JPH09183724A - Naphthalene carboxylic acid derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a uzedicine useful as an antitumor agent and expected as an antitumor medicine having high safety and little side effect by compounding a specific naphthalene carboxylic acid derivative obtained from propolis or its salt as an active component. SOLUTION: This medicine contains 1,2,3,4,4a,7,8,8a-octahydro-1,4a,-5- trimethyl-1-(3-methyl-5-oxo-3-pentenyl)-2-naphthalene carboxylic acid of the formula or its salt as an active component. A compound of the formula is preferably obtained by a new method consisting of the extraction and purification from propolis. The below-mentioned processes are preferable for this purpose. At first, the solvent of an ethanol extract of propolis is removed by evaporation. The residue is extracted with a mixed solution of water and ethyl acetate, and subsequently the residue of the obtained ethyl acetate extract is extracted with methanol. The extract is purified serially with liquid chromatography using a reverse phase column, liquid chromatography using a absorption column and liquid chromatography using a molecular sieve column.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to known substances 1, 2, 3, 4, 4a, 7, 8, 8a represented by the chemical formula (I).
-Octahydro-1,4a, 5-trimethyl-1- (3
-Methyl-5-oxo-3-pentenyl) -2-naphthalenecarboxylic acid (hereinafter abbreviated as naphthalenecarboxylic acid derivative) and a novel method for producing the isomer thereof, and a novel use thereof as a pharmaceutical, particularly as an antitumor agent Regarding

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[0002]

2. Description of the Related Art Antitumor agents used for the treatment of cancer can be broadly classified into chemotherapeutic agents and immunotherapeutic agents. The chemotherapeutic agent is not only administered to unresectable cancer, but is also used in combination with surgery by being administered before or after surgery. For example, cancer chemotherapeutic agents include alkylating agents (nitrodiene mustards, ethyleneimines, sulfonates, etc.), antimetabolites (folic acid antagonists, pyrimidine antagonists, etc.), plant mitotic poisons (colcemid, Vinblastine, etc.), antibiotics (sarcomycin, carcinophylline, mitomycin, etc.), hormonal agents (corticosteroids, male hormones, female hormones, etc.), and porphyrin complex salts (morphylin, copp) etc. are clinically used. However, chemotherapeutic agents are often cytotoxic substances, not only attacking cancer cells but also acting on normal cells, and generally have strong side effects. For example, side effects such as vomiting, nausea, loss of appetite, malaise, neuropathy, bone marrow disorder (leukopenia), hair loss and stomatitis may be observed. Therefore, caution is required for long-term administration.

An immunotherapeutic agent is mainly an immunostimulant, which enhances immunity to cause immune response cells to recognize cancer cells as foreign substances and treat them. Although immunostimulants have few side effects, they generally have a mild antitumor effect and are often used as an adjunct therapy.

As described above, conventional antitumor agents have advantages and disadvantages, and therefore have high antitumor activity and few side effects.
It has been desired to develop an antitumor agent that can be administered for a long period of time. Further, since the antitumor agent is generally expensive, it is also desired that the antitumor agent be supplied at a low price in order to reduce the financial burden on the patient.

Propolis, which is used as a folk remedy, is a resinous substance in which the components of plants collected by bees and saliva, beeswax, pollen, etc. are mixed, and they have pharmacological actions such as antibacterial action and anti-inflammatory action. It is known. In addition, it is known that when propolis is taken as a dietary supplement, an antitumor effect is exhibited, and attempts have been made to extract and purify an antitumor active substance from propolis (JP-A-5-589).
43, JP-A-5-271031), and the extract is recognized to have an antibacterial action and an antitumor action.

The present inventor has also extracted and purified from propolis in order to discover a novel physiologically active substance having an antitumor effect,
As a substance having antitumor activity, a naphthalenecarboxylic acid derivative represented by the chemical formula (I) was found.

[0007]

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The naphthalenecarboxylic acid derivative represented by the chemical formula (I) of the present application is a plant of SYMPHYOPAPPUS species (PHYTO
CHEMISTRY, 20 (7), 1657-1663, 1981) and Physcomitrella patens (P
HYTOCHEMISTRY, 28 (12), 3415-3419, 1989). Further, the chemical formulas (II) and (III), which are the isomers, have RN = 126239-79-
0 and RN = 126239-80-3 CHEMICAL A
Registered in BSTRACT.

However, it is not known what kind of pharmacological action this compound has and whether it can be used as a medicine. In particular, it is a novel finding that this compound has an antitumor effect.

[0010]

DISCLOSURE OF THE INVENTION The present invention provides a drug, particularly an antitumor agent, which contains a naphthalenecarboxylic acid derivative represented by the chemical formula (I) and its isomers (II) and (III) as a main component. With the goal. Another object is to provide a novel method for producing the present naphthalenecarboxylic acid derivative.

[0011]

The present invention provides a naphthalenecarboxylic acid derivative represented by the following chemical formulas (I), (II) and (III) and a drug mainly comprising the isomer thereof, particularly an antitumor agent. . Further, the present invention provides a production method for extracting and purifying the naphthalenecarboxylic acid derivative (I) (II) (III) from propolis.

[Chemical 6]

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[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is used as a drug, particularly an antitumor agent, containing a naphthalenecarboxylic acid derivative or a salt thereof as an active ingredient. The salts include pharmaceutically acceptable salts.

Further, the present invention (1) removes the insoluble matter of the ethanol suspension of propolis to obtain an ethanol extract of propolis, and then evaporates and removes the solvent of the obtained ethanol extract to obtain a residue. Extract with a mixture of water and ethyl acetate to obtain an ethyl acetate extract, evaporate and remove the solvent of the obtained ethyl acetate extract, disperse the obtained residue in methanol, remove insolubles, and remove methanol of propolis. A step of obtaining an extract, (2) subjecting the methanol extract to liquid chromatography using a reverse phase column,
Gradient elution with 100% methanol concentration gradient
A step of collecting a fraction having a methanol concentration of 90 to 95%,
(3) The solvent of the 90-95% methanol concentration fraction was evaporated and removed, then the obtained residue was dissolved in chloroform, and the obtained solution was subjected to liquid chromatography using an adsorption system column and eluted with chloroform. Fraction collecting step, (4) The solvent of the above fraction is removed by evaporation, then the obtained residue is dissolved in chloroform, and the obtained solution is subjected to liquid chromatography using a molecular sieve system column and eluted with chloroform. It is also a production method for extracting and purifying a naphthalenecarboxylic acid derivative from propolis, which comprises a step of collecting a main fraction and removing a solvent by evaporation to obtain a naphthalenecarboxylic acid derivative.

The column used in the step (2) is for high performance liquid chromatography, and a commercially available ODS type silica gel column can be used as the reversed phase column, and in the present application, it is equilibrated with 70% methanol. ODS 80TM
A column (manufactured by Tosoh Corporation) is preferable.

The column used in the step (3) is for high performance liquid chromatography, and a commercially available Inertsil type column can be used as the adsorption system column. In particular, in the present application, Inertsil S equilibrated with chloroform is used.
IL column (manufactured by GL Sciences Inc.) is preferable.

The column used in step (4) is for high performance liquid chromatography, and a commercially available GPC column can be used as the molecular sieve column, and in the present application, Shodex GPC-H20 equilibrated with chloroform is used.
00 column (manufactured by Showa Denko KK) is preferable.

In steps (3) and (4) above, the isomers (II) and (III) are eluted as different peaks that are close to each other but have different retention times. In order to isolate and purify the isomer (II), in step (3) (I
The purified isomer (II) is isolated by collecting the peak of I) and removing a small amount of (III) remaining as an impurity in step (4). Further, if the step (3) and the step (4) are repeated twice, further purification (I
I) is obtained.

In order to isolate and purify the isomer (III), the peak of (III) is collected in the step (3) in reverse to the above, and a small amount of the remaining (II) is removed as an impurity in the step (4). The purified isomer (III)
Is isolated. Further, if the step (3) and the step (4) are repeated twice, a further purified (III) can be obtained.

When it is not necessary to isolate the isomers (II) and (III), (II) and (III) are used in step (3).
The whole peak containing is collected, and in step (4), (II) (I
By removing remaining impurities other than II) and purifying, the purified naphthalenecarboxylic acid derivative (I) is obtained as a mixture of isomers (II) and (III).

By the above operation, the purified naphthalenecarboxylic acid derivative (I) is obtained as an oily substance, and the isomers (II) and (III) are obtained as crystalline powders.

Since the naphthalenecarboxylic acid derivative of the present invention exhibits an antitumor cell action on tumor cells as shown in Examples, it is considered that the naphthalenecarboxylic acid derivative exhibits a very effective antitumor effect when administered in various modes. The method for administering the present compound may be parenteral administration or oral administration, and the composition to be administered may have a therapeutically effective amount of the present compound and a pharmacologically acceptable diluent, stabilizer, excipient, etc. Is contained. Dosage forms include intravenous injection, subcutaneous injection, intramuscular injection, suppository,
Examples include parenteral administration methods such as ointments and tinctures, and oral administration methods such as tablets, powders, capsules and granules.

Since the naphthalenecarboxylic acid derivative of the present invention is obtained as an oily substance or a crystalline powder, it is preferable to use the isomer on the powder when it is administered in a dry form such as a tablet or a powder. If it is administered, it can be used as long as it is dissolved or suspended in the form of oil or powder and it is suitable for the administration form. Further, even if it is oily, it can be used as a powder if it is in the form of a double powder.

[0023] At present, the safety of administering the present naphthalenecarboxylic acid derivative to humans is unknown. But,
No toxicity was observed when propolis itself was orally administered to dogs, rats and guinea pigs for 10 months at 10 to 15 g / kg (PROPOLIS: 2ND ed., Y.
According to DONADEU, 1983), it is considered that the safety is high even if the naphthalenecarboxylic acid derivative is administered to humans.

Further, alcohol extract of propolis is known to have antibacterial action, antioxidative action, antiinflammatory action, virus growth inhibiting action, macrophage activating action, hair growth action and the like (JP-A-5-271031 and JP-A-5-31103). -31696
8, JP-A-7-298833), and those effects can be expected in the naphthalenecarboxylic acid derivative of the present invention obtained by purifying an alcohol extract of propolis.

The present invention will be described in more detail based on the following examples. The following examples are for illustrative purposes only and do not limit the present invention in any way.

【Example】

Example 1 Extraction of the Present Compound from Propolis Extraction and purification of a naphthalenecarboxylic acid derivative from propolis were carried out through the following [Step 1] to [Step 5]. [Step 1] 100 g of propolis is added to 10 times amount of 9
It was mixed with 9.5% ethanol and stirred at room temperature using a magnetic stirrer. The obtained suspension was filtered under reduced pressure to obtain an ethanol extract of propolis. Then, the solvent of the obtained ethanol extract was removed by evaporation using a rotary evaporator, about 30 g of the obtained residue was dispersed in a mixed solution of water and ethyl acetate (1: 1), and the upper ethyl acetate layer was collected to remove propolis. An ethyl acetate extract was obtained. The solvent of the obtained ethyl acetate extract was removed by evaporation with a rotary evaporator, about 20 g of the obtained residue was dispersed in 99.5% methanol, and the insoluble matter was removed by low-speed centrifugation to obtain a methanol extract of propolis.

[Step 2] ODS 8 column for high performance liquid chromatography, ODS 8 in which the methanol extract obtained in Step 1 was equilibrated with 70% methanol
Injected into a 0TM column (manufactured by Tosoh Corporation), 70% to 100
Gradient elution was performed with a% methanol concentration gradient. Fractions having a methanol concentration of 90 to 95% were collected.

[Step 3] Fractions having a methanol concentration of 90 to 95% obtained in Step 2 were collected into one, and the solvent was removed by rotary evaporation to obtain a residue of about 2 g.
I got The resulting residue is then dissolved in chloroform,
Inertsil-based column for high performance liquid chromatography in which the obtained solution was equilibrated with chloroform, Iner
It was injected into a tsil SIL column (manufactured by GL Sciences) and two fractions having different retention times were collected.

[Step 4] The solvent of the two fractions obtained in Step 3 was removed by evaporation using a rotary evaporator to obtain residues of about 70 mg and 60 mg, respectively. Next, each of the obtained residues was dissolved in chloroform, and the obtained solution was equilibrated with chloroform, a GPC system column for high performance liquid chromatography, Shodex GPC-H200.
It was injected into 0 column (manufactured by Showa Denko KK), and each main fraction was collected.

[Step 5] The solvent of each of the main fractions obtained in Step 4 was removed by evaporation with a rotary evaporator to give about 50 mg of each residue as a colorless crystalline substance,
40 mg were obtained. Due to the physicochemical properties of the residue, it is 1,2,3,4,4a, 7,8,8a-octahydro-
It was confirmed to be isomers (II) and (III) of 1,4a, 5-trimethyl-1- (3-methyl-5-oxo-3-pentenyl) -2-naphthalenecarboxylic acid.

Further, in step 3, the isomers were not separated, and the subsequent operation was carried out in the same manner to obtain the compound (I). Compound (I) was obtained as an oily substance.

Example 2 Physicochemical properties of naphthalenecarboxylic acid derivative (II) Compound name: 2-Naphthalenecarboxylic acid, 1,2,3,4,4
a, 7,8,8a-octahydro-1,4a, 5-trimethyl-1- (3-methyl-5-
oxo-3-pentenyl)-, [1R- [1.alpha. (Z), 2.beta., 4a.bet
a., 8a.alpha.]]-(9CI) (CA INDEX NAME) CA registration number: 126239-79-0 Molecular weight: 318.46 Molecular formula: C ₂₀ H ₃₀ O ₃

Chemical formula:

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Properties: colorless needle crystals or crystalline powder,
Smell has fragrance. Solubility: Easily soluble in chloroform and dimethylformamide,
Soluble in ethanol and methanol, insoluble in water and cyclohexane

Mass spectrum: Instrument: Finnigan MAT INCOS 50 Ionization: EI Accelation VOLTAGE: 70 eV The results are shown in FIG.

NMR spectrum: Instrument: JEOL JNM-GSX400 Resonance: ¹ H (400 MHz) Sample phase: 7.7 mg / 0.55 ml CDCl ₃ Internal reference: TMS Temperature: 30 ° C. The results are shown in FIG.

HPLC: Instrument: SHIMADZU CL-4A Separation conditions and retention time are as follows.

[Table 1]

From the analysis of these data, the naphthalenecarboxylic acid derivative of the present invention was identified as the compound represented by the chemical formula (II).

Example 3 Physicochemical properties of naphthalenecarboxylic acid derivative (III) Compound name: 2-Naphthalenecarboxylic acid, 1,2,3,4,4
a, 7,8,8a-octahydro-1,4a, 5-trimethyl-1- (3-methyl-5-
oxo-3-pentenyl)-, [1R- [1.alpha. (E), 2.beta., 4a.bet
a., 8a.alpha.]]-(9CI) (CA INDEX NAME) CA registration number: 126239-80-3 Molecular weight: 318.46 Molecular formula: C ₂₀ H ₃₀ O ₃

Chemical formula:

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Properties: colorless needle crystals or crystalline powder,
Smell has fragrance. Solubility: Easily soluble in chloroform and dimethylformamide,
Soluble in ethanol and methanol, insoluble in water and cyclohexane

Mass spectrum: Instrument: Finnigan MAT INCOS 50 Ionization: EI Accelelation VOLTAGE: 70 eV The results are shown in FIG.

NMR spectrum: Instrument: JEOL JNM-GSX400 Resonance: ¹ H (400 MHz) Sample phase: 4.05 mg / 0.55 ml CDCl ₃ Internal reference: TMS Temperature: 25 ° C. The results are shown in FIG.

HPLC: Instrument: SHIMADZU CL-4A Separation conditions and retention time are as follows.

[Table 2]

From the analysis of these data, the naphthalenecarboxylic acid derivative of the present invention was identified as the compound represented by the chemical formula (III).

Example 4 Antitumor Cell Action on Cultured Tumor Cells The naphthalenecarboxylic acid derivative (I) obtained in Example 1
Using (II) and (III) as test substances, a tumor cell cytotoxicity test was performed as follows. 9
Human liver cancer HuH13 strain treated with trypsin (J. CELLULAR PHYS) was added to each well of a 6-well microtiter plate after adding 0.1 ml of a test substance appropriately diluted with MEM medium containing 10% fetal bovine serum and 2 mM glutamine.
YOL., VOL 148, 290-294, 1991) with the above culture medium 3 × 1
It was adjusted to 0 ⁴ pieces / ml and dispensed in 0.05 ml portions.

After culturing the plate in a carbon dioxide gas incubator at 37 ° C. for 72 hours, the culture supernatant was removed and
0.1 ml of a culture solution containing 2% neutral red was added to each well, and the cells were stained at 37 ° C. for 1 hour in a carbon dioxide gas incubator to stain the cells. After removing the culture supernatant, the residue was washed once with physiological saline. Then, the dye was extracted with 0.001 N hydrochloric acid / 30% ethanol, and the absorbance at 550 nm was measured with a microplate reader. The absorbance of untreated cells and cells treated with a test substance at a known concentration were compared to calculate the cell growth inhibition rate according to the following formula.

[0048]

[Equation 1]

From the obtained growth inhibition rate, the cell growth rate was determined to be 5
The test substance concentration at which 0% inhibition (ID ₅₀ ) was calculated. The results are shown in Table 1. As is clear from Table 1, the naphthalenecarboxylic acid derivatives (I), (II) and (III) showed an excellent growth inhibitory effect on tumor cells.

[0050]

[Table 3]

[0051]

INDUSTRIAL APPLICABILITY The naphthalenecarboxylic acid derivative (1,2,2) of the present invention easily obtained by extraction from propolis
3,4,4a, 7,8,8a-octahydro-1,4
a, 5-Trimethyl-1- (3-methyl-5-oxo-
3-Pentenyl) -2-naphthalenecarboxylic acid and its isomers exhibit antitumor activity and are useful as antitumor agents. It can also be expected as an antitumor agent with high safety and few side effects.

[Brief description of the drawings]

FIG. 1 Mass spectrum of naphthalenecarboxylic acid derivative (II)

FIG. 2 ¹ H- of naphthalenecarboxylic acid derivative (II)
NMR spectrum

FIG. 3 Mass spectrum of naphthalenecarboxylic acid derivative (III)

FIG. 4 ¹ H of naphthalenecarboxylic acid derivative (III)
-NMR spectrum

Claims (9)

[Claims] 1. A drug containing a naphthalenecarboxylic acid derivative represented by the chemical formula (I) or a salt thereof as an active ingredient. 2. A drug comprising a naphthalenecarboxylic acid derivative represented by the chemical formula (II), which is an isomer of a compound of the formula (I), or a salt thereof as an active ingredient. 3. A drug comprising a naphthalenecarboxylic acid derivative represented by the chemical formula (III), which is an isomer of a compound of the formula (I), or a salt thereof as an active ingredient. 4. An antitumor agent containing a naphthalenecarboxylic acid derivative represented by the chemical formula (I) or a salt thereof as an active ingredient. 5. An antitumor agent containing as an active ingredient a naphthalenecarboxylic acid derivative represented by the chemical formula (II), which is an isomer of a compound of the formula (I), or a salt thereof. 6. An antitumor agent comprising as an active ingredient a naphthalenecarboxylic acid derivative represented by chemical formula (III), which is an isomer of a compound of formula (I), or a salt thereof. 7. A production method for extracting and purifying a naphthalenecarboxylic acid derivative represented by the chemical formula (I) from propolis through the following steps: (1) Insoluble matter in an ethanol suspension of propolis is removed, and ethanol of propolis is removed. An extract was obtained, and then the solvent of the obtained ethanol extract was removed by evaporation, and the obtained residue was extracted with a mixed solution of water and ethyl acetate to obtain an ethyl acetate extract, and the solvent of the obtained ethyl acetate extract. Is removed by evaporation, the resulting residue is dispersed in methanol, insoluble matter is removed,
Step (2) of obtaining a methanol extract of propolis, the methanol extract is subjected to liquid chromatography using a reverse phase column, and gradient elution is performed with a methanol concentration gradient of 70% to 100% to obtain a methanol concentration of 90 to 95.
% Fractionation step, (3) The solvent of the 90 to 95% methanol concentration fraction is evaporated and removed, then the obtained residue is dissolved in chloroform, and the obtained solution is liquid by an adsorption system column. Chromatography, eluting with chloroform, and collecting the main fraction, (4) The solvent of the fraction is removed by evaporation, then the obtained residue is dissolved in chloroform, and the obtained solution is a molecular sieve column. By liquid chromatography according to 1., eluting with chloroform, collecting a main fraction, and removing the solvent by evaporation to obtain a naphthalenecarboxylic acid derivative represented by the chemical formula (I). 8. The method according to claim 7, wherein the obtained naphthalenecarboxylic acid derivative has the chemical formula (II). 9. The method according to claim 7, wherein the obtained naphthalenecarboxylic acid derivative has the chemical formula (III).