CN101830963A - Bufogenin compound and application thereof in preparing anti-tumor medicaments - Google Patents

Abstract

The invention relates to the technical field of medicine, and relates to the application of a bufolide compound in the preparation of antitumor drugs. The bufalide compound refers to cinobufagin alcohol and its derivatives, including their pharmaceutically acceptable salts. The general chemical structure is as follows: , wherein: the groups R ₁ , R ₂ , R ₃ , and R ₄ are respectively selected from hydrogen, halogen, C ₁ -C ₆ linear or branched saturated or unsaturated hydrocarbon groups, C ₃ -C ₇ cycloalkyl groups, benzyl , aromatic group or 5-7 membered heterocyclic group. In vitro cytology experiments show that the compound of the invention can significantly inhibit the proliferation of liver cancer and lung cancer cells, and therefore can be used to prepare drugs for inhibiting tumor growth.

Description

A kind of bufolide compound and its application in the preparation of antitumor drugs

technical field

The invention relates to the technical field of medicine, and relates to a bufolide compound and its application in the preparation of antitumor drugs. The bufalide compound refers to cinobufagin alcohol and its derivatives, including their pharmaceutically acceptable salts.

Background technique

There are more than 250 species of toads distributed all over the world. There are nearly 10 species in my country, and the main three are: Toad Sinensis, Toad Sinina, and Toad Sinensis. Ancient prescriptions are widely used in toad detoxification and swelling, ketone killing insects, strengthening the heart and diuresis. At present, there are Chansu Analgesic Ointment, Chansu Injection, Cinobufacin Injection and Compound Chanpi Capsules and other preparations on the market, which are used in clinical anti-tumor, analgesic, diuresis and swelling, and enhance the body's immunity. The clinical use of toad preparations in adjuvant treatment of tumors has the advantages of treating both symptoms and root causes, and can significantly inhibit tumor metastasis and improve the body's immunity. Compared with chemotherapy drugs, it can reduce the pain of tumor patients, significantly improve the quality of life, and prolong the life cycle. It can also be used in combination with chemotherapeutic drugs to enhance the curative effect. However, the current toad preparations all use crude extracts as medicines, and the ingredients are extremely complex. In addition to bufagenin, bufotoxin, and indole alkaloids, they also contain amino acids, reducing sugars, steroids, peptides, etc. types of chemical constituents. Therefore, in clinical use, toad preparations have the following two main disadvantages: low rate of killing tumor cells; allergic reactions occur. This is all due to failing to remove invalid and adverse reaction ingredients, and failing to control the real active ingredients.

Commonly used Chinese medicinal materials based on toads include toad venom, toad skin, and dried toad. Indole alkaloids. At present, the compounds that have been studied more include bufafelin, bufafetalin, bufafetalin, cinobufafalin, bufafagenin and so on.

So far, there is no report on the use of cinobufaginol and its derivatives in the preparation of tumor growth inhibitory drugs.

Contents of the invention

One of the objects of the present invention is to provide a class of cinobufagin alcohol derivatives; the second object is to provide a class of cinobufagin alcohol and its derivatives or pharmaceutically acceptable salts thereof for use in antitumor drugs. The general chemical structure formula of cinobufagin alcohol and its derivatives of the present invention is as follows:

Wherein: group R ₁ represents hydrogen, halogen, C ₁ -C ₆ straight chain or branched saturated or unsaturated hydrocarbon group, C ₃ -C ₇ cycloalkyl group, benzyl group, aromatic group or 5-7 membered heterocyclic group, R ₂ represents hydrogen, halogen, C ₁ -C ₆ straight chain or branched saturated or unsaturated hydrocarbon group, C ₃ -C ₇ cyclic hydrocarbon group, benzyl, aromatic group or 5-7 membered heterocyclic group, R ₃ represents hydrogen , halogen, C ₁ -C ₆ linear or branched saturated or unsaturated hydrocarbon group, C ₃ -C ₇ cycloalkyl group, benzyl, aryl or 5-7 membered heterocyclic group, R ₄ represents hydrogen, halogen, C ₁ -C ₆ straight chain or branched saturated or unsaturated hydrocarbon group, C ₃ -C ₇ cycloalkyl group, benzyl group, aryl group or 5-7 membered heterocyclic group;

Said halogen is selected from fluorine, chlorine, bromine or iodine; said aromatic group is selected from phenyl, substituted phenyl, naphthyl or biphenyl; said substituted phenyl includes 1 to 4 substituents, the The substituent is selected from halogen, C ₁ -C ₆ straight chain or branched chain hydrocarbon group, cyano, nitro, amino, hydroxyl, hydroxymethyl, trifluoromethyl, trifluoromethoxy, carboxyl, C ₁ -C ₄ Alkoxy, mercapto and C ₁ -C ₄ acyl; said 5-7 membered heterocyclic group contains 1-3 heteroatoms selected from oxygen, sulfur or nitrogen, and/or is divided by phenyl in the structural formula combined, and/or contain one or more selected from halogen, C ₁ -C ₆ straight chain or branched hydrocarbon group, cyano group, nitro group, amino group, hydroxyl group, hydroxymethyl group, trifluoromethyl group, trifluoromethoxy Substituents of radical, carboxyl, C ₁ -C ₄ alkoxy, mercapto C ₁ -C ₄ acyl and aryl.

Said pharmaceutically acceptable salt is selected from organic acids such as propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, and aspartic acid , Glutamic acid and other acidic amino acids form esters and then form salts with inorganic bases, such as sodium, potassium, calcium, aluminum salts and ammonium salts, or salts with organic bases, such as methylamine salts, ethylamine salts, ethanolamine salts etc.; or form esters with basic amino acids such as lysine, arginine, ornithine, and then react with inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, or with formic acid, acetic acid, bitter acid, salts formed from organic acids such as methanesulfonic acid and ethanesulfonic acid.

In vitro cytology experiments show that the compound of the present invention can cause various tumor cell cycle arrests such as liver cancer, lung cancer, prostate cancer and leukemia, and significantly inhibit cell proliferation, so it can be used to prepare inhibitors for the growth of various tumors including liver cancer. drug.

Description of drawings

Fig. 1 is that 60 kinds of bufolide compounds of the present invention (4 * 10 ^-4 mg/ml) are to SMMC-7721 cell inhibitory rate graph

Fig. 2 is a graph showing the inhibitory rate of 60 kinds of bufolide compounds (4×10 ^-4 mg/ml) of the present invention on A549 cells

Fig. 3 is the comparative figure of cinobufagin alcohol in the present invention to SMMC-7721 cell inhibitory rate

Fig. 4 is the effect diagram of cinobufagin alcohol acting on normal cells L02 and 293T in the present invention

Fig. 5 is the therapeutic curve of cinobufagin alcohol in the present invention to tumors in vivo

Detailed ways

The present invention will be described in detail in conjunction with the accompanying drawings and embodiments.

Example 1. Preparation of Cinobufagin Alcohol

(1) Preparation of bufolide extract

Take 10Kg of toad skin, extract 3 times with 95% ethanol as usual, the amount of alcohol used each time is 10 times the weight of toad skin, the extraction time is 120 minutes, combine the filtrate, concentrate the filtrate under reduced pressure to 10L, and apply XAD-4 macroporous resin (Rohm and Haas), first wash off impurities with water and 30% ethanol successively, then elute with 95% ethanol, collect the eluate, recover the solvent under reduced pressure, and dry to obtain 125 g of bufolide extract.

(2) Preparation of cinobufagin alcohol

Take 75g of the bufolide extract obtained in (1), and separate it by preparative HPLC (Waters Xterra C18, A: 0.1% formic acid water; B: 0.1% formic acid acetonitrile, gradient conditions: 5% B to 25% B, 30min; 25%B～50%B, 22min; 50%B～95%B, 18min; 95%B, 5min, 300nm), divided into 60 components (Fr.1～Fr.60).

Fr.16 recovered solvent, separated by HPLC (functionalized chromatographic column, gradient condition: 95% B ~ 60% B, 40min, wherein A: 0.1% formic acid water; B: 0.1% formic acid acetonitrile, detection wavelength 300nm.), Collect wherein main chromatographic peak, reclaim solvent, obtain compound I 11mg, HPLC detection content is 99%, physicochemical determination data and literature (Linde H., Hofer P., Meyer K., 1966, Cinobufaginol-Uber Krotengifte.32., HELVETICA CHIMICA ACTA, 49, 1243-&.) The compound cinobufaginol reported is basically the same, so it can be considered that compound I is cinobufaginol.

Embodiment 2.60 kinds of bufolide compound compound are to the cytotoxicity test of SMMC-7721 and A549

Cells: SMMC-7721 and A549 tumor cell lines, both international general cell lines, were obtained from the Oriental Institute of Hepatobiliary Surgery, Second Military Medical University of the Chinese People's Liberation Army

Bufa lactone compounds: prepared in Example 1 (the same below)

Using SMMC-7721 and A549 as cell models, 60 compounds were initially screened with CCK-8 cell proliferation kit (product of Dojin Chemical Research Institute, Japan). The experimental design is as follows:

The SMMC-7721 and A549 cells in the logarithmic growth phase were adjusted to 5× ¹⁰ cells/ml in DMEM medium with 10% calf serum, and the control group and the experimental group were divided into three duplicate wells, and 100 μl of each was inoculated in 96 In the well plate, place it in an incubator containing 5% CO ₂ at 37°C. After 24 hours of adherence, replace with 100 μl of 10% calf serum DMEM medium, and add 4×10 ⁻⁴ mg/ml of each compound to the experimental group. After 48 hours, follow the steps of the CCK-8 kit, suck off the medium, add 100 μl of 0.1% calf serum DMEM medium to each well, then add 10 μl of CCK-8 reagent, and add 0.1% calf serum to three blank wells. 100 μl of serum DMEM medium and 10 μl of CCK-8 reagent were placed in an incubator containing 5% _CO2 at 37°C and incubated for 90 minutes, and the absorbance value (A value) at 450 nm was measured with a multi-functional microplate reader. The wells were set to zero, and the cell proliferation inhibition rate was calculated. Inhibition rate=(1-A value of experimental group/A value of control group)×100%. The results are shown in Figure 1 and Figure 2.

It can be seen from Figure 1 and Figure 2 that cinobufagin alcohol has obvious inhibitory effect on the proliferation of liver cancer cell line SMMC-7721 and lung cancer cell line A549, and the inhibition rate has reached 4×10 ^-4 mg/ml at the concentration of At saturation level (90%), other small molecules also have some inhibitory effect (10-20%) at this concentration.

Example 3. Cytotoxicity test of cinobufaginol on SMMC-7721

Cells: SMMC-7721 tumor cell line, which is an international general cell line, was obtained from the Oriental Institute of Hepatobiliary Surgery, Second Military Medical University of the Chinese People's Liberation Army

Cinobufaginol: Preparation in Example 1 (the same below)

Cinobufacin Injection: Anhui Jinchan Biochemical Co., Ltd. (the same below)

Using SMMC-7721 as the cell model, CCK-8 cell proliferation kit was used to screen the inhibition rate of different concentrations of cinobufaginol. The experimental design was as follows:

SMMC-7721 cells in the logarithmic growth phase were adjusted to 5×10 ⁴ cells/ml with 10% calf serum DMEM medium, and a control group was set up separately (the concentration of the equivalent crude drug of cinobufacin injection was 0.5×10 ^-3 g/ml respectively. , 1×10 ^-3 g/ml, 2×10 ^-3 g/ml, 4×10 ^-3 g/ml, 8×10 ^-3 g/ml, 16×10 ^-3 g/ml, corresponding to Figure 3 A1-A6) and experimental groups (cinobufaginol 0.25×10 ^-4 mg/ml, 0.5×10 ^-4 mg/ml, 1.0×10 ^-4 mg/ml, 2.0×10 ^-4 mg/ml, 3.0 ×10 ^-4 mg/ml, 4.0×10 ^-4 mg/ml, the equivalent crude drug concentration is 0.375×10 ^-3 g/ml, 0.75×10 ^-3 g/ml, 1.5×10 ^-3 g/ml, 3× 10 ^-3 g/ml, 4.5×10 ^-3 g/ml, 6×10 ^-3 g/ml, corresponding to B1-B6 in Fig. 3), 3 replicate wells in each group, each 100 μl was inoculated in a 96-well plate, They were placed in an incubator at 37°C containing 5% CO ₂ , and after 24 hours of adherence, 100 μl of DMEM medium with 10% calf serum was changed, and drugs were added to both groups according to the predetermined plan. After 48 hours, follow the steps of the CCK-8 kit, suck off the medium, add 100 μl of 0.1% calf serum DMEM medium to each well, then add 10 μl of CCK-8 reagent, and add 0.1% calf serum to three blank wells. 100 μl of serum DMEM medium and 10 μl of CCK-8 reagent were placed in an incubator containing 5% _CO2 at 37°C and incubated for 90 minutes, and the absorbance value (A value) at 450 nm was measured with a multi-functional microplate reader. The wells were set to zero, and the cell proliferation inhibition rate was calculated. Inhibition rate=(1-A value of experimental group/A value of control group)×100%. The result is shown in Figure 3.

It can be seen from Figure 3 that cinobufaginol has reached the saturation level (90%) at 4×10 ^-4 mg/ml, equivalent to a crude drug concentration of 8×10 ^-4 g/ml; The injection requires 160×10 ^-4 g/ml (converted based on the concentration of the crude drug), and the concentration difference between the two is very large.

Example 4. Toxic effect of cinobufaginol on non-tumor cell lines

Cells: L02 and 293T, both international general cell lines, obtained from the Oriental Institute of Hepatobiliary Surgery, Second Military Medical University of the Chinese People's Liberation Army

Cinobufaginol: Preparation in Example 1 (the same below)

In order to exclude whether cinobufagin alcohol has non-specific cytotoxicity, non-tumor cell lines L02 and 293T were selected for detection, and the experimental design was as follows:

Normal liver epithelial cells L02 and human embryonic kidney epithelial cells 293T in the logarithmic growth phase were adjusted to 5× ¹⁰ cells/ml in 10% calf serum DMEM medium, and the control group and the experimental group were divided into three replicate wells , each 100 μl was inoculated in a 96-well plate, placed in an incubator at 37°C containing 5% CO ₂ , and after 24 hours of adherence, 100 μl of 10% calf serum DMEM medium was replaced, and the experimental group was added with drugs so that the final concentration was 1×10 ^-4 mg/ml, 2×10 ^-4 mg/ml, 3×10 ^-4 mg/ml, 4×10 ^-4 mg/ml. After 48 hours, follow the steps of the CCK-8 kit, suck off the medium, add 100 μl of 0.1% calf serum DMEM medium to each well, then add 10 μl of CCK-8 reagent, and add 0.1% calf serum to three blank wells. 100 μl of serum DMEM medium and 10 μl of CCK-8 reagent were placed in an incubator containing 5% _CO2 at 37°C and incubated for 90 minutes, and the absorbance value (A value) at 450 nm was measured with a multi-functional microplate reader. The wells were set to zero, and the cell proliferation inhibition rate was calculated. Inhibition rate=(1-A value of experimental group/A value of control group)×100%. The results are shown in Figure 4 (A is L02 cells, B is 293T cells).

It can be seen from Figure 4 that in non-tumor cell lines L02 and 293T, cinobufaginol has no obvious effect on cell proliferation, and there is no specific dose-effect relationship, and the inhibitory effect at low and high concentrations is basically the same. The inhibition rate of the highest concentration can only reach 20%, indicating that its inhibition on tumor cells is not caused by non-specific toxicity (if it is toxicity, the survival rate should decrease with the increase of concentration).

Example 5. The Inhibitory Effect of Cinobufagin Alcohol on Tumor

Animals: Nude mice, provided by the Animal Center of the Second Military Medical University; SMMC-7721 tumor cell line, an international general cell line, was obtained from the Oriental Hepatobiliary Surgery Institute of the Second Military Medical University of the Chinese People’s Liberation Army

Cinobufaginol: Preparation in Example 1 (the same below)

In order to detect the anti-tumor activity of cinobufaginol in animals, Nude mice were selected as models, and cinobufacin injection was used as a positive reference to observe its therapeutic effect on tumors. The experimental design is as follows:

Take 8-week-old Nude mice, 6 males with an average weight of about 25 grams, subcutaneously inject 2× ¹⁰⁶ liver cancer cell line SMMC-7721 cells, and successfully bear the tumor after 3 weeks, and randomly divide them into 2 groups, with 3 mice in the control group. Group 3 only. The experimental group was continuously injected with 0.5 mg*kg ^-1 *d ^-1 cinobufaginol into the tumor, and the control group was continuously injected with the same amount of normal saline, and observed for 4 weeks. The result is shown in Figure 5.

It can be seen from Fig. 5 that compared with the control group, the tumor volume of the experimental group was significantly reduced. Statistical analysis was performed by paired t-test, P<0.05, indicating that cinobufagin alcohol has a significant therapeutic effect on liver cancer.

Claims (2)

1. toad lactone compound and pharmacy acceptable salt thereof, chemical structure of general formula is as follows:

Wherein: radicals R ₁Represent hydrogen, halogen, C ₁-C ₆Saturated or the unsaturated alkyl of straight or branched, C ₃-C ₇Cyclic hydrocarbon radical, benzyl, aromatic base or 5-7 unit heterocyclic radical, R ₂Represent hydrogen, halogen, C ₁-C ₆Saturated or the unsaturated alkyl of straight or branched, C ₃-C ₇Cyclic hydrocarbon radical, benzyl, aromatic base or 5-7 unit heterocyclic radical, R ₃Represent hydrogen, halogen, C ₁-C ₆Saturated or the unsaturated alkyl of straight or branched, C ₃-C ₇Cyclic hydrocarbon radical, benzyl, aromatic base or 5-7 unit heterocyclic radical, R ₄Represent hydrogen, halogen, C ₁-C ₆Saturated or the unsaturated alkyl of straight or branched, C ₃-C ₇Cyclic hydrocarbon radical, benzyl, aromatic base or 5-7 unit heterocyclic radical;

Said halogen is selected from fluorine, chlorine, bromine or iodine; Said aromatic base is selected from phenyl, substituted-phenyl, naphthyl or xenyl; Said substituted-phenyl comprises 1～4 substituting group, and this substituting group is selected from halogen, C ₁-C ₆Straight or branched alkyl, cyano group, nitro, amino, hydroxyl, methylol, trifluoromethyl, trifluoromethoxy, carboxyl, C ₁-C ₄Alkoxyl group, sulfydryl and C ₁-C ₄Acyl group; Said 5-7 unit heterocyclic radical is to contain 1-3 heteroatoms that is selected from oxygen, sulphur or nitrogen, and/or by the phenyl in the structural formula and close, and/or contain one or more halogen, C of being selected from ₁-C ₆Straight or branched alkyl, cyano group, nitro, amino, hydroxyl, methylol, trifluoromethyl, trifluoromethoxy, carboxyl, C ₁-C ₄Alkoxyl group, sulfydryl C ₁-C ₄The substituting group of acyl group and aromatic base;

Said pharmacy acceptable salt be selected from propionic acid, oxalic acid, propanedioic acid, succsinic acid, fumaric acid, toxilic acid, lactic acid, oxysuccinic acid, tartrate, citric acid, etc. organic acid and acidic amino acids such as aspartic acid, L-glutamic acid form behind the esters again the salt that forms with mineral alkali, as sodium, potassium, calcium, aluminium salt and ammonium salt, or the salt that forms with organic bases, as methylamine salt, ethylamine salt, ethanolamine salt etc.; Or form behind the esters again and mineral acids such as hydrochloric acid, Hydrogen bromide, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid with basic aminoacidss such as Methionin, arginine, ornithine, or with formic acid, acetate, the salt that organic acids such as picric acid, methylsulfonic acid, ethyl sulfonic acid form.

2. described toad lactone compound of claim 1 and pharmacy acceptable salt thereof the application in the preparation antitumor drug.

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