CN113480590A

CN113480590A - Wilforinupinone, its preparation method and application in medicine

Info

Publication number: CN113480590A
Application number: CN202110924931.0A
Authority: CN
Inventors: 李香梅; 姜先俊; 魏国柱; 王飞; 文世珍; 任丽花
Original assignee: Yunnan Biobiaopha Technology Co ltd
Current assignee: Yunnan Biobiaopha Technology Co ltd
Priority date: 2021-08-12
Filing date: 2021-08-12
Publication date: 2021-10-08
Anticipated expiration: 2041-08-12
Also published as: CN113480590B

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

Wilforinupinone, its preparation method and application in medicine

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 of Celastraceae (Celastraceae), and the plant height is 1-3 m. Produced in Taiwan, Fujian, Jiangsu, Zhejiang, Anhui, Hubei, Hunan and Guangxi. 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 called 3- (4, 5-dimethylthiozol-2-yl) -5- (3-carboxymethylethoxy-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 RMPI1640) containing 10% fetal calf serum, inoculating 3000-15000 cells in each hole to a 96-well plate, wherein each hole 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; setting 3 blank multiple wells (mixed solution of 20 mu L MTS solution and 100 mu L culture solution), and continuing incubation for 2-4 hours to ensure that the light absorption value is measured after the reaction is fully performed.

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)₅₀The 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)₅₀The value is obtained.

The results are shown in Table 1:

TABLE 1. Wilfordii Euphorbiaceae (wilfordeu)Phone) half-growth inhibitory concentration IC on tumor cell lines₅₀(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 wilfordiumukurone (wilfordiumphone) 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 C₃₀H₄₈O₃The 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 with a mixed solvent (1:1) of chloroform and methanol, mixing the dissolved extract with 2 times of silica gel (1300g), separating with 4 times of silica gel (2600g), performing gradient elution sequentially with petroleum ether/acetone solvent (20:1, 10:1, 6:1, 4:1, 2:1, 1:1, 0:1) systems, combining TLC point plates, collecting 7 parts of eluents, and sequentially naming the eluates as Fr.1-Fr.7, wherein target fraction Fr.3 is obtained at the gradient elution stages of petroleum ether/acetone 4:1 and 2: 1.

Normal phase silica gel column material: 80-100 mesh, Qingdao Kangshenxin pharmaceutical silica gel desiccant GmbH;

the flow rate of the eluent is 10 mL/min;

3) fr.3(20g) 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 (400g), 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 Kangshenxin pharmaceutical silica gel desiccant GmbH;

eluent: petroleum ether/acetone 10:1

The flow rate of the eluent is 10 mL/min;

4) after obtaining Fr.3-2(2g), the Fr.3-2 was subjected to medium pressure preparative liquid column chromatography using C₁₈Separating 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 C₁₈(40～75μm,Fuji Silysia Chemical Ltd.,Japan)；

The flow rate of the eluent is 10 mL/min;

5) after Fr.3-2-4(0.2g) 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 2 mL/min;

the eluent is chloroform/methanol-1: 1;

the wilfordinum euphorbia ketone (wilfordinuophone) structure data obtained by the separation process are as follows:

white needle crystals (chloroform-methanol); moleculeFormula C₃₀H₄₈O₃；

(c 0.10,MeOH)；ESIMS(pos.):m/z 479 [M+Na]⁺；HRESIMS:m/z 479.3496[M+Na]⁺(479.3496calcd for C₃₀H₄₈O₃Na)；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 stereo structure of the compound is preliminarily identified by ROESY, and finally the stereo configuration of the compound is identified by an X-single crystal diffraction technology.

TABLE 2 NMR spectroscopy data (CDCl) for wilfordinum wilfordii euphorbia₃Middle 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:C₃₀H₄₈O₃,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 R₁ values were 0.1738(I>2σ(I)).The final wR(F²)values were 0.4337(I> 2σ(I)).The final R₁ values were 0.2734(all data).The final wR(F²)values were 0.5023(all data). The goodness offit onF² 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 10 mg.

Example 3:

and (3) 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:

an 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

1. A compound of the following structural formula I:

2. a process for the preparation of a compound of formula I according to claim 1, which process comprises the steps of:

14.0kg of dried roots of tripterygium wilfordii are crushed and then extracted by alcohol, the extracting solution is merged and decompressed and concentrated 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 (20:1, 10:1, 6:1, 4:1, 2:1, 1:1, 0:1) system, the fractions are spotted and combined at about 4:1 and 2:1 elution sections of petroleum ether/acetone to obtain target fractions, and the fractions are further recrystallized by silica gel (petroleum ether/acetone 10:1 elution), medium pressure preparation (methanol/water 60% elution), Sephadex LH-20 (chloroform/methanol 1:1 elution) and chloroform-methanol solvent to obtain 118mg of pure wilfordeuphone (compound shown in formula I) product.

3. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) as claimed in claim 1 or a pharmaceutically acceptable salt, ester or stereoisomer thereof.

4. The medicament of claim 3, wherein the mass fraction of wilforonidone or its pharmaceutically acceptable salts, esters or stereoisomers in the medicament is 0.1-99%.

5. The use of a compound of formula I according to claim 1 for the preparation of a medicament for the treatment and prophylaxis of tumors.

6. The medicament of claim 3 or the use of claim 5, wherein the medicament further comprises a pharmaceutically acceptable excipient.