CN1616428A - Acenaphthoheterocyclic Compounds and Their Applications in Inducing Cell Apoptosis and Antitumor - Google Patents

Abstract

The present invention relates to a kind of amino group or halogen substituted 8-oxy-8H-acenaphthenee[1, 2-b] pyrrolyl-9-nitrile derivatives and their biological use. They are applied mainly in intracorporeal and extracorporeal inducing cell apoptosis and as anticancer compound. These compounds are 3, 6-disubstituted, 3-disubstituted or 6-substituted 8-oxy-8H-acenaphthenee[1, 2-b] pyrrolyl-9-nitrile derivatives. They can induce tumor cell apoptosis and block cell period in the dosage dependent mode within the wide concentration range of 0.01-10 micro mole. The highest activity compound B1 has IC50=0.17 micro mole (0.56 micro gram). When they are used in animal tumor model body, they can inhibit tumor growth obviously in inducing cell apoptosis mode. Therefore, the present invention is one kind of apoptosis inducing agent and antitumor compound with high activity.

Description

Acenaphthoheterocyclic Compounds and Their Applications in Inducing Cell Apoptosis and Antitumor

technical field

The present invention relates to a class of amino or halogen substituted derivatives of 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile and their in vivo and in vitro induction of cell apoptosis and their anticancer compounds application.

Background technique

With the in-depth research on the process and mechanism of cell death, apoptosis has become a new target of anti-tumor drug research. Because the characteristic of tumor cells is to resist apoptosis and achieve "immortality" by evading the regulation of organism signals. Therefore, effective apoptosis inducers will fundamentally inhibit the survival of tumor cells and the continued atypia of precancerous lesions. Apoptosis inducers are of great value no matter as a tool for basic research in oncology or as a drug for clinical treatment of tumors. At present, many biological companies including Calbiochem (German), Merck (German), Promage (USA) and so on have developed and continue to develop apoptosis-inducing products. Some apoptosis inducers have become clinical chemotherapy drugs, such as camptothecin (Y Pommier. Diversity of DNA topoisomerases I and inhibitors. Biochimie, 1998, 80; 255-270.). These products all belong to vitamin A, non-steroidal anti-inflammatory drugs, polyphenols, vanilloids, and topoisomerase inhibitors. It can be seen that they are still mainly chemical synthesis products of natural medicines or their analogues, and there are sources Insufficient, expensive, low selectivity, large side effects, unsatisfactory effect and other problems. In order to discover new chemotherapeutic drugs, The National Cancer Institute has randomly screened the antitumor activity of 300,000 different kinds of compounds (John N. Weinstein, Timothy GMyers, Patrick M. O'Connor, et al. Aninformation-intensive approach to the molecular Pharmacology of cancer. Science, 1997, 275; 3430-349.). In recent years, Qian Xuhong and others also synthesized, detected and reported a variety of anticancer compounds. The goal of this field of research is to discover compounds with new structures, lower median lethal concentration, apoptosis-inducing effect, and anti-tumor activity both in vivo and in vitro.

Contents of the invention

The present invention is based on the successful preparation of new fluorescent chromophore 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile and its derivatives (ZL021484007, Qian Xuhong, Xiao Yi) , Introducing a new active group on the parent 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile, creating a new type of apoptosis inducer and anti-tumor compound involved in the present invention.

The present invention introduces various primary and secondary amines and halogens at the 3 or 6 positions of 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile to generate 3,6-disubstituted, 3- Substituted or 6-substituted 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile, which is a class of compounds with the effect of inducing apoptosis and antitumor activity, and its general molecular structure formula:

(1) R ¹ =NHR ⁴ , R ² =H,

a. R ⁴ ＝X SR ⁵ , X ＝ C ₂ -C ₆ straight chain or branched chain alkyl, R ⁵ ＝CH ₃ , C ₂ H ₅ ;

b. R ⁴ =YCOO(CH ₂ ) _m CH ₃ , Y=C ₂ -C ₆ straight chain or branched chain alkyl, m=0-3;

c. R ⁴ =(CH ₂ ) _n Ar, n=1-3, Ar=2'-CF ₃ Ph-, 3'-CF ₃ Ph-, 4'-CF ₃ Ph-, 2'-FPh-,

        3'--FPh-, 4'-FPh-;

d. R ⁴ =(CH ₂ ) _x CF ₃ , x=2,3;

e. R ⁴ ＝X S(O)R ₆ , X ＝ C ₂ -C ₆ straight or branched chain alkyl, R ⁶ ＝CH ₃ , C ₂ H ₅ ;

f. R ⁴ ＝X S(O2)R ₆ , X＝C ₂ -C ₆ straight or branched chain alkyl, 6 ⁵ ＝CH ₃ , C ₂ H ₅

(2) R ² = pyrrolidinyl, piperidinyl, piperazinyl, N'-methylpiperazinyl, morpholinyl, thiomorpholinyl;

(3) R ¹ =F, Cl, Br, R ² =H;

(4) R ¹ =H, R ² =F, Cl, Br;

(5) R ¹ =R ² =F, Cl, Br.

The present invention uses 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (ZL021484007) with good rigid coplanarity and strong electron deficiency as raw material, and nucleophile primary amine or Aromatic hydrogen nucleophilic substitution reaction occurs in the amine to obtain 3,6-disubstituted, 3-substituted or 6-substituted 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (molecular structure general Formula A):

8-Oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile in a solvent (tetrahydrofuran, acetonitrile, pyridine, dimethylformamide or dimethyl sulfoxide), with an equimolar amount of primary amine , secondary amine nucleophile reaction, the temperature is 20-100 ° C, the reaction time is 0.5 to 24 hours. After cooling, evaporate part of the solvent under reduced pressure, filter or direct column chromatography to obtain product 3 or 6-monosubstituted amino-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile .

And the synthetic method of 3,6-disubstituted amino-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile is: 8-oxo-8H-acenaphtho[1,2-b] Pyrrole-9-carbonitrile reacts with an excess (4 to 5 times the molar amount) of secondary amines in a solvent (tetrahydrofuran, acetonitrile, pyridine, dimethylformamide or dimethyl sulfoxide) for a long time of 6 to 48 hours, The temperature is 20-100°C, and the disubstituted product is obtained after the same treatment as above.

The synthesis method of 3 or 6-halogen substituted 8-oxo-8 hydrogen-acenaphtho[1,2-b]pyrrole-9-carbonitrile (5,6,7 in general molecular formula A) is base-catalyzed ring closure reaction : starting material 2-(2-oxo-2hydro-haloacenaphthene)-malononitrile in a solvent (tetrahydrofuran, acetone, pyridine, dimethylformamide, acetonitrile, ethanol, methanol or dimethylsulfoxide) , in the presence of a basic catalyst (potassium carbonate, triethylamine or pyridine), at a temperature of 40-120° C., stirring for 10-100 minutes. After cooling, evaporate part of the solvent under reduced pressure, and then filter to obtain the product.

A series of compounds, and B series compounds whose structures have been disclosed as fluorescent chromophores in patent ZL021484007:

(1) R ¹ =thiomorpholinyl, pyrrolidinyl, R ² =H;

(2) R ¹ =NHR ⁴ , R ² =H, R ⁴ =C ₁ -C ₁₂ branched alkyl;

(3) R ¹ = R ² = pyrrolidinyl, thiomorpholinyl;

(4) R ¹ =NH(CH ₂ ) _y NR ⁵ , y=2,3, R ⁵ =CH ₃ , C ₂ H ₅ , R ² =H;

(5) B1 (when R ¹ =thiomorpholinyl, R ² =H)

Applications as in vivo and in vitro apoptosis inducers and antitumor compounds. The method is as follows: firstly, the IC ₅₀ is determined by the MTT method. Select HELA cells as target cells, dilute the cells with RPMI1640 culture medium to a cell suspension with a density of about 5×10 ⁵ cells/ml, add them to a 96-well cell culture plate, and add 200 μl of tumor cell suspension to each well. 100% DMSO solutions of compounds A and B were added respectively, with final concentrations of 100, 50, 10, 5, 0.5, 0.1, 0.05, and 0.01 μM, and a cell control group and a positive drug cyclophosphamide control group were set at the same time. After incubation in a _CO2 incubator for 24 hrs, 20 μl of MTT solution was added to each well, the optical density (OD) value at 570 nm was measured with a microplate reader, and the survival rate of tumor cells in each well was calculated according to the optical density value.

The results showed that the IC ₅₀ of the most cytotoxic compound B1 was 0.17 μM (0.56 μg). Other compounds had IC ₅₀ values between 0.4-6.8 μM.

The experimental method of anti-tumor activity in vivo is as follows: firstly, culture the liver cancer cell line H22, inoculate 200 μl/only under the skin of the right axilla of the mouse, and prepare a tumor animal model. Tumor masses formed 5 days after inoculation. Begin to intravenously or locally inject 30% DMSO solution of compound A and B once a day according to the concentration gradient. During the experiment period, the long diameter (a) and the short diameter (b) perpendicular to it were measured twice a week, and the tumor volume was calculated according to the formula 1/2ab ² . Observe the animal survival time. The results show that both the A series compounds and the B series compounds can inhibit tumor growth to varying degrees and prolong the survival time of tumor model animals.

The present invention also detects the effects of A and B series compounds on inducing cell apoptosis and arresting cell cycle in vivo and in vitro by flow cytometry. The method is to select HeLa cells as target cells in the in vitro test, dilute the cells into a cell suspension with a density of about 5×10 ⁵ cells/ml with RPMI1640 culture medium, add them to a 96-well cell culture plate, and add 200 μl of tumor cells to each well Suspension. Compounds A and B were added according to the concentration gradient, and a cell control group and a positive drug cyclophosphamide control group were set at the same time. After incubating in a CO ₂ incubator for 24 hours, fix with 70% cold ethanol; in the in vivo test, take the tumor tissue of the tumor model animal treated with the compound to prepare a single cell suspension, and set the untreated tumor model animal group as control. Fix with 70% cold ethanol. Before testing on the machine, the fixative was washed away, stained with propidium iodide, and tested by flow cytometry. The computer automatically analyzes the position of each peak, the height of the peak, and the percentage of each phase of the cell cycle. The results showed that compound B1 with the best effect induced apoptosis in a dose-dependent manner between 0.01-10 μM, and the apoptosis rate was between 9% and 31%. Cell cycle arrest in S phase. Other compounds induced apoptosis in cultured cells in a dose-dependent manner.

Both A and B series compounds are used as anti-tumor compounds or cell apoptosis inducers, and inhibit tumor growth in vivo and in vitro by inducing cell apoptosis.

Description of drawings

Figure 1: Flow cytometry results. The abscissa is the DNA concentration, and the ordinate is the cell number. Compound B1 was co-incubated with cultured cells at a final concentration of 0.01 μM. After 24 hours, flow cytometry detection, computer analysis of DNA concentration and cell cycle. Figure: Apoptotic peak can be seen, the percentage of apoptosis is 9.4%, and the results of cell cycle analysis show that the cells in S phase account for 6.7%.

Figure 2: Flow cytometry results. Compound B1 was co-incubated with cultured cells at a final concentration of 0.1 μM. After 24 hours, flow cytometry detection. Results: The apoptotic peak was seen, and the apoptotic ratio was 15.6%. The cell cycle analysis showed that the cells in S phase accounted for 26.5%.

Figure 3: Flow cytometry results. Compound B1 was co-incubated with cultured cells at a final concentration of 10 μM. After 24 hours, flow cytometry detection. Results: The apoptotic peak was seen, and the proportion of apoptotic cells was 31.3%. The results of cell cycle analysis showed that the cells in S phase accounted for 32.4%.

Figure 4a: Flow cytometry results. Subcutaneous tumor-bearing mice were treated with compound B1, and the tumor block was taken to prepare cell suspension, which was detected by flow cytometry. Results: The apoptotic peak was seen, and the apoptotic ratio was 13.1%.

Figure 4b: Flow cytometry results. For the subcutaneous tumor-bearing mice in the control group (not treated with compounds), the tumor block was taken to prepare cell suspension, which was detected by flow cytometry. Result: No apoptotic peak.

Detailed ways

Example 1

        

Add 0.46 g of 3-mercaptomethyl-propylamine to 50 ml of acetonitrile in 1 g of 8-oxo-8H acenaphtho[1,2-b]pyrrole-9-carbonitrile, stir at room temperature for 2 hours, evaporate part of the solvent, and precipitate the product 3- (3'-Mercaptomethyl-propyl)amino-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile A1, yield 60%. Mp266-268°C; ¹ H NMR (400M, DMSO): δ9.604 (br s, -NH-, 1H), 8.5-8.54 (d, J=7.6Hz, 1H), 8.2-8.21 (d, J= 7.2Hz, 1H), 7.75-79(d, J=8.8Hz, 1H), 7.57-7.6(t, J=7.8Hz, 1H), 6.9-6.92(d, J=9.2Hz, 1H), 3.24( br s, -NHCH ₂ CH ₂ -, 2H), 2.82-2.84 (m, -NHCH ₂ CH ₂ CH ₂ -, 2H), 2.42 (br s, -SCH ₃ -, 3H), 1.89-1.91 (m, _{-NHCH2CH2CH2-} , _2H ); ESI-MS: [M+H] ^- (334m/ _z ).

Example 2

   

Add 0.54 g of ethyl 4-aminobutyrate to 50 ml of acetonitrile in 1 g of 8-oxo-8H acenaphtho[1,2-b]pyrrole-9-carbonitrile, stir at room temperature for 30 minutes, evaporate part of the solvent, and precipitate the product 3- (3'-Carboxylate ethyl propyl)amino-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile A2, yield 65%. Mp>300°C; ¹ H NMR (400M, DMSO): δ9.20 (br s, -NH-, 1H), 8.84-8.86 (d, J=7.6Hz, 1H), 8.64-8.66 (d, J= 7.2Hz, 1H), 7.95=7.97(d, J=8.8Hz, 1H), 7.74-7.78(t, J=7.8Hz, 1H), 6.80-6.82(d, J=9.2Hz, 1H), 4.14- 4.19 (q, -COOCH ₂ CH ₃ , 2H), 3.62-3.67 (m, -NHCH ₂ CH ₂ CH ₂ -, 2H), 2.52-2.55 (m, -NHCH ₂ CH ₂ CH ₂ COO-, 2H), 2.10-2.13(m, -NHCH ₂ CH ₂ CH ₂ COO-, 2H), 1.35-1.38(t, -COOCH ₂ CH ₃ , 3H); ESI-MS: [M+H] ^- (360m/z).

Example 3

    

Add 0.45 g of thiomorpholine to 50 ml of acetonitrile in 1 gram of 8-oxo-8H acenaphtho[1,2-b]pyrrole-9-carbonitrile, stir at room temperature for 2 hours, and separate the product 3-thiomorpholino by column chromatography -8-Oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile and 6-thiomorpholino-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile A3 , and the yields were 40% and 30%, respectively. A3: Mp decomposed at 225°C; ¹ H NMR (400M, DMSO): 8.69-8.71 (d, J=8.0Hz, 1H), 8.42-8.45 (d, J=8.2Hz, 1H), 7.98-7.96 (d, J =8.0Hz, 1H), 7.88-7.92(t, J=8.0Hz, 1H), 7.04-7.02(d, J=8.2Hz, 1H), 3.68(br s, -N(CH ₂ CH ₂ ) ₂ S , 4H), 3.05 (br s, -N(CH ₂ CH ₂ ) ₂ S, 4H); ESI-MS: [MH] ^- (330m/z).

Example 4

     

0.43 gram of N-methylpiperazine was added to 50 milliliters of acetonitrile of 1 gram of 8-oxo-8H acenaphtho[1,2-b]pyrrole-9-carbonitrile, stirred at room temperature for 2 hours, and the product 6-( N'-methylpiperazinyl)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile A4, yield 30%. A4: Mp decomposed at 240°C; ¹ H NMR (400M, DMSO): 8.69-8.71 (d, J=8.0Hz, 1H), 8.42-8.45 (d, J=8.2Hz, 1H), 7.98-7.96 (d, J =8.0Hz, 1H), 7.88-7.92(t, J=8.0Hz, 1H), 7.04-7.02(d, J=8.2Hz, 1H), 3.45(br s, -N(CH ₂ CH ₂ ) ₂ NCH ₃ , 4H), 2.95 (br s, -N(CH ₂ CH ₂ ) ₂ NCH ₃ , 4H), 2.43 ((br s, -N(CH ₂ CH ₂ ) ₂ NCH ₃ , 3H); ESI-MS: [M+H] ^- (329m/z).

Example 5

Add 0.82 g of 2-(4′-trifluoromethylphenyl)ethylamine to 50 ml of acetonitrile in 1 g of 8-oxo-8H acenaphtho[1,2-b]pyrrole-9-carbonitrile, stir at room temperature for 30 minutes, Part of the solvent was evaporated, and the product 3-2-(4'-trifluoromethylphenyl)ethylamino-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile A5 was precipitated, with a yield of 55% . A5: Mp254-256°C; ¹ H NMR (400M, DMSO): δ9.13-9.11 (d, J=7.6Hz, -NH-, 1H), 9.03-9.01 (d, J=7.6Hz, 1H), 8.54-8.52(d, J=7.2Hz, 1H), 7.90-7.87(d, J=9.2Hz, 1H), 7.87-7.83(d, J=8.0Hz, 2H), 7.27-7.25(d, J= 8.0Hz, 2H), 7.22-7.20(t, J=8.0Hz, 1H), 7.09-7.07(d, J=9.2Hz, 1H), 3.60-3.59(br s, -NHCH ₂ CH ₂ -, 2H) , 2.80-2.78 (d, -NHCH ₂ CH ₂ , 2H); ESI-MS: [M+H] ^- (418m/z).

Example 6

 

Add 0.50 g of 2-trifluoromethylethylamine to 50 ml of acetonitrile in 1 g of 8-oxo-8H acenaphtho[1,2-b]pyrrole 9-carbonitrile, stir at room temperature for 30 minutes, evaporate part of the solvent, and precipitate the product 3- (2'-trifluoromethylethyl)amino-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile A6, yield 55%. Mp266-268°C; ¹ H NMR (400M, DMSO): δ9.450 (br s, -NH-, 1H), 8.6-8.62 (d, J=7.6Hz, 1H), 8.42-8.45 (d, J= 7.2Hz, 1H), 7.80-7.82(d, J=8.8Hz, 1H), 7.57-7.6(t, J=7.8Hz, 1H), 7.12-7.14(d, J=9.2Hz, 1H), 3.16( br s, -NHCH ₂ CH ₂ -, 2H), 2.32-2.34 (m, -NHCH ₂ CH ₂ CF ₃ -, 2H); ESI-MS: [M+H] ^- (342m/z).

Example 7

          

1 g of 2-(2-oxo-2hydro-acenaphthene)-malononitrile, 0.2 g of potassium carbonate, and 10 ml of dimethyl sulfoxide were heated to 100°C, stirred for 15 minutes, and cooled to precipitate crystals. After filtering, washing and drying with water, the product 8-oxo-8hydro-acenaphtho[1,2-b]pyrrole-9-carbonitrile A7 was obtained as a yellow-brown crystal, with a yield of 80%. ¹ H NMR (400M, DMSO): δ8.71-8.69 (d, J=8.0Hz, 1H), 8.67-8.65 (d, J=7.6Hz, 1H), 8.64-8.62 (d, J=8.0Hz, 1H), 8.42-8.40 (d, J=7.6Hz, 1H), 7.99-7.95 (t, J=7.8Hz, 1H); ¹³ C NMR (100M, DMSO): δ177.48, 138.26, 137.73, 134.40, 132.72, 131.82, 131.37, 128.91, 127.94, 127.37, 126.13, 122.22, 119.72, 113.82, 113.38; IR(KBr)cm ^-1 : 2231, 1643, 1577; ESI-MS: M+Na ⁺ (331, m/z ).

Example 8

Antitumor activity test in vitro. The antitumor activity experiments of the A and B series compounds were carried out by cell culture method. _IC50 was first determined by the MTT method. Select HeLa cells as target cells, dilute the cells into a cell suspension with a density of about 5×10 ⁵ cells/ml with RPMI1640 culture medium, add them to a 96-well cell culture plate, and add 200 μl of tumor cell suspension to each well. Add the 100% DMSO solution of A and B series compounds respectively, the final concentration is 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01 μ M, and set up the cultured tumor cell control group and the anticancer drug cyclophosphamide positive control at the same time Group. After incubating in a _CO2 incubator for 24 hrs, 20 μl of MTT solution was added to each well, and the optical density (OD) value at 570 nm was measured with a microplate reader, and the survival rate of tumor cells in each well was calculated according to the optical density value. The experiment was repeated 3 times, and the average value was taken. IC ₅₀ was calculated according to the formula logIC ₅₀ =Xm-I[P-1/4(3-Pm-Pn)].

Among them, Xm: log maximum concentration, I: log (maximum concentration/relative concentration), P: sum of positive rates, Pm: maximum positive rate, Pn: minimum positive rate. The results showed that the IC ₅₀ of the most cytotoxic compound B1 was 0.17 μM (0.56 μg). (Table 1). Other compounds had IC ₅₀ values between 0.4-6.8 μM.

The percentage of apoptosis and cell cycle arrest were detected by flow cytometry. Select HELA cells as target cells, dilute the cells into a cell suspension with a density of about 5×10 ⁵ cells/ml with RPMI1640 culture medium, add them to a 96-well cell culture plate, and add 200 μl of tumor cell suspension to each well. Compounds were added respectively, and a cell control group and a positive drug cyclophosphamide control group were set up simultaneously. After 24 hours of incubation in a CO ₂ incubator, fix with cold ethanol. Wash off the fixative before using the machine, take a single cell suspension, stain with propidium iodide, and detect by flow cytometry. The computer automatically analyzes the position of each peak, the height of the peak, and the percentage of each phase of the cell cycle. The results showed that compound B1 with the best effect induced apoptosis in a dose-dependent manner between 0.01-10 μM, and the apoptosis rate was between 9% and 31%. Cell cycle arrest in S phase (Fig. 1-3).

All other compounds induced apoptosis in cultured cells in a dose-dependent manner, and the percentage of apoptosis was about 15%.

Example 9

In vivo antitumor activity assay. The Chinese Kunming mice were selected and randomly divided into groups, 10 in each group. The cultured liver cancer cells H22 were inoculated subcutaneously in the right axilla of mice at 200 μl/mouse. After 5 days of tumor bearing, a subcutaneous tumor mass formed. Intravenous or topical injections of Compounds A, B in 30% DMSO were initiated. Taking compound B1 as an example, it is divided into 0.03 mg/kg body weight group and 0.3 mg/kg body weight group. During the experiment period, the long diameter (a) and the short diameter (b) perpendicular to it were measured twice a week, and the tumor volume was calculated according to the formula 1/2ab ^2. The survival time of the animals was observed. On the 14th day of the experiment, the tumor inhibition rate was calculated according to the tumor volume. The tumor inhibition rate of 0.03mg/kg body weight is 50%, and the tumor inhibition rate of 0.3mg/kg body weight is 70%. All other compounds have different degrees of inhibition of tumor growth, and the inhibition rate is about 20-50%.

The average lifespan of animals in the control group was 18 days, and the average lifespan of the compound group was 26 days. The results of statistical processing showed that P<0.05, it was considered that these compounds had the effect of prolonging the survival time of tumor animals.

After 7 days of the experiment, the subcutaneous tumors of the mice were removed, and the tissue was homogenized with physiological saline at a volume of 1:3 to prepare a cell suspension, and the apoptosis rate was measured by flow cytometry as above. The results showed that these compounds all have the effect of inducing tumor tissue cell apoptosis (Fig. 4a, 4b). The percentage of apoptosis is between 10% and 50%.

Table 1 Compound B1 inhibits cultured HeLa cells Concentration (μM) 0.01 0.05 0.1 0.2 0.4 0.5 1 2 Inhibition rate (%) (3 times) 2 6.7 31.5 53.5 68 78 83.8 89.8 1.2 6 31 52.2 67.5 76.6 83.4 87.8 1.4 7.1 31.4 51.2 67.6 76.4 83.6 88.2 Average inhibition rate (%) 1.53 6.6 31.3 52.3 67.7 77.0 83.6 88.6

Claims (3)

1, the also amino or the halogen substituted derivative of [1,2-b] pyrroles-9-nitrile of a class 8-oxygen-8H-acenaphthene is characterized in that this derivative is 3, and 6-two replaces, and 8-oxygen-8H-acenaphthene that 3-replaces or 6-replaces is [1,2-b] pyrroles-9-nitrile also, general formula of molecular structure A:

Among the general formula A, (1) R ¹=NHR ⁴, R ²=H,

A, R ⁴=XSR ⁵, X=C ₂-C ₆The straight or branched alkyl, R ⁵=CH ₃, C ₂H ₅

B, R ⁴=YCOO (CH ₂) _nCH ₃, Y=C ₂-C ₆The straight or branched alkyl, n=0-3;

c、R ⁴＝(CH ₂) _xAr，x＝1-3，Ar＝2’-CF ₃Ph-，3’-CF ₃Ph-，4’-CF ₃Ph-，2’-FPh-，

3’--FPh-，4’-FPh-；

d、R ⁴＝(CH ₂) _yCF ₃，y＝2，3；

E, R ⁴=XS (O) R ⁶, X=C ₂-C ₆The straight or branched alkyl, R ⁶=CH ₃, C ₂H ₅

F, R ⁴=XS (O ₂) R ⁶, X=C ₂-C ₆The straight or branched alkyl, R ⁶=CH ₃, C ₂H ₅

(2) R ²=pyrrolidyl, piperidyl, piperazinyl, N '-methylpiperazine base, morpholinyl, parathiazan base, R ¹=H;

(3)R ¹＝F，Cl，Br，R ²＝H；

(4)R ¹＝H，R ²＝F，Cl，Br；

(5)R ¹＝R ²＝F，Cl，Br。

2, according to the application of the described compd A of claim 1 and compd B as cell death inducer, it is characterized in that: the 100%DMSO solution of A, B two compounds, hatched altogether 24 hours with the mammalian tumor cell of cultivating, concentration gradient is under the condition of 0.01 μ M, 0.1 μ M, 1 μ M, 10 μ M, fix through 70% ethanol, after the propidium iodide dyeing, flow cytometer detects, the result proves that they can be with dose-dependent mode inducing apoptosis of tumour cell, the apoptosis ratio and can be blocked the cell cycle in the S phase between 9%-31%.

3, according to the application of the described compd A of claim 1 and compd B as anticancer compound, it is characterized in that: the 30%DMSO solution of A, B two compounds, local injection or intravenous injection are in the tumor model animal body, through about 30 days observation period, prove that they can prolong the lifetime of tumor model animal (P＜0.05), the mean lifetime of experimental group was at 26 days, the mean lifetime of 30%DMSO control group was at 18 days, measure tumour major diameter (a) and perpendicular minor axis (b), by formula 1/2ab twice weekly in the observation process ²Calculate gross tumor volume, the result proves that they can suppress the growth of tumor tissues (P＜0.01); Observe and finish back execution animal, strip tumor tissues, detect by flow cytometer after the homogenate, prove their cell death inducing in vivo, the apoptosis ratio is between 10%-50%.

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