JPH0656657A - Antitumor agent and anti-AIDS agent containing fucosidase inhibitor - Google Patents

Abstract

PURPOSE:To provide the subject low toxic agent excellent in antimetastatic and anti-HIV activity, containing a specific substance as active ingredient. CONSTITUTION:This antitumor/AIDS agent contains, as active ingredient, a chemical substance of the formula obtained by isolating and purifying a material extracted from the seed skin of Anacardium occidentum L. using an organic solvent such as hexane. It is preferable that the dose of this agent be 0.1-1.0g a day per adult.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antitumor agent and an anti-AIDS (AIDS) agent containing a substance having a fucosidase inhibitory activity as an active ingredient.

[0002]

2. Description of the Related Art Recently, it has been suggested that the sugar chain structure of glycolipids and glycoproteins present on the cell surface is important for cancer metastasis, virus infection, differentiation and proliferation. For example, it has been reported that changes in the sugar chain structure of glycolipids on the cell surface associated with canceration of cells, mainly fucosylation and sialylation ^(1-4) . In addition, castanospermine ⁽⁵⁾ and swainsonine, which are believed to inhibit the biosynthetic process of sugar chain structure,
Glycolytic enzyme inhibitors such as ⁽⁶⁾ are used in mouse melanoma cells B
It was reported to inhibit the experimental metastasis of 16 / F10.

HIV (human immunodeficiency virus), a causative virus of AIDS (acquired immunodeficiency syndrome), infects T4 antigen-expressing cells, but formation of syncytia and cytotoxicity due to HIV infection is gp120, which is a membrane glycoprotein of the virus. It depends on the binding of T4 molecules on the cell surface ⁽⁷⁾ and is inhibited by castanospermine ⁽⁸⁾ and deoxydinomycin ⁽⁹⁾ , which are inhibitors of glucosidase I and II. It was shown that the normal glycation of gp120 was inhibited by the addition of these glycolytic enzyme inhibitors ^{(8) (9)} .

Therefore, the anti-HIV activity of such glycolytic enzyme inhibitors has been of interest, and the research of structure-activity relationship has been advanced to seek a highly active form from existing inhibitor derivatives.
^{(10) (11)} Among the derivatives exhibiting anti-HIV activity, those exhibiting α-L-fucosidase inhibitory activity were found ⁽¹¹⁾ .

[0005]

Therefore, the present inventors
As a substance expected to have anti-metastatic activity and anti-HIV activity, α
-L-fucosidase inhibitor is screened to inhibit bovine kidney α-L-fucosidase (IC ₅₀ : about 43μ)
g / ml) material was isolated and purified.

[0006]

The present invention provides a tropical plant Anac.
It is based on the discovery that a substance isolated and purified from ardium occidentale has fucosidase inhibitory activity.

Anacardium occidentale is a tropical plant belonging to the family Rhusidae, and the emulsion of its peel is used as a protozoan family or dye, and the seeds (or fruit) remaining after removing the peel are used as food. .

That is, the substance used in the present invention has a structural formula:

[0009]

[Chemical 2]

It is a type of substance having a substance known as cardol.

Hereinafter, in the present specification, the substance having the above structural formula is simply referred to as "cardol".

Cardol is isolated and purified from the organic solvent extract of the seed coat of Anacardium Occidental.

As Anacardium Occidental, Anacardi Occidentale L ( Anacardi
um occidentale L.) is preferred.

The raw material Anacardium occidentale is preferably dried and crushed to prevent spoilage during storage.

As the hydrocarbon solvent used in the extraction step, hydrocarbons such as pentane, hexane, heptane, octane, petroleum ether, petroleum benzine, ligroin, benzene, toluene, xylene, ethylbenzene; halogenated hydrocarbons Examples thereof include chloroform, methylene chloride, carbon tetrachloride, etc .; alcohols such as methanol, ethanol, butanol; etc .; or a mixed solvent thereof, and hexane, chloroform and methanol are particularly preferable. Of course, other solvents with similar extraction capabilities other than these solvents can be used.

The amount of the organic solvent is not particularly limited, but may be any amount capable of extracting cardol, and is preferably 100 to 1000 ml per 50 g (dry weight) of seed (or fruit) containing the skin of Anacardium occidentale. The extraction temperature is not particularly limited as long as it can extract the isolated substance of the present invention, and is preferably room temperature to 65 ° C, more preferably room temperature to 45 ° C. The extraction time varies depending on the temperature, but is 1 to 12 hr when the extraction is performed at room temperature to 45 ° C, for example. The pressure at the extraction stage is not particularly limited, but the extraction is usually performed at normal pressure. Note that the extraction is 2 depending on the extraction rate.
May be repeated more than once.

In order to further isolate cardol from the obtained extract, the extract is directly or once concentrated and then purified by liquid chromatography.

According to an embodiment of the present invention, liquid chromatography may include silica gel chromatography, molecular sieve chromatography and HPLC.

More specifically, the extract obtained in the extraction step is once concentrated and then subjected to silica gel column chromatography using a column packed with Kieselgel 60 and a developing system of chloroform; the developing system is toluene: acetic acid. Perform chromatography in the same manner instead of ethyl = 5: 1; use a column packed with Sephadex LH-20 and perform molecular sieve chromatography with methanol as a developing system; and acetonitrile: H ₂ O = 8: Cardol can be purified by performing HPLC using 2 as the developing system.

The cardol used in the present invention has fucosidase inhibitory activity, and its IC ₅₀ is estimated to be about 43 μg / ml.

Further, when 50 μg / ml of cardol was added to the culture medium of mouse melanoma cells B16 / F10 and the culture medium cells were counted for 2 days, no difference was observed from the control, so that the cytotoxicity was substantially There is no such thing.

The antitumor agent and anti-AIDS agent of the present invention are mainly administered orally, but may be administered intravenously in case of emergency, and the present invention is not particularly limited by the administration method. The daily dose for an adult varies depending on the administration method. In the case of oral administration, which is the main method of administration,
Cardol 0.1-1.0 g is the preferred daily dose. However, the present invention is not limited by dose. In addition, as described above, cardol, which is an active ingredient, has extremely low toxicity to cells.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.

[0024]

【Example】

[I] Fucusida from Anacardium Occidentale
After drying in the preparation sun substances having over peptidase inhibitory activity, heater seeds 50g including pericarp milled Anakarujiumu-Okushidentare el (Anacardium occidentale L.), glass container of 1L equipped with a stirrer and a condenser , Add 200 ml of hexane,
The mixture was extracted at room temperature with stirring for half a day. After extraction, the extract was separated into a solid residue by filtration, and the solid residue was extracted twice more by the same procedure. Hexane was distilled off by heating under reduced pressure, and the resulting residue was freeze-dried to obtain 16 g of a dark brown viscous liquid substance. This liquid substance is called a hexane extract.

Next, 16 g of this liquid substance was added to Kieselgel 60.
(Merck) 180 g silica gel column (34
φ × 62 mm) and eluted with chloroform solvent to collect active fractions. The active fractions were collected and the solvent was evaporated to dryness under reduced pressure.

0.9593 g of residue obtained Kiesel as above
Place on a column packed with gel 60 (50 g), elute with a mixed solvent of toluene: ethyl acetate (5: 1),
The active fraction was collected. The eluate containing the active fraction was dried under reduced pressure to obtain 80 mg of residue.

Furthermore, 20.4 mg of the obtained residue was used as Sephad.
Place on a column (19φ x 580 mm) packed with 100 ml of ex LH-20 (Pharmacia), perform molecular sieve chromatography using methanol as a solvent, collect active fractions, and dry under reduced pressure. 17 mg of residue was obtained.

Further, the residue was subjected to HPLC (high pressure liquid chromatography) to give acetonitrile: H ₂ O (8:
Elution was performed using the mixed solvent of 2), the active fractions were collected, and dried under reduced pressure to obtain about 5 mg of the final substance.

The respective physical properties of the obtained substance are as shown in the drawings attached to this specification.

The assignments of ¹ H-NMR and ¹³ C-NMR spectra are summarized in Table 1 below.

[0031]

[Table 1]

From these physical properties, the final substance had a molecular weight of 316, a molecular formula: C ₂₁ H ₃₂ O ₂ and a structural formula:

[0033]

[Chemical 3]

Was identified as a type of cardol having

[II] Fucosidase Inhibitory Activity (Materials and Methods) The fucosidase reaction and its measurement were 96.
A well tissue culture plate (Falcon) was used.

To one well of the culture plate, 3 μl of DMSO
Cardol dissolved in 10 µl of substrate solution [15 mM
p-nitrophenyl-α-L-fucopyranoside, 25 mM
Sodium acetate (pH 5.3)] was added and the mixture was stirred. To this mixture, add 20 μl of enzyme solution [0.8 μg bovine kidney α-L-
Fucosidase (Boehringer Mannheim), 25 mM
Sodium acetate (pH 5.3)] was added and the reaction was carried out at 37 ° C for 10 minutes. 0.4M glycine buffer (pH 10.4)
The reaction was stopped by adding 150 μl of. 450 after reaction
The absorbance at nm was measured by an auto microplate reader (Tosoh Corporation).

The results are shown in Table 2.

From these results, the IC ₅₀ was about 43 μg / ml.
Was estimated.

[0039]

[Table 2]

As is apparent from the above results, cardol has fucosidase inhibitory activity and can be used as an antitumor agent for suppressing cancer metastasis and an anti-AIDS agent having anti-HIV activity.

[0041]

[References]

(1) Matyas, GR, et al., Proc. Natl. Acad. Sc
i. USA , 84, 6065 (1987). (2) Singhal, AK et al. Cancer Res ., 47, 55
66 (1987). (3) Hakomori, SI, et al. J. Biol. Chem . 259,
4672 (1984). (4) Fukushi, Y. et al. J. Biol. Chem . 259, 1
0511 (1984). (5) Humphries, MJ et al. Cancer Res ., 46,
5215 (1986). (6) Humphries, MJ et al. Proc. Natl. Acad.
Sci. USA , 83, 1752 (1986). (7) Haserutin, W. et al. Science 231, 382
(1986). (8) Walker, BD et al. Proc. Natl. Acad. Sci.
USA , 84, 8120 (1987). (9) Gruters, RA et al. Nature 330, 74 (198)
7). (10) Abraham, K. et al. Proc. Natl. Acad. Sci.
USA , 85, 9229 (1988). (11) Fleet, GWJ et al. FEBS Letter , 237,
128 (1988).

[Brief description of drawings]

FIG. 1 is ¹ H-NMR data of cardol.

FIG. 2 is data of ¹³ C-NMR of cardol.

FIG. 3 is data of ultraviolet absorption spectrum of cardol.

FIG. 4 is data of mass spectral analysis of cardol.

FIG. 5 is data of mass spectral analysis of cardol.

FIG. 6 is data of mass spectral analysis of cardol.

FIG. 7: Cardol HMBC data.

Front page continued (72) Inventor Tomio Takeuchi 5-1-11 Higashigotanda, Shinagawa-ku, Tokyo (72) Inventor Takashi Oyano 1-3-1 Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Tonen Research Institute ( 72) Inventor Tomoko Hara 1-3-1 Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Prefecture Tonen Corporation Research Institute

Claims (2)

[Claims] 1. A structural formula: An antitumor agent containing a substance represented by as an active ingredient. 2. An anti-AIDS agent containing the substance according to claim 1 as an active ingredient.