JPH0967376A - Suppressant for cancer gene function - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound, available from an extract of a leaf of a tropical plant Aglaia odorata LOUR, having inhibiting activities against actions of a ras cancer gene product, manifesting suppressing activities against cancer gene functions and useful as a suppressant for the cancer gene functions, an anticancer agent, etc. SOLUTION: This new compound of the formula (Me is methyl). The compound has inhibiting activities against actions of a ras cancer gene product and is useful as an active ingredient of a suppressant for cancer gene functions capable of manifesting suppressing activities against the cancer gene functions and further as an anticancer agent, etc., of a new concept. The new compound is obtained by drying a leaf of a tropical plant Aglaia odorata LOUR in an oven at 60 deg.C, pulverizing the dried leaf, then adding chloroform thereto, stirring the resultant mixture at ambient temperature for 5hr, concentrating the prepared extract solution under a reduced pressure, dissolving the extract in toluene, adding the obtained solution onto a silica get column, eluting the adsorbed substance with a mixed solvent of toluene/acetone, further treating the resultant active fraction by the gel filtration, high-performance liquid chromatography, etc., and purifying the active fraction.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an anticancer agent containing the compound of the present invention having an oncogene function suppressing activity as an active ingredient.

[0002]

2. Description of the Related Art In the process of carcinogenesis and promotion of cells,
It is known that oncogenes play an important role. Oncogenes are generated when abnormalities such as point mutations, translocations, and amplifications occur in proto-oncogenes on chromosomal DNA of normal cells, and more than 70 types have been found so far. Among them, the ras oncogene is colon cancer, breast cancer,
It is a typical one found in leukemia and the like, and is said to be present in about 20% of all human cancer tissues. So this r
A substance that inhibits the action of the as oncogene product is expected as a new therapeutic drug for cancer. from now on,
Examples of such inhibitors include oxanosine isolated from actinomycetes (O.Ito et al., Cancer Research, vol.49,996-100.
0,1989), compactin isolated from mold (N. Matsud
a, Cellular Pharmacology, vol.1, 219-223, 1994) and the like are known.

[0003]

However, all of the ras oncogene action inhibitor substances discovered so far have problems in stability and toxicity, and none of them are clinically applicable yet. Therefore, there is a demand for the development of a drug that has a mechanism of action that is not found in the inhibitors known to date and is clinically applicable.

[0004]

[Means for Solving the Problems] In order to obtain an inhibitor that can be clinically applied, the present inventors have screened by using the action of normalizing the morphology of cancer cells as an index,
As a result of conducting a search for a substance that inhibits the action of the ras oncogene product derived from a plant, the activity of inhibiting the action of the ras oncogene product was found in the extract of leaves of the tropical plant Aglaia odorata LOUR, A compound represented by the chemical formula (1) is obtained by specifying the active ingredient,
The present invention has been completed.

[0005]

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[0006] That is, the present invention provides an anticancer agent containing a compound represented by the chemical formula (1) as an active ingredient and optionally a pharmaceutically acceptable carrier. The compound of the present invention represented by the chemical formula (1) isolated from Aglaia odorata (hereinafter, this substance is referred to as “aglaiastatin C” for convenience in this specification) has a molecular weight of 526 (FAB-MS).
Colorless powder (according to spectrum measurement). The Rf values in silica gel thin layer chromatography were 0.42 (developing solvent: chloroform: methanol = 20: 1) and 0.49 (developing solvent: toluene: acetone = 1: 1). As a result of elemental analysis, the molecular formula was C ₃₁ H ₃ 0N ₂ O ₆ .
The ultraviolet absorption spectrum is shown in FIGS. 1 to 3, and the infrared absorption spectrum is shown in FIG. The ¹ H-NMR spectrum and ¹³ C-NMR measurement results are shown in FIGS. 5 and 6. As a result of the investigation, it was supported that the compound of the present invention isolated from Aglaia odorata has the structure represented by the above chemical formula (1).

[0007] When aglaiastatin C was given to a cancer cell having a ras oncogene, the cell morphology was normalized and its growth was suppressed. Therefore, the present compound may have an effect of inhibiting the action of the oncogene product. found. Examples of the pharmaceutically acceptable carrier used in the anticancer agent of the present invention include excipients such as natural or synthetic aluminum silicate, microcrystalline cellulose, talc, dextrin, starch and lactose, and diluents such as vegetable oil and propylene glycol. Is mentioned. In addition to this, pharmaceutically acceptable stabilizers, coating agents, suspending agents, emulsifying agents, solubilizing agents, preservatives, buffering agents, sweetening agents and the like may be present as other optional ingredients. Known additives can be used in appropriate combination as these additives. The dosage form may be any form such as powder, granules, tablets, capsules and injections. The anticancer agent of the present invention is mainly orally administered, but the present invention is not limited by the administration method. The daily dose for an adult varies depending on the administration method, medical condition and the like. Oral administration, which is the main method of administration, is 10 per adult per day.
The usual dosage is 0-500 mg. However, the present invention is not limited by dose.

[0008] Aglaia odorata is a small tree of the genus Asteraceae, which grows naturally in Southeast Asia, or is partly cultivated and used as an ornamental or fragrance. The raw material plant may be dried for reasons such as storage and transportation. In that case, it is preferable to dry it in the sun or in an oven at 60 ° C. or lower. In addition, crushed ones are preferable for extraction.
Any solvent can be used for the extraction as long as it can stably extract aglaiastatin C, but a nonpolar solvent is preferable, and a hydrocarbon solvent or a halogenated hydrocarbon solvent is particularly preferable. Examples of the hydrocarbon solvent include pentane, hexane, heptane, octane, benzene, toluene, xylene and the like, and toluene is particularly preferable.
Examples of the halogenated hydrocarbon solvent include chloroform, methylene dichloride, carbon tetrachloride and the like, and chloroform is particularly preferable. Of course, the types of these solvents are not limited. The amount of solvent, temperature, time, etc. during extraction are not particularly limited as long as aglaiastatin C is obtained in a stable and high yield without decomposition.

The method for isolating and purifying aglaiastatin C is not particularly limited as long as the compound can be stably obtained from the extract. Chromatography can be mentioned as an example of a suitable isolation and purification method. Chromatography may preferably include column chromatography, gel filtration chromatography, high performance liquid chromatography, thin layer chromatography or combinations thereof. Silica gel, alumina, etc. can be used as the carrier used in column chromatography, and hydrocarbon solvents, halogenated hydrocarbons, alcohols, etc. can be used as the developing solvent. For gel filtration chromatography, Toyopearl HW-40 (manufactured by Tosoh Corporation) as a carrier and alcohols as a solvent can be used. However, the types of chromatography carrier and solvent are not limited to those described above. And aglaiastatin
As a result of analyzing the structure of C, it was found to be represented by the chemical formula (1). Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples.

[0010]

[Example] 1. Extraction of Active Substances from Aglaia odorata After crushing 167 g of leaves of Aglaia odorata dried in an oven at 60 ° C., it was put in a 2 L Erlenmeyer flask,
1000 ml of chloroform was added and the mixture was stirred at room temperature for 5 hours. The solvent was renewed and the same operation was repeated, and extraction was performed a total of 3 times. The three extracts were combined and chloroform was distilled off under reduced pressure to obtain 5.7 g of dark green syrup. This syrup was used as a raw material for the next purification.

[0011] 2. Purification of active substance from extract Dissolve 3.9 g of chloroform extract in a small amount of toluene,
120 g of silica gel packed in a column (trade name: Merck s
ilicagel 60, manufactured by Merck & Co., Inc.), and toluene: acetone = 20: 1, 16: 1, 6: 1, 3: 1, 2: 1,
Elution with a 1: 1 solvent was carried out sequentially, and about 20 ml fractions were fractionated. When each fraction was subjected to the cell morphological change induction test used in "4. Effect of aglaiastatin C on morphological change induction in various cells", the morphological normalization effect was obtained in the toluene: acetone = 3: 1 to 1: 1 elution fraction. Was recognized. The active fraction was collected and weighed 639 mg. About 2 ml of this active fraction was fractionated with Toyopearl HW-40 equilibrated with methanol, and the column was packed with Merck silica gel 60 (sila).
nisied) and eluted with 70% methanol to fractionate about 4 ml each. Each fraction was subjected to a cell morphological change induction test, and the fractions in which activity was observed were collected. Furthermore, HPLC fractionation of this active fraction (column: PEGASIL ODS, manufactured by Senshu Kagaku Co., Ltd., elution solvent: 65% methanol, flow rate: 5 ml / m)
in, detection: UV210 nm) and fractionated by about 1 ml each. When each fraction was subjected to a cell morphology change induction test, activity was confirmed in the fraction eluted at a retention time of 42 minutes.
This fraction was collected and collected as a colorless powder (aglaiastatin C 2.1m
g) was obtained. When silica gel thin layer chromatography was performed, this fraction was a single spot.

[0012] 3. Physicochemical properties of aglaiastatin C Various physicochemical properties of colorless powder (aglaiastatin C) were measured. The Rf values of this compound in silica gel thin layer chromatography were 0.42 (developing solvent: chloroform: methanol = 20: 1) and 0.49 (developing solvent: toluene: acetone = 1: 1). The melting point was 157 to 160 ° C. The results of measurement of various spectra are shown in FIGS. 1 to 6.

[0013] 4. aglaiastatin C for various cells
Shape change inducing effect a. Culturing of various cells K-ras ^ts -NRK cells (NIH, USA) are Dulbecco's modified cells containing 5% fetal calf serum (manufactured by Gibco Laboratories) at 33 ° C. showing the morphology of cancer cells and 39 ° C. showing the morphology of normal cells. Method Eagle medium (Nissui Pharmaceutical Co., Ltd.) glutamine 0.6g / l, kanamycin 0.2g / l, NaHCO ₃ 2.25g / l
The culture solution (hereinafter, the culture broth of "medium") plus as were cultured in 5% CO ₂ incubator. In addition, normal rat kidney cells NRK-49 cells (Dainippon Pharmaceutical Co., Ltd.) and mouse NIH3T3 cells (J
CRS cell bank) and cancer cells K-ras-N
RK cells (Showa Pharmaceutical University), K-ras-NIH3T3 cells, H-ras-NIH3T3 cells, N-ras-NIH3
T3 cells (Institute of Medical Science, University of Tokyo) were similarly cultured at 37 ° C. In addition, the cells contained calcium-magnesium-free phosphate buffer (PBS ^(-) : NaCl 8.0 g / l, Na ₂ PO ₄ .12H ₂ O).
After washing with 2.31 g / l, KH ₂ PO ₄ 0.2 g / l; pH 7.4), trypsin-EDTA solution (trypsin 0.5 g / l, NaCl 8.0 g / l)
l, glucose 1.0g / l, phenolred 0.02g / l, KH ₂ PO ₄ 0.35g
/ l, EDTA 0.2g / l), remove the cells from the culture flask,
Passage was carried out by transferring into a flask containing fresh medium.

B. Observation of morphological change inducing effect Various cells were seeded on a 48-well plastic plate (manufactured by Coaster) with 1.0 × 10 ⁴ cells / 0.5 ml medium / well and 5% CO 2
And cultured for one day at ₂ incubator in Better. The next day, the test substance was added, and the morphological changes of the cells were observed until the third day. i) aglaiastatin C for K-ras ^ts -NRK cells
Effects of K-ras ^ts -NRK cells show several thin projections from the cells at 33 ° C, and the cells overlap each other to show the morphology of cancer cells, and at 39 ° C, the cells are flat and do not show projections and overlap. Without any exception, it shows the morphology of normal cells. Then, as a result of adding aglaiastatin C 10 ng / ml at 33 ° C. and culturing for 3 days, the morphology of normal cells without protrusions and flat and not overlapping was shown. At 39 ° C, there was no significant change in cell morphology. ii) Effect of aglaiastatin C on K-ras-NRK cells K-ras-NRK cells, which are cancer cells, have a rounded and rounded shape, and aglaiastatin C 20 ng / ml is added thereto and cultured at 37 ° C for 3 days. As a result, the morphology of the cells was flattened and the adhesion surface was increased to show the morphology of normal cells. iii) aglaiastati for K-ras-NIH3T3 cells
Effect of n C K-ras-NIH3T3 cells, which are cancer cells, each have an elongated protrusion-like morphology, and the cells overlap each other. When 20 ng / ml of aglaiastatin C was added thereto and cultured at 37 ° C. for 3 days, it was observed that the cells became flat and the adhesive surface increased, and the cells were in the form of normal cells, NIH3T3 cells.

5. aglaiastatin C for various cells
Cell Growth Inhibitory Effect Various cells were seeded on a 48-well plastic plate (manufactured by Coaster) at 1.0 × 10 ⁴ cells / 0.5 ml medium / well and cultured for 1 day in a 5% CO ₂ incubator at an appropriate temperature. Next day aglaia
After adding statin C and culturing for another 3 days and washing twice with PBS ^(-) , trypsin was added at 100 μl / well each, and 0% C was added for several minutes.
The cells were removed in an incubator under O ₂ conditions, and the medium was added at 900 μl / well to homogenize. The cell concentration of this solution was measured using a Coulter counter (ZM type), and the concentration at which cell growth was inhibited by 50% (IC ₅₀ ) was determined. The results are shown in Table 1, and ag for cancer cells
IC ₅₀ of Laiastatin C is 1.0~5.6ng / ml.

[Table 1] Cell name IC ₅₀ (ng / ml) K-ras-NRK 2.8 K-ras-NIH3T3 5.6 H-ras-NIH3T3 1.0 N-ras-NIH3T3 1.8 NRK 2.1 NIH3T3 4.1

[0017]

The compound of the present invention extracted from the tropical plant Aglaia odorata represented by the chemical formula (1) has the activity of inhibiting the growth of cancer cells and normalizing the morphology thereof,
It can be effectively used as an anti-cancer agent.

[Brief description of drawings]

FIG. 1 UV absorption spectrum neutral condition (MeOH)
Measurement results at

[Figure 2] UV absorption spectrum acidic conditions (0.1N HCl-M
eOH) measurement results

Fig. 3 UV absorption spectrum basic conditions (0.1N NaO
H-MeOH) measurement results

FIG. 4 Infrared absorption spectrum measurement results

FIG. 5: ¹ H-NMR spectrum measurement results

FIG. 6 Results of ¹³ C-NMR spectrum measurement

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takashi Oyano 1500 Inoguchi, Nakai-cho, Ashigaraue-gun, Kanagawa Terumo Corporation (72) Inventor, Kazuo Umezawa 3-1-2-505 Hiroo, Shibuya-ku, Tokyo

Claims (2)

[Claims] 1. A compound represented by the chemical formula (1). Embedded image 2. An oncogene function inhibitor comprising a compound represented by the chemical formula (1) as an active ingredient. Embedded image