CN103142584B - Application of polysubstituted 5-hydroxypyrrolidone compounds in preparation of antitumor medicines - Google Patents

Abstract

The invention relates to the technical field of medicines, and provides an application of polysubstituted 5-hydroxypyrrolidone compounds in the preparation of antitumor medicines. Animal tests prove that the polysubstituted 5-hydroxypyrrolidone compounds have antitumor activities. The polysubstituted 5-hydroxypyrrolidone compounds inhibit the tumor growth in in-vivo and in-vitro in a tumor cell apoptosis mode, and have obvious antitumor effects and small toxic side effects.

Description

Application of multi-substituted 5-hydroxypyrrolone compounds in the preparation of antitumor drugs

technical field

The invention relates to the technical field of medicine, in particular to the application of multi-substituted 5-hydroxypyrrolone compounds in the preparation of antitumor drugs.

Background technique

Cancer has a high mortality rate worldwide, ranking second only to cardiovascular and cerebrovascular diseases. The total number of cancer patients in the world is more than 6 million. In my country, 1.2 million new cases are added every year, and about 900,000 people die. The incidence of tumors is increasing year by year, seriously affecting the quality of life of patients. Among them, the highest incidence is gastric cancer, followed by lung cancer, liver cancer and breast cancer. In recent years, research on anticancer drugs in various countries has paid great attention. With the development of molecular oncology and molecular pharmacology, the nature of tumors is gradually being clarified. At present, there are various anti-tumor drugs on the market, most of which belong to vitamin A class, non-steroidal anti-inflammatory drugs, polyphenols, vanilloids and topoisomerase inhibitors, etc. Growth or apoptosis to play a role (Lichter and Lawrence, 1995). For example, camptothecin (Y Pommier. Diversity of DNA topoisomerases I and inhibitors. Biochimie, 1998, 80; 255-270.) as an apoptosis inducer has become a clinical chemotherapy drug. However, these drugs have problems such as insufficient sources, high prices, low selectivity, high toxicity and side effects, unsatisfactory effects, and drug dependence after long-term use. With the development of the field of drug synthesis, new anti-tumor compounds are gradually approaching people's field of vision. The goal of this research field is to discover compounds with new structures, low median lethal concentration, low toxicity and anti-tumor activity both in vivo and in vitro, so as to bring good news to cancer patients.

Multi-substituted 5-hydroxypyrrolone compounds are amino acetal compounds, and their skeletons exist in many natural products and compounds with biological activity. For example, epolactaene was isolated from Penicilliumsp.BM1689-P fungal stem cells by Osada et al. It has the potential to promote the growth of human neuroblast neurite. Other 5-hydroxypyrrolone compounds and their analogues also exhibit very important therapeutic activity in the treatment of mental and nervous weakness, memory loss, decline or nervousness.

The patent application number is 200810225731.0 discloses the preparation method of multi-substituted 5-hydroxypyrrolidone compounds, in which it is only briefly explained that the multi-substituted 5-hydroxypyrrolidone compound Ia is effective in inhibiting human neuroblastoma cell line SH-SY5Y The IC50 of cell growth is 400μM, and its inhibitory effect and medicinal efficacy on human neuroblastoma cells need further investigation.

At present, there has never been any report on the anti-tumor effects of multi-substituted 5-hydroxypyrrolone compounds, especially anti-colon cancer and gastric cancer.

Contents of the invention

The object of the present invention is to provide the application of multi-substituted 5-hydroxypyrrolone compounds in the preparation of antitumor drugs.

The invention provides the application of multi-substituted 5-hydroxypyrrolone compounds in the preparation of antitumor drugs.

The multi-substituted 5-hydroxypyrrolone compound of the present invention has a chemical structural formula as follows:

In the chemical structural formula of the above polysubstituted 5-hydroxypyrrolone compounds, Bn is benzyl, Ph is phenyl, Me is methyl, PMB is p-methoxybenzyl, and Allyl is allyl.

The multi-substituted 5-hydroxypyrrolone compounds can be prepared with reference to the patent application number 200810225731.0.

Preferably, the substituted 5-hydroxypyrrolone compound is 1d, wherein R ¹ is Bn, R ² is 4-Cl-C ₆ H ₄ , and R ³ is Ph.

Preferably, the tumor is colon or gastric cancer.

The invention provides the application of multi-substituted 5-hydroxypyrrolone compounds in the preparation of anti-colon cancer or gastric cancer drugs.

Preferably, the present invention provides the use of the multi-substituted 5-hydroxypyrrolone compound 1d in the preparation of anti-colon cancer or gastric cancer drugs.

Furthermore, the anti-tumor drug of the present invention is made solely from polysubstituted 5-hydroxypyrrolone compounds or its active ingredients are polysubstituted 5-hydroxypyrrolidone compounds.

The antitumor drug contains medically acceptable pharmaceutical auxiliary materials.

The anti-tumor medicine is in liquid dosage form, injection dosage form, tablet, pill, granule or capsule etc.

The method for preparing the anti-tumor drug is a conventional method in the field. The method is to mix the multi-substituted 5-hydroxypyrrolone compound and the medically acceptable pharmaceutical excipients uniformly, and prepare different dosage forms.

In the present invention, the multi-substituted 5-hydroxypyrrolidone compound is subjected to an in vitro anti-tumor experiment, and its IC50 is determined by the MTT method; and the multi-substituted 5-hydroxypyrrolidone compound is tested for cell apoptosis and in vivo anti-tumor activity.

In vitro experiments were performed to detect that the compounds had the effect of inducing tumor cell apoptosis by flow cytometry. The method is as follows: co-incubate the cultured cells with different concentrations of the compound, and set up the cell control group (the compound solvent DMSO is co-incubated with the cells). After incubating for 24 hours in a CO ₂ incubator at 37°C, stain with AnnexinV for 10 minutes, and then add iodide After staining with propidium for 5 min, the cells were detected by flow cytometry. The percentage of cells stained with AnnexinV and propidium iodide was automatically analyzed by computer.

In vivo anti-tumor activity experiment, the method is as follows: cultured colon cancer HCT-116 cells or gastric cancer AGS cells were inoculated in the subcutaneous right axilla of nude mice at 6×10 ⁶ cells/only, and tumor masses formed after 4 days. The DMSO solution of the compound was injected intraperitoneally every day, and after one week, the administration was given every two days, and the administration was terminated after 18 days. At the same time, a control group was established, and the control group was injected with the same dose of DMSO in the same way. During the experiment, the long diameter (a) and the short diameter (b) perpendicular to it were measured every three days, and the tumor volume was calculated according to the formula 0.5236ab ² , and the curve was drawn. At the same time, their body weight and diet were measured, and other organs of the nude mice, such as heart, liver, spleen, lung, and kidney, were examined histopathologically.

In the cell activity test of the multi-substituted 5-hydroxypyrrolidone compound disclosed in the patent application number 200810225731.0 to the human neuroblastoma cell line SH-SY5Y, it is only briefly explained that the multi-substituted 5-hydroxypyrrolidone compound Ia The IC50 for inhibiting the growth of human neuroblastoma cell line SH-SY5Y cells is 400 μM. On the one hand, the IC50 value is too large, and its inhibitory effect and medicinal efficacy on human neuroblastoma cells need further investigation; on the other hand, , the inhibitory effect of multi-substituted 5-hydroxypyrrolidone compounds on human neuroblastoma cells has no correlation with its inhibitory effect on colon cancer or gastric cancer cells. The pathogenesis and treatment methods of different tumor cells are very different, so we cannot Only through the inhibitory effect of the multi-substituted 5-hydroxypyrrolidone compound Ia on human neuroblastoma cells, it is simply inferred that the multi-substituted 5-hydroxypyrrolidone compound of the present invention has anti-tumor properties, especially anti-colon cancer and gastric cancer role.

The animal test of the present invention shows that the multi-substituted 5-hydroxypyrrolone compound has anti-tumor activity, especially the obvious anti-colon cancer and gastric cancer effect. The multi-substituted 5-hydroxypyrrolidone compound inhibits tumor growth in vivo and in vitro by inducing tumor cell apoptosis, has obvious antitumor effect, and has little toxic and side effects.

Description of drawings

Figure 1 shows the results of flow cytometry; the box in the lower left corner is the cells not stained by AnnexinV and propidium iodide, the box in the upper left corner is the cells stained by propidium iodide, and the box in the lower right corner is the cells stained by propidium iodide AnnexinV-stained cells, cells stained with AnnexinV and propidium iodide in the box at the upper right corner.

Figure 2 shows the results of flow cytometry; the box in the lower left corner is the cells not stained by AnnexinV and propidium iodide, the box in the upper left corner is the cells stained by propidium iodide, and the box in the lower right corner is the cells stained by propidium iodide. AnnexinV-stained cells, cells stained with AnnexinV and propidium iodide in the box at the upper right corner.

Fig. 3 is the curve of tumor volume; wherein, the abscissa is the time and days (the first day is the time of inoculation of tumor cells, and the administration starts on the fourth day), and the ordinate is the tumor volume (mm ³ ).

Figure 4 shows the weight measurement results of nude mice; wherein, the abscissa is the time and days (the first day is the time of inoculation of tumor cells, and the fourth day is the start of administration), and the ordinate is the average body weight (g) of each nude mouse.

Fig. 5 is the measurement result of the food intake of nude mice; wherein, the abscissa is the time and days (the first day is the time of inoculation of tumor cells, and the fourth day begins to administer the drug), and the ordinate is the average daily food intake of each nude mouse ( g).

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

If not specified, all reagents used in the examples and experimental examples of the present invention are commercially available reagents. If not specified, the technical means used in the examples of the present invention are conventional means well known to those skilled in the art.

Example 1

The preparation process of multi-substituted 5-hydroxypyrrolone compound 1d:

Under the protection of argon, add magneton, enamide IId (188mg, 0.5mmol) to a dry and clean two-necked flask in sequence, then add calcium hydride and dry dichloromethane 25ml, add anhydrous ferric chloride after the dissolution is complete (1.6mg, 0.01mmol), after 30 minutes of reaction at room temperature, TLC analysis showed that the raw material IId was completely consumed. Add 20 mL of saturated sodium bicarbonate solution to the reaction system to quench the reaction. Extract with dichloromethane (25ml×3 times), combine the organic phases, dry over anhydrous sodium sulfate, remove the solvent by rotary evaporation after filtration, mix the sample with crude silica gel, perform 200-300 mesh silica gel column chromatography, mix ethyl acetate and petroleum ether Solvent (1:3) rinse. 1d180mg was isolated with a yield of 96%.

The multi-substituted 5-hydroxypyrrolone compounds of the present invention are prepared with reference to the patent application number 200810225731.0.

Experimental example

The cell lines used in the experiment of the present invention are all international general people's tumor cell lines, namely:

Colon cancer HCT-116 cells and gastric cancer AGS cells.

Experimental example 1 Antitumor activity experiment of multi-substituted 5-hydroxypyrrolone compounds 1a-1k

The antitumor activity of multi-substituted 5-hydroxypyrrolone compounds 1a-1k was tested by cell culture method. IC50 was determined by MTT method.

Colon cancer HCT-116 cells were selected as target cells. Dilute the cells with DMEM culture medium to a cell suspension with a density of about 10 ³ ~10 ⁴ cells/ml, add 100 μl of HCT-116 cell suspension per well into a 96-well plate, and after 12 hours of cell attachment, put the cell plate in the original Aspirate the liquid; add the 100% DMSO solution of multi-substituted 5-hydroxypyrrolidone compound 1a to the DMEM medium, so that the concentration gradient is 1, 2.5, 5, 7.5, 10, 25, 50, 75, 100 μg/ml , and mix evenly to obtain a mixed solution. At the same time, a cultured cell control group (co-incubation of colon cancer HCT-116 cells with the same concentration of DMSO) was established. Carefully add 200 μl of the above mixture into the cells per well. After incubating in a CO ₂ incubator at 37°C for 48 hours, add 20 μl of MTT solution (5 mg/ml, prepared in PBS, pH=7.4) to each well (200 μl medium), continue to incubate for 4 hours, terminate the culture, and carefully aspirate and discard the culture on the well. For the supernatant, add 150 μl DMSO to each well, and shake on a decolorizing shaker for 10 minutes to fully dissolve the crystals. Select a wavelength of 490nm, measure the light absorption value of each well on an enzyme-linked immunosorbent monitor, and record the results. The survival rate of tumor cells in each well was calculated according to the light absorbance value. The experiment was repeated three times, and the average value was taken. Plot the logarithm of the dose of the compound with the cell viability, and calculate the IC50 value (the concentration of the compound required when the cell proliferation rate is reduced to 50%).

The same method was used to conduct anti-tumor activity experiments on multi-substituted 5-hydroxypyrrolone compounds 1b-1k, and the results are shown in Table 1.

Table 1 Antitumor activity of different compounds

It can be seen from Table 1 that polysubstituted 5-hydroxypyrrolone compounds 1a-1k have anti-tumor activity against colon cancer HCT-116 cells, with IC50 between 21 and 226 μM.

Taking compound 1d as an example, the antitumor activity experiment was carried out in different tumor cells. IC50 was determined by MTT method. Colon cancer HCT-116 cells and gastric cancer AGS cells were used as target cells, the method was the same as above, and the results are shown in Table 2.

Table 2 Antitumor activity of compound 1d on different tumor cells

tumor cells Cytotoxicity (IC50, μM) HCT-116 twenty one AGS 10.7

Experimental example 2 Induction of tumor cell apoptosis by compound 1d

The apoptosis of tumor cells by compound 1d was detected by flow cytometry. Colon cancer HCT-116 cells and gastric cancer AGS cells were selected as target cells, taking colon cancer HCT-116 cells as an example, and the method was the same for other cells.

1. Compound 1d induces apoptosis of colon cancer cells

Colon cancer HCT-116 cells were suspended in DMEM medium and inoculated into a 6-well plate. After 15-20 hours, the density grew to about 50%, and a 100% DMSO solution of compound 1d was added to the DMEM medium. Compound 1d The final concentration of DMSO was 26.7μM, and a cultured cell control group was set up (the same concentration of DMSO was co-incubated with colon cancer HCT-116 cells). After incubating in a CO ₂ incubator at 37°C for 24 hours, wash twice with pre-cooled PBS, then digest with trypsin, collect the cells, wash away the trypsin, wash twice with pre-cooled PBS, and stain with AnnexinV for 10 min. Propidium iodide was then added for staining for 5 min, and detected by flow cytometry. The percentage of cells stained with AnnexinV and propidium iodide was automatically analyzed by computer. The result is shown in Figure 1.

In Figure 1, compound 1d was co-incubated with colon cancer HCT-116 cells at a final concentration of 26.7 μM, and detected by flow cytometry after 24 hours. The four squares in the figure, the lower left corner is the cells not stained by AnnexinV and propidium iodide, the upper left corner is the cells stained by propidium iodide, the lower right corner is the cells stained by AnnexinV, and the upper right corner is the cells stained by AnnexinV and iodine Propidium-stained cells. The ratio of the four parts in the grid is automatically generated by computer, and the apoptotic ratio is the ratio of cells in the upper right corner and lower right corner to the cells in the four parts. It can be seen from Figure 1 that the colon cancer HCT-116 cells obviously undergo apoptosis, and the percentage of apoptosis is 60.59%.

2. Compound 1d induces apoptosis of gastric cancer cells

By the same method, gastric cancer AGS cells were used as target cells, and the apoptosis of tumor cells by compound 1d was detected by flow cytometry, and the results are shown in FIG. 2 .

In Figure 2, compound 1d was co-incubated with gastric cancer AGS cells at a final concentration of 26.7 μM. After 24 hours, it was detected by flow cytometry. The four squares in the figure, the lower left corner is the cells not stained by AnnexinV and propidium iodide, the upper left corner is the cells stained by propidium iodide, the lower right corner is the cells stained by AnnexinV, and the upper right corner is the cells stained by AnnexinV and iodine Propidium-stained cells. The apoptotic ratio is the ratio of cells in the upper right corner and lower right corner to the cells in the four parts. It can be seen from Figure 2 that the gastric cancer AGS cells obviously undergo apoptosis, and the apoptosis rate is 36.62%.

3. Summary

Figure 1 and Figure 2 show that the multi-substituted 5-hydroxypyrrolidone compound 1d can obviously induce the apoptosis of colon cancer HCT-116 cells and gastric cancer AGS cells, and has the effect of obviously inducing the apoptosis of tumor cells (especially colon cancer and gastric cancer) Effect.

Experimental example 3 In vivo anti-tumor activity detection of compound 1d

1. Experimental method

Compound 1d was selected for in vivo anti-tumor activity experiment in living animals, the specific method is as follows:

Six-week-old female Balb/c nude mice were selected and randomly divided into experimental group and control group, with 10 mice in each group. The cultured colon cancer HCT-116 cells were inoculated subcutaneously in the right axilla of nude mice at 6×10 ⁶ cells/mouse (suspended in 100 μl PBS), and a tumor mass formed 4 days later. Nude mice with tumors of the same size were selected and randomly assigned to the experimental group and the control group, with 6 mice in each group. The experimental group was injected intraperitoneally with a DMSO solution of compound 1d at a dose of 25 mg/kg every day. After one week, the drug was administered every two days, and the drug was terminated after 18 days. The control group was injected with the same dose of DMSO in the same way. During the experiment, the long diameter (a) and the short diameter (b) perpendicular to it were measured every three days, and the tumor volume was calculated according to the formula 0.5236ab ² , and the curve was drawn. At the same time, their body weight and diet were measured, and other organs of the nude mice, such as heart, liver, spleen, lung, and kidney, were examined histopathologically.

2. Results

2.1 The in vivo antitumor activity test results of compound 1d are shown in Table 3.

Table 3 In vivo anti-tumor activity test results of compound 1d

It can be seen from Table 3 that compared with the control group, the experimental group has an obvious effect of inhibiting tumor growth.

2.2 The curve of tumor volume is shown in Figure 3, where the abscissa is the time and days (the first day is the time of inoculation of tumor cells, and the administration begins on the fourth day), and the ordinate is the tumor volume (mm ³ ), which can be seen in the experimental group Compared with the control group, it has obvious effect of inhibiting tumor growth.

2.3 The body weight of the nude mice was measured every three days on average. The body weight measurement results of the nude mice are shown in Figure 4, where the abscissa is the time and days (the first day is the time of inoculation of tumor cells, and the administration starts on the fourth day) , and the ordinate is the average body weight of each nude mouse (g). Since the excess body weight of the control group over the experimental group is basically equal to the excess tumor weight of the control group over the experimental group, it can be seen that the experimental group has no significant impact on the body weight of the nude mice compared with the control group.

2.4 The diet of nude mice was measured once every three days on average. The measurement results of the diet of nude mice are shown in Figure 5, where the abscissa is the time and days (the first day is the time of inoculation of tumor cells, and the fourth day begins to give drug), the ordinate is the average daily diet of each nude mouse (g), it can be seen that the diet of the nude mice has no obvious effect on the experimental group compared with the control group.

2.5 The results of anatomical observation and histological examination of the main organs of nude mice are as follows:

(1) Anatomical observation: Nude mice were observed with the naked eye. Compared with the control group, the main organs of the experimental group, such as the heart, liver, spleen, lung, and kidney, had no pathological changes such as congestion, swelling, and necrosis.

(2) Histological examination: The animal organ specimens of each group were processed routinely, and the heart, liver, spleen, lung, kidney and other organs were observed under an optical microscope. The results are as follows:

Myocardial tissue: There were no abnormal changes in myocardial cells, endocardium, and connective tissue in the myocardium in the experimental group and the control group.

Liver tissue: There were no abnormal changes in liver cells, liver lobules and interlobular structures in the experimental group and the control group.

Spleen tissue: The cells and tissue structures in the red pulp and white pulp of the experimental group and the control group were normal.

Lung tissue: There was no abnormality in the bronchial tissue and epithelium at all levels in the experimental group and the control group, and there was no morphological change in the alveoli, alveolar sacs, and alveolar walls.

Kidney tissue: There were no abnormal changes in the morphology of the renal corpuscles and renal tubules in the experimental group and the control group.

The results show that: compared with the control group, the experimental group has no pathological changes, indicating that the compound 1d has little toxic and side effects, and is safe and reliable.

3. Summary: The above results show that compound 1d can significantly inhibit the growth of tumors (especially colon cancer), and has no toxic side effects on other organs.

4. By the same method, the cultured gastric cancer AGS cells were inoculated into nude mice for in vivo anti-tumor activity experiments, and the same effect was obtained, indicating that compound 1d had a significant inhibitory effect on the growth of tumors (especially gastric cancer), and had Other organs have no toxic side effects.

Experimental summary: Multi-substituted 5-hydroxypyrrolidone compound 1d can significantly induce the apoptosis of colon cancer HCT-116 cells and gastric cancer AGS cells; it can significantly inhibit the growth of tumors (especially colon cancer and gastric cancer) in animals, and has no effect on other The organ has no toxic and side effects, is safe and reliable, and can prepare a class of novel anti-tumor drugs with high activity.

At the same time, through the same experimental method, other types of multi-substituted 5-hydroxypyrrolidone compounds were tested for inducing tumor cell apoptosis, and for anti-tumor activity experiments in living animals, all of which could achieve similar effects, indicating that other types of 5-hydroxypyrrolone compounds Multi-substituted 5-hydroxypyrrolone compounds also have obvious effects of inhibiting the growth of tumors (especially colon cancer and gastric cancer), and have no toxic side effects on other organs.

Although, the present invention has been described in detail with general description, specific implementation and test above, but on the basis of the present invention, some modifications or improvements can be made to it, which will be obvious to those skilled in the art . Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (3)

1. The application of multi-substituted 5-hydroxypyrrolone compounds in the preparation of antitumor drugs, characterized in that the tumor is colon cancer; The chemical structural formula of described polysubstituted 5-hydroxypyrrolone compound is as follows: Wherein, Bn is benzyl, Ph is phenyl, Me is methyl, PMB is p-methoxybenzyl, and Allyl is allyl. 2. The application according to claim 1, wherein the multi-substituted 5-hydroxypyrrolone compound is 1d, wherein R ¹ is Bn, R ² is 4-Cl-C ₆ H ₄ , R ³ for Ph. 3. The application of multi-substituted 5-hydroxypyrrolone compounds in the preparation of anti-gastric cancer drugs; The chemical structural formula of described polysubstituted 5-hydroxypyrrolone compound is as follows: