JPH1135569A - Polyphenol compound and drug containing the same - Google Patents

Abstract

(57) Abstract: Nepalencinol A, B and C, and a medicament containing the same as an active ingredient. Embedded image [Effect] Has excellent topoisomerase II inhibitory action,
Useful as an antitumor agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel plant-derived polyphenol compound and a medicament represented by an antitumor agent containing the same.

[0002]

2. Description of the Related Art Plants have been known to contain various physiologically active substances. For example, alkaloids such as vinplastin and vincristine, colsemide analog of cohachitin, camptothecin derivative CPT
-11 and the like are known to have an antitumor effect.

[0003]

However, conventional plant-derived medicines are not yet sufficiently satisfactory.
Further, new physiologically active substances are desired. An object of the present invention is to provide a novel plant-derived compound useful as a medicine.

[0004]

Accordingly, the present inventors have focused on topoisomerase II, and have a drug that exerts an antitumor effect by acting on the topoisomerase II to inhibit the distribution (M phase) of DNA whose replication has been completed. In search of various plant extracts, we found that three polyphenol compounds isolated from Cyperaceae plants have excellent topoisomerase II inhibitory activity and are useful as antitumor agents. The invention has been completed.

That is, the present invention provides the compounds nepalencinol A, B and C represented by the following formula or salts thereof.

[0006]

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[0007]

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[0008]

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The present invention also provides a medicine containing these compounds as active ingredients.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The salt of the compound of the present invention includes inorganic acid salts such as hydrochloride, hydrobromide, sulfate and perchlorate;
Sodium salts, potassium salts and the like can be mentioned. In the present invention, a sodium salt is preferable. In the present invention, hydrates and solvates of these compounds or salts thereof can also be used.

The compound of the present invention can be obtained by extracting and isolating the compound from the Cyperaceae plant. The cyperaceae plant used is Koges
ia), Beetle (Maltifolia), Beetle (Morr)
owii), Foliosissima, Azusa (Thunbergii), Kasagusuge (Dispalata), Kokusuge (Reinii), Ciliato-morginata, and the like. The extraction site is not particularly limited, and one or two or more sites (hereinafter, referred to as “the original material”) among the fruits, pericarp, bark, root bark, leaves, root materials and the like of these plants are used.

[0012] The extraction method is not particularly limited, either, and the extraction can be performed by extracting the starting material with a solvent and isolating. For example, the drug substance is cut into small pieces, dried and pulverized, extracted with an organic solvent such as benzene, ethyl acetate, methanol, and acetone, and concentrated under reduced pressure to obtain an extract. The obtained extract is purified by recrystallization, silica gel column chromatography or the like, and the components can be isolated. In the present invention, as long as it contains an effective amount of the compound, it can be used at a stage during the above-mentioned extraction operation, for example, at the stage of the extract.

The compound of the present invention or a salt thereof is useful as an antitumor agent because it exhibits excellent topoisomerase II inhibitory activity.

The medicament of the present invention can be administered either orally or parenterally (intramuscularly, subcutaneously, intravenously, suppositories, etc.).

When preparing an oral preparation, excipients and, if necessary, a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent and the like are added, and then tablets and coated tablets are prepared in a conventional manner. ,
Granules, capsules, solutions, syrups, elixirs, oily or aqueous suspensions, etc. As an excipient, for example, lactose, corn starch, sucrose, glucose, sorbite, crystalline cellulose, and the like, as a binder, for example, polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose , Hydroxypropyl starch, polyvinylpyrrolidone and the like.

Disintegrators include, for example, starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextran, pectin and the like. Lubricants include, for example, magnesium stearate, talc, polyethylene glycol. , Silica, hydrogenated vegetable oils, etc. are permitted to be added to pharmaceuticals as coloring agents, but as flavoring agents, cocoa powder, peppermint brain, aromatic acid, peppermint oil, dragon brain, cinnamon powder and the like are used. Can be In these tablets, the granules may be sugar-coated, gelatin-coated, or appropriately coated as needed.

When preparing an injection, a pH adjuster, a buffer, a stabilizing agent, a preservative and the like are added as necessary, and a subcutaneous, intramuscular or intravenous injection is prepared by a conventional method. The injection may be prepared as a solid preparation by freeze-drying or the like after storage of the solution in a container, and may be prepared at the time of use. One dose may be stored in a container, or multiple doses may be stored in the same container.

The dose of the medicament of the present invention, as nepalencinol A, B or C, is generally 0.01 to 1000 mg, preferably 0.1 to 100 mg per day for an adult in humans. In the case of animals, the dose is usually in the range of 0.001 to 1000 mg, preferably 0.1 to 100 mg, per kg of body weight of the animal to be treated. This daily dose is administered once a day or divided into 2 to 4 times.

[0019]

Hereinafter, the present invention will be described with reference to examples.
The present invention is not limited to these examples.

Example 1 A cyperaceae (Kobresia) of the family Cyperaceae was washed with water, air-dried, pulverized, and extracted with methanol. The solvent was distilled off from the methanol extract under reduced pressure, and the obtained residue was fractionated into n-hexane, ethyl acetate and butanol-soluble parts, and the obtained ethyl acetate-soluble part was filled with Kieselgel 60 (manufactured by Merck). After silica gel column chromatography, ODS medium pressure column chromatography and preparative column chromatography were performed to obtain 70 mg of nepalencinol A.
I got

Red-brown powder [α] _D = -161.7 ° (c = 0.2 MeOH) UVλ _max 276.0, 220.4 nm (MeOH) IRν _max 3200-3600, 1612 cm ^-1 (KBr) ¹ H-MMR (400 MHz, acetone-d ₆ ) δ: 2.89 (1H, s), 3.05 (1H,
d, 9.6), 3.87 (1H, d, 3.6), 4.16 (1H, s), 4.34 (1H, dd, 3.5,
9.7), 4.95 (1H, d, 7.2), 5.48 (1H, d, 7.2), 5.90 (2H, d, 2.
1), 6.60 (1H, t, 2.1), 6.30 (1H, t, 2.2), 6.40 (3H, s, H-10,
14,12, overlaped), 6.54 (2H, d, 8.7), 6.58 (2H, d, 8.7),
6.77 (2H, d, 8.5), 6.87 (2H, d, 8.6), 6.93 (2H, d, 8.4), 7.
31 (2H, d, 8.6) ¹³ C-NMR (100 MHz, acetone-d ₆ ) δ: 55.8, 57.5, 57.7, 5
8.2, 77.8, 93.6, 96.0, 100.5, 101.6, 105.5, 108.0,
114.7, 115.0, 115.4, 119.9, 122.7,127.7, 128.4, 12
8.8, 133.4, 136.0, 136.8, 145.0, 146.7, 150.5, 154.
7, 155.7, 156.6, 157.5, 158.2, 158.4, 161.4

Example 2 The above-ground part of Kobresia neparensis collected in the Himalaya Highland (over 3000 m) in Nepal was washed with water, air-dried (dry weight: about 1.5 kg), and immersed in methanol for about one month. The concentrate (about 400 g) was fractionated into hexane, ethyl acetate, and butanol-soluble parts, and the ethyl acetate-soluble part (about 50 g) was subjected to silica gel column chromatography, and eluted with a chloroform: methanol system. Crude fractionation was performed. The obtained fraction was further subjected to a silica gel column, Sephadex LH-20 and high performance liquid chromatography (HPLC) to repeat nepalencinol B
And nepalencinol C were obtained.

(1) Nepalencinol B ¹ H-NMR (400 MHz, acetone-d ₆ ) δ: 3.97 (1H, s), 4.29 (1H,
s), 4.31 (1H, d, 1.7), 5.31 (1H, d, 1.7), 6.29 (2H, brs),
6.31 (1H, t, 2.0), 6.56 (2H, d, 8.5), 6.75 (2H, d, 8.5), 7.1
1 (2H, d, 8.5) ¹³ C-NMR (100 MHz, acetone-d ₆ ) δ: 49.6, 56.4, 59.9, 9
3.4, 96.1, 101.6, 106.0, 114.8, 115.4, 115.6, 125.
9, 126.6, 128.6, 134.0, 137.9, 143.9, 148.0, 154.9,
155.3, 157.3, 159.5, 162.6

(2) Nepalencinol C ¹ H-NMR (400 MHz, acetone-d ₆ ) δ: 3.54 (1H, dd, 11.1, 9.
9), 3.98 (1H, dd, 11.3, 9.4), 4.32 (1H, d, 6.0), 4.50 (1H,
d, 9.5), 5.28 (1H, d, 6.0), 5.33 (1H, d, 9.2), 5.86 (2H, d,
2.1), 6.02 (1H, d, 1.9), 6.08 (1H, t, 2.1), 6.12 (1H, d, 1.
9), 6.29 (2H, d, 2.1), 6.41 (1H, t, 2.1), 6.64 (2H, d, 8.5),
6.76 (2H, d, 8.5), 6.88 (2H, d, 8.5), 7.04 (2H, d, 8.4), 7.
06 (2H, d, 8.5), 7.16 (2H, d, 8.5)

Test Example 1 Measurement of Topoisomerase Type II Inhibitory Activity (A) Extraction of Crude Topoisomerase II from Rat Infant Brain Nuclei (IBN) 4.2 μl of IBN (11.98 mg / ml in 50% glycerol) was taken and centrifuged ( (3,000 xg, 1 minute) to remove glycerol. Nuclear extr pellet
action buffer (20mM Tris-HCl <pH7.5>, 0.3M NaCl, 14
(10 mM 0 mM β-Me, 50 ml / ml BSA, 20 mM PMSF) and incubated on ice for 30 minutes. Next, centrifuge (1
(000 × g, 10 minutes) to obtain a crude topoisomerase II. This was diluted according to the activity.

(B) Purified topoisomerase II (Top GEN,
Inc.) topoisomerase II (Top GEN, Inc)
It was diluted with Nuclear extration buffer to t / ml.

Place a 1 μl sample in an Eppendorf tube.
1, required amount of Milli Q, 4 × topoisomerase II buffe
r (250 mM Tris-HCl <pH7.9>, 600 mM KCl, 50 mM MgCl ₂ ,
0.5mMEDTA, 2.5mM ATP, 150mg / ml BSA), kDNA
(312 ng / ml) mixed with 18 μl of buffer,
Vortexing and light centrifugation were performed. Next, 1 μl of diluted crude topoisomerase II or purified purified topoisomerase II was added, and vortexing and light centrifugation were performed again (however, Milli Q, Milli Q, 1 μl and 1 μl of 50% DMSO, and 1 μl of 50% DMSO instead of the xanthone derivative-free reaction).

The above Ependorf tube was left at 30 ° C. for crude topoisomerase II and at 37 ° C. for purified topoisomerase II for 30 minutes, and then placed on ice for SDS / P.
4 μl of K / BJ was added. Thereafter, in each case, the mixture was left at 50 ° C. for 5 minutes, then vortexed and lightly centrifuged, left again at 50 ° C. for 30 minutes, and then vortexed and lightly centrifuged.

(C) Agarose gel electrophoresis and densitometry 1 × TBE buffer for electrophoresis (89 mM Tris Base / 89 mM boric acid)
/ 2mM EDTA2Na) for agarose gel electrophoresis (-EtB
r 50 V), and after electrophoresis, 1 × TBE (0.1 ml / ml EtB)
The gel was stained in r), and after decolorization of the gel, UV irradiation was performed and the band was observed. 0.8% agarose gel
%.

The reaction amount of the enzyme was quantified by quantifying the band density of the agarose gel using a computer <Gel Plotting Macros> attached to NIH Image. The band of the minicircle DNA was loaded into a computer, quantified and calculated to obtain a residual activity rate (%). Table 1 shows the results.

[0031]

[Table 1]

From Table 1, it is apparent that the compound of the present invention has topoisomerase II inhibitory activity.

[0033]

Industrial Applicability The compound of the present invention exhibits excellent topoisomerase II inhibitory activity and is useful as an antitumor agent.

Claims (5)

[Claims] 1. A compound represented by the following formula: Neparensinol A or a salt thereof. Embedded image 2. A compound represented by the following formula: Neparensinol B or a salt thereof. Embedded image 3. A compound represented by the following formula: Neparensinol C or a salt thereof. Embedded image A pharmaceutical comprising the compound according to any one of claims 1 to 3 as an active ingredient. 5. The medicament according to claim 4, which is an antitumor agent.