BIFLAVONE COMPOUND AND USES THEREOF FOR TREATING CANCERS AND PREPARING DRUGS

Abstract

Provided in the present invention is a compound having these structure of formula I or a pharmaceutically acceptable salt thereof. Also provided in the present invention is a pharmaceutical composition containing the compound, and a use of the compound for treating cancers.

##STR00001##

Claims

1. (canceled)

2. (canceled)

3. A pharmaceutical composition for treating cancer, which comprises a compound of Formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier: ##STR00008## wherein R.sub.1, R.sub.1 and R.sub.3 are each independently selected from H, hydroxy, lower alkyl, lower alkenyl, lower alkoxy, halo, amino, hydroxyalkyl, aminoalkyl, nitro, aryl and heteroaryl, and wherein the pharmaceutical composition is sterile.

4. The pharmaceutical composition according to claim 3, wherein said cancer is selected from skin cancer, lung cancer, Kaposi's sarcoma, testicular cancer, lymphoma, leukemia, esophageal cancer, stomach cancer, colon cancer, breast cancer, endometrial cancer, ovarian cancer, central nervous system cancer, liver cancer and prostate cancer.

5. The pharmaceutical composition according to claim 4, wherein said cancer is lung cancer.

6. A method for treating cancer, which comprises administering to a subject in need thereof a treatment effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof: ##STR00009## wherein R.sub.1, R.sub.1 and R.sub.3 are each independently selected from H, hydroxy, lower alkyl, lower alkenyl, lower alkoxy, halo, amino, hydroxyalkyl, aminoalkyl, nitro, aryl and heteroaryl.

7. The method according to claim 6, wherein said cancer is selected from skin cancer, lung cancer, Kaposi's sarcoma, testicular cancer, lymphoma, leukemia, esophageal cancer, stomach cancer, colon cancer, breast cancer, endometrial cancer, ovarian cancer, central nervous system cancer, liver cancer and prostate cancer.

8. The method according to claim 7, wherein said cancer is lung cancer.

9. (canceled)

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is formulated for intravenous administration.

15. The pharmaceutical composition according to claim 3, which comprises a compound ##STR00010## or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition according to claim 5, wherein said cancer is non-small cell lung cancer.

17. The method according to claim 6, which comprises administering to a subject in need thereof a treatment effective amount of a compound ##STR00011## or a pharmaceutically acceptable salt thereof.

18. The method according to claim 8, wherein said cancer is non-small cell lung cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 illustrates HPLC chromatogram of preparation of the compound of the present invention.

[0052] FIG. 2 illustrates Analysis HPLC chromatograms for purity analysis of compound of the present invention. FIG. 2(A) is the chromatogram for the 70% ethanol extraction crude material; FIG. 2(B) is the chromatogram for the ethyl acetate extraction crude material; FIG. 2(C) is an diagram showing the overlapping chromatograms for each of the separated compounds: 1(I), 2(II), 3(III), 4(IV), 5.

[0053] FIG. 3 shows photos illustrating the change of the size of tumor volume in nude mice under continuous administration in the in vivo anti-tumor activity evaluation of compound of the present invention on animal model. Animal groups: high dosage 30 mg/kg, middle dosage 15 mg/kg, low dosage 5 mg/kg; positive control group 2 mg/kg Doxorubicin.

[0054] FIG. 4 illustrates the change of the size of tumor volume in nude mice under continuous administration in the in vivo anti-tumor activity evaluation of compound of the present invention on animal model. Animal groups: high dosage 30 mg/kg, middle dosage 15 mg/kg, low dosage 5 mg/kg; positive control group 2 mg/kg Doxorubicin.

DETAILED DESCRIPTION OF THE INVENTION

[0055] The present invention is explained in greater detail on its contents and advantages in the following non-limiting examples.

Example 1 Preparation of Compound of Formula I

[0056] 1. Handling of Shishangbai Sample

[0057] Shishangbai, the dry whole plant of Selaginella doederleinii Hieron was cut into small pieces of the length of smaller than 5 mm and then extracted under reflux with 70% ethanol. The extracted solution was then concentrated by rotary evaporation under a low temperature (40-50° C.) to give yield to an ethanol extraction crude material. The ethanol extraction crude material was then resuspended with 10× volume double stilled water, followed by successive extraction with petroleum ether, then dichloromethane, and finally ethyl acetate. The extracted solution was then concentrated by rotary evaporation under a low temperature to give yield to an ethyl acetate extraction crude material. The obtained ethyl acetate extraction crude material is then used for preparing the compound of the present invention as mentioned below.

[0058] 2. Preparation by HPLC Separation

[0059] The HPLC separation was carried out with an Agilent SB-C18 column (250 mm×21.2 mm, 7 μm). The mobile phase was comprised of acetonitrile-water (44:56, v/v). The flow rate was 7 mL/min and the detection wavelength was set at 270 nm. The HPLC was carried out at RT. The injection volume was 100 μL. After the HPLC separation, as shown in FIG. 1 (preparation HPLC chromatogram), Compound III, Compound IV, Compound 5 and fraction Fr.4-1 were obtained.

[0060] Compound 5 was a powder with the color of pale yellow.

[0061] 3. Purity Analysis of Compound 5 by HPLC.

[0062] FIG. 2 provides the analysis HPLC chromatograms for purity analysis of compound of the present invention. FIG. 2(A) is the chromatogram for the ethanol extraction crude material; FIG. 2(B) is the chromatogram for the ethyl acetate extraction crude material; FIG. 2(C) is a diagram showing the overlapping of the chromatograms for each of the seven separated compounds.

[0063] The HPLC settings included: A Shimadzu LC-20A HPLC system was used for the chromatographic analysis. All separations were carried out on an Ultimate XP-C18 column (Welch Materials Inc.; 4.6 mm×250 mm, 5.0 μm). The detection wavelength was set at 203 nm and 254 nm. The injection volume was 10 μL, the flow rate was 1 ml/min and column temperature is 30° C. The solvent system for the HPLC is acetonitrile-water, using a gradient elution of 10-42% (v/v), 0-30 min; 42-60% (v/v), 30-60 min; 100% (v/v), 60-70 min. The re-equilibration time was 10 min.

[0064] As calculated by the peak area normalization method, the purity of Compound 5 is higher than 95%.

[0065] Structural Identification of Compound 5

[0066] Compound 5 was subjected to thin-layer chromatography (TLC), a single round point was observed. Compound 5 was subjected to HPLC analysis using different elution system and all showed a single peak. Compound 5 was subjected to different types of spectra analysis (UV, IR, NMR, MS), the results were as shown below.

[0067] MS: (−) ESI-MS 537.2 EMHf, which means MW=538; the deduced formula is C.sub.30H.sub.18O.sub.10; Ω=22;

[0068] UV λ.sub.min.sup.ET: 210 nm, 268 nm, 330 nm, which are characteristic absorption peak of flavonoids;

[0069] IR λ.sub.max.sup.KBr (cm.sup.−1): 3588 (OH in an aromatic ring); 3423 (C—H in an aromatic ring); 1654 (OH in an aromatic ring); 1610 (C═C in an aromatic ring); 1500 (OH in an aromatic ring); 1289 (C—O); 1028 (C—O); 838 (aryl para substitution).

[0070] 1H NMR showed there were 13 Hs in the aromatic area; 13C NMR showed there were 2 carbonyl carbons (δ82.3, 6176.5) and 28 aromatic carbons, which implied that the compound is a biflavonoid. 1H NMR showed there were 2 hydroxy H (1H, s) and 12.24 (1H, s). Deduced according to the coupling constant, 8.01 (2H, d, J=8.4 Hz), 7.86 (2H, d, J=8.8 Hz), 7.26 (2H, d, J=8.8 Hz) and 6.91 (2H, d, J=8.8 Hz) constructed two AA′BB′ systems; 6.56 (1H, s, J=1.2 Hz), 6.50 (1H, s, J=1.2 Hz), 6.27 (1H, s, J=1.6 Hz) and 5.88 (1H, s, J=1.6 Hz) constructed two meta coupling H, the rest was a single H 6.85 (1H, s). 13C NMR showed δ72.9, δ99.4, δ94.7 and δ99.4, which showed C6 and C8 on the two flavone mother ring A are unsubstituted.

[0071] Based on the 1H NMR and 13C NMR data (as given in Table 1), Compound 5 was identified to have the following structure:

##STR00007##

TABLE-US-00001 TABLE 1 NMR data of Compound 5 (DMSO-d6, 400 MHz) position δC δH 2 161.1 3 72.9 4 176.5 5 161.9 12.86 (s, —OH) 6 99.4 6.27 (d, J = 1.6 Hz) 7 165.1 8 94.6 6.56 (d, J = 1.2 Hz) 9 157.3 10  104.6 .sup. 1′ 120.2 2′, 6′ 129.0 7.86 (d, J = 8.8 Hz) 3′, 5′ 116.3 6.91 (d, J = 8.8 Hz) .sup. 4′ 157.6  2″ 163.5  3″ 104.3 6.85 (s)  4″ 182.3  5″ 161.6 12.24 (s, —OH)  6″ 99.4 5.88 (d, J = 1.6 Hz)  7″ 164.8  8″ 94.7 6.50 (d, J = 1.2 Hz)  9″ 157.9 10″ 104.6 .sup.  1′″ 125.4 2′″, 6′″ 130.7 8.01 (d, J = 8.4 Hz) 3′″, 5′″ 116.2 7.26 (d, J = 8.8 Hz) .sup.  4′″ 160.0

Example 2 In Vitro Anti-Tumor Activity Evaluation of Compound 5

[0072] Compound 5 obtained in Example 1 was subjected to the in vitro anti-tumor activity evaluation.

[0073] 1. Preparing test solutions: taking a certain amount of compounds 5, using dimethyl sulfoxide (DMSO) to dissolve the compound, and using cell growth medium for each corresponding cell line to dilute the solution to 5 concentrations of test solutions: 500, 250, 125, 62.5, 31.25 μg/mL. The positive control doxorubicin were diluted with cell growth medium for each corresponding cell line to 3 concentrations: 5, 2.5, 1.25 μM.

[0074] 2. Cell lines and cell culture

[0075] Seven human cancer cell lines, including five adhesive cancer cell lines: human gastric cancer cell line (MKN-45), non-small cell lung cancer cell line (A549), nasopharyngeal carcinoma cell line (CNE1, CNE2), large cell lung cancer cell line (PC-9); and two suspensive cancer cell lines: human promyelocytic leukemia cell line (HL60), human chronic myelogenous leukemia cell line (K562). All seven human cancer cell lines are purchased from Shanghai Chinese Academy of Sciences Cell Bank and stored in School of Medicine of Fujian Medical University.

[0076] The aforementioned seven cancer cell lines were recovered from the liquid nitrogen, and then cultured in 25 cm.sup.2 flasks under the corresponding culture conditions of each cell lines (see Table 2). When the cells were grown 6-8 times of passages and the cell coverage rate in the bottom was about 80%, i.e., the cells were in the logarithmic growth phase, the cells were digested with 0.25% trypsin solution and used for passage. According to various types of cell growth rate, cell suspension were adjusted to the appropriate concentration and inoculated in 96 well plates.

TABLE-US-00002 TABLE 2 Seven cancer cell lines and the culturing conditions. Cancer Full name of Grow Culture cell lines the cell lines status condition K562 human chronic myelogenous suspensive 5% FBS-RPMI1640, 5% CO.sub.2, 37□ leukemia cell line HL-60 human promyelocytic leukemia cell suspensive 8% FBS-RPMI1640, 5% CO.sub.2, 37□ line CNE1 Human highly differentiated adhesive 10% CS-RPMI1640, 5% CO.sub.2, 37□ nasopharyngeal carcinoma cells CNE2 Human lowly differentiated adhesive 8% FBS-RPMI1640, 5% CO.sub.2, 37□ nasopharyngeal carcinoma cells A549 Human non-small cell lung cancer adhesive 6% FBS-RPMI1640, 5% CO.sub.2, 37□ cell line PC-9 human cell lung cancer cell line adhesive 10% FBS-RPMI1640, 5% CO.sub.2, 37□ MKN-45 human gastric cancer cell line adhesive 10% CS-RPMI1640, 5% CO.sub.2, 37□

[0077] Determination of Inhibition Rate to Seven Types of Cancer Cell Lines by MTT Method

[0078] Test solutions of Compound 5 prepared as aforementioned were used to treat the seven types of cancer cell lines for 72 hs. 104, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml) were added to each well and continued cultivation for 4 hs. After removal of the solutions in the wells, 2000 μL DMSO was added, the plates were well shook for complete dissolve. OD was measured with microplate reader under wavelength of 492 nm and then the inhibition rate was calculated. The inhibitory effect of each sample solution on each cancer cell line was assayed three times in parallel.

[0079] Software program Origin 7.5 was used for analyzing the OD data obtained in the testing wells. IC50 to each human cancer cell lines are calculated and shown in the following Table 3.

TABLE-US-00003 TABLE 3 Inhibition effects of Compound 5 on human cancer cell lines Cell line IC50 (μg/mL) A549  13.2 ± 0.91 PC-9 21.8 ± 1.3 K562 16.8 ± 2.8 HL60 18.3 ± 3.0 CNE1 49.29 ± 2.8  CNE2 13.3 ± 2.6 MKN-45 33.2 ± 3.1

[0080] Compound 5 had shown significant inhibitory effect on all the seven types of human cancer cell lines. IC50 was in the range of 13.2±0.91 to 49.29±2.8 μg/mL. It shows Compound 5 can inhibit a broad scope of cancers, among which, Compound 5 had the best inhibitory effect on A549.

Example 3 In Vivo Anti-Tumor Activity Evaluation of Compound 5 on Animal Model

[0081] Compound 5 obtained in Example 1 was subjected to the in vitro anti-tumor activity evaluation.

[0082] 1. Cell Line and Laboratory Animal

[0083] Human non-small cell lung cancer cell line (A549) was purchased from Shanghai Chinese Academy of Sciences Cell Bank and stored in School of Medicine of Fujian Medical University.

[0084] 3-4-week-old nude BALB-c mice weighted 18±2 g, purchased from SLRC laboratory Animal Co. Ltd., Shanghai, China (), Laboratory Animal License: SOCK () 2012-0002, Animal License: 2007000537438).

[0085] The nude mice were fed in the SPF-system, six mice in a cage. The nude mice were provided with standard food and water, maintained in the light and dark cycling conditions (12/12 h), room temperature 25±2° C., humidity 50±10%. The water and beddings were steriled and changed every day. The mice were fed in the maintenance room for a week before the experiments.

[0086] 2. Preparation of Test Solution of Compound 5

[0087] Compound 5 was poorly dissolved in water. Its water solution (or saline solution) was acidic. When the pH of the solution was adjusted to be with the injection acceptable range (6.0-8.0), the solubility was enhanced a little, but it was still quite low, thus led to precipitation. Suitable co-solvents were selected to enhance the solubility. Studied the solubility enhancing effects of Tween-80 (final concentration 0.02-2%, v/v), PEG-400 (final concentration <40%, v/v), 1, 2-propanediol (final concentration <40%, v/v), glycerol (final concentration <30%, v/v), and CMC-Na (final concentration 1-5%, w/v). It was found that, within the injection acceptable range, Tween-80 and PEG-400 showed a better solubility enhancing effect. Considering formulations comprising tween compounds may lead to hemolysis, PEG-400 was selected as the co-solvent. After comparing solubility enhancing effects of different ratio of PEG-400, it was found that 10% PEG-400 had the best solubility enhancing effect.

[0088] Accurately weighed 6 mg of compound 5 powder, added cosolvent PEG-400 (100 μL), vortex and treated with ultrasonic. After being fully dissolved, a small amount of physiological saline was gradually added into the solution, adjusted the pH to around 7.4, and made the solution to 1 mL.

[0089] 3. Preparation of Positive Control Solution

[0090] Accurately weighed a certain amount of injection grade doxorubicin powder, added saline to obtain a 0.4 mg/mL test solution. The test solution is prepared avoiding light and handy for use.

[0091] 4. Construction of Human Tumor Cell A549 Allograft Nude Mouse Model

[0092] 4.1 Cell Culture

[0093] Human non-small cell lung cancer cell line A549 was resuscitated. The ratio of FBS in the culture was increased from 6% to 10%, so as to make the cells proliferated rapidly. When the cell coverage in the culture flask reached 80% or so (i.e., logarithmic growth phase), the A549 cells were digested and suspended. Using autoclaved PBS to wash the cells twice to remove the residual culture medium. And then the cells were resuspended with PBS and adjusted the cells concentration to 1×10.sup.7 cells/mL.

[0094] 4.2 Construction of Nude Mouse Model

[0095] After the mice accustomed to the environment, under steriled condition, using disposable syringe to transfer A549 cells (1×10.sup.7/mL) and subcutaneously inoculated in the flank of nude mice. 0.2 ml of suspension of A549 cells was injected into each nude mice and the procedure was finished within 30 min. The rest of the cells were subjected to culture and observed for any contamination, so as to provide information on whether the construction of animal model was success.

[0096] 4.3 Grouping of the Testing Animals

[0097] After the inoculation of cancer cells was success and the volume of xenograft tumors reached 50 to 100 mm.sup.3, the animals were divided randomly into 5 groups (high dosage, middle dosage, low dosage, positive control, blank control) and each group comprised 6 mice.

[0098] 5. Study on In Vivo Anti-Tumor Effect of Compound 5

[0099] 5.1 Animal Grouping

[0100] According to the animal grouping, compound 5 testing solutions were injected via tail vein, the corresponding injection dosage were: high dosage 30 mg/kg, middle dosage 15 mg/kg, low dosage 5 mg/kg. The animals were administered once per day. The injection volume was adjusted according to the weight measured. The animals were administered for 12 days. Positive control group was injected with 2 mg/kg Doxorubicin. The animals were administered once every 3 days, and 4 times in the 12-day period. Blank control group was injected with saline solution.

[0101] 5.2 Measurement of Tumor Size

[0102] During the administration period, the living and mental state of the mice were observed every day. Body weight and tumor size was measured and recorded. The tumor size was measured using Vernier caliper and tumor volumes were calculated using the formula: V (mm.sup.3)=½×L (mm)×W.sup.2 (mm.sup.2) in which L is the length and W is the width of the solid tumor nodules.

[0103] 24 hrs after the last administration, the nude mice were sacrificed and the tumor was removed for volume measurements. Tumor inhibition rate was calculated. Tumor inhibition rate (%)=(1−tumor weight of testing group/tumor weight of blank control group)×100%.

[0104] 5.3 Treatment of Mice During Administration Period

[0105] Each mouse was weighted 24 hours after administration. In the end of the experiment, the mice were sacrifice by treating at the neck area. The tumor was removed for volume measurements and took photos.

[0106] Animals were handled according to Regulations on the administration of laboratory animals (Order No. second of the State Science and Technology Commission of the People's Republic of China, No. 1988).

[0107] 5.4 Statistical Analysis

[0108] The experimental data were analyzed by SPSS 13.0 software Comparison between groups were carried out by one-way ANOVA or two-sample comparison of U-test. Results were provided in mean±SE, difference with P<0.05 was deemed significant.

[0109] During the 12 days of continuous treatment process on the 5 groups of animals, the nude mice food intake were normal and no discomfort were observed, There was a slight increase in body weight on the mice. The growth rate of the tumor in the control group was significantly faster than that in the other groups. The inhibition of the tumor growth was obvious in the four groups of nude mice treating with drugs, and no obvious side effects were observed. The tumor volume change curve of the nude mice was plotted according to the tumor volume calculated from the daily tumor size.

[0110] As can been seem from FIG. 3, the separated ventral subcutaneous transplanted tumor specimens were a single tumor mass and had no adhesion to the skin. The out appearance was red and white and had an irregular round or oval shape. The membrane of the tumors were intact. The surface had rich vascular distribution. There were no basal adhesion to the ventral muscles. The tissue texture were tough and hard, and their cutting surface was gray. There were no obvious local infiltration.

[0111] The average tumor inhibition rate after drug treatment was calculated and the results were as below: Positive control group (Doxorubicin) 56.8±5.6%, Compound 5 high dosage group 62.2±21.3%, Compound 5 middle dosage group 43.7±19.8%, Compound 5 low dosage group 36.9±6.1%. The results were shown in Table 4 below.

TABLE-US-00004 TABLE 4 Drug dosage, weights of before and after treatment, tumor weights and tumor weight-inhibitions of e in vivo experiments of Compound 5, (n = 6) Weights (g) Animal Dosage Before After Tumor Tumor Weight- Groups (mg/kg) treatment treatment weights (g) inhibitions(%) Blank Conrtol NS + 10% PEG-400 23.7 ± 1.1 25.4 ± 1.2 0.471 ± 0.092 — Doxorubicin  2 mg/kg 22.7 ± 1.9 23.6 ± 1.1 0.220 ± 0.037 56.8 ± 5.6  High 30 mg/kg 22.4 ± 1.2 24.4 ± 1.6 0.193 ± 0.056 62.2 ± 21.3 Middle 15 mg/kg 23.9 ± 1.5 24.9 ± 1.0 0.287 ± 0.101 43.7 ± 19.8 Low  5 mg/kg 22.8 ± 0.8 23.4 ± 0.7 0.305 ± 0.039 36.9 ± 6.1 

[0112] The change of the size of tumor volume in nude mice under continuous administration was shown in FIG. 3 and FIG. 4. There was significant difference between the different dosage of Compound 5 treatment groups, as well as between the treatment group and the positive control group and the negative control group (P<0.05).

[0113] In the allograft nude mouse model, Compound 5 could significantly inhibit the proliferation of A549 cells xenograft and reduce the tumor growth rate. The high dosage (30 mg/kg) of Compound 5 had a better inhibitory effect than the positive control drug doxorubicin. Doxorubicin is one of the clinically commonly used drugs for lung cancer. In the allograft nude mouse model, the middle dosage of compound 5 (15 mg/kg) and low dosage (5 mg/kg) also showed good inhibition of tumor cell proliferation effect. The tumor volume of the high dosage group was obviously smaller than that of the low-dosage group, indicating a dose-dependent effect.

[0114] Therefore, it can be seem that Compound 5 could effectively inhibit the growth of human non-small cell lung cancer cells A549 solid tumor in nude mice. The toxicity thereof was low and nude mice were well tolerated to the drugs. Therefore, Compound 5 is a potential highly efficient, low toxicity of anti-tumor active ingredient.

[0115] The compounds provided by the present invention showed significant anti-tumor effects on multiple human cancer cell lines, including lung cancer (such as non-small cell lung cancer and small cell lung cancer), stomach cancer, leukemia (Including acute promyelocytic leukemia and chronic myeloid leukemia) and esophageal cancer.

[0116] These compounds also showed promising anti-tumor effects in the animal bodies. The experimental results showed that the compounds of the present invention are effective in inhibiting various cancer cells, particularly lung cancers, such as non-small cell lung cancer in the animal bodies.

[0117] The above description of the present invention can not be construed as limiting the present invention. Unless otherwise indicated, the practice of the present invention will use conventional techniques such as organic chemistry, polymer chemistry, biotechnology, etc. And it is obviously that the invention may be practiced otherwise than as specifically described in the foregoing description and examples. Other aspects and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. Many changes and variations are possible in accordance with the teachings of the present invention and are therefore within the scope of the invention.

[0118] Unless otherwise indicated, the unit of temperature, i.e., degree as present herein refers to Celsius degree, i.e., ° C.