Sterine derivatives and synthesis and use thereof

FIELD: chemistry.

SUBSTANCE: invention relates to a compound CL168 of general structural formula I where R is oxygen. The invention also relates to a method of producing a compound of formula I and use of the compound of formula I to produce a medicinal agent for preventing or treating tumorous and immunological diseases.

EFFECT: compound of formula I for producing a medicinal agent for preventing or treating tumorous and immunological diseases.

4 cl, 11 tbl, 19 ex

 

The SCOPE of the INVENTION

This invention relates to pharmaceutical compounds in the field of chemical and biological Sciences, in particular, to anticancer drugs CL168 with the General structural formula I and II and their method of synthesis and application.

BACKGROUND of the INVENTION

The tumor is one of the main diseases that are harmful to human health, and becomes the second in mortality among different diseases. A large number of clinical experiments prove that when the destruction of tumor cells chemical treatment and radiation treatment at the same time are characterized by a significant damaging effect on normal cells. These treatments lead to serious damage to the hematopoietic system and the immune function of the human body and can easily lead to death of patients. All tumor cells depend on blood vessels, and angiogenesis is an important step in the growth and metastasis of tumors. As for primary tumors and secondary tumors angiogenesis occurs after the tumor grows to a diameter of more than 2 mm, and then the tumors grow faster and occurs metastasis (Folkman J. what is the evidence that tumors are angiogenesis department? J Nati Cancer Inst. 1990, 82: 4-6).

Currently, medicines for the treatment of tumors mainly may be edeleny into three types: cytotoxic drugs, adjuvant radiotherapy and chemotherapy and angiogenesis inhibitors. Currently, inhibitors of angiogenesis are very promising anticancer drugs.

A BRIEF DESCRIPTION of the INVENTION

The compounds of this invention called CL168 represent the type of compounds with novel structural skeletons, explicit activities and specific antitumor targets selected from structural modifications of the hundreds of natural products on the basis of the principle of selection on the model ITSELF (Ribatti D, Vacca A, et al. The chick embryo chorioallantoic membrane as a model for in vivo research on anti-angiogenesis. Curr Pharm Biotechnol. 2000 Jul;l(l):73-82.) and VEGF (Gretten TF. Korangy F, et al. Molecular therapy for the treatment of hepatocellular carcinoma.Br J Cancer. 2009 Jan 13; 100(1): 19-23.). Pharmacological experiments show that these compounds possess significant antitumor effects, where CL 168-6 has antitumor therapeutic index 49,3, and can be used for getting medicines for prevention and treatment of cancer such as liver cancer, lung cancer, etc. and immunological diseases.

One of the aims of this invention is the provision of path synthesis for obtaining CL168, which presents the General formula I, and intermediate chemical compounds.

The second purpose of this invention is to provide the tion of the synthesis pathway for obtaining CL168, it is represented by the General formula P, and chemical intermediates.

The third objective of this invention is the provision of applications CL168 General formulas I and II and their chemical intermediates in studies of antitumor activity and immunity.

The purpose of the present invention can be implemented using the following procedures.

Connection CL168 represented by the General structural formula I, can be obtained by a process comprising the following steps:

(1) dissolution of cholesterol (cholesterol-5-EN-3-ol), (compound 2) in an organic solvent to obtain cholesterolaemia (compound 3) by the reaction of cholesterol with acetic anhydride in the catalysis of the catalyst at a certain temperature;

(2) dissolving compound 3 in an organic solvent to obtain 7-bronholitin-3-Alzette (compound 4) by reaction of compound 3 with bromidum reagent during the catalysis of the catalyst at a certain temperature;

(3) the dissolution of compound 4 in an organic solvent to obtain 7-dehydrocholesterol-3-Alzette (compound 5) by reaction off with the base at a certain temperature;

(4) dissolving compound 5 in an organic solvent to obtain 7-dehydrocholesterol (compound 6) by hydroly the connections 5 with the base at a certain temperature;

(5) the dissolution of compound 6 in an organic solvent to obtain 5A,8A-apidocs-6-cholesten-3-ol (compound 7) by oxidation of compound 6 oxidant at a certain temperature;

(6) the dissolution of compound 7 in an organic solvent to obtain 5A,8A-apidocs-6-cholesten-3-one (compound 1, i.e CL168-6) by oxidation of compound 7 oxidant at a certain temperature;

The reactions in the above steps (1)to(6) are as follows:

Where on the stage (1) an organic solvent selected from the group comprising the following compounds, each of which contains 2-20 carbon atoms: ester, alcohol, alkane, aromatic hydrocarbon, ketone, alkylhalogenide, amide, nitrile, ester or a mixture of different relations of concentrations of components of the mixture; the temperature ranges from -20°C to 250°C; the catalyst is a protonic acid, such as sulfuric acid, or an organic base, such as pyridine; the molar ratio of compound 2 to acetic anhydride is 1:1-20.

Where on the stage (2) an organic solvent selected from the group comprising the following compounds, each of which contains 2-20 carbon atoms: aromatic hydrocarbon, alkane, ether, ketone, alkylhalogenide, amide, nitrile, ester or a mixture of different different from what oszeniami concentrations of the components of the mixture; temperature ranges from -10°C to 150°C; bromidum reagent is bromosuccinimide (NBS), bromovalerate or acetylmuramic; the catalyst is triphenylphosphine or a light source with a wavelength of 290-800 nm; molar ratio of compound 3 to breedname reagent is 1:1-10.

Where on the stage (3) an organic solvent selected from the group comprising the following compounds, each of which contains 2-20 carbon atoms: aromatic hydrocarbon, alkane, ether, ketone, alkylhalogenide, amide, nitrile, ester or a mixture of different relations of concentrations of components of the mixture; the temperature is from 0°C to 150°C; catalyst is an organic base, such as pyridine or triethylamine; the molar ratio of compound 4 to the organic base is 1:1-10.

Where on the stage (4) an organic solvent selected from the group comprising the following compounds, each of which contains 2-20 carbon atoms: ester, alcohol, alkane, aromatic hydrocarbon, ketone, alkylhalogenide, amide, nitrile, ester or a mixture of different relations of concentrations of components of the mixture; the temperature is from 0°C to 150°C; catalyst is a protonic acid, a sulfuric acid, or proton base, which is represented by sodium hydroxide.

Where on the stage (5) organic dissolve the spruce is chosen from the group includes the following compounds, each of which contains 2-20 carbon atoms:

aromatic hydrocarbon, alkane, ether, ketone, alkylhalogenide, amide, nitrile, ester or a mixture of different relations of concentrations of components of the mixture; the temperature is from 0°C to 150°C; catalyst is eosin Y, triphenylphosphine or a light source with a wavelength of 290-800 nm.

Where on the stage (6) an organic solvent selected from the group comprising the following compounds, each of which contains 2-20 carbon atoms:

aromatic hydrocarbon, alkane, ether, ketone, alkylhalogenide, amide, nitrile, ester or a mixture of different relations of concentrations of components of the mixture; the temperature ranges from -20°C to 100°C, sulfuric acid or a solution of chromic acid are illustrative oxidizing agent.

Connection CL168 represented by the General structural formula II may be obtained by a process comprising the following steps:

(1) dissolution of cholesterol (compound 2) in an organic solvent to obtain compound 3 by the reaction of cholesterol with a reagent serving as a donor R (R represents a C2-25alkyl group, aryl group, aryl group, substituted by an electron-donating group or electron-withdrawing group, With3-6alkylamino group, alkenylphenol group3- cycloalkyl group3-9substituted geterotsyklicescoe group1-20fatty acid acyl group, aromatic acyl group, sulfonyl, cynnamoyl, caffeoyl, galloyl, feruloyl, benzoyl, L-aliphatic aminoacyl or L-aromatic aminoacyl), during the catalysis of the catalyst at a certain temperature;

(2) dissolving compound 3 in an organic solvent to obtain compound 4 by reaction of compound 3 with bromidum reagent during the catalysis of the catalyst at a certain temperature;

(3) the dissolution of compound 4 in an organic solvent to obtain compound 5 by reaction off with the base at a certain temperature;

(4) dissolving compound 5 in an organic solvent to obtain compound 6 by oxidation of compound 5 oxidant at a certain temperature;

Reaction equations in the above steps (1)to(4) are as follows:

Where on the stage (1) an organic solvent selected from the group comprising the following compounds, each of which contains 2-20 carbon atoms: ester, alcohol, alkane, aromatic hydrocarbon, ketone, alkylhalogenide, amide, nitrile, ester or a mixture of different relations of concentrations of components of the mixture; the temperature ranges from -20°C to 250°C; catalyst which is a proton acid, such as sulfuric acid, or an organic base, such as pyridine; the molar ratio of compound 2 to acetic anhydride is 1:1 -20;

Where on the stage (2) an organic solvent selected from the group comprising the following compounds, each of which contains 2-20 carbon atoms: aromatic hydrocarbon, alkane, ether, ketone, alkylhalogenide, amide, nitrile, ester or a mixture of different relations of concentrations of components of the mixture; the temperature is from -10°C to 150°C; bromidum reagent is bromosuccinimide (NBS), bromovalerate or acetylmuramic; the catalyst is triphenylphosphine or a light source with a wavelength of 290-800 nm; molar ratio of compound 3 to breedname reagent is 1:1-10;

Where on the stage (3) an organic solvent selected from the group comprising the following compounds, each of which contains 2-20 carbon atoms: aromatic hydrocarbon, alkane, ether, ketone, alkylhalogenide, amide, nitrile, ester or a mixture of different relations of concentrations of components of the mixture; the temperature is from 0°C to 150°C; catalyst is an organic base that is represented by pyridine and triethylamine; the molar ratio of compound 4 to the organic base is 1:1-10;

Where on the stage (4) an organic solvent selected from the group comprising, after the ith connection, each of which contains 2-20 carbon atoms: aromatic hydrocarbon, alkane, ether, ketone, alkylhalogenide, amide, nitrile, ester or a mixture of different relations of concentrations of components of the mixture; the temperature is from 0°C to 150°C; catalyst is eosin Y, triphenylphosphine or a light source with a wavelength of 290-800 nm.

Obtained above CL 168-6 (5α,8α-apidocs-6-cholesten-3-one) has a significant ingibiruet effect on the proliferation of cells human hepatoma HepG2 cells and human lung cancer A in vitro;

Obtained above CL168-6 (5α,8α-apidocs-6-cholesten-3-one) has a significant inhibitory activity on tumor angiogenesis;

Obtained above CL168-6 (5α,8α-apidocs-6-cholesten-3-one) can effectively inhibit the growth of S180 tumor, prolong the survival time of mice with tumors and increase the splenic index mice;

Obtained above CL168-6 (5α,8α-apidocs-6-cholesten-3-one) shows low toxicity in vivo, and LD50mice is 1479 mg/kg;

Obtained above CL168-6 (5A,8A-apidocs-6-cholesten-3-one) can be made into dosage forms for oral, injectable and topical administration and be used for the prevention and treatment of tumors.

A brief description of Graphic materials

Figure 1 illustrates the effect CL168-6 vessels in ITSELF.

Figure 2 illustrates the curves vyzhe the needs of mice.

Figure 3 illustrates the tumor of mice of groups with different doses.

Figure 4 illustrates the results of determining VEGF blood eye mouse.

Figure 5 illustrates the results of a 24-hour determination of caspase-3, 8, 9.

Figure 6 illustrates the results of the 48-hour determination of caspase-3, 8, 9.

Figure 7 illustrates a standard curve of protein.

Figure 8 illustrates the level of p53 expression (n=3).

Figure 9 illustrates the level of bcl-2 expression (n=3).

Figure 10 illustrates the level of expression of VEGF (n=3).

Figure 11 illustrates the level of expression of P21 (n=3).

A detailed description of the preferred embodiments

Without limitation, some examples of this invention will be described by way of illustration the push in this document.

An example of obtaining 1; synthesis of cholesterolaemia (compound 3)

Cholesterol (11,58 g, 30,00 mmol), toluene (60 ml), acetic anhydride (5,67 ml, 60,00 mmol) and pyridine (1 ml, 12,41 mmol) were placed in a 100 ml reaction flask, stirred with a magnetic stirrer, and was heated to 114°C., was heated under reflux, and the reaction was carried out as long as there was raw material. The reaction liquid was cooled, washed twice with 2-fold amount of hydrochloric acid (0,10%), twice washed 2 times the amount of saturated sodium chloride solution, twice washing the 2-fold amount of distilled water, was dried using anhydrous sodium sulfate, and then subjected to vacuum distillation with the separation of toluene, eventually received a white solid (12,84 g). Output equal to 100.0%, and the NMR spectra of hydrogen and carbon compounds 3 are as follows:

1H NMR (500 MHz, CDCl3): of 0.68 (s, 3H, H-18), 0,86 (d, 3H, J=2 Hz, H-26), of 0.87 (d, 3H, J=2 Hz, H-27), of 0.91 (d, 3H, J=4.4 Hz, H-21), of 1.02 (s, 3H, H-19), 2,03 (s, 3H, H-2'), 2,32 (dd, 2H, H-4), 4,60 (m, 1H, H-3), of 5.39 (t, 1H, H-6);

13C NMR (500 MHz, CDCl3): 36,6 (C-1), FOR 31.9 (C-2), OF 74.0 (C-3), AND 39.7 (C-4), 140,0 (C-5), THE 122.7 (C-6), 28,2 (C-7), OF 31.8 (C-8), 50,0 (C-9), 38,1 (C-10), OR 21.0 (C-11), 37,0 (C-12), 42,3 (C-13), OR 56.1 (C-14), 24,3 (C-15), WHILE 27.8 (C-16), OF 56.7 (C-17), 11,9 (C-18), AND 19.3 (C-19), A 35.8 (C-20), TO 18.7 (C-21), AND 36.2 (C-22), TO 23.8 (C-23), 39,5 (C-24), 28,0 (C-25), AND 22.6 (C-26), AND 22.8 (C-27), 170,6 (C-1'), 21,5 (C-2').

Example of getting a 2: synthesis of 7-dehydrocholesterol-3-Alzette (connection5)

Cholesterylester (4,28 g, 10,00 mmol), carbon tetrachloride (30 ml) and NBS (1.78 g, 10,00 mmol) were placed in a 50 ml reaction flask was exposed to fluorescent light, was heated under reflux at 74°C, and the reaction was carried out as long as there was raw material. The reaction liquid was cooled, subjected to filtration with air pump, washed with a small amount of carbon tetrachloride, was subjected to vacuum distillation with the release of carbon tetrachloride, eventually got an orange-yellow butter-like liquid.

Oran is Evo-yellow butter-like liquid was added to toluene (50 ml) and 2,6-dimethylpyridine (5 ml), were placed in a 100 ml reaction flask, stirred with a magnetic stirrer, and was heated to 114°C., was heated under reflux, and the reaction was carried out as long as there was raw material. The reaction liquid was cooled, washed twice with 2-fold amount of hydrochloric acid (0,10%), twice washed 2 times the amount of saturated sodium chloride solution, twice washed 2 times with distilled water, dried using anhydrous sodium sulfate, and then subjected to vacuum distillation for separation of toluene, was dissolved in absolute ethanol, were subjected to re-crystallization with the receipt of 3.54 g of a white solid with a yield of 85.5%. NMR spectra of hydrogen and carbon compounds 5 are as follows:

1H NMR (500 MHz, CDCl3): of 0.62 (s, 3H, H-18), of 0.87 (d, 3H, J=2 Hz, H-26), of 0.87 (d, 3H, J=2 Hz, H-27), were 0.94 (d, 3H, J=4.4 Hz, H-21), 0,99 (s, 3H, H-19), 2,04 (s, 3H, H-2'), of 2.38 (m, 1H, H-4A), of 2.50 (m, 1H, H-4b), 4,70 (m, 1H, H-3), of 5.39 (d, 1H, J=2 Hz, H-7), 5,59 (d, 1H, J=2 Hz, H-6)

13With NMR (500 MHz, CDCl3): 36,6 (C-1), 28,1 (C-2), FOR 72.8 (C-3), OF 37.9 (C-4), 141,6 (C-5), 120,2 (C-6), TO 116.2 (C-7), ON 138.5 (C-8), 46,0 (C-9), TO 39.5 (C-10), A 21.5 (C-11), 37,1 (C-12), 42,9 (C-13), A 55.4 (C-14), OF 23.9 (C-15), 28,1 (C-16)AND 55.8 (C-17), AND 11.8 (C-18), AN 18.4 (C-19), AND 36.2 (C-20), AND 16.2 (C-21), 36,1 (C-22), 23,0 (C-23), 39,1 (C-24), 28,1 (C-25), AND 22.6 (C-26), AND 22.8 (C-27), 170,6 (C-1'), 21,0 (C-2').

An example of retrieving C: synthesis of 7-dehydrocholesterol (compound 6)

7-dehydrocholesterol-3-olacetat (compound 5)(2,07 g, 5 mmol), ethanol (50 ml) and sodium hydroxide solution (10%, 50 ml) were placed in a reaction flask of 250 ml, the reaction was conducted at 80°C as long as there was raw material. The reaction liquid was subjected to vacuum distillation with the release of the remaining ethanol was extracted once with a single quantity of ethyl acetate, washed with distilled water to neutrality and dried using anhydrous sodium sulfate. After the separation of the ethyl acetate the resulting solid was subjected to re-crystallization with ethanol to obtain 1.85 g of 5,7-Dien-cholesterol with out 96,4%. The NMR spectrum of the hydrogen compound 6 is as follows:

1H NMR (500 MHz, CDCl3): of 0.62 (s, 3H, H-18), 0,86 (d, 3H, J 2 Hz, H-26), to 0.88 (d, 3H, J=2 Hz, H-27), were 0.94 (s, 3H, H-19), to 1.22 (d, 3H, J=12 Hz, H-21), 2,33 (dd, 1H, H-4a), 2,49 (dd, 1H, H-4b), 3,66 (m, 1H, H-3), a 4.03 (m, 1H, 3-OH), of 5.39 (m, 1H, H-7), of 5.68 (dd, 1H, H-6).

Example 4: synthesis of 5α,8α-apidocs-6-cholesten-3-ol (compound 7)

5,7-Dien-cholesterol (1,15 g, approximately 3 mmol), eosin Y (200 mg, approximately 0.31 mmol, dissolved in alcohol) and absolute ethanol (100 ml, 10,00 mmol) were placed in a 250 ml reaction flask. After insufflation of air into the reaction liquid was last exposed to fluorescent light, the reaction was carried out as long as there was raw material, the person to distil for the selection of absolute ethanol, while the e has reached a certain volume, kept for crystallization with the receipt of 0.94 g of 5α,8α-apidocs-6-cholesten-3-ol with a yield of 75.3%of.

1H NMR (500 MHz, CDCl3): 0,80 (s, 3H, H-18)to 0.85 (d, 3H, J 2 Hz, H-26), to 0.88 (d, 3H, 3=2 Hz, H-27), to 0.88 (s, 3H, H-19), of 0.91 (d, 3H, J 12 Hz, H-21), of 3.97 (m, 1H, H-3), 6,23 (d, 1H, J-7 Hz, H-7), 6,51 (d, 1H, J=7 Hz, H-6);

13With NMR (500 MHz, CDCl3): 36,0 (C-1), AND 28.3 (C-2), WITH 66.5 (C-3), TO 39.4 (C-4), AND 82.2 (C-5), 135,4 (C-6), 130, 8MM (C-7), OF 79.5 (C-8), OF 51.1 (C-9), OF 35.2 (C-10), TO 20.6 (C-11), TO 34.7 (C-12), WITH 44.8 (C-13), WITH 52.0 (C-14), OF 23.4 (C-15), TO 30.1 (C-16), IS 56.4 (C-17), 12.7MM (C-18), AND 18.2 (C-19), 36,9 (C-20), 19,0 (C-21), 36,9 (C-22), TO 23.8 (C-23), 37,0 (C-24), 28,0 (C-25), AND 22.6 (C-26), AND 22.8 (C-27).

An example of obtaining 5: synthesis of 5A,8A-apidocs-6-cholesten-3-one (compound 1, CL168-6)

5α,8α-apidocs-6-cholesten-3-ol (compound 7) (0,62 g, approximately 1.5 mmol) was dissolved in acetone (50 ml) in a 100 ml reaction flask was slowly added dropwise a solution of chromic acid (1.6 mmol) in a bath with a mixture of water and ice, the reaction was carried out as long as there was raw material. The reaction liquid was poured into a mixture of water and ice (600 ml), stirred, stood in for a whole night and then subjected to filtration and air pump. The precipitate on the filter was subjected to re-crystallization from ethanol with the receipt of 0.62 g of 5α,8α-apidocs-6-cholesten-3-one with output 96,1%.

1H NMR (500 MHz, CDCl3): 0,85 (s, 3H, H-19), to 0.88 (d, 3H, J=2 Hz, H-26), to 0.89 (d, 3H, J 2 Hz, H-27), to 0.92 (d, 3H, J-6.5 Hz, H-21), with 1.07 (s, 3H, H-18), of 3.97 (m, 1H, H-3), of 6.29 (d, 1H, J-8.5 Hz, H-7), 6,59 (d, 1H, J-8.5 Hz, H-6);

1 With NMR (500 MHz, CDCl3): TO 36.7 (C-1), 35,3 (C-2), 207,0 (C-3), WAS 43.6 (C-4), OF 83.4 (C-5), 134,2 (C-6), HUMIDITY 131.6 (C-7), 80,0 (C-8), OF 51.1 (C-9), TO 39.4 (C-10), 20,5 (C-11), 37,3 (C-12), WITH 44.9 (C-13), 51,4 (C-14), 23,8 (C-15), 28,2 (C-16), IS 56.4 (C-17), 12,8 (C-18), AND 18.5 (C-19), 35,2 (C-20)AND 17.5 (C-21), OR 35.9 (C-22), 23.5CM (C-23), OR 39.3 (C-24), 28,0 (C-25), TO 22.5 (C-26), AND 22.8 (C-27).

The final example 1: impact assessment CL168-6 on cell proliferation of human hepatoma HepG2 cells human lung cancer A cells and immortalized human fibroblasts NIH3T3 by MTT analysis

1. Materials

1.1 Tumor lines

Cells human hepatoma HepG2 cells and human lung cancer A was cultivated by the infectious diseases Institute of the people's liberation army of China to assess the viability, whereas cells immortalized human fibroblasts NIH3T3 were purchased from Academy of military medical Sciences.

1.2 Experimental drugs CL168-6, obtained independently, which had a purity equal to or greater than 98%, evaluated using liquid chromatography high pressure (HPLC), and, therefore, meets the requirements of the experiment. Powder specified CL168-6, which is pre-tightly corked and kept at 4°C, was dissolved in dimethyl sulfoxide (DMSO) to obtain the initial solution with a concentration of 1 ml/mg for later use.

2. Way

2.1 Cultivation of cells

Cells human hepatoma is and HepG2, cancer cells of the human lung A and NIH3T3 cells were recovered and subculturally in flasks for cultivation. Once the cells reached the logarithmic phase of growth, the experiment can begin. Cells were treated with trypsin, filtered under high pressure to get suspensions containing cells were stained for 3 minutes with 0.4% Trifanova blue and then were counted using the count of blood cells (live cells were not stained, whereas dead cells were stained blue). The percent of living cells, identified by exclusion Trypanosoma blue all amounted to more than 98%.

2.2 experiments on the inhibition of cell multiplication

Three types of cells in logarithmic phase were inoculated in 96-well tablets with a density of 1×104/ml (200 μl/well) and then were cultured for 24 hours at 37°C suitable for cultivation box with 5% CO2. Nutrient medium aspirated and removed. Added 200 μl CL168-6 different concentrations (final concentration 10 μg/ml, 5 μg/ml, 2.5 μg/ml and 0 μg/ml, obtained from the culture medium from 4% calf serum in DMEM), where for each concentration was 6 parallel wells. After culturing for 24 hours and 48 hours, 100 μl of supernatant carefully aspirated and removed, respectively. Added MTS (20 μl/well) and evenly CME is ivali. The mixture was cultured for 1 hour at 37°C suitable for cultivation box with 5% CO2. The absorption at 492 nm was measured by a quantitative enzyme linked immunosorbent assay (ELISA). The experiment was repeated three times. The degree of growth inhibition was calculated as follows. The degree of growth inhibition (%)=[(average OD value of control group - average OD value of the processing group)/mean OD of control group]×100%

3. Results

CL168-6 has a significant inhibitory effect on cell proliferation of human hepatoma HepG2 cells and human lung cancer A, cultured in vitro, and shows the dependence of the dose; the corresponding results are summarized in Table 1-1. When processing of the medicinal product with a concentration of 2.5 µg/ml for 24 hours, the degree of inhibition equal 52,85% and 48,69% for HepG2 cells and cells A, respectively. The degree of inhibition increases with increasing concentration of the drug; if the drug concentration of 10 µg/ml, the degree of inhibition in relation to the above two types of cells equal 64.39% 62,40%, respectively; if after 48 hours and at the dosage of 2.5 mg/ml, the rate of inhibition of HepG2 cells and A equal to 59,83% and 51,91%, respectively; if the concentration is 10 μg/ml, the degree towards HepG2 and A Rav is and 73,67% and 69,67%, respectively. Compared with the situation with NIH3T3 cells inhibition CL168-6 against HepG2 and A shows a significant difference in the groups with different concentration and time of treatment (p<0,05). The results of the experiment showed that the inhibitory effect CL168-6 in the cells has good selectivity, and the effect is positively correlated with the concentration of drug and the time of treatment.

Table 1-1
The inhibitory effect of CL 168-6 on reproduction 3 types of cell lines
ConcentrationThe degree of inhibition 24 h (%): The degree of inhibition of 48 hours (%)
(ág/ml)HepG2A549NIH3T3HepG2A549NIH3T3
02,552,85*48,69*a 3.8759,83*51,91*0,75
5,058,51* 55,78*5,6969,08*59,75*1,97
10,064,39*62,40*8,4573,67*69,37*lower than the 5.37
Note: *P<0.05 compared with 0,0 mg/ml

4. Conclusion

CL168-6 can significantly inhibit the cell proliferation of human hepatoma HepG2 cells and human lung cancer A549.

The final example 2: impact assessment CL168-6 on tumor angiogenesis through the analysis ITSELF

1. Materials

1.1 Animals

Eggs breed German Roman embryo (each weighing 50-60 g, obtained from the Experimental center for the study of embryos China agricultural University).

1.2 Experimental drugs

CL168-6, obtained independently, which had a purity equal to or greater than 98%, evaluated using liquid chromatography high pressure (HPLC), and, therefore, meets the requirements of the experiment. Powder specified CL168-6 tightly corked and kept at 4°C.

Suramin provided by the company SIGMA.

2. Way

2.1 the Method of obtaining the evaluated samples

Erased the optimum gelatin sponge, previously made in the form of a disk with a diameter of 5 mm using a hole punch, used as a media sample. CL168-6 was dissolved in 70% ethanol to obtain a solution with a concentration of 2 mg/5 ml Were 3 groups of doses, where 5 μl (group with low dose volume dose of 2 μg/embryo), 10 μl (group with an average dose volume dose of 4 μg/embryo) and 20 μl (group with high dose volume dose of 8 μg/embryo) solution was added respectively in sections gelatin sponge with quantitative transfer fluid. Part of the sponge was dried in a sterile environment.

2.2 Incubation of the embryo and the process of removing air chamber in the egg with embryo

Sterilized eggs were placed in an incubator at 37°C With his air chamber, upward. On day 7 embryos were placed in an ultra-clean laminar flow Cabinet, and sterilized with ethanol, and then drilled a drill with the formation of small holes at the top of the embryo. Egg shell and shell membrane around the hole was carefully removed with the formation of cracks approximately 1.2 cm × 1.2 cm; after determining the site of addition of the sample, the membrane air chamber carefully pierced in the place of separation between the camera and the yolk needle of the syringe, through the puncture hole was administered 1-2 drops of sterilized water, which made the membrane chamber and the membrane ITSELF divided; after careful UD the population of the upper layer of the membrane chamber forceps membrane ITSELF in the lower layer was opened.

2.3 the Process of adding sample

The media containing the drug, was placed on the transition region of the membrane ITSELF and the membrane of the yolk SAC to the site where it was least of all vessels, and then have sealed using sterilized transparent adhesive tape with continued incubation for 72 hours.

2.4 Measurement of vessels

After incubation transparent adhesive tape, sealing the top of the air chamber was carefully removed using tweezers; was carefully added to a mixture of methanol/acetone (1:1, 1-2 ml); the room temperature remained constant for 10 minutes. Then, the membrane ITSELF was carefully taken away millimetre and placed on a glass slide for observation and receive photos. The effects of compounds on angiogenesis was assessed by counting the number of large, medium, small blood vessels, erodirovannyj near media.

2.5 Statistical processing

All data were analyzed statistically using the software package SPSS 11,0; comparing count data was confirmed using the test x2. Since P<0,05, all data were statistically significant.

3. Results

Figure 1 illustrates the impact of CL 168-6 on the vessel ITSELF and idle control.

The number of small vessels three groups doses CL168-6 was compared with that of blank group, according to the public; for all three groups were found to have significant differences and shows a dependence on dose. The results are summarized in table 2-1, which shows that the connection has a significant inhibitory effect on the growth and angiogenesis.

Table 2-1
Statistical table of approximate impact CL168-6 on the vessel ITSELF with the analysis of blood vessels [X(number of vessels) ±s]
GroupThe number of embryosLarge vesselsMedium-sized vesselsSmall vessels
CL168-6203,5±1,813,2±3,78,8±2,3**

(2 μg/embryo)
CL 168-6(4 μg/embryo)203,6±1,911,2 who 3,2 6,5±2,5**
CL168-6 (8 μg/embryo)202,5±2,012,6±5,64,0±2,8**
Suramin (4 μg/embryo)203,6±2,012,4±3,86,1±3,0**
The control group is empty204,7±3,118,1±5,511,6±2,2
Notes: compared with group blank control, *P<0,05, **P<0,01.

4. Conclusion

CL168-6 has a significant inhibitory effect on tumor angiogenesis, and the effect depends on number of drugs.

The final example 3: the antitumor effect CL168-6 against ascitic mice bearing S180 tumor, n

1. Materials

1.1 Experimental animals and tumor line Healthy female Balb/C mice, each weighing 18-22 g, were purchased from the Center of experimental animals of the Academy of military medical Sciences (certificate number SCXK-(Military)2007-004). Cell hepatotomy mouse n were cultured in the laboratory to assess viability; cell mouse sarcoma S180 was is provided by the Institute of Chinese medicine of the 302 hospital; ascites of mice bearing S180 tumor, n, subculturally once every 7 days.

1.2 Experimental drugs

CL168-6, obtained independently, had a purity equal to or more than 98%, evaluated using liquid chromatography high pressure (HPLC) and therefore met the requirements of the experiment.

Powder specified CL168-6 tightly corked and kept at 4°C.

Cyclophosphamide for injection: made Shanxi Pude Pharmaceutical Co., Ltd.

2. Process

2.1 Development of animal models

Ascitic S180 mice and n, which were vaccinated for 7 days, were killed by the collapse of the neck, of them received ascites in sterile conditions, respectively; the resulting ascites twice washed with culture medium RPMI1640, and obtained a suspension of 2×107/ml in sterile physiological saline. Suspension of S180 cells were inoculable subcutaneously in the right axillary fossa 40 mice (0.1 ml/mouse) for models of solid tumor; whereas the cell suspension n was inoculable in the abdominal cavity 40 mice (0.2 ml/mouse) in the usual way under sterile conditions to obtain models of ascitic tumors.

2.2 Experimental groups

90 mice randomly divided into 9 groups of 3 types, where a group of first type was a group of normal control groups of the second type were solid tumor S180,including the group positive (cyclophosphamide) control, group negative control group, low dose group and high dose, and groups of the third type were the group n ascites, including the group positive (cyclophosphamide) control group, negative control group, low dose group and high dose. Each group contained 10 mice. The experiment was repeated three times.

2.3 Introduction of the medicinal product

CL168-6 was obtained in the form of emulsions in corn oil. The group with low dose was administered parenteral dose of 16 mg/kg, the group with the high dose was administered parenteral dose of 32 mg/kg, the negative control group was injected injecting the same volume of corn oil, and the positive control group was administered parenteral cyclophosphamide injection solution with a dose of 0.02 g/(kg day). The applied volume of injection was 0.1 ml/mouse, and the injection was given in the form of intraperitoneal injections every other day for 15 days. Within 15 days of daily observed for total activity, hair, excrement, etc. mice. 24 hours after the last injection of mice that were inoculable subcutaneously S180, squashed by folding the neck, then removed the tumor, thymus and spleen. After the last injection mice inoculated in the abdominal cavity n, continuously fed by a common way, weighed and observed survival time each day d is as long while all mice negative group were not dead.

2.4 Calculation of the degree of prolongation of survival of mice

24 hours after inoculation of ascites measured the weight and size of the circumference of the abdomen of the mice in groups ascitic models. The introduction of a drug and feeding continued, respectively, for different groups, weight and abdominal circumference were measured daily until one day before the killing. Expected weight gain (g) and increased abdominal circumference (cm). The experiment began on the day of tumor inoculation and finished on the day when all the mice in the negative control group were dead; the time of death was recorded and used to calculate the degree of prolongation of survival.

The degree of prolongation of survival (%)=[(average number of days of survival of the processing group - average days of survival of the group of negative control)/mean days of survival of the control group]×100%

2.5 Calculation of the splenic index and liver index

After injection, all mice in the group of solid tumors, the mice were killed and their spleen and liver were weighed on electronic scales. Splenic index was equivalent weight of spleen (mg) of mice in each group divided by the weight of the mice, whereas the hepatic index was equivalent weight of liver (100 g), divided by the weight (g) mice.

2.6 calculation of the degree in which euromania tumor

The next day after the abolition of medicines mice in the negative control group with a solid tumor weighed and were killed by the collapse of the neck, where the serum was collected by phlebotomy from the eye for further use in subsequent experiments. Tumor tissue was dissected and weighed on electronic scales. The degree of inhibition of tumor was calculated as follows.

The degree of inhibition of tumor (%)=[(average tumor weight of negative control group - average tumor weight of the processing group)/average tumor weight of control group]×100%

2.7 Statistical processing

All data were statistically analyzed using the software package SPSS 11,0; comparing count data was confirmed using the test x2. Since P<0,05, all data were statistically significant.

3. The results of the experiment

3.1 Change in weight, abdominal circumference and the time of survival of mice in groups

As shown in Table 3-1, mice in groups CL 168-6 with low and high dose had a longer survival time than in the negative control group, and corresponding indicators of the degree of prolongation of survival was 37,11% 51,55%, respectively. Mouse in the processing group had a smaller increase in circumference of the abdomen than in the negative group, but the maximum growth of such treated mice was not affected by the specified magnification. The weight of mice in the group injected parenterally cyclophosphamide increased slowly with the increase only of 3.42 g, whereas mice in the normal group had a weight gain for 6,72, Figure 2 illustrates the statistical timeline the survival rate in ascitic mice.

Table 3-1

Impact CL168-6 by weight, abdominal circumference and the survival time of mice in different groups (x±s)

Table 3-1
Impact CL168-6 by weight, abdominal circumference and the survival time of mice in different groups (x±s)
GroupWeight gain (g)The increase in abdominal circumference (cm)The survival time (days)The degree of extension survive one hundred (%)
Group normal control6,76±1,560,58±0,09--
Group negative control4,85±1,213,17±0,339,7±2,7-
Group ciclopista the IDA 3,42±1,67②④ 2,30±0,66②④ 15,3±1,0657,73
Group CL168-6 low dose7,16±1,392,84±0,34②③ 13,3±1,7737,11
Group CL168-6 with a high doseof 5.05±1,882,32±0,71②④ 14,7±1,4151,55
Notes: 1. Compared with the group of normal control:P<0,05,P<0,01;
2. Compared with negative control group:P<0,05,P<0,01.

3.2 Change of hepatic and splenic index index of mice in different groups

As shown in Table 3-2, as the liver index and splenic index ascitic mice in the group of cyclophosphamide were respectively lower than those of mice in the negative control group, so they had statistical significance. All indexes of groups CL168-6 with low and high doses were greater than in the negative control.

Table 3-2
Influence CL168-6 on hepatic and splenic index index ascitic mice (x±s)
GroupLiver index (100 g/d)Splenic index (mg/g)
The group of single controlAndroid 4.04±0,473,45±0,58
Group negative control4,32±0,143,84±0,56
Group cyclophosphamide3,26±0,27①④ 2,45±0,34①④
Group CL 168-6 group with low dose4,9±0,24①④ 6,53±1,29②④
Group CL 168-6 with a high dose5,19±0,64②④ 5,76±0,96②④
Notes: 1. Compared with the group of normal control:p<0,05,P<0,01;
2. Compared with negative control group:P<0,05,P<0,01.

3.3 Anticancer activity CL168-6 invivo

CL168-6 had no effect on the increase in the mass of mouse sarcoma S180, the hen as the increase in the weight of mice in the positive control group was insignificant. The degree of inhibition of tumor mice bearing tumor sarcoma S180, in the group with low and high dose was 44,33% 54,58%, respectively; compared with negative control group, the group with the highest dose were statistically significant. The results are shown in table 3-3. The main parts of the tumors of the mice in all groups are shown in figure 3.

Table 3-3
The inhibitory effect of CL 168-6 on S-180 sarcoma in mice (x±s)
GroupThe increase in mass (g)Weight of tumor (g)The degree of inhibition

tumor (%)
The control group is empty6,76±1,56
Group negative control6,92±1,231,385±0,440
Group cyclophosphamide4,76±1,01①③ 0,518±0,23162,63%
Group CL 168-6 low doseof 6.71±1,220,771±0,40744,33%
Group CL 168-6 with a high dose6,00±1,110,629±of 0.13354,58%
Notes: 1. Compared with the group of normal control:P<0,05,P<0,01;
2. Compared with negative control group:P<0,05,P<0,01.

3.4 Alteration of hepatic and splenic index S180 mice in groups

As shown in Table 3-4, hepatic and splenic indices S180 mice in the cyclophosphamide group is lower than in the negative group, and, thus, have statistical significance. Although splenic indices mice in groups CL 168-6 low and high doses higher than those of mice in the negative group, and between them there is a significant difference; however, between the corresponding hepatic indices no significant difference.

Table 3-4
Influence CL168-6 splenic index and hepatic index S 180-mice (x±s)
Liver index (100 g/d)Splenic index (mg/g)
The control group is emptyAndroid 4.04±0,473,45±0,58
Group negative control4,21±0,453,83±0,63
Group cyclophosphamide3,09±0,31①④ was 2.76±0,64①④
Group CL 168-6 low dose4,45±0,195,32±0,68②④
Group CL 168-6 with a high doseto 4.38±0,13of 5.34±0,73②④
Notes: 1. Compared with the group of normal control:P<0,05;P<0,01;
2. Compared with negative control group:P<0,05,P<0,01.

4. Conclusion

4.1 CL168-6 can significantly inhibit the growth of mouse sarcoma S180.

4.2 CL168-6 can improve the splenic index mouse bearing a tumor.

4.3 CL168-6 can extend the survival of mice bearing a tumor.

4.4 CL168-6 may dissipate ascites or delay ascites produced in mouse n./p>

The final example 4: study of antitumor mechanism CL168-6

It was a hot topic in the study of tumors to study the mechanisms of apoptosis of tumor cells and signal transduction, to selectively block transmission of the signal of tumor cells and destroy their own controlling growth regulatory mechanisms. P53, Bcl-2, P21, VEGF are key molecules in the growth or apoptosis of tumor cells. In this experiment studied the antitumor mechanism CL168-6 at the molecular level.

I. Experimental materials (for details, see the final examples 1 and 3)

II. The process of the experiment

1. Determination of VEGF in the serum of the eye mouse (for details, see the final examples 1 and 3)

In front of the fence tumor blood collected by phlebotomy from the eyes of mice in four groups, i.e. the group of negative control group, positive CTX (cyclophosphamide) control group with high dose (32 mg/kg) and the group with low dose (16 mg/kg).

1.1 Obtaining reagents, samples and standards;

1.2 Adding the samples and standards and interoperability for 90 minutes at 37°C;

1.3 the Washing tablet twice, add the working solution of the biotinylated antibody and the interaction within 60 minutes at 37°C;

1.4 the Washing tablet three times, adding R the working solution ABC (complex of avidin-Biotin-horseradish peroxidase) and interaction within 30 min at 37°C;

1.5 Washing tablet five times, adding a coloring liquid TMB (tetramethylbenzidine) and the interaction in the dark for 15 minutes at 37°C;

1.6 Adding TMB stop solution and measuring the OD values at 450 nm.

2. The determination of the activity of caspase 3, 8, 9

2.0 ág/ml CL 168-6 impacted HepG2 cells for 24 hours and 48 hours.

2.1 Collection of samples

2.1.1 HepG2 Cells were treated with trypsin in the medium for cell cultivation. The resulting mixture (600 g) was centrifuged for 5 minutes at 4°C to collect the cells. The supernatant carefully aspirated and poured. The remains were washed once with PBS, was added to the lysate (100 ál/2×106cells); and then after resuspendable sludge ice pricked for 15 minutes.

2.1.2 the resulting mixture (16000 g) was centrifuged at 4°C for 15 minutes.

2.1.3 the Supernatant was transferred into a centrifuge tube, which is pre-cooled in an ice bath. 2.2 Determination of the activity of caspase 3

2.2.1 Took pNA (paranitroaniline) and an appropriate amount of Ac-DEVD-PNA (2 mm), put them in an ice bath for later use;

2.2.2 Reaction system was as follows:

Idle speed controlSamples
Definition is offered with buffer solution 90 µl80 ál
The samples that you want to define0 ál10 ál
Ac-DEVD-PNA(2 mm)10 ál10 ál
Total100 µl100 µl

2.2.3 Incubated at 37°C for 120 minutes, was determined by caspase 3 A;

2.2.4 Added Ac-IETD-PNA (2 mm) and Ac-LEHD-PNA (2 mm), respectively, for determination of caspase 8, 9

3. The determination of the activity of tumor cell VEGF, P53, P21, BC1-2 (took β-actin as internal standard)

Using electrophoresis of proteins (Western blotting)

3.1 Extraction of total cellular protein

Pre isecheno tumor tissue the size of a bean was ground and placed in pre-cooled homogenizer tissue. Added 400 μl of solution for lysis of tissue. After 10 minutes of careful homogenization, the mixture was transferred into a 1.5 ml centrifuge tube and then after intensive mixing on a vortex-mixer tubes for 15 seconds, the mixture was placed on ice for 10 minutes and then subjected to shaking. After 20 minutes of centrifugation (12000 rpm) the supernatant was collected, transferred to seeing what Ino chilled Eppendorf tube and then separately downloaded and stored frozen at -80°C. to determine the activity level of different antibodies.

3.2 Determination of protein: using ICA (bicinchoninic acid) protein analysis

3.2.1 preparation of a series of standards (table 4-1): a Standard set of bred in order in accordance with its description to obtain standard solutions with a concentration of 2000 ág/ml 1500 ág/ml 1000 ág/ml 750 ág/ml, 500 ág/ml 250 ág/ml 125 ág/ml and 25 µg/ml

Table 4-1
Preparation of standards
No. tubesThe amount of deionized waterThe number of standard BSAThe final concentration of BSA
And0300 ál of uterine2000 µg/ml
solution of BSA
In125 ál375 ál of uterine1500 mcg/ml
solution of BSA
325 ál 325 ál of uterine1000 µg/ml
solution of BSA
D175 ál175 ál of diluent In750 µg/ml
E325 ál325 ál of diluent500 mg/ml
F325 ál325 ál of diluent E250 mcg/ml
G325 ál325 ál of diluent F125 µg/ml
N400 ál100 μl of diluent25 mg/ml
G
I400 ál00 mcg/ml

3.2.2 Preparation of working reagent: solution a and solution B (50:1) was mixed for subsequent application is.

3.2.3 Sample, you need to determine diluted to 20-fold dilution. 10 μl sample and 10 μl of the standard was added to three wells of 96-well plate to ELISA. 200 μl of working solution was added and mixed. The mixture was reacted at 37°C for 30 minutes.

3.2.4 Tablet for ELISA were removed, cooled to room temperature and was measured value of the absorption at 562 nm. A standard curve was constructed according to the standards and the protein content measured samples was calculated using a standard curve. 3.3 protein Electrophoresis

3.3.1 sample Preparation protein: an appropriate amount of each sample was added to the buffer 4 to the protein sample and buffer 1 for protein sample was mixed with receipt of each volume of 300 μl and the concentration of 3.7 ág/ál, was heated for 3-5 minutes in hot water for denaturation, centrifuged in a short period of time with high speed and then kept at -20°C for later use.

3.3.2 Preparation of 12% separating gel and 4% concentrating gel and pouring gel: see table 4-2.

Table 4-2
Preparation of separating gel and concentrating gel
Separating gel (ml) Concentrating gel (ml)
30% solution of acrylamide40,5
1.5 mmol/l Tris (pH 8,8)2,5-
1.0 mmol/l Tris (pH 6,8)-0,38
10% SDS (sodium dodecyl sulphate)0,10,03
Deionized water3,32,1
10% ammonium persulfate (AP)0,10,03
5% TEMED0,0040,003
(tetramethylethylenediamine)

Upon receipt of the separating gel and concentrating gel to prevent premature solidification of the gel TEMED should be added before pouring the gel. First, the separating gel was poured in a layer between the glass; then the upper surface of the gel was poured into distilled water to maintain the flat surface of the gel. Secondly, when the separating gel was hardened, and distiller the bathroom the water was absorbed, the mixture was washed with distilled water several times to remove acrylamide, which is not polymerizable. Finally, when distilled water was again absorbed, put the comb to make the sample, and its front part was 0.5 cm from the separating gel.

3.3.3 Adding sample: When the gel is hardened at room temperature, electrophoretic buffer was poured into the tank for electrophoresis; after extracting combs for sample loading, the appropriate volumes of samples to be studied, respectively, was added to the wells made by the comb, using a micropipette in accordance with the concentration of cytoplasmic protein.

3.3.4 Electrophoresis: After adding the sample was connected to the power supply, set the voltage to 120 V, and during this period the sample was aligned in a line in the concentrating gel, and the leading edge of the sample included in the separating gel; then set the voltage to 100 V and during this period electrophoresis was performed at constant voltage for 120 minutes; at the end, turn off the power supply when the indicator bromophenol blue reached the bottom edge of the gel.

3.4 Analysis using Western blotting

3.4.1 Electroblotting: Film of PVDF (polyvinylidene fluoride) was first soaked in absolute methanol for 30 seconds, h is ordinary to make it transparent, and then soaked in distilled water for 10 minutes, at the end soaked with Whatman paper in the buffer for blotting. The gel was removed. Filter paper, gel, film of PVDF and filter paper were placed in order to prevent short circuit by ensuring that the dimensions of both sides of the paper, recorded and inserted in the tank for electroblotting, providing the gel towards the cathode, and a film of PVDF in the direction of the anode. Electroblotting was carried out for 3 hours at 80 V, 4°C.

3.4.2 Blocking: After electroblotting film of PVDF were removed, blocked with 5% blocking buffer BSA (bovine serum albumin) (dissolved in a solution of 1 TBST) at room temperature for 3 hours and then washed with TBST shaking 3 times, each for 10 minutes.

3.4.3 Reaction with the first antibody: the First antibody was diluted with TBST solution at a ratio of 1:200 and then brought in contact with the film of PVDF at 4°C over night. The next day after equilibration at room temperature for one hour, the reaction mixture was washed with TBST 3 times.

3.4.4 Reaction with the second antibody: Appropriate NKR(horseradish peroxidase)-conjugated second antibody was diluted with TBST solution at a ratio of 1:2000, was brought into interaction with a film of PVDF at room temperature for 2 the aces and then washed with TBST 3 times.

3.4.5 developing and fixing: the Solutions of the substrates a and b were mixed in a 1:1 ratio. The mixture was applied to a film of PVDF, incubated 5 minutes at room temperature, and after removal of excess liquid on the film, the remaining substance was covered with cling film to prevent the occurrence of bubbles between the film of PVDF and a polymer film. The following procedures were included layering, maintaining, developing, 1 minute rinsing with bidistilled water and 1 minute of recording.

3.4.6 Scanned manifest pictures were subjected to analysis of optical density using software Alpha Ease FC4.0, which took an average light intensity of the bands of β-actin as an internal standard values, and showed the results as ratios of the corresponding protein to β-actin.

4. Statistical processing

All data was statistically analyzed using the software package SPSS 11.0; data with normal distribution and homogeneity of variance was determined using t-test. If the data did not conform to a normal distribution or homogeneity of variance, was used nonparametric test.

III. The results of the experiment

1. Determination of VEGF in the serum of the eye mouse

As shown in table 4-3 and Figure 4, the value of optical density (meant is e OP) serum VEGF mice in the group with high dose was 0,1937 when compared with the value of OP 0,2200 group negative control, P=0,0037; for groups with a low dose when compared with negative control group, P=0,0259, both have statistical significance.

Table 4-3
The results of determining the activity of VEGF (x±s)
GroupNegative controlThe positive control16 mg/kg32 mg/kg
The OD value0,2200±0,01740,2205±0,02380,2064±0,01140,1937±0,0170
Notes: compared to the negative control,P<0,05,P<0,01.

2. The determination of the activity of caspase 3, 8, 9

After steps CL168-6 in HepG2 cells for 24 hours and 48 hours was determined by the activity of caspase 3, 8, 9, and the corresponding results are shown in table 4-4 and Figure 6.

Table 4-4
value OP caspase 3, 8, 9
24 hours48 h
0 mcg2.0 µg0 mcg2.0 µg
The caspase 30,1630,1900,1650,185
The caspase 80,3620,4130,3680,408
The caspase 90,1300,1560,1320,146

Activity three caspase not significantly increased and therefore had no statistical significance.

3. Electrophoresis protein

3.1 Determination of protein concentrations in cell extracts (Figure 7) Standard curve of protein was constructed according to the concentrations (x) and the values of absorbance (y) of the BSA standards. The linear equation Y=2,22x-0,02 was obtained using the standard curve, where Y represents the concentration of the diluted samples, and x represents the absorption values. As shown in Figure 3.3 that the corresponding coefficient r=1,00 means a good correlation in a linear equation. Corresponding to concentrate the radio squirrel (x) was calculated on the basis of the specific absorption values (y).

3.2 the Level of P53 protein expression

During electrophoresis, the protein amounts of total protein samples in each well correspond to each other. Three groups consisted of a group of negative control group, positive control (MMS) and experimental group CL168-6. β-actin was a sample internal standard. For the experimental group relative expression level of P53 was equal to 0.74±0,03; whereas for the negative control group it was equal 0,32±0,05; there was a significant difference between the two groups, P=0.002. (Figure 8)

3.3 the Level of expression of bcl-2

For the experimental group relative level of expression of bcl-2 was equal to 0.75±0,04; whereas for the negative control group it was equal 0,31±0,01; P=0,000; the values of both of the two groups had statistical significance. (Figure 9)

3.4 the Level of expression of VEGF

For the experimental group relative expression level of VEGF was equal 0,46=1=0,08; whereas for the negative control group it was equal to 0.71±0,05; there were no significant differences between the two groups, P-0,04. (Figure 10)

3.5 the Level of expression of P21

For the experimental group relative expression level of P21 was equal 0,79±0,07; whereas for the negative control group it was equal 0,76±0,06; there were no significant differences between the two groups, P=0,73. (Figure 11)

IV. Conclusion

The present experimental study shows that CL 168-6 may reduce the level of VEGF in the blood eye tumor bearing mice; at the same time the level of expression of VEGF protein in the tumor tended to decrease. CL 168-6 can inhibit the proliferation of a tumor by reducing the activity of VEFG.

The experiment showed that CL 168-6 had no significant effect on the activity of caspase 3, 8, 9. It is assumed that the induced CL168-6 apoptosis in HepG2 cells may depend on the activation of these three enzymes.

In this experiment, because the levels of expression of P21 protein in the control group and group treatment had no statistical significance, you can come to the preliminary conclusion that the effect of inhibiting tumor CL168-6 is not achieved through the path of signal transduction of P53→P21.

In the experiment it was found that the expression level of the protein Bcl-2 group was significantly lower than in the negative control group. We can conclude that CL168-6 can achieve the effect of inhibiting tumor by reducing the expression level of the protein Bcl-2. A specific mechanism should be further investigated.

Gene P53 has two types. One is a p53 wild-type, i.e. wtp53, the other is a p53 mutant type, i.e. mtp53. Wtp53, which is a suppressor gene tumor growth, can is to be included in the regulation of cell cycle and play an important role in the maintenance of normal cell growth and inhibition of proliferation of the tumor. The results of this experiment showed that the expression level of P53 protein to the processing group is significantly higher than in the negative control group. Consequently, it is possible to come to the preliminary conclusion that CL168-6 can increase the level of expression of wtp53 in tumor bearing mice and to inhibit the expression of protein BC1-2 and VEGF in order to achieve an anti-tumor effect, etc.

A case study of the toxicity of 1

I. Subject of the experiment: intraperitoneal injection of Gozo for mouse (a method according to Kerber)

II. Experimental materials

Mouse ICR [male/female=1/1, 18-22 g, the Number of licenses for the use of animals: SCXK (Beijing)2007-0001]; CL168, white powder (obtained independently), diluted with vegetable oil, cooked before using.

III. Method experiment

Used ICR mice (SPF) with a range of weight 18-22 g, which has provided Vital River Laboratories (VRL) in Beijing, China. Just had 5 treatment groups, where 2000 mg/kg was the highest dose, while others were reduced to 0.8 fold, i.e. 1600 mg/kg 1280 mg/kg, 1024 mg/kg and 819,2 mg/kg Mice were randomly allocated into groups. Each group contained 10 mice - half males and half females. Each mouse was injected intraperitoneally (0.2 ml/10 g). Observed 3-7 days, recorded the varied reactions of animals and the number of dead mice in each group. LD50and trust PR the outside was calculated using a modified method according to the Cerberus (Cerberus) according to the mortality of animals in each group.

IV. The result of the experiment

12 hours after the introduction of the death not occurred. The situation with the death of mice in each group after 7 days set forth in the following table.

Table example: Intraperitoneal injection LD50for CL168-mice

td align="center"> 3,20
no groupThe number of animalsDose administration (mg/kg)The logarithm of the dose (X)The death of animalsThe mortality rateR2
110819,22,91000
21010243,0110,10,01
31012803,1130,30,09
410160060,60,36
51020003,3080,80,64

V. a Brief conclusion

LD50calculated according to the modified method of Cerberus (Cerberus), for CL 168-6 was 1479,11 mg/kg; 95% confidence interval is 13 04,19-1677,49 mg/kg

A case study of the toxicity of 2

I. Subject of the experiment: antitumor therapeutic index CL168

II. Experimental materials: see the final example 2 and example toxicity studies 1.

III. The method of experiment: see the final example 2 and example toxicity studies 1.

IV. The result of the experiment: see the final example 2 and example toxicity studies 1.

V. the final example 2 you may find that when you use a high dose (30 mg/kg) CL168, the degree of inhibition of solid tumor S180 was 54,58%, the survival rate ascitic mice with cancer n was 51,55%; using a low dose (15 mg/kg) CL168 the degree of inhibition of solid tumor S180 was 44,33%, the survival rate ascitic mice with cancer n was 37,11%; in combination with an example of research toxicness the 1 you can see what LD50CL168 equal 1479,11 mg/kg, and its therapeutic index (TI) is approximately equal to 49.3.

VI. A short summary

The fact that therapeutic index (TI) CL168 was approximately 49,3, indicates that CL168 has a high safety and a significant antitumor effect.

Example composition 1

10 g CL168-6 and suitable excipients for injection (including dried powder and dry powder in sterile packaging) were mixed and prepared in the form of anti-tumor injection through the process of receiving injections (including dried powder and dry powder in a sterile package).

Example composition 2

10 g CL168-6 and suitable excipients for tablets (including tablet slow release matrix tablet, coated tablet, dispersible tablet, etc.) were mixed and prepared in the form of anticancer tablets using the process of obtaining tablets (including tablet slow release matrix tablet, coated tablet, dispersible tablet, etc.).

An example of a structure of 3

10 g CL168-6 and suitable fillers for capsules were mixed and prepared in the form of antitumor capsules using the process of getting capsules.

Example of part 4

10 g CL168-6 and suitable fillers for emulsions (including microemulsion, a nanoemulsion, etc.) were mixed and prepared in the form against the tumor emulsions using the process of producing emulsions (including microemulsion, the nanoemulsion and so on).

Example of part 5

10 g CL168-6 and suitable fillers for the granules were mixed and prepared in the form of antitumor granules by a process of granulation.

Example of compound 6

10 g CL168-6 and suitable excipients for drug controlled release was mixed and prepared in the form of anticancer drugs controlled release through the process of obtaining the drug controlled release.

Example of compound 7

10 g CL168-6 and suitable excipients for oral fluid was mixed and prepared in the form of oral anticancer liquids through the process of obtaining oral fluid.

Example of compound 8

10 g CL168-6 and suitable fillers for liposomes were mixed and prepared in the form of antitumor liposomes through the process of obtaining liposomes.

1. Connection CL168 represented by the General structural formula I,

where R represents oxygen (i.e. the connection CL 168-6).

2. A method of obtaining a compound according to claim 1, characterized in that it includes
the steps are:
(1) dissolve cholesterol (compound 2) in toluene to obtain cholesterolaemia (compound 3) by the interaction of cholesterol with acetic anhydride in presence of pyridine at a temperature of 114°C., and the molar ratio of compound 2 to acetic acid is in the anhydride is 1:2;
(2) dissolve the specified connection 3 in carbon tetrachloride to obtain 7-bronholitin-3-Alzette (compound 4) by reacting compound 3 with bromosuccinimide (NBS) when exposed to fluorescent light at a temperature of 74°C, and the molar ratio of compound 3 to bromosuccinimide is 1:1;
(3) dissolve the specified compound 4 in toluene to obtain 7-dehydrocholesterol-3-Alzette (compound 5) by reaction off with dimethylpyridine at a temperature of 114°C., and the molar ratio of compound 4 to dimethylpyridine is 1:1;
(4) dissolve the specified connection 5 in ethanol to obtain 7-dehydrocholesterol (compound 6) by hydrolysis of the compounds 5 with sodium hydroxide at a temperature of 80°C;
(5) dissolve the specified connection 6 in ethanol to obtain 5α,8α-apidocs-6-cholesten-3-ol (compound 7) by oxidation of the compounds 6 eosin Y when exposed to fluorescent light;
(6) dissolve the specified connection 7 in acetone to obtain 5α,8α-apidocs-6-cholesten-3-one (compound 1, i.e CL168-6) by oxidation of compound 7 chromic acid while cooling;
where the reaction equations for the above stages(1), (2), (3), (4), (5) and (6) how are the following:

3. The use of compounds according to claim 1 when getting medicines DL the prevention and treatment of neoplastic diseases.

4. The use of compounds according to claim 1 when getting medicines for the prevention and treatment of immunological diseases.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: method of producing moronic acid involves treating 3β,28-diacetoxyolean-18(19)-ene with an alcoholic solution of an alkali while boiling, followed by separation of the formed 3β,28-dihydroxyolean-18(19)-ene, which is treated with a Jones reagent with molar ratio of 3β,28-dihydroxyolean-18(19)-ene to Jones reagent equal to 1:10 in acetone, mixing the reaction mixture for 4-5 hours at temperature of 0 - +5°C and separating the end product, where 3β,28-diacetoxyolean-18(19)-ene is obtained by reacting allobetulin with acetic acid anhydride in the presence of a catalytic amount of perchloric acid with molar ratio allobetulin: acetic anhydride: perchloric acid equal to 1:100:0.1, while boiling for 15-20 hours and then separating the product.

EFFECT: high efficiency of use.

2 cl, 1 ex

FIELD: chemistry.

SUBSTANCE: described is a method of producing 7,8-dihydro-analogues of ecdysteroids via catalytic hydrogenation of ecdysteroids in methanol solution containing 1:6-fold excess Na metal in the presence of a Pd/C catalyst (1:1 to the weight of the substrate).

EFFECT: obtained 7,8-dihydro-analogues of ecdysteroids are structurally similar to castasterone type brassinosteroids - highly active phytohormones, plant growth and development stimulants.

3 cl, 4 ex

FIELD: veterinary.

SUBSTANCE: claimed is method, which allows to separate from pregnant horse urine by hard-phase extraction mixture of conjugated estrogens, depleted of phenol urine components and non-conjugated lipophilic compounds from group including non-conjugated flavonoids, non-conjugated isoflavonoides, non-conjugated norisoprenoids, non-conjugated steroids, first of all, androstane and preganane steroids, and comparable with them non-conjugates compounds.

EFFECT: improved method of obtaining extract, containing natural mixture of conjugated horse estrogens.

16 cl, 3 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: preparation belongs to gestagen class as 17α-acetoxy-3β-butanoyloxy-6-methyl-pregna-4,6-dien-20-on allowing peroral application. The compound shows no toxic activity and has no androgenic side effect. It is found to be better than Depo-Provera gestagen used in clinical practice and possesses chemosensitizing activity. The preparation is obtained by megestrol acetate reduction with sodium borohydride with following product etherification with butyric acid anhydride. The reaction mixture is treated with ammonia solution after etherification with butyric acid excess being removed to increase output and target product separation.

EFFECT: enhanced effectiveness of treatment.

3 cl, 3dwg, 1 tbl

The invention relates to an improved method of direct esterification of Stanlow/sterols interaction of stanol/sterols and acid taken in stoichiometric ratio, in the presence of a sufficient amount of catalyst, which can be acidic or basic, and in the presence of a sufficient amount of decolorizing agent, preferably activated carbon

The invention relates to the field of biologically active substances from plant material, in particular to a method of simultaneously receiving 20-hydroxyecdysone but also of inokosterone, ecdysone, magisteria And having the following structural formula:

20-hydroxyecdysone - R1=OH, R2=H, R3=OH, R4=H
The invention relates to medicine and agriculture, in particular to a method of extraction of biologically active compounds from plant material
The invention relates to a method of extraction of biologically active compounds from plant material
The invention relates to a method of extraction of biologically active compounds from plant material

FIELD: biotechnologies.

SUBSTANCE: in general formula (I), R1 and R2 that have been taken jointly are a group of formula (II), R3 represents atom of hydrogen or F and R4 represents F; R1, R2, R3 and R4 are connected to 17, 16, 6 and 9 carbon atoms of steroid structure in position α or β; R represents (III) or (IV), and the rest values of radicals are given in the description.

EFFECT: improving use efficiency of compositions for curing of eye diseases, such as diabetic macular edema, diabetic retinopathy, macular degeneration, age-related macular degeneration and other macular and retina diseases.

11 cl, 4 dwg, 5 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to organic chemistry and chemistry of natural products, more specifically to a method for preparing a new compound, a 20-hydroxyecdysone derivate conjugated with a short-chain vitamin E analogue, promising for medicine and pharmacology, namely to a method for preparing a 20-hydroxyecdysone conjugate by a reaction thereof with (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)acetaldehyde in ethyl acetate at room temperature in the absence of an acid catalyst (TsOH or phosphonomolybdic acid) for 24 h, debenzylation of the prepared intermediate conjugate in the ethanol solution in the presence of the catalyst Pd-C. The invention also refers to using such compound as an antioxidant agent inhibiting the lipid peroxidation process.

EFFECT: prepared hybride compounds appears to be promising in medicine and pharmacology as the new geroprotective antioxidants.

3 cl, 3 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention describes compounds applicable for treating or relieving a neurodegenerative disorder. There are also described methods of treating or relieving said disorders with said method involving the introduction a compound according to the present invention or a composition containing said compound in a patient.

EFFECT: method may be applicable for treating or relieving eg Alzheimer's disease.

21 cl, 9 ex, 22 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing a novel compound, a derivative of diacetonide 20-hydroxyecdysone with a novel regrouped 18-methyl group. Described is a method of producing a C27-steroid with a regrouped methyl group via hydrogenation over a palladium catalyst (10% Pd/C) in a solution of an alcohol (preferably methanol) in the presence of an alcoholate of an alkali metal (preferably sodium methylate), diacetonide 9α, 14α-epoxy-14-desoxy-20-hydroxyecdysone or diacetonide 14α-hydroperoxy-20-hydroxyecdysone, or Δ8(14) - 14-desoxy-20-hydroxyecdysone, followed by treatment of the reaction mixture with ammonium chloride solution and extraction of the desired 2,3:20,22-diacetonide 9α, 13α-epoxy-14β-methyl-13-demethyl-14-desoxy-7,8-dihydro-20-hydroxyecdysone using known techniques with output of 60-65%.

EFFECT: obtaining a novel useful compound.

1 cl, 3 ex

FIELD: medicine.

SUBSTANCE: invention also describes methods of treatment or reduction of such malfunctions severity, including introduction into patient's organism of compound according to claimed invention or composition, which contains it.

EFFECT: method is useful for treatment or reduction of difficulty, for example, Alzheimer's disease.

20 cl, 2 ex, 22 dwg

FIELD: chemistry.

SUBSTANCE: invention describes substituted derivatives of steroids which are selective inhibitors of type I 17β-hydroxysteroid dehydrogenase 17β-HSD1 and which can also be steroid sulphatase inhibitors, as well as salts thereof, pharmaceutical agents containing said compounds. The invention also describes use of said novel substituted derivatives of steroids for treatment, especially their use in treating or preventing steroid hormone dependent diseases or disorders, such as those for which inhibition of the 17β-HSD1 and/or STS is necessary and/or for which reduction of concentration of endogenic 17β-oestradiol is necessary.

EFFECT: more effective treatment.

35 cl, 164 ex, 19 tbl

FIELD: chemistry.

SUBSTANCE: invention describes a method of producing Timosaponin BII, which uses traditional Chinese medicinal agent Rhizoma Anemarrhenae or Anemarrhena asphodeloides Bge coronal root as raw material and involves extraction of Timosaponin BII using one or more processes selected from solvent extraction, polymer resin adsorption, polyamide chromatography, inverted phase column chromatography, column chromatography with Sephadex LH-20 etc, combined with the corresponding method of drying, e.g. low pressure drying, freeze drying, spray drying etc.

EFFECT: Timosaponin BII obtained using the said method has purity of over 90%, and the method is simple, practical and suitable for industrial application.

10 cl, 7 ex, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention refers to production of new tritiated analogues of physiologically active compounds - triterpene glycosides of holothurians Cucumaria of formula: .

EFFECT: there are produced new tritiated analogues of physiologically active compounds.

2 cl, 2 ex

FIELD: chemistry, immunology.

SUBSTANCE: extraction of dried cook water of sea cucumber Cucumaria frondosa or dried powder tissues of Cucumaria frondosa by chloroform and methanol mix is performed during boiling with reflux condenser to obtain organic extract; extract is evaporated, evaporated extract is re-dissolved in water, obtained colloid solution is decanted, ethylacetate is added to solution, water phase is decanted and undergoes chromatography in teflon or silica gel columns. Immune system activity of mammals, including lizosomic activity, phagocitosis and active oxygen form generation in macrophags of mammals, infected with bacterial cells, viruses and protozoa, is stimulated by oral, intraperitoneal or intramuscular or combined introduction of frondosite A dosage of 1 to 15 mcg/kg, preferably 10 mcg/kg.

EFFECT: implementation of claimed objective.

11 cl, 18 ex

FIELD: medicine; pharmacology.

SUBSTANCE: described method of stereo-specific production of 3(3-hydroxy-5β-H steroid sapogen or its derivatives by reduction of 3-keto,5β-H-steroid sapogenine with steric unfavoured organ-borane. 3β-Hydroxy,5β-H- steroid sapogenine or its derivative can be produced by reduction of 3-keto,5β-H- steroid sapogenine with relatively high steric unfavoured organ-borane reagent used as reducing agent, or by SN2 transformation of 3α-hydroxy,5β-H- steroid sapogenine or its derivative. Method represents easy way of steroid sapogenine production, such as sarsasapogenine, epysarsasapogenine, smylagenine, epysmylagenine andtheir esters using available or easy-made original substances (e.g., diosgenone, preferably of diosgenine).

EFFECT: improved method of composition production.

43 cl, 11 ex, 3 tbl, 1 dwg

Iimmunomodulator // 2504371

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to an immunomodulator. An immunomodulator for the immunocorrection accompanying the integrated treatment of chronic non-specific pulmonary diseases, chronic obstructive pulmonary disease, bronchial obstruction syndrome, chronic bronchial pneumonia, pulmonary fibrosis, tracheobronchitis, chronic laryngitis, pulmonary, tracheal and pharyngeal cancer; the immunomodulator is prepared by mixing a water infusion of rose bay leaves and a water infusion of yellow melilot taken in equal proportions, with a cattle lung and larynx powder, settling the prepared mixture, keeping on a boiling water bath, cooling; further, the mixture is filtered; the prepared solution is added with cattle blood serum containing leukaemia oncovirus antibodies, hemlock infusion, ascorbic and sorbic acids until all the ingredients fully dissolved; the prepared solution is placed in the water bath, cooled, filtered, sterilised under certain conditions.

EFFECT: above preparation provides higher effectiveness and reduces the length of treating the above diseases, and ensures the higher immunobiological properties of the human body.

3 ex

Up!