in the HMBC spectrum
H1.82 (δ
C19.1) and δ
C144.8 (C-15), 114.0 (C-16), 74.8 have long-range being correlated with, and there is structure fragment 1 in prompting, and δ is described
H1.82 link to each other, be 17 methyl signals with terminal double link.δ
H0.84 (δ
C13.8) methyl hydrogen signal and δ
C35.7 36.4,73.1 have long-range being correlated with, and release structure fragment 2, δ
H0.95 (δ
C15.9) methyl hydrogen signal and δ
C34.1 31.9,74.1 have long-range being correlated with, its chemical environment is similar to structure fragment 2, wouldn't belong to δ
H0.84 with 0.95 methyl signals be 18 or 19 methyl.H-14 (δ
H4.10) shift to High-Field than compound genkwanin II, illustrate that 14 do not connect substituting group, simultaneously H-14 (δ
H4.10) and δ
C35.7 73.1,44.5,81.0 carbon signal has long-range relevant, releases structure fragment 3, illustrates and δ
C35.7 73.1 have relevant δ
H0.84 be 18 methyl hydrogen signals, δ
H0.96 be 19 methyl signals.(4.82 1H, d, J=12, H β-20) and δ
H4.41 (1H, d, J=12, H α-20) shifts to low than compound genkwanin II, illustrating has replacement on the hydroxyl, and sees and δ
C167.2 the ester carbonyl group carbon signal have relevantly, proved that benzoyl links to each other with C-20; δ
H4.23 (H-3) and δ
C15.9 (C-19), 34.1 (C-1), 31.9 (C-2), 90.8 (C-4), 83.1 (C-5) have long-range relevant, δ
H5.02 (H-5) and δ
C47.7 (C-10), 83.0 (C-6) have long-range relevant, δ
H4.16 (H-7) and δ
C67.4 (C-20), 80.3 (C-6), 72.0 (C-14) have long-range relevant; Also see δ in addition
H4.16 (H-7) and δ
C90.8 (C-4) have three strong long-range relevant, promptly 7 are connected by Sauerstoffatom with 4, illustrate that 7 and 4 s' hydroxyl is dehydrated into the oxygen ring, C-4, C-5, C-6, the chemical shift of C-7 is all shifted to low than compound genkwanin II, illustrates that also it forms five yuan of oxygen rings; The derivation structure meets HR-ESIMS and provides molecular weight, matches with degree of unsaturation 10, has determined the structure of compound thus.Through sci finder literature search, do not appear in the newspapers, be a new compound, called after genkwanineIII.
Its
1H NMR spectrum,
13C NMR spectrum signal ownership and HMBC see Table 2, and relevant collection of illustrative plates is seen accompanying drawing 5-Fig. 8.
Table 2 gcnkwaninIII's
1H NMR composes (600MHz, CDCl
3) and
13C NMR composes (150MHz, CDCl
3) data
Experimental example 3
GenkwaninIV, white crystals (methyl alcohol) is soluble in chloroform, methyl alcohol.[α]
D 22-14.9(c0.022，CHCl
3)。
1H NMR (600MHz, CDCl
3) information provides the basic parent nucleus feature of daphane diterpene,
1δ in the H NMR spectrum
H8.00 (2H, m), 7.55 (1H, m) and 7.45 (prompting has a monosubstituted phenyl in the molecule for 2H, existence m); Three methyl feature hydrogen signal δ
H1.92 (3H, s), 0.96 (3H, d, J=6.6), 0.98 (3H, d, J=7.2); A methylol feature hydrogen signal δ who is connected on the quaternary carbon
H3.47 (1H, d, J=11.4) and 3.95 (1H, d, J=11.4); Four hydrogen signal δ on the oxygen carbon even
H4.23 (1H, d, J=10.2), 4.59 (1H, s), 3.90 (1H, s), 5.79 (1H, s); And 2 feature hydrogen signal δ that are connected on the terminal double link
H5.20 (1H, s, H-16) and 5.19 (1H, s, H-16).
13Can see 27 carbon signals, three methyl carbon signal δ in the C NMR spectrum
C15.9,13.6,19.2; A pair of terminal double link carbon signal δ
C144.8 (C-15) and 114.9 (C-16), the carbon signal (δ on one group of phenyl ring
C129.6 * 2,128.6 * 2,130.0,133.0); Wherein the feature quaternary carbon blackout of daphane diterpene ortho ester be the substitute is δ
C166.6 the ester carbonyl group carbon signal, illustrate the oxygen ring opening of ortho ester to form a benzoyloxy, δ
C80.5 shift to low than genkwanin II with company's oxygen carbon signal of 83.8, point out 6,7 oxygen ring openings, its chemical displacement value is similar to genkwaninIII.
In the HMBC spectrum, methyl hydrogen signal δ
H1.92 with δ
C144.8 (C-15), 114.9 (C-16), 74.2 have long-range being correlated with, and there is structure fragment 1 in prompting, and δ is described
H1.92 link to each other, be 17 methyl signals with terminal double link.δ
H0.96 (δ
C13.6) methyl hydrogen signal and δ
C35.9 35.8,71.8 have long-range being correlated with, and release structure fragment 2, δ
H0.98 (δ
C15.9) methyl hydrogen signal and δ
C34.9 31.9,74.3 have long-range being correlated with, its chemical environment is similar to structure fragment 2, wouldn't belong to δ
H0.84 with 0.95 methyl signals be 18 or 19 methyl.H-14 (δ
H5.79) and δ
C166.6 long-range being correlated with arranged, prompting benzoyloxy and C-14 (δ
C72.2) be connected while H-14 (δ
H5.79) go back and δ
C35.9 71.8,45.8,80.5,74.2 carbon signal has long-range relevant, releases structure fragment 3, illustrates and δ
C35.9 71.8 have relevant δ
H0.96 be 18 methyl hydrogen signals, δ
H0.98 be 19 methyl signals; Do reference, δ with compound GenkwanineIII chemical displacement value
H4.23 (1H, d J=10.2) are defined as H-3; δ
H4.59 (1H s) is defined as H-5; δ
H1.59 (1H m) is defined as H-11; δ
H1.91 with δ
C34.9 (C-1), 71.8 (C-9), 45.8 (C-8) are relevant, are defined as H-10; δ
H1.84 with δ
C71.8 (C-9), 74.3 (C-3) have relevant, determine that it is H-1 β, δ
H1.18 with 31.9 (C-2) 49.0 (C-10), 71.8 (C-9) have relevant, are defined as H-1 α; While δ
H1.18 with δ
C15.9 have relevantly, determine δ
C15.9 be C-19; Because δ
H4.16 with C-20 (δ
C67.4), C-6 (δ
C80.3), C-14 (δ
C72.2) long-range being correlated with arranged, determine δ
H4.16 be H-7; GenkwanineIII is similar to compound, also see H-7 and C-4 have three strong long-range relevant, 7 are connected by Sauerstoffatom with 4, the hydroxyl that 7 and 4 are described is dehydrated into the oxygen ring, C-4, C-5, C-6, the chemical shift of C-7 is all shifted to low than compound genkwaninII, illustrates that it forms five yuan of oxygen rings; Determined the structure of compound to be the isomers of genkwanineIII by above analysis.Through the scifinder literature search, do not see the pertinent literature report, be a new compound, called after genkwaninIV.
Its
1H NMR spectrum,
13C NMR spectrum signal ownership and HMBC see Table 3, and relevant collection of illustrative plates is seen accompanying drawing 9-Figure 11.
Table 3 genkwaninIV's
1H NMR composes (600MHz, CDCl
3) and
13C NMR composes (150MHz, CDCl
3) data
Experimental example 4
GenkwaninV: white crystals (methyl alcohol) is soluble in chloroform, methyl alcohol.
1H NMR (600MHz, CDCl
3) information provides the basic parent nucleus feature of daphane diterpene,
1δ in the H NMR spectrum
H8.02 (2H, m), 7.55 (1H, m) and 7.42 (prompting has a monosubstituted phenyl in the molecule for 2H, existence m); Three methyl feature hydrogen signal δ
H1.82 (3H, s), 0.95 (3H, d, J=6.6), 0.91 (3H, d, J=7.2); A methylol feature hydrogen signal δ who is connected on the quaternary carbon
H3.70 (1H, d, J=12) and 3.90 (1H, d, J=12); Four hydrogen signal δ on the oxygen carbon even
H4.22 (1H, d, J=10.2), 5.50 (1H, s), 4.36 (1H, s), 5.91 (1H, s); And 2 feature hydrogen signal δ that are connected on the terminal double link
H5.09 (1H, s, H-16) and 5.07 (1H, s, H-16).
13Can see 27 carbon signals, three methyl carbon signal δ in the C NMR spectrum
C15.7,15.4,19.1; A pair of terminal double link carbon signal δ
C144.3 (C-15), the carbon signal (δ on 114.9 (C-16), one group of phenyl ring
C129.9 * 2,128.4 * 2,130.2,133.3); Wherein the feature quaternary carbon blackout of daphane diterpene ortho ester be the substitute is δ
C166.8 the ester carbonyl group carbon signal, illustrate the oxygen ring opening of ortho ester to form a benzoyloxy.
Hsqc spectrum has carried out directly related to all the hydrocarbon signals except that quaternary carbon; δ in the HMBC spectrum
H0.95 (δ
C15.4) methyl hydrogen signal and δ
C33.5 36.1,77.0 have long-range being correlated with, and release structure fragment 1, δ
H0.91 (δ
C15.7) methyl hydrogen signal and δ
C35.1 31.6,73.5 have long-range being correlated with, its chemical environment is similar to structure fragment 1, wouldn't belong to δ
H0.95 with 0.91 methyl signals be 18 or 19 methyl.δ
H1.82 with δ
C74.9,144.3 (C-15), 114.9 (C-16) have relevant; δ
H4.36 (δ
C75.329) and δ
C33.5 74.9,77.0,41.2 carbon signal has long-range relevant; δ
H5.91 (δ
C75.330) and δ
C77.0,73.6,86.1 have relevant, in addition also and δ
C166.1 the carbonyl carbon signal, and δ
C63.2 (C-20) have relevantly, release structure fragment 2; Structure fragment 2 explanation δ
H5.91 be 7 hydrogen signals, and benzoyl links to each other in C-7; H-7 and δ
C77.0 (C-9) related description δ is arranged
H0.95 be 18 methyl hydrogen signals, 0.91 is 19 methyl hydrogen signals; Determined the structure of compound by above analysis.Through the scifinder retrieval, do not see the pertinent literature report, be a new compound, called after genkwanin V.
Its
1H NMR spectrum,
13C NMR spectrum signal ownership and HMBC see Table 4, and relevant collection of illustrative plates is seen accompanying drawing 12-Figure 15.
Table 4 genkwaninV's
1H NMR composes (600MHz, CDCl
3) and
13C NMR composes (150MHz, CDCl
3) data
Experimental example 5
The unformed powder of GenkwaninVI (methyl alcohol) is soluble in chloroform, methyl alcohol.
1H NMR (600MHz, CDCl
3) information provides the basic parent nucleus feature of daphane diterpene,
1Two groups of monosubstituted phenyl signals, δ can be seen in low place in the HNMR spectrum
H8.06 (2H, m), 7.56 (1H, m) and 7.44 (2H m) is a benzoyl signal, δ
H7.74 (2H, m), 7.34 (3H m) is the phenyl signal that links to each other with daphane diterpene ortho ester quaternary carbon; Three methyl characteristic signal δ
H1.84 (3H, s), 1.31 (J=6.6), 1.05 (3H, d J=6.6), judge that according to the chemical displacement value of similar compound in the document it is respectively 17,18,19 methyl signals for 3H, d; A methylol feature hydrogen signal δ who is connected on the quaternary carbon
H4.02 (1H, d, J=12) and 5.09 (1H, d J=12) are 20 methylol signals, shift to low than compound genkwaninIV, illustrate that 20 are connected with substituting group, 2 feature hydrogen signal δ that are connected on the terminal double link
H5.07 (1H, s, H-16) and 4.92 (1H, s, H-16).
13In the CNMR spectrum, three methyl carbon signal δ
C19.2,20.8,13.0; A pair of terminal double link carbon signal δ
C146.5 (C-15) and 111.2 (C-16), the carbon signal (δ on two groups of phenyl ring
C129.8 * 3,129.2 * 2,128.4 * 2,127.9 * 2,126.1 * 2,133.2,136.0); δ
C117.0 be the feature quaternary carbon signal of daphane diterpene ortho ester; δ
C166.6 be the carbonyl carbon signal of benzoyloxy; δ
C60.2 connect the oxygen carbon signal with 64.2 feature, point out 6,7 to become the oxygen rings.
δ in the HMBC spectrum
H1.84 (CH3-17) and δ
C146.5 (C-15), 111.2 (C-16), 84.3 (C-13) have long-range relevant, release structure fragment 1; δ
H1.31 (CH
3-18) and δ
C35.3 (C-11), 35.9 (C-12), 80.0 (C-9) have long-range relevant, release structure fragment 2; δ
H1.05 (CH
3-19) and δ
C34.3 (C-1), 36.7 (C-2), 78.3 (C-3) have long-range relevant, release structure fragment 3; 20 hydrogen signal δ
H5.09 shift to low, and and δ
C166.6 long-range being correlated with arranged, illustrates that benzoyl links to each other δ with 20
H4.52 (H-14) and δ
C80.0 (C-9), 117.0 (C=O) have relevant; δ
H3.43 (H-7) and δ
C36.5 (C-8), 80.0 (C-9), 82.5 (C-14) have long-range relevant; δ
H3.05 (1H, d, J=3.0, H-8) and δ
C60.2 (C-7), 64.2 (C-6) have long-range relevant; δ
H2.77 (H-10) with 80.1 (C-4), 71.3 (C-5), 35.3 (C-11) have long-range relevant; δ
H2.77 (H-11) and δ
C48.6 (C-10), 35.3 (C-11), 20.8 (C-18) have long-range relevant; Will by HMBC
1H,
13The C signal belongs to, and has determined the structure of compound; Carry out literature search by sci finder, do not appear in the newspapers, illustrate that this compound is a new compound, called after genkwaninVI.
Its
1H NMR spectrum,
13C NMR spectrum signal ownership and HMBC see Table 5, and relevant collection of illustrative plates is seen accompanying drawing 16-Figure 18.
Table 5 genkwaninVI's
1H NMR composes (600MHz, CDCl
3) and
13C NMR composes (150MHz, CDCl
3) data
Experimental example 6
The unformed powder of GenkwaninVII (methyl alcohol) is soluble in chloroform, methyl alcohol.
1H NMR (600MHz, CDCl
3) information provides the basic parent nucleus feature of daphane diterpene,
1Two groups of monosubstituted phenyl signals, δ can be seen in low place in the HNMR spectrum
H8.06 (2H, m), 7.63 (1H, m) and 7.50 (2H m) is a benzoyl signal, δ
H7.75 (2H, m), 7.36 (3H m) is the phenyl signal that links to each other with daphane diterpene ortho ester quaternary carbon; Three methyl characteristic signal δ
H1.84 (3H, s), 1.35 (J=6.6), 1.14 (3H, d J=6), judge that according to the chemical displacement value of similar compound in the document it is respectively 17,18,19 methyl signals for 3H, d; A methylol feature hydrogen signal δ who is connected on the quaternary carbon
H3.81 (1H, d, J=12) and 3.86 (1H, d are 20 methylol signals J=12), and genkwaninVI shifts to High-Field than compound, illustrate that 20 do not connect substituting group; 2 feature hydrogen signal δ that are connected on the terminal double link
H5.03 (1H, s, H-16) and 4.90 (1H, s, H-16); δ
H4.84 (H-3) shift to low, illustrate that 3 are connected with substituting group than compound genkwaninVI;
13In the C NMR spectrum, three methyl carbon signal δ
C19.2,20.8,13.0; A pair of terminal double link carbon signal δ
C146.5 (C-15) and 111.2 (C-16), the carbon signal (δ on two groups of phenyl ring
C129.8 * 3,129.2 * 2,128.4 * 2,127.9 * 2,126.1 * 2,133.2,136.0); δ
C117.0 be the feature quaternary carbon signal of daphane diterpene ortho ester; δ
C166.6 be the carbonyl carbon signal of benzoyloxy; δ
C60.2 connect the oxygen carbon signal with 64.2 feature, point out 6,7 to become the oxygen rings.
In the HMBC spectrum, δ
H1.81 (CH
3-17) and δ
C146.6 (C-15), 111.4 (C-16), 84.6 (C-13) have long-range relevant, release structure fragment 1; δ
H1.50 (CH
3-18) and δ
C35.5 (C-11), 36.3 (C-12), 80.5 (C-9) have long-range relevant, δ
H1.14 (CH
3-19) and δ
C36.1 (C-1), 36.55 (C-2), 82.9 (C-3) have long-range relevant, δ
H2.88 (H-10) with 36.1 (C-1), 80.5 (C-9), 81.8 (C-5) have long-range relevant, 3 hydrogen signal δ
H4.84 shift to low, and and δ
C168.8 long-range being correlated with arranged, illustrates that benzoyl links to each other with 3.More than analyze and release structure fragment 2; δ
H4.49 (H-14) and δ
C80.5 (C-9), 117.6 (C=O) have relevant; δ
H3.43 (H-7) and δ
C36.59 (C-8), 80.5 (C-9), 82.7 (C-14), 66.3 (C-20) have relevant; δ
H4.13 (H-5) and δ
C82.9 (C-3), 60.7 (C-6), 64.2 (C-8) have long-range relevant; By more than
1H NMR spectrum,
13C NMR spectrum, the structure of compound has been determined in the analysis that reaches the HMBC spectrum, and signal is belonged to; Through sci finder literature search, do not appear in the newspapers, illustrate that this compound is a new compound, called after genkwaninVII.
Its
1H NMR spectrum,
13C NMR spectrum signal ownership and HMBC see Table 6, and relevant collection of illustrative plates is seen accompanying drawing 19-Figure 21.
Table 6 genkwaninVII's
1H NMR composes (600MHz, CDCl
3) and
13C NMR composes (150MHz, CDCl
3) data
Claims (7)
1. new daphane diterpene-kind compound in the lilac daphne, it is characterized in that: this compounds has following structural formula:
2. the preparation method of new daphane diterpene-kind compound in the lilac daphne as claimed in claim 1, it is characterized in that: this method is:
Selecting the dry flower 4000～6000g of thymelaeceae plant lilac daphne (Daphne genkwa.et zucc) for use is raw material, the volume ratio of 5～8 times of amounts is that cold soaking extracted 15～20 days under 60%～95% industrial alcohol room temperature, per 24～48h hour concentrating under reduced pressure once, the total medicinal extract 500～600g of accumulative total, total medicinal extract with 3～5L water suspendible after, use chloroform respectively, propyl carbinol extracts with the volume ratio of 1:1, extract 3～5 times, the chloroform layer dry extract 200～300g that obtains, n-butanol layer dry extract 200～300g, and concentrate back water liquid 10～15L;
Chloroform layer medicinal extract carries out the fast decompression column chromatography or the normal pressure column chromatography is separated, boiling range is 30～60 ℃, or 60～90 ℃ sherwood oil-acetone 50:1～2:1 (V/V) mixed solvent gradient elution, per 300～500ml volume is a cut, collect 40～50 cuts altogether, know through the silica gel thin-layer chromatography inspection, merge 6 cuts that obtain F1～F6
Cut F4 is through chloroform-methanol 100:0～2:1 or methylene chloride-methanol 100:0～2:1 mixed solvent gradient elution, obtain 6 cuts of F4-1～F4-6, F4-4 removes chlorophyll through MCI gel column chromatography methanol-water system gradient elution respectively, silica gel column chromatography methylene chloride-methanol system wash-out separates, C
18Reversed-phase silica gel column chromatography and preparative high-performance liquid chromatographic methanol-water system are gradient elution separation, and Rotary Evaporators reclaims solvent and obtains white powder or transparence material, compound Genkwanin VI and Genkwanin VII;
Cut F6 is through chloroform-methanol 80:0～2:1 or methylene chloride-methanol 80:0～2:1 mixed solvent gradient elution, obtain 4 cuts of F6-1～F6-4, F6-2 removes chlorophyll through MCI gel column chromatography methanol-water system gradient elution respectively, silica gel column chromatography methylene chloride-methanol system wash-out separates, C
18Reversed-phase silica gel column chromatography and preparative high-performance liquid chromatographic methanol-water system are gradient elution separation, and Rotary Evaporators reclaims solvent and obtains white powder or transparence material, compound Genkwanin II, GenkwaninIII, Genkwanin IV, Genkwanin V.
3. the preparation method of new daphane diterpene-kind compound in the lilac daphne according to claim 2, it is characterized in that: this method is:
The chloroform layer dry extract that described extraction and extracting process obtain obtains cut F1-F6 behind the sherwood oil-50:1～2:1 of acetone system (V/V) mixed solvent gradient elution silica gel column chromatography.
Cut F3 is through sherwood oil-acetone 80:0～2:1 or methylene chloride-methanol 100:0～2:1 mixed solvent gradient elution, obtain 7 cuts of F3-1～F3-7, cut F3-3 with methanol-water or alcohol-water system gradient elution, collects the cut of 20～50% wash-outs, through C through MCI gel column chromatography
18Reversed-phase silica gel column chromatography is collected the cut of 50～80% wash-outs with methanol-water system gradient elution, separates through anti-phase preparative high-performance liquid chromatographic, adopt methanol-water system gradient elution, behind the recovery solvent, obtain compound Genkwanin II altogether, GenkwaninIV, Genkwanin V.
4. the preparation method of new daphane diterpene-kind compound in the lilac daphne according to claim 2, it is characterized in that: the 30:1 of the part chloroform-methanol system～20:1 that contains the daphane diterpene in the cut behind the wash-out launches, the Rf value is 0.1～0.8,254nm uviol lamp identification or developer are differentiated, developer can be 10% sulfuric acid developer, or the inclined to one side alum acid of 10% sulfuric acid ammonium-developer, or 10% sulfuric acid-Vanillin developer.
5. daphane diterpene-kind compound as claimed in claim 1 is characterized in that: the drug regimen that described compound can be formed with one or more pharmaceutically acceptable carriers, make acceptable forms clinically.
6. daphane diterpenes chemical combination is in the purposes of preparation in the cancer therapy drug.
7. purposes according to claim 6, described cancer therapy drug can be the medicines at HL-60 people's acute myeloid leukaemia cell cancer.
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