CN101531644B - 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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- 229930004069 diterpene Natural products 0.000 title claims abstract description 24
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- -1 daphnane diterpene compounds Chemical class 0.000 title abstract description 19
- 150000001875 compounds Chemical class 0.000 claims abstract description 70
- SDTOABMYDICPQU-UHFFFAOYSA-N Genkwanin Natural products C=1C(C)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(O)C=C1 SDTOABMYDICPQU-UHFFFAOYSA-N 0.000 claims abstract description 39
- 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 39
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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
Isolated From Thymelaeaceae Species lilac daphne (Daphne genkwa sieb.et Zucc) is traditional removing water retention by purgation medicine.The aspect researchs such as at present, induced labor antitumor to it, kobadrin, desinsection, anti-inflammatory, immunologic function adjusting are more.Chinese scholars from the flower and alabastrum of Isolated From Thymelaeaceae Species 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 study hotspot, 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 seek 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 Chinese Drug, yuan Hua, 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 Structural Identification 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, GenkwaninIV, Genkwanin V, GenkwaninVI, GenkwaninVII) is as follows:
(2) extraction, the preparation method of new daphane diterpene-kind compound of the present invention (Genkwanine II, GenkwaninIII, GenkwaninIV, Genkwanin V, GenkwaninVI, GenkwaninVII) are as follows:
Selecting the dry flower 4000～6000g of Isolated From Thymelaeaceae Species lilac daphne (Daphne genkwa.et zucc) 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 respectively chloroform, propyl carbinol extracts with 1: 1 volume ratio, extract 3～5 times, the chloroform layer dry extract 200～300g that obtains, n-butanol layer dry extract 200～300g, and water liquid 10～15L after concentrated;
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 50: 1～2: 1 (V/V) mixed solvents of sherwood oil-acetone of 60～90 ℃ gradient elution, per 300～500ml volume is a cut, collect altogether 40～50 cuts, know through the silica gel thin-layer chromatography inspection, merge 6 cuts that obtain F1～F6
Cut F4 is through 100: 0～2: 1 mixed solvent gradient elutions of chloroform-methanol, obtain 6 cuts of F4-1～F4-6, 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 GenkwaninVI and GenkwaninVII;
Cut F6 is through chloroform-methanol 80: 0～2: 1 or 80: 0～2: 1 mixed solvent gradient elutions of methylene chloride-methanol, 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, Genkwanin V.
The chloroform layer dry extract that obtains according to aforesaid extraction and extracting process obtains cut F 1-F6 behind sherwood oil-acetone system 50: 1～2: 1 (V/V) mixed solvents gradient elution silica gel column chromatography.
Cut F3 is through sherwood oil-acetone 80: 0～2: 1 or 100: 0～2: 1 mixed solvent gradient elutions of methylene chloride-methanol, obtain 7 cuts of F3-1～F3-7, cut F3-3 with methanol-water or ethanol-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 altogether compound Genkwanin II, GenkwaninIV, Genkwanin V.
The part that contains the daphane diterpene in the cut behind the wash-out was launched with the chloroform-methanol system in 30: 1～20: 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, Genkwanin V, GenkwaninVI, GenkwaninVII) has preferably anti-tumor activity.
1. experiment material
1.1 be subjected to test product: compound Genkwanine II, GenkwaninIII, GenkwaninIV, Genkwanin V, GenkwaninVI, GenkwaninVII.
1.2 experimental cell strain and source HL-60: people's acute myeloid leukemia cells in children
2. experimental technique
2.1 drug treating
The dissolving of medicine
Compound Genkwanine II, GenkwaninIII, GenkwaninIV, Genkwanin V, 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 tests.When the sample of DMSO configuration was tested, 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 processed
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.
2.2MTT the measuring method of method
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, uses microplate reader to measure its optical density(OD) OD value in 490nm fully after the dissolving.Take blank group OD value as 100%, calculate and respectively organize cell inhibitory rate at last.
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 Genkwanine II, GenkwaninIII, GenkwaninIV is though Genkwanin V 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 leukemia cells in children, and may have preferably restraining effect to other tumour cells, such 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 for clinical chemotherapy stage medicine.
Preparation method of the present invention is simple, favorable reproducibility, and dna purity is high.The compound that obtains has preferably anti-tumor 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
Preparation Example 1
Selecting the dry flower 6000g of Isolated From Thymelaeaceae Species lilac daphne (Daphne genkwa.et zucc) 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 respectively chloroform, propyl carbinol extracts with 1: 1 volume ratio, extracts the chloroform layer dry extract 245g that obtains 4 times, n-butanol layer dry extract 250g, and water liquid 15L after concentrated;
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 elutions, every 500ml volume is a cut, collect altogether 46 cuts, know through the silica gel thin-layer chromatography inspection, merge 6 cuts that obtain F1～F6
Cut F4 was through chloroform-methanol 100: 0,30: 1,8: 1,3: 1,2: 1 (V/V) mixed solvent gradient elutions, obtain 6 cuts of F4-1～F4-6, F4-4 removes chlorophyll, silica gel column chromatography methylene chloride-methanol system 30: 1,8: 1 through MCI gel column chromatography methanol-water system gradient elution respectively, (V/V) wash-out separated in 2: 1, 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 GenkwaninVI and GenkwaninVII;
Cut F6 was through chloroform-methanol 80: 1,30: 1,8: 1,2: 1 (V/V) mixed solvent gradient elutions obtain 4 cuts of F6-1～F6-4, and F6-2 removes chlorophyll through MCI gel column chromatography methanol-water system gradient elution respectively, silica gel column chromatography methylene chloride-methanol system 30: 1,10: 1, (V/V) wash-out separated in 2: 1, 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.
Preparation Example 2
Selecting the dry flower 6000g of Isolated From Thymelaeaceae Species lilac daphne (Daphne genkwa.et zucc) 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 with 1: 1, extract 4 times, the chloroform layer dry extract 245g that obtains.
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 elutions, every 500ml volume is a cut, collect altogether 46 cuts, know through the silica gel thin-layer chromatography inspection, merge 6 cuts that obtain F1～F6
Cut F3 was 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 elutions obtain 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 altogether compound Genkwanin II, GenkwaninIV, Genkwanin V.
The structure elucidation of new daphane diterpene-kind compound of the present invention (Genkwanine II, GenkwaninIII, GenkwaninIV, Genkwanin V, GenkwaninVI, GenkwaninVII)
Experimental example 1:
Genkwanine II, white amorphous powder (methyl alcohol) is soluble in chloroform, methyl alcohol.[α]
D 22-57.3 (c 0.012, 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
H(8.12 2H, m), the existence of 7.56 (1H, m) and 7.45 (2H, m) has a monosubstituted phenyl in the prompting molecule; Three methyl feature proton signal δ
H(1.87 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
H(3.70 1H, d, J=12) and 3.93 (1H, d, J=12); Four hydrogen signal δ on the oxygen carbon even
H(4.13 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
H(5.17 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 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 distant relation, and there is structure fragment 1 in prompting, and δ is described
H1.87 link to each other with terminal double link, be 17 methyl signals.δ
H1.00 (δ
C15.4) methyl hydrogen signal and δ
C33.9 36.1,73.1 have distant relation, pushing-out structure fragment 2, δ
H0.96 (δ
C15.7) methyl hydrogen signal and δ
C35.1 31.4,75.5 have distant relation, 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 distant relation is 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 distant relation, pushing-out 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 distant relation, δ
H4.52 (H-5) and δ
C54.4 (C-10), 64.5 (C-6) have distant relation; δ
H3.24 (H-7) and δ
C68.0 (C-20), 64.5 (C-6), 39.5 (C-8), 77.2 (C-14) have distant relation; 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, have no report, 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 1genkwanin 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
H(8.00 2H, m), the existence of 7.55 (1H, m) and 7.42 (2H, m) has a monosubstituted phenyl in the prompting molecule; Three methyl feature hydrogen signal δ
H(1.82 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) shifts to low than compound genkwanin II; Four hydrogen signal δ on the oxygen carbon even
H(4.23 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
H(5.12 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 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 distant relation, and there is structure fragment 1 in prompting, and δ is described
H1.82 link to each other with terminal double link, be 17 methyl signals.δ
H0.84 (δ
C13.8) methyl hydrogen signal and δ
C35.7 36.4,73.1 have distant relation, pushing-out structure fragment 2, δ
H0.95 (δ
C15.9) methyl hydrogen signal and δ
C34.1 31.9,74.1 have distant relation, 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 distant relation, pushing-out 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 distant relation, δ
H5.02 (H-5) and δ
C47.7 (C-10), 83.0 (C-6) have distant relation, δ
H4.16 (H-7) and δ
C67.4 (C-20), 80.3 (C-6), 72.0 (C-14) have distant relation; Also see in addition δ
H4.16 (H-7) and δ
C90.8 (C-4) three strong distant relations are arranged, namely 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 thus the structure of compound.Through sci finder literature search, have no report, 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 2genkwaninIII'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(c 0.022，CHCl
3)。
1H NMR (600MHz, CDCl
3) information provides the basic parent nucleus feature of daphane diterpene,
1δ in the H NMR spectrum
H(8.00 2H, m), the existence of 7.55 (1H, m) and 7.45 (2H, m) has a monosubstituted phenyl in the prompting molecule; Three methyl feature hydrogen signal δ
H(1.92 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
H(3.47 1H, d, J=11.4) and 3.95 (1H, d, J=11.4); Four hydrogen signal δ on the oxygen carbon even
H(4.23 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
H(5.20 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 with company's oxygen carbon signal of 83.8 than genkwanin II, 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 distant relation, and there is structure fragment 1 in prompting, and δ is described
H1.92 link to each other with terminal double link, be 17 methyl signals.δ
H0.96 (δ
C13.6) methyl hydrogen signal and δ
C35.9 35.8,71.8 have distant relation, pushing-out structure fragment 2, δ
H0.98 (δ
C15.9) methyl hydrogen signal and δ
C34.9 31.9,74.3 have distant relation, 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 distant relation is 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 distant relation, pushing-out 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) is 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) distant relation is arranged, determine δ
H4.16 be H-7; GenkwanineIII is similar to compound, see that also H-7 and C-4 have three strong distant relations, 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; By above Analysis deterrmination the structure of compound, be the isomers of genkwanineIII.Through the scifinder literature search, have no 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 3genkwaninIV's
1H NMR composes (600MHz, CDCl
3) and
13C NMR composes (150MHz, CDCl
3) data
Experimental example 4
Genkwanin V: 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
H(8.02 2H, m), the existence of 7.55 (1H, m) and 7.42 (2H, m) has a monosubstituted phenyl in the prompting molecule; Three methyl feature hydrogen signal δ
H(1.82 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
H(3.70 1H, d, J=12) and 3.90 (1H, d, J=12); Four hydrogen signal δ on the oxygen carbon even
H(4.22 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
H(5.09 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 quaternary carbon; δ in the HMBC spectrum
H0.95 (δ
C15.4) methyl hydrogen signal and δ
C33.5 36.1,77.0 have distant relation, pushing-out structure fragment 1, δ
H0.91 (δ
C15.7) methyl hydrogen signal and δ
C35.1 31.6,73.5 have distant relation, 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 distant relation; 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 relevant, pushing-out 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; By above Analysis deterrmination the structure of compound.Through the scifinder retrieval, have no 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 4genkwaninV'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) are a benzoyl signal, δ
H7.74 (2H, m), 7.34 (3H, m) are the phenyl signal that links to each other with daphane diterpene ortho ester quaternary carbon; Three methyl characteristic signal δ
H(1.84 3H, s), 1.31 (3H, d, 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; 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) is 20 methylol signals, shifts to low than compound genkwaninIV, illustrates that 20 are connected with substituting group, 2 feature hydrogen signal δ that are connected on the terminal double link
H(5.07 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 (CH
3-17) and δ
C146.5 (C-15), 111.2 (C-16), 84.3 (C-13) have distant relation, pushing-out structure fragment 1; δ
H1.31 (CH
3-18) and δ
C35.3 (C-11), 35.9 (C-12), 80.0 (C-9) have distant relation, pushing-out structure fragment 2; δ
H1.05 (CH
3-19) and δ
C34.3 (C-1), 36.7 (C-2), 78.3 (C-3) have distant relation, pushing-out structure fragment 3; 20 hydrogen signal δ
H5.09 shift to low, and and δ
C166.6 distant relation is 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 distant relation; δ
H(3.05 1H, d, J=3.0, H-8) and δ
C60.2 (C-7), 64.2 (C-6) have distant relation; δ
H2.77 (H-10) with 80.1 (C-4), 71.3 (C-5), 35.3 (C-11) have distant relation; δ
H2.77 (H-11) and δ
C48.6 (C-10), 35.3 (C-11), 20.8 (C-18) have distant relation; Will by HMBC
1H,
13The C signal belongs to, and has determined the structure of compound; Carry out literature search by sci finder, have no report, 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 5genkwaninVI'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) are a benzoyl signal, δ
H7.75 (2H, m), 7.36 (3H, m) are the phenyl signal that links to each other with daphane diterpene ortho ester quaternary carbon; Three methyl characteristic signal δ
H(1.84 3H, s), 1.35 (3H, d, 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; A methylol feature hydrogen signal δ who is connected on the quaternary carbon
H3.81 (1H, d, J=12) and 3.86 (1H, d, J=12) is 20 methylol signals, genkwaninVI shifts to High-Field than compound, illustrates that 20 do not connect substituting group; 2 feature hydrogen signal δ that are connected on the terminal double link
H(5.03 1H, s, H-16) and 4.90 (1H, s, H-16); δ
H4.84 (H-3) shift to low than compound genkwaninVI, illustrate that 3 are connected with substituting group;
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 distant relation, pushing-out structure fragment 1; δ
H1.50 (CH
3-18) and δ
C35.5 (C-11), 36.3 (C-12), 80.5 (C-9) have distant relation, δ
H1.14 (CH
3-19) and δ
C36.1 (C-1), 36.55 (C-2), 82.9 (C-3) have distant relation, δ
H2.88 (H-10) with 36.1 (C-1), 80.5 (C-9), 81.8 (C-5) have distant relation, 3 hydrogen signal δ
H4.84 shift to low, and and δ
C168.8 distant relation is arranged, illustrates that benzoyl links to each other with 3.More than analyze pushing-out 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 distant relation; By more than
1H NMR spectrum,
13C NMR spectrum, and the Analysis deterrmination of HMBC spectrum the structure of compound, and signal belonged to; Through sci finder literature search, have no report, 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 6genkwaninVII's
1H NMR composes (600MHz, CDCl
3) and
13C NMR composes (150MHz, CDCl
3) data
Claims (7)
1. the daphane diterpene-kind compound in the lilac daphne, it is characterized in that: this compounds has following structural formula:
GenkwanineⅡ GenkwaninⅢ
GenkwanineⅣ GenkwanineⅤ
GenkwaninⅥ GenkwaninⅦ 。
2. the preparation method of the daphane diterpene-kind compound in the lilac daphne as claimed in claim 1, it is characterized in that: the method is:
Select the Isolated From Thymelaeaceae Species lilac daphne
Daphne genkwa.etDry flower 4000 ~ 6000g of zucc 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 respectively chloroform, propyl carbinol extracts with the volume ratio of 1:1, extracts the chloroform layer dry extract 200 ~ 300g that obtains 3 ~ 5 times, n-butanol layer dry extract 200 ~ 300g, and water liquid 10 ~ 15L after concentrated;
Chloroform layer medicinal extract carries out the fast decompression column chromatography or the normal pressure column chromatography is separated, and boiling range is 30 ~ 60 ℃, or 60 ~ 90 ℃ sherwood oil-acetone 50:1 ~ 2:1(
V/V) the mixed solvent gradient elution, per 300 ~ 500ml volume is a cut, collects altogether 40 ~ 50 cuts, knows through the silica gel thin-layer chromatography inspection, merges 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, Genkwanin III, Genkwanin IV, Genkwanin V.
3. the preparation method of daphane diterpene-kind compound in the lilac daphne according to claim 2, it is characterized in that: the method is:
The chloroform layer dry extract that described extraction and extracting process obtain is through sherwood oil-50:1 ~ 2:1(of acetone system
V/V) obtain cut F1-F6 behind the 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 ethanol-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 altogether compound Genkwanin II, the Genkwanin IV, the Genkwanin V.
4. the preparation method of daphane diterpene-kind compound in the lilac daphne according to claim 2, it is characterized in that: contain the 30:1 of part chloroform-methanol system of daphane diterpene ~ 20:1 in the cut behind the wash-out and launch, 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 form with one or more pharmaceutically acceptable carriers, make clinically acceptable formulation.
6. daphane diterpenes chemical combination claimed in claim 1 is in the purposes of preparation in the cancer therapy drug.
7. purposes according to claim 6, described cancer therapy drug is the medicine for HL-60 people's acute myeloid leukemia cells in children cancer.
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| CN102532250B (en) * | 2011-11-07 | 2013-09-25 | 中国科学院昆明植物研究所 | Przewalsikone A compound with anti-tumor activity and preparation method and application thereof |
| CN105585574A (en) * | 2015-12-28 | 2016-05-18 | 吴芊葭 | Diterpene compound and preparation method thereof |
| CN110538243A (en) * | 2018-05-29 | 2019-12-06 | 复旦大学 | Preparation method of active total diterpene in Thymelaeaceae plant and application thereof in pharmacy |
| CN110305093B (en) * | 2019-04-11 | 2021-03-23 | 沈阳药科大学 | Guaiane type sesquiterpenes and preparation method and application thereof |
| CN110305092B (en) * | 2019-04-11 | 2021-03-23 | 沈阳药科大学 | Guaiane sesquiterpenes and preparation and application thereof |
| CN111606787B (en) * | 2020-07-02 | 2022-08-23 | 西安市红会医院 | Fatty alcohol compound and preparation method thereof |
| CN112538088B (en) * | 2020-12-22 | 2022-02-22 | 中山大学 | Daphnane diterpene for resisting prostatic cancer and preparation method thereof |
| CN114031579B (en) * | 2021-11-11 | 2023-03-07 | 沈阳药科大学 | Preparation and application of daphnane diterpenoid compounds in lilac daphne flower buds |
| CN116854704B (en) * | 2023-07-06 | 2024-05-07 | 沈阳药科大学 | Daphnane diterpenoid derivative with anti-liver cancer activity and preparation method and application thereof |
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| Bo-Young Park等.Daphnane diterpene esters isolated from flower buds of Daphne genkwa induce apoptosis in human myelocytic HL-60 cells and suppress tumor growth in Lewis lung carcinoma (LLC)-inoculated mouse model.《Journal of Ethnopharmacology》.2006,第111卷496-503. * |
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