JPH07138165A - Anticancer agent - Google Patents

Abstract

PURPOSE:To provide an anticancer agent containing ellagitannin as an active ingredient and having a topoisomerase I-inhibiting action. CONSTITUTION:An anticancer agent contains a compound of the formula or its salt as an active ingredient. The compound of the formulas is a known compound, a kind of ellagitannin called chebulanin, and is obtained by extracting and separating a galenical KASHI (the dry fruit of terminalia chebula) and subsequently purifying the separated compound. The anticancer agent can be prepared in the form of oral or parenteral administration preparations, and is administered at a does of 1-500mg/kg/day for an adult. The galenical KASHI is the commercially available dry fruit of Terminalia chebula which is a tall tree of the Combretaceae, is used for astriction, stegnosis, hemostasis or antitussis, and contains many tannin compounds.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anticancer agent containing ellagitannin having a topoisomerase I inhibitory activity as an active ingredient.

[0002]

2. Description of the Related Art Camptothecin (camptotheci) is used as a compound that exerts an anticancer effect by inhibiting topoisomerase I.
n; hereinafter referred to as CPT) is known (Biochemistry) 1989, 28, 4629-4638.
page). In addition, chebulagic acid (hereinafter referred to as CBA) is known as a compound having the strongest topoisomerase I inhibitory activity reported so far (journal of organic chemistry).
(Journal of Organic Chemistry) 1992, Volume 57, 420-
422). It is reported to have 10 to 50-fold topoisomerase I inhibitory activity against CPT. There is a report of chebulinic acid as a compound having an anticancer activity due to topoisomerase II inhibitory activity (Journal of Pharmaceutical Sciences, 1993, 82).
Vol. 5, p. 487-492).

[0003]

DISCLOSURE OF THE INVENTION The present invention provides an excellent anticancer agent having a higher topoisomerase I inhibitory activity than conventional compounds.

[0004]

Means for Solving the Problems As a result of intensive investigations by the present inventors for the purpose of discovering a substance having an excellent anticancer activity, the following formula (I):

[Chemical 3] It was found that the compound represented by the formula (1) has an excellent topoisomerase I inhibitory activity, and completed the present invention.

The compound (I) according to the present invention is a known compound and is a kind of ellagitannin called chebulanin. It can be obtained by extracting, separating and purifying from herbs of crude drug.

It has long been known that a compound having a topoisomerase inhibitory activity is useful as an anticancer agent,
CPT, which is a strong topoisomerase I inhibitor, has been proved to exhibit anticancer activity in clinical trials (Cancer Chemotherapy Reports 1972, 56, 515-52).
1). Kebulanin (I) has higher topoisomerase I than CBA, which is said to be stronger than CPT.
It had inhibitory activity.

[0007] The herbal medicine "Rinja" is a dried fruit of Takanashi Terminalia chebula of the family Rhinoceros and is generally commercially available. It is used for astringent, antidiarrheal, hemostasis, antitussive, and is known to contain a lot of tannins.

[0008] Kebulanin (I) can be obtained by subjecting commercially available silkworms to known extraction / isolation / purification means.

Suitable solvents commonly used for extraction,
For example, purified water, methanol, ethanol, acetone or the like can be used alone or in combination. The finely crushed silkworms are mixed with an appropriate amount of solvent, and extraction is carried out at room temperature or under heating, preferably near room temperature for several hours to several days. After the extraction is completed, the solid content is preferably removed with a filter paper or the like. Then, if necessary, concentration is carried out. In that case, the solvent may be distilled off at a room temperature to about 40 ° C. by a commonly used means.

Various methods are conceivable for removing contaminants from the thus obtained extract or residue, but washing by the extraction operation is simple. For example, after adding a suitable alkali to the extract to adjust the liquidity to around pH 7,
If shaken appropriately with an organic solvent, the fat-soluble contaminants move to the organic solvent layer. Then, the organic solvent layer is removed, the aqueous layer is adjusted to acidic, and then extracted with a suitable organic solvent. After that, the organic solvent layer is heated or concentrated under reduced pressure or the like, and subjected to column chromatography to obtain kebranine (I) having a relatively high purity. If further purification is required, for example, high performance liquid chromatography (HPL
Separation, recrystallization, and phase transfer by C) may be performed once to several times. As the above-mentioned organic solvent for extraction, any solvent having a high solubility for quebranine (I) can be used, and examples thereof include ethyl acetate, butanol, methyl ethyl ketone and the like, and ethyl acetate is particularly preferable. For adjusting the pH, hydrochloric acid, sulfuric acid, sodium carbonate, sodium hydroxide or the like may be appropriately used.

[0012] Kebulanin (I) can be easily obtained as a salt by a usual salt forming reaction. Specifically, for example, alkali metal salts such as sodium or potassium, alkaline earth metal salts such as calcium or magnesium, heavy metal salts such as zinc, and organic amine salts such as ammonium, triethylamine, pyridine, ethanolamine or basic amine. Is mentioned. These can be obtained by mixing with a basic substance such as sodium hydroxide, calcium carbonate or ammonium acetate depending on the desired salt and shaking.

[0012] The dosage form of quebranine (I) or a salt thereof includes powders, granules, tablets, pills, capsules, buccal agents or liquids such as oral agents, inhalants, suppositories, transdermal agents or injectable agents. It may be parenteral. The effective amount of the present compound can be mixed with pharmaceutical additives such as excipients, binders, wetting agents, disintegrating agents and lubricants suitable for the dosage form, if necessary, to prepare a pharmaceutical preparation. . In the case of an injection, it is sterilized with an appropriate carrier to prepare a preparation.

Specifically, as the excipient, lactose, sucrose,
Glucose, starch, calcium carbonate, crystalline cellulose and the like, binders such as methyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, gelatin and polyvinylpyrrolidone, and disintegrants such as carboxymethyl cellulose and carboxy. Methylcellulose sodium, starch, sodium alginate, agar powder, sodium lauryl sulfate and the like, and examples of lubricants include talc, magnesium stearate and macrogol. As the base of the suppository, cacao butter, macrogol, methylcellulose or the like can be used. In addition, in the case of preparing as a liquid preparation, an emulsion injection or a suspension injection, a solubilizing agent, a suspending agent, an emulsifier, a stabilizer, a preservative, an isotonic agent and the like which are usually used are appropriately used It may be added, and in the case of oral administration, flavoring agents, aromatic agents, etc. may be added.

For coating tablets, sugar coating may be used to facilitate administration, and film coating, enteric coating, multi-layer coating or compression coating may be used to enhance drug efficacy. Is.

The dose varies depending on the administration method, disease state, age and weight of the patient, etc., but when orally administered, it is usually 1 to 500 mg / kg / day for an adult, preferably 50 to 50 200 mg / kg / day may be administered once or in several divided doses.

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited thereto.

[0017]

Example 1 (Extraction method) 150 g of crude drug extract was cold-extracted and extracted with acetone: water (1: 1) for 5 days, and half of the extract was concentrated under reduced pressure and adjusted to pH 7 with an aqueous sodium hydrogen carbonate solution. It was extracted with ethyl acetate. The aqueous layer was adjusted to pH 3 with hydrochloric acid, extracted with ethyl acetate, and the ethyl acetate layer was dried under reduced pressure to give 3.571 g of a residue. 1.010 g of it was eluted with 85% ethanol using a Sephadex LH-20 column (22 × 262 mm). A 126 mg residue obtained by concentrating the fraction eluted with 70 ml after elution with 150 ml under reduced pressure was used as a column (1) of Capsule Pack C18, SG120 (manufactured by Shiseido Co., Ltd.).
Separation was performed by high performance liquid chromatography using 0 to 25 μm, 20 × 250 mm). 30% methanol (0.
It was eluted with 05% trifluoroacetic acid) and the fraction having a retention time of about 30 minutes was concentrated under reduced pressure and recrystallized from water to obtain 47 mg of quebranine (I).

. [0018] Colorless _{needles, mp 215~219 ℃ [α] D} : +9.1 (c = 0.51) UVλmax (MeOH) nm (logε): 221 (4.63), 278.5 (4.30) IRνmax (KBr) cm - ¹ : 3406, 1795, 1707, 1615, 1223 LSIMS (m / z): 675 (M + Na) ⁺ , 483, 246, 154, 137 HRLSIMS (m / z): C ₂₇ H ₂₄ O ₁₉ Na (M + Na) ⁺ and a theoretical: 675.0801 ^{Found: 675.0807 600MHz 1 H-NMR (} d 6 -acetone) δ: 2.18 (dd, J = 17.0, 4.7H
z), 2.21 (dd, J = 17.0, 10.5Hz), 3.90 (ddd, J = 10.5, 4.
7, 1.6Hz), 4.00 (dd, J = 11.3, 5.6Hz), 4.15 (dd, J = 11.
3, 6.7Hz), 4.31 (m), 4.84 (m), 4.90 (m), 4.95 (d, J = 7.
2Hz), 5.19 (dd, J = 7.2, 1.6Hz), 5.24 (m), 6.36 (m), 7.
21 (s, 2H), 7.51 (s)

Test Example 1 (Inhibition of DNA topoisomerase I)
Action test) Topoisomerase I (Takara Shuzo Co., Ltd.) 0.5un
reaction solution (35 mM Tris-HCl (pH 8.0), 72 mM potassium chloride, 5 mM magnesium chloride, 5 mM DTT, 5 mM spermidine, 0.
It was diluted with 01% bovine serum albumin), and 2 μl was dispensed to a 96-well microplate. The test sample diluted with the same reaction solution to a predetermined concentration was added in an amount of 10 μl each. Further diluted substrate DNA (pBR322: 2
50 ng / 8 μl) was added in 8 μl increments. 45 at 37 ° C
After reacting for a minute, the reaction stop solution (4.2% sodium dodecyl sulfate (SDS), 21% phycol 4) was added.
00, 0.2 mg / ml bromophenol blue,
0.33 mg / ml proteinase K) was added in 6 μl portions. After performing agarose electrophoresis (1%, 100 V, 1 hour) in tris borate buffer, ethidium bromide staining was performed, and closed circular DNA and open circular DNA which were fluorescently colored by UV irradiation were detected. The minimum concentration of sample that inhibits the transfer of closed circular DNA to open circular DNA was determined. The test results compared with CBA are shown in Table 1.

[0020]

[Table 1]

[0021]

EFFECTS OF THE INVENTION The quebranine according to the present invention has an extremely excellent topoisomerase I inhibitory activity and is useful as an anticancer agent.

Claims (2)

[Claims] 1. The following formula (I): An anticancer agent containing the compound represented by or a pharmaceutically acceptable salt thereof as an active ingredient. 2. The following formula (I): A topoisomerase I inhibitor containing as an active ingredient a compound represented by: or a pharmaceutically acceptable salt thereof.