WO2001060359A1 - Activite physiologique d'un derive de benzopyrane issu de la propolis - Google Patents

Activite physiologique d'un derive de benzopyrane issu de la propolis Download PDF

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Publication number
WO2001060359A1
WO2001060359A1 PCT/JP2000/000896 JP0000896W WO0160359A1 WO 2001060359 A1 WO2001060359 A1 WO 2001060359A1 JP 0000896 W JP0000896 W JP 0000896W WO 0160359 A1 WO0160359 A1 WO 0160359A1
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WIPO (PCT)
Prior art keywords
methanol
benzopyran derivative
propolis
reduced pressure
under reduced
Prior art date
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PCT/JP2000/000896
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English (en)
Japanese (ja)
Inventor
Mikako Hirota
Original Assignee
Mikako Hirota
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to JP10309339A priority Critical patent/JP3035846B2/ja
Application filed by Mikako Hirota filed Critical Mikako Hirota
Priority to PCT/JP2000/000896 priority patent/WO2001060359A1/fr
Publication of WO2001060359A1 publication Critical patent/WO2001060359A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the use and production of 2,2-dimethyl-8- (3-methyl-2-buteninole) benzopyran-16-cis-propionic acid represented by the chemical formula (I) as an antitumor agent About the method.
  • Antineoplastic agents are used to treat cancer and are broadly divided into chemotherapeutic agents and immunotherapy agents.
  • Chemotherapeutic agents include alkylating agents (nitrogen mustard, melpheran, cyclophosphamide), anticancer antibiotics (adriamycin, bleomycin, nercarzinostatin), and antimetabolites (metabolic agents).
  • Immunotherapeutic agents having an immunostimulatory effect.
  • Immunotherapeutic agents enhance immunity, act on immune response cells, and exert anticancer effects. Compared with chemotherapeutic agents, immunotherapy has fewer side effects but mild antitumor effects. Therefore, it is used as a rescue therapy.
  • Propolis is a glue made from the mixture of vegetable fats collected by honeybees and their 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 Unexamined Patent Publication No. Hei 5-58943 Japanese Unexamined Patent Publication No. Hei 5-271031, Japanese Unexamined Patent Publication No. Hei 9-1143179
  • An object of the present invention is to provide a novel benzopyran derivative, an antitumor agent comprising 2,2-dimethyl-8- (3-methyl-2-butul) benzopyran-16-cis-1-propionic acid as a main component. .
  • the benzopyran derivative of the present invention 2,2-dimethyl-8- (3-methyl-12-butyr) benzopyran-16-cis-propionic acid, is represented by the chemical formula (I).
  • the present invention provides an antitumor agent comprising a benzopyran derivative, 2,2-dimethyl-8- (3-methyl-2-butul) benzopyran-16-cis-propionic acid and a salt thereof as an active ingredient. Used. Salts include pharmaceutically acceptable salts.
  • the present invention provides (1) pulverizing propolis with a mixer, extracting with methanol, removing impurities in a methanol suspension, obtaining a methanol extract of propolis, and then removing a solvent of the obtained methanol extract. Is concentrated under reduced pressure to obtain a methanol extract of propolis.
  • the methanol extract is applied to an adsorption system using a silica gel and a 100% methyl chloride. Gradient elution with a mixture of methyl chloride and methanol (95%: 5% v / v) followed by a mixture of methyl chloride and methanol (90%: 10% v / v).
  • the solvent acetonitril content of the solution was distilled off under reduced pressure while shielding the light, and the resulting residue was converted into a 50% v / v methanol solution, which was used for liquid chromatography using an ODS column to perform liquid chromatography.
  • the silica gel used in step (2) is commercially available. Particularly, silica gel of 75 to 150 ⁇ m (manufactured by Kanto Chemical Co., Ltd.) is preferable in the present application.
  • step (3) and step (5) are for high-performance liquid chromatography, and commercially available ODS-based silica gel columns can be used for the reversed-phase column.
  • ODS-based silica gel columns can be used for the reversed-phase column.
  • a 5-ODS-H column (Chemcobond) equilibrated with acetonitrile is preferred.
  • FIG. 1 shows the results of liquid cloning chromatography, Z-mass analysis, and ionization (ESI) of the present benzopyran derivative.
  • Figure 2 shows the results of gas chromatography mass spectrometry and ionization (EI, m / z) of the benzopyran derivative.
  • Fig. 3 shows the results of photodiode array (UV measurement) of the benzopyran derivative.
  • the symbol (1) in this figure is the absorbance of the present benzopyran derivative, and the symbol (2) is the absorbance of the photoisomer of the substance.
  • FIG. 4 is a diagram showing the result of 1 H-NMR spectrum (500 MHz) of the present benzopyran derivative.
  • FIG. 4 is a diagram showing the result of 1 H-NMR spectrum (500 MHz) of the present benzopyran derivative.
  • FIG. 5 is a diagram of the result of a “C-NMR spectrum (125.655 MHz) of the present benzopyran derivative.
  • FIG. 6 is a two-dimensional NMR spectrum of the present benzopyran derivative.
  • DOO Norre NO ESY ( 'H-NMR 500 MHz, 13 C - NMR 125.65MH z).
  • diagrams the results of Figure 7 is of the base Nzopiran derivative two-dimensional NMR space-vector HMB C (1 H - NMR 500 H z, 13 C - .
  • NMR 125.65MHz) is a partial view of the results of the invention bEST mODE fOR CARRYING OUT tHE
  • Step 1 250 g of propolis was ground with a mixer, mixed with 1500 mL of 99.5% methanol, and left at room temperature for about one month. Obtained suspension The 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 Approximately 1 g of the residue obtained in Step 1 is converted into a methanol solution, and is sucked into 100 g of gel. And gradient elution was performed with a concentration gradient of a mixture of methyl chloride and methanol (95%: 5% v / v) and then a mixture of methyl chloride and methanol (90%: 10% v / v). Then, it was spotted on a thin layer plate and developed with a developing solvent of methyl chloride and methanol (97%: 3% v / v), and elution fractions having an R f 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. The resulting residue was then converted to a 50% methanol solution, and the resulting solution was applied to an ODS silica gel column for high-performance liquid chromatography and equilibrated with acetonitrile, and a 5-ODS-H column (Chemco pound). It was injected and the main fraction was collected.
  • Step 4 The fraction obtained in Step 3 together with the solvent was directly irradiated with ultraviolet light for 10 minutes using a transilluminator with a wavelength of 366 ° C, and the solvent was distilled off under reduced pressure while shielding the acetonitril content of the solvent from light.
  • Step 5 The residue solution obtained in Step 4 was converted to a 50% methanol solution, and the obtained solution was equilibrated with acetonitrile.
  • Step 6 The acetonitrile component of each solution obtained in Step 5 was distilled off under reduced pressure while shielding 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 the light, to obtain about 1.1 mg of a residue. Due to the physicochemical properties of this residue, it is 2,2-dimethyl-8- (3-methyl-12-butenyl) benzopyra It was confirmed to be 1-6-cis-propionic acid.
  • the present benzopyran derivative and a photoisomer substance undergo photoisomerization under visible light.
  • the reaction reached a complete equilibrium by UV irradiation of the 366 nm transilluminator, and the UV, mass spectrum, NMR measurement, etc. agreed with the optical equilibrium.
  • This benzopyran derivative is stable under light shielding.
  • This benzopyran derivative is insoluble in water and soluble in methanol, ethanol, acetonitril, chloroform, and ethyl acetate.
  • TM S Fig. 4 shows the results. The assignment of each peak is shown in the table below.
  • Fig. 5 shows the results. The assignment of each peak is shown in the following table c (Table 2) The 13 C-NMR assignments
  • Fig. 6 shows the results.
  • Fig. 7 shows the results.
  • the benzopyran derivative showed a single peak at the retention times indicated under the conditions (1) and (2) above.
  • the benzopyran derivative of the present invention was found to have 2,2-dimethyl-8- (3-methyl-2-buturyl) benzopyran-6-cis-propinoic acid represented by the chemical formula (1) (molecular formula: C 19 H 22 0 3, molecular weight: 2 9 8.3) and was identified.
  • Example 1 Using the present benzopyran derivative obtained in Example 1 as a test substance, a cytotoxicity test was performed using the inhibition of tumor cell proliferation as an index as follows.
  • Human lung cancer cultured cells trypsinized in the logarithmic growth phase (HL C-2 strain) was prepared in 1 X 1 0 5 cells / m L, 0. 0 5 mL each of 9 6-well microtiter plates Ueru Each was seeded and pre-cultured.
  • a MEM- ⁇ medium (Gibco) containing 10% fetal bovine serum and L-glutamic acid supplemented with antibiotics was used as a culture solution.
  • the test substance should be shielded with the same culture medium as appropriate. After dilution to each strain, 0.05 mL was added to the same well to make the total volume 0.5 mL.
  • HLC—2 shares 1 8
  • the benzopyran derivative of the present invention 2,2-dimethyl-8- (3-methyl-12-butyr) benzopyran-16-cis-propenoic acid, exhibits antitumor activity and can be used as an antitumor agent . It is also expected to be an antitumor agent with high safety and few side effects.
  • the benzopyran derivative of the present invention 2,2-dimethyl-8- (3-methyl-2-butur) benzopyran-16-cis-propinoic acid, has an antitumor cell effect on tumor cells as shown in Example 3. Therefore, it is considered that administration in various forms shows a very effective antitumor cell effect.
  • the method of administering the compound may be parenteral or oral, and the composition administered may include a therapeutically effective amount of the compound and a pharmaceutically acceptable diluent, stabilizer, or excipient. Etc. are contained.
  • the dosage form include intravenous injection, subcutaneous injection, intramuscular injection, parenteral administration such as suppositories and ointments, and oral administration using tablets, powders, capsules, granules and the like.
  • the propolis extract has various effects such as anti-inflammatory, anti-ulcer, anti-tumor, macrophage activation, cancer metastasis suppression, active oxygen elimination, virus growth suppression, and hair growth.
  • Japanese Unexamined Patent Publication No. 5-2711031 Japanese Unexamined Patent Publication No. 5-3166968, etc.
  • the action of the benzopyran derivative of the present invention 2,2-dimethyl-8- (3-methyl-2-butenyl) benzopyran-16-cis-propionic acid, can be expected.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Pyrane Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

L'invention concerne des agents antitumoraux contenant en tant qu'ingrédient actif le composé (I), de l'acide 2,2-diméthyl-8-(3-méthyl-2-butényl)benzopyrane-6-cis-propénoïque, ou des sels pharmaceutiquement acceptables dudit composé. Ce composé (I) est un dérivé de benzopyrane obtenu à partir de la propolis par purification. Il est utilisé comme agent antitumoral et présente un degré d'innocuité élevé.
PCT/JP2000/000896 1998-09-28 2000-02-17 Activite physiologique d'un derive de benzopyrane issu de la propolis WO2001060359A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP10309339A JP3035846B2 (ja) 1998-09-28 1998-09-28 プロポリス由来のベンゾピラン誘導体の生理活性
PCT/JP2000/000896 WO2001060359A1 (fr) 1998-09-28 2000-02-17 Activite physiologique d'un derive de benzopyrane issu de la propolis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP10309339A JP3035846B2 (ja) 1998-09-28 1998-09-28 プロポリス由来のベンゾピラン誘導体の生理活性
PCT/JP2000/000896 WO2001060359A1 (fr) 1998-09-28 2000-02-17 Activite physiologique d'un derive de benzopyrane issu de la propolis

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WO2001060359A1 true WO2001060359A1 (fr) 2001-08-23

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Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3035846B2 (ja) * 1998-09-28 2000-04-24 三佳子 廣田 プロポリス由来のベンゾピラン誘導体の生理活性
JP2006213609A (ja) * 2005-02-01 2006-08-17 Iwate Univ 免疫担当細胞の活性化剤、これを用いたネコ免疫不全ウイルス感染の予防方法ならびにネコ免疫不全ウイルスの駆逐方法および癌発生の予防方法ならびに癌の駆逐方法
KR100618497B1 (ko) 2005-02-28 2006-08-31 거창군 (관리부서 : 거창군 농업기술센터) 프로폴리스에서 퀘르세틴을 추출하는 방법

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998029404A1 (fr) * 1995-11-24 1998-07-09 Eiken Kagaku Kabushiki Kaisha Derive de benzopyranne obtenu a partir de propolis
JP2000103789A (ja) * 1998-09-28 2000-04-11 Mikako Hirota プロポリス由来のベンゾピラン誘導体の生理活性

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998029404A1 (fr) * 1995-11-24 1998-07-09 Eiken Kagaku Kabushiki Kaisha Derive de benzopyranne obtenu a partir de propolis
JP2000103789A (ja) * 1998-09-28 2000-04-11 Mikako Hirota プロポリス由来のベンゾピラン誘導体の生理活性

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
G.BOUDOUROVA-KRASTEVA ET AL: "Phenolics from Brazilian Propolis", Z. NATURFORSCH., vol. 52, no. 9/10, 1997, pages 676 - 679, XP002931785 *

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