NOVEL INDOLE DERIVATIVE AND ANTI-CANCER CONPOSITION CONTAINING SAME

Abstract

The present invention relates to: an indole derivative, which is a novel cell mitosis inhibitor; a stereoisomer thereof or a pharmaceutically acceptable salt thereof; a use thereof as a therapeutic agent; a composition containing the same and a treatment method using the composition; and a preparation method therefor. According to the present invention, the indole derivative, the stereoisomer thereof or the pharmaceutically acceptable salt thereof, inhibits the tubulin polymerization during mitosis so as to induce apoptosis, and has an excellent anti-cancer effect also in cancer cells having multiple drug resistance.

Claims

1. An indole compound of the following Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: ##STR00002##

2. A composition comprising the indole compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof according to claim 1, as an active ingredient, and a carrier.

3. The composition according to claim 2, which is a pharmaceutical composition.

4. The composition according to claim 2, which is a food composition.

5. The composition according to claim 2, which is a cosmetic composition.

6. A method for preventing and/or treating a cancer in a subject in need thereof, comprising administering to the subject a composition comprising an indole compound of the following Formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: ##STR00003##

7. The method according to claim 6, wherein the cancer is selected from the group consisting of rectal cancer, breast cancer, lung cancer, gastric cancer, liver cancer, leukemia, glioma, skin cancer, and cervical cancer.

8. The method according claim 6, wherein the composition is a cosmetic composition and the cancer is skin cancer.

9. The method according to claim 6, wherein the composition is a pharmaceutical composition or a food composition.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0041] FIG. 1 shows the effect of the compound of Example 1 (WCI-1031) on the spindle fibers and chromosomes of the mitotic cells depending on the concentration of the compound examined by immunofluorescence staining.

[0042] FIG. 2 is a graph showing changes in weights after treatment with the compound in a human-derived skin cancer cell line (A431) implantation nude mouse model.

[0043] FIG. 3 is a graph showing changes in tumor sizes after treatment with the compound in a human-derived skin cancer cell line (A431) implantation nude mouse model.

[0044] FIG. 4 provides photographs showing the tumor sizes of the mice on the last day (day 23) after treatment with the compound in a human-derived skin cancer cell line (A431) implantation nude mouse model.

[0045] FIG. 5 is a graph showing the tumor weights after treatment with the compound in a human-derived skin cancer cell line (A431) implantation nude mouse implantation model.

[0046] FIG. 6 provides photographs showing the tumors isolated from the mice on the last day (day 23) after treatment with the compound in a human-derived skin cancer cell line (A431) implantation nude mouse model.

[0047] FIG. 7 is a graph showing the changes in plasma concentration over time after intravenous administration of the compound of Example 1 (WCI-1031).

[0048] FIG. 8 is a solubility curve of the compound of Example 1 (WCI-1031) at pH 6.5.

[0049] FIG. 9 is a solubility curve of the compound of Example 1 (WCI-1031) at pH 7.4.

BEST MODE FOR CARRYING OUT THE INVENTION

Mode for the Invention

[0050] Hereinafter, the present invention is explained in detail by Examples. The following Examples are intended to further illustrate the present invention without limiting its scope.

<Example 1> Preparation of methyl (E)-2-(2-methyl-3-((2-(naphtho[2,1-b]furan-2-carbonyl) hydrazono)methyl)-1H-indole-1-yl)acetate (WCI-1031)

[0051] Naphtho[2,1-b]furan-2-carbohydrazide (100 mg, 0.44 mmol) and methyl 2-(3-formyl-2-methyl-1H-indol-1-yl)acetate (102 mg, 0.44 mmol) and a solution of a catalytic amount of acetic acid (0.1 ml) in ethanol (5 ml) were stirred at 90° C. for 2 hours. After confirming that the starting substances were exhausted by TLC, the reaction mixture was cooled to room temperature and added to ice water. The separated solid mass was filtered, washed with water and then dried. The residue thus produced was purified by silica gel flash column chromatography using a mixture of hexane and ethyl acetate at a mixing ratio of 1:1, to obtain the desired compound, methyl (E)-2-(2-methyl-3-((2-(naphtho[2,1-b]furan-2-carbonyl) hydrazono)methyl)-1H-indole-1-yl)acetate (174 mg, 90%) as a pale yellow solid.

[0052] ESIMS found: m/z 440.36[M+H].sup.+, 879.16 [2M+H].sup.+.

<Experimental Example 1> Examination of Anti-Proliferative Activity of Indole Derivative of the Present Invention

[0053] <1-1> Examination of Anti-Proliferative Activity in HeLa Cell Line

[0054] The following experiments were conducted to examine the anti-proliferative activity of the indole derivatives of the present invention.

[0055] HeLa cells (ATCC, USA), which are a human cervical cancer cell line, were dispensed into a 96-well plate at 2×10.sup.3 cells/well, and the cells were treated with DMSO or the indole derivatives of the present invention, and then allowed to grow for 4 days. And then, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent was added to the wells at 10 μl/well. After 2 hours, the absorbance was measured at “OD 450” and statistical values were obtained using the plizm6 program. The data represent mean values of the results of two repeated assays (Table 1).

TABLE-US-00001 TABLE 1 Treatment substance IC.sub.50 (μM) DMSO — Example 1 WCI-1031 0.653

[0056] As shown in Table 1, the indole derivatives of the present invention were found to have excellent anti-proliferative activity in the HeLa cell line (cervical cancer cell line).

[0057] <1-2> Examination of Anti-Proliferative Activity in Other Cancer Cell Lines

[0058] The following experiment was conducted to determine whether the indole derivative of the present invention shows anti-proliferative activity in other cancer cells as well as in the HeLa cell line (cervical cancer cell line).

[0059] Specifically, various cancer cell lines were cultured on a microtiter plate (1-3×10.sup.3 cells/well), and the cells were treated with the indole derivative of the present invention and cultured for 4 days. Cytotoxicity was examined by the MTT assay by the same method as in Experimental Example 1-1, and the IC.sub.50 was obtained by the log-dose response curve. The data represent mean values of the results of three repeated assays (Table 2).

TABLE-US-00002 TABLE 2 Origin of Example 1 Cell line Tumor WCI-1031 HeLa Uterine 0.297 cercix Hep3B Liver 0.503 HepG2 Liver 2.402 A172 Brain 1.117 U373MG Brain 0.262 A431 Skin 0.186 MCF7 Breast 1.321 PC3 Prostate 0.549 NCIH-125 Lung 0.333 NCIH-460 Lung 0.671 NCIH-1299 Lung 0.376 A 549 Lung 0.495 SNU 484 Stomach 0.493 Wehi 3 Bone 0.290 marrow K562 Bone 0.549 marrow

[0060] As shown in Table 2, it was found that the indole derivative of the present invention has an excellent anti-proliferative activity in various cancer cell lines as well.

[0061] <1-3> Examination of Anti-Proliferative Activity in Cancer Cells Showing Multiple Drug Resistance

[0062] The following experiment was conducted to examine whether the indole derivatives of the present invention are effective in cancer cells showing multiple drug resistance.

[0063] Specifically, K562 and MCF7 (Bio Evaluation Center, Korea Research Institute of Bioscience and Biotechnology, Korea), and K562/ADR and MCF7/ADR (Bio Evaluation Center, Korea Research Institute of Bioscience and Biotechnology, Korea), which are multiple drug-resistant cell lines of the above mentioned cell lines, respectively, were cultured on a microtiter plate at 1-3×10.sup.3 cells/well. Then, the cells were treated with WCI-1031 (compound of Example 1), an indole derivative of the present invention, taxol, doxorubicin, vinblastine, or colchicine, and cultured for 4 days. The cytotoxicity was examined by the MTT assay by the same method as in Experimental Example 1-1, and the IC.sub.50 was obtained by the log-dose response curve (unit: nM). The data represent mean values of the results of three repeated assays (Tables 3 and 4).

[0064] The resistance factor of a cell line exhibiting multiple drug resistance is the ratio of the IC.sub.50 of the multiple drug-resistant cell line to the IC.sub.50 of a non-resistant parent cell line.

TABLE-US-00003 TABLE 3 WCI-1031 Taxol Doxorubicin Vinblastine colchicine K562 532 1.347 2.848 17.13 15.86 K562/ADR 317.8 699.9 2187 267.8 363.4 Resistance 0.60 519.6 767.91 15.63 22.91 factor

TABLE-US-00004 TABLE 4 WCI-1031 Taxol Doxorubicin Vinblastine colchicine MCF7 1321 2.345 15.38 5.275 2.71 MCF7/ 481.1 1028 2608 95.87 92.54 ADR Resistance 0.36 438.38 169.57 18.17 34.15 factor

[0065] As shown in the Tables 3 and 4, the multiple drug-resistant cell lines exhibited strong resistance to the conventional anti-cancer agents, with resistance factors ranging from several tens to several hundreds. However, the resistance factors against the compound of Example 1, an indole derivative of the present invention, ranged from 0.36 to 0.60, indicating that the indole derivative of the present invention has a stronger cytotoxic effect on the cancer cells which show multiple drug resistance, as compared to the conventional anti-cancer agents.

<Experimental Example 2> Examination of Effect of Indole Derivative of the Present Invention on Cell Cycle Progression

[0066] Several cancer cell lines were cultured on a 12 well plate (3×10.sup.4 cells/well), and treated with DMSO or WCI-1031, an indole derivative of the present invention, for 17 hours, and then added with a propidium iodine dye to stain the cellular DNA, and subjected to measurement using FACS. The concentrations at which cells gathered in the G2/M phase and numbers of the cells represented as percentages were shown (Table 5).

TABLE-US-00005 TABLE 5 Cell line effect μM (% of G2/M) HeLa 0.5 (82.36) Hep3B 0.5 (68.13) A172 1.0 (70.10) A431 0.5 (66.32) MCF7 0.5 (59.08) MCF7 ADR 0.5 (52.37) PC3 0.5 (65.35) NCIH-125 1.0 (48.10) EL4 0.5 (50.84) K562 0.5 (76.72)

[0067] With regard to WCI-1031, an indole derivative of the present invention, cells gathered in the G2/M phase at the concentrations of 0.5 to 1.0 μM, and the numbers of the cells were 50% or higher, as shown in Table 5. Therefore, it was found that the indole derivative of the present invention suppresses the cell cycle in G2 and M phases.

<Experimental Example 3> Examination of Effect of Indole Derivative of the Present Invention on Tubulin Polymerization

[0068] In order to examine the effect of the indole derivative of the present invention on intracellular microtubules, HeLa cells were treated with DMSO or WCI-1031 (0.1 μM, 0.5 μM, and 1.0 μM) for 16 hours. The cells were fixed, and stained with an anti-tubulin antibody and Alexa Fluor 488, and stained with an anti-centrosome antibody and Texas Red, and then the nuclei of the cells were immunostained using Hoechst 33342 to examine the α-tubulin, centrosome and DNA (FIG. 1).

[0069] As shown in FIG. 1, when the cells were treated with the indole derivative of the present invention, as the concentration of the derivative became higher, the shape of the tubulin became loose and shorter, as compared to the DMSO control. And the pattern of the DNA which deviated from the central array was increasingly found.

[0070] Therefore, it was found that the indole derivative of the present invention is a formulation which depolymerizes microtubules.

<Experimental Example 4> Examination of Anti-Cancer Effect in a Human-Derived Skin Cancer Cell (A431) Implantation Model

[0071] <4-1> Culture of Cancer Cells and Implantation of Cancer Cells

[0072] The human cancer cell line A431 which was kept frozen in liquid nitrogen was thawed and cell culture was conducted. Cells were cultured in a CO2 incubator (Forma, USA) at 37° C. and 5% CO2 for an appropriate period of time.

[0073] On the last day of culture, all cancer cells were collected and counted and the cell concentration was adjusted to 1×10.sup.7 cells/ml using serum free media. The culture solution thus prepared was injected subcutaneously in the axillary region between the scapula and chest wall in an amount of 0.3 ml per BALB/C female nude mouse (5 weeks old, Nara Biotech) (3×10.sup.6 cells/mouse).

[0074] <4-2> Method of Preparation and Administration of Samples

[0075] WCI-1031 (Example 1), an indole derivative compound of the present invention, was used as a test substance, and 5-FU and colchicine were used as positive control substances.

[0076] The compounds were used after dissolving in DMAC (dimethyl acetamide) 20%+Tween80 5%+20% HPbCD (2-hydroxypropyl-beta-cyclodextrin) 75% to appropriate concentrations immediately before administration. 5-FU and colchicine used as the positive control substances were prepared for use in the concentrations of 2 and 0.007 mg/ml using normal saline and PBS, respectively.

[0077] The prepared substances were repeatedly administered intraperitoneally and orally at 0.2 ml per 20 g of the mouse (10 ml/kg) according to the dosage schedule below. [0078] carrier, WCI-1031 (10, 20 mg/kg), colchicine (0.07 mg/kg): days 0-23 [0079] 5-FU: days 0-2, 5-9, 12-16, 19-23

[0080] <4-3> General Symptom and Weight Change Verification

[0081] To examine the toxicity levels of the repeated intraperitoneal administration of WCI-1031 to A431 cancer cell implantation nude mice, general symptoms and weight changes of the animals were observed during the administration period.

[0082] As a result, no statistically significant weight loss was observed in all drug-treated groups as compared to the solvent control group, and no significant general symptoms were observed during the test period (FIG. 2).

[0083] <4-4> Examination of Tumor Size Changes

[0084] After the cancer cell implantation, the tumor size of each animal was measured in three directions using a vernier caliper from the point when the average tumor size reached 57.0 mm.sup.3 until day 23, for a total of 11 times, which was expressed by the equation of length×width×height/2.

[0085] On the last day (day 23), tumor growth inhibitions of 9.3% and 37.2% (p<0.001) were observed in the groups administered with WCI-1031 at 10 and 20 mg/kg, respectively, as compared to the solvent administration control group. The tumor growth inhibitions in the 5-FU and colchicine administration groups, the positive control groups, were 20.7% (p<0.01) and 17.6% (p<0.05), respectively (FIGS. 3 and 4).

[0086] <4-5> Examination of Tumor Weight Changes

[0087] On day 23 after the start of the drug administration, blood was taken from the mice through ophthalmic veins 2 hours after the last administration, and the mice were sacrificed using CO2 gas. Then the mice were photographed and the tumors was isolated and weighed in a chemical balance. After taking photographs, each tumor was divided in half, and the resultant tumors were fixed to liquid nitrogen and formalin, respectively.

[0088] On day 16 after the start of administration of the drug, A431 tumors were excised and weighed. The tumor weight reductions of 10.7% and 38.0% (p<0.001) were observed in the groups administered with WCI-1031 at 10 and 20 mg/kg, respectively. The tumor weight reductions in the 5-FU and colchicine administration groups, the positive control groups, were 20.8% (p<0.05) and 17.6% (p<0.05), respectively (FIGS. 5 and 6).

<Experimental Example 5> Examination of Stability of Indole Derivative of the Present Invention in Blood Plasma

[0089] In order to examine the stability of the indole derivative of the present invention in vivo, the compound of Example 1 (WCI-1031) was injected intravenously and the plasma concentration was observed over time (FIG. 7).

[0090] As shown in FIG. 7, the stability of WCI-1031 in the blood plasma was found to be excellent.

<Experimental Example 6> Examination of Solubility of Indole Derivative of the Present Invention

[0091] In order to examine the solubility of the indole derivative of the present invention, the solubility of the compound of Example 1 (WCI-1031) was examined at pH 6.5 or pH 7.4.

[0092] Specifically, a solid sample (˜1 mg) of the test substance was placed on Whatman Syringeless filter (PVDF membrane, 0.45 μm pore size) and mixed with 0.5 ml of a phosphate buffer (pH 6.5 or 7.4). The mixture was sonicated for 15 minutes and vortexed for 1 hour at room temperature. The equilibrated mixture was filtered and the filtrate was analyzed with a multi-wavelength UV plate reader (FIGS. 8 and 9).

[0093] The data of the solubility and the like of WCI-1031 are shown in Table 6 below.

TABLE-US-00006 TABLE 6 Solubility Ka SIWV SITT MAD pH (μg/ml) (min.sup.−1) (ml) (ml) (mg) 6.5 0.9 0.03 250 270 1.8 7.4 0.4 0.03 250 270 0.8 (Ka: intestinal absorption rate constant SIWV: small intestinal water volume (~250 ml) SITT: small intestinal transit time (~270 min) MAD : maximum absorbable dose in human)