JPH09143179A - Benzopyran derivative derived from propolis - Google Patents
Benzopyran derivative derived from propolisInfo
- Publication number
- JPH09143179A JPH09143179A JP7329583A JP32958395A JPH09143179A JP H09143179 A JPH09143179 A JP H09143179A JP 7329583 A JP7329583 A JP 7329583A JP 32958395 A JP32958395 A JP 32958395A JP H09143179 A JPH09143179 A JP H09143179A
- Authority
- JP
- Japan
- Prior art keywords
- solvent
- chloroform
- propolis
- benzopyran derivative
- methanol
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
Classifications
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、化学式（Ｉ）で表
わされる３−［２−ジメチル−８−（３−メチル−２−
ブテニル）ベンゾピラン］−６−プロペノイック酸（以
下ベンゾピラン誘導体と略す）、及びその抗腫瘍剤とし
ての用途及び製造方法に関する。TECHNICAL FIELD The present invention relates to 3- [2-dimethyl-8- (3-methyl-2-) represented by the chemical formula (I).
The present invention relates to butenyl) benzopyran] -6-propenoic acid (hereinafter abbreviated as benzopyran derivative), its use as an antitumor agent, and a production method.
【化２】 Embedded image
【０００２】[0002]
【従来の技術】癌の治療に用いられる抗腫瘍剤は大きく
化学療法剤と免疫療法剤に別けられる。化学療法剤は切
除不能な癌に投与されるだけでなく、術前や術後に投与
することにより、外科療法と組合せても使用されてい
る。例えば、癌化学療法剤としては、アルキル化剤（ニ
トロジエンマスタード類、エチレンイミン類、スルホン
酸エステル類等）、代謝拮抗物質（葉酸拮抗剤、ピリミ
ジン拮抗剤等）、植物性核***毒（コルセミド、ビンブ
ラスチン等）、抗生物質（ザルコマイシン、カルチノフ
ィリン、マイトマイシン等）、ホルモン剤（副腎皮質ス
テロイド、男性ホルモン、女性ホルモン等）、及びポル
フィリン錯塩（モーフィリン、copp）等が臨床上使用さ
れている。しかし、化学療法剤は細胞毒性物質であるこ
とが多く、癌細胞を攻撃するのみでなく、正常細胞にも
作用し、一般に強い副作用を伴う。例えば、嘔吐、悪
心、食欲不振、倦怠感、神経障害、骨髄障害（白血球減
少）、脱毛、口内炎等の副作用がみられる場合もある。
そのため、長期投与にあたっては注意が必要である。2. Description of the Related Art Antitumor agents used for treating cancer are largely divided 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.
【０００３】免疫療法剤は主に免疫賦活剤で、免疫力を
高めることにより、免疫応答細胞に癌細胞を異物として
認識させ、これを治療する。しかし免疫賦活剤は副作用
は少ないが、一般的には抗腫瘍効果が穏やかで、補助的
療法として用いられることが多い。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.
【０００４】このように従来の抗腫瘍剤には一長一短が
有り、それゆえ、抗腫瘍活性が高く、副作用が少なく、
長期投与が可能な抗腫瘍剤の開発が望まれていた。また
抗腫瘍剤は一般に高価であるので、患者の経済的負担を
軽減するために、それが安価に供給されることも望まれ
ている。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 inexpensively supplied in order to reduce the financial burden on the patient.
【０００５】民間療法剤として用いられているプロポリ
スは、蜜蜂が集めた草木の成分と唾液、蜜蝋、花粉等が
混合された樹脂状物質で、それに抗菌作用、抗炎症作用
などの薬理作用があることが知られている。また、プロ
ポリスを健康補助食品として飲用すると抗腫瘍効果が現
れることが知られており、プロポリスより抗腫瘍性活性
物質を抽出・精製する試みがなされ（特開平５−５８９
４３号、特開平５−２７１０３１号）、抽出物には抗腫
瘍作用が認められている。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).
No. 43, JP-A-5-271031), the extract has an antitumor effect.
【０００６】本発明者も抗腫瘍作用を有する新規生理活
性物質を発見すべく、プロポリスより抽出精製を行い、
抗腫瘍活性を有する物質として化学式（Ｉ）で表わされ
るベンゾピラン誘導体を見出した。The present inventor has also extracted and purified from propolis in order to discover a novel physiologically active substance having an antitumor effect,
The benzopyran derivative represented by the chemical formula (I) was found as a substance having antitumor activity.
【０００７】本発明のベンゾピラン誘導体の類似物質
は、ＰＨＹＴＯＣＨＥＭＩＳＴＲＹ，２５（４），８８
３−８８９，１９８６ 及び ＡＵＳＴ．Ｊ．ＣＨＥ
Ｍ．，２２（１０），１９６９ 等の文献で報告され、
既に数多くの誘導体が知られている。しかし本発明のベ
ンゾピラン誘導体は未だ知られておらず、またそれが抗
腫瘍活性を有することは当然のことながら新規な知見で
ある。The analogs of the benzopyran derivative of the present invention are PHYTOCHEMISTRY, 25 (4), 88.
3-889, 1986 and AUST. J. CHE
M. , 22 (10), 1969 etc.,
Many derivatives are already known. However, the benzopyran derivative of the present invention has not been known yet, and it is a novel finding that it has antitumor activity.
【０００８】[0008]
【発明が解決しようとする課題】本発明は、新規なベン
ゾピラン誘導体を提供し、それを主成分とする抗腫瘍剤
を提供することを目的とする。さらにベンゾピラン誘導
体の新規な製造方法を提供することも目的とする。DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel benzopyran derivative and an antitumor agent containing it as a main component. Another object is to provide a novel method for producing a benzopyran derivative.
【０００９】[0009]
【課題を解決するための手段】本発明のベンゾピラン誘
導体は次の化学式（Ｉ）で表される。The benzopyran derivative of the present invention is represented by the following chemical formula (I).
【化３】 Embedded image
【００１０】[0010]
【発明の実施の形態】本発明は、ベンゾピラン誘導体ま
たはその塩を有効成分として含有してなる抗腫瘍剤とし
て用いられる。塩としては製薬学的に許容される塩類が
含まれる。BEST MODE FOR CARRYING OUT THE INVENTION The present invention is used as an antitumor agent containing a benzopyran derivative or a salt thereof as an active ingredient. The salts include pharmaceutically acceptable salts.
【００１１】また本発明は（１） プロポリスのエタノ
ール懸濁液の不溶物を除去し、プロポリスのエタノール
抽出液を得、次いで得られたエタノール抽出液の溶媒を
蒸発除去し、得られた残渣を水と酢酸エチルの混液で抽
出し、酢酸エチル抽出液を得、得られた酢酸エチル抽出
液の溶媒を蒸発除去し、得られた残渣をメタノールに分
散させ、不溶物を除去し、プロポリスのメタノール抽出
液を得る工程、（２） 前記メタノール抽出液を逆相系
カラムによる液体クロマトグラフィーにかけ、７０％〜
１００％のメタノール濃度勾配による傾斜溶離を行い、
メタノール濃度９０〜９５％の画分を分取する工程、
（３） 前記メタノール濃度９０〜９５％画分の溶媒を
蒸発除去し、次いで得られた残渣をクロロホルムに溶解
し、得られた溶液を吸着系カラムによる液体クロマトグ
ラフィーにかけ、クロロホルムで溶出し、主画分を分取
する工程、（４） 前記画分の溶媒を蒸発除去し、次い
で得られた残渣をクロロホルムに溶解し、得られた溶液
を分子篩系カラムによる液体クロマトグラフィーにか
け、クロロホルムで溶出し、主画分を分取し、溶媒を蒸
発除去し、無色の針状結晶物質としてベンゾピラン誘導
体を得る工程よりなる、プロポリスからベンゾピラン誘
導体を抽出・精製する製造方法でもある。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 methanol concentration 90-95% fraction was removed by evaporation, 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, (4) The solvent of the 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 benzopyran derivative from propolis, which comprises a step of collecting a main fraction, removing a solvent by evaporation to obtain a benzopyran derivative as a colorless needle-shaped crystal substance.
【００１２】工程（２）で使用するカラムは高速液体ク
ロマトグラフィー用であり、逆相系カラムとしては市販
のＯＤＳ系シリカゲルカラムが使用可能であり、特に本
願には７０％メタノ−ルで平衡化したＯＤＳ ８０ＴＭ
カラム（東ソー社製）が好ましい。The column used in the step (2) is for high performance liquid chromatography, and a commercially available ODS silica gel column can be used as the reverse phase column, and in the present application, it is equilibrated with 70% methanol. ODS 80TM
A column (manufactured by Tosoh Corporation) is preferable.
【００１３】工程（３）で使用するカラムは高速液体ク
ロマトグラフィー用であり、吸着系カラムとしては市販
のＩｎｅｒｔｓｉｌ系カラムが使用可能であり、特に本
願にはクロロホルムで平衡化したＩｎｅｒｔｓｉｌ Ｓ
ＩＬカラム（ジーエルサイエンス社製）が好ましい。The column used in the step (3) is for high performance liquid chromatography, and a commercially available Inertsil type column can be used as an 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.
【００１４】工程（４）で使用するカラムは高速液体ク
ロマトグラフィー用であり、分子篩系カラムとしては市
販のＧＰＣ系カラムが使用可能であり、特に本願にはク
ロロホルムで平衡化したＳｈｏｄｅｘ ＧＰＣ−Ｈ２０
００カラム（昭和電工社製）が好ましい。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.
【００１５】本発明のベンゾピラン誘導体は実施例に示
すように腫瘍細胞に対し抗腫瘍細胞作用を有するので、
様々な態様で投与することにより極めて有効な抗腫瘍効
果を示すと考えられる。本化合物を投与するための方法
は非経口投与、または経口投与が考えられ、投与される
組成物には治療上有効量の本化合物と薬理上許容される
希釈剤、安定剤、賦形剤等が含有される。投与形態とし
ては、静脈内注射、皮下注射、筋肉注射、座薬、軟膏等
の非経口投与法、錠剤、散剤、カプセル剤、顆粒剤等に
よる経口投与法が挙げられる。Since the benzopyran derivative of the present invention has an antitumor cell action on tumor cells as shown in Examples,
It is considered that when it is administered in various modes, it exhibits an extremely effective antitumor effect. 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. Examples of the dosage form include intravenous injection, subcutaneous injection, intramuscular injection, suppository, ointment and other parenteral administration methods, and tablets, powders, capsules, granules and other oral administration methods.
【００１６】現時点では本ベンゾピラン誘導体をヒトに
投与した場合の安全性は不明である。しかし、プロポリ
スそのもの１０〜１５ｇ／ｋｇをイヌ、ラットおよびモ
ルモットに数ヶ月間経口投与しても毒性は見られなかっ
た（ＰＲＯＰＯＬＩＳ：２ＮＤ ｅｄ．，Ｙ．ＤＯＮＡ
ＤＩＥＵ，１９８３）ことより、ベンゾピラン誘導体を
ヒトに投与しても安全性は高いと考えられる。[0016] At present, the safety of administering this benzopyran derivative to humans is unknown. However, 10 to 15 g / kg of propolis itself was orally administered to dogs, rats and guinea pigs for several months without showing toxicity (PROPOLIS: 2ND ed., Y. DONA.
According to DIEU, 1983), it is considered that the safety is high even if the benzopyran derivative is administered to humans.
【００１７】また前述の如くプロポリス抽出物には抗菌
作用、抗酸化作用、抗炎症作用、ウイルス増殖抑制作
用、マクロファージ活性化作用、育毛作用等が知られて
おり（特開平５−２７１０３１、特開平５−３１６９６
８等）、プロポリスより抽出された本発明のベンゾピラ
ン誘導体にもそれらの作用が期待できる。As described above, the propolis extract 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-5-271031). 5-31696
8), and those effects can be expected in the benzopyran derivative of the present invention extracted from propolis.
【００１８】以下、実施例に基づき本発明をさらに詳細
に説明する。なお、下記実施例は単に説明のためのもの
であり、本発明を何ら限定するものではない。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.
【００１９】実施例１ プロポリスからベンゾピラン誘
導体の抽出 プロポリスから３−［２−ジメチル−８−（３−メチル
−２−ブテニル）ベンゾピラン］−６−プロペノイック
酸の精製は、次の［ステップ １］〜［ステップ ５］
を通して実施した。 ［ステップ １］プロポリス１００ｇを１０倍量の９
９．５％エタノールと混合し、マグネチックスターラー
を用いて室温下で攪拌した。得られた懸濁液を減圧ろ過
してプロポリスのエタノール抽出液を得た。次いで得ら
れたエタノール抽出液の溶媒をローターリーエバポレー
ターを用い蒸発除去し、得られた残渣約３０ｇを水と酢
酸エチル混液（１：１）に分散し、上層の酢酸エチル層
を分取しプロポリスの酢酸エチル抽出液を得た。得られ
た酢酸エチル抽出液の溶媒をロータリーエバポレーター
で蒸発除去し、得られた残渣約２０ｇを９９．５％メタ
ノールに分散させ、不溶物を低速遠心で取り除き、プロ
ポリスのメタノール抽出液を得た。Example 1 Extraction of Benzopyran Derivative from Propolis The purification of 3- [2-dimethyl-8- (3-methyl-2-butenyl) benzopyran] -6-propenoic acid from propolis was carried out by the following [Step 1] to [Step 5]
Through. [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, and 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 of 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.
【００２０】［ステップ ２］ステップ １で得られた
メタノール抽出液を７０％メタノールで平衡化した高速
液体クロマトグラフィー用ＯＤＳ系カラム、ＯＤＳ ８
０ＴＭカラム（東ソー社製）に注入し、７０％〜１００
％のメタノール濃度勾配による傾斜溶離を行った。メタ
ノール濃度９０〜９５％の画分を分取した。[Step 2] ODS 8 column for high performance liquid chromatography in which the methanol extract obtained in Step 1 was equilibrated with 70% methanol, ODS 8
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.
【００２１】［ステップ ３］ステップ ２で得られた
メタノール濃度９０〜９５％の画分を１つに集め、溶媒
をロータリーエバポレーターで蒸発除去し、残渣約２ｇ
を得た。次いで得られた残渣をクロロホルムに溶解し、
得られた溶液をクロロホルムで平衡化した高速液体クロ
マトグラフィー用Ｉｎｅｒｔｓｉｌ系カラム、Ｉｎｅｒ
ｔｓｉｌ ＳＩＬカラム（ジーエルサイエンス社製）に
注入し、主画分を分取した。[Step 3] Fractions having a methanol concentration of 90 to 95% obtained in Step 2 were collected, and the solvent was removed by evaporation using a rotary evaporator.
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 the main fraction was separated.
【００２２】［ステップ ４］ステップ ３で得られた
画分を１つに集め、溶媒をロータリーエバポレーターで
蒸発除去し、残渣約５０ｍｇを得た。次いで得られた残
渣をクロロホルムに溶解し、得られた溶液をクロロホル
ムで平衡化した高速液体クロマトグラフィー用ＧＰＣ系
カラム、Ｓｈｏｄｅｘ ＧＰＣ−Ｈ２０００カラム（昭
和電工社製）に注入し、主画分を分取した。[Step 4] The fractions obtained in Step 3 were combined and the solvent was removed by evaporation on a rotary evaporator to give a residue of about 50 mg. Next, the obtained residue was dissolved in chloroform, and the obtained solution was injected into a GPC system column for high performance liquid chromatography, Shodex GPC-H2000 column (manufactured by Showa Denko KK) equilibrated with chloroform, and the main fraction was separated. I took it.
【００２３】［ステップ ５］ステップ ４で得られた
画分を１つに集め、溶媒をロータリーエバポレーターで
蒸発除去し、無色の針状結晶物質約１５ｍｇを得た。そ
の針状晶物質の実施例２に示す理化学的性質より、それ
が３−［２−ジメチル−８−（３−メチル−２−ブテニ
ル）ベンゾピラン］−６−プロペノイック酸であること
を確認した。[Step 5] The fractions obtained in Step 4 were combined and the solvent was removed by evaporation on a rotary evaporator to give about 15 mg of colorless needle-like crystalline substance. From the physicochemical properties of the acicular substance shown in Example 2, it was confirmed that it was 3- [2-dimethyl-8- (3-methyl-2-butenyl) benzopyran] -6-propenoic acid.
【００２４】実施例２ ベンゾピラン誘導体の理化学的
性質 ２−１ 融点 １１３〜１１５℃Example 2 Physicochemical properties of benzopyran derivative 2-1 Melting point 113-115 ° C.
【００２５】２−２ 溶解性 本ベンゾピラン誘導体は、水に不溶でアルカリで可溶化
し、酸性では溶解しない。またメタノール、エタノー
ル、アセトン、エーテルに僅かに溶け、アセトニトリル
に可溶、クロロホルム、酢酸エチル、ヂメチルホルムア
ミドに易溶、ｎ−ヘキサン、石油エーテルに不溶であ
る。2-2 Solubility This benzopyran derivative is insoluble in water, solubilized by alkali, and not soluble in acid. It is slightly soluble in methanol, ethanol, acetone and ether, soluble in acetonitrile, easily soluble in chloroform, ethyl acetate and dimethylformamide, and insoluble in n-hexane and petroleum ether.
【００２６】２−３ マススペクトル 装置：Finnigan ＭＡＴ ＩＮＣＯＳ ５０ イオン化法：ＥＩ （７０ｅＶ） 結果を図１に示す。2-3 Mass spectrum device: Finnigan MAT INCOS 50 ionization method: EI (70 eV) The results are shown in FIG.
【００２７】２−４ 1Ｈ−ＮＭＲスペクトル（４００
ＭＨｚ） 装置：JEOL ＪＮＭ−ＧＳＸ４００ サンプル：３．２７ｍｇ／０．５５ｍｌ ＣＤＣＬ3 内部標準：ＴＭＳ 結果を図２に示す。また各ピークの帰属を下表に示す2-4 1 H-NMR spectrum (400
MHz) Device: JEOL JNM-GSX400 Sample: 3.27 mg / 0.55 ml CDCL 3 internal standard: TMS The results are shown in FIG. The table below shows the attribution of each peak.
【００２８】[0028]
【表１】 [Table 1]
【００２９】２−５ ＨＰＬＣ 装置：Shimadzu ＣＬ−４Ａ （１）カラム：ＯＤＳ ８ＯＴＭ ２１．５×３００ｍ
ｍ 溶媒：７０→１００％メタノール グラジエント（１２
０分） 流速：４ｍｌ／分 保持時間：約８０分 （２）カラム：Ｉｎｅｒｔｓｉｌ ＳＩＬ １０×２５
０ｍｍ 溶媒：クロロホルム 流速：１０ｍｌ／分 保持時間：５．５〜６分 （３）カラム：ＧＰＣ−Ｈ２０００ ２０×５００ｍｍ 溶媒：クロロホルム 流速：３．５ｍｌ／分 保持時間：約２０分 上記（１）〜（３）の条件でそれぞれ示した保持時間に
本ベンゾピラン誘導体は単一のピークを示した。2-5 HPLC apparatus: Shimadzu CL-4A (1) Column: ODS 8OTM 21.5 × 300 m
m solvent: 70 → 100% methanol gradient (12
Flow rate: 4 ml / min Retention time: about 80 minutes (2) Column: Inertsil SIL 10 × 25
0 mm Solvent: Chloroform Flow rate: 10 ml / min Retention time: 5.5 to 6 minutes (3) Column: GPC-H2000 20 × 500 mm Solvent: Chloroform Flow rate: 3.5 ml / min Retention time: Approximately 20 minutes (1) to The benzopyran derivative showed a single peak at the retention times shown under the condition (3).
【００３０】これらのデータの解析より本発明のベンゾ
ピラン誘導体は化学式（Ｉ）で示される３−［２−ジメ
チル−８−（３−メチル−２−ブテニル）ベンゾピラ
ン］−６−プロペノイック酸（分子式：Ｃ19Ｈ22Ｏ3、
分子量：２９８．３８）と同定した。From the analysis of these data, the benzopyran derivative of the present invention shows that 3- [2-dimethyl-8- (3-methyl-2-butenyl) benzopyran] -6-propenoic acid (molecular formula: C 19 H 22 O 3 ,
Molecular weight: 298.38) was identified.
【００３１】実施例３ 培養腫瘍細胞に対する抗腫瘍細
胞作用 実施例１で得られたベンゾピラン誘導体を被験物質とし
て用いて、以下のようにして腫瘍細胞の細胞損傷活性試
験を行った。９６穴マイクロタイタープレートの各ウエ
ルに１０％ウシ胎児血清及び２ｍＭグルタミンを含むＭ
ＥＭ培地により適宜希釈した被験物質０．１ｍｌずつ添
加後、トリプシン処理したヒト肝ガン ＨｕＨ１３株
（Ｊ． ＣＥＬＬＵＬＡＲ ＰＨＹＳＩＯＬ．， ＶＯ
Ｌ １４８， ２９０−２９４， １９９１）を上記培
養液で３×１０4個／ｍｌに調製し、０．０５ｍｌずつ
分注した。Example 3 Antitumor Cell Action on Cultured Tumor Cells Using the benzopyran derivative obtained in Example 1 as a test substance, a tumor cell cytotoxicity test was conducted as follows. M containing 10% fetal bovine serum and 2 mM glutamine in each well of a 96-well microtiter plate
Human liver cancer HuH13 strain (J. CELLURAL PHYSIOL., VO) treated with trypsin after adding 0.1 ml each of the test substances appropriately diluted with EM medium
L 148, 290-294, 1991) was prepared at 3 × 10 4 cells / ml with the above-mentioned culture solution and dispensed in 0.05 ml portions.
【００３２】該プレートを炭酸ガスインキュベーター内
で３７℃、７２時間培養後、培養上清を除去し、０．０
２％ニュートラルレッドを含む培養液を０．１ｍｌずつ
各ウエルに加え、３７℃で１時間炭酸ガスインキュベー
ター内で培養し、細胞を染色した。培養上清を除去後、
残渣を生理食塩水で１回洗浄した。次いで０．００１規
定塩酸／３０％エタノールで色素を抽出後、マイクロプ
レートリーダーにより５５０ｎｍの吸光度を測定した。
無処理細胞と既知濃度の被験物質で処理した細胞との吸
光度を比較して次式に従って細胞の増殖阻止率を算出し
た。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.
【００３３】[0033]
【数１】 (Equation 1)
【００３４】得られた増殖阻止率から、細胞の増殖を５
０％阻害する被験物質濃度（ＩＤ50）を算出した。結果
を表２に示す。表２から明らかなようにベンゾピラン誘
導体は腫瘍細胞に対し、優れた増殖阻止作用を示した。From the obtained growth inhibition rate, the cell growth was determined to be 5
The concentration of the test substance that caused 0% inhibition (ID 50 ) was calculated. Table 2 shows the results. As is clear from Table 2, the benzopyran derivative showed an excellent growth inhibitory action on tumor cells.
【００３５】[0035]
【表２】 [Table 2]
【００３６】[0036]
【発明の効果】本発明のベンゾピラン誘導体（３−［２
−ジメチル−８−（３−メチル−２−ブテニル）ベンゾ
ピラン］−６−プロペノイック酸）は、抗腫瘍活性を示
し、抗腫瘍剤として使用できる。また安全性が高く副作
用の少ない抗腫瘍剤として期待できる。The benzopyran derivative (3- [2
-Dimethyl-8- (3-methyl-2-butenyl) benzopyran] -6-propenoic acid) exhibits antitumor activity and can be used as an antitumor agent. It can also be expected as an antitumor agent with high safety and few side effects.
【図１】ベンゾピラン誘導体のマススペクトルFIG. 1 Mass spectrum of benzopyran derivative
【図２】ベンゾピラン誘導体の1Ｈ−ＮＭＲスペクトルFIG. 2 1 H-NMR spectrum of benzopyran derivative
Claims (4)
誘導体またはその塩 【化１】 1. A benzopyran derivative represented by the chemical formula (I) or a salt thereof:
誘導体またはその塩を有効成分として含有してなる医薬
品2. A drug containing a benzopyran derivative represented by the chemical formula (I) or a salt thereof as an active ingredient.
誘導体またはその塩を有効成分として含有してなる抗腫
瘍剤3. An antitumor agent comprising a benzopyran derivative represented by the chemical formula (I) or a salt thereof as an active ingredient.
（Ｉ）で表わされるベンゾピラン誘導体を抽出・精製す
る製造方法 （１） プロポリスのエタノール懸濁液の不溶物を除去
し、プロポリスのエタノール抽出液を得、次いで得られ
たエタノール抽出液の溶媒を蒸発除去し、得られた残渣
を水と酢酸エチルの混液で抽出し、酢酸エチル抽出液を
得、得られた酢酸エチル抽出液の溶媒を蒸発除去し、得
られた残渣をメタノールに分散させ、不溶物を除去し、
プロポリスのメタノール抽出液を得る工程、（２） 前
記メタノール抽出液を逆相系カラムによる液体クロマト
グラフィーにかけ、７０％〜１００％のメタノール濃度
勾配による傾斜溶離を行い、メタノール濃度９０〜９５
％の画分を分取する工程、（３） 前記メタノール濃度
９０〜９５％画分の溶媒を蒸発除去し、次いで得られた
残渣をクロロホルムに溶解し、得られた溶液を吸着系カ
ラムによる液体クロマトグラフィーにかけ、クロロホル
ムで溶出し、主画分を分取する工程、（４） 前記画分
の溶媒を蒸発除去し、次いで得られた残渣をクロロホル
ムに溶解し、得られた溶液を分子篩系カラムによる液体
クロマトグラフィーにかけ、クロロホルムで溶出し、主
画分を分取し、溶媒を蒸発除去し、化学式（Ｉ）で表わ
されるベンゾピラン誘導体を得る工程4. A production method for extracting and purifying a benzopyran derivative represented by the chemical formula (I) from propolis via the following steps: (1) An insoluble matter of an ethanol suspension of propolis is removed, and an ethanol extract of propolis is obtained. Then, the solvent of the obtained ethanol extract was removed by evaporation, the obtained residue was extracted with a mixture of water and ethyl acetate to obtain an ethyl acetate extract, and the solvent of the obtained ethyl acetate extract was evaporated. The resulting residue was dispersed in methanol to remove insoluble materials,
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 to collect 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 benzopyran derivative represented by the chemical formula (I).
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7329583A JPH09143179A (en) | 1995-11-24 | 1995-11-24 | Benzopyran derivative derived from propolis |
| PCT/JP1996/003860 WO1998029404A1 (en) | 1995-11-24 | 1996-12-27 | Benzopyran derivative originating in propolis |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7329583A JPH09143179A (en) | 1995-11-24 | 1995-11-24 | Benzopyran derivative derived from propolis |
| PCT/JP1996/003860 WO1998029404A1 (en) | 1995-11-24 | 1996-12-27 | Benzopyran derivative originating in propolis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH09143179A true JPH09143179A (en) | 1997-06-03 |
Family
ID=26437287
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7329583A Pending JPH09143179A (en) | 1995-11-24 | 1995-11-24 | Benzopyran derivative derived from propolis |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPH09143179A (en) |
| WO (1) | WO1998029404A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006213609A (en) * | 2005-02-01 | 2006-08-17 | Iwate Univ | Activating agent of immunocompetent cell, method for preventing feline immunodeficiency virus infection by using the same, method for eliminating feline immunodeficiency virus, method for preventing cancer growth and method for eliminating cancer |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3035846B2 (en) * | 1998-09-28 | 2000-04-24 | 三佳子 廣田 | Bioactivity of benzopyran derivatives derived from propolis |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0749365B2 (en) * | 1991-07-03 | 1995-05-31 | 日本プロポリス株式会社 | Method for producing propolis extract |
| JPH0819039B2 (en) * | 1991-08-29 | 1996-02-28 | 哲也 松野 | New bioactive substance derived from propolis |
| JPH07330596A (en) * | 1994-06-01 | 1995-12-19 | Eiken Chem Co Ltd | Antitumor agent |
-
1995
- 1995-11-24 JP JP7329583A patent/JPH09143179A/en active Pending
-
1996
- 1996-12-27 WO PCT/JP1996/003860 patent/WO1998029404A1/en active Application Filing
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006213609A (en) * | 2005-02-01 | 2006-08-17 | Iwate Univ | Activating agent of immunocompetent cell, method for preventing feline immunodeficiency virus infection by using the same, method for eliminating feline immunodeficiency virus, method for preventing cancer growth and method for eliminating cancer |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1998029404A1 (en) | 1998-07-09 |
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