CN108553476A - A kind of preparation method of hinokiflavone derivative and the application of anti-breast cancer - Google Patents
A kind of preparation method of hinokiflavone derivative and the application of anti-breast cancer Download PDFInfo
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- CN108553476A CN108553476A CN201810648480.0A CN201810648480A CN108553476A CN 108553476 A CN108553476 A CN 108553476A CN 201810648480 A CN201810648480 A CN 201810648480A CN 108553476 A CN108553476 A CN 108553476A
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- hinokiflavone
- breast cancer
- acetyl
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- WTDHMFBJQJSTMH-UHFFFAOYSA-N hinokiflavone Chemical class C1=CC(O)=CC=C1C1=CC(=O)C2=C(O)C(OC=3C=CC(=CC=3)C=3OC4=CC(O)=CC(O)=C4C(=O)C=3)=C(O)C=C2O1 WTDHMFBJQJSTMH-UHFFFAOYSA-N 0.000 title claims abstract description 58
- 206010006187 Breast cancer Diseases 0.000 title claims abstract description 46
- 208000026310 Breast neoplasm Diseases 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- HLFVFEBKCLAGBY-UHFFFAOYSA-N hinokiflavone Natural products Oc1ccc(cc1)C2=COc3cc(O)c(Oc4ccc(cc4)C5=CC(=O)c6c(O)cc(O)cc6O5)c(O)c3C2=O HLFVFEBKCLAGBY-UHFFFAOYSA-N 0.000 claims abstract description 43
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- 238000000605 extraction Methods 0.000 claims abstract description 5
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 16
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 16
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 15
- -1 acetyl tert-butyl Chemical group 0.000 claims description 15
- 238000001035 drying Methods 0.000 claims description 15
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 12
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- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 8
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- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- WGYKZJWCGVVSQN-UHFFFAOYSA-N propylamine Chemical class CCCN WGYKZJWCGVVSQN-UHFFFAOYSA-N 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- CHQMHPLRPQMAMX-UHFFFAOYSA-L sodium persulfate Substances [Na+].[Na+].[O-]S(=O)(=O)OOS([O-])(=O)=O CHQMHPLRPQMAMX-UHFFFAOYSA-L 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- 229960004355 vindesine Drugs 0.000 description 1
- UGGWPQSBPIFKDZ-KOTLKJBCSA-N vindesine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(N)=O)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1N=C1[C]2C=CC=C1 UGGWPQSBPIFKDZ-KOTLKJBCSA-N 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C07—ORGANIC CHEMISTRY
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- C07H1/00—Processes for the preparation of sugar derivatives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
- C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
- C07H5/06—Aminosugars
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Abstract
The invention belongs to pharmaceutical technology fields, the preparation method of hinokiflavone derivative WG020 a kind of and the application in anti-breast cancer, wherein hinokiflavone is the extract from Rock lily plant selaginella doederleinii, and hinokiflavone derivative WG020 is synthesized on the basis of extraction obtains hinokiflavone.WG020 has been carried out cytotoxicity experiment (mtt assay) by the present invention, flow cytometer showed (FCM) Apoptosis of Breast Cancer is tested, protein immunoblot (WB) analyzes Apoptosis mechanism experiment and Nasopharyngeal neoplasms Inhibition test, the result shows that WG020 has time dependence and concentration dependent to breast cancer cell activity influence, there can be good inhibiting effect to breast cancer cell transfer with induced breast cancer apoptosis.Therefore, hinokiflavone derivative WG020 can be used as the lead compound for developing new anti-breast cancer, and the present invention provides a kind of new source to seek anti-breast cancer medicines.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to the preparation of hinokiflavone derivative WG020 and its anti-
Application in breast cancer medicines.
Background technology
Currently, seeking antitumor medicine is still the master that the mankind obtain antitumor drug from natural products or derivatives thereof
Want means.According to Demain etc., between 1981~2002 years, 74% antitumor drug is from natural products, semi-synthetic
Product or using natural products as the fully synthetic product of model.Taxol, hydroxycamptothecin, colchicin, oxaliplatin, vincristine and
Eldisine etc. is all the anticancer first-line treatment drug extracted from plant.Therefore, it is found from natural plants efficient, special
The anticancer compound of anisotropic strong, non-toxic or low-toxic structure novel, or new anticancer drug is formulated as lead compound using it
It is of great significance.
From the hinokiflavone of Chinese medicine selaginella doederleinii extraction, there is antitumor, anti-HIV-1 reverse transcriptase, anti influenza
Viral sialidases and the bioactivity such as anti-oxidant etc..Since hinokiflavone is insoluble in, water, external activity be relatively low, body-internal-circulation
The disadvantage that time is short, oral dose is high, bioavilability is low limits its application clinically.Therefore it is with hinokiflavone
Lead compound obtains noval chemical compound WG020 to its structural modification, and inquires into WG020 and inhibit the cell Proliferation of breast cancer, induction
Apoptosis of tumor cells and inhibition migration and invasion equimolecular mechanism, are of great significance.
Invention content
In order to overcome the problems referred above, the purpose of the present invention is to provide a kind of hinokiflavone derivatives in anti-breast cancer medicines
In application, obtained hinokiflavone derivative WG020 has certain anti-breast cancer effect.
In order to realize that above-mentioned target, the present invention adopt the following technical scheme that：
Hinokiflavone derivative WG020, structural formula are Formulas I：
On the basis of hinokiflavone tert-butyl diphenyl Japan cypress pair is obtained with TBDPSCl reaction selectivities protection phenolic hydroxyl group
Flavones, tert-butyl diphenyl hinokiflavone obtain four acetyl tert-butyl diphenyl hinokiflavones through aceticanhydride acetylation again, then
Removing silicon substrate protects to obtain four acetyl hinokiflavones, four acetyl hinokiflavones to be reacted with ring succinic anhydride under pyridine effect
Four acetyl hinokiflavone ketobutyric acids act on obtaining intermediate 6 through EDCl, NHS, and compound 6 reacts to obtain amide 7 with amino sugar,
Removing acetyl group is acted on through sodium methoxide to protect to obtain target product WG020, synthetic route is as follows：
(1) precision weighs hinokiflavone, and acetonitrile dissolving is added, TBSCl is added until completely dissolved and imidazoles, room temperature are anti-
1h is answered, is quenched and is reacted with saturated ammonium chloride solution.It being extracted 3 times with dichloromethane, combining extraction liquid is dried with anhydrous sodium sulfate,
Through silica gel column chromatography (chloroform after concentration：Acetone=10:1) yellow solid product tert-butyl diphenyl hinokiflavone, is obtained, it is dry
It is spare.
(2) yellow solid product in step (1) is taken, acetic anhydride and pyridine is added, is heated to reflux 1h, filters, obtains yellow
Four acetyl tert-butyl diphenyl hinokiflavone of solid product, drying for standby.
(3) at room temperature, the yellow solid product in step (2) is taken, is dissolved in THF, then TBAF is added dropwise, 3h is reacted, is filtered,
Obtain four acetyl hinokiflavone of yellow solid product, drying for standby.
(4) four acetyl hinokiflavones are dissolved in organic solvent, pyridine and ring succinic anhydride are added, is heated to reflux for 24 hours,
It filters, obtains four acetyl hinokiflavone ketobutyric acid of yellow solid product, drying for standby.
(5) four acetyl hinokiflavone ketobutyric acids are dissolved in anhydrous tetrahydro furan, stir and N- hydroxysuccinimidyls is added
Acid imide and EDCl react at room temperature 12h under nitrogen protection.It collects filtrate and is spin-dried for, dichloromethane extracts and uses saturated salt solution
Washing 3 times, the drying of organic phase anhydrous magnesium sulfate are concentrated to give crude product, and (eluant, eluent is crude product purified by silica gel column chromatography for separation
Chloroform：Acetone=10:1).Obtain yellow solid intermediate 6, drying for standby.
(6) intermediate 6 is dissolved in DMF, 2- Glucosamines is added, room temperature reaction overnight, filters, obtains intermediate 7, dry
It is spare.
(7) yellow solid product in step (6) is taken, is dissolved in methanol, and metallic sodium is added, reacts 2h at room temperature, is taken out
Filter, obtains WG020, drying for standby.
The preparation of hinokiflavone is prepared by following method：Using industrialization counter-current chromatograph (Britain
Dynamic Extract companies) applied to selaginella doederleinii crude extract hinokiflavone is isolated and purified, dicyandiamide solution is by pentane-
The two-phase system of acetate-methanol-water composition, is injected in column with the rate of 600mL/min, finally from selaginella doederleinii crude extract
Middle acquisition hinokiflavone monomer overcomes countercurrent chromatography disengaging time length, the technological difficulties of low separation efficiency, realizes
Adverse current chromatogram quickly, on a large scale prepares the technology of high economic value monomeric compound.
WG020 has concentration dependent and time dependence to breast cancer cell activity influence.Simultaneously.WG020 is to normal
The toxicity of cell is obviously not so good as breast cancer cell.Since WG020 is to the IC of normal cell50Value is more than breast cancer cell, explanation
The toxicity of WG020 has selectivity.WG020 has good inhibiting effect to the Clone formation of breast cancer cell, and this
Effect has concentration dependent.WG020 can be with induced breast cancer apoptosis, and this apoptosis induction has centainly dense
Spend dependence.This illustrates that WG020 has breast cancer cell a strong apoptosis-induced effect, and this effect have concentration according to
Lai Xing.WG020 can inhibit the expression of anti-apoptotic proteins Bcl-2, while promote the expression of pro apoptotic protein Bax, cause apoptosis
Activation pro-caspase3 completes apoptotic process again afterwards.This further illustrates WG020 to inhibit breast cancer by apoptosis-induced by
Cell.
Hinokiflavone derivative WG020 provided by the invention proves it with certain anti-breast cancer through active testing
Effect can be used as a kind of anti-breast cancer medicines of high-efficiency low-toxicity.The synthesis technology of the present invention is selectively good, and raw material is easy to get, cost
Cheap, synthetic route is simple, implementation easy to operation, and synthesizes products therefrom small toxicity, and yield is high, and product purity is high, thus
The technique has the characteristics that efficient, convenient, inexpensive.
Description of the drawings
Fig. 1 is the structural formula of hinokiflavone derivative WG020 of the present invention.
Fig. 2 is that WG020 of the present invention inhibits breast cancer cell WTT results.
Fig. 3 is that WG020 of the present invention inhibits Apoptosis of Breast Cancer result.
Fig. 4 is breast cancer cell mitochondrial membrane potential and active oxygen testing result.
Fig. 5 is migration and the invasion figure that WG020 of the present invention inhibits breast cancer cell 4T1 and MDA-MB-231.
Specific implementation mode
The separation of 1 hinokiflavone of embodiment
It is detached applied to selaginella doederleinii crude extract using industrialization counter-current chromatograph (Dynamic Extract companies of Britain)
Hinokiflavone is purified, dicyandiamide solution is by 8:8:9:The two-phase system of 7 pentane-acetate-methanol-water composition, by this
Ratio injects two phase solvent system with the rate of 600mL/min in column through different pumps respectively, rotating speed 600r/min, every time
The applied sample amount of selaginella doederleinii crude extract is 80g, final to obtain hinokiflavone monomer 10g, entire disengaging time only 30min, head
It is secondary to breach countercurrent chromatography disengaging time length, the technological difficulties of low separation efficiency, realize adverse current chromatogram quickly, on a large scale
Prepare the technology of high economic value monomeric compound.
Hinokiflavone, yellow powder are soluble in DMSO.ESI-MSm/z：537[M+H]-, 256~258 DEG C of mp,1HNMR
(d-DMSO, 400MHz), δ：6.84 (IH, s, H-3), 5.95 (1H, s, H-6), 6.18 (IH, s, H-8), 7.83 (1H, dd, J=
8.4Hz, H-2 '), 6.93 (1H, dd, J=8.4Hz, H-3 '), 6.93 (1H, dd, J=8.4Hz, H-5 '), 7.83 (1H, dd, J
=8.4Hz, H-6 '), 6.84 (IH, s, H-3 "), 6.28 (IH, s, H-8 "), 7.70 (1H, d, J=8.4Hz, H-2 " '), 6.94
(1H, d, J=8.4Hz, H-3 " '), 6.94 (1H, d, J=8.4Hz, H-5 " '), 7.70 (1H, d, J=8.4Hz, H-6 " ').13C
NMR (d-DMSO, 400MHz), δ：182.2 (C-4 "), 181.9 (C-4), 164.2 (C-2 "), 163.2 (C-2), 164.4 (C-
7), 153.9 (C-7 "), 161.5 (C-5), 153.2 (C-5 "), 157.4 (C-9), 157.4 (C-9 "), 124.8 (C-6 "), 99.7
(C-6), 103.9 (C-3), 102.6 (C-3 "), 94.0 (C-8), 94.7 (C-8 "), 104.1 (C-10), 104.0 (C-10 "),
121.2 (C-l " '), 124.3 (C-l '), 128.7 (C-2 " '), 128.4 (C-2 '), 115.4 (C-3 '), 116.1 (C-3 " '),
160.7 (C-4 '), 161.4 (C-4 " '), 115.4 (C-5 '), 116.1 (C-5 " '), 128.4 (C-6 '), 128.7 (C-6 " ').
The synthesis of 2 hinokiflavone derivative WG020 of embodiment
(1) precision weighs hinokiflavone 10g, and dry acetonitrile 50ml dissolvings are added, imidazoles is added until completely dissolved
2ml is added dropwise TBSCl 5.5g, reacts 1h after being added dropwise at room temperature at room temperature.Dichloromethane 100ml is added after completion of the reaction
With water 100ml, layering, water layer extracts 3 times (50ml is primary) with dichloromethane, and combining extraction liquid is washed with water 100ml, is saturated
Saline solution 100ml washings, organic layer is dried with anhydrous sodium sulfate, through silica gel column chromatography (chloroform after concentration：Acetone=10:1) it, obtains
Yellow solid product tert-butyl diphenyl hinokiflavone 7.8g, drying for standby.
N, N- dimethyl methyls also can be used other than acetonitrile in organic solvent in above-described embodiment for dissolving hinokiflavone
Any one of amide, dimethyl sulfoxide (DMSO), tetrahydrofuran replace；The reaction temperature can be between 20 DEG C~35 DEG C.
(2) the yellow solid product tert-butyl diphenyl hinokiflavone 5.0g in step (1) is taken, pyridine 50ml is added,
Acetic anhydride 3ml is instilled at room temperature, reacts at room temperature 2h.Ethyl acetate 100ml and water 50ml is added after completion of the reaction, layering has
Machine layer is washed with water (50ml*3) and saturated salt solution (50ml*3) respectively, and organic layer is dried with anhydrous sodium sulfate, evaporated under reduced pressure
Solvent obtains four acetyl tert-butyl diphenyl hinokiflavone 5.4g of yellow solid product.
(3) the four acetyl tert-butyl diphenyl hinokiflavone hinokiflavone 5g of yellow solid in step (2) is taken, is dissolved in
In THF 50ml and acetic acid 10ml, TBAF 6.9g, the reaction was continued 3h are added at room temperature.Ethyl acetate is added after completion of the reaction
100ml and water 50ml, layering, organic layer are washed with water (50ml*3) and saturated salt solution (50ml*3) respectively, organic layer nothing
Aqueous sodium persulfate is dried, and evaporated under reduced pressure solvent obtains four acetyl hinokiflavone 3.4g of yellow solid product.
(4) four acetyl hinokiflavone 5.0g are dissolved in pyridine 50ml, instill succinic anhydride 1.0g, heating at room temperature
Back flow reaction 4h.After completion of the reaction be added ethyl acetate 100ml and water 50ml, layering, organic layer respectively use water (50ml*2) and
Saturated salt solution (50ml*3) washs, and organic layer is dried with anhydrous sodium sulfate, and evaporated under reduced pressure solvent obtains yellow solid product tetrem
Acyl hinokiflavone ketobutyric acid 5.1g.
(5) four acetyl hinokiflavone ketobutyric acid 5.0g are dissolved in anhydrous THF 50ml, stir and N- hydroxyls is added
Succinimide 0.8g and EDCl 1.3g reacts at room temperature 12h under nitrogen protection.Filtering is collected filtrate and is spin-dried for, dichloro is added
Methane 100ml, with saturated common salt water washing (50ml*3), organic phase is dried with anhydrous sodium sulfate, is concentrated to give crude product.Thick production
With silica gel column chromatography separation, (eluant, eluent is chloroform to object：Acetone=10:1) 6 4.5g of yellow solid intermediate, is obtained, drying is standby
With.
(6) 6 4.5g of intermediate is dissolved in DMF 20ml, 2- Glucosamine 1.0g is added, room temperature reaction overnight, is taken out
Filter, is washed with water (20ml*3) and ether (20ml*2), dries to obtain 7 4.4g of intermediate respectively.
(7) the yellow solid product 4.0g in step (6) is taken, is dissolved in methanol 30ml, with the methanol solution of sodium methylate of 1M
PH8~9 are adjusted, react 1h at room temperature.A small amount of resin cation is added, it is 7 to make system pH, filters, concentrates the filtrate to
It is dry, obtain WG020 3.0g.
WG020, yellow powder,1H NMR (400MHz, d-DMSO) δ 9.72 (t, J=9.2,1H, H-5 " "), 8.05 (d, J
=5.2Hz, 1H, H-3 " "), 7.98 (dd, J=19.0,8.1Hz, 4H, H-3', 4', 5', 6'), 6.99 (dd, J=38.1,
7.9Hz,4H,H-2”',3”',5,”',6”'),6.72(s,2H,H-6,8),6.48(s,1H,H-7),6.20(s,1H,H-5),
4.53 (t, 2H, J=7.2Hz, H-4 " "), 4.14 (t, 1H, J=7.8Hz, H-6 " "), 3.30 (t, 2H, J=7.8Hz, H-
), 7 " " 3.25 (t, 2H, J=7.8Hz, H-8 " "), 3.10 (t, 2H, J=7.8Hz, H-9 " ").13C NMR (400MHz, d-
DMSO), δ：207.7(C-6””),202.2(C-4),193.0(C-4”),180.2(C-1),178.9(C-4””),173.0(C-
7),163.4(C-1”),163.2(C-1),162.2(C-5),160.5(C-6”),160.4(C-7”),159.7(C-2),157.4
(C-4””),141.2(C-4'),127.7(C-2”),126.4(C-6”),122.8(C-3”'),121.3(C-5”),119.1(C-
8 "), 117.1 (C-3'), 114.4 (C-5'), 105.1 (C-3 "), 99.5,94.7 (C-6), 94.1 (C-8), 72.5 (C-9 " "),
71.2(C-8””),69.2(C-8””),66.2(C-8””),64.2(C-10””)29.9(C-2””).29.0(C-3””).
3 hinokiflavone derivative WG020 of embodiment inhibits Cells Proliferation of Human Breast Cancer to influence
One, experiment material
Trial drug hinokiflavone derivative WG020, buff powder, 98% purity, DMSO dissolvings, filtration sterilization are protected
It deposits, is diluted with cell culture fluid with preceding.
The breast cancer cell MDA-MB-231 and MCF-7 of reagent humanized, the breast cancer cell 4T1 of mouse, culture medium,
Pancreatin, top grade fetal calf serum, tetramethyl azo file blue powder (MTT powder).
Two, the micro adjustable pipette of laboratory apparatus, cell counter, CO2Incubator, Millipore companies pure water meter are low
Warm centrifuge, enzyme mark detector, -80 DEG C of ultra low temperature freezers, constant temperature mixer, inverted microscope.
Three, above-mentioned 3 kinds of cells are equably layered in 96 orifice plates by experimentation when cells growth activity tends towards stability,
The culture medium that 100 μ L contain certain amount cell, number of cells is added to be made according to the speed and drug of vitro growth rates per hole
It is adjusted with the difference of time.And a series of working concentration of hinokiflavones is set, and 6 multiple holes are arranged in each concentration.
After 96 orifice plates are placed in incubator for 24 hours, the hinokiflavone WG020 of respective concentration is added.Drug effect is for 24 hours, every after 48h, 72h
20 μ LMTT (5mg/mL) are added in hole, and supernatant is siphoned away after being incubated 4h, the first a ceremonial jade-ladle, used in libation for adding 150 μ L/ hole DMSO dissolvings to generate, in 570nm
Lower measurement OD values are with indirect reaction cell viability.
Four, experimental result (such as Fig. 2) shows that WG020 has concentration dependent and time to breast cancer cell activity influence
Dependence.After WG020 effects for 24 hours, MDA-MB-231,4T1, the IC of tri- kinds of cells of MCF-750It is 36.4 μM respectively, 34.4 μM, 35
μM；IC after effect 48h50It is 25.1 μM, 18.1 μM, 20.0 μM respectively；It is 18.5 μM, 21.0 μM, 18. μM respectively after effect 72h.
The IC of 48h and 72h50Difference is little, illustrates that WG020 may have reached the maximum function and effect in 48h, when extension acts on after 48h
Between cell activity is influenced it is little.
4 hinokiflavone derivative WG020 of embodiment influences Apoptosis of Breast Cancer
One, experiment material
Trial drug hinokiflavone derivative WG020 preserves dilution process with embodiment 3.
The breast cancer cell MDA-MB-231 and MCF-7 of reagent humanized, the breast cancer cell 4T1 of mouse, culture medium,
Pancreatin, top grade fetal calf serum, tetramethyl azo file blue powder (MTT powder).
Two, the micro adjustable pipette of laboratory apparatus, cell counter, CO2Incubator, Millipore companies pure water meter are low
Warm centrifuge, enzyme mark detector, -80 DEG C of ultra low temperature freezers, constant temperature mixer, inverted microscope.
Three, experimentation
MDA-MB-231,4T1 and MCF-7 cell inoculation add the working solution of WG020 a series of, often afterwards for 24 hours in 6 orifice plates
3 multiple holes are arranged in a concentration.Drug effect siphons away supernatant afterwards for 24 hours, will be under cell dissociation with pancreatin after washing 2 times with precooling PBS
Come, cell is finally collected into centrifugation (2500rpm, 5min) in corresponding streaming pipe, is washed twice with the PBS of precooling.It is added 300
In the buffer solution to every pipe of μ L, AnnexinV-FITC dye liquors are added in each pipe (5 μ L/ pipes), are protected from light dyeing
Propidium iodide (PI) dyestuff (5 μ L/ pipes) is added after 15min, is protected from light machine testing on dyeing 10min.
Four, experimental result flow cytometer showed result (such as Fig. 3) shows that the breast cancer cell after WG020 effects occurs obviously
Apoptosis.Apoptosis rate (the sum of early apoptosis rate and late apoptic rate) is with respect to blank control group under the WG020 effects of low concentration
Do not increase significantly, but with the increase of concentration, apoptosis rate is also dramatically increased therewith.After 3 repeated experiments, we
From result：When WG020 is 50 μM a concentration of, the mean apoptotic rate of MDA-MB-231 cells reaches 18.5% (Figure
2b), the mean apoptotic rate of 4T1 cells is up to 19% (Figure 2a), and the mean apoptotic rate of MCF-7 cells reaches 15.3%
(Figure 2c).This illustrates that WG020 has breast cancer cell strong apoptosis-induced effect, and this effect is with dense
Spend dependence.
5 hinokiflavone derivative WG020 of embodiment explores breast cancer cell mechanism of inducing apoptosis
One, experiment material
Trial drug hinokiflavone derivative WG020 preserves dilution process with embodiment 3.
The breast cancer cell MDA-MB-231 and MCF-7 of reagent humanized, the breast cancer cell 4T1 of mouse, culture
Base, pancreatin, top grade fetal calf serum, trishydroxymethylaminomethane hydrochloric acid TrisHCl, bovine serum albumin(BSA) BSA, benzyl sulfonephthalein fluorine
PMSF, protease inhibitors PI, polyacrylamide solution, tetraethylethylenediamine TDMED, ammonium persulfate, albumen pre-dyed Marker,
Internal reference β-actin primary antibodies.
Two, the micro adjustable pipette of laboratory apparatus, cell counter, CO2Incubator, Millipore companies pure water meter are low
Warm centrifuge, enzyme mark detector, -80 DEG C of ultra low temperature freezers, constant temperature mixer, inverted microscope, flow cytometer, SDS-PAGE
Vertial electrophorestic tank and electrophoresis membrane-transferring device, fluorescence imaging system, image analysis software Imagetool2.0 analysis softwares, laser are total
Focusing microscope.
Three, experimentation, which passes on 4T1 cells, expands culture, three WG020 concentration gradients is arranged, 3 ware of each concentration is thin
Born of the same parents, i.e. each cell add blank control group totally 4 groups, 12 ware cells.When cell growth is to certain density, it is dense that correspondence is added
The hinokiflavone working solution (culture medium configuration) of degree.After effect for 24 hours, the supernatant collection of each concentration is managed to corresponding BD
In, it is washed 2 times with the PBS of precooling, with being collected into after trypsin digestion cell in corresponding BD pipes, precooling PBS is washed 3 times.It will be in BD pipes
Cell be transferred in corresponding EP pipes, precooling PBS wash 1 time, fully siphon away supernatant.Often the Ripa cells of 500 μ L are added in pipe
Lysate (adds protease inhibitors cocktail) using preceding according to v/v=100/1.1h is cracked on ice, is used per 5min
Gently fully piping and druming is primary for insulin syringe.The cell ultrasonication of 2min is carried out after the completion of cracking.Then 4 degree of lower progress
Ultracentrifugation (13300rpm, 15min).It collects supernatant and carries out protein quantification.Each drug concentration of each cell acts on
The albumen collected afterwards will carry out trim according to concentration.A certain amount of 5 × SDS is added in albumen after trim and is boiled in boiling water
5min.It is stored in spare under -20 degree.
The poly- propyl amides gel electrophoresis of the albumen of each concentration is detached, and destination protein is gone into corresponding pvdf membrane
On.5% skim milk closes 2h, and TBST cleans 20min (5min/ times), corresponding primary antibody is incubated overnight under 4 degree.Second day
Primary antibody is recycled, 20min (5min/ times) is cleaned with TBST.Then it is incubated corresponding secondary antibody 1h down for 37 degree, TBST cleans 50min
(10min/ times).It finally carries out albumen exposure and preserves exposure data.
Four, after experimental result WB data (such as Fig. 3 d) show WG020 effects 48h, the Bax in 4T1 cells and cleaved-
The expression of caspase3 is raised, and the expression of Bcl-2 has dropped.This illustrates that WG020 can inhibit anti-apoptotic proteins Bcl-2
Expression, while promoting the expression of pro apoptotic protein Bax, cause after apoptosis that activation pro-caspase3 completes apoptotic process again.
This further illustrates WG020 to inhibit breast cancer cell by apoptosis-induced by.
The detection of 6 mitochondrial membrane potential of embodiment (MMP) and active oxygen (ROS)
One, experiment material
Trial drug hinokiflavone derivative WG020 preserves dilution process with embodiment 3.
The breast cancer cell MDA-MB-231 and MCF-7 of reagent humanized, culture medium, pancreatin, top grade fetal calf serum, three hydroxyls
Aminomethane hydrochloric acid TrisHCl, bovine serum albumin(BSA) BSA, benzyl sulfonephthalein fluorine PMSF, protease inhibitors PI, poly- third
Acrylamide solution, tetraethylethylenediamine TDMED, ammonium persulfate, albumen pre-dyed Marker, internal reference β-actin primary antibodies.
Two, the micro adjustable pipette of laboratory apparatus, cell counter, CO2Incubator, Millipore companies pure water meter are low
Warm centrifuge, enzyme mark detector, -80 DEG C of ultra low temperature freezers, constant temperature mixer, inverted microscope, flow cytometer, SDS-PAGE
Vertial electrophorestic tank and electrophoresis membrane-transferring device, fluorescence imaging system, image analysis software Imagetool2.0 analysis softwares, laser are total
Focusing microscope.
Three, experimentation is in order to further study the mechanism that WG020 generates apoptosis, breast cancer after we act on WG020
The mitochondrial membrane potential of cell and intracellular reactive oxygen species are detected.
MDA-MB-231 the and MCF-7 cells of certain density are layered in 6 orifice plates, a series of concentration are added afterwards for 24 hours
WG020 working solutions, effect siphon away supernatant, are washed 2 times with the PBS of precooling afterwards for 24 hours, and prepared Rh123 (detection mitochondrias are added
Film potential) or 500 holes μ L/ of DCFH-DA (detection reactive oxygen species) dyestuff.It is protected from light under 37 degree and is incubated 30min.Collection has been hanged
In floating cell to corresponding streaming pipe, is washed 2 times with precooling PBS, finally go up machine testing mitochondrial membrane potential and reactive oxygen species.
Four, experimental result flow cytometer detection result (such as Fig. 4) shows after WG020 effects for 24 hours, MDA-MB-231 cells
It is decreased obviously with the mitochondrial membrane potential of MCF-7 cells, this reduction trend also has concentration dependent.ROS levels are in MDA-MB-
It is also to be decreased obviously, but but have the variation of non-concentration dependant in MCF-7 cells in 231 cells：In the WG020 of low concentration
Under effect, ROS levels rise with the rising of concentration, but when having arrived high concentration, ROS levels are really with the rising of concentration
And decline, imply that the WG020 of various concentration has broken the balance of ROS levels.
In summary all mechanism is explored as a result, we can obtain a conclusion substantially：WG020 passes through mitochondria way
The Apoptosis access that diameter mediates carrys out induced apoptosis to play the role of anti-breast cancer.
The metastasis suppressor of 7 breast cancer cell of embodiment is tested
One, experiment material
Trial drug hinokiflavone derivative WG020 preserves dilution process with embodiment 3.
The breast cancer cell MDA-MB-231 and 4T1 of reagent humanized, culture medium, pancreatin, top grade fetal calf serum, three hydroxyl first
Base aminomethane hydrochloric acid TrisHCl, bovine serum albumin(BSA) BSA, benzyl sulfonephthalein fluorine PMSF, protease inhibitors PI, polypropylene
Amide solution, tetraethylethylenediamine TDMED, ammonium persulfate, albumen pre-dyed Marker, internal reference β-actin primary antibodies.
Two, the micro adjustable pipette of laboratory apparatus, cell counter, CO2Incubator, Millipore companies pure water meter are low
Warm centrifuge, enzyme mark detector, -80 DEG C of ultra low temperature freezers, constant temperature mixer, inverted microscope, flow cytometer, SDS-PAGE
Vertial electrophorestic tank and electrophoresis membrane-transferring device, fluorescence imaging system, image analysis software Imagetool2.0 analysis softwares, laser are total
Focusing microscope.
Three, experimentation simulates the environment investigation WG020 of in-vivo tumour necessary being to MDA-MB-231 and 4T1 in vitro
The influence of cell transfer ability.In migration experiment, cell is put into 24 orifice plates, the cell that the 50 certain density of μ L are added in upper chamber is outstanding
Liquid (2 × 106/ mL, double without antibody of serum-free prepare without culture medium) and 50 μ L with double without the certain density of culture medium preparation
WG020 liquids, lower room adds a certain concentration WG020 working solutions that 600 μ L are prepared with complete medium, and blank control group is arranged.
In Matrigel, 60 μ L matrigels are added in upper chamber, and (use pair is without culture medium according to volume ratio 1:5 dilutions), wait for that matrigel fully solidifies
Afterwards, the cell suspension (1 × 10 of the 50 certain density of μ L is added in upper chamber6/ mL, serum-free are double without culture medium preparation without antibody) and 50
The double certain density WG020 liquids prepared without culture medium of μ L, 600 μ L complete mediums are added in lower room, and blank pair is arranged
According to group.Migration is identical with the post-processing of Matrigel, that is, after being incubated 24 hours, siphons away the liquid of upper chamber and lower room, small with cotton swab
The heart cleans the cell that upper chamber is not migrated or invaded.Cell is placed in the 600 pre- cold methanols of μ L after washing cell 2 times with precooling PBS solid
Determine 15min, then washed 2 times with the PBS of precooling, cell is placed in 600 μ L Crystal Violet Dyes (mass concentration 0.5%), is protected from light
Dye 2h.Finally with the PBS wash clean backgrounds of precooling, film is carefully cut off with glycerine mounting, observe and count under the microscope.
Four, experimental result passes through migration and Matrigel (such as Fig. 5), after WG020 is handled, MDA-MB-231 and 4T1
The number that cell is migrated and invaded greatly reduces, and drug effect concentration is higher, and the MDA-MB-231 of migration invasion occurs
It is fewer with 4T1 cell numbers.Migrate Matrigel the result shows that, WG020 has good suppression to the transfer of breast cancer cell
It makes and uses.
Claims (3)
1. a kind of applications of hinokiflavone derivative WG020 in anti-breast cancer medicines, the hinokiflavone derivative
The structural formula of WG020 is Formulas I：
2. the preparation method of hinokiflavone derivative WG020 as described in claim 1, which is characterized in that closed according to following
It is carried out at route：To obtain tert-butyl diphenyl flat with TBDPSCl reaction selectivities protection phenolic hydroxyl group on the basis of hinokiflavone
Cypress biflavone, tert-butyl diphenyl hinokiflavone obtain four acetyl tert-butyl diphenyl hinokiflavones through aceticanhydride acetylation again,
Then removing silicon substrate protects to obtain four acetyl hinokiflavones, four acetyl hinokiflavones anti-with ring succinic anhydride under pyridine effect
Deserved four acetyl hinokiflavone ketobutyric acid acts on obtaining midbody compound 6 through EDCl, NHS, and compound 6 and amino sugar are anti-
Deserved amide 7 acts on removing acetyl group through sodium methoxide and protects to obtain target product WG020；It is i.e. as follows：
3. the preparation method of hinokiflavone derivative WG020 as claimed in claim 2, which is characterized in that the specific steps are：
(1) precision weighs hinokiflavone, and acetonitrile dissolving is added, and TBSCl and imidazoles, room temperature reaction are added until completely dissolved
1h is quenched with saturated ammonium chloride solution and is reacted.It is extracted 3 times with dichloromethane, combining extraction liquid is dried with anhydrous sodium sulfate, dense
Through silica gel column chromatography (chloroform after contracting：Acetone=10：1) yellow solid product tert-butyl diphenyl hinokiflavone, is obtained, drying is standby
With；
(2) yellow solid product in step (1) is taken, acetic anhydride and pyridine is added, is heated to reflux 1h, filters, obtains yellow solid
Four acetyl tert-butyl diphenyl hinokiflavone of product, drying for standby；
(3) at room temperature, the yellow solid product in step (2) is taken, is dissolved in THF, then TBAF is added dropwise, 3h is reacted, filters, obtains yellow
Four acetyl hinokiflavone of color solid product, drying for standby；
(4) four acetyl hinokiflavones are dissolved in organic solvent, pyridine and ring succinic anhydride is added, be heated to reflux for 24 hours, take out
Filter, obtains four acetyl hinokiflavone ketobutyric acid of yellow solid product, drying for standby；
(5) four acetyl hinokiflavone ketobutyric acids are dissolved in anhydrous tetrahydro furan, stir and N- hydroxysuccinimidyls acyl is added and is sub-
Amine and EDCl react at room temperature 12h under nitrogen protection.It collects filtrate and is spin-dried for, dichloromethane extracts and uses saturated common salt water washing 3
Secondary, organic phase anhydrous magnesium sulfate, which is dried, is concentrated to give crude product, crude product purified by silica gel column chromatography for separation (eluant, eluent is chloroform:
Acetone=10: 1).Obtain yellow solid intermediate 6, drying for standby；
(6) intermediate 6 is dissolved in DMF, 2- Glucosamines is added, room temperature reaction overnight, filters, and obtains intermediate 7, and drying is standby
With；
(7) yellow solid product in step (6) is taken, is dissolved in methanol, and metallic sodium is added, reacts 2h at room temperature, filters, obtains
WG020, drying for standby.
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| CN104473936A (en) * | 2014-11-11 | 2015-04-01 | 济南星懿医药技术有限公司 | Pharmaceutical composition used for treating liver cancer |
| CN108299366A (en) * | 2018-03-09 | 2018-07-20 | 遵义医学院 | A kind of application of the preparation method and melanoma of hinokiflavone derivative |
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| CN104473936A (en) * | 2014-11-11 | 2015-04-01 | 济南星懿医药技术有限公司 | Pharmaceutical composition used for treating liver cancer |
| CN108299366A (en) * | 2018-03-09 | 2018-07-20 | 遵义医学院 | A kind of application of the preparation method and melanoma of hinokiflavone derivative |
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| Title |
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| 齐妍: "卷柏抗肿瘤转移的活性成分", 《中成药》 * |
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