CN105175475A - Synthetic method for 11-oxooleanolic acid - Google Patents
Synthetic method for 11-oxooleanolic acid Download PDFInfo
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- CN105175475A CN105175475A CN201510447782.8A CN201510447782A CN105175475A CN 105175475 A CN105175475 A CN 105175475A CN 201510447782 A CN201510447782 A CN 201510447782A CN 105175475 A CN105175475 A CN 105175475A
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Abstract
The present invention discloses a synthetic method for 11-oxooleanolic acid. The method comprises: taking oleanolic acid as a lead compound, carrying out functional group protection, and potassium dichromate oxidation and deprotection on the lead compound, thereby the obtaining 11-oxooleanolic acid. According to the method of the invention, the operation is safe and simple, reaction time is short, yield is high, and antitumor activity is preferable.
Description
[technical field]
The present invention relates to the technical field of the synthetic method of 11-oxo Oleanolic Acid.
[background technology]
Pentacyclic triterpenoid is that a class is extensively present in nature, and has the micromolecular compound of multiple pharmacologically active, comprises anti-HIV, anti-inflammatory, antitumor pharmacological action of Denging.The active lead compound of this compounds Chang Zuowei, by structural modification or transformation, therefrom finding low, that curative effect the is high compound of toxicity is pharmaceutical chemists important research content.
Oleanolic Acid is that one is extensively present in plant, belongs to volatile oil pentacyclic triterpenoid, and research shows to have multiple pharmacologically active, comprises and protecting the liver, anti-inflammatory, anti-HIV, antitumor isoreactivity.In the last few years, anti-tumor activity was paid close attention to well, can produce restraining effect to kinds of tumor cells such as colorectal carcinoma, mammary cancer, liver cancer, lung cancer and osteosarcoma.OA can Tumor suppression growth in various degree, comprises the suppression of breeding the generation of tumour cell, and can the differentiation of T suppression cell.
[summary of the invention]
Object of the present invention solves the problems of the prior art exactly, proposes a kind of synthetic method of 11-oxo Oleanolic Acid, and operational safety of the present invention is simple, reaction times short, yield and anti-tumor activity all better.
For achieving the above object, the present invention proposes a kind of synthetic method of 11-oxo Oleanolic Acid, comprise step successively:
A) Oleanolic Acid, DMAP (DMAP) and acetic anhydride are dissolved in tetrahydrofuran (THF) (THF) solution at room temperature react 2.5h, then underpressure distillation is gone out tetrahydrofuran (THF) and is obtained solid substance, solid substance 30ml acetic acid ethyl dissolution obtains organic layer, the saturated NaCl solution washing of organic layer, then uses anhydrous Na
2sO
4drying, filters Na
2sO
4remove desolventizing by the method for underpressure distillation afterwards and obtain white solid, white solid carries out separation by column chromatography and obtains compound 1, and ethyl acetate is the eluent in column chromatography, and silica gel is the stationary phase in column chromatography;
B) by step a) in compound 1 acetic acid that obtains, add potassium bichromate, 5 ~ 7h is stirred under the condition of 80 ~ 100 DEG C, after question response terminates, reaction solution is carried out decompression and obtain concentrated solution, concentrated solution saturated sodium bicarbonate is neutralized to neutrality, wash with saturated NaCl with the organic layer after ethyl acetate (EA) extraction, then use anhydrous Na
2sO
4drying, filters Na
2sO
4, obtain faint yellow solid by underpressure distillation, faint yellow solid obtains compound 2 by column chromatography for separation, and ethyl acetate is the eluent in column chromatography, and silica gel is the stationary phase in column chromatography;
C) by step b) the compound dissolve with ethanol that obtains, add the potassium hydroxide aqueous solution of 10%, 6h is stirred under 60 DEG C of conditions, underpressure distillation removes desolventizing and obtains light yellow solid, the pH that light yellow solid adopts rare HCl to be adjusted to mixture is 2 ~ 3, extract by ethyl acetate again, then wash with saturated NaCl, the organic phase anhydrous Na of gained
2sO
4drying, filter, then underpressure distillation obtains 11-oxo Oleanolic Acid, and 11-oxo Oleanolic Acid high performance liquid chromatography (HPLC) of gained is further purified, for biological test;
The synthesis type of 11-oxo Oleanolic Acid is as follows:
As preferably, described step a) in organic layer saturated NaCl solution washing 2 ~ 5 times.
As preferably, described step b) in by step a) in compound 1 acetic acid that obtains, add potassium bichromate, under the condition of 90 DEG C, stir 6h; 2 ~ 5 times are extracted by ethyl acetate (EA).
As preferably, described step c) in high performance liquid chromatography adopt the methanol solution containing 0.01% formic acid to be eluent, high performance liquid chromatography adopts wavelength to be 210, the UV-light of 250nm is as detection chromatogram.
Beneficial effect of the present invention: the invention discloses a kind of easy, efficient 11-oxo Oleanolic Acid synthetic route, and purified by HPLC, carry out antitumor activity to it, operational safety of the present invention is simple, reaction times short, yield and anti-tumor activity all better.
[embodiment]
The synthetic method of a kind of 11-oxo of the present invention Oleanolic Acid, comprises step successively:
A) Oleanolic Acid, DMAP (DMAP) and acetic anhydride are dissolved in tetrahydrofuran (THF) (THF) solution at room temperature react 2.5h, then underpressure distillation is gone out tetrahydrofuran (THF) and is obtained solid substance, solid substance 30ml acetic acid ethyl dissolution obtains organic layer, the saturated NaCl solution washing of organic layer, then uses anhydrous Na
2sO
4drying, filters Na
2sO
4remove desolventizing by the method for underpressure distillation afterwards and obtain white solid, white solid carries out separation by column chromatography and obtains compound 1, and ethyl acetate is the eluent in column chromatography, and silica gel is the stationary phase in column chromatography;
B) by step a) in compound 1 acetic acid that obtains, add potassium bichromate, 5 ~ 7h is stirred under the condition of 80 ~ 100 DEG C, after question response terminates, reaction solution is carried out decompression and obtain concentrated solution, concentrated solution saturated sodium bicarbonate is neutralized to neutrality, wash with saturated NaCl with the organic layer after ethyl acetate (EA) extraction, then use anhydrous Na
2sO
4drying, filters Na
2sO
4, obtain faint yellow solid by underpressure distillation, faint yellow solid obtains compound 2 by column chromatography for separation, and ethyl acetate is the eluent in column chromatography, and silica gel is the stationary phase in column chromatography;
C) by step b) the compound dissolve with ethanol that obtains, add the potassium hydroxide aqueous solution of 10%, 6h is stirred under 60 DEG C of conditions, underpressure distillation removes desolventizing and obtains light yellow solid, the pH that light yellow solid adopts rare HCl to be adjusted to mixture is 2 ~ 3, extract by ethyl acetate again, then wash with saturated NaCl, the organic phase anhydrous Na of gained
2sO
4drying, filter, then underpressure distillation obtains 11-oxo Oleanolic Acid, and 11-oxo Oleanolic Acid high performance liquid chromatography (HPLC) of gained is further purified, for biological test;
The synthesis type of 11-oxo Oleanolic Acid is as follows:
Described step a) in organic layer saturated NaCl solution washing 2 ~ 5 times, described step b) in by step a) in compound 1 acetic acid that obtains, add potassium bichromate, under the condition of 90 DEG C, stir 6h; Extract 2 ~ 5 times by ethyl acetate (EA), described step c) in high performance liquid chromatography to adopt containing the methanol solution of 0.01% formic acid be eluent, high performance liquid chromatography adopts wavelength to be 210, the UV-light of 250nm is as detection chromatogram.
Embodiment:
0.5g (1.095mmol) Oleanolic Acid, DMAP (0.026g, 0.22mmol, 0.2eq) and acetic anhydride (500 μ l, 1.64mmol, 1.5eq) are dissolved in 10mlTHF solution and at room temperature react 2.5h.THF is gone out in underpressure distillation, solid substance 30ml acetic acid ethyl dissolution.The saturated NaCl solution washing of organic layer 20ml 3 times, anhydrous Na
2sO
4drying, filters, and underpressure distillation removes desolventizing and obtains white solid, and ethyl acetate is eluent column chromatography, obtains compound 10.52g (95%);
0.4g (0.80mmol) compound 1 is used 10ml acetic acid, add potassium bichromate (0.71g, 2.41mmol, 3eq), 90 DEG C are stirred 6h, and question response terminates the concentration of reaction solution that reduces pressure, and is neutralized to neutrality with saturated sodium bicarbonate, extract 3 times with 20mlEA, the saturated NaCl of organic layer wash, anhydrous Na
2sO
4drying, filter, underpressure distillation obtains faint yellow solid, and column chromatography (EA: PE=1: 3) is separated and obtains compound 2, and weight is 0.30g (productive rate 73%);
0.2g (0.39mmol) compound 2 is used 3ml dissolve with ethanol, adds the potassium hydroxide aqueous solution (3eq) of 10%, under 60 DEG C of conditions, stir 6h.Underpressure distillation removes desolventizing and obtains light yellow solid, and rare HCl regulates the PH=2-3 of mixture, then extracts by ethyl acetate, washs, gained organic phase anhydrous Na with saturated NaCl
2sO
4drying, filter, underpressure distillation obtains faint yellow solid 0.18g (productive rate 97%).Gained 11-oxo Oleanolic Acid HPLC is further purified (methanol solution (volume fraction 80%) containing 0.01% formic acid is eluent, 210,250nm is determined wavelength), for biological test.
The anti-tumour cell proliferative experiment of 11-oxo Oleanolic Acid
Selected cell strain is Hela cell, and the vegetative period tumour cell of taking the logarithm makes certain density cell suspension inoculation in 96 orifice plates, and every hole adds certain density testing compound, and contrast sky does not add medicine.Cultivate after 24h, use CCK-8 test kit, microplate reader measures absorbancy, the drug level (IC50) when suppressing 50% Growth of Cells by SPSS computed in software.In triplicate, the growth of 11-oxo Oleanolic Acid to Hela cell has obvious restraining effect (IC50=9.6 μM), and inhibition comparatively Oleanolic Acid (IC50=108.6 μM) adds 11 times in experiment.
11-oxo Oleanolic Acid promotes apoptosis of tumor cells experiment
Collecting cell after Hela cell 24h cultivated by 11-oxo Oleanolic Acid and Oleanolic Acid under the concentration of 10 μMs, after the two dye of PI/AnnexinV-FITC, and flow cytometer detection result, Oleanolic Acid group apoptosis is early stage/and the per-cent of late cell is 8.0%/5.5%; 11-oxo Oleanolic Acid group apoptosis is early stage/and the per-cent of late cell is 25.8%/17.4%, improves the apoptotic cell per-cent of nearly 5 times compared with OA group, presents short anti-apoptotic activity very well.WesternBlot has investigated the regulation and control situation of compound to Bcl-2 apoptotic proteins family, and compared with Oleanolic Acid group, the regulation and control degree of 11-oxo Oleanolic Acid to Bcl-2 family apoptotic proteins obviously increases.The apoptosis of compound promoted cell, by the cascade reaction regulating the expression amount of Bcl-2 family protein to cause next step, causes cell to move towards apoptosis.
The neat morphological change of pier cell death inducing of 11-oxo and the retardance of cell cycle
The morphological change of induced Hcla cell apoptosis, use Hoechst33342 dyeing, inverted fluorescence microscope imaging is used after culturing cell 24h under same concentrations (10 μMs), the nucleus of control group Hela is even blue light, color is slightly light, connect closely evenly between cell, in good condition; The core of apoptotic cell is in the fine and close dense bright blue light or in chunky shape of having hair dyed.OA does not find obvious phenomena of apoptosis; 11-oxo Oleanolic Acid group observes that the nucleus of obvious fine and close dense dye and cracked particulate state are dashed forward light, and has apoptotic body to occur.
To the cycle regulating situation of Hela cell, use PI mono-dye, the Flow cytometry cell cycle.After compound 10 μMs process Hela cell 24h, period profile is compared with 0.1%DMSO control group, the cell quantity of S, G2/M phase increases, illustrate compound can arresting cell cycle in S, G2/M phase, and in the effect of 11-oxo Oleanolic Acid after 24 hours, the cell proportion of S, G2/M phase is in up to 16.68%, 15% in cell, compared with OA, cell-cycle arrest effect also significantly strengthens.
11-oxo Oleanolic Acid is to the regulation and control of ROS and promote apoptosis by mitochondria pathway
Hela cell is after compound 10 μMs process 24h, use active oxygen detection probes DCFH, detect the content of ROS in cell, mitochondrial probe rhodamine123 marks, the fluorescence intensity of flow cytomery rhodamine123, increase burst size and the mitochondrial membrane degree of injury of ROS in cell in various degree, in compound promoted Hela apoptosis process, the decline of the generation heap sum Δ ψ m of ROS is as apoptotic critical event, both mutually promote, and play a significant role in apoptosis.
Probe into the induction situation of compound to the related apoptosis factor of mitochondria pathway further, the expression amount of cytochromec and caspase enzyme is investigated, after the Oleanolic Acid of same concentrations (10 μMs) and 11-oxo Oleanolic Acid process HeLa cell, compared with the control, the burst size of cytochromec obviously increases, the enzyme of caspase-9, caspase-8 and caspase-3 is lived and is all increased.Generally speaking, the 11-oxo Oleanolic Acid after structural modification carrys out the apoptosis of triggering tumor cell by mitochondria pathway, its activity comparatively primer is significantly improved.
Above-described embodiment is to explanation of the present invention, is not limitation of the invention, anyly all belongs to protection scope of the present invention to the scheme after simple transformation of the present invention.
Claims (4)
1. a synthetic method for 11-oxo Oleanolic Acid, comprises step successively:
A) Oleanolic Acid, DMAP (DMAP) and acetic anhydride are dissolved in tetrahydrofuran (THF) (THF) solution at room temperature react 2.5h, then underpressure distillation is gone out tetrahydrofuran (THF) and is obtained solid substance, solid substance 30ml acetic acid ethyl dissolution obtains organic layer, the saturated NaCl solution washing of organic layer, then uses anhydrous Na
2sO
4drying, filters Na
2sO
4remove desolventizing by the method for underpressure distillation afterwards and obtain white solid, white solid carries out separation by column chromatography and obtains compound 1, and ethyl acetate is the eluent in column chromatography, and silica gel is the stationary phase in column chromatography;
B) by step a) in compound 1 acetic acid that obtains, add potassium bichromate, 5 ~ 7h is stirred under the condition of 80 ~ 100 DEG C, after question response terminates, reaction solution is carried out decompression and obtain concentrated solution, concentrated solution saturated sodium bicarbonate is neutralized to neutrality, wash with saturated NaCl with the organic layer after ethyl acetate (EA) extraction, then use anhydrous Na
2sO
4drying, filters Na
2sO
4, obtain faint yellow solid by underpressure distillation, faint yellow solid obtains compound 2 by column chromatography for separation, and ethyl acetate is the eluent in column chromatography, and silica gel is the stationary phase in column chromatography;
C) by step b) the compound dissolve with ethanol that obtains, add the potassium hydroxide aqueous solution of 10%, 6h is stirred under 60 DEG C of conditions, underpressure distillation removes desolventizing and obtains light yellow solid, the pH that light yellow solid adopts rare HCl to be adjusted to mixture is 2 ~ 3, extract by ethyl acetate again, then wash with saturated NaCl, the organic phase anhydrous Na of gained
2sO
4drying, filter, then underpressure distillation obtains 11-oxo Oleanolic Acid, and 11-oxo Oleanolic Acid high performance liquid chromatography (HPLC) of gained is further purified, for biological test;
The synthesis type of 11-oxo Oleanolic Acid is as follows:
2. the synthetic method of a kind of 11-oxo Oleanolic Acid as claimed in claim 1, is characterized in that: described step a) in the saturated NaCl solution washing of organic layer 2 ~ 5 times.
3. the synthetic method of a kind of 11-oxo Oleanolic Acid as claimed in claim 1, is characterized in that: described step b) in by step a) in compound 1 acetic acid that obtains, add potassium bichromate, under the condition of 90 DEG C, stir 6h; 2 ~ 5 times are extracted by ethyl acetate (EA).
4. the synthetic method of a kind of 11-oxo Oleanolic Acid as claimed in claim 1, it is characterized in that: described step c) in high performance liquid chromatography to adopt containing the methanol solution of 0.01% formic acid be eluent, high performance liquid chromatography adopts wavelength to be 210, the UV-light of 250nm is as detection chromatogram.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1724556A (en) * | 2005-03-11 | 2006-01-25 | 南京大学 | Oleanolic acid and its derivative, preparation method and use |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1724556A (en) * | 2005-03-11 | 2006-01-25 | 南京大学 | Oleanolic acid and its derivative, preparation method and use |
Non-Patent Citations (6)
| Title |
|---|
| HAREGEWEIN ASSEFA等: "SYNTHESIS AND EVALUATION OF POTENTIAL COMPLEMENT INHIBITORY SEMISYNTHETIC ANALOGS OF OLEANOLIC ACID", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
| SHOJI SHIBATA等: "Chemical Modification of Glycyrrhetinic Acid in Relation to the Biological Activities", 《CHEM. PHARM. BULL.》 * |
| SIEWERT,BIANKA等: "The chemical and biological potential of C ring modified triterpenoids", 《EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY》 * |
| YUAN ZHANG等: "Synthesis and activity of oleanolic acid derivatives, a novel class of inhibitors of osteoclast formation", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
| 孟艳秋等: "齐墩果酸衍生物的合成及抗肿瘤活性的研究", 《药学学报》 * |
| 郑彦云主编: "《化学检验技术》", 31 May 2014 * |
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Application publication date: 20151223 |
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