JP4810642B2 - Triterpene derivative for inhibiting cancer metastasis and composition for inhibiting cancer metastasis using the triterpene derivative - Google Patents

Description

  The present invention relates to a use of a triterpene derivative, and more specifically, a cancer-metastasis of a cancer-metastasis-suppressing triterpene derivative having a cancer metastasis-suppressing ability that is expressed based on a specific function, and a pharmaceutical or health functional food using the compound. The present invention relates to a composition for suppression.

  The process of cancer cells moving to tissues or organs far away from the primary lesion is as follows: (i) cancer cells leave the primary lesion, (ii) break the basement membrane and infiltrate surrounding tissues, (iii) blood vessels and lymph Follow the flow of entering a tube and being transported to a distant tissue by the flow of blood or lymph, or (iv) re-entering and proliferating into a tissue from a blood vessel or lymphatic vessel to form a new metastasis (for example, non-patent literature) 1). As long as the cancer stays in the primary lesion, it is easy to handle by surgery or the like, but when metastasis occurs, the prognosis is generally poor, and cancer that is prone to metastasis is considered malignant.

  Malignant melanoma is often cured by surgical excision in the very early stages, but radiation therapy and chemotherapy are used in combination with the progression of the disease state. Malignant melanoma is prone to metastasis, and it is said that the effects of these treatments are low among cancers. As anticancer agents, there are dacarbazine and the like, and immunotherapy is also expected and attempted, but in reality the treatment has not yet been established.

Thus, cancer metastasis is one of the fields that have not been elucidated most, and no cancer metastasis inhibitor that directly acts on a part of the metastasis process and has an effect has not been put into practical use.
"α-Melanocyte-stimulating hormone blocks invasion of reconstituted basement membrane (Matrigel) by murine B16 melanoma cells"; Jun Murata, Koichi Ayukawa, Masaru Ogasawara, Hideaki Fujii, Ikuo Saiki; Invasion Metastasis (1997), 17, pp.82- 93.

  The present invention has been developed in view of the above circumstances, and its purpose is cancer having an excellent inhibitory effect on metastasis of tumor cells such as malignant melanoma, neuroblastoma, glioblastoma and astrocytoma An object of the present invention is to provide a metastasis inhibitor and a composition containing the same.

  As a result of diligent studies to solve the above problems, the present inventors have suppressed the motility of cancer cells when the depolymerization of actin polymers in the cancer cells is promoted, and thereby have a very high cancer metastasis suppressing effect. At the same time, the present inventors have found that a specific triterpene derivative has an ability to suppress cancer metastasis based on such a function, and completed the present invention.

That is, the present invention relates to a triterpene derivative for inhibiting cancer metastasis represented by formula (I) or (II), or a pharmaceutically acceptable salt thereof.

In the formula, R represents a hydroxyl group or —O—CO—R ′, and R ′ represents an alkyl group having 1 to 23 carbon atoms or an aromatic ring-containing group having 6 to 23 carbon atoms.

  In the present invention, the triterpene derivative for suppressing cancer metastasis has a function of suppressing cancer cell motility, and the function of suppressing cancer cell motility is a depolymerization of actin polymer in cancer cells. It is characterized by suppressing the motility of cancer cells by promoting.

  In one embodiment of the present invention, the cancer cell is at least one selected from malignant melanoma, neuroblastoma, glioblastoma and astrocytoma.

  The present invention also relates to a cancer metastasis inhibitor containing the above-mentioned triterpene derivative for inhibiting cancer metastasis or a pharmaceutically acceptable salt thereof as an active ingredient.

  The present invention also relates to a health functional food containing the triterpene derivative for suppressing cancer metastasis or a pharmaceutically acceptable salt thereof as an active ingredient.

  INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a cancer metastasis-suppressing drug and a health food that are particularly effective for suppressing metastasis of malignant melanoma, neuroblastoma, glioblastoma, astrocytoma and the like.

Hereinafter, the present invention will be described in detail.
The triterpene derivative for suppressing cancer metastasis according to the present invention is represented by the following formula (I) or (II).

In the formula, R represents a hydroxyl group or —O—CO—R ′, and R ′ represents an alkyl group having 1 to 23 carbon atoms or an aromatic ring-containing group having 6 to 23 carbon atoms. Specific examples of R ′ include phenyl (C ₆ H ₅ —), tolyl (CH ₃ C ₆ H ₅ —), benzyl (C ₆ H ₅ CH ₂ —), phenethyl (C ₆ H ₅ CH ₂ CH ₂ —). ), Styryl (C ₆ H ₅ CH═CH—), naphthyl (C ₁₀ H ₇ —) and the like.

  As described above, the cancer metastasis-suppressing action of the triterpene derivative for suppressing cancer metastasis according to the present invention and a pharmaceutically acceptable salt thereof (hereinafter referred to as “the triterpene derivative of the present invention”) is actin in cancer cells. It is expressed based on the suppression of cancer cell motility by promoting the depolymerization of the polymer, and the cancer cell motility suppression effect according to the present invention is shown in malignant melanoma, neuroblastoma, nerve Particularly effective against cancer cells such as glioblastoma and astrocytoma.

  The triterpene derivative of the present invention is used as it is, or together with a conventional carrier or the like, as a medicine for medical treatment for suppressing cancer metastasis (hereinafter referred to as “cancer metastasis inhibitor”), or in the food field, a health functional food Etc.

  First, the cancer metastasis inhibitor of the present invention relating to pharmaceutical use will be described. The cancer metastasis inhibitor of the present invention can be administered, for example, orally or parenterally, and can be administered by injection, transdermal administration, rectal administration, intraocular administration, etc. Depending on the case, it can be formulated in combination with an appropriate pharmaceutically acceptable excipient or diluent. The dosage form suitable for oral administration is not particularly limited, but includes those in a solid, semi-solid, liquid, etc. Specifically, tablets, capsules, powders, granules, Examples include solutions, suspensions, and syrups. The dose of the cancer metastasis inhibitor of the present invention can be appropriately set and adjusted according to the administration form, administration route, the degree and stage of the target epidemic, etc. About 0.0001 to 5000 mg, preferably 0.001 to 500 mg, more preferably 0.01 to 100 mg, and 0.01 mg, 0.2 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg as a single dose, Although 250 mg, 500 mg, etc. are set, it is not limited to these.

  As a means for formulating the cancer metastasis inhibitor of the present invention into tablets, capsules, powders, granules, solutions, suspensions, etc., known formulation methods can be used, and the triterpene derivatives of the present invention Can be mixed with excipients, lubricants and the like, and further mixed with diluents, buffers, wetting agents, preservatives and the like as necessary. Specific examples of these additive components include starch, sugars such as lactose, cellulose derivatives such as hydroxypropylcellulose, gelatin, talc, magnesium stearate and the like.

  As the administration by injection, it can be administered subcutaneously, intradermally, intravenously, intramuscularly, and the like, and a generally used method can be used as a method for formulating an injectable preparation used in such an administration method. Specifically, the triterpene derivative of the present invention is dissolved or suspended or emulsified in an aqueous or non-aqueous solvent such as vegetable oil, synthetic fatty acid glyceride, higher fatty acid ester, propylene glycol, and further if necessary. Can be formulated with conventionally used additives such as solubilizers, osmotic pressure regulators, emulsifiers, stabilizers and preservatives.

  As transdermal administration, it can be administered as an ointment, emulsifier, pasta agent, haptic agent, liniment agent, lotion agent, suspension agent, etc. depending on the state of the target skin, etc., and is used in such an administration method. As a method for formulating a preparation for transdermal administration, a commonly used method can be used. For example, the ointment can be formulated by kneading the triterpene derivative of the present invention with a hydrophobic substrate such as petrolatum or paraffin or a hydrophilic substrate such as hydrophilic petrolatum or polyethylene glycol by a known method.

  For rectal administration, for example, it can be administered as a suppository, and a commonly used method can be used as a suppository formulation method. Specifically, the triterpene derivative of the present invention can be formulated by mixing with an excipient such as cocoa butter, carbon wax, polyethylene glycol, etc., which melts at body temperature but solidifies at room temperature, and is molded. it can.

Next, the composition for suppressing cancer metastasis of the present invention used as a health functional food will be described.
The triterpene derivative of the present invention is also useful as a use for so-called health functional foods, and can be widely applied as beverages such as confectionery and soft drinks, processed vegetables or fruits, livestock meat products, seasonings, and the like. The form is not particularly limited, and may be, for example, powder, solid, solution, or the like. When the triterpene derivative of the present invention is blended in the foods as described above, the triterpene derivative of the present invention is blended as a purified chemical substance, and plants such as Asteraceae containing the triterpene derivative of the present invention Or a processed product thereof, an extract thereof with an appropriate solvent, or a product obtained by fractionating the extract by an appropriate method. The blending amount of the triterpene derivative of the present invention in such health functional food can be appropriately set according to the purpose, product form and the like. In general, in the case of a solution or suspension of a drink or the like, for example, in 30 ml, the active ingredient is 0.001 to 100 mg, preferably 0.01 to 10 mg, more preferably 0.1 to 5 mg. It is. Moreover, in the case of powder solid products, such as a tablet, it is 0.001-50 mg as an active ingredient in 300 mg, for example, Preferably it is 0.01-10 mg, More preferably, it is 0.1-2 mg.

  When the triterpene derivative of the present invention is used as a health functional food, various additions such as sweeteners, acidulants, preservatives, fragrances, colorants, excipients, stabilizers, wetting agents, absorption enhancers, pH adjusters, etc. Ingredients can be blended. Specific examples of these additive components include various food extract extracts such as mushroom extract and carrot extract, various food extract extracts such as mushroom extract and carrot extract, honey, cyclic oligosaccharides, reduced maltose, and trehalose. , Lactose, sucrose fatty acid ester and the like.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.

[Preparation of triterpene derivatives]
Preparation examples of triterpene derivatives 1 to 3 according to the present invention are shown below.

(Preparation Example 1) Preparation of Triterpene Derivative 1 (Lupeol) Triterpene derivative 1 (Lupeol; a compound in which R is a hydroxyl group in formula (I)) according to the present invention was extracted and isolated by the following means.
After crushing 350 g of dandelion root crude drug (Tatsumi Koei, Uchida Wakayaku), extraction was performed using 5 L of ethanol, and this was filtered. After this extraction operation was further repeated 4 times, the obtained filtrate was dried under reduced pressure to obtain a crude extract (24.6 g).

The obtained crude extract was subjected to a liquid separation operation with chloroform-methanol-distilled water (4: 4: 3, 1 L), and separated into a chloroform layer and an aqueous layer. The fraction of the chloroform layer in which the mouse melanoma cell B16-derived α-MSH highly sensitive strain B16 2F2 differentiation-inducing activity selected and established by the present inventors was detected was dried under reduced pressure (7.5 g). Furthermore, the obtained extract was subjected to silica gel chromatography using a solvent of n-hexane-chloroform (1: 1), and n-hexane-chloroform (1: 1), (1: 3), chloroform- After sequentially eluting with methanol (3: 1) and (1: 1), the activity was examined. Since the activity was detected mainly in the fraction eluted with n-hexane-chloroform (1: 3) (1.2 g), this fraction was further purified with Cosmosil using chloroform-methanol (1: 9). Purification by reverse phase silica gel column chromatography by 75C ₁₈ (Nacalai Tesque, Inc.) gave colorless needle-shaped lupeol (89.6 mg).

  The physical property values and various spectrum results of the obtained lupeol are shown below.

Melting point 213-214 ° C; [α] ²⁵ _D + 26.6 ° (c 1.00, chloroform); IR (KBr) ν _max 3600-3100, 3000-2800, 1639, 1454, 1379, 1295, 1187, 1041, 883 cm ^-1 ; ¹ H-NMR (CDCl ₃ ) δ 4.69, 4.57 (each 1H, br. S, H2-29), 3.20 (1H, dd, J = 10.8, 4.4 Hz, 3α-H), 2.38 (1H, m, H2-2), 1.69 (3H, s, H3-30), 1.04 (3H, s), 0.98 (3H, s), 0.96 (3H, s), 0.85 (3H, s), 0.80 (3H, s), 0.77 (3H, s); ¹³ C-NMR (CDCl ₃ ) δ 150.9 (s, C-20), 109.3 (t, C-29), 79.0 (d, C-3), 55.3 (d, C-5), 50.5 (d, C-9), 48.3 (d, C-19), 48.0 (d, C-18), 43.0 (s, C-14), 42.9 (s, C-17) 40.8 (s, C-8), 40.0 (t, C-16), 38.9 (s, C-4), 55.3 (d, C-5), 38.8 (t, C-1), 38.1 (d, C- 13), 37.2 (s, C-10), 36.6 (t, C-22), 34.3 (t, C-7), 29.9 (t, C-15), 28.0 (q, C-23), 27.5 ( t, C-2), 27.4 (t, C-21), 25.2 (t, C-12), 21.0 (t, C-11), 19.3 (q, C-30), 18.3 (t, C-6 ), 18.0 (q, C-28), 16.1 (q, C-25), 16.0 (q, C-26), 15.4 (q, C-24), 14.6 (q, C-27); EIMS m / z 426 (4), 370 (2), 316 (10), 257 (8), 207 (58), 189 (50), 135 (45), 109 (100); HREIMS m / z 426.3866 (C ₃₀ H ₅₀ O ₁ calcd: 426.3861).

The chemical structure of lupeol is considered to be a triterpene compound from the results of HREIMS, and is a double tablet derived from 3α protons (J = 10.8, 4.4, corresponding to the root of a typical hydroxyl group at δ 3.20 ppm, characteristic of ¹ H-NMR. Hz) proton appears to suggest the presence of β-hydroxyl group at the 3-position, and from the results of multiplicity measurement by ¹³ C-NMR, lupeol [= lup-20 (29 ) -en-3β-ol]. Therefore, loupeol as a reagent was directly compared with the mixing test and IR, ¹ H-NMR and ¹³ C-NMR spectra to confirm the chemical structure.

(Preparation Example 2) Preparation of Triterpene Derivative 2 (Lupenyl Palmitate) Triterpene Derivative 2 (Lupenyl Palmitate) According to the Present Invention; In Formula (I), R is —O—CO— (CH ₂ ) ₁₄ CH ₃ ( Palmitic acid ester) in which R ′ is a C15 alkyl group) was synthesized and purified by the following means.

  Lupeol (30 mg) and palmitic acid (40 mg) were dissolved in 3 mL of dry tetrahydrofuran at room temperature (25 ° C.), 0.5 mL of concentrated sulfuric acid was added, and this was allowed to stand for 1 hour. The obtained reaction solution was poured into saturated brine under ice cooling, and extraction was repeated twice using chloroform. The obtained chloroform solution was washed successively with aqueous sodium hydrogen carbonate and saturated brine, and then dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure to obtain luphenyl palmitate (colorless columnar crystal substance, 65 mg).

In the formula (I), an ester derivative (lupenyl acetate) in which R is —O—CO—CH ₃ (R ′ is a methyl group) is obtained by acetylating lupeol with acetic anhydride-pyridine. Other ester derivatives according to the present invention (in the formula (I), R is —O—COR ′) are commercially available free acids of alkyl acids, aryl acids, and benzyl acids having 2 to 23 carbon atoms. A compound or a methyl ester compound and lupeol can be obtained by ester-bonding by the aforementioned concentrated sulfuric acid treatment.

  The physical data of rupenyl palmitate is shown below.

Melting point 94-96 ° C; [α] ²⁵ _D + 52.7 ° (c 0.30, chloroform); IR (KBr) ν _max 3000-2800, 1728, 1640, 1465, 1380, 1320, 1255, 1179, 1147, 1116, 1038 , 977, 879 cm ^-1 ; ¹ H-NMR (CDCl ₃ ) δ 4.69, 4.57 (each 1H, br s, H ₂ -29), 4.49 (1H, dd, J = 10.8, 5.6 Hz, 3 (H) , 2.38 (1H, m, H ₂ -2), 2.29 (2H, t, J = 7.2 Hz, H ₂ -2 '), 1.69 (3H, s, H ₃ -30), 1.30-1.20 (palmitic acid moiety Alkyl chain proton), 1.05 (3H, s), 0.96 (3H, s), 0.87 (3H, t, J = 8.4 Hz, H ₃ -16 '), 0.85 (6H, s), 0.80 (3H, s) ; ¹³ C-NMR (CDCl ₃ ) δ 179.6 (s, C-1 '), 150.8 (s, C-20), 109.4 (t, C-29), 80.6 (d, C-3), 55.4 (d , C-5), 50.4 (d, C-9), 48.3 (d, C-19), 48.0 (d, C-18), 43.0 (s, C-14), 42.9 (s, C-17) , 40.9 (s, C-8), 40.0 (t, C-16), 38.4 (t, C-1), 38.0 (d, C-13), 37.1 (s, C-10), 35.6 (t, C-22), 34.8 (t, C-2 '), 34.2 (t, C-7), 31.9 (t, C-15'), 29.9 (t, C-15), 29.7-29.6 (12C, each t, C-3 'to C-14'), 28.0 (q, C-23), 27.5 (t, C-21), 25.2 (2C, each t, C-2, C-12), 21.0 (t , C-11), 19.3 (q, C-30), 18.2 (t, C-6), 18.0 (q, C-2 8), 16.6 (q, C-16 '), 16.2 (q, C-25), 16.0 (q, C-26), 14.5 (C-27), 14.0 (C-24); EIMS m / z 664 (25), 649 (13), 408 (40), 368 (40), 256 (55), 236 (60), 189 (70), 149 (85), 123 (90), 109 (92), 95 (100); HREIMS m / z 664.6125 (C 46 H 80 O 2, calcd: 664.6158).

(Preparation Example 3) Preparation of Triterpene Derivative 3 (Lupeon) Triterpene derivative 3 (Lupeon; compound represented by formula (II)) according to the present invention was synthesized and purified by the following means.
To a solution of lupeol (100 mg) dissolved in anhydrous dichloromethane (2 mL) was added pyridinium chlorochromate (50 mg) and stirred at room temperature for 2 hours. The reaction product was dissolved in diethyl ether (5 mL), passed through a Florisil column and eluted with 10 volumes of diethyl ether. The solvent was removed from the obtained eluate by concentration under reduced pressure to obtain lupeon (colorless needle-like crystal substance, 60 mg). The chemical structure was confirmed by directly comparing Lupeon as a reagent with a mixing test and IR and ¹ H-NMR and ¹³ C-NMR spectra.

(Example 1)
B16 2F2 was cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum. The Boyden chamber was a 24-well culture plate using a mold with an 8 μm pore size polycarbonate filter insert as an upper chamber (manufactured by Corning; trade name COSTARTRANSWELL). B16 2F2 cells were prepared to 1 × 10 ⁶ cells / mL in Dulbecco's modified Eagle medium supplemented with 0.1% bovine serum albumin, and the lupeol produced in Preparation Example 1 was added to a final concentration of 1 to 20 μM. A cell suspension of 5 × 10 ⁵ cells / mL was prepared. The lower chamber well was preliminarily filled with 600 μL of Dulbecco's modified Eagle's medium supplemented with 0.1% bovine serum albumin containing lupeol at the same concentration as the upper chamber, and then the upper chamber was set. 100 μL of B16 2F2 cells 5 × 10 ⁵ cells / mL were placed in the upper chamber, and incubated at 37 ° C. in a 5% CO ₂ atmosphere for 6 hours. Thereafter, the filter was taken out, and B16 2F2 cells that had migrated to the lower surface of the filter were fixed and then stained with a hematoxylin staining solution, and the number of migrated cells was calculated under a microscope.

  The results are shown in FIG. In FIG. 1, Migration Index (%) represents the percentage when the number of migratory cells in the additive-free group is 100. In the following, the ability of the triterpene derivative of the present invention to inhibit B16 2F2 cell migration is It is evaluated by this Migration Index (%). Moreover, in FIG. 1, * mark shows that there is a significant difference with a risk factor of less than 0.01 compared to the non-added section.

  1 that the triterpene derivative of the present invention has a function of suppressing the motility of cancer cells.

(Example 2)
For Example 1, the experiment was conducted in the same manner as in Example 1 except that as the triterpene derivative of the present invention, lupeon and lupenyl acetate produced according to the above preparation example were used instead of lupeol. As a result, the migration index at a concentration of 10 μM of lupeon and lupenyl acetate was 29.2% and 39.7%, respectively, and it was revealed that in this case also, the B16 2F2 cell migration inhibitory ability was excellent.

(Example 3)
Using 10 μM lupeol, human-derived melanoma, neuroblastoma, glioblastoma, astrocytoma, lung adenocarcinoma, renal adenocarcinoma, fibrosarcoma, osteosarcoma, gastric cancer, cervical cancer, pancreatic cancer, bladder cancer The ability to inhibit the migration ability was evaluated in the same manner as in Example 1. The data is shown by the average value of 4 holes. As a result, among the cancer cells, melanoma (Migration Index = 40.8%), neuroblastoma (Migration Index = 39.7%), glioblastoma (Migration Index = 39.8%), and astrocytoma (Migration Index) = 43.3%), it was revealed that it exhibited particularly excellent migration inhibition ability.

Example 4
B16 2F2 cells were prepared to 1 × 10 ⁶ cells / mL (1 mL) in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, and lupeol was added to a final concentration of 10 μM. Incubated for 12 hours at 37 ° C., 5% CO ₂ atmosphere. After fixing B16 2F2 cells, the cells were stained with Alexa-Fluora 488-Phalloidin solution, and intracellular actin polymer (= stress fiber) was observed under a fluorescence microscope. The results are shown in FIG.

  In FIG. 2, actin polymerization (white arrow in the figure) is observed in untreated B16 2F2 cells, whereas no actin polymer is observed in lupeol-treated B16 2F2 cells, and actin polymer depolymerization is observed. It was. From this result, it is considered that the function of suppressing the motility of B16 2F2 cells by lupeol is based on the promotion of the depolymerization of the actin polymer.

(Example 5)
The cancer metastasis inhibitor for injection according to the present invention can be prepared, for example, by the following means.

Ingredient Compounding amount Triterpene derivative of the present invention 10 mg
Polyoxyethylene hydrogenated castor oil 60 400 mg
Amount required to make 1 mL of ethanol The triterpene derivative of the present invention and polyoxyethylene hydrogenated castor oil 60 are dissolved in ethanol by a conventional method to prepare a solution having the above component composition. The resulting solution can be diluted with an appropriate amount of saline and administered by intravenous infusion.

( Reference example )
The health functional food (tablet) containing the triterpene derivative of the present invention can be produced, for example, in the following component composition.

Triterpene derivative of the present invention 0.5 mg
Reduced maltose 87.8 mg
Trehalose 41.2 mg
Lactose 16.5 mg
Sucrose fatty acid ester 6.2 mg
Dextrin 147.8 mg

The graph which shows the evaluation result of the B16 2F2 cell migration inhibitory ability by lupeol. A photomicrograph showing the depolymerization of actin polymer by lupeol.

Claims (4)

A cancer metastasis inhibitor comprising a triterpene derivative represented by formula (I) or (II) or a pharmaceutically acceptable salt thereof as an active ingredient.
In the formula, R represents a hydroxyl group.
  The cancer metastasis inhibitor according to claim 1, wherein the triterpene derivative has a function of suppressing motility of cancer cells.   The cancer metastasis inhibitor according to claim 2, wherein the cancer cell motility suppressing function suppresses the motility of cancer cells by promoting depolymerization of actin polymers in the cancer cells.   The cancer metastasis inhibitor according to claim 2 or 3, wherein the cancer cell is at least one selected from malignant melanoma, neuroblastoma, glioblastoma and astrocytoma.