JP5201603B2 - Ryokyo-derived malignant tumor therapeutic agent - Google Patents

Description

  The present invention relates to a malignant tumor therapeutic agent, and more particularly to a malignant tumor therapeutic agent containing a plant-derived component as an active ingredient.

  Plants have attracted attention as a pharmaceutical resource since ancient times, and various pharmaceuticals or pharmaceutical materials have been found in plants. Plant-derived malignant tumor therapeutic agents include paclitaxel (Taxol), which is an extract from a yew family plant; camptothecin, an alkaloid contained in the black-headed crabs, cyperaceae; Some vincristine, vinblastine, etc. are known.

  It has been known for a long time that Ryokyo, which is a herbal medicine derived from the rhizome of Ginger family, has an antiemetic action. In addition, Ryokyo contains 1,8-cineole, methyl cinnamate, eugenol, pinene, and kadinene as essential oil components, and is known to contain galangol as a pungent component. .

On the other hand, curcumin is a pigment component having a yellow color contained in turmeric, and is known to have antitumor activity in addition to actions such as improvement of liver function (Non-patent Document 1).
Anticancer Res. , 2004 Mar-Apri; 24 (2B): 987-998.

However, it is not known that malignant tumor activity is present in the extracted components of Ryokyo. Moreover, the growth inhibitory activity (EC ₅₀ ) of curcumin on neuroblastoma cells was about 10 μM, and it could not be used as a therapeutic agent for malignant tumors.

  An object of the present invention is to search for novel malignant tumor therapeutic agents from plants.

  When the present inventor screened Ryokyo extract using antitumor activity as an index, a compound having a structure similar to curcumin has significantly superior antitumor activity compared to curcumin, and treatment for malignant tumors The present invention was completed by finding it useful as a medicine.

  That is, the present invention relates to the general formula (1)

(Wherein R ¹ represents a hydrogen atom, a hydroxy group or a methoxy group; R ² represents a hydrogen atom or a hydroxy group; R ³ represents a hydrogen atom or a hydroxy group; R ⁴ represents a hydrogen atom or a methoxy group; The dashed line indicates that the moiety may be a double bond; R ⁵ is a hydrogen atom or formula (a);

(In the formula, an arrow indicates a binding site, and R ¹ , R ² , R ³ , R ⁴ and a broken line are the same as described above.)
Represents a group represented by
The present invention provides a therapeutic agent for malignant tumors comprising a compound represented by the formula:
Moreover, this invention provides use of the compound represented by the said General formula (1) for manufacture of a malignant tumor therapeutic agent.
The present invention also provides a compound represented by the above general formula (1) for use in the treatment of malignant tumors.
Furthermore, the present invention provides a method for treating a malignant tumor, which comprises administering an effective amount of the compound represented by the general formula (1).

  In addition, among the above general formula (1), some compounds are novel compounds. Accordingly, the present invention provides the following formulas (1d) to (1f)

The compound represented by these is provided.

  The therapeutic agent for malignant tumor of the present invention has excellent antitumor activity, and its action is due to its ability to induce apoptosis on cancer cells. Therefore, it is useful as a new malignant tumor therapeutic agent.

The extraction figure of this invention compound from Ryoko is shown. It is a figure which shows the cytotoxic activity of the compound (1a) with respect to IMR-32 which is a neuroblastoma cell (In the figure, Norm is the neuroblastoma cell which is not made to act at all, Veh shows the control which added only the solvent. same as below.). It is a figure which shows the cytotoxic activity of the compound (1b) with respect to IMR-32 which is a neuroblastoma cell. It is a figure which shows the cytotoxic activity of the compound (1c) with respect to IMR-32 which is a neuroblastoma cell. It is a figure which shows the cytotoxic activity of compound (1a) and (1b). It is a figure which shows the phase-contrast image and fluorescence image of IMR-32 cell which made the compound (1b) act.

The active ingredient of the therapeutic agent for malignant tumor of the present invention is represented by the general formula (1). In formula (1), R ¹ is preferably a methoxy group or a hydrogen atom. R ² is preferably a hydrogen atom. R ³ is preferably a hydroxy group, and R ⁴ is preferably a methoxy group. R ⁵ is preferably a hydrogen atom.

  Of the compounds represented by the general formula (1), the compounds represented by the following formulas (1a) to (1f) are preferable, the compounds represented by the formulas (1a) to (1c) are more preferable, and particularly the formula The compound represented by (1a) or (1b) is preferable.

  The compound represented by the general formula (1) can be obtained by extraction from, for example, Ryokyo. For example, examples of the extraction solvent include lower alcohols such as methanol, ethanol, n-propanol, isopropanol, and aqueous media and ethers thereof. These may be used alone or in combination. Preferred is methanol, ethanol or ether. Ethyl acetate and acetone are also preferable extraction solvents. The compound according to the present invention can be obtained by separation and purification from the extraction solvent.

  As will be apparent from Examples described later, the compound of the general formula (1) has an excellent cytotoxic activity against neuroblastoma cells. Moreover, the cytotoxic activity is based on apoptosis-inducing activity. Therefore, the compound of the general formula (1) or the Ryokyo extract containing these compounds is useful as a therapeutic agent for malignant tumors in mammals including humans.

  Malignant tumors that are targets of the malignant tumor therapeutic agent of the present invention include blood and hematopoietic tissue tumors such as leukemia and lymphoma, and solid tumors. Examples of solid tumors include neuroblastoma, epithelial cell cancer such as skin cancer, lung cancer, colon cancer, stomach cancer, breast cancer, prostate cancer, thyroid cancer; and sarcomas such as leiomyosarcoma and osteosarcoma.

  The medicament of the present invention comprises the compound of formula (1) or the above extract with an excipient, binder, lubricant, disintegrant, coating agent, emulsifier, suspending agent, solvent, stabilizer, absorption aid. It is obtained by adding one or more pharmaceutically acceptable carriers as appropriate, such as ointments and ointment bases, and formulating them into dosage forms for oral administration, injection administration, rectal administration, external use, etc. by conventional methods. It is done.

  Preparations for oral administration include granules, tablets, dragees, capsules, pills, liquids, emulsions, suspensions, etc .; preparations for injection administration include intravenous injection, intramuscular injection, subcutaneous injection, infusion Preparations for injection and the like; As preparations for rectal administration, suppository soft capsules and the like are preferable.

  The medicament of the present invention can be administered to mammals including humans as a preparation as described above.

  The medicament of the present invention is preferably administered as a compound of formula (1) at about 1 to 500 mg / kg once to 4 times per day.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples at all.

Example 1
Hereinafter, an example of a method for separating and purifying six kinds of diallylheptanoid compounds from Ryokan will be described with reference to FIG.

(1) The methanol extract of Ryokyo was first subjected to solvent fractionation with ethyl acetate-water (1: 1) to obtain an ethyl acetate fraction (collected amount: 25.7 g) and an aqueous layer.
Next, for the ethyl acetate fraction, Sephadex LH-20-column chromatography (hereinafter, column chromatography is abbreviated as “CC”) [Sephadex LH-20 usage: 120 g, eluent: chloroform-methanol (1: 1 )] And fractionated into 7 fractions (fractions 1-7).
Among the above fractions, the fraction (hereinafter abbreviated as “Fr.”) 4 (recovered amount: 1.5 g) was separated into 10 fractions by silica gel-CC (silica gel use amount: 300 g, elution solvent: chloroform-methanol). Fractions were fractionated (fractions 4-1 to 4-10).

(2) Fr. 4-2 (recovered amount: 5.6 mg) was obtained by repeating preparative [octadecyl silica (hereinafter abbreviated as “ODS”)]-high performance liquid chromatography (hereinafter abbreviated as “HPLC”) to obtain alpinoids. A (compound 1d; recovered amount: 0.8 mg) was obtained.
1d: [α] ²⁵ _D : −6.08 ^o (CHCl ₃ )
FAB MS m / z: [M + Na] ⁺ 705
HR FABMS m / z: [M + Na] ⁺ 705.34035 (calcd. For 705.34096)

(3) Fr. 4-5 (recovered amount: 62 mg) was obtained by fractionation of 5 (fraction 4-5-1-4 with silica gel-CC [silica gel use amount: 100 g, elution solvent: n-hexane-ethyl acetate (4: 1)]. -5-5). Fr. 4-5-2 (recovered amount: 3.2 mg) was obtained by repeating preparative ODS-HPLC to obtain alpinoid C (compound 1e; recovered amount: 1.5 mg).
1e: [α] ²⁵ _D : -3.92 ^o (CHCl ₃ )
^{EI MS m / z: [M} ] + 280
HR EIMS m / z: [M] ⁺ 280.14673 (calcd. For 280.14632)

(4) On the other hand, Fr. 4-5-4 (recovery amount: 41 mg) was obtained by repeating preparative ODS-HPLC to give 7- (4 "-hydroxy-3" -methoxyphenyl) -1-phenylhept-4-en-3-one (compound 1a; recovered amount: 36 mg).

(5) Fr. 4-7 (recovered amount: 313 mg) was obtained by adding 3 fractions (fractions 4-7-1 to 4) according to silica gel-CC [silica gel use amount: 200 g, elution solvent: n-hexane-ethyl acetate (4: 1)]. -7-3). Fr. 4-7-2 (recovery amount: 275 mg) was obtained by repeating preparative ODS-HPLC to give 5-methoxy-7- (4 "-hydroxy-3" -methoxyphenyl) -1-phenyl-3-heptaneone (compound 1b; recovered amount: 270 mg).

(6) Fr. 4-8 (recovery amount: 112 mg) was obtained by fractionation of 5 (fractions 4-8-1 to 4) according to silica gel-CC [silica gel use amount: 200 g, elution solvent: n-hexane-ethyl acetate (4: 1)]. It fractionated into -8-5). Fr. 4-8-2 (recovery amount: 99.5 mg) was obtained by repeating preparative ODS-HPLC to give 5-hydroxy-7- (4 "-hydroxy-3" -methoxyphenyl) -1-phenyl-3-heptone. (Compound 1c; recovery amount: 97 mg) was obtained. On the other hand, Fr. 4-8-4 (recovery amount: 12 mg) obtained alpinoid B (compound 1f; recovery amount: 10.3 mg) by repeating preparative ODS-HPLC.

  Table 1 shows the NMR spectra of the compounds (1d), (1f) and (1e).

Example 2 (Anti-tumor activity)
Antitumor activity was measured by the MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) method.
A 96-well plate was inoculated with 100 μL of tumor cells (1 × 10 ⁴ cells / well) suspended in RPMI 1640 medium and cultured for 24 hours (5% CO ₂ , 37 ° C. under humid conditions). Thereafter, 0.2 μL each of the test compound (final concentration 1 × 10 ⁻⁴ to 1 × 10 ⁻⁸ M) and DMSO as a control were added and allowed to act on tumor cells for 48 hours. Next, 10 μL of 0.5% MTT solution was added, and after 3 hours, 100 μL of a reaction stop solution (0.04N HCl / isopropanol) was added to stop the reaction. After well pipetting, the absorbance at 570 nm (top) and 655 nm (bottom) was measured with a microplate reader. The cell viability at each concentration of each specimen was determined as a percentage of the control group, and EC ₅₀ values were calculated therefrom (FIGS. 2 to 5). As tumor cells, IMR-32, which is a neuroblastoma cell, was used.

Example 3 (Apoptosis induction ability)
Apoptosis induction ability was tested by nuclear staining with Hoechst 33342 staining method.
2 mL of cells (IMR-32: 2 × 10 ⁵ cells / well) suspended in RPMI 1640 medium were seeded on a 6-well plate, and cultured for 24 hours (5% CO ₂ , 37 ° C. under saturated conditions). Thereafter, 4 μL each of the test compound (final concentration 1 × 10 ⁻⁶ M) and DMSO as a control were added and allowed to act on the cells for 24 hours. Next, 100 μL of a 0.025% Hoechst 33342 solution was added, and after 15 minutes, a phase contrast image and a fluorescence image were photographed with a fluorescence microscope to observe changes in cell morphology. As a result, compound (1b) was found to have nuclear aggregation and fragmentation at 1 μM, indicating that cytotoxic activity against IMR-32 cells was due to apoptosis induction (FIG. 6).

As a result, as shown in FIGS. 2 to 6, the compound of the formula (1) had strong antitumor activity from a concentration as low as 10 ⁻⁷ M. The cytotoxic activity was due to apoptosis induction.

Claims (1)

The following formulas (1a) to (1c)

A therapeutic agent for neuroblastoma comprising a compound represented by the formula:

Images