CN113480590B - Wilforinupinone, its preparation method and application in medicine - Google Patents
Wilforinupinone, its preparation method and application in medicine Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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Abstract
The invention provides euphorbia alkane type triterpenoid Tripterygium wilfordii ketone (compound shown in formula I), a method for extracting the compound from Tripterygium wilfordii, a pharmaceutical composition which takes the compound shown in formula I as an active ingredient and is used for treating and preventing tumors, and application of the compound and the pharmaceutical composition thereof in preparing medicaments for treating and preventing tumors.
Description
Technical Field
The invention belongs to the field of pharmaceutical compounds and pharmacology, and particularly relates to a novel compound wilfordinum euphorbia ketone (wilfordiuuphone), a preparation method thereof, a pharmaceutical composition taking the compound as an active ingredient, and application of the compound and the pharmaceutical composition thereof in preparing medicaments for treating tumors.
Background
Tripterygium wilfordii (Tripterygium wilfordii) is an annual vine plant of Tripterygium genus (Tripterygium) of Celastraceae family, and the plant height is 1-3 m. It is produced in Taiwan, fujian, jiangsu, zhejiang, anhui, hubei, hunan, guangxi, etc. Growing in the forest of mountain land in the place of yin-dampness. Tripterygium wilfordii is a common Chinese herbal medicine in traditional Chinese medicine, and has the effects of resisting inflammation, suppressing immunity, resisting fertility, resisting tumor, resisting bacteria, relieving swelling and pain, and the like.
The tumor refers to abnormal hyperplasia caused by the loss of normal regulation and control of cell growth due to gene change of cells under the action of tumorigenic factors. At present, surgical therapy (operation), chemotherapy (chemotherapy), radiotherapy (radiotherapy), traditional Chinese medicine treatment, and modern minimally invasive treatment and biological treatment methods are mainly used for treating tumors. With the rapid advance of nano molecular medicine and molecular biology technology in recent years, the elucidation of tumor generating mechanism, the search of anti-tumor target points, the development of novel anti-tumor drugs and the innovation and the comprehensive application of treatment means all have great progress, and the survival time of tumor patients is obviously prolonged. However, the treatment of solid tumors, which are more than 90% of malignant tumors and seriously threaten human life and health, has not yet achieved satisfactory curative effect, and a large number of tumor patients finally fail to treat the tumors because of no response or drug resistance to the treatment. Therefore, the discovery and development of novel antitumor drugs is still a problem to be continuously faced.
In the prior art, reports of wilfordinump (a compound of formula (I) related to the invention and its effective component in treating tumors and reports of antitumor activity of the compound are not found.
Disclosure of Invention
The invention aims to provide wilfordinum wilfordii knoponone (wilfordinuphone) with medicinal value.
Another objective of the invention is to provide a method for extracting the compound of the invention from tripterygium wilfordii which is a medicinal plant.
The invention further aims to provide a pharmaceutical composition for treating and preventing tumors by using the compound shown in the formula I as an active ingredient, and application of the compound and the composition in preparing medicaments for treating tumors.
In order to achieve the purpose, the invention provides the following technical scheme:
the medicine for treating tumor contains effective tumor treating amount of the compound in the formula I or its pharmaceutically acceptable salt, ester or stereoisomer and pharmaceutically acceptable carrier and/or excipient.
The compound triptonide (wilfordiuboards wilfordii) can be converted into pharmaceutically acceptable derivatives. For example, the hydroxyl group can be acylated (e.g., formylated, acetylated, benzoylated, sulfonylated), halogenated, oxidized, etc., by a known method.
When used as a medicament, the compounds of the present invention may be used as such or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90% of the compound of the invention, and the balance of pharmaceutically acceptable salts, or pharmaceutically acceptable carriers and/or excipients which are non-toxic and inert to humans and animals.
The pharmaceutically acceptable carrier or excipient is one or more selected from solid, semi-solid and liquid diluents, fillers and pharmaceutical adjuvants. The pharmaceutical composition is administered in the form of a dose per unit body weight. The medicine of the invention can be administered by oral administration and injection (intravenous injection and intramuscular injection).
It can be administered orally in the form of solid or liquid, such as powder, tablet, coated preparation, capsule, solution, syrup, drop pill, etc.
The injection can be solid or liquid preparation, such as powder injection, solution injection, etc.
In order to better understand the nature of the invention, the results of the biological activity of the compounds of formula I according to the invention will be used to illustrate their use in the medical field.
The MTS method is used for detecting the cell activity:
MTS is a novel MTT analogue, is totally called 3- (4,5-dimethylthiozol-2-yl) -5- (3-carboxymethyloxy-phenyl) -2- (4-sulfophenyl) -2H-tetrazolium, and is a yellow dye. Succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTS to generate soluble formazan (formazan) compounds, and the content of the formazan can be measured at 490nm by using an enzyme labeling instrument. Since the formazan production amount is generally proportional to the number of living cells, the number of living cells can be estimated from the optical density OD value.
The experimental method comprises the following steps:
1. inoculating cells: preparing single cell suspension by using culture solution (DMEM or RMPI 1640) containing 10% fetal bovine serum, inoculating 3000-15000 cells in each hole to a 96-well plate, wherein each hole volume is 100 mu L, and the adherent cells are inoculated and cultured 12-24 hours in advance.
2. A solution of test compound (fixed concentration 40. Mu.M primary screen, compound at this concentration which inhibits tumor cell growth by around 50% rescreened at a concentration gradient of 40. Mu.M, 8. Mu.M, 1.6. Mu.M, 0.32. Mu.M, 0.064. Mu.M) was added to a final volume of 200. Mu.L per well, with 3 replicates per treatment.
3. Color development: after culturing for 48 hours at 37 ℃, removing culture solution in the hole of the adherent cells, and adding 20 mu L of MTS solution and 100 mu L of culture solution in each hole; discarding 100 mu L of culture supernatant of suspension cells, and adding 20 mu L of MTS solution into each well; after incubating for 2 to 4 hours with 3 blank double wells (mixed solution of 20. Mu.L MTS solution and 100. Mu.L culture medium) to allow the reaction to proceed sufficiently, the light absorption value was measured.
4. Color comparison: selecting 492nm wavelength, reading light absorption value of each hole with multifunctional microplate reader (MULTISKAN FC), recording result, drawing cell growth curve with concentration as abscissa and cell survival rate as ordinate after data processing, and calculating IC of compound by two-point method (Reed and Muench method) 50 The value is obtained.
5. Positive control compound: cisplatin (DDP) is taken as a positive compound in each experiment, a cell growth curve is drawn by taking the concentration as the abscissa and the cell survival rate as the ordinate, and the IC of the compound is calculated by using a two-point method (Reed and Muench method) 50 The value is obtained.
The results are shown in Table 1:
TABLE 1 half growth inhibitory concentration IC of Wilfordii euphorbiaceae (wilfordiurouping) on tumor cell lines 50 (μM)
The test results show that wilfordii euphorbia ketone (wilfordiurouping) has obvious inhibition effect on the growth of various tumor cell strains, and the anticancer activity of the wilfordii euphorbia ketone is equivalent to that of a clinical antitumor drug cisplatin.
Drawings
FIG. 1 shows the crystal structure of X-ray single crystal diffraction measurement of wilforinulinone (wilfordeuplone) compound of the present invention.
Detailed Description
The following examples further illustrate the essence of the present invention, but the present invention is not limited thereto.
Example 1:
this example provides an euphorbiane triterpene compound extracted from Tripterygium wilfordii, which is a white crystal with a molecular weight of 456 and a molecular formula of C 30 H 48 O 3 The specific structural formula is as follows:
the compound is obtained by separating and purifying tripterygium wilfordii dry roots, and the specific extraction steps are as follows:
1) Taking 14.0kg of dried roots of tripterygium wilfordii as raw materials, crushing, heating and refluxing for 3 times by using ethanol water solution with volume fraction of 95% and 6 times of the weight of the raw materials, and then carrying out reduced pressure concentration until no alcohol exists to obtain 650g of extract.
2) Dissolving the extract by using a mixed solvent (1:1) of chloroform and methanol, stirring the dissolved extract by using 2 times of silica gel (1300 g), separating by using 4 times of silica gel (2600 g), sequentially carrying out gradient elution by using petroleum ether/acetone solvent (20.
Normal phase silica gel column material: 80-100 mesh, qingdao Kang Yexin pharmaceutical silica gel desiccant GmbH;
the flow rate of the eluent is 10mL/min;
3) Fr.3 (20 g) was obtained, and the obtained Fr.3 was subjected to normal phase silica gel column chromatography, further eluted with 20 times the amount of silica gel (400 g), and 5 fractions of the eluate were collected and sequentially named as Fr.3-1 to Fr.3-5.
Normal phase silica gel column material: 200-300 mesh, qingdao Kang Yexin pharmaceutical silica gel desiccant GmbH;
eluent: petroleum ether/acetone =10
The flow rate of the eluent is 10mL/min;
4) After obtaining Fr.3-2 (2 g), the Fr.3-2 was subjected to medium pressure preparative liquid column chromatography using C 18 Separating by reverse phase silica gel column chromatography, eluting with 60% methanol water solution, collecting 5 eluates, and sequentially naming as Fr.3-2-1-Fr.3-2-5.
Reversed phase silica gel material: chromatorex C 18 (40～75μm,Fuji Silysia Chemical Ltd.,Japan)；
The flow rate of the eluent is 10mL/min;
5) After Fr.3-2-4 (0.2 g) is obtained, sephadex LH-20 gel column chromatography is adopted to separate and concentrate the Fr.3-2-4; collecting one part of chromatographic solution every 5mL, merging TLC point plates, detecting target material flow parts, placing the flow parts containing the target material at room temperature, and separating out crystals after 24 hours to finally obtain 118mg of the pure wilforonidine.
Gel material Sephadex LH-20: 40-70 μm (GE Healthcare Bio-Sciences AB, uppsala, sweden);
the flow rate of the eluent is 2mL/min;
eluent chloroform/methanol =1:1;
the wilfordinum euphorbia ketone (wilfordinuophone) structure data obtained by the separation process are as follows:
white needle crystals (chloroform-methanol); molecular formula C 30 H 48 O 3 ；
(c 0.10,MeOH)；ESIMS(pos.):m/z 479 [M+Na] + ；HRESIMS:m/z 479.3496[M+Na] + (479.3496calcd for C 30 H 48 O 3 Na)；IR(KBr)ν max : 3430,2968,2948,1708,1630,1452,1386,1005,cm –1 (ii) a The NMR spectrum data are shown in Table 2. The spatial structure of the compound is preliminarily identified by ROESY, and finally the spatial configuration of the compound is subjected to X-single crystal diffraction technologyAnd (5) confirming.
TABLE 2 NMR spectroscopy data (CDCl) for wilfordinum wilfordii euphorbia 3 Middle assay)
Crystallization conditions are as follows: dissolving 15mg of sample in a 10mL ampule with acetonitrile solution, covering the bottle mouth with a plastic film, pricking 2 small holes on the film, and volatilizing at room temperature to obtain needle crystal (the single crystal structure is shown in the attached figure of the specification). The data of X-ray single crystal diffraction are as follows:
Crystal data for wilfordeuphone:C 30 H 48 O 3 ,M＝456.68,
α＝90°,β＝126.803(5)°,γ＝90°,
T＝100.(2)K,space group C121, Z＝4,μ(Cu Kα)＝0.560mm -1 ,12938reflections measured,4574independent reflections(R int ＝0.3557).The final R 1 values were 0.1738(I>2σ(I)).The final wR(F 2 )values were 0.4337(I> 2σ(I)).The final R 1 values were 0.2734(all data).The final wR(F 2 )values were 0.5023(all data). The goodness offit onF 2 was 1.503.Flackparameter＝-0.4(9).
example 2:
and (3) tablet preparation: 10mg of the compound obtained in example 1, 180mg of lactose, 55mg of starch, 5mg of magnesium stearate;
the preparation method comprises the following steps: the compound, lactose, starch were mixed, uniformly moistened with water, the moistened mixture was sieved and dried, and sieved again, magnesium stearate was added, and the mixture was compressed into tablets weighing 250mg each, of which the compound content was 10mg.
Example 3:
and (4) capsule preparation: 100mg of the compound obtained in example 1, 100mg of starch, and an appropriate amount of magnesium stearate;
the preparation method comprises the following steps: mixing the compound with adjuvants, sieving, mixing in suitable container, and encapsulating the obtained mixture into hard gelatin capsule.
Example 4:
ampoule agent: 2mg of the compound obtained in example 1, 10mg of sodium chloride;
the preparation method comprises the following steps: the compound and sodium chloride are dissolved in an appropriate amount of water for injection, filtered, and the resulting solution is aseptically filled into ampoules.
Claims (6)
1. Tripterygium wilfordii euphorbia ketone, which is characterized in that the tripterygium wilfordii euphorbia ketone is a compound with the following structural formula I:
2. the method of preparing the wilfordii ingenone compound of claim 1, comprising the steps of:
14.0kg of dried roots of Tripterygium wilfordii are pulverized and extracted with alcohol, the extracts are combined and concentrated under reduced pressure to obtain 650g of crude extract, the obtained extract is separated by normal phase silica gel column chromatography, gradient elution is carried out by a petroleum ether/acetone solvent 20.
3. A pharmaceutical composition comprising a therapeutically effective amount of the wilfordii ingenone or pharmaceutically acceptable salt thereof of claim 1.
4. The pharmaceutical composition according to claim 3, wherein the mass fraction of wilfordii ingenone or its pharmaceutically acceptable salt in the pharmaceutical composition is 0.1-99%.
5. The use of the wilfordii ingenione of claim 1 in the manufacture of a medicament for the treatment and prevention of tumors.
6. The pharmaceutical composition of claim 3, further comprising a pharmaceutically acceptable excipient.
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