JP2007223902A - New isochroman compound and utilization thereof for anti-cancer agent or the like - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To find out a new naturally occurring compound having a useful physiological activity, to find out, in particular a new compound having anti-cancer actions and to provide an anti-cancer agent, a medicine, a functional food, etc., utilizing the compound. <P>SOLUTION: The new isochroman compound pseudodeflectusin isolated and purified from a marine microorganism parasitizing on seaweeds has a chemical structure represented by formula (1) and exhibits selectively a high growth inhibitory activity for human carcinoma cell strains. The compound as a new anti-cancer substance is useful in a pharmaceutical, a functional food, etc. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel isochroman compound and use thereof. The novel compound of the present invention exhibits selective toxicity to human cancer cell types and can be used as a pharmaceutical such as an anticancer agent, and can also be used as a functional food effective for the prevention and treatment of cancer. Is.

  So far, many novel compounds having useful physiological activities have been separated and purified from terrestrial microorganisms, and these compounds have been used in pharmaceuticals, foods and drinks, cosmetics and the like. Thus, a compound obtained from nature has a relatively high safety to the human body and is likely to be applied to pharmaceuticals and the like. Furthermore, recently, research studies aimed at isolating and discovering useful new substances from metabolites of marine microorganisms have also become active (see, for example, Non-Patent Documents 1 and 2 below).

Pietra, F. Nat. Prod. Rep. 1997, 14, 453-464 Cuomo, V .; Palomba, I .; Perretti, A .; Guerriero, A .; D'Ambrosio, M .; Pietra, F. J. Mar. Biotechnol. 1995, 2, 199

  An object of the present invention is to find a novel compound that exists in nature and exhibits useful physiological activity. In particular, focusing on marine microorganisms that have not yet been fully explored, industrially useful novel compounds from marine microorganisms have been developed. To isolate and identify.

  In addition, we have discovered new compounds with anti-cancer activity, especially novel compounds with high anti-cancer activity selectively according to the type of cancer, and we have developed anti-cancer drugs, pharmaceuticals, functional foods, etc. Providing is also an object of the present invention.

  As a result of diligent research in view of the above problems, the inventor of the present invention is highly selective for a novel isochroman compound (ie, pseudodeflectusin described later) isolated from a marine microorganism (Aspergillus pseudodeflectus) against human cancer cell types. Completed the present invention by showing growth inhibition, LDH-cytotoxicity test, and the like that the compound shows cytotoxicity against cancer cells and has the effect of reducing intracellular glutathione level. I came to let you.

  That is, the present invention includes the following inventions A) to E) as industrially and medically useful inventions.

A) A compound represented by the following formula (1) (that is, pseudodeflectusin described below: 9-hydroxy-7-methyl-2- (methylethylidene) -furano [3,2-H] isochroman-3-one), or Its pharmacologically acceptable salt.

B) The anticancer agent which uses the compound represented by the said Formula (1), or its pharmacologically acceptable salt as an active ingredient.

C) A pharmaceutical composition comprising a compound represented by the above formula (1) or a pharmacologically acceptable salt thereof as an active ingredient.

D) Edible composition containing the compound represented by the above formula (1) or a pharmacologically acceptable salt thereof.

E) Aspergillus pseudodeflectus Hiji005 strain (FERM AP-20008) which produces the compound represented by the above formula (1).

As shown in the examples below, the novel isochroman compound of the above formula (1) has an anticancer activity. Therefore, the same substance as well as its pharmacologically acceptable salt is a pharmaceutical, It can be used for functional foods. In particular, since the isochroman compound according to the present invention showed a strong growth inhibitory activity against floating cancer cells, it is effective as an anticancer agent against cancer such as leukemia (lymphoma / hematologic malignancy). Can be expected.
In addition, the isochroman compound according to the present invention may have useful physiological activity in addition to anticancer activity, and can be expected to be used in various industries such as pharmaceuticals, foods, and cosmetics. is there.

Hereinafter, specific embodiments of the present invention will be described in detail.
The present inventor purified a compound from an extract of the marine microorganism Aspergillus pseudodeflectus Hiji005 strain (reception number FERM AP-20008) that parasitizes seaweed, and analyzed the structure by high-resolution electrospray ionization mass spectrometry ( HR-ESIMS), ¹ H-nuclear magnetic resonance (NMR) spectrum, ¹³ C-nuclear magnetic resonance spectrum, etc., and as a result, it was determined that the compound was a novel isochroman compound having the structure of the formula (1). The new substance obtained from pseudodeflectus was named “pseudodeflectusin”.

  The purification method of pseudodeflectusin will be briefly described. First, a fungus (Aspergillus sp.) Is isolated from a seaweed (Sargassum fusiforme) collected in Izu on a potato dextrose agar medium manufactured by Difco, and the resulting strain is extracted from a potato dextrose medium ( 24 g / 1 L), and cultured for 3 weeks in the dark and at rest. Then, 4 L of the culture solution was extracted with methylene chloride to obtain 442.3 mg of a crude extract. This crude extract was separated and purified by silica gel column chromatography with hexane: ethyl acetate = 4: 1 to 1: 4 to obtain 3.4 mg of fraction A and 284.2 mg of fraction B. Next, fraction B was subjected to silica gel column chromatography with chloroform: methanol = 19: 1, followed by silica gel column chromatography with chloroform: methanol = 99: 1 to 95: 5, and 1.2 mg of compound was obtained in fraction C. Separated and purified. From the results of structural analysis, this compound was determined to be a novel isochroman compound having the structure of formula (1) (ie, pseudodeflectusin).

  As described above, the novel isochroman compound pseudodeflectusin of the present invention can be separated and purified from naturally occurring microorganisms such as Aspergillus sp., But the production method of the compound of the present invention is not limited thereto. It may be synthesized, or may be produced by subjecting a substance obtained from a natural product as a starting material to a treatment or the like.

  When the above-mentioned pseudodeflectusin was examined for the growth inhibitory activity against human cancer cells by MTT assay, it showed a high growth inhibitory activity selectively according to the cancer cell type (see FIG. 1). Specifically, among the four types of human cancer cells used (NUGC-3 (gastric cancer cells), HeLa (uterine cancer cells), HL-60 (white blood cells), A549 (lung cancer cells)), HL-60 On the other hand, it showed the strongest growth inhibitory activity and also showed growth inhibitory activity against NUGC-3 and HeLa (50% inhibitory concentrations were 39 μM, 49 μM and 47 μM, respectively). On the other hand, no growth inhibitory activity was observed against A549, which is a squamous cell.

  Since the novel compound pseudodeflectusin of the present invention selectively inhibits the growth of human cancer cell types (cell lines), it can be expected to be an anticancer agent with few side effects. Furthermore, as an anticancer agent having a novel function, a synergistic effect of cancer treatment is expected when used in combination with an existing anticancer agent.

  When the cytotoxicity of the above pseudodeflectusin was examined by LDH-cytotoxicity test, the pseudodeflectusin showed strong cytotoxicity against HeLa cells which are cancer cells (see FIG. 2). Moreover, pseudodeflectusin had the effect | action which reduces the amount of intracellular glutathione with respect to a HeLa cell (refer FIG. 3). There has been a report that intracellular glutathione is related to resistance to apoptosis inducers and anticancer agents, and it is considered that the decrease in the amount of intracellular glutathione may lead to the expression of cytotoxicity.

  Thus, since the novel compound pseudodeflectusin of the present invention induces cell death, it is also useful as an apoptosis inducer. The novel compound of the present invention may have other useful physiological activities, and can be expected to be used in various industries such as pharmaceuticals, foods, and cosmetics. Of course, the novel compound of the present invention can also be used as a biochemical reagent.

  The present invention also includes pharmacologically acceptable salts of the above pseudodeflectusin. Examples of such pharmacologically acceptable salts include hydrohalides such as hydrofluoride and hydrochloride, sulfates and nitrates. Examples thereof include inorganic acid salts such as sodium salts, alkali metal salts such as sodium salts and potassium salts, sulfonic acid salts, and organic acid salts.

  When the compound of the present invention (pseudodeflectusin or a pharmacologically acceptable salt thereof) is used for a pharmaceutical, as one aspect thereof, the compound of the present invention may be used as a lead compound in a pharmaceutical development process.

  An example in which the compound of the present invention is used for a pharmaceutical product (pharmaceutical composition) will be described. The compound of the present invention can be administered to humans (or animals) as it is or as a pharmaceutical composition together with a conventional pharmaceutical preparation carrier. The dosage form of the pharmaceutical composition is not particularly limited and may be appropriately selected as necessary.For example, oral preparations such as tablets, capsules, granules, fine granules, powders, injections, Non-oral agents such as suppositories and coating agents can be mentioned.

  Oral preparations such as tablets, capsules, granules, fine granules, powders and the like are produced according to conventional methods using, for example, starch, lactose, sucrose, trehalose, mannitol, carboxymethylcellulose, corn starch, inorganic salts and the like. The compounding quantity of the compound of this invention in these formulations is not specifically limited, It can set suitably. In this type of preparation, binders, disintegrants, surfactants, lubricants, fluidity promoters, corrigents, colorants, fragrances and the like can be appropriately used.

  In the case of a parenteral preparation, the dose is adjusted according to the age, weight, disease severity, etc. of the patient, and for example, intravenous administration, intravenous infusion, subcutaneous injection, intramuscular injection or the like is used. This parenteral preparation is produced according to a conventional method, and distilled water for injection, physiological saline and the like can be generally used as a diluent. Furthermore, you may add a disinfectant, antiseptic | preservative, and a stabilizer as needed. In addition, from the viewpoint of stability, this parenteral preparation can be frozen after filling into a vial or the like, the water can be removed by ordinary freeze-drying treatment, and the liquid preparation can be re-prepared from the freeze-dried product immediately before use. Furthermore, you may add an isotonic agent, a stabilizer, an antiseptic | preservative, and a soothing agent as needed. The compounding quantity of the compound of this invention in these formulations is not specifically limited, It can set arbitrarily. Examples of other parenteral agents include liquid preparations for external use, coating agents such as ointments, suppositories for rectal administration, etc., and these are also produced according to conventional methods.

  For example, the compound of the present invention may be encapsulated in a carrier such as a liposome and administered in the body using a known DDS (drug delivery system). At this time, if a carrier that specifically recognizes cancer cells or the like at the target site is used, the compound of the present invention can be efficiently carried to the target site, which is effective.

  When the compound of the present invention is used in foods (edible compositions), the compound of the present invention is added to food and drink as raw materials for various beverages and various processed foods, or if necessary, shaping such as dextrin, lactose, starch, etc. It can be processed into pellets, tablets, granules, etc. together with agents, fragrances, pigments, etc., or coated with gelatin and molded into capsules for use as health foods, health foods, and the like.

  Examples of the present invention will be described below with reference to the drawings, but the present invention is not limited to these examples.

[Example 1: Isolation and purification of a novel isochroman compound from marine microorganisms parasitic on seaweed]
The novel isochroman compound of the present invention was extracted and purified from marine microorganisms parasitic on seaweed by the following procedure, and its structure was determined.

  First, the seaweed (Sargassum fusiforme) collected in the Izu Miura Peninsula was washed with sterilized water, cut into small pieces, immersed in 3.6% saline, cultured in Difco potato dextrose agar medium, and the fungus (Aspergillus pseudodeflectus Samon & Mouchacca) was isolated. The isolated strain was further cultured in a potato dextrose medium (24 g / 1 L) for 3 weeks in the dark and at rest. Thereafter, 4 L of the culture solution was filtered through a Buchner funnel to remove the cells, and then extracted with methylene chloride to obtain 442.3 mg of a crude extract from the organic layer.

  The crude extract was subjected to silica gel column chromatography three times (manufactured by Kanto Chemical Co., Inc., silica gel 60N spherical / neutral) to separate and purify the contained substances. Specifically, first, silica gel column chromatography was performed with hexane: ethyl acetate = 4: 1 to 1: 4 to obtain 3.4 mg of fraction A and 284.2 mg of fraction B. Next, silica gel column chromatography was performed on the fraction B with chloroform: methanol = 19: 1, and then silica gel column chromatography was performed with chloroform: methanol = 99: 1 to 95: 5. Solid compound 1 was obtained. As a result of the structural analysis described later, this compound 1 was determined to be a novel isochroman compound having the chemical structure of the formula (1), and the present inventor named this novel compound “pseudodeflectusin”.

On the other hand, as a result of structural analysis, the above fraction A contains compound 2 (7-methyl-2- (1-methylethylethlidene) -furo [3,2-H] isoquinoline-3 having the chemical structure of the following formula (2). -one) was identified.

  The structure of Compound 2 is as follows from Aspergillus urtus in the literature Kohno, J .; Hiramatsu, H .; Nishio, M .; Sakurai, M .; Okuda, T .; Komatsubara, S. Tetrahedron 1999, 55, 11247. It was the same as the structure of the substance reported as the released TMC-120B and was a known substance. In the examples described later, this compound 2 was used to compare the physiological activity with the compound 1.

[Example 2: Determination of structure of novel isochroman compound]
The optical rotation of Compound 1 obtained by the above method was measured to obtain the optical rotation [α] _D ^23.2 = + 11 ° (c = 0.18, MeOH).

In order to determine the structure of Compound 1, the compound was subjected to high-resolution electrospray ionization mass spectrometry (HR-ESIMS). The result of m / z 283.0950 (M + H ⁺ ), theoretical value 283.0940 showed that Compound 1 has the molecular formula C ₁₅ H ₁₆ O ₄ .

For compound 1, ¹ H-NMR and ¹³ C-NMR were measured using CDCl ₃ as a solvent and TMS as an internal standard, and the data listed in Table 1 below were obtained.

  Further, when the IR spectrum of Compound 1 was measured, the following values were obtained. IR (film): 3338.18, 3019.98, 1690.30, 1644.98, 1607.38, 1436.71, 1289.18, 1119.48, 1079.94, 1022.09, 855.28, 669.18.

  As a result of structural analysis by HMBC spectrum analysis and COSY spectrum analysis based on the data thus obtained, it was determined that Compound 1 has the following structure (in addition, each numerical value in the formula is shown in Table 1). Corresponding to the number of carbon atoms).

[Example 3: Human-derived cancer cell growth inhibitory effect of Compound 1 and Compound 2]
It was examined whether Compound 1 and Compound 2 had a human cancer cell growth inhibitory effect. In the experiment, four types of cancer cells, NUGC-3 cell, a human gastric cancer cell line, HeLa cell, a human uterine cancer cell line, HL-60 cell, a human leukocyte cell line, and A549 cell, a human lung cancer cell line, were used. In use, various concentrations of Compound 1 or Compound 2 were added and incubated, and after 48 hours, the viability of each cancer cell was determined by MTT assay.

  FIG. 1 is a graph showing the experimental results. In each graph, black circles indicate the results of Compound 1, and white circles indicate the results of Compound 2. (A)-(d) is the result of human origin cancer cell lines NUGC-3, HeLa, HL-60, and A549, respectively. Compound 1 exhibited a cell growth inhibitory effect in a concentration-dependent manner on three types of human cancer cell lines, NUGC-3, HeLa, and HL-60. Among them, HL-60 was most strongly inhibited at a 50% inhibitory concentration of 39 μM. On the other hand, A549 cells are squamous epithelial cells and did not show growth inhibition. Compound 1 is believed to have selectivity for human-derived cancer cell types.

  Compound 2 did not show growth inhibition against all human cancer cell lines.

[Example 4: Measurement of free lactate dehydrogenase (LDH) activity]
After compound 1 was added to the medium and HeLa cells were cultured for 48 hours, the LDH activity in the culture supernatant and the total LDH amount of the remaining cells were measured. Thereby, both cell death in culture and cell growth inhibition can be measured. The LDH activity was measured using an LDH-cytotoxicity test Wako manufactured by Wako Pure Chemical Industries, Ltd. The LDH activity liberated in the culture supernatant of cells cultured in the presence and absence (control) of Compound 1 was shown as a percentage of the total LDH amount, with the total LDH amount being 100% (see FIG. 2).

  As shown in the figure, free LDH activity increased by culturing in the presence of Compound 1, indicating an approximately 4-fold increase compared to the control group. From the above results, it was revealed that Compound 1 exhibits cytotoxicity against cancer cells.

[Example 5: Measurement of total intracellular glutathione (GSH) amount]
Intracellular glutathione was measured by enzymatic cycling. After compound 1 was added to the medium and HeLa cells were cultured for 48 hours, the cells were collected and a crude cell extract was prepared using a 5% TCA solution. This crude cell extract was neutralized with 0.5N NaOH, and the reaction solution (dissolved in 0.2 mM DTNB, 0.3 mM NADPH, 1.5 mU glutathione reductase, 0.1 M phosphate buffer (pH 7.5)) The reaction was started by adding and the absorbance change rate at 405 nm was measured. The amount of glutathione was determined by calculating from a calibration curve.

  The experimental results are shown in FIG. As shown in the figure, assuming that the total intracellular GSH amount in the control (compound 1 non-addition) group was 100%, the cells to which compound 1 was added decreased to about 70%. There are reports that intracellular glutathione is associated with resistance to apoptosis-inducing agents and anticancer agents, suggesting that a decrease in intracellular glutathione level leads to the development of cytotoxicity.

  From the above results, it is considered that treatment of cells with compound 1 decreased the amount of intracellular glutathione, induced cell death, and increased LDH activity.

  As described above, the present invention relates to a novel isochroman compound. As described above, the present invention can be used as an anticancer agent, and can be widely used industrially as a pharmaceutical, food, biochemical reagent and the like. is there.

(A)-(d) is a graph which shows the result of having investigated the growth inhibitory effect of the novel isochroman compound of this invention with respect to human origin cancer cell lines NUGC-3, HeLa, HL-60, and A549, respectively. Each data is the average of 4 experiments performed independently. It is a graph which shows the result of having examined the cytotoxicity of the novel isochroman compound of this invention by LDH-cytotoxicity test. Data are the average of 4 experiments performed independently. It is a graph which shows the result of having examined the presence or absence of the reduction | decrease in the amount of intracellular glutathione by the novel isochroman compound of this invention. Data are the average of 4 experiments performed independently.

Claims (5)

A compound represented by the following formula (1) or a pharmacologically acceptable salt thereof.

  The anticancer agent which uses the compound represented by Formula (1) of Claim 1, or its pharmacologically acceptable salt as an active ingredient.   A pharmaceutical composition comprising a compound represented by formula (1) according to claim 1 or a pharmacologically acceptable salt thereof as an active ingredient.   An edible composition comprising a compound represented by the formula (1) according to claim 1 or a pharmacologically acceptable salt thereof. Aspergillus pseudodeflectus Hiji005 strain (FERM AP-20008).

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