JP2000103789A - Physiological activity of benzopyran derivatives derived from propolis - Google Patents

Abstract

(57) [Problem] An object of the present invention is to provide a novel benzopyran derivative which is useful as an antitumor agent of a photoisomer substance purified from propolis and which is excellent in safety. SOLUTION: The present invention relates to 2,2 represented by the chemical formula (I).
It comprises 2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid and an antitumor agent comprising this compound and a pharmaceutically acceptable salt as active ingredients. Embedded image

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid represented by the formula (I) and an antitumor agent thereof And a production method.

Embedded image

[0002]

2. Description of the Related Art Antitumor agents are used for treating cancer and are roughly classified into chemotherapeutic agents and immunotherapy agents. As chemotherapeutic agents, alkylating agents (nitrogen mustard, melpheran, cyclophosphamide), anticancer antibiotics (adriamycin, bleomycin, nelcarzinostatin), antimetabolites (methotrexate, 5-fluorouracil, 6- Mercaptopurines, hormonal agents (estrogens, androgens, prostins), plant-derived substances (vinblastine, camptothecin, etoposide), enzymes (asparaginase), and others (procarpazine, hydroxyurea, cisplatin) are mainly used clinically. However, chemotherapeutics are used for a wide range of cancers,
Despite its effects, no true anticancer drug has yet been developed that selectively attacks only cancer cells, has no toxicity in normal cells, and thus has no side effects. As a result, care must be taken for long-term administration.

[0003] Recently, research and development of drugs or therapeutic methods that change the host's responsiveness to tumor cells with the expectation of a therapeutic effect on cancer have been proposed and development has begun. This type includes immunotherapeutic agents having an immunostimulatory effect. Immunotherapeutic agents enhance immunity, act on immune response cells, and exert anticancer power. Compared with chemotherapeutic agents, immunotherapeutic agents have fewer side effects, but their antitumor effect is mild. Therefore, it is used as an adjuvant therapy.

As described above, conventional antitumor agents have many problems. Therefore, it is desired to develop an inexpensive antitumor agent which has a high antitumor effect, has few side effects, and is highly safe to enhance immunity.

[0005] Propolis is a gelatinous substance formed by mixing plant-derived resins collected by honeybees with their own secretions. Since ancient times, it has been used as a folk remedy showing pharmacological activities such as antibacterial and anti-inflammatory activities mainly in Eastern European countries.
Recently, propolis has been consumed as a dietary supplement. As a result, an antitumor effect was recognized, and an antitumor active substance was isolated and identified from the extract. (Japanese Patent Laid-Open No. 5-
58943, JP-A-5-27031, JP-A-9-
No. 143179)

The present invention also extracts and purifies from propolis in order to discover a novel physiologically active substance having an antitumor activity. As a substance having an antitumor activity, 2,2-dimethyl-8- represented by the chemical formula (I) is used. (3-methyl-2-butenyl)
Benzopyran-6-cis-propenoic acid was found.

An analog of the benzopyran derivative of the present invention, 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid, is PHY
TOCHMISTRY, 25 (4), 883-889,
1986 and 22 (10), 1969S4K, many derivatives are already known. However, the benzopyran derivative of the present invention, 2,2-dimethyl-8-
(3-Methyl-2-butenyl) benzopyran-6-cis-propenoic acid is not yet known, and it is a new finding that it has antitumor activity.

[0008]

SUMMARY OF THE INVENTION The present invention provides a novel benzopyran derivative, 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid, which comprises It is intended to provide an antitumor agent as a component. Benzopyran derivatives, 2,
The present invention provides a novel method for producing 2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid, and proves that it is a photoisomer produced by a photoreaction under visible light. The purpose is to do.

[0009]

The benzopyran derivative of the present invention, 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid, is represented by the chemical formula (I).

Embedded image

[0010]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a benzopyran derivative,
2,2-dimethyl-8- (3-methyl-2-butenyl)
It is used as an antitumor agent containing benzopyran-6-cis-propenoic acid and a salt thereof as an active ingredient. Salts include pharmaceutically acceptable salts.

[0011] The present invention also provides (1) pulverizing propolis with a mixer, extracting with methanol, removing impurities in a methanol suspension, obtaining a methanol extract of propolis, and then solvent of the obtained methanol extract. Is concentrated under reduced pressure to obtain a methanol extract of propolis,
(2) The above methanol extract is applied to an adsorption column using a silica gel column, and from 100% methyl chloride, a mixed solution of methyl chloride and methanol (95%: 5% v / v), and then methyl chloride and methanol (90%: 10% v / v) gradient elution with a concentration gradient using a mixed solution, and fractionation of each fraction, (3) the methyl chloride concentration of 100% to 95%
% Fraction, developing solvents methyl chloride and methanol (95%: 5
% V / v), the solvent was concentrated under reduced pressure, and the resulting residue was dissolved in a mixture of methanol and distilled water (50%: 50% v / v). The resulting solution was subjected to liquid chromatography using an ODS-based column, and eluted with a mixture of acetonitrile and 50 mM ammonium formate (52%: 48% v / v).
A step of collecting a main fraction, (4) a step of irradiating the solution of the main fraction with ultraviolet light with a transilluminator at 366 nm for 10 minutes to obtain a photoisomer fraction, and (5) a step of obtaining a solution of the fraction. The solvent acetonitrile was distilled off under reduced pressure while shielding the light, and the resulting residue was converted to a 50% v / v methanol solution, subjected to liquid chromatography using an ODS column, and acetonitrile and 50 mM ammonium formate (50%: 50% v / v) were used. / V) eluted with a mixture of
A step of fractionating each of the photoisomer fractions while shielding light, (6)
The solvent acetonitrile in the solution of each of the above fractions was distilled off under reduced pressure while shielding from light, and 100% methyl chloride was added thereto to carry out liquid separation.
An organic solvent layer is obtained, and the process is repeated three times. The collected organic solvent layer is concentrated under reduced pressure while shielding the organic solvent layer from light to obtain a benzopyran derivative represented by the chemical formula (I). It is also a production method for extracting and purifying a benzopyran derivative from propolis.

The silica gel used in step (2) is commercially available. Especially in the present application, silica gel 75-150 μm
(Manufactured by Kanto Chemical Co.) is preferred.

The columns used in the steps (3) and (5) are for high performance liquid chromatography, and a commercially available ODS silica gel column can be used as the reversed phase column. In the present invention, in particular, the column is equilibrated with acetonitrile. 5-
ODS-H columns (Chemco Bond) are preferred.

As shown in the Examples, the benzopyran derivative of the present invention, 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid, is an antitumor cell Since it has an action, it is considered that it shows a very effective antitumor cell effect by administration in various modes. The method for administering the present compound may be parenteral administration or oral administration. The composition to be administered contains a therapeutically effective amount of the compound and a pharmaceutically acceptable diluent, stabilizer, excipient, etc. Is contained. Examples of the administration form include parenteral administration such as intravenous injection, subcutaneous injection, intramuscular injection, suppository, and ointment, and oral administration using tablets, powders, capsules, granules and the like.

At present, the present benzopyran derivative, 2,2
The safety of -dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid when administered to humans is unknown. However, toxicity was not observed when 10 to 15 g / kg of propolis itself was orally administered to dogs, rats and guinea pigs for several months (PR
OPOLIS: 2ND ed. , Y. Donadie
U, 1983), it is considered that the safety is high even if the benzopyran derivative is administered to humans.

As described above, it has been reported that the propolis extract has various effects such as anti-inflammatory, anti-ulcer, anti-tumor, macrophage activation, cancer metastasis suppression, elimination of active oxygen, virus growth inhibition, and hair growth. (Japanese Patent Laid-Open No. 5-2
71031, JP-A-5-316968), the benzopyran derivative of the present invention extracted from propolis,
2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid can also be expected to have these effects.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are for illustrative purposes only and do not limit the present invention in any way.

【Example】

Example 1 Extraction of benzopyran derivative from propolis 2,2-dimethyl-8- (3-methyl-
Purification of 2-butenyl) benzopyran-6-cis-propenoic acid is carried out in the following [Step 1] to [Step 1].
5]. [Step 1] 250 g of propolis is pulverized with a mixer, mixed with 1500 mL of 99.5% methanol, and mixed for about 1 hour.
Stored at room temperature for months. The resulting suspension was filtered under reduced pressure to obtain a methanol extract of propolis. Next, the solvent of the obtained methanol extract was distilled off under reduced pressure.

[Step 2] About 1 g of the residue obtained in Step 1 was converted into a methanol solution, and 100 g of silica gel was used.
After that, 300 mL of 100% methyl chloride, which is three times the column volume, was flowed, and methyl chloride and methanol (9
Gradient elution with a mixture of 5%: 5% v / v) followed by a gradient of methyl chloride and methanol (90%: 10% v / v), spotting on a thin layer plate, The mixture was developed with a developing solvent of methyl and methanol (97%, 3% v / v), and elution fractions having an Rf value of 0.3 were collected.

[Step 3] The solvent of the fraction obtained in Step 2 was distilled off under reduced pressure to obtain about 20 mg of a residue. Then, the obtained residue was made into a 50% methanol solution, and the obtained solution was equilibrated with acetonitrile, and an ODS-based silica gel column for high performance liquid chromatography was used.
The mixture was injected into an H column (Chemco Bond), and the main fraction was collected.

[Step 4] The fraction obtained in Step 3 together with the solvent was directly irradiated with ultraviolet light by a transilluminator at a wavelength of 366 nm for 10 minutes, and the solvent acetonitrile in this solution was distilled off under reduced pressure while shielding from light.

[Step 5] The residue solution obtained in Step 4 was converted into a 50% methanol solution, and the obtained solution was equilibrated with acetonitrile. The ODS silica gel column for high performance liquid chromatography, the 5-ODS-H column (Chemcobond) ), And the photoisomer fractions were fractionated while shielding light.

[Step 6] The acetonitrile content of each solution obtained in Step 5 was distilled off under reduced pressure while shielding from light, and methyl chloride was added to carry out a liquid separation to obtain an organic solvent layer. It was repeated three times. The collected organic solvent layer was distilled off under reduced pressure while shielding from light to obtain about 1.1 mg of a residue. Due to the physicochemical properties of this residue, it was found that it was 2,2-dimethyl-8- (3
-Methyl-2-butenyl) benzopyran-6-cis-propenoic acid.

Example 2 Physicochemical Properties of Benzopyran Derivative 2-1 Photosteady Reaction The present benzopyran derivative and a photoisomer substance (JP-A-9-14)
No. 3179) causes photoisomerization under visible light.
In fact, it was confirmed that the reaction reached a complete equilibrium by UV irradiation of a 366 nm transilluminator.
Mass spectrum, NMR measurement, and the like were consistent with optical equilibrium. The present benzopyran derivative is stable under light shielding. 2-2 Solubility The present benzopyran derivative is insoluble in water and soluble in methanol, ethanol, acetonitrile, chloroform, and ethyl acetate.

2-3 Mass Spectrum Apparatus: API165 Perkin Elmer Ionization method: ESI 297.3 ([MH] ⁻ ) The results are shown in FIG.

2-4 Mass Spectrum Apparatus: JEOL JMS-AX505HA Ionization method: EI m / z [M] (70 eV) The results are shown in FIG.

2-5 Photodiode Array (UV
Measurement) Apparatus: JASCO MD-910 The results are shown in FIG.

2-6 ¹ H-NMR spectrum (500
MHz) Apparatus: JEOL ALPHA-500 Sample: 1.1 mg / 0.55 mL CDCL ₃ internal standard: TMS The results are shown in FIG. The assignment of each peak is shown in the table below.

[0029]

[Table 1]

2-7 ¹³ C-NMR spectrum (1
25.65 MHz) Apparatus: JEOL ALPHA-500 Sample: 1.1 mg / 0.55 mL CDCL ₃ internal standard: TMS The results are shown in FIG. The assignment of each peak is shown in the table below.

[0031]

[Table 2]

2-8 Two-dimensional NMR spectrum NOE
SY ( ¹ H-NMR 500 MHz, ¹³ C-NMR 1
25.65 MHz) Apparatus: JEOL ALPHA-500 Sample: 1.1 mg / 0.55 mL CDCL ₃ internal standard: TMS The results are shown in FIG.

2-9 Two-dimensional NMR spectrum HMB
C ( ¹ H-NMR 500 MHz, ¹³ C-NMR 1
25.65 MHz) Apparatus: JEOL ALPHA-500 Sample: 1.1 mg / 0.55 mL CDCL ₃ internal standard: TMS The results are shown in FIG.

2-10 HPLC apparatus: Toso-Tokyo CCPS (1) Column: 5-ODS-H 10 × 150 (W)
C / N H7A55 Solvent: acetonitrile / 50 mM ammonium formate Isocratec: 50% acetonitrile Flow rate: 3 mL / min Retention time: 20 minutes Detector: UV wavelength: 254 nm (2) Column: 5-ODS-H 10 × 150 ( W)
C / N H7A55 Solvent: acetonitrile / 50 mM ammonium formate Isocratec: 52% acetonitrile Flow rate: 3 mL / min Retention time: 20 minutes Detector: UV wavelength: 254 nm to 370 nm Each of the above conditions (1) to (370) At the indicated retention times, the benzopyran derivative showed a single peak.

2-11 Photodiode Array (U
V measurement) Apparatus: JASCO MD-910 Column: S-C18 250 mm × 4.6 φ Solvent: acetonitrile / 50 mM ammonium formate Gradient: 40% to 60% acetonitrile Flow rate: 1 mL / min Retention time: 20 minutes Detector: UV Wavelength: 254 nm

From these data analysis, it was found that the benzopyran derivative of the present invention is 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid represented by the chemical formula (1) (molecular formula: C ₁₉ H ₂₂
O ₃ , molecular weight: 298.3).

Example 3 Antitumor Cell Action on Cultured Tumor Cells Using the present benzopyran derivative obtained in Example 1 as a test substance, a cytotoxicity test using the cell growth inhibition of tumor cells as an index was performed as follows. went. Trypsin-treated human lung cancer cultured cells in the logarithmic growth phase (HLC-2 strain)
Was adjusted to 1 × 10 ⁵ cells / mL, and 0.05 mL was inoculated into each well of a 96-well microplate, and pre-cultured overnight. MEM-α medium (Gibco) containing 10% fetal bovine serum and L-glutamic acid supplemented with antibiotics
Was used. The test substance was appropriately diluted to each concentration while being shielded from light with the same culture solution, and 0.05 mL was added to each well to make the total volume 0.1 mL.

After culturing the plate in a carbon dioxide gas incubator at 37 ° C. for 72 hours, a cell proliferation measurement kit (C
(Etn Counting Kit, Dotite), 0.01 mL of each of the same color reagents was added while shielding from light, and a color reaction was performed for 1 to 2 hours in a 37 ° C. carbon dioxide incubator. Then, with a microplate reader,
The absorbance was measured at a wavelength of 450 nm. The absorbance of untreated cells was compared with that of cells treated with a test substance at a known concentration, and the cell growth inhibition rate was calculated according to the following equation.

[0039]

(Equation 1)

From the obtained growth inhibition rate, the cell growth was determined to be 5
The concentration of the test substance that inhibits 0% (IC ₅₀ ) was calculated. Table 3 shows the results. As is clear from Table 3, the present benzopyran derivative showed an excellent growth inhibitory effect on tumor cells.

[0041]

[Table 3]

The benzopyran derivative of the present invention, 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid, exhibits antitumor activity and can be used as an antitumor agent. In addition, it can be expected as an antitumor agent with high safety and few side effects.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of liquid chromatography / mass spectrometry and ionization (ESI) of the present benzopyran derivative.

FIG. 2 is a graph showing the results of gas chromatography / mass spectrometry and ionization (EI, m / z) of the present benzopyran derivative.

FIG. 3 is a diagram showing the results of a photodiode array (UV measurement) of the present benzopyran derivative.

FIG. 4 is a diagram showing a result of ¹ H _- NMR spectrum (500 MHz) of the present benzopyran derivative.

FIG. 5 is a diagram showing a result of a ¹³ C _- NMR spectrum (125.65 MHz) of the present benzopyran derivative.

[6] of the benzopyran derivatives two-dimensional NMR spectra _{^{NOESY (1 H - NMR500MHz, 13}} C -
It is a figure of the result of NMR 125.65MHz).

[7] of the benzopyran derivatives two-dimensional NMR spectra _{^{HMBC (1 H - NMR500MHz),}} 13 C -
FIG. 15 is a partial view of the result of NMR (125.65 MHz).

[Explanation of symbols]

 1 Absorbance of this benzopyran derivative 2 Absorbance of photoisomeric substance

[Procedure amendment]

[Submission date] August 23, 1999 (1999.8.2
3)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction contents]

[Document Name] Statement

Title of the Invention Bioactivity of Propolis-Derived Benzopyran Derivatives

[Claims]

Embedded image

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an antitumor agent of 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid represented by the formula (I): It relates to uses and manufacturing methods.

Embedded image

[0002]

2. Description of the Related Art Antitumor agents are used for treating cancer and are roughly classified into chemotherapeutic agents and immunotherapy agents. As chemotherapeutic agents, alkylating agents (nitrogen mustard, melpheran, cyclophosphamide), anticancer antibiotics (adriamycin, bleomycin, nelcarzinostatin), antimetabolites (methotrexate, 5-fluorouracil, 6- Mercaptopurines, hormonal agents (estrogens, androgens, prostins), plant-derived substances (vinblastine, camptothecin, etoposide), enzymes (asparaginase), and others (procarpazine, hydroxyurea, cisplatin) are mainly used clinically. However, chemotherapeutics are used for a wide range of cancers,
Despite its effects, no true anticancer drug has yet been developed that selectively attacks only cancer cells, has no toxicity in normal cells, and thus has no side effects. As a result, care must be taken for long-term administration.

[0003] Recently, research and development of drugs or therapeutic methods that change the host's responsiveness to tumor cells with the expectation of a therapeutic effect on cancer have been proposed and development has begun. This type includes immunotherapeutic agents having an immunostimulatory effect. Immunotherapeutic agents enhance immunity, act on immune response cells, and exert anticancer power. Compared with chemotherapeutic agents, immunotherapeutic agents have fewer side effects, but their antitumor effect is mild. Therefore, it is used as an adjuvant therapy.

As described above, conventional antitumor agents have many problems. Therefore, it is desired to develop an inexpensive antitumor agent which has a high antitumor effect, has few side effects, and is highly safe to enhance immunity.

[0005] Propolis is a gelatinous substance formed by mixing plant-derived resins collected by honeybees with their own secretions. Since ancient times, it has been used as a folk remedy showing pharmacological activities such as antibacterial and anti-inflammatory activities mainly in Eastern European countries.
Recently, propolis has been consumed as a dietary supplement. As a result, an antitumor effect was recognized, and an antitumor active substance was isolated and identified from the extract. (Japanese Patent Laid-Open No. 5-
58943, JP-A-5-27031, JP-A-9-
No. 143179)

The present invention also extracts and purifies from propolis in order to discover a novel physiologically active substance having an antitumor activity. As a substance having an antitumor activity, 2,2-dimethyl-8- represented by the chemical formula (I) is used. (3-methyl-2-butenyl)
Benzopyran-6-cis-propenoic acid was found.

An analog of the benzopyran derivative of the present invention, 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid, is PHY
TOCHMISTRY, 25 (4), 883-889,
1986 and 22 (10), 1969S4K, many derivatives are already known. However, the benzopyran derivative of the present invention, 2,2-dimethyl-8-
The physiological activity of (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid is not yet known,
It is a novel finding that it has antitumor activity.

[0008]

The present invention relates to a benzopyran derivative, 2,2-dimethyl-8- (3-methyl-2-
An object of the present invention is to provide (butenyl) benzopyran-6-cis-propenoic acid and an antitumor agent containing the same as a main component. Further, the present invention provides a novel method for producing a benzopyran derivative, 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid, and a photoisomer produced by a photoreaction under visible light. The purpose is to prove that it is a substance.

[0009]

The benzopyran derivative of the present invention, 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid, is represented by the chemical formula (I).

Embedded image

[0010]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a benzopyran derivative,
2,2-dimethyl-8- (3-methyl-2-butenyl)
It is used as an antitumor agent containing benzopyran-6-cis-propenoic acid and a salt thereof as an active ingredient. Salts include pharmaceutically acceptable salts.

[0011] The present invention also provides (1) pulverizing propolis with a mixer, extracting with methanol, removing impurities in a methanol suspension, obtaining a methanol extract of propolis, and then solvent of the obtained methanol extract. Is concentrated under reduced pressure to obtain a methanol extract of propolis,
(2) The above methanol extract is applied to an adsorption column using a silica gel column, and from 100% methyl chloride, a mixed solution of methyl chloride and methanol (95%: 5% v / v), and then methyl chloride and methanol (90%: 10% v / v) gradient elution with a concentration gradient using a mixed solution, and fractionation of each fraction, (3) the methyl chloride concentration of 100% to 95%
% Fraction, developing solvents methyl chloride and methanol (95%: 5
% V / v), the solvent was concentrated under reduced pressure, and the resulting residue was dissolved in a mixture of methanol and distilled water (50%: 50% v / v). The resulting solution was subjected to liquid chromatography using an ODS-based column, and eluted with a mixture of acetonitrile and 50 mM ammonium formate (52%: 48% v / v).
A step of collecting a main fraction, (4) a step of irradiating the solution of the main fraction with ultraviolet light with a transilluminator at 366 nm for 10 minutes to obtain a photoisomer fraction, and (5) a step of obtaining a solution of the fraction. The solvent acetonitrile was distilled off under reduced pressure while shielding the light, and the resulting residue was converted to a 50% v / v methanol solution, subjected to liquid chromatography using an ODS column, and acetonitrile and 50 mM ammonium formate (50%: 50% v / v) were used. / V) eluted with a mixture of
A step of fractionating each of the photoisomer fractions while shielding light, (6)
The solvent acetonitrile in the solution of each of the above fractions was distilled off under reduced pressure while shielding from light, and 100% methyl chloride was added thereto to carry out liquid separation.
An organic solvent layer is obtained, and the process is repeated three times. The collected organic solvent layer is concentrated under reduced pressure while shielding the organic solvent layer from light to obtain a benzopyran derivative represented by the chemical formula (I). It is also a production method for extracting and purifying a benzopyran derivative from propolis.

The silica gel used in step (2) is commercially available. Especially in the present application, silica gel 75-150 μm
(Manufactured by Kanto Chemical Co.) is preferred.

The columns used in the steps (3) and (5) are for high performance liquid chromatography, and a commercially available ODS silica gel column can be used as the reversed phase column. In the present invention, in particular, the column is equilibrated with acetonitrile. 5-
ODS-H columns (Chemco Bond) are preferred.

As shown in the Examples, the benzopyran derivative of the present invention, 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid, is an antitumor cell Since it has an action, it is considered that it shows a very effective antitumor cell effect by administration in various modes. The method for administering the present compound may be parenteral administration or oral administration. The composition to be administered contains a therapeutically effective amount of the compound and a pharmaceutically acceptable diluent, stabilizer, excipient, etc. Is contained. Examples of the administration form include parenteral administration such as intravenous injection, subcutaneous injection, intramuscular injection, suppository, and ointment, and oral administration using tablets, powders, capsules, granules and the like.

At present, the present benzopyran derivative, 2,2
The safety of -dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid when administered to humans is unknown. However, toxicity was not observed when 10 to 15 g / kg of propolis itself was orally administered to dogs, rats and guinea pigs for several months (PR
OPOLIS: 2ND ed. , Y. Donadie
U, 1983), it is considered that the safety is high even if the benzopyran derivative is administered to humans.

As described above, it has been reported that the propolis extract has various effects such as anti-inflammatory, anti-ulcer, anti-tumor, macrophage activation, cancer metastasis suppression, elimination of active oxygen, virus growth inhibition, and hair growth. (Japanese Patent Laid-Open No. 5-2
71031, JP-A-5-316968), the benzopyran derivative of the present invention extracted from propolis,
2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid can also be expected to have these effects.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are for illustrative purposes only and do not limit the present invention in any way.

【Example】

Example 1 Extraction of benzopyran derivative from propolis 2,2-dimethyl-8- (3-methyl-
Purification of 2-butenyl) benzopyran-6-cis-propenoic acid is carried out in the following [Step 1] to [Step 1].
5]. [Step 1] 250 g of propolis is pulverized with a mixer, mixed with 1500 mL of 99.5% methanol, and mixed for about 1 hour.
Stored at room temperature for months. The resulting suspension was filtered under reduced pressure to obtain a methanol extract of propolis. Next, the solvent of the obtained methanol extract was distilled off under reduced pressure.

[Step 2] About 1 g of the residue obtained in Step 1 was converted into a methanol solution, and 100 g of silica gel was used.
After that, 300 mL of 100% methyl chloride, which is three times the column volume, was flowed, and methyl chloride and methanol (9
Gradient elution with a mixture of 5%: 5% v / v) followed by a gradient of methyl chloride and methanol (90%: 10% v / v), spotting on a thin layer plate, The mixture was developed with a developing solvent of methyl and methanol (97%, 3% v / v), and elution fractions having an Rf value of 0.3 were collected.

[Step 3] The solvent of the fraction obtained in Step 2 was distilled off under reduced pressure to obtain about 20 mg of a residue. Then, the obtained residue was made into a 50% methanol solution, and the obtained solution was equilibrated with acetonitrile, and an ODS-based silica gel column for high performance liquid chromatography was used.
The mixture was injected into an H column (Chemco Bond), and the main fraction was collected.

[Step 4] The fraction obtained in Step 3 together with the solvent was directly irradiated with ultraviolet light by a transilluminator at a wavelength of 366 nm for 10 minutes, and the solvent acetonitrile in this solution was distilled off under reduced pressure while shielding from light.

[Step 5] The residue solution obtained in Step 4 was converted into a 50% methanol solution, and the obtained solution was equilibrated with acetonitrile. The ODS silica gel column for high performance liquid chromatography, the 5-ODS-H column (Chemcobond) ), And the photoisomer fractions were fractionated while shielding light.

[Step 6] The acetonitrile content of each solution obtained in Step 5 was distilled off under reduced pressure while shielding from light, and methyl chloride was added to carry out a liquid separation to obtain an organic solvent layer. It was repeated three times. The collected organic solvent layer was distilled off under reduced pressure while shielding from light to obtain about 1.1 mg of a residue. Due to the physicochemical properties of this residue, it was found that it was 2,2-dimethyl-8- (3
-Methyl-2-butenyl) benzopyran-6-cis-propenoic acid.

Example 2 Physicochemical Properties of Benzopyran Derivative 2-1 Photosteady Reaction The present benzopyran derivative and a photoisomer substance (JP-A-9-14)
No. 3179) causes photoisomerization under visible light.
In fact, it was confirmed that the reaction reached a complete equilibrium by UV irradiation of a 366 nm transilluminator.
Mass spectrum, NMR measurement, and the like were consistent with optical equilibrium. The present benzopyran derivative is stable under light shielding. 2-2 Solubility The present benzopyran derivative is insoluble in water and soluble in methanol, ethanol, acetonitrile, chloroform, and ethyl acetate.

2-3 Mass Spectrum Apparatus: API165 Perkin E1mer Ionization method: ESI 297.3 ([MH] ⁻ ) The results are shown in FIG.

2-4 Mass spectrum Apparatus: JEOL JMS-AX505HA Ionization method: EI m / z [M] (70 eV) The results are shown in FIG.

2-5 Photodiode Array (UV
Measurement) Apparatus: JASCO MD-910 The results are shown in FIG.

2-6 ¹ H-NMR spectrum (500
MHz) Apparatus: JEOL ALPHA-500 Sample: 1.1 mg / 0.55 mL CDCL ₃ internal standard: TMS The results are shown in FIG. The assignment of each peak is shown in the table below.

[0029]

[Table 1]

2-7 ¹³ C-NMR spectrum (1
25.65 MHz) Apparatus: JEOL ALPHA-500 Sample: 1.1 mg / 0.55 mL CDCL ₃ internal standard: TMS The results are shown in FIG. The assignment of each peak is shown in the table below.

[0031]

[Table 2]

2-8 Two-dimensional NMR spectrum NOE
SY ( ¹ H-NMR 500 MHz, ¹³ C-NMR
125.65 MHz) Apparatus: JEOL ALPHA-500 Sample: 1.1 mg / 0.55 mL CDCL ₃ internal standard: TMS The results are shown in FIG.

2-9 Two-dimensional NMR spectrum HMB
C ( ¹ H-NMR 500 MHz, ¹³ C-NMR
125.65 MHz) Apparatus: JEOL ALPHA-500 Sample: 1.1 mg / 0.55 mL CDCL ₃ internal standard: TMS The results are shown in FIG.

2-10 HPLC apparatus: Toso-Tokyo CCPS (1) Column: 5-ODS-H 10 × 150 (W)
C / N H7A55 Solvent: acetonitrile / 50 mM ammonium formate Isocratec: 50% acetonitrile Flow rate: 3 mL / min Retention time: 20 minutes Detector: UV wavelength: 254 nm (2) Column: 5-ODS-H 10 × 150 ( W)
C / N H7A55 Solvent: acetonitrile / 50 mM ammonium formate Isocratec: 52% acetonitrile Flow rate: 3 mL / min Retention time: 20 minutes Detector: UV wavelength: 254 nm to 370 nm Each of the above conditions (1) to (370) At the indicated retention times, the benzopyran derivative showed a single peak.

2-11 Photodiode Array (UV
Measurement) Apparatus: JASCO MD-910 Column: S-C18 250 mm × 4.6 φ Solvent: acetonitrile / 50 mM ammonium formate Gradient: 40% to 60% acetonitrile Flow rate: 1 mL / min Retention time: 20 minutes Detector: UV wavelength : 254 nm

From these data analysis, it was found that the benzopyran derivative of the present invention is 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid represented by the chemical formula (1) (molecular formula: C ₁₉ H ₂₂
O ₃ , molecular weight: 298.3).

Example 3 Antitumor Cell Action on Cultured Tumor Cells Using the present benzopyran derivative obtained in Example 1 as a test substance, a cytotoxicity test using the cell growth inhibition of tumor cells as an index was performed as follows. went. Trypsin-treated human lung cancer cultured cells in the logarithmic growth phase (HLC-2 strain)
Was adjusted to 1 × 10 ⁵ cells / mL, and 0.05 mL was inoculated into each well of a 96-well microplate, and pre-cultured overnight. MEM-α medium (Gibco) containing 10% fetal bovine serum and L-glutamic acid supplemented with antibiotics
Was used. The test substance was appropriately diluted to each concentration while being shielded from light with the same culture solution, and 0.05 mL was added to each well to make the total volume 0.1 mL.

After culturing the plate in a carbon dioxide gas incubator at 37 ° C. for 72 hours, a cell proliferation measurement kit (C
(Etn Counting Kit, Dotite), 0.01 mL of each of the same color reagents was added while shielding from light, and a color reaction was performed for 1 to 2 hours in a 37 ° C. carbon dioxide incubator. Then, with a microplate reader,
The absorbance was measured at a wavelength of 450 nm. The absorbance of untreated cells was compared with that of cells treated with a test substance at a known concentration, and the cell growth inhibition rate was calculated according to the following equation.

[0039]

(Equation 1)

From the obtained growth inhibition rate, the cell growth was determined to be 5
The concentration of the test substance that inhibits 0% (IC ₅₀ ) was calculated. Table 3 shows the results. As is clear from Table 3, the present benzopyran derivative showed an excellent growth inhibitory effect on tumor cells.

[0041]

[Table 3]

The benzopyran derivative of the present invention, 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-6-cis-propenoic acid, exhibits antitumor activity and can be used as an antitumor agent. In addition, it can be expected as an antitumor agent with high safety and few side effects.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of liquid chromatography / mass spectrometry and ionization (ESI) of the present benzopyran derivative.

FIG. 2 is a graph showing the results of gas chromatography / mass spectrometry and ionization (EI, m / z) of the present benzopyran derivative.

FIG. 3 is a diagram showing the results of a photodiode array (UV measurement) of the present benzopyran derivative.

[Explanation of symbols] 1. Absorbance of this benzopyran derivative 2. Absorbance of photoisomer substance

FIG. 4 is a diagram of a ¹ H-NMR spectrum (500 MHz) of the present benzopyran derivative.

FIG. 5 is a diagram showing a result of a ¹³ C-NMR spectrum (125.65 MHz) of the present benzopyran derivative.

6 two-dimensional NMR spectra NOESY of the benzopyran derivatives ^{(1 H-NMR500MHz, 13} C
-NMR 125.65 MHz).

[7] the two-dimensional NMR spectra HMBC of the benzopyran derivatives ^{(1 H-NMR500MHz,} 13 C-
FIG. 15 is a partial view of the result of NMR (125.65 MHz).

Claims (4)

[Claims] 1. A benzopyran derivative represented by the formula (I) and a salt thereof: 2. A pharmaceutical comprising a benzopyran derivative represented by the formula (I) and a salt thereof as an active ingredient. 3. An antitumor agent comprising a benzopyran derivative represented by the formula (I) and a salt thereof as an active ingredient. 4. A production method for extracting and purifying a benzopyran derivative represented by the chemical formula (I) from propolis through the following steps: (1) Propolis is pulverized with a mixer, methanol is extracted, and methanol suspension is obtained. Removing the impurities to obtain a methanol extract of propolis, and then concentrating the solvent of the obtained methanol extract under reduced pressure to obtain a methanol extract of propolis; (2) adsorbing the methanol extract with a silica gel column; From a 100% methyl chloride column, methyl chloride and methanol (95%: 5% v /
v), then methyl chloride and methanol (90%:
10% v / v) gradient elution with a concentration gradient using a mixed solution, and fractionation of each fraction, (3) the above-mentioned 100% to 95% methyl chloride concentration fraction, a developing solvent methyl chloride and methanol ( 95%: 5% v / v) (Rf value of 0.
3) was collected, the solvent was concentrated under reduced pressure, and the obtained residue was dissolved in a mixed solution of methanol and distilled water (50%: 50% v / v). Chromatography with acetonitrile and 50
mM ammonium formate (52%: 48% v /
e) eluting with the mixed solution of v) and collecting a main fraction, (4)
A step of irradiating the solution of the main fraction with ultraviolet light for 10 minutes using a transilluminator at a wavelength of 366 nm to obtain a photoisomer fraction; , Then the resulting residue 5
A 0% v / v methanol solution was prepared and subjected to liquid chromatography using an ODS-based column.
0 mM ammonium formate (50%: 50% v /
e) eluting with the mixture of v) and separating each photoisomer fraction while shielding the light; (6) distilling off the solvent acetonitrile in each solution of each of the fractions under reduced pressure while shielding from light to obtain 100% A step of obtaining an organic solvent layer by adding methyl chloride and separating the mixture, repeating the process three times, and concentrating the collected organic solvent layer under reduced pressure while shielding light to obtain a benzopyran derivative represented by the chemical formula (I).